Recombinant human antibody fragment against tetanus toxoid produced by phage display

PubMed Central

Neelakantam, B.; Sridevi, N. V.; Shukra, A. M.; Sugumar, P.; Samuel, S.

2014-01-01

Phage display technology is a powerful in vitro method for the identification of specific monoclonal antibodies (antibody fragments) to an antigenic target and allows the rapid generation and selection of high affinity, fully human antibodies directed toward any disease target appropriate for antibody therapy. In the present study, we exploited the phage display technology for the selection of an antigen binding fragment (Fabs) toward tetanus toxoid using human naïve phage antibody library constructed from peripheral blood lymphocytes of naïve human donors. The phages displaying Fab were subjected to three rounds of bio-panning with tetanus toxoid as antigen on a solid phase. The high affinity antibody fragments were expressed in HB2151 strain of Escherichia coli and purified by immobilized metal affinity chromatography. The binding activity and specificity of the antibody fragment was established by its reactivity toward tetanus toxoid and non-reactivity toward other related toxins as determined by enzyme-linked immunosorbent assay and immunoblot analysis. The selected Fab fragment forming the antigen-binding complexes with the toxoid in flocculation assay indicates that the Fab may have a potential neutralizing ability toward antigen. PMID:24678405

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Identification and Characterization of Anti-Platelet Antibodies in Idiopathic Thrombocytopenic Purpura Patients

PubMed Central

Aghabeigi, N; Lindsey, N; Zamani, A; Shishaeyan, B

2012-01-01

Background: The autoimmune disease known as Idiopathic (immune thrombocytopenic purpura thrombocytopenic purpura (ITP) is clinically defined by a low numbers of platelets in the circulation blood. This study aimed to isolate autoantibodies made against the platelet glycoproteins using platelets from healthy volunteers, to determine their specificity and further elucidate their effects on platelet function. Methods: This study used a phage display system to recognize Fab anti-platelet antibodies. Anti-platelet After isolation, the anti-platelet Fab-expressing phage was characterized by ELISA and Western blotting. The Fab-bearing phage pool obtained from five rounds of panning was analysed in order to determine its anti-platelet reactivity. Of the phage colonies obtained, 100 colonies of different sizes were randomly selected for reaction with whole platelets, using M13 phage as a negative control. Results: Twelve colonies of them had strong reactions against the whole platelet preparation, but only four colonies showed substantial reactivity against the lysed platelet preparation (lysate). Three of the four colonies showed three bands representing proteins with different molecular weights. The fourth colony showed only a single band. The final experiment to characterise the protein isolated from the phage library was a DNA gel agarose test. Conclusion: Each colony showed a DNA band that corresponded with the molecular size marker for 5.4 kbase pairs, and this suggested the presence of heavy and light antibody chains in the phage. PMID:23113135

Engineered antibodies for monitoring of polynuclear aromatic hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karu, A.E.; Roberts, V.A.; Li, Q.X.

1998-06-01

'The long-term goal of this project is to develop antibodies and antibody-based methods for detection and recovery of polynuclear aromatic hydrocarbons (PAHs) and PAH adducts that are potential biomarkers in environmental and biological samples. The inherent cross-reactivity will be exploited by pattern recognition methods. Dr. Karu''s laboratory uses new haptens representing key PAHs to derive recombinant Fab (rFab) and single-chain Fv (scFv) antibodies from hybridoma lines and combinatorial phage display libraries. Computational models of the haptens and combining sites made by Dr. Roberts''s group are used to guide antibody engineering by mutagenesis. Dr. Li''s laboratory develops enzyme immunoassays (EIAs), sensors,more » and immunoaffinity methods that make use of the novel haptens and antibodies for practical analytical applications in support of DOE''s mission. This report summarizes work completed in one and one-half years of a 3-year project, with close collaboration between the three research groups. Dr. Alexander Karu''s laboratory: the authors proceeded with the two strategies described in the original proposal. Site-directed mutagenesis was used to correct differences in the rFab N-terminal amino acids that were introduced by the degenerate PCR primers used for gene amplification. The binding constants of the rFabs with the corrected sequences will be compared with those of the parent MAbs, and should be very similar. The 4D5 and 10C10 heavy and light chain sequences are being moved to the pCOMB3H phagemid vector to facilitate selection of new engineered mutants.'« less

Designed Electroresponsive Biomaterials: Sequence-Controlled Behavior

DTIC Science & Technology

2010-06-29

protein of the M13 . Traditional phage and yeast display methodologies indicate that peptide sequences with high affinities for electrode materials...drug delivery. The original vision for this work was to employ combinatorial tools such as phage and yeast display under electrical selection pressure...and drug delivery. The original vision for this work was to employ combinatorial tools such as phage and yeast display under electrical selection

A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

PubMed

Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

2014-03-01

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer. © 2013 UICC.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

PubMed

Kulmala, Antti; Huovinen, Tuomas; Lamminmäki, Urpo

2017-06-19

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

Construction of Rabbit Immune Antibody Libraries.

PubMed

Nguyen, Thi Thu Ha; Lee, Jong Seo; Shim, Hyunbo

2018-01-01

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Selection and maturation of antibodies by phage display through fusion to pIX.

PubMed

Tornetta, Mark; Reddy, Ramachandra; Wheeler, John C

2012-09-01

Antibody discovery and optimization by M13 phage display have evolved significantly over the past twenty years. Multiple methods of antibody display and selection have been developed - direct display on pIII or indirect display through a Cysteine disulfide linkage or a coiled-coil adapter protein. Here we describe display of Fab libraries on the smaller pIX protein at the opposite end of the virion and its application to discovery of novel antibodies from naive libraries. Antibody selection based on pIX-mediated display produces results comparable to other in vitro methods and uses an efficient direct infection of antigen-bound phages, eliminating any chemical dissociation step(s). Additionally, some evidence suggests that pIX-mediated display can be more efficient than pIII-mediated display in affinity selections. Functional assessment of phage-derived antibodies can be hindered by insufficient affinities or lack of epitopic diversity. Here we describe an approach to managing primary hits from our Fab phage libraries into epitope bins and subsequent high-throughput maturation of clones to isolate epitope- and sequence-diverse panels of high affinity binders. Use of the Octet biosensor was done to examine Fab binding in a facile label-free method and determine epitope competition groups. A receptor extracellular domain and chemokine were subjected to this method of binning and affinity maturation. Parental clones demonstrated improvement in affinity from 1-100nM to 10-500pM. Copyright © 2012 Elsevier Inc. All rights reserved.

A universal phage display system for the seamless construction of Fab libraries.

PubMed

Nelson, Renae S; Valadon, Philippe

2017-11-01

The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-11-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed Central

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-01-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis. Images PMID:1438192

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

PubMed

Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

2011-01-01

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.

The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.

PubMed

Huovinen, Tuomas; Syrjänpää, Markku; Sanmark, Hanna; Seppä, Titta; Akter, Sultana; Khan, Liton Md Ferdhos; Lamminmäki, Urpo

2014-09-19

Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins. In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection. The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.

Nanotube Interactions with Nanoparticles and Peptides

DTIC Science & Technology

2008-01-01

combinatorial phage display technique. We find a tryptophan rich binding motif to nanotubes on solid silicon substrates. The motif resembles an alpha helix...CHAPTER 2. DIELECTROPHORESIS AND PHAGE DISPLAY 2.1. Dielectrophoresis (DEP) 12 2.2. Phage display 14 References...104 5.3. Conclusions 105 5.4. Experimental Section 105 5.4.1. Nanotube synthesis 105 5.4.2. Phage display

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

PubMed Central

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

Next generation phage display by use of pVII and pIX as display scaffolds.

PubMed

Løset, Geir Åge; Sandlie, Inger

2012-09-01

Phage display technology has evolved to become an extremely versatile and powerful platform for protein engineering. The robustness of the phage particle, its ease of handling and its ability to tolerate a range of different capsid fusions are key features that explain the dominance of phage display in combinatorial engineering. Implementation of new technology is likely to ensure the continuation of its success, but has also revealed important short comings inherent to current phage display systems. This is in particular related to the biology of the two most popular display capsids, namely pIII and pVIII. Recent findings using two alternative capsids, pVII and pIX, located to the phage tip opposite that of pIII, suggest how they may be exploited to alleviate or circumvent many of these short comings. This review addresses important aspects of the current phage display standard and then discusses the use of pVII and pIX. These may both complement current systems and be used as alternative scaffolds for display and selection to further improve phage display as the ultimate combinatorial engineering platform. Copyright © 2012 Elsevier Inc. All rights reserved.

Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro.

PubMed

Czerwinski, Marcin; Krop-Watorek, Anna; Wasniowska, Kazimiera; Smolarek, Dorota; Spitalnik, Steven L

2009-11-30

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kay, B. K.; Castagnoli, L.; Biosciences Division

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

Molecular Determinants of Estrogen Receptor Alpha Stability

DTIC Science & Technology

2008-07-01

presence of E2. This question can be addressed by a T7 phage display screen using a breast cancer cell library and DNA-bound ERα in the presence of...conformation of ERα induced by 27HC versus E2. To accomplish this, we performed combinatorial phage display using a modified M13 phage display screen

Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

PubMed

Bailey, Lucas J; Sheehy, Kimberly M; Dominik, Pawel K; Liang, Wenguang G; Rui, Huan; Clark, Michael; Jaskolowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A

2018-02-02

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biomolecular Recognition of Semiconductors and Magnetic Materials to Assemble Nanoparticle Heterostructures

DTIC Science & Technology

2002-01-01

shown that engineered viruses can recognize specific semiconductor surfaces through the selection by combinatorial phage display method. These specific... phage display libraries. The screening method selected for binding affinity of a population of random peptides displayed as part of the pIII minor coat...shorter spacing than expected distance ( M13 phage length: 880 nm) corresponds to the length scale imposed by the phage which formed the tilted

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

PubMed

Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

2011-10-28

Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library.

PubMed

Pitaksajjakul, Pannamthip; Lekcharoensuk, Porntippa; Upragarin, Narin; Barbas, Carlos F; Ibrahim, Madiha Salah; Ikuta, Kazuyoshi; Ramasoota, Pongrama

2010-05-14

Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development-three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin-Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

DTIC Science & Technology

2010-10-01

1012 Fabs displayed on the surface of phage amplified from a large naive library [35] were suspended in PBS with 2% dry milk and applied to wells coated...antigens were first incubated with the same amount of phage as in the plate format in 1 ml of PBS+2% dry milk suspension at room temperature for 2 hour...Fifteen ml of Dynabeads MyOne Streptavidin T1(Invitro- gen Dynal AS, Oslo, Norway) pre-blocked with PBS+2% dry milk was then added to the antigen/phage

Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat.

PubMed

Zhou, Bin; Wirsching, Peter; Janda, Kim D

2002-04-16

A naive, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of Bacillus subtilis IFO 3336 suspended in solution. A direct in vitro panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of B. subtilis IFO 3336 vs. a panel of spores from 11 other Bacillus species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with B. licheniformis 5A24. The Fab-phage clone 52 was improved compared with 5B and was comparable to 7E in binding B. subtilis IFO 3336 vs. B. licheniformis 5A24, yet showed a distinctive crossreactivity pattern with other spores. We also developed a method to directly detect individual spores by using fluorescently labeled antibody-phage. Finally, a variety of "powders" that might be used in deploying spores of B. anthracis were examined for antibody-phage binding. The strategies described provide a foundation to discover human antibodies specific for native spores of B. anthracis that can be developed as diagnostic and therapeutic reagents.

Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface.

PubMed

Hillig, Roman C; Urlinger, Stefanie; Fanghänel, Jörg; Brocks, Bodo; Haenel, Cornelia; Stark, Yvonne; Sülzle, Detlev; Svergun, Dmitri I; Baesler, Siegfried; Malawski, Guido; Moosmayer, Dieter; Menrad, Andreas; Schirner, Michael; Licha, Kai

2008-03-14

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.

Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization.

PubMed

Saito, Misa; Kondo, Masahiro; Ohshima, Motohiro; Deguchi, Kazuki; Hayashi, Hideki; Inoue, Kazuyuki; Tsuji, Daiki; Masuko, Takashi; Itoh, Kunihiko

2014-04-01

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

PubMed

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C Ronald; Sidhu, Sachdev S

2017-05-19

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis▿‡

PubMed Central

Chen, Zhaochun; Chumakov, Konstantin; Dragunsky, Eugenia; Kouiavskaia, Diana; Makiya, Michelle; Neverov, Alexander; Rezapkin, Gennady; Sebrell, Andrew; Purcell, Robert

2011-01-01

Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case. PMID:21345966

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

PubMed

Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

2016-01-01

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

Phage display for the discovery of hydroxyapatite-associated peptides.

PubMed

Jin, Hyo-Eon; Chung, Woo-Jae; Lee, Seung-Wuk

2013-01-01

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures. © 2013 Elsevier Inc. All rights reserved.

Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

NASA Astrophysics Data System (ADS)

Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

1993-05-01

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

PubMed

Tsurushita, N; Fu, H; Warren, C

1996-06-12

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

Selection of stably folded proteins by phage-display with proteolysis.

PubMed

Bai, Yawen; Feng, Hanqiao

2004-05-01

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae.

PubMed

Zhou, Yikang; Li, Gang; Dong, Junkai; Xing, Xin-Hui; Dai, Junbiao; Zhang, Chong

2018-05-01

Facing boosting ability to construct combinatorial metabolic pathways, how to search the metabolic sweet spot has become the rate-limiting step. We here reported an efficient Machine-learning workflow in conjunction with YeastFab Assembly strategy (MiYA) for combinatorial optimizing the large biosynthetic genotypic space of heterologous metabolic pathways in Saccharomyces cerevisiae. Using β-carotene biosynthetic pathway as example, we first demonstrated that MiYA has the power to search only a small fraction (2-5%) of combinatorial space to precisely tune the expression level of each gene with a machine-learning algorithm of an artificial neural network (ANN) ensemble to avoid over-fitting problem when dealing with a small number of training samples. We then applied MiYA to improve the biosynthesis of violacein. Feed with initial data from a colorimetric plate-based, pre-screened pool of 24 strains producing violacein, MiYA successfully predicted, and verified experimentally, the existence of a strain that showed a 2.42-fold titer improvement in violacein production among 3125 possible designs. Furthermore, MiYA was able to largely avoid the branch pathway of violacein biosynthesis that makes deoxyviolacein, and produces very pure violacein. Together, MiYA combines the advantages of standardized building blocks and machine learning to accelerate the Design-Build-Test-Learn (DBTL) cycle for combinatorial optimization of metabolic pathways, which could significantly accelerate the development of microbial cell factories. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display.

PubMed

Baskar, Sivasubramanian; Suschak, Jessica M; Samija, Ivan; Srinivasan, Ramaprasad; Childs, Richard W; Pavletic, Steven Z; Bishop, Michael R; Rader, Christoph

2009-11-12

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

PubMed

Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik; Jensen, Anders A; Gill, Avinash; Madden, Dean R; Kastrup, Jette S; Skottrup, Peter D

2016-11-01

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

Mapping protease substrates using a biotinylated phage substrate library.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scholle, M. D.; Kriplani, U.; Pabon, A.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobicmore » is the scissile bond.« less

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

2016-01-01

The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

PubMed

Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

2018-05-01

Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis

NASA Astrophysics Data System (ADS)

Ghosh, Eshan; Srivastava, Ashish; Baidya, Mithu; Kumari, Punita; Dwivedi, Hemlata; Nidhi, Kumari; Ranjan, Ravi; Dogra, Shalini; Koide, Akiko; Yadav, Prem N.; Sidhu, Sachdev S.; Koide, Shohei; Shukla, Arun K.

2017-12-01

Beta-arrestins (βarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of βarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of βarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted βarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting βarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

PubMed Central

Staquicini, Fernanda I.; Ozawa, Michael G.; Moya, Catherine A.; Driessen, Wouter H.P.; Barbu, E. Magda; Nishimori, Hiroyuki; Soghomonyan, Suren; Flores, Leo G.; Liang, Xiaowen; Paolillo, Vincenzo; Alauddin, Mian M.; Basilion, James P.; Furnari, Frank B.; Bogler, Oliver; Lang, Frederick F.; Aldape, Kenneth D.; Fuller, Gregory N.; Höök, Magnus; Gelovani, Juri G.; Sidman, Richard L.; Cavenee, Webster K.; Pasqualini, Renata; Arap, Wadih

2010-01-01

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors. PMID:21183793

Tumor-targeting peptides from combinatorial libraries*

PubMed Central

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S.

2018-01-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges infighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. PMID:27210583

Tumor-targeting peptides from combinatorial libraries.

PubMed

Liu, Ruiwu; Li, Xiaocen; Xiao, Wenwu; Lam, Kit S

2017-02-01

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges in fighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors. Copyright © 2017. Published by Elsevier B.V.

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

Synthetic Fab Fragments that Bind the HIV-1 gp41 Heptad Repeat Regions

PubMed Central

Liu, Yanyun; Regula, Lauren K.; Stewart, Alex; Lai, Jonathan R.

2011-01-01

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F. A. Fellouse, B. Li, D. M. Compaan, A. A. Peden, S. G. Hymowitz, and S. S. Sidhu, J. Mol. Biol., 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications. PMID:21925149

Characterization of novel Staphylococcus aureus lytic phage and defining their combinatorial virulence using the OmniLog® system

PubMed Central

Estrella, Luis A.; Quinones, Javier; Henry, Matthew; Hannah, Ryan M.; Pope, Robert K.; Hamilton, Theron; Teneza-mora, Nimfa; Hall, Eric; Biswajit, Biswas

2016-01-01

ABSTRACT Skin and soft tissue infections (SSTI) caused by methicillin resistant Staphylococcus aureus (MRSA) are difficult to treat. Bacteriophage (phage) represent a potential alternate treatment for antibiotic resistant bacterial infections. In this study, 7 novel phage with broad lytic activity for S. aureus were isolated and identified. Screening of a diverse collection of 170 clinical isolates by efficiency of plating (EOP) assays shows that the novel phage are virulent and effectively prevent growth of 70–91% of MRSA and methicillin sensitive S. aureus (MSSA) isolates. Phage K, which was previously identified as having lytic activity on S. aureus was tested on the S. aureus collection and shown to prevent growth of 82% of the isolates. These novel phage group were examined by electron microscopy, the results of which indicate that the phage belong to the Myoviridae family of viruses. The novel phage group requires β-N-acetyl glucosamine (GlcNac) moieties on cell wall teichoic acids for infection. The phage were distinct from, but closely related to, phage K as characterized by restriction endonuclease analysis. Furthermore, growth rate analysis via OmniLog® microplate assay indicates that a combination of phage K, with phage SA0420ᶲ1, SA0456ᶲ1 or SA0482ᶲ1 have a synergistic phage-mediated lytic effect on MRSA and suppress formation of phage resistance. These results indicate that a broad spectrum lytic phage mixture can suppress the emergence of resistant bacterial populations and hence have great potential for combating S. aureus wound infections. PMID:27738555

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

Phage protein-targeted cancer nanomedicines

PubMed Central

Petrenko, V.A.; Jayanna, P.K.

2015-01-01

Nanoencapsulation of anticancer drugs improves their therapeutic indices by virtue of the enhanced permeation and retention effect which achieves passive targeting of nanoparticles in tumors. This effect can be significantly enhanced by active targeting of nanovehicles to tumors. Numerous ligands have been proposed and used in various studies with peptides being considered attractive alternatives to antibodies. This is further reinforced by the availability of peptide phage display libraries which offer an unlimited reservoir of target-specific probes. In particular landscape phages with multivalent display of target-specific peptides which enable the phage particle itself to become a nanoplatform creates a paradigm for high throughput selection of nanoprobes setting the stage for personalized cancer management. Despite its promise, this conjugate of combinatorial chemistry and nanotechnology has not made a significant clinical impact in cancer management due to a lack of using robust processes that facilitate scale-up and manufacturing. To this end we proposed the use of phage fusion protein as the navigating modules of novel targeted nanomedicine platforms which are described in this review. PMID:24269681

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

PubMed

Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

2017-10-06

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli , had unprecedentedly high binding affinities ( K d ∼10 -12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

Using Phage Display to Create Recombinant Antibodies.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors. The phagemid is packaged into phage particles with the assistance of a helper phage to produce the antibody display phage. This protocol describes a method for creating a phagemid library. The multiple cloning site (MCS) of the pBluescript KS(-) phagemid vector is replaced by digestion with the restriction enzyme BssHII, followed by the insertion of four overlapping oligonucleotides to create a new MCS within the vector. Next, the 3' portion of gene III (from M13mp18) is amplified and combined with an antibody sequence using overlap extension PCR. This product is inserted into the phagemid vector to create pPDS. Two helper plasmids are also created from the modified pBluescript vector: pLINK provides the linker between the heavy and light chains, and pFABC provides the CH1 domain of the heavy chain. An antibody cDNA library is constructed from the RNA of interest and ligated into pPDS. The phagemid library is electroporated into Escherichia coli cells along with the VCS-M13 helper phage. © 2017 Cold Spring Harbor Laboratory Press.

Affinity maturation of a portable Fab–RNA module for chaperone-assisted RNA crystallography

PubMed Central

Koirala, Deepak; Shelke, Sandip A; Dupont, Marcel; Ruiz, Stormy; DasGupta, Saurja; Bailey, Lucas J; Benner, Steven A; Piccirilli, Joseph A

2018-01-01

Abstract Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography. PMID:29309709

Phage display as a promising approach for vaccine development.

PubMed

Aghebati-Maleki, Leili; Bakhshinejad, Babak; Baradaran, Behzad; Motallebnezhad, Morteza; Aghebati-Maleki, Ali; Nickho, Hamid; Yousefi, Mehdi; Majidi, Jafar

2016-09-29

Bacteriophages are specific antagonists to bacterial hosts. These viral entities have attracted growing interest as optimal vaccine delivery vehicles. Phages are well-matched for vaccine design due to being highly stable under harsh environmental conditions, simple and inexpensive large scale production, and potent adjuvant capacities. Phage vaccines have efficient immunostimulatory effects and present a high safety profile because these viruses have made a constant relationship with the mammalian body during a long-standing evolutionary period. The birth of phage display technology has been a turning point in the development of phage-based vaccines. Phage display vaccines are made by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response. Also, the ability to produce combinatorial peptide libraries with a highly diverse pool of randomized ligands has transformed phage display into a straightforward, versatile and high throughput screening methodology for the identification of potential vaccine candidates against different diseases in particular microbial infections. These libraries can be conveniently screened through an affinity selection-based strategy called biopanning against a wide variety of targets for the selection of mimotopes with high antigenicity and immunogenicity. Also, they can be panned against the antiserum of convalescent individuals to recognize novel peptidomimetics of pathogen-related epitopes. Phage display has represented enormous promise for finding new strategies of vaccine discovery and production and current breakthroughs promise a brilliant future for the development of different phage-based vaccine platforms.

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Immunodiagnosis of Canine Visceral Leishmaniasis Using Mimotope Peptides Selected from Phage Displayed Combinatorial Libraries

PubMed Central

Toledo-Machado, Christina Monerat; Machado de Avila, Ricardo Andrez; NGuyen, Christophe; Granier, Claude; Bueno, Lilian Lacerda; Carneiro, Claudia Martins; Menezes-Souza, Daniel; Carneiro, Rubens Antonio; Chávez-Olórtegui, Carlos; Fujiwara, Ricardo Toshio

2015-01-01

ELISA and RIFI are currently used for serodiagnosis of canine visceral leishmaniasis (CVL). The accuracy of these tests is controversial in endemic areas where canine infections by Trypanosoma cruzi may occur. We evaluated the usefulness of synthetic peptides that were selected through phage display technique in the serodiagnosis of CVL. Peptides were chosen based on their ability to bind to IgGs purified from infected dogs pooled sera. We selected three phage clones that reacted only with those IgGs. Peptides were synthesized, polymerized with glutaraldehyde, and used as antigens in ELISA assays. Each individual peptide or a mix of them was reactive with infected dogs serum. The assay was highly sensitive and specific when compared to soluble Leishmania antigen that showed cross-reactivity with anti-T. cruzi IgGs. Our results demonstrate that phage display technique is useful for selection of peptides that may represent valuable synthetic antigens for an improved serodiagnosis of CVL. PMID:25710003

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

PubMed Central

Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

2016-01-01

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier.

PubMed

Uematsu, Shuta; Tabuchi, Yudai; Ito, Yuji; Taki, Masumi

2018-06-01

A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein-ligand docking simulation.

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

PubMed

Koenig, Patrick; Sanowar, Sarah; Lee, Chingwei V; Fuh, Germaine

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K D in the sub-nanomolar range. However, it also exhibits significant (K D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Targeted binding of the M13 bacteriophage to thiamethoxam organic crystals.

PubMed

Cho, Whirang; Fowler, Jeffrey D; Furst, Eric M

2012-04-10

Phage display screening with a combinatorial library was used to identify M13-type bacteriophages that express peptides with selective binding to organic crystals of thiamethoxam. The six most strongly binding phages exhibit at least 1000 times the binding affinity of wild-type M13 and express heptapeptide sequences that are rich in hydrophobic, hydrogen-bonding amino acids and proline. Among the peptide sequences identified, M13 displaying the pIII domain heptapeptide ASTLPKA exhibits the strongest binding to thiamethoxam in competitive binding assays. Electron and confocal microscopy confirm the specific binding affinity of ASTLPKA to thiamethoxam. Using atomic force microscope (AFM) probes functionalized with ASTLPKA expressing phage, we found that the average adhesion force between the bacteriophage and a thiamethoxam surface is 1.47 ± 0.80 nN whereas the adhesion force of wild-type M13KE phage is 0.18 ± 0.07 nN. Such a strongly binding bacteriophage could be used to modify the surface chemistry of thiamethoxam crystals and other organic solids with a high degree of specificity. © 2012 American Chemical Society

Using the QCM Biosensor-Based T7 Phage Display Combined with Bioinformatics Analysis for Target Identification of Bioactive Small Molecule.

PubMed

Takakusagi, Yoichi; Takakusagi, Kaori; Sugawara, Fumio; Sakaguchi, Kengo

2018-01-01

Identification of target proteins that directly bind to bioactive small molecule is of great interest in terms of clarifying the mode of action of the small molecule as well as elucidating the biological phenomena at the molecular level. Of the experimental technologies available, T7 phage display allows comprehensive screening of small molecule-recognizing amino acid sequence from the peptide libraries displayed on the T7 phage capsid. Here, we describe the T7 phage display strategy that is combined with quartz-crystal microbalance (QCM) biosensor for affinity selection platform and bioinformatics analysis for small molecule-recognizing short peptides. This method dramatically enhances efficacy and throughput of the screening for small molecule-recognizing amino acid sequences without repeated rounds of selection. Subsequent execution of bioinformatics programs allows combinatorial and comprehensive target protein discovery of small molecules with its binding site, regardless of protein sample insolubility, instability, or inaccessibility of the fixed small molecules to internally located binding site on larger target proteins when conventional proteomics approaches are used.

Biased selection of propagation-related TUPs from phage display peptide libraries.

PubMed

Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

2017-08-01

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

PubMed

Tullila, Antti; Nevanen, Tarja K

2017-05-31

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

PubMed Central

Tullila, Antti; Nevanen, Tarja K.

2017-01-01

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

Progress in the development of immunoanalytical methods incorporating recombinant antibodies to small molecular weight biotoxins.

PubMed

Kavanagh, Owen; Elliott, Christopher T; Campbell, Katrina

2015-04-01

Rapid immunoanalytical screening of food and environmental samples for small molecular weight (hapten) biotoxin contaminations requires the production of antibody reagents that possess the requisite sensitivity and specificity. To date animal-derived polyclonal (pAb) and monoclonal (mAb) antibodies have provided the binding element of the majority of these assays but recombinant antibodies (rAb) isolated from in vitro combinatorial phage display libraries are an exciting alternative due to (1) circumventing the need for experimental animals, (2) speed of production in commonly used in vitro expression systems and (3) subsequent molecular enhancement of binder performance. Short chain variable fragments (scFv) have been the most commonly employed rAb reagents for hapten biotoxin detection over the last two decades but antibody binding fragments (Fab) and single domain antibodies (sdAb) are increasing in popularity due to increased expression efficiency of functional binders and superior resistance to solvents. rAb-based immunochromatographic assays and surface plasmon resonance (SPR) biosensors have been reported to detect sub-regulatory levels of fungal (mycotoxins), marine (phycotoxins) and aquatic biotoxins in a wide range of food and environmental matrices, however this technology has yet to surpass the performances of the equivalent mAb- and pAb-based formats. As such the full potential of rAb technology in hapten biotoxin detection has yet to be achieved, but in time the inherent advantages of engineered rAb are set to provide the next generation of ultra-high performing binder reagents for the rapid and specific detection of hapten biotoxins.

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

PubMed

Enshell-Seijffers, D; Smelyanski, L; Gershoni, J M

2001-05-15

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

Genetic Selection for Improved Abzymes in E. Coli

DTIC Science & Technology

1994-08-03

immunoassay to scemen antibody phage libraries directly for catalysis of a bimolecular Diels - Alder reaction and have identified several active clones...sensitive growth selection assay in F coli for catalysts with chorismate mutase activity; and (3) we identified new abzymes for a Diels - Alder ...generated combinatorial antibody libraries from mRNA isolated from the spleens of mice hyperimmunized with transition state analogs for Diels - Alder and

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

RNA recognition by a human antibody against brain cytoplasmic 200 RNA

PubMed Central

Jung, Euihan; Lee, Jungmin; Hong, Hyo Jeong; Park, Insoo; Lee, Younghoon

2014-01-01

Diverse functional RNAs participate in a wide range of cellular processes. The RNA structure is critical for function, either on its own or as a complex form with proteins and other ligands. Therefore, analysis of the RNA conformation in cells is essential for understanding their functional mechanisms. However, no appropriate methods have been established as yet. Here, we developed an efficient strategy for panning and affinity maturation of anti-RNA human monoclonal antibodies from a naïve antigen binding fragment (Fab) combinatorial phage library. Brain cytoplasmic 200 (BC200) RNA, which is also highly expressed in some tumors, was used as an RNA antigen. We identified MabBC200-A3 as the optimal binding antibody. Mutagenesis and SELEX experiments showed that the antibody recognized a domain of BC200 in a structure- and sequence-dependent manner. Various breast cancer cell lines were further examined for BC200 RNA expression using conventional hybridization and immunoanalysis with MabBC200-A3 to see whether the antibody specifically recognizes BC200 RNA among the total purified RNAs. The amounts of antibody-recognizable BC200 RNA were consistent with hybridization signals among the cell lines. Furthermore, the antibody was able to discriminate BC200 RNA from other RNAs, supporting the utility of this antibody as a specific RNA structure-recognizing probe. Intriguingly, however, when permeabilized cells were subjected to immunoanalysis instead of purified total RNA, the amount of antibody-recognizable RNA was not correlated with the cellular level of BC200 RNA, indicating that BC200 RNA exists as two distinct forms (antibody-recognizable and nonrecognizable) in breast cancer cells and that their distribution depends on the cell type. Our results clearly demonstrate that anti-RNA antibodies provide an effective novel tool for detecting and analyzing RNA conformation. PMID:24759090

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

PubMed

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries.

PubMed

Souriau, Christelle; Rothacker, Julie; Hoogenboom, Hennie R; Nice, Edouard

2004-09-01

Antibodies to EGFR have been shown to display anti-tumour effects mediated in part by inhibition of cellular proliferation and angiogenesis, and by enhancement of apoptosis. Humanised antibodies are preferred for clinical use to reduce complications with HAMA and HAHA responses frequently seen with murine and chimaeric antibodies. We have used depletion and subtractive selection strategies on cells expressing the EGFR to sample two large antibody fragment phage display libraries for the presence of human antibodies which are specific for the EGFR. Four Fab fragments and six scFv fragments were identified, with affinities of up to 2.2nM as determined by BIAcore analysis using global fitting of the binding curves to obtain the individual rate constants (ka and kd). This overall approach offers a generic screening method for the identification of growth factor specific antibodies and antibody fragments from large expression libraries and has potential for the rapid development of new therapeutic and diagnostic reagents.

Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

PubMed

Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

2018-03-13

Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Hua; Jiang Lifang; Fang Danyun

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosenmore » for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.« less

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

PubMed

Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; Schütte, Mark; Hust, Michael

2015-02-19

Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication.

PubMed

Brödel, Andreas K; Jaramillo, Alfonso; Isalan, Mark

2017-09-01

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Synthetic antibodies: concepts, potential and practical considerations.

PubMed

Miersch, S; Sidhu, S S

2012-08-01

The last 100 years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories. Copyright © 2012 Elsevier Inc. All rights reserved.

Structure-Guided Combinatorial Engineering Facilitates Affinity and Specificity Optimization of Anti-CD81 Antibodies.

PubMed

Nelson, Bryce; Adams, Jarrett; Kuglstatter, Andreas; Li, Zhijian; Harris, Seth F; Liu, Yang; Bohini, Sandya; Ma, Han; Klumpp, Klaus; Gao, Junjun; Sidhu, Sachdev S

2018-07-06

Hepatitis C viral infection is the major cause of chronic hepatitis that affects as many as 71 million people worldwide. Rather than target the rapidly shifting viruses and their numerous serotypes, four independent antibodies were made to target the host antigen CD81 and were shown to block hepatitis C viral entry. The single-chain variable fragment of each antibody was crystallized in complex with the CD81 large extracellular loop in order to guide affinity maturation of two distinct antibodies by phage display. Affinity maturation of antibodies using phage display has proven to be critical to therapeutic antibody development and typically involves modification of the paratope for increased affinity, improved specificity, enhanced stability or a combination of these traits. One antibody was engineered for increased affinity for human CD81 large extracellular loop that equated to increased efficacy, while the second antibody was engineered for cross-reactivity with cynomolgus CD81 to facilitate animal model testing. The use of structures to guide affinity maturation library design demonstrates the utility of combining structural analysis with phage display technologies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Detection of biological threats. A challenge for directed molecular evolution.

PubMed

Petrenko, Valery A; Sorokulova, Iryna B

2004-08-01

The probe technique originated from early attempts of Anton van Leeuwenhoek to contrast microorganisms under the microscope using plant juices, successful staining of tubercle bacilli with synthetic dyes by Paul Ehrlich and discovery of a stain for differentiation of gram-positive and gram-negative bacteria by Hans Christian Gram. The technique relies on the principle that pathogens have unique structural features, which can be recognized by specifically labeled organic molecules. A hundred years of extensive screening efforts led to discovery of a limited assortment of organic probes that are used for identification and differentiation of bacteria. A new challenge--continuous monitoring of biological threats--requires long lasting molecular probes capable of tight specific binding of pathogens in unfavorable conditions. To respond to the challenge, probe technology is being revolutionized by utilizing methods of combinatorial chemistry, phage display and directed molecular evolution. This review describes how molecular evolution methods are applied for development of peptide, antibody and phage probes, and summarizes the author's own data on development of landscape phage probes against Salmonella typhimurium. The performance of the probes in detection of Salmonella is illustrated by a precipitation test, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS) and fluorescent, optical and electron microscopy.

Improving the solubility of anti-LINGO-1 monoclonal antibody Li33 by isotype switching and targeted mutagenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.

2010-11-15

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for itsmore » propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.« less

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

PubMed

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae

PubMed Central

Guo, Yakun; Dong, Junkai; Zhou, Tong; Auxillos, Jamie; Li, Tianyi; Zhang, Weimin; Wang, Lihui; Shen, Yue; Luo, Yisha; Zheng, Yijing; Lin, Jiwei; Chen, Guo-Qiang; Wu, Qingyu; Cai, Yizhi; Dai, Junbiao

2015-01-01

It is a routine task in metabolic engineering to introduce multicomponent pathways into a heterologous host for production of metabolites. However, this process sometimes may take weeks to months due to the lack of standardized genetic tools. Here, we present a method for the design and construction of biological parts based on the native genes and regulatory elements in Saccharomyces cerevisiae. We have developed highly efficient protocols (termed YeastFab Assembly) to synthesize these genetic elements as standardized biological parts, which can be used to assemble transcriptional units in a single-tube reaction. In addition, standardized characterization assays are developed using reporter constructs to calibrate the function of promoters. Furthermore, the assembled transcription units can be either assayed individually or applied to construct multi-gene metabolic pathways, which targets a genomic locus or a receiving plasmid effectively, through a simple in vitro reaction. Finally, using β-carotene biosynthesis pathway as an example, we demonstrate that our method allows us not only to construct and test a metabolic pathway in several days, but also to optimize the production through combinatorial assembly of a pathway using hundreds of regulatory biological parts. PMID:25956650

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

PubMed

Falk, Shaun P; Weisblum, Bernard

2013-03-01

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Developing recombinant antibodies for biomarker detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.

2010-10-01

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune librariesmore » provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.« less

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library

PubMed Central

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S.; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-01-01

We constructed a large naïve human Fab library (3 × 1010 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity. PMID:28927259

Generation, Diversity Determination, and Application to Antibody Selection of a Human Naïve Fab Library.

PubMed

Kim, Sangkyu; Park, Insoo; Park, Seung Gu; Cho, Seulki; Kim, Jin Hong; Ipper, Nagesh S; Choi, Sun Shim; Lee, Eung Suk; Hong, Hyo Jeong

2017-09-30

We constructed a large naïve human Fab library (3 × 10 10 colonies) from the lymphocytes of 809 human donors, assessed available diversities of the heavy-chain variable (VH) and κ light-chain variable (VK) domain repertoires, and validated the library by selecting Fabs against 10 therapeutically relevant antigens by phage display. We obtained a database of unique 7,373 VH and 41,804 VK sequences by 454 pyrosequencing, and analyzed the repertoires. The distribution of VH and VK subfamilies and germline genes in our antibody repertoires slightly differed from those in earlier published natural antibody libraries. The frequency of somatic hypermutations (SHMs) in heavy-chain complementarity determining region (HCDR)1 and HCDR2 are higher compared with the natural IgM repertoire. Analysis of position-specific SHMs in CDRs indicates that asparagine, threonine, arginine, aspartate and phenylalanine are the most frequent non-germline residues on the antibody-antigen interface and are converted mostly from the germline residues, which are highly represented in germline SHM hotspots. The amino acid composition and length-dependent changes in amino acid frequencies of HCDR3 are similar to those in previous reports, except that frequencies of aspartate and phenylalanine are a little higher in our repertoire. Taken together, the results show that this antibody library shares common features of natural antibody repertoires and also has unique features. The antibody library will be useful in the generation of human antibodies against diverse antigens, and the information about the diversity of natural antibody repertoires will be valuable in the future design of synthetic human antibody libraries with high functional diversity.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

PubMed

Khlusevich, Yana; Matveev, Andrey; Baykov, Ivan; Bulychev, Leonid; Bormotov, Nikolai; Ilyichev, Ivan; Shevelev, Georgiy; Morozova, Vera; Pyshnyi, Dmitrii; Tikunova, Nina

2018-04-01

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies. Copyright © 2018. Published by Elsevier B.V.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Selection of binding targets in parasites using phage-display and aptamer libraries in vivo and in vitro.

PubMed

Tonelli, R R; Colli, W; Alves, M J M

2012-01-01

Parasite infections are largely dependent on interactions between pathogen and different host cell populations to guarantee a successful infectious process. This is particularly true for obligatory intracellular parasites as Plasmodium, Toxoplasma, and Leishmania, to name a few. Adhesion to and entry into the cell are essential steps requiring specific parasite and host cell molecules. The large amount of possible involved molecules poses additional difficulties for their identification by the classical biochemical approaches. In this respect, the search for alternative techniques should be pursued. Among them two powerful methodologies can be employed, both relying upon the construction of highly diverse combinatorial libraries of peptides or oligonucleotides that randomly bind with high affinity to targets on the cell surface and are selectively displaced by putative ligands. These are, respectively, the peptide-based phage display and the oligonucleotide-based aptamer techniques. The phage display technique has been extensively employed for the identification of novel ligands in vitro and in vivo in different areas such as cancer, vaccine development, and epitope mapping. Particularly, phage display has been employed in the investigation of pathogen-host interactions. Although this methodology has been used for some parasites with encouraging results, in trypanosomatids its use is, as yet, scanty. RNA and DNA aptamers, developed by the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment), were described over two decades ago and since then contributed to a large number of structured nucleic acids for diagnostic or therapeutic purposes or for the understanding of the cell biology. Similarly to the phage display technique scarce use of the SELEX process has been used in the probing of parasite-host interaction. In this review, an overall survey on the use of both phage display and aptamer technologies in different pathogenic organisms will be discussed. Using these techniques, recent results on the interaction of Trypanosoma cruzi with the host will be highlighted focusing on members of the 85 kDa protein family, a subset of the gp85/TS superfamily.

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

PubMed

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ 50 , p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inorganic binding peptides designed by phage display techniques for biotechnology applications

NASA Astrophysics Data System (ADS)

Liao, Chih-Wei

Biomacromolecules play an important role in the control of hard tissue structure and function via specific molecular recognition interactions between proteins of the matrix and inorganic species of the biomineral phase. During the construction of the tissue, biomacromolecules are usually folded into a certain comformation, analogous to a "lock" for fitting with other proteins or smaller molecules as a "key". Currently, the rational design of molecular recognition in biomacro-molecules is still hard to accomplish because the protein conformation is too complex to precisely predict based on the existing conformational information of proteins found in biological systems. In the past two decades, the combinatorial approach (e.g. phage display techniques) has been used to select short binding peptides with molecular recognition to an inorganic target material without a prior knowledge of the amino acid sequence required for the specific binding. The technique has been referred to as "biopanning" because bacteriophages are used to "screen" for peptides that exhibit strong binding to a target material of interest. In this study, two diverse applications were chosen to demonstrate the utility of the biopanning approach. In one project, phage display techniques were used to pan for Indium Zinc Oxide (InZnO) binding peptides to serve as linkers between transducer devices and biosensing elements for demonstration of the feasibility of reversibly electro-activated biosensors. The amorphous InZnO, with its homogeneous surface, led to three consensus peptide sequences, AGFPNSTHSSNL, SHAPDSTWFALF, and TNSSSQFVVAIP. In addition, it was demonstrated that some selected phage clones of the InZnO binding peptides were able to be released from the InZnO surface after applying a voltage of 1400 mV on an electro-activated releasing device. In the second project, phage display techniques were used to select phage clones that bind specifically to francolite mineral in order to achieve separation of francolite particles from dolomitic particles within Florida phosphate ore. A phage clone with a 12-mer francolite binding peptide of WSITTYHDRAIV was able to concentrate the content of francolite from 25% to 42% in a bench-top flotation process of mixed minerals. The first system demonstrates an advanced technology application of the biopanning approach for the development of novel biosensors, while the second system demonstrates application of the biotechnology approach to a commodity industry.

A phage display-selected peptide inhibitor of Agrobacterium vitis polygalacturonase.

PubMed

Warren, Jeremy G; Kasun, George W; Leonard, Takara; Kirkpatrick, Bruce C

2016-05-01

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage-displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase-inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Selection of peptides for binding semiconductor and magnetic materials for the purpose of organizing nanoscaled materials

NASA Astrophysics Data System (ADS)

Whaley, Sandra Renee

A peptide combinatorial approach, also known as phage display, was used to isolate peptides with the ability to bind semiconductor (GaAs, GaN, and InP) and magnetic (Fe2O3 and Fe3O4) materials. The commercially available combinatorial libraries contain randomized peptides either twelve (Ph.D-12(TM)) or seven (Ph.D-C7C(TM)) amino acids in length. The peptides are displayed on the pIII protein of M13 bacteriophage, which have been imaged by atomic force microscopy and transmission electron microscopy. After seven rounds of phage selection with a constrained seven amino acid sequence library (Ph.D-C7C(TM)), two sequences were isolated for binding Fe3O4 (MG-127 and MG-78). The haematite surface was screened with the same library and four unique sequences were isolated after six rounds of selection (HM-95, HM-101, HM-103, and HM-111). According to binding experiments (MG-78 v. MG-127 on Fe3O 4, MG-127 v. HM-95 on Fe3O4 and Fe2O 3, and MG-127 v. HM-95 on gamma-Fe2O3), the MG-127 clone had the highest affinity for iron oxide surfaces (magnetite, haematite, and maghemite) among the clones tested. The Fe3O 4 clone MG-127 displayed the ability to organize Fe3O 4 nanoparticles along bundles of phage. The synthetic peptide analog of this clone was used in the organization of nanoparticles onto the surface of latex beads. The surfaces of the III-V semiconductors were studied using x-ray photoelectron spectroscopy to determine their reactivity in the aqueous conditions used for phage selection. The GaN surface was shown to oxidize the least under these conditions, aiding in the ability to isolate a consensus amino acid sequence responsible for binding to this surface. The G1-3 clone isolated for binding the GaAs (100) surface displayed preferential binding to the GaAs (100) surface over Si (100), GaAs (111) A, GaAs (111) B, and AlGaAs. The synthetic peptide analog of the G12-3 clone was found to preferentially bind to GaAs (100) over either GaAs (111) surfaces or InP (100). This peptide was used to immobilize 10 nm gold particles onto the surface of GaAs within ten minutes. From these results we have shown that it is possible to isolate peptides with high affinities for binding technologically relevant materials, even those not found in nature. These peptides can be used for the organization of pre-formed nanoparticles in solution and on the surface of semiconductor materials.

A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

PubMed

Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

2015-09-01

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

PubMed Central

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

PubMed

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

Determination of allergen specificity by heavy chains in grass pollen allergen-specific IgE antibodies.

PubMed

Gadermaier, Elisabeth; Flicker, Sabine; Lupinek, Christian; Steinberger, Peter; Valenta, Rudolf

2013-04-01

Affinity and clonality of allergen-specific IgE antibodies are important determinants for the magnitude of IgE-mediated allergic inflammation. We sought to analyze the contribution of heavy and light chains of human allergen-specific IgE antibodies for allergen specificity and to test whether promiscuous pairing of heavy and light chains with different allergen specificity allows binding and might affect affinity. Ten IgE Fabs specific for 3 non-cross-reactive major timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) obtained by means of combinatorial cloning from patients with grass pollen allergy were used to construct stable recombinant single chain variable fragments (ScFvs) representing the original Fabs and shuffled ScFvs in which heavy chains were recombined with light chains from IgE Fabs with specificity for other allergens by using the pCANTAB 5 E expression system. Possible ancestor genes for the heavy chain and light chain variable region-encoding genes were determined by using sequence comparison with the ImMunoGeneTics database, and their chromosomal locations were determined. Recombinant ScFvs were tested for allergen specificity and epitope recognition by means of direct and sandwich ELISA, and affinity by using surface plasmon resonance experiments. The shuffling experiments demonstrate that promiscuous pairing of heavy and light chains is possible and maintains allergen specificity, which is mainly determined by the heavy chains. ScFvs consisting of different heavy and light chains exhibited different affinities and even epitope specificity for the corresponding allergen. Our results indicate that allergen specificity of allergen-specific IgE is mainly determined by the heavy chains. Different heavy and light chain pairings in allergen-specific IgE antibodies affect affinity and epitope specificity and thus might influence clinical reactivity to allergens. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

PubMed

Rhaiem, Rafik Ben; Houimel, Mehdi

2016-07-01

Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency during the screening procedure, best inhibits L. major growth kinetics in vitro, and reduces cutaneous lesions in BALB/c mice, thus showing great promise in the development of new therapeutic molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Biomining of MoS2 with Peptide-based Smart Biomaterials.

PubMed

Cetinel, Sibel; Shen, Wei-Zheng; Aminpour, Maral; Bhomkar, Prasanna; Wang, Feng; Borujeny, Elham Rafie; Sharma, Kumakshi; Nayebi, Niloofar; Montemagno, Carlo

2018-02-20

Biomining of valuable metals using a target specific approach promises increased purification yields and decreased cost. Target specificity can be implemented with proteins/peptides, the biological molecules, responsible from various structural and functional pathways in living organisms by virtue of their specific recognition abilities towards both organic and inorganic materials. Phage display libraries are used to identify peptide biomolecules capable of specifically recognizing and binding organic/inorganic materials of interest with high affinities. Using combinatorial approaches, these molecular recognition elements can be converted into smart hybrid biomaterials and harnessed for biotechnological applications. Herein, we used a commercially available phage-display library to identify peptides with specific binding affinity to molybdenite (MoS 2 ) and used them to decorate magnetic NPs. These peptide-coupled NPs could capture MoS 2 under a variety of environmental conditions. The same batch of NPs could be re-used multiple times to harvest MoS 2 , clearly suggesting that this hybrid material was robust and recyclable. The advantages of this smart hybrid biomaterial with respect to its MoS 2 -binding specificity, robust performance under environmentally challenging conditions and its recyclability suggests its potential application in harvesting MoS 2 from tailing ponds and downstream mining processes.

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection.

PubMed

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-09-07

Hepatitis B virus (HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic (vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspectives to the growing field of bacteriophage researches directing towards HBV infection.

Bacteriophages and their applications in the diagnosis and treatment of hepatitis B virus infection

PubMed Central

Bakhshinejad, Babak; Sadeghizadeh, Majid

2014-01-01

Hepatitis B virus (HBV) infection is a major global health challenge leading to serious disorders such as cirrhosis and hepatocellular carcinoma. Currently, there exist various diagnostic and therapeutic approaches for HBV infection. However, prevalence and hazardous effects of chronic viral infection heighten the need to develop novel methodologies for the detection and treatment of this infection. Bacteriophages, viruses that specifically infect bacterial cells, with a long-established tradition in molecular biology and biotechnology have recently been introduced as novel tools for the prevention, diagnosis and treatment of HBV infection. Bacteriophages, due to tremendous genetic flexibility, represent potential to undergo a huge variety of surface modifications. This property has been the rationale behind introduction of phage display concept. This powerful approach, together with combinatorial chemistry, has shaped the concept of phage display libraries with diverse applications for the detection and therapy of HBV infection. This review aims to offer an insightful overview of the potential of bacteriophages in the development of helpful prophylactic (vaccine design), diagnostic and therapeutic strategies for HBV infection thereby providing new perspectives to the growing field of bacteriophage researches directing towards HBV infection. PMID:25206272

Modification of Antibody Function by Mutagenesis.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

PubMed Central

Amir, Shahzada; Hartvigsen, Karsten; Hansen, Lotte F.; Woelkers, Douglas; Tsimikas, Sotirios; Binder, Christoph J.; Kipps, Thomas J.; Witztum, Joseph L.

2013-01-01

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells. PMID:23840319

Epitope mapping of anti-interleukin-13 neutralizing antibody CNTO607.

PubMed

Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L

2009-05-29

CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

Synthetic Human Monoclonal Antibodies toward Staphylococcal Enterotoxin B (SEB) Protective against Toxic Shock Syndrome*

PubMed Central

Karauzum, Hatice; Chen, Gang; Abaandou, Laura; Mahmoudieh, Mahta; Boroun, Atefeh R.; Shulenin, Sergey; Devi, V. Sathya; Stavale, Eric; Warfield, Kelly L.; Zeitlin, Larry; Roy, Chad J.; Sidhu, Sachdev S.; Aman, M. Javad

2012-01-01

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 μg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development. PMID:22645125

Resolving the Orientation of Cylinders and Cuboids from Projected Area Measurements

DTIC Science & Technology

2016-05-01

fabs ( e1 ) + Ay * fabs ( e2...Az * fabs ( e3 ) - A_xy; 90 g = Ax * fabs ( e4 ) + Ay * fabs ( e5 ) + Az * fabs ( e6 ) - A_xz; 91 h = Ax * fabs ( e7 ) + Ay * fabs ( e8 ) + Az * fabs ( e9...b23 * h; 128 del_r = b31 * f + b32 * g + b33 * h; 129 130 p -= del_p; 131 y -= del_y; 132 r -= del_r; 133 134 if ( fabs ( del_p ) < TOL && fabs (

Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.

PubMed

Zhang, Hongkai; Torkamani, Ali; Jones, Teresa M; Ruiz, Diana I; Pons, Jaume; Lerner, Richard A

2011-08-16

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor.

PubMed

Mitchell, Michael S; Rao, Venigalla B

2006-01-06

Packaging of double-stranded DNA into bacteriophage capsids is driven by one of the most powerful force-generating motors reported to date. The phage T4 motor is constituted by gene product 16 (gp16) (18 kDa; small terminase), gp17 (70 kDa; large terminase), and gp20 (61 kDa; dodecameric portal). Extensive sequence alignments revealed that numerous phage and viral large terminases encode a common Walker-B motif in the N-terminal ATPase domain. The gp17 motif consists of a highly conserved aspartate (Asp255) preceded by four hydrophobic residues (251MIYI254), which are predicted to form a beta-strand. Combinatorial mutagenesis demonstrated that mutations that compromised hydrophobicity, or integrity of the beta-strand, resulted in a null phenotype, whereas certain changes in hydrophobicity resulted in cs/ts phenotypes. No substitutions, including a highly conservative glutamate, are tolerated at the conserved aspartate. Biochemical analyses revealed that the Asp255 mutants showed no detectable in vitro DNA packaging activity. The purified D255E, D255N, D255T, D255V, and D255E/E256D mutant proteins exhibited defective ATP binding and very low or no gp16-stimulated ATPase activity. The nuclease activity of gp17 is, however, retained, albeit at a greatly reduced level. These data define the N-terminal ATPase center in terminases and show for the first time that subtle defects in the ATP-Mg complex formation at this center lead to a profound loss of phage DNA packaging.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

PubMed Central

Ruha, Anne-Michelle; Seifert, Steven A.; Morgan, David L.; Lewis, Brandon J.; Arnold, Thomas C.; Clark, Richard F.; Meggs, William J.; Toschlog, Eric A.; Borron, Stephen W.; Figge, Gary R.; Sollee, Dawn R.; Shirazi, Farshad M.; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D.; Boyer, Leslie V.

2015-01-01

Background. Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm3, fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation. PMID:25361165

Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: a prospective, blinded, multicenter, randomized clinical trial.

PubMed

Bush, Sean P; Ruha, Anne-Michelle; Seifert, Steven A; Morgan, David L; Lewis, Brandon J; Arnold, Thomas C; Clark, Richard F; Meggs, William J; Toschlog, Eric A; Borron, Stephen W; Figge, Gary R; Sollee, Dawn R; Shirazi, Farshad M; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D; Boyer, Leslie V

2015-01-01

Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm(3), fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation.

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.

PubMed

Feng, Youjun; Cronan, John E

2011-04-01

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid composition analyses demonstrated that V. cholerae FabR was a functional repressor of unsaturated fatty acid synthesis. However, in contrast to E. coli, gel mobility shift assays indicated that neither E. coli nor V. cholerae FabRs bound the V. cholerae fabB promoter, although both proteins efficiently bound the V. cholerae fabA promoter. This asymmetry was shown to be due to the lack of a FabR binding site within the V. cholerae fabB promoter region. © 2011 Blackwell Publishing Ltd.

Detection of biomarkers using recombinant antibodies coupled to nanostructured platforms

PubMed Central

Kierny, Michael R.; Cunningham, Thomas D.; Kay, Brian K.

2012-01-01

The utility of biomarker detection in tomorrow's personalized health care field will mean early and accurate diagnosis of many types of human physiological conditions and diseases. In the search for biomarkers, recombinant affinity reagents can be generated to candidate proteins or post-translational modifications that differ qualitatively or quantitatively between normal and diseased tissues. The use of display technologies, such as phage-display, allows for manageable selection and optimization of affinity reagents for use in biomarker detection. Here we review the use of recombinant antibody fragments, such as scFvs and Fabs, which can be affinity-selected from phage-display libraries, to bind with both high specificity and affinity to biomarkers of cancer, such as Human Epidermal growth factor Receptor 2 (HER2) and Carcinoembryonic antigen (CEA). We discuss how these recombinant antibodies can be fabricated into nanostructures, such as carbon nanotubes, nanowires, and quantum dots, for the purpose of enhancing detection of biomarkers at low concentrations (pg/mL) within complex mixtures such as serum or tissue extracts. Other sensing technologies, which take advantage of ‘Surface Enhanced Raman Scattering’ (gold nanoshells), frequency changes in piezoelectric crystals (quartz crystal microbalance), or electrical current generation and sensing during electrochemical reactions (electrochemical detection), can effectively provide multiplexed platforms for detection of cancer and injury biomarkers. Such devices may soon replace the traditional time consuming ELISAs and Western blots, and deliver rapid, point-of-care diagnostics to market. PMID:22833780

FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

2014-01-01

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

PubMed Central

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2015-01-01

SUMMARY In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids. PMID:23972938

Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

PubMed

Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

2015-05-27

Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen-food syndrome and to study the IgG epitope of the allergens.

Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen

PubMed Central

1995-01-01

To gain insight into the mechanism and limitations of antibody affinity maturation leading to memory B cell formation, we generated a phage display library of random mutants at heavy chain variable (V) complementarity determining region 2 positions 58 and 59 of an anti-p- azophenylarsonate (Ars) Fab. Single amino acid substitutions at these positions resulting from somatic hypermutation are recurrent products of affinity maturation in vivo. Most of the ex vivo mutants retained specificity for Ars. Among the many mutants displaying high Ars-binding activity, only one contained a position 58 and 59 amino acid combination that has been previously observed among the monoclonal antibodies (mAbs) derived from Ars-immunized mice. Affinity measurements on 14 of the ex vivo mutants with high Ars-binding activity showed that 11 had higher intrinsic affinities for Ars that the wild-type V region. However, nine of these Fabs also bound strongly to denatured DNA, a property neither displayed by the wild-type V region nor observed among the mutants characteristic of in vivo affinity maturation. These data suggest that ex vivo enhancement of mAb affinity via site-directed and random mutagenesis approaches may often lead to a reduction in antibody specificity that could complicate the use of the resulting mAbs for diagnostic and therapeutic applications. Moreover, the data are compatible with a hypothesis proposing that increased specificity for antigen, rather than affinity per se, is the driving force for formation of the memory B cell compartment. PMID:7650481

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

PubMed Central

Levenhagen, Marcelo Arantes; de Almeida Araújo Santos, Fabiana; Fujimura, Patrícia Tiemi; Caneiro, Ana Paula; Costa-Cruz, Julia Maria; Goulart, Luiz Ricardo

2015-01-01

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis. PMID:25994608

Investigation of the effect of physical parameters on the design of tumour targeting agents

NASA Astrophysics Data System (ADS)

Casey, Joanne Lois

Tumour targeting using radiolabelled antibodies for radioimmunodetection (RAID) and radioimmunotherapy (RIT) has been studied for many years. The main factors that have limited clinical success are low tumour uptake, immunogenicity and poor therapeutic ratios. This thesis has applied current technology to make advances in this area of research. The effect of physical parameters (antibody size, valency, affinity and charge) on the design of tumour targeting agents was studied by constructing divalent (DFM) and trivalent (TFM) forms of the murine anti-CEA antibody A5B7 Fab' by chemical cross-linking. This involves partial reduction of the hinge disulphides to expose thiol (-SH) groups and subsequent reaction with a maleimide cross-linker to form a thioether bond at the hinge region. Previous studies have suggested that the stability of thioether bonds is superior to naturally occurring disulphide bonds present at the hinge region of IgG and F(ab')2. The aim was to compare the functional affinities and in vivo tumour targeting in nude mice bearing human tumour xenografts of DFM and TFM to similar sized parent IgG and F(ab')2. Radiolabelling with 131I and 90Y was also compared with a view to determine which combination would be optimal for RIT. Results clearly demonstrated a significantly faster on-rate of DFM compared to all other antibody forms and estimated dosimetry analysis suggested that DFM would be the most suitable antibody form radiolabelled with 131I for RIT. Both F(ab')2 and DFM showed high kidney uptake levels on labelling with which is unacceptable for RIT. Despite the improved tumour: blood ratios for TFM, the increased estimated dose to normal tissues and lower therapeutic effect in RIT studies suggests that the most promising combination with the radionuclide appears to be IgG. A humanised version of A5B7 hFab' has been constructed previously in order to reduce its immunogenicity in man. The in vivo stability of hDFM proved to be superior to hF(ab')2 in the nude mouse xenograft model. To study the safety, stability and tumour targeting of hDFM a clinical trial using 131I was described here including details of production, characterisation, pharmacokinetics and dosimetry. ScFv's are known to have favourable tumour targeting characteristics compared to whole antibodies for RAID. To evaluate the clinical potential of a scFv, the methodology to prepare a phage derived scFv with the aid of a subcloned hexahistidine tail was described here. To enhance the clinical potential of scFv's a construct consisting of a hinge region containing a single cysteine residue was constructed. This enabled site-specific 99mTc-labelling and could facilitate multimerisation. One of the major limitations revealed by this and other studies is the problem associated with renal accretion of antibody fragments. Various modification techniques and blocking effects were used here in attempt to reduce the kidney uptake levels in mouse models. Reduction of the pi of A5B7 Fab by attachment of NHS-ester groups was effective in lowering kidney uptake levels, but losses in immunoreactivity could limit this approach. Attachment of PEG (5kD) to DFM did not adversely affect immunoreactivity and increased the circulation time of DFM in vivo. This has implications for reducing kidney uptake levels at early time points, in addition PEG is known to reduce immunogenicity of proteins.

Reagents for astatination of biomolecules. 2. Conjugation of anionic boron cage pendant groups to a protein provides a method for direct labeling that is stable to in vivo deastatination.

PubMed

Wilbur, D Scott; Chyan, Ming-Kuan; Hamlin, Donald K; Vessella, Robert L; Wedge, Timothy J; Hawthorne, M Frederick

2007-01-01

Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination, as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of 211At-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with 125I, and that material was coinjected with the 211At-labeled Fab'. For comparison, direct labeling of the Fab' with 125I and 211At was conducted. Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[211At]At and Chloramine-T resulted in a yield of <1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[211At]astatobenzoate NHS ester, [211At]1c-Fab', and (separately) with p-[125I]iodobenzoate NHS ester, [125I]1b-Fab'. An evaluation in athymic mice demonstrated that [211At]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [125I]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [211At]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([125I]2-Fab'/ [211At]2-Fab') and the bis-nido-carborane (VFC) ([125I]3-Fab'/[211At]3-Fab') had very different in vivo distributions than the control [125I]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([125I]4-Fab'/[211At]4-Fab', [125I]6-Fab'/[211At]6-Fab', and [125I]7-Fab'/[211At]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [125I]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([125I]5-Fab'/[211At]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct 211At labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)*

PubMed Central

Yao, Jiangwei; Maxwell, John B.; Rock, Charles O.

2013-01-01

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Kiapp increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step. PMID:24189061

Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.

PubMed

Toleman, Mark A; Walsh, Timothy R

2011-09-01

The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

DOE PAGES

McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

2016-01-28

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. This, coupled with their low homology and difference in organization compared to the equivalent system in humans, makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. Additionally, we have discoveredmore » two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. These results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

PubMed

Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

2013-12-31

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH).

PubMed

Li, Yongli; Florova, Galina; Reynolds, Kevin A

2005-06-01

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (approximately 70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a deltafabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

PubMed

Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

2013-10-01

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies.

PubMed

Welch, Brett D; Paduch, Marcin; Leser, George P; Bergman, Zachary; Kors, Christopher A; Paterson, Reay G; Jardetzky, Theodore S; Kossiakoff, Anthony A; Lamb, Robert A

2014-10-01

Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.

{beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rankmore » order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.« less

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Controlled Fab installation onto polymeric micelle nanoparticles for tuned bioactivity

NASA Astrophysics Data System (ADS)

Chen, Shaoyi; Florinas, Stelios; Teitgen, Abigail; Xu, Ze-Qi; Gao, Changshou; Wu, Herren; Kataoka, Kazunori; Cabral, Horacio; Christie, R. James

2017-12-01

Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

PubMed

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-06-18

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes*

PubMed Central

Nelson, Alfreda D.; Hoffmann, Michele M.; Parks, Christopher A.; Dasari, Surendra; Schrum, Adam G.; Gil, Diana

2012-01-01

Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes. PMID:23109335

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

PubMed

Hou, Jing; Zheng, Heping; Tzou, Wen-Shyong; Cooper, David R; Chruszcz, Maksymilian; Chordia, Mahendra D; Kwon, Keehwan; Grabowski, Marek; Minor, Wladek

2018-06-19

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-ACP synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most Gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH and six vcFabH2 structures determined in either apo-form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

PubMed

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-01-01

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Short-term outcomes after Fab antivenom therapy for severe crotaline snakebite.

PubMed

Lavonas, Eric J; Kokko, Jamie; Schaeffer, Tammi H; Mlynarchek, Sara L; Bogdan, Gregory M; Dart, Richard C

2011-02-01

We seek to determine the short-term outcomes associated with the use of Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) therapy for severe crotaline snake envenomation and to better define the incidence of hypersensitivity reactions associated with FabAV use. We conducted a multicenter observational case series study of patients who received FabAV at 17 US hospitals in 2002 to 2004. A 7-point score incorporating local, systemic, and hematologic venom effects was used to grade envenomation severity before and after FabAV therapy. The primary outcome for response to therapy was the change in overall envenomation severity after FabAV administration. The primary safety outcomes were the rates of immediate hypersensitivity reactions and serum sickness. The outcome-evaluable population included 209 patients, of whom 28 had severe envenomation. All severely envenomated patients improved after receiving FabAV. The median severity scores of severely envenomated patients were 5 (interquartile range [IQR] 5 to 5) before FabAV, 1 (IQR 1 to 2) at the last FabAV loading dose, and 1 (IQR 0 to 1) at the last clinical observation. The proportion of patients with progressive pain, progressive swelling, cardiovascular effects, respiratory effects, neurologic effects, gastrointestinal effects, coagulopathy, and thrombocytopenia all improved after FabAV therapy. The safety population included 247 patients. Immediate hypersensitivity reactions were reported in 6.1% (95% confidence interval 3.4% to 9.8%) of patients. Serum sickness was reported in 5% (95% confidence interval 0.6% to 17%) of patients with a minimum of 6 days of follow-up after the last dose of FabAV. FabAV therapy is associated with clinical improvement in severe crotaline snake envenomation. Immediate hypersensitivity and serum sickness rates may be less than described in the FabAV prescribing information. Copyright © 2010. Published by Mosby, Inc.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

PubMed Central

Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

2015-01-01

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

PubMed

Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

2015-01-01

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

PSMA-targeted bispecific Fab conjugates that engage T cells.

PubMed

Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

Molecular biomimetics: nanotechnology through biology.

PubMed

Sarikaya, Mehmet; Tamerler, Candan; Jen, Alex K-Y; Schulten, Klaus; Baneyx, François

2003-09-01

Proteins, through their unique and specific interactions with other macromolecules and inorganics, control structures and functions of all biological hard and soft tissues in organisms. Molecular biomimetics is an emerging field in which hybrid technologies are developed by using the tools of molecular biology and nanotechnology. Taking lessons from biology, polypeptides can now be genetically engineered to specifically bind to selected inorganic compounds for applications in nano- and biotechnology. This review discusses combinatorial biological protocols, that is, bacterial cell surface and phage-display technologies, in the selection of short sequences that have affinity to (noble) metals, semiconducting oxides and other technological compounds. These genetically engineered proteins for inorganics (GEPIs) can be used in the assembly of functional nanostructures. Based on the three fundamental principles of molecular recognition, self-assembly and DNA manipulation, we highlight successful uses of GEPI in nanotechnology.

The use and tolerability of Crotalidae Polyvalent Immune FAB (Ovine) in pediatric envenomations.

PubMed

Farrar, Henry C; Grayham, Taylor; Bolden, Branson; Vyas, Dileep; Graham, James; James, Laura P

2012-10-01

There are limited data on the use of Crotalidae Polyvalent Immune FAB-Ovine (CroFab) in the management of crotalid envenomations in children. Thus, the primary objective of this retrospective chart review was to evaluate the safety and tolerability of CroFab in a pediatric population. Over an 8-year time period at this institution, there were 204 admissions for snakebite of which 82 received CroFab. Children who received CroFab were more often associated with bites to the hands and fingers and tended to have more significant envenomations as indicated by longer hospital stays, greater tissue injury, and a tendency to require surgery more often. Six (7.3%) of the 82 patients who received CroFab experienced an adverse drug reaction. Reactions consisted of allergic symptoms that were mild, responded to minimal interventions, and did not limit the subsequent use of CroFab. It is concluded that CroFab use is typically well tolerated in pediatric patients.

Identification of basic/helix-loop-helix transcription factors reveals candidate genes involved in anthocyanin biosynthesis from the strawberry white-flesh mutant.

PubMed

Zhao, Fengli; Li, Gang; Hu, Panpan; Zhao, Xia; Li, Liangjie; Wei, Wei; Feng, Jiayue; Zhou, Houcheng

2018-02-09

As the second largest transcription factor family in plant, the basic helix-loop-helix (bHLH) transcription factor family, characterized by the conserved bHLH domain, plays a central regulatory role in many biological process. However, the bHLH transcription factor family of strawberry has not been systematically identified, especially for the anthocyanin biosynthesis. Here, we identified a total of 113 bHLH transcription factors and described their chromosomal distribution and bioinformatics for the diploid woodland strawberry Fragaria vesca. In addition, transcription profiles of 113 orthologous bHLH genes from various tissues were analyzed for the cultivar 'Benihoppe', its white-flesh mutant 'Xiaobai', and the 'Snow Princess' from their fruit development to the ripening, as well as those under either the ABA or Eth treatment. Both the RT-PCR and qRT-PCR results show that seven selected FabHLH genes (FabHLH17, FabHLH25, FabHLH27, FabHLH29, FabHLH40, FabHLH80, FabHLH98) are responsive to the fruit anthocyanin biosynthesis and hormone signaling according to transcript profiles where three color modes are observed for strawberry's fruit skin and flesh. Further, prediction for the protein interaction network reveals that four bHLHs (FabHLH25, FabHLH29, FabHLH80, FabHLH98) are involved in the fruit anthocyanin biosynthesis and hormone signaling transduction. These bioinformatics and expression profiles provide a good basis for a further investigation of strawberry bHLH genes.

20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

Code of Federal Regulations, 2010 CFR

2010-04-01

... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of such...

Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

PubMed

Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

2015-02-02

We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

The reliability and validity of the Turkish version of Fullerton Advanced Balance (FAB-T) scale.

PubMed

Iyigun, Gozde; Kirmizigil, Berkiye; Angin, Ender; Oksuz, Sevim; Can, Filiz; Eker, Levent; Rose, Debra J

2018-06-04

The aim of this study was to evaluate the reliability and validity of the Turkish version of the FAB(FAB-T) scale in the older Turkish adults. The reliability and validity of the scale was tested on 200 community-dwelling older adults. FAB-T scale was scored by different physiotherapists on different days to evaluate inter-rater and intrarater reliability. The Berg Balance Scale (BBS) was used for the evaluation of convergent validity, and the content validity of the FAB-T scale was investigated. The FAB-T scale showed very high inter- and intra-rater reliability. For inter-rater agreement, on the individual test items and total score ICC values were 0.92 (95 %CI; 0.90-0.94) and 0.96 (95% CI; 0.95-0.97) respectively. The intra-rater agreement, on the individual test items and total score ICC values were 0.93 (95 %CI; 0.91- 0.95) and 0.96 (95% CI; 0.95- 0.97) respectively. There was a good agreement between the FAB-T and BBS scales. A high correlation was found between the BBS and FAB-T scales [rho = 0.70 (%95 CI; 0.62-0.76)] indicating good convergent validity. Considering the content validity of the FAB-T scale, no floor (floor score: 0%) or ceiling (ceiling score: 6.5%) effect was detected. The FAB-T scale was successfully translated from the original English version (FAB) and demonstrated strong psychometric features. It was found that the FAB-T scale has very high inter-rater and intra-rater reliability. Considering the convergent validity, the scale has high correlation with the BBS. The FAB-T has no floor and ceiling effect. Copyright © 2018 Elsevier B.V. All rights reserved.

Initial experience with Crotalidae polyvalent immune Fab (ovine) antivenom in the treatment of copperhead snakebite.

PubMed

Lavonas, Eric J; Gerardo, Charles J; O'Malley, Gerald; Arnold, Thomas C; Bush, Sean P; Banner, William; Steffens, Mark; Kerns, William P

2004-02-01

Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) effectively treats patients bitten by rattlesnakes. The copperhead snake (Agkistrodon contortrix) caused 37% of venomous snakebites reported to US poison centers in 2001 and is the major envenomating reptile in the southeastern United States. FabAV has not been tested in human beings envenomated by copperhead snakes. In this preliminary study, we performed a retrospective chart review of all copperhead snake envenomations reported to the Carolinas Poison Center that were treated with FabAV. Progression of limb swelling, coagulopathy, and hemodynamic status before and after FabAV administration, adverse effects of FabAV therapy, and recurrence phenomena were recorded. Of approximately 400 copperhead envenomation cases reported to the poison center during the study period, 32 received FabAV and were included. Most patients had moderate envenomation. The median time to FabAV administration was 4.0 hours. The median time to achieve initial control was 1.0 hour, with a median dose of 4 vials of FabAV. A rapid initial response, defined as cessation of the progression of local tissue injury within 4 hours of FabAV administration, occurred in 28 cases (88%; 95% confidence interval [CI] 76% to 99%). Four cases (13%; 95% CI 1% to 24%) were considered treatment failures. Recurrent swelling occurred in 6 cases (19%; 95% CI 5% to 32%). The incidence of recurrent swelling was not reduced by administration of repeated doses of antivenom on a planned schedule. One patient developed late-onset coagulopathy. One minor allergic reaction was observed. In this select group of patients bitten by copperhead snakes, local tissue effects of envenomation halted promptly after FabAV treatment in most cases. Treatment failures occurred, and recurrence of swelling and defibrination syndrome was sometimes problematic. Time to return to work and long-term limb function were not assessed. A controlled trial with long-term follow-up is needed to define the role of FabAV treatment for copperhead envenomation.

Topical ocular treatment with monoclonal antibody Fab fragments targeting Japanese cedar pollen Cry j 1 inhibits Japanese cedar pollen-induced allergic conjunctivitis in mice.

PubMed

Mizutani, Nobuaki; Nabe, Takeshi; Yoshino, Shin

2017-03-05

Fab fragments (Fabs) of antibodies having the ability only to bind to specific allergens lack effector functions due to the absence of the Fc portion. In the present study, we examined whether IgG1 monoclonal antibody (mAb) Fabs targeting Japanese cedar pollen (JCP) Cry j 1 were able to regulate JCP-induced allergic conjunctivitis in mice. BALB/c mice actively sensitized with JCP were repeatedly challenged by topical administration of JCP eye drops. Fabs prepared by the digestion of anti-JCP IgG1 mAbs (P1-3 and P1-8) with papain were applied to the eye 15min before the JCP challenges followed by measurement of the clinical conjunctivitis score. In the in vitro experiments, P1-3 and P1-8 showed specific binding to JCP Cry j 1. Furthermore, intact P1-3 binding to Cry j 1 was inhibited by P1-3 Fabs, but not P1-8 Fabs; additionally, P1-8 Fabs, but not P1-3 Fabs, suppressed the intact P1-8 binding, suggesting that the epitopes of Cry j 1 recognized by P1-3 and P1-8 were different. Topical ocular treatment with P1-3 Fabs or P1-8 Fabs was followed by marked suppression of JCP-induced conjunctivitis (P<0.01). In histological evaluation, P1-8 Fabs showed a reduction in eosinophil infiltration in the conjunctiva (P<0.01). These results demonstrated that topical ocular treatment with IgG1 mAb Fabs to Cry j 1 was effective in suppressing JCP-induced allergic conjunctivitis in mice. Furthermore, it suggests the possibility that some epitopes recognized by Fabs could be used as a tool to regulate allergic conjunctivitis. Copyright © 2017 Elsevier B.V. All rights reserved.

75 FR 52724 - Procurement List Additions

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-27

...: 8405-00-FAB-0201--Man's, S/S, Air Force Shade 1550, Size 13\\1/ 2\\ NSN: 8405-00-FAB-0202--Man's, S/S, Air Force Shade 1550, Size 14 NSN: 8405-00-FAB-0203--Man's, S/S, Air Force Shade 1550, Size 14 \\1/ 2\\ NSN: 8405-00-FAB-0204--Man's, S/S, Air Force Shade 1550, Size 15 NSN: 8405-00-FAB-0212--Man's, S/S...

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

PubMed

Shatz, Whitney; Hass, Philip E; Mathieu, Mary; Kim, Hok Seon; Leach, Kim; Zhou, Michelle; Crawford, Yongping; Shen, Amy; Wang, Kathryn; Chang, Debby P; Maia, Mauricio; Crowell, Susan R; Dickmann, Leslie; Scheer, Justin M; Kelley, Robert F

2016-09-06

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

PubMed

Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

2015-01-01

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures.

PubMed

Ukkonen, Kaisa; Veijola, Johanna; Vasala, Antti; Neubauer, Peter

2013-07-29

Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization.

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

PubMed Central

2013-01-01

Background Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Results Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Conclusions Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These findings have practical implications for screening applications and small-scale Fab production, and highlight the importance of maintaining consistent aeration conditions during scale-up to avoid changes in product yield and localization. On the other hand, the dependency of Fab leakage on cultivation conditions provides a practical way to manipulate Fab localization. PMID:23895637

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

PubMed

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Tracheal intubation prevented with administration of Fab antivenom after severe crotaline envenomation.

PubMed

Bebarta, Vikhyat S; Ferre, Robinson M; Peck, Michael

2010-07-01

Crotaline snake envenomations are common, but severe crotaline envenomations are infrequent. Death from severe envenomation is usually from upper airway edema and respiratory failure. Published reports of severe respiratory compromise and anaphylactoid reactions are rare. Currently, FabAV (Crotalidae polyvalent immune Fab [Ovine] [CroFab]) is the mainstay of crotaline envenomation treatment; however, FabAV has been approved for only mild and moderate envenomations. We describe a case of a male with severe systemic effects and airway compromise after crotaline envenomation. The patient's systemic effects and upper airway edema substantially improved after antivenom infusion and before epinephrine administration. Endotracheal intubation was averted, clinical deterioration was avoided, and improvement occurred after prompt FabAV use. Fab antivenom likely prevented endotracheal intubation in our case of severe crotaline envenomation. Published by Elsevier Inc.

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

PubMed

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa.

PubMed

Kimber, Matthew S; Martin, Fernando; Lu, Yingjie; Houston, Simon; Vedadi, Masoud; Dharamsi, Akil; Fiebig, Klaus M; Schmid, Molly; Rock, Charles O

2004-12-10

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

PubMed

Nanson, Jeffrey D; Forwood, Jade K

2015-01-01

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

Practical Nonlinearities

DTIC Science & Technology

2016-07-01

All Initial Designs for Final Fab Run Month 29 Masks and wafers prepared for Final Fab Run Month 30 Start of Final Fab Run Month 35 Completion of...Final Fab Run Month 36 Delivery of devices based on designs from other DEFYS performers Because of momentum from efforts prior to the start of...report (June 2016), our project is completed, with most tasks completed ahead of schedule. For example, the 3rd Fab Run started 5 months early and was

Trusted Fabrication through 3D Integration

DTIC Science & Technology

2017-03-01

contiguous and thus identifiable. The concept of a “smart partitioner” is introduced for a second experiment. Keywords: Trusted Fab ; VLSI; 3DIC...to the fabrication facility. One solution is the split- fab concept in which the design is split into two separate fabs early in the metal stack, and...possible solution is proposed herein whereby a three chip stack is formed, two built in normal semiconductor fabs and one in an interposer fab . This

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.

Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. In this paper, we compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, themore » Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Finally, additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.« less

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1*

PubMed Central

Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; Dhoke, Reema R.; Nath, Samir K.; Nihalani, Deepak; Ashish

2014-01-01

Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. We compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability. PMID:25331945

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

DOE PAGES

Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; ...

2014-10-20

Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. In this paper, we compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, themore » Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Finally, additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability.« less

FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

ERIC Educational Resources Information Center

Pagano, John

2015-01-01

This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

PubMed

Caballero, Armando R; Foletti, Davide L; Bierdeman, Michael A; Tang, Aihua; Arana, Angela M; Hasa-Moreno, Adela; Sangalang, Emma Ruth B; O'Callaghan, Richard J

2015-08-01

To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

PubMed Central

Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M.; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G. Marius; Wlodawer, Alexander

2013-01-01

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design. PMID:24244293

1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

PubMed Central

He, Xin; Reeve, Anne McElwee; Desai, Umesh R.; Kellogg, Glen E.; Reynolds, Kevin A.

2004-01-01

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered. PMID:15273125

Structural and dynamical aspects of Streptococcus gordonii FabH through molecular docking and MD simulations.

PubMed

Shamim, Amen; Abbasi, Sumra Wajid; Azam, Syed Sikander

2015-07-01

β-Ketoacyl-ACP-synthase III (FabH or KAS III) has become an attractive target for the development of new antibacterial agents which can overcome the multidrug resistance. Unraveling the fatty acid biosynthesis (FAB) metabolic pathway and understanding structural coordinates of FabH will provide valuable insights to target Streptococcus gordonii for curing oral infection. In this study, we designed inhibitors against therapeutic target FabH, in order to block the FAB pathway. As compared to other targets, FabH has more interactions with other proteins, located on the leading strand with higher codon adaptation index value and associated with lipid metabolism category of COG. Current study aims to gain in silico insights into the structural and dynamical aspect of S. gordonii FabH via molecular docking and molecular dynamics (MD) simulations. The FabH protein is catalytically active in dimerization while it can lock in monomeric state. Current study highlights two residues Pro88 and Leu315 that are close to each other by dimerization. The active site of FabH is composed of the catalytic triad formed by residues Cys112, His249, and Asn279 in which Cys112 is involved in acetyl transfer, while His249 and Asn279 play an active role in decarboxylation. Docking analysis revealed that among the studied compounds, methyl-CoA disulfide has highest GOLD score (82.75), binding affinity (-11 kcal/mol) and exhibited consistently better interactions. During MD simulations, the FabH structure remained stable with the average RMSD value of 1.7 Å and 1.6 Å for undocked protein and docked complex, respectively. Further, crucial hydrogen bonding of the conserved catalytic triad for exhibiting high affinity between the FabH protein and ligand is observed by RDF analysis. The MD simulation results clearly demonstrated that binding of the inhibitor with S. gordonii FabH enhanced the structure and stabilized the dimeric FabH protein. Therefore, the inhibitor has the potential to become a lead compound. Copyright © 2015 Elsevier Inc. All rights reserved.

Crotalidae polyvalent immune Fab (ovine) antivenom is effective in the neutralization of South American viperidae venoms in a murine model.

PubMed

Richardson, William H; Tanen, David A; Tong, Tri C; Betten, David P; Carstairs, Shaun D; Williams, Saralyn R; Cantrell, Frank L; Clark, Richard F

2005-06-01

Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) is used in the treatment of symptomatic crotaline envenomations in North America. Unlike Antivenin (Crotalidae) Polyvalent, which is approved for treatment of crotaline envenomation in North and South America, FabAV is manufactured using only venoms from crotaline snakes native to the United States. This study was designed to evaluate the efficacy of FabAV in the neutralization of venom from 2 South American crotaline snakes: Crotalus durissus terrificus (tropical rattlesnake) and Bothrops atrox (fer-de-lance). A randomized, blinded, placebo-controlled murine model of intraperitoneal venom injection was used. Venom potency was determined in preliminary median lethal dose (LD 50) dosing studies. Study animals were then divided into 7 groups: (1) C durissus terrificus venom (Sigma-Aldrich Co.)+FabAV, (2) C durissus terrificus venom (Sigma-Aldrich Co.)+0.9% normal saline solution, (3) C durissus terrificus venom (Biotoxins Inc.)+FabAV, (4) C durissus terrificus venom (Biotoxins Inc.)+normal saline solution, (5) B atrox venom+FabAV, (6) B atrox venom+normal saline solution, and (7) FabAV+normal saline solution. Twice the estimated LD 50 was the chosen venom dose, and the amount of FabAV injected was 10 times the amount needed for venom neutralization. Statistical analysis included Fisher's exact test and log-rank testing to compare survival rates and times. The venom LD 50 was found in preliminary studies to be 0.9 mg/kg and 1.35 mg/kg for the C durissus terrificus venom obtained from Sigma-Aldrich Co. and Biotoxins Inc., respectively. The LD 50 for B atrox venom was 5.0 mg/kg. All animals receiving venom only and saline solution died. Animals receiving FabAV together with either venom survived to the end of the 24-hour observation period ( P <.001). Comparison of survival times between groups demonstrated a significant difference in time to death between venom-only control groups and the FabAV+venom groups (P <.001). All animals in the FabAV+normal saline solution group survived to the conclusion of the study. FabAV, when premixed with venom, decreases lethality in a murine model of intraperitoneal venom injection of the South American pit vipers, C durissus terrificus and B atrox .

Imaging Potential Evaluation of Fab Derived from the Anti-EGFRvIII Monoclonal Antibody 4G1.

PubMed

Jing, Shen; He, Yujia; He, Yanqiong; Wang, Liang; Jia, Jianhua; Shan, Xiaomin; Liu, Shuang; Tang, Min; Peng, Zhiping; Liu, Xujie

2018-05-31

As one of the most crucial epidermal growth factor receptor (EGFR) variants, EGFRvIII can be detected in various tumors but rarely in normal tissues, making it an ideal target for prognosis, diagnosis or immune therapy. The recently developed anti-EGFRvIII monoclonal antibody (mAb), 4G1, has been validated as a promising molecular probe to detect EGFRvIII expression in tumors by single-photon emission computed tomography/computed tomography imaging. To overcome shortcomings associated with the whole antibody, including long-term retention, circulation and enhanced permeability and retention effects, the Fab fragment of 4G1 (Fab-4G1) was generated, labeled with 131 I and evaluated in vitro and in vivo to test its potential application in molecular imaging. Whole mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment, Fab-4G1. Next, SDS-PAGE, Western blot, indirect fluorescence assay, flow cytometry and enzyme-linked immunosorbent assay were performed to verify molecular weight, specificity and affinity of Fab-4G1. Finally, biodistribution planar gamma imaging was performed by injection of 131 I-labeled Fab-4G1 into xenografted EGFRvIII-overexpressed tumors in nude mice. Parallel studies were also performed with intact 4G1. The molecular weight of Fab was determined to be 35-40 kDa by SDS-PAGE. In vitro tests confirmed both intact 4G1 and Fab-4G1 specifically bound EGFRvIII but not wild-type EGFR, and Fab-4G1 showed decreased affinity. Compared to 131 I-4G1, biodistribution studies showed lower tumor uptake of 131 I-Fab-4G1 at all time points, but much faster elimination in all normal organs. As for planar gamma imaging, 131 I-Fab-4G1 and 31 I-4G1 showed similar imaging effect at 2 h after injection of tracer, while 131 I-Fab-4G1 was eliminated more quickly with time, suggesting radiolabeled Fab-4G1 could be potentially used for imaging of EGFRvIII-positive tumors at early time points. Radiolabeled Fab-4G1 would be a promising nuclear probe for future clinical EGFRvIII tumor detection.

Performance on the Frontal Assessment Battery is sensitive to frontal lobe damage in stroke patients.

PubMed

Kopp, Bruno; Rösser, Nina; Tabeling, Sandra; Stürenburg, Hans Jörg; de Haan, Bianca; Karnath, Hans-Otto; Wessel, Karl

2013-11-16

The Frontal Assessment Battery (FAB) is a brief battery of six neuropsychological tasks designed to assess frontal lobe function at bedside [Neurology 55:1621-1626, 2000]. The six FAB tasks explore cognitive and behavioral domains that are thought to be under the control of the frontal lobes, most notably conceptualization and abstract reasoning, lexical verbal fluency and mental flexibility, motor programming and executive control of action, self-regulation and resistance to interference, inhibitory control, and environmental autonomy. We examined the sensitivity of performance on the FAB to frontal lobe damage in right-hemisphere-damaged first-ever stroke patients based on voxel-based lesion-behavior mapping. Voxel-based lesion-behavior mapping of FAB performance revealed that the integrity of the right anterior insula (BA13) is crucial for the FAB global composite score, for the FAB conceptualization score, as well as for the FAB inhibitory control score. Furthermore, the FAB conceptualization and mental flexibility scores were sensitive to damage of the right middle frontal gyrus (MFG; BA9). Finally, the FAB inhibitory control score was sensitive to damage of the right inferior frontal gyrus (IFG; BA44/45). These findings indicate that several FAB scores (including composite and item scores) provide valid measures of right hemispheric lateral frontal lobe dysfunction, specifically of focal lesions near the anterior insula, in the MFG and in the IFG.

Performance on the Frontal Assessment Battery is sensitive to frontal lobe damage in stroke patients

PubMed Central

2013-01-01

Background The Frontal Assessment Battery (FAB) is a brief battery of six neuropsychological tasks designed to assess frontal lobe function at bedside [Neurology 55:1621-1626, 2000]. The six FAB tasks explore cognitive and behavioral domains that are thought to be under the control of the frontal lobes, most notably conceptualization and abstract reasoning, lexical verbal fluency and mental flexibility, motor programming and executive control of action, self-regulation and resistance to interference, inhibitory control, and environmental autonomy. Methods We examined the sensitivity of performance on the FAB to frontal lobe damage in right-hemisphere-damaged first-ever stroke patients based on voxel-based lesion-behavior mapping. Results Voxel-based lesion-behavior mapping of FAB performance revealed that the integrity of the right anterior insula (BA13) is crucial for the FAB global composite score, for the FAB conceptualization score, as well as for the FAB inhibitory control score. Furthermore, the FAB conceptualization and mental flexibility scores were sensitive to damage of the right middle frontal gyrus (MFG; BA9). Finally, the FAB inhibitory control score was sensitive to damage of the right inferior frontal gyrus (IFG; BA44/45). Conclusions These findings indicate that several FAB scores (including composite and item scores) provide valid measures of right hemispheric lateral frontal lobe dysfunction, specifically of focal lesions near the anterior insula, in the MFG and in the IFG. PMID:24237624

Frontal assessment battery (FAB) performance following traumatic brain injury hospitalized in an acute care setting.

PubMed

Rojas, Natalia; Laguë-Beauvais, Maude; Belisle, Arielle; Lamoureux, Julie; AlSideiri, Ghusn; Marcoux, Judith; Maleki, Mohammed; Alturki, Abdulrahman Y; Anchouche, Sonia; Alquraini, Hanan; Feyz, Mitra; Guise, Elaine de

2018-01-19

The Frontal Assessment Battery (FAB) has been shown to be useful in several clinical settings. The aim of the present study was to examine the performance of patients with traumatic brain injury (TBI) on the FAB and to predict their acute outcome. The FAB was administered to 89 patients with mild (27 = uncomplicated and 39 = complicated) and moderate (n = 23) TBI during hospitalization in an acute care setting. The length of stay in days (LOS), Glasgow Outcome Scale-Revised score (GOSE) and Disability Rating Scale (DRS) score were collected. Results showed no significant differences between the three groups on the FAB score, but age and education were significantly associated with the FAB score. Parietal lesions were associated with lower total FAB score, and with the Similarities, Motor series and Conflicting instructions subscales, while frontal lesions were associated with lower performance on the Motor series and Conflicting instructions subscales. Total FAB score was significantly correlated with all outcome measures, and together the FAB total score and the Glasgow Coma Scale (GCS) score explained 30.8% of the variance in the DRS score. The FAB may be useful clinically to acutely assess frontal and parietal lobe functions at bedside in patients with TBI and, in combination with the GCS score to measure TBI severity, can enable clinicians to predict early outcome.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

PubMed

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

PubMed

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

PubMed Central

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT.

PubMed

Chan, Conrad; Scollard, Deborah A; McLarty, Kristin; Smith, Serena; Reilly, Raymond M

2011-08-17

Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab. We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab.

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT

PubMed Central

2011-01-01

Background Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Methods Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. Results 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab. Conclusions We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab. PMID:22214307

21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that consists...

Crotalidae polyvalent immune Fab for the treatment of pediatric crotaline envenomation.

PubMed

Goto, Collin S; Feng, Sing-Yi

2009-04-01

Crotaline snakebites occur frequently in children, often resulting in significant morbidity. Crotalidae Polyvalent Immune Fab antivenom (FabAV) became available for clinical use in the US in 2000 and is currently the standard of care for the treatment of crotaline envenomation. The pediatric emergency care provider should be familiar with FabAV because its judicious use in affected children can greatly decrease morbidity caused by crotaline snakebites. This article will review the use of FabAV for the treatment of pediatric crotaline envenomation.

A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

PubMed

Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

2016-12-01

Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-23

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,642] Intel Corporation, Fab 20... of Intel Corporation, Fab 20 Division, including on-site leased workers of Volt Technical Resources... Precision, Inc. were employed on-site at the Hillsboro, Oregon location of Intel Corporation, Fab 20...

Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volkov, V. V., E-mail: vvo@ns.crys.ras.ru; Lapuk, V. A.; Shtykova, E. V.

The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that formore » Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.« less

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

PubMed

Crivianu-Gaita, Victor; Thompson, Michael

2015-08-15

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Site-specific PEGylation of an anti-CEA/CD3 bispecific antibody improves its antitumor efficacy

PubMed Central

Pan, Haitao; Liu, Jiayu; Deng, Wentong; Xing, Jieyu; Li, Qing; Wang, Zhong

2018-01-01

Introduction Bispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies. Materials and methods In this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab. Results The half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab. Conclusion This study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies. PMID:29881272

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

PubMed

Valjakka, J; Hemminki, A; Teerinen, T; Takkinen, K; Rouvinen, J

2000-02-01

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

The fading affect bias shows positive outcomes at the general but not the individual level of analysis in the context of social media.

PubMed

Gibbons, Jeffrey A; Horowitz, Kyle A; Dunlap, Spencer M

2017-08-01

Unpleasant affect fades faster than pleasant affect (e.g., Walker, Vogl, & Thompson, 1997); this effect is referred to as the Fading Affect Bias (FAB; Walker, Skowronski, Gibbons, Vogl, & Thompson, 2003a). Research shows that the FAB is consistently related to positive/healthy outcomes at a general but not at a specific level of analysis based on event types and individual differences (e.g., Gibbons et al., 2013). Based on the positive outcomes for FAB and negative outcomes for social media (Bolton et al., 2013; Huang, 2010), the current study examined FAB in the context of social media events along with related individual differences. General positive outcomes were shown in the form of robust FAB effects across social media and non-social media events, a larger FAB for non-social media events than for social media events, negative correlations of FAB with depression, anxiety, and stress as well as a positive correlation of FAB with self-esteem. However, the lack of a negative correlation between FAB and anxiety for social media events in a 3-way interaction did not show positive outcomes at a specific level of analysis. Rehearsal ratings mediated the 3-way interaction. Implications are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

PubMed

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

PubMed Central

Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

2012-01-01

Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

Physical functional outcome assessment of patients with major burns admitted to a UK Burn Intensive Care Unit.

PubMed

Smailes, Sarah T; Engelsman, Kayleen; Dziewulski, Peter

2013-02-01

Determining the discharge outcome of burn patients can be challenging and therefore a validated objective measure of functional independence would assist with this process. We developed the Functional Assessment for Burns (FAB) score to measure burn patients' functional independence. FAB scores were taken on discharge from ICU (FAB 1) and on discharge from inpatient burn care (FAB 2) in 56 patients meeting the American Burn Association criteria for major burn. We retrospectively analysed prospectively collected data to measure the progress of patients' physical functional outcomes and to evaluate the predictive validity of the FAB score for discharge outcome. Mean age was 38.6 years and median burn size 35%. Significant improvements were made in the physical functional outcomes between FAB 1 and FAB 2 scores (p<0.0001). 48 patients were discharged home, 8 of these with social care. 8 patients were transferred to another hospital for further inpatient rehabilitation. FAB 1 score (≤ 9) is strongly associated with discharge outcome (p<0.006) and as such can be used to facilitate early discharge planning. FAB 2 score (≤ 26) independently predicts discharge outcome (p<0.0001) and therefore is a valid outcome measure to determine discharge outcome of burn patients. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Persian version of frontal assessment battery: Correlations with formal measures of executive functioning and providing normative data for Persian population.

PubMed

Asaadi, Sina; Ashrafi, Farzad; Omidbeigi, Mahmoud; Nasiri, Zahra; Pakdaman, Hossein; Amini-Harandi, Ali

2016-01-05

Cognitive impairment in patients with Parkinson's disease (PD) mainly involves executive function (EF). The frontal assessment battery (FAB) is an efficient tool for the assessment of EFs. The aims of this study were to determine the validity and reliability of the psychometric properties of the Persian version of FAB and assess its correlation with formal measures of EFs to provide normative data for the Persian version of FAB in patients with PD. The study recruited 149 healthy participants and 49 patients with idiopathic PD. In PD patients, FAB results were compared to their performance on EF tests. Reliability analysis involved test-retest reliability and internal consistency, whereas validity analysis involved convergent validity approach. FAB scores compared in normal controls and in PD patients matched for age, education, and Mini-Mental State Examination (MMSE) score. In PD patients, FAB scores were significantly decreased compared to normal controls, and correlated with Stroop test and Wisconsin Card Sorting Test (WCST). In healthy subjects, FAB scores varied according to the age, education, and MMSE. In the FAB subtest analysis, the performances of PD patients were worse than the healthy participants on similarities, fluency tasks, and Luria's motor series. Persian version of FAB could be used as a reliable scale for the assessment of frontal lobe functions in Iranian patients with PD. Furthermore, normative data provided for the Persian version of this test improve the accuracy and confidence in the clinical application of the FAB.

Recurrent coagulopathy and thrombocytopenia in children treated with crotalidae polyvalent immune fab: a case series.

PubMed

Miller, Alexander D; Young, Michael C; DeMott, Megan C; Ly, Binh T; Clark, Richard F

2010-08-01

Recurrent signs and symptoms after initial treatment and control of coagulopathy and thrombocytopenia after American pit viper (crotaline) envenomations have been previously described in patients treated with Crotalidae polyvalent immune Fab antivenom (FabAV). The significance and necessity of treatment of these recurrent abnormalities are uncertain. Our goal was to further characterize recurrent coagulopathy or thrombocytopenia in pediatric patients. All cases presenting to our Toxicology Consult Service, which covers 6 hospitals in a metropolitan area, from May 2007 to April 2008 with recurrent coagulopathy after initial control with FabAV were included and retrospectively reviewed. Four cases of pediatric patients are presented who presented with recurrent coagulopathy and/or thrombocytopenia after initial control with FabAV. The patients were all treated with delayed administration of FabAV with variable results. Blood products administered without concurrent FabAV were of limited use. The laboratory abnormalities took up to 18 days to resolve in one case. One patient developed hemodynamically significant spontaneous bleeding. The cases presented here suggest administration of FabAV may correct delayed coagulopathy associated with crotaline envenomations. The first 3 cases illustrate that in the face of severe derangements in laboratory values, most envenomated patients treated with FabAV do not develop significant bleeding. These cases may respond to additional antivenom alone. However, case 4 illustrates that hemodynamically significant spontaneous bleeding can occur. Until more data are available, readministration of FabAV is a reasonable first-line therapy for delayed coagulopathy associated with crotaline envenomations.

Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

PubMed

Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

2016-01-01

Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

Deciphering the key residues in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase responsible for interactions with Plasmodium falciparum acyl carrier protein.

PubMed

Karmodiya, Krishanpal; Modak, Rahul; Sahoo, Nirakar; Sajad, Syed; Surolia, Namita

2008-10-01

The type II fatty acid synthase (FAS) pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials, due to its intrinsic differences from the typeI pathway operating in humans. beta-Ketoacyl acyl carrier protein (ACP) reductase (FabG) performs the NADPH-dependent reduction of beta-ketoacyl-ACP to beta-hydroxyacyl-ACP, the first reductive step in the elongation cycle of fatty acid biosynthesis. In this article, we report intensive studies on the direct interactions of Plasmodium FabG and Plasmodium ACP in solution, in the presence and absence of its cofactor, NADPH, by monitoring the change in intrinsic fluorescence of P.falciparum FabG (PfFabG) and by surface plasmon resonance. To address the issue of the importance of the residues involved in strong, specific and stoichiometric binding of PfFabG to P.falciparum ACP (PfACP), we mutated Arg187, Arg190 and Arg230 of PfFabG. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The affinities of all the PfFabG mutants for acetoacetyl-ACP (the physiological substrate) were reduced to different extents as compared to wild-type PfFabG, but were equally active in biochemical assays with the substrate analog acetoacetyl-CoA. Kinetic analysis and studies of direct binding between PfFabG and PfACP confirmed the identification of Arg187 and Arg230 as critical residues for the PfFabG-PfACP interactions. Our studies thus reveal the significance of the positively charged/hydrophobic patch located adjacent to the active site cavities of PfFabG for interactions with PfACP.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Crotaline Fab antivenom appears to be effective in cases of severe North American pit viper envenomation: An integrative review

PubMed Central

Lavonas, Eric J; Schaeffer, Tammi H; Kokko, Jamie; Mlynarchek, Sara L; Bogdan, Gregory M

2009-01-01

Background In 2000, the United States Food and Drug Administration approved Crotalidae Polyvalent Immune Fab (Ovine) (hereafter, FabAV), "for the management of patients with minimal to moderate North American Crotalid envenomation." Because whole-IgG pit viper antivenom is no longer available in the United States, FabAV is currently the only specific treatment option available to United States clinicians treating snakebite victims of any severity. No clinical trial data are available concerning the effectiveness of FabAV for treatment of severe snakebite, but several published articles describe its use in this setting. Methods We performed a comprehensive review of the English-language medical literature to identify all publications (1996 to July, 2008) containing data about the administration of FabAV. Two trained reviewers separately extracted case-level data concerning the administration of FabAV to patients with severe envenomation by North American crotaline snakes to a standardized form. Descriptive statistics were used. In addition, we hand-searched the US National Poison Data System reports for the years 2000–2006 to identify and describe any reports of death that occurred after FabAV administration. Results The literature review found 147 unique publications regarding FabAV. Twenty-four evaluable cases of severe human envenomation treated with FabAV were identified in 19 publications. Seven cases were described in five cohort studies, and 17 cases were described in 14 single patient case reports or non-cohort case series. Sixty-five specific severe venom effects were reported in these 24 patients, of which 50 effects (77%) improved or resolved after FabAV therapy. Initial control of all severe venom effects was achieved in 12 patients (50%). The rate at which initial control was achieved was significantly higher among patients reported in the cohort series than in the case series and non-cohort reports (100% vs. 29%, P = 0.005). The median dose of FabAV used to obtain initial control was 6 vials (range: 4 – 18 vials). Nine patients had severe venom effects that persisted despite FabAV therapy. Recurrent and/or delayed-onset severe defibrination syndrome occurred in 12 patients, most of whom did not receive recommended maintenance FabAV dosing. No patient developed systemic bleeding. Conclusion In this structured literature review, FabAV appears to be effective in the management of severe crotaline snake envenomation. Incomplete response to therapy, recurrence of venom effects, and delayed-onset venom effects were reported in case reports, but not reported in cohort studies. PMID:19545426

78 FR 19017 - Agency Information Collection Activities; Submission for OMB Review; Comment Request; Annual...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-28

... (FAB) 2013-01 concerning the new disclosure requirements mandated the MAP-21. The FAB addresses a need...), as amended by the MAP-21. The FAB sets forth technical questions and answers and provides a model... the FAB is necessary for plan administrators to satisfy the annual funding notice requirements. The...

Crotalidae polyvalent immune Fab: in patients with North American crotaline envenomation.

PubMed

Keating, Gillian M

2011-04-01

Crotalidae polyvalent immune Fab is an antivenom comprising purified, sheep-derived, Fab IgG fragments and is indicated for use in patients with North American crotaline envenomation. Crotalidae polyvalent immune Fab is produced using four North American snake venoms: Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus, and Agkistrodon piscivorus. Intravenous crotalidae polyvalent immune Fab was effective in patients aged ≥10 years who had minimal or moderate envenomation by a North American crotaline, who presented within 6 hours of the snakebite, and who had progression of the envenomation syndrome, according to the results of two prospective trials. One trial was a noncomparative, multicenter pilot study and the other trial was a randomized, open-label, multicenter trial in which patients received scheduled or 'as needed' administration of crotalidae polyvalent immune Fab after initial control had been achieved. A prospective, postmarketing trial demonstrated the efficacy of crotalidae polyvalent immune Fab in confirmed Crotalus viridis helleri envenomation (indicating cross-protection against a venom not used in its production). Results of these prospective trials are supported by the findings of additional (mainly retrospective) studies demonstrating the efficacy of crotalidae polyvalent immune Fab in patients with crotaline envenomation, including patients with severe envenomation, pediatric patients, and patients with symptoms of neurotoxicity. Despite treatment with crotalidae polyvalent immune Fab, patients may experience delayed-onset or recurrent venom effects (e.g. coagulopathy). Intravenous crotalidae polyvalent immune Fab was generally well tolerated; acute hypersensitivity reactions (e.g. urticaria, rash, pruritus) were the most commonly occurring adverse event. © 2011 Adis Data Information BV. All rights reserved.

Recurrent hemorrhage after western diamondback rattlesnake envenomation treated with crotalidae polyvalent immune fab (ovine).

PubMed

Fazelat, Joyia; Teperman, Sheldon H; Touger, Michael

2008-11-01

Recurrent coagulopathy has been observed in patients after rattlesnake envenomation treated with Crotalidae Polyvalent Immune Fab (ovine) [FabAV]. While recurrent coagulopathy is well documented in the literature, clinically significant sequelae have not been reported. We present a case of recurrent thrombocytopenia after western diamondback envenomation treated with FabAV, resulting in an extensive recurrent local hemorrhage. A 24-year-old male presented to our emergency department several hours after western diamondback envenomation. He sustained bites to both hands and the right flank by leaning over his pet "snake enclosure." On presentation, the patient was hypotensive, tachycardic, and thrombocytopenic with a platelet count of 17/nl. Antivenom therapy was initiated according to the standard FabAV protocol. However, sixteen hours after completion of the recommended FabAV infusion, the patient experienced a recurrent thrombocytopenia with a dramatic seventeen point drop in hematocrit. The source of bleeding was clinically attributed to an expanding hematoma at the site of envenomation. FabAV has become the standard treatment for symptomatic crotalid envenomation. However, the pharmacokinetics of this drug predispose it to recurrent coagulopathies. While studies have shown persistent and recurrent coagulopathic derangements after FabAV therapy, no clinically significant sequelae have been reported. This report highlights the potential for recurrent local hemorrhagic complications following rattlesnake envenomation, even after treatment guided by the current FabAV protocol. Recurrent coagulopathy following FabAV therapy can result in clinically significant hemorrhage, supporting the observation that extended repeat dosing may be necessary to adequately treat subjects of rattlesnake envenomation.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

PubMed Central

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

PubMed

Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

2009-01-01

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the FAB...

Antivenom therapy for snakebites in children: is there evidence?

PubMed

Schmidt, James M

2005-04-01

A new Fab fragment antivenom (CroFab) for the treatment of crotaline envenomation, the predominant venomous snakebite in the United States, has drastically changed snakebite management since its release in December 2000. This review examines the evidence supporting the use of CroFab, with particular attention on the pediatric population. The published experience with CroFab in humans consists of six studies and some case reports. These publications demonstrate that CroFab is highly efficacious in treating both the local and systemic toxic effects of crotaline envenomation. They identify an important phenomenon of recurrent or delayed toxicity in some patients. The studies report a very low incidence of acute or delayed hypersensitivity reactions to the antivenom. They suggest comparable efficacy and safety of CroFab in the pediatric population as in adults. Based on limited data, CroFab has been shown to be a safe and efficacious antivenom for use in children as well as adults. Further studies are needed to refine our understanding of its efficacy, safety, indications, and dosing.

Individual differences in emotional processing and autobiographical memory: interoceptive awareness and alexithymia in the fading affect bias.

PubMed

Muir, Kate; Madill, Anna; Brown, Charity

2017-11-01

The capacity to perceive internal bodily states is linked to emotional awareness and effective emotional regulation. We explore individual differences in emotional awareness in relation to the fading affect bias (FAB), which refers to the greater dwindling of unpleasant compared to pleasant emotions in autobiographical memory. We consider interoceptive awareness and alexithymia in relation to the FAB, and private event rehearsal as a mediating process. With increasing interoceptive awareness, there was an enhanced FAB, but with increasing alexithymia, there was a decreased FAB. Further, the effects of interoceptive awareness were partially mediated by private rehearsal of pleasant events. We provide novel evidence that capacity for emotional awareness and thus effective processing is an important factor predictive of the FAB. Moreover, our results imply an important role for maintaining positive affect in the FAB. Our findings offer new insights into the effects of interoception and alexithymia on autobiographical memory, and support concepts of the FAB emerging as a result of adaptive emotional regulation processes.

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Methyl-branched fatty acids, inhibitors of enoyl-ACP reductase with antibacterial activity from Streptomyces sp. A251.

PubMed

Zheng, Chang-Ji; Sohn, Mi-Jin; Chi, Seung-Wook; Kim, Won-Gon

2010-05-01

Bacterial enoyl-ACP reductase (FabI) has been demonstrated to be a novel antibacterial target. In the course of our screening for FabI inhibitors we isolated two methyl-branched fatty acids from Streptomyces sp. A251. They were identified as 14-methyl-9(Z)-pentadecenoic acid and 15-methyl-9(Z)-hexadecenoic acid by MS and NMR spectral data. These compounds inhibited Staphylococcus aureus FabI with IC50 of 16.0 and 16.3mu M, respectively, while didn't affect FabK, an enoyl-ACP reductase of Streptococcus pneumonia, at 100muM. Consistent with their selective inhibition for FabI, they blocked intracellular fatty acid synthesis as well as the growth of S. aureus, while didn't inhibit the growth of S. pneumonia. Additionally, these compounds showed reduced antibacterial activity against fabI-overexpressing S. aureus compared to the wild-type strain. These results demonstrate that the methyl-branched fatty acids showed antibacterial activity by inhibiting FabI in vivo.

75 FR 39497 - Procurement List; Proposed Additions and Deletion

Federal Register 2010, 2011, 2012, 2013, 2014

2010-07-09

... U.S. Coast Guard as aggregated by the U.S. Coast Guard. NSN: 8415-00-FAB-5722--Kit, Pre-Cut Fabric, ACU Coat, XS-XS. NSN: 8415-00-FAB-5723--Kit, Pre-Cut Fabric, ACU Coat, XS-S. NSN: 8415-00-FAB-5724--Kit, Pre-Cut Fabric, ACU Coat, XS-R. NSN: 8415-00-FAB-5725--Kit, Pre-Cut Fabric, ACU Coat, S-XXS. NSN...

20 CFR 30.316 - How does the FAB issue a final decision on a claim?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final decision...

Prophylactic or therapeutic administration of Agaricus blazei Murill is effective in treatment of murine visceral leishmaniasis.

PubMed

Valadares, Diogo G; Duarte, Mariana C; Ramírez, Laura; Chávez-Fumagalli, Miguel A; Martins, Vivian T; Costa, Lourena E; Lage, Paula S; Ribeiro, Tatiana G; Castilho, Rachel O; Fernandes, Ana Paula; Régis, Wiliam C B; Soto, Manuel; Tavares, Carlos A P; Coelho, Eduardo A F

2012-10-01

The present study aimed to investigate the in vitro antileishmanial activity of five fractions obtained from Agaricus blazei water extract (AbM), namely, Fab1, Fab2, Fab3, Fab4, and Fab5; and use the selected leishmanicidal fraction to treat BALB/c mice infected with Leishmania chagasi. A curve dose-titration was performed to obtain the concentration to be test in infected animals. In this context, Fab5 fraction and AbM were used in the doses of 20 and 100 mg/kg/day, respectively, with the product been administered once a day. The effect induced by a chemo-prophylactic regimen, based on the administration Fab5 fraction and AbM 5 days before infection, and maintained for an additional 20 days post-infection was compared to a therapeutic regimen, in which the compounds were administered from 0 to 20 days of infection. Control animals were either treated with amphotericin B deoxycholate (AmpB) or received distilled water. All groups were followed up for 10 weeks post-infection, when parasitological and immunological parameters were analyzed. The Fab5 presented the best results of in vitro leishmanicidal activity. In the in vivo experiments, the use of Fab5 or AbM, as compared to control groups, resulted in significant reduced parasite burdens in the liver, spleen, and draining lymph nodes of the infected animals, as compared to control groups. A Type 1 immune response was observed in the Fab5 or AbM treated animals. No significant toxicity was observed. The chemo-prophylactic regimen proved to be more effective to induce theses responses. In this context, the data presented in this study showed the potential of the purified Fab5 fraction of AbM as a therapeutic alternative to treat visceral leishmaniasis. In addition, it can be postulated that this fraction can be also employed in a chemo-prophylactic regimen associated or not with other therapeutic products. Copyright © 2012 Elsevier Inc. All rights reserved.

A comparison of capture antibody fragments in cardiac troponin I immunoassay.

PubMed

Hyytiä, Heidi; Järvenpää, Marja-Leena; Ristiniemi, Noora; Lövgren, Timo; Pettersson, Kim

2013-08-01

To compare cardiac troponin I (cTnI) values measured from 32 normal plasma specimens with a two-site cTnI research assay exploiting different molecular forms of a capture antibody. The current research assay consists of two capture antibodies immobilized on streptavidin-well surface and one detection antibody attached to highly fluorescent europium(III)-chelate-doped nanoparticles. Four different molecular forms of one of the capture antibodies (intact monoclonal (Mab), F(ab')2 fragment, Fab fragment and chimeric Fab fragment (cFab)) were tested. The developed immunoassays were evaluated in terms of their analytical sensitivities and assay kinetics. Furthermore, cTnI concentrations were measured from 32 heparin plasma samples from apparently healthy donors (mean age 32; range 24-60 years). The differences in the measured cTnI concentrations (corrected for the buffer-based zero calibrator) between the Mab and the three fragmented forms were highly significant (P<0.0001). Replacing the intact Mab with the antibody fragments also reduced the required antibody amount from 100 ng to 66 ng (F(ab')2) and 16.5 ng (Fab and cFab). Furthermore, the limit of detection was improved when Fab fragments were employed (Mab: 0.90 ng/L, Fab: 0.69 ng/L and cFab: 0.41 ng/L). The apparent normal range median (minimum/maximum) of the 32 healthy subjects was reduced from 7.28 ng/L (2.64/116 ng/L) with Mab to 1.80 ng/L (0.746/10.6 ng/L) for the cFab. Eliminating the Fc-part from one of the two capture antibodies in an immunofluorometric cTnI assay substantially reduced the measured cTnI concentrations, simultaneously improving the assay sensitivity and reducing the reagent consumption. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

A Cryo-Electron Microscopy Study Identifies the Complete H16.V5 Epitope and Reveals Global Conformational Changes Initiated by Binding of the Neutralizing Antibody Fragment

PubMed Central

Lee, Hyunwook; Brendle, Sarah A.; Bywaters, Stephanie M.; Guan, Jian; Ashley, Robert E.; Yoder, Joshua D.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.

2014-01-01

ABSTRACT Human papillomavirus 16 (HPV16) is a worldwide health threat and an etiologic agent of cervical cancer. To understand the antigenic properties of HPV16, we pursued a structural study to elucidate HPV capsids and antibody interactions. The cryo-electron microscopy (cryo-EM) structures of a mature HPV16 particle and an altered capsid particle were solved individually and as complexes with fragment of antibody (Fab) from the neutralizing antibody H16.V5. Fitted crystal structures provided a pseudoatomic model of the virus-Fab complex, which identified a precise footprint of H16.V5, including previously unrecognized residues. The altered-capsid–Fab complex map showed that binding of the Fab induced significant conformational changes that were not seen in the altered-capsid structure alone. These changes included more ordered surface loops, consolidated so-called “invading-arm” structures, and tighter intercapsomeric connections at the capsid floor. The H16.V5 Fab preferentially bound hexavalent capsomers likely with a stabilizing effect that directly correlated with the number of bound Fabs. Additional cryo-EM reconstructions of the virus-Fab complex for different incubation times and structural analysis provide a model for a hyperstabilization of the capsomer by H16.V5 Fab and showed that the Fab distinguishes subtle differences between antigenic sites. IMPORTANCE Our analysis of the cryo-EM reconstructions of the HPV16 capsids and virus-Fab complexes has identified the entire HPV.V5 conformational epitope and demonstrated a detailed neutralization mechanism of this clinically important monoclonal antibody against HPV16. The Fab bound and ordered the apical loops of HPV16. This conformational change was transmitted to the lower region of the capsomer, resulting in enhanced intercapsomeric interactions evidenced by the more ordered capsid floor and “invading-arm” structures. This study advances the understanding of the neutralization mechanism used by H16.V5. PMID:25392224

Toxicokinetic and toxicodynamic analyses of Androctonus australis hector venom in rats: Optimization of antivenom therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hammoudi-Triki, D.; Laboratoire de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Universite des Sciences et de la Technologie 'Houari Boumedienne' Bab Ezzouar, Alger, Algerie; Laboratoire de Recherche et de Developpement sur les Venins, Institut Pasteur d'Algerie, Algerie

2007-02-01

This paper reports the simultaneous determination of toxicokinetic and toxicodynamic properties of Androctonus australis hector venom, in the absence and presence of antivenom (F(ab'){sub 2} and Fab), in envenomed rats. After subcutaneous injection of the venom, toxins showed a complete absorption phase from the site of injection associated with a distribution into a large extravascular compartment. The injection of Fab and F(ab'){sub 2} induced the neutralization of venom antigens in the blood compartment, as well as the redistribution of venom components from the extravascular compartment to the blood compartment. Interestingly, F(ab'){sub 2} and Fab showed distinct efficiencies depending on theirmore » route of injection. F(ab'){sub 2} induced a faster venom neutralization and redistribution than Fab when injected intravenously. Fab was more effective than F(ab'){sub 2} by the intramuscular route. The hemodynamic effects of Aah venom were further investigated. Changes in mean arterial pressure and heart rate were observed in parallel with an upper airway obstruction. Fab was more effective than F(ab'){sub 2} for preventing early symptoms of envenomation, whatever their route of administration. Intraperitoneal injection of F(ab'){sub 2} and Fab was similar for the prevention of the delayed symptoms, even after a late administration. Fab was more effective than F(ab'){sub 2} in the inhibition of airway resistance, independent of the route and time of administration. These results show that the treatment for scorpion stings might be improved by the intravascular injection of a mixture of Fab and F(ab'){sub 2}. If antivenom cannot be administered intravenously, Fab might be an alternative as they are more effective than F(ab'){sub 2} when injected intramuscularly.« less

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

PubMed

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Acute hypersensitivity reaction to Crotalidae polyvalent immune Fab (CroFab) as initial presentation of galactose-α-1,3-galactose (α-gal) allergy.

PubMed

Rizer, Justin; Brill, Kaitlin; Charlton, Nathan; King, Joshua

2017-08-01

Crotalidae polyvalent immune Fab antivenom (CroFab), commonly used for the treatment of clinically significant North American crotalinae envenomation, is generally well-tolerated. A novel form of anaphylaxis due to an IgE antibody response to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) has been established following red-meat consumption as well as IV administration of cetuximab, which contain the α-gal epitope. We present a case of α-gal allergy discovered after acute hypersensitivity reaction to FabAV. A 61-year-old healthy female was bitten on her left ankle by Agkistrodon contortrix. Given the patient's rapid progression of pain and swelling, she was given FabAV. During infusion of FabAV, she developed diffuse hives over her entire body and itching, but denied respiratory or gastrointestinal symptoms and her vital signs remained stable. The FabAV was immediately discontinued and she received intravenous diphenhydramine and famotidine with gradual resolution of symptoms. On further discussion, she denied a history of α-gal or papaya allergy but rarely ate red meat and endorsed sustaining frequent tick bites. Subsequent antibody testing was significant for an α-1,3-galactose IgE concentration of 45,000 U/L (normal <3500 U/L), confirming α-gal allergy. To our knowledge, this is the first report of FabAV hypersensitivity associated with an underlying α-gal allergy.

Initial postmarketing experience with crotalidae polyvalent immune Fab for treatment of rattlesnake envenomation.

PubMed

Ruha, Anne-Michelle; Curry, Steven C; Beuhler, Michael; Katz, Ken; Brooks, Daniel E; Graeme, Kimberlie A; Wallace, Kevin; Gerkin, Richard; Lovecchio, Frank; Wax, Paul; Selden, Brad

2002-06-01

We describe our postmarketing experience with patients receiving Crotalidae polyvalent immune Fab (CroFab; FabAV) antivenom for treatment of rattlesnake envenomation. The charts of 28 patients admitted between March 1 and September 9, 2001, with rattlesnake envenomation and treated with FabAV were reviewed for demographic information, time until antivenom treatment, laboratory findings, evidence of hypersensitivity reaction, length of hospital stay, and readmission to the hospital. All patients had swelling, 20 patients had elevated prothrombin times (>14 seconds), 12 patients had low fibrinogen levels (<170 mg/dL), and 6 patients had thrombocytopenia (platelet count <120,000/mm(3)) on presentation. The total dose of FabAV ranged from 10 to 47 vials per patient. Hypofibrinogenemia was resistant to FabAV in some patients. On follow-up, recurrence of coagulopathy was detected in 3 patients, and recurrence of thrombocytopenia was detected in 1 patient. Two patients demonstrated delayed-onset severe thrombocytopenia. Recurrence or delayed-onset toxicity might have been underestimated because of incomplete follow-up in some patients. No acute hypersensitivity reactions occurred. Two patients reported mild symptoms of possible serum sickness on follow-up. FabAV effectively controlled the effects of envenomation; however, initial control of coagulopathy was difficult to achieve in some cases, and recurrence or delayed-onset hematotoxicity was common. When initially managing hematotoxicity, a trend toward normalization of laboratory values might be a more reasonable end point for FabAV treatment than attainment of normal reference values in nonbleeding patients.

Humoral anti-OV-TL 3 response after the intravenous administration of radiolabelled Fab' or F(ab')2 fragments in ovarian cancer patients.

PubMed

Tibben, J G; Thomas, C M; Massuger, L F; Segers, M F; Schijf, C P; Corstens, F H; Boerman, O C

1995-10-01

The human anti-mouse antibody (HAMA) response was determined in the serum of patients suspected of having ovarian cancer who underwent radioimmunoscintigraphy with either 99Tcm-OV-TL 3 Fab' (n = 20) or 111In-DTPA-OV-TL 3 F(ab')2 (n = 73). Blood samples were collected prior to and at several time points post-intravenous injection. The detection of HAMA was performed with an in-house OV-TL 3 F(ab')2-based sandwich-type immunoradiometric assay (IRMA). The homologous IRMA demonstrated that 8 of 20 (40%) patients had developed HAMA responses after injection of Fab' fragments and that 14 of 73 (19%) patients had developed HAMA responses after F(ab')2 administration. The subclass of the measured HAMA was analysed in a limited number of samples, showing IgG or IgM as well as mixed responses. The kinetics of the HAMA responses varied greatly. Our study showed the relevance of the sampling time and frequency: HAMA responses can be easily underestimated with a low sampling frequency. The homologous IRMA described in this study was able to quantify the OV-TL 3-specific HAMA responses. With additional assays, the subclass of the HAMA could be further analysed. Remarkably, the fraction of HAMA responders after injection of OV-TL 3 Fab' fragments was in the same range as the proportion of HAMA responders after F(ab')2 administration.

A novel antibody-drug conjugate anti-CD19(Fab)-LDM in the treatment of B-cell non-Hodgkin lymphoma xenografts with enhanced anticancer activity.

PubMed

Jiang, Linlin; Yang, Ming; Zhang, Xiaoyun; Bao, Shiqi; Ma, Li; Fan, Dongmei; Zhou, Yuan; Xiong, Dongsheng; Zhen, Yongsu

2016-01-01

Rituximab is widely used in clinical setting for the treatment of B malignant lymphoma and has achieved remarkable success. However, in most patients, the disease ultimately relapses and become resistant to rituximab. To overcome the limitation, there is still a need to find novel strategy for improving therapeutic efficacy. To construct genetically engineered antibody anti-CD19(Fab)-LDM, and verify the anticancer activity targeted toward B-lymphoma. The anticancer activity of anti-CD19(Fab)-LDM in vitro and in vivo was examined. In vitro, the binding activity and internalization of anti-CD19(Fab)-LDP were measured. Using comet assay and apoptosis, the cytotoxicity of energized fusion proteins was observed. From in vivo experiments, targeting of therapeutic effect and anticancer efficacy bythe fusion protein was verified. Data showed that anti-CD19(Fab)-LDM does not only binding the cell surface but is also internalized into the cell. The energized fusion proteins anti-CD19(Fab)-LDM can induce DNA damage. Furthermore, significant in vivo therapeutic efficacy was observed. The present study demonstrated that the genetically engineered antibody anti-CD19(Fab)-LDM exhibited enhanced cytotoxicity compared to LDM alone. One of the most powerful advantages of anti-CD19(Fab)-LDM, however, is that it can be internalized within the cells and carry out cytotoxic effects. Therefore, anti-CD19(Fab)-LDM may be as a useful targeted therapy for B-cell lymphoma.

Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen‐induced allergic rhinitis

PubMed Central

Mizutani, N

2016-01-01

Background and Purpose Fab fragments (Fabs) of antibodies have the ability to bind to specific allergens but lack the Fc portion that exerts effector functions via binding to receptors including FcεR1 on mast cells. In the present study, we investigated whether intranasal administration of the effector function‐lacking Fabs of a monoclonal antibody IgG1 (mAb, P1‐8) to the major allergen Cry j1 of Japanese cedar pollen (JCP) suppressed JCP‐induced allergic rhinitis in mice. Experimental Approach Balb/c mice sensitized with JCP on days 0 and 14 were challenged intranasally with the pollen on days 28, 29, 30 and 35. Fabs prepared by the digestion of P1‐8 with papain were also administered intranasally 15 min before each JCP challenge. Key Results Intranasal administration of P1‐8 Fabs was followed by marked suppression of sneezing and nasal rubbing in mice with JCP‐induced allergic rhinitis. The suppression of these allergic symptoms by P1‐8 Fabs was associated with decreases in mast cells and eosinophils and decreased hyperplasia of goblet cells in the nasal mucosa. Conclusions and Implications These results demonstrated that intranasal exposure to P1‐8 Fabs was effective in suppressing JCP‐induced allergic rhinitis in mice, suggesting that allergen‐specific mAb Fabs might be used as a tool to regulate allergic pollinosis. PMID:26895546

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

PubMed

Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

2016-12-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316, and...

20 CFR 30.317 - Can the FAB request a further response from the claimant or return a claim to the district office?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB request a further response from....317 Can the FAB request a further response from the claimant or return a claim to the district office? At any time before the issuance of its final decision, the FAB may request that the claimant submit...

Increasing the utility of the Functional Assessment for Burns Score: Not just for major burns.

PubMed

Smailes, Sarah T; Engelsman, Kayleen; Rodgers, Louise; Upson, Clara

2016-02-01

The Functional Assessment for Burns (FAB) score is established as an objective measure of physical function that predicts discharge outcome in adult patients with major burn. However, its validity in patients with minor and moderate burn is unknown. This is a multi-centre evaluation of the predictive validity of the FAB score for discharge outcome in adult inpatients with minor and moderate burns. FAB assessments were undertaken within 48 h of admission to (FAB 1), and within 48 h of discharge (FAB 2) from burn wards in 115 patients. Median age was 45 years and median burn size 4%. There were significant improvements in the patients' FAB scores (p<0.0001), 98 patients were discharged home (no social care) and 17 patients discharged to further inpatient rehabilitation or home with social care. FAB 1 score (≤ 14) is strongly associated with discharge to inpatient rehabilitation or home with social care (p=0.0001) and as such can be used to facilitate early discharge planning. FAB 2 (≤ 30) independently predicts discharge outcome to inpatient rehabilitation or home with social care (p<0.0001), increasing its utility to patients with minor and moderate burns. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

PubMed

Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Comparative study of thiophilic functionalised matrices for polyclonal F(ab')2 purification.

PubMed

Kumpalume, Peter; Slater, Nigel K H

2004-01-02

Thiophilic adsorbents have been developed using divinyl sulfone or epoxy activated Streamline quartz base matrix. Their capacity and selectivity for binding polyclonal F(ab')2 fragments generated by whole serum proteolysis was tested. Except for epoxy activated guanidine, all the adsorbents displayed high selectivity for F(ab')2 with dynamic binding capacities ranging from 3 to 10 mg/ml of adsorbent. Thiol immobilised ligands adsorbed more F(ab')2 and the recovery was equal to or more than that from amino immobilised ligands. All adsorbents showed good selectivity for IgG and the dynamic binding capacities were better than for F(ab')2.

Recurrent coagulopathy with delayed significant bleeding after crotaline envenomation.

PubMed

O'Brien, Nicole F; DeMott, Megan C; Suchard, Jeffrey R; Clark, Richard F; Peterson, Bradley M

2009-07-01

Report of delayed significant coagulopathy, thrombocytopenia, and bleeding after Crotaline envenomation. Recurrent coagulopathy and thrombocytopenia have been described after treatment of Crotaline envenomation with Crotalidae polyvalent immune Fab (CroFab). Until now, there have been no reports of significant spontaneous bleeding despite these abnormalities. Crotalidae polyvalent immune Fab has a relatively short half-life compared with previous antivenoms used to treat snake bite. This shorter half-life allows for recurrence of venom effects. Therefore, patients with Crotaline envenomation should undergo close monitoring for recurrence of coagulopathy or thrombocytopenia after treatment with CroFab. If coagulopathy or thrombocytopenia recurs, retreatment with CroFab should be considered to prevent significant bleeding.

Continuous IV Crotalidae Polyvalent Immune Fab (Ovine) (FabAV) for selected North American rattlesnake bite patients.

PubMed

Bush, Sean P; Seifert, Steven A; Oakes, Jennifer; Smith, Susan D; Phan, Tammy H; Pearl, Sarah R; Reibling, Ellen T

2013-07-01

In patients bitten by North American rattlesnakes and treated with Crotalidae Polyvalent Immune Fab (Ovine) (FabAV), late hematologic abnormalities-persistent, recurrent, or late, new onset of hypofibrinogenemia, prolonged PT/INR, prolonged PTT, and/or thrombocytopenia beyond 48 h post-envenomation-are common, difficult to manage, and may result in morbidity and mortality are common, difficult to manage, and may result in morbidity and mortality. The optimal management of late hematologic abnormalities, particularly the use of further treatment with antivenom, has not been well defined. The current FabAV treatment regimen is to give antivenom as a bolus dose over a one-hour period. We describe our experience using a continuous intravenous infusion of FabAV for late hematologic effects and/or associated bleeding complications in rattlesnake envenomation. This is a retrospective, observational case series of patients envenomated by North American rattlesnakes at three medical centers managed with a continuous intravenous infusion of FabAV for late hematologic abnormalities and/or associated bleeding complications. Indications, dilution and infusion protocols, and duration of therapy were individualized. Five cases were identified between July 2010 and September 2011. All patients had profound late hematologic abnormalities and/or were associated with bleeding complications. Several patients had received repeat bolus infusions of FabAV, with or without human blood products, with either inadequate or only transient beneficial response. All patients were then managed with a continuous intravenous infusion of FabAV and all appeared to respond to the continuous intravenous infusion of FabAV, titrated to effect, with cessation of progression and, in most cases, improvement in hematologic abnormalities. Rates of infusion varied from 2 to 4 vials per 24 h (mean = 3.1 ± 0.4 vials/day). The termination of FabAV infusion was between day 6 and day 14 from the time of envenomation (mean = 10 ± 3 days), after which hematologic values were normalized or were normalizing in all patients and continued to do so. The use of FabAV as a continuous intravenous infusion, particularly after the acute phase of envenomation has passed, provides a continuous source of circulating antibodies to neutralize venom components reaching circulation from tissue stores and allows natural replenishment of hematologic factors such as platelets and/or fibrinogen. This method is an efficient use of FabAV, avoiding the wasteful excess of a bolus dose, may be more effective, eliminating the potential for destruction of hematologic factors when protective antivenom levels are lost between bolus FabAV doses, and appears to be safe. Further assessments of the stability and sterility of the product during infusion are needed. The need to continue hospitalization is the major drawback, but continued observation and inpatient care may be needed for other indications (e.g. bleeding) in this subset of patients. A continuous intravenous infusion of FabAV between 2 and 4 vials per day, titrated to effect, and continued for 6-14 days post-envenomation appeared to be associated with reversal of late hematologic effects of rattlesnake envenomation and, when combined with indicated human blood products, control of significant bleeding. Continuous intravenous infusion of FabAV may be safer, more efficacious, and more cost-effective than observation without FabAV treatment or as-needed bolus dosing in selected patients with late hematologic abnormalities. Copyright © 2013 Elsevier Ltd. All rights reserved.

Immediate hypersensitivity reaction associated with the rapid infusion of Crotalidae polyvalent immune Fab (ovine).

PubMed

Holstege, Christopher P; Wu, Jeffrey; Baer, Alexander B

2002-06-01

A 16-year-old boy presented to the emergency department with rapidly progressing extremity pain, edema, and ecchymosis after envenomation by a copperhead. Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) was infused. Six vials were placed in 250 mL of normal saline solution, and the infusion was gradually increased. Fifty minutes after beginning, the infusion was increased to 640 mL/h. Within minutes of the rate increase, the patient experienced full-body urticaria, facial edema, voice change, and tachycardia. The infusion was stopped. Hydroxyzine pamoate, famotidine, methylprednisolone, and a 1-L bolus of normal saline solution were administered intravenously. The symptoms abated, and the remaining FabAV was infused at a slower rate without return of this reaction. This immediate hypersensitivity reaction was most likely a rate-related anaphylactoid reaction that has not been previously reported with FabAV.[Holstege CP, Wu J, Baer AB. Immediate hypersensitivity reaction associated with the rapid infusion of Crotalidae polyvalent immune Fab (ovine). Ann Emerg Med. June 2002;39:677-679.

Crotalidae polyvalent immune Fab: a guide to its use in North American crotaline envenomation.

PubMed

Keating, Gillian M; Lyseng-Williamson, Katherine A

2012-08-01

Intravenous crotalidae polyvalent immune Fab (CroFab(®)) is effective in patients with minimal, moderate or severe crotaline envenomation, halting the progression of local venom effects and ameliorating haematological and other systemic abnormalities of envenomation. Despite treatment, patients may experience delayed-onset or recurrent venom effects (e.g. coagulopathy). Intravenous crotalidae polyvalent immune Fab is generally well tolerated, with acute hypersensitivity reactions being the most commonly occurring adverse event.

20 CFR 30.312 - What will the FAB do if the claimant objects to the recommended decision but does not request a...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false What will the FAB do if the claimant objects....312 What will the FAB do if the claimant objects to the recommended decision but does not request a... period of time allotted in § 30.310 but does not request a hearing, the FAB will consider any objections...

[FAB immunoglobulin fragments. I. The comparative characteristics of the serological and virus-neutralizing properties of a gamma globulin against tick-borne encephalitis and of the FAB fragments isolated from it].

PubMed

Barban, P S; Minaeva, V M; Pantiukhina, A N; Startseva, M G

1976-06-01

A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a marked virus-neutralizing activity. The mean value of a logarithm of the neutralization index was 2.65 +/- 0.2 for Fab-fragments and 3.74 +/- 0.38 for gamma-globulin (P less than 0.01).

Pygmy rattlesnake envenomation treated with Crotalidae Polyvalent Immune Fab Antivenom.

PubMed

King, Andrew M; Crim, William S; Menke, Nathan B; Pizon, Anthony F

2012-12-01

Documented envenomations by the pygmy rattlesnake (Sistrurus miliarius barbouri) are rare. While there have been no documented fatalities, several older case reports describe significant morbidity. We describe the first known case of pygmy rattlesnake envenomation that was treated with Crotalidae Polyvalent Immune Fab Antivenom (CroFab®). A 28-year-old man with no significant past medical history presented after being envenomated on the right hand by his friend's pet pygmy rattlesnake. He developed swelling and pain in his hand and forearm. He responded well to a ten vial loading dose and a 18 h maintenance protocol of CroFab and was discharged the following day without developing any hematological or electrolyte derangements. This is the first documented use of CroFab for S. m. barbouri envenomation. The outcome of this case suggests that CroFab is a safe treatment modality in this setting. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biphasic rattlesnake venom-induced thrombocytopenia.

PubMed

Offerman, Steven R; Barry, J David; Schneir, Aaron; Clark, Richard F

2003-04-01

Thrombocytopenia is a common occurrence in moderate to severe crotaline envenomation. The exact mechanism by which rattlesnake venom leads to thrombocytopenia is unclear, but aggressive treatment with crotaline-specific antivenom often leads to resolution of this disorder. Crotalinae Polyvalent Immune Fab (CroFab(TM), Protherics Inc., Nashville, TN) (crotaline Fab) is now available for the treatment of symptomatic rattlesnake envenomation. Although recurrence of thrombocytopenia has been reported in patients after envenomation treated with crotaline Fab, cases refractory to this therapy have not been described. We report a case of severe crotaline envenomation that appears to have exhibited two separate episodes of thrombocytopenia, only one of which responded to antivenom. The second, later phase was refractory to both crotaline Fab as well as traditional Antivenin (Crotalinae) Polyvalent (Wyeth-Ayerst Pharmaceuticals, Philadelphia, PA) (ACP). By reviewing the literature regarding venom-induced thrombocytopenia, we attempt to explain this "biphasic" phenomenon and the inability of crotaline Fab to reverse this toxic effect.

Human Anti-Plague Monoclonal Antibodies Protect Mice from Yersinia pestis in a Bubonic Plague Model

PubMed Central

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L.; Wormald, Michael M.; Fast, Randy L.; Worsham, Patricia L.; Cote, Christopher K.; Amemiya, Kei; Dimitrov, Dimiter S.

2010-01-01

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans. PMID:20976274

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

PubMed

Xiao, Xiaodong; Zhu, Zhongyu; Dankmeyer, Jennifer L; Wormald, Michael M; Fast, Randy L; Worsham, Patricia L; Cote, Christopher K; Amemiya, Kei; Dimitrov, Dimiter S

2010-10-13

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

PubMed Central

Eraso, Jesus M.; Olsen, Randall J.; Beres, Stephen B.; Kachroo, Priyanka; Porter, Adeline R.; Nasser, Waleed; Bernard, Paul E.; DeLeo, Frank R.

2016-01-01

To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here. PMID:27600505

Crotalidae polyvalent immune Fab (ovine) antivenom is efficacious for envenomations by Southern Pacific rattlesnakes (Crotalus helleri).

PubMed

Bush, Sean P; Green, Steven M; Moynihan, James A; Hayes, William K; Cardwell, Michael D

2002-12-01

Southern Pacific rattlesnake (Crotalus helleri ) venom is not 1 of the 4 venoms used to produce Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV). There is currently no published clinical experience regarding the efficacy of this new antivenom for confirmed C helleri envenomation, and animal data suggest greatly diminished efficacy. We assessed the efficacy of FabAV for patients with confirmed C helleri envenomation. We conducted a prospective observational study of 23 consecutive rattlesnake envenomations that were treated with FabAV at our center. Patients were excluded if the species of snake could not be confirmed, if FabAV antivenom was not given, or if Antivenin (Crotalidae) polyvalent (equine) was given. We collected serial physical examination and laboratory data over a 24-hour period to serially evaluate the severity score and performed follow-up to evaluate delayed reactions. There were 15 patients who received FabAV and had the species of rattlesnake confirmed (9 C helleri, 4 C scutulatus scutulatus, 1 C mitchellii pyrrhus, 1 C ruber ruber ). C helleri envenomations demonstrated similar improvement in serial snakebite severity scores to those of other species. Three patients treated with scheduled dosing had recurrence of progressive swelling (2 C helleri and 1 C mitchellii pyrrhus ) during the 24-hour study period. We observed similar improvement in FabAV-treated patients with C helleri envenomation compared with those of other species and conclude that this treatment in standard doses appears efficacious for bites by this species. Progressive swelling may recur despite scheduled dosing.

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

PubMed Central

Shibazaki, Chie; Adachi, Motoyasu; Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Tahara, Tomoyuki; Kato, Takashi; Miyazaki, Hiroshi; Blaber, Michael; Kuroki, Ryota

2016-01-01

Abstract Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody–hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen‐binding fragment (Fab) derived from the TN1 antibody (TN1‐Fab). To clarify the mechanism by which hTPO is recognized by TN1‐Fab the conformation of free TN1‐Fab was determined to a resolution of 2.0 Å using X‐ray crystallography and compared with the hTPO‐bound form of TN1‐Fab determined by a previous study. This structural comparison revealed that the conformation of TN1‐Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen‐binding site (paratope) of TN1‐Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (−1.52 ± 0.05 kJ mol−1 K−1) differed significantly from calculations based upon the X‐ray structure data of the hTPO‐bound and unbound forms of TN1‐Fab (−1.02 ∼ 0.25 kJ mol−1 K−1) suggesting that hTPO undergoes an induced‐fit conformational change combined with significant desolvation upon TN1‐Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition. PMID:27419667

Impact of FAB classification on predicting outcome in acute myeloid leukemia, not otherwise specified, patients undergoing allogeneic stem cell transplantation in CR1: An analysis of 1690 patients from the acute leukemia working party of EBMT.

PubMed

Canaani, Jonathan; Beohou, Eric; Labopin, Myriam; Socié, Gerard; Huynh, Anne; Volin, Liisa; Cornelissen, Jan; Milpied, Noel; Gedde-Dahl, Tobias; Deconinck, Eric; Fegueux, Nathalie; Blaise, Didier; Mohty, Mohamad; Nagler, Arnon

2017-04-01

The French, American, and British (FAB) classification system for acute myeloid leukemia (AML) is extensively used and is incorporated into the AML, not otherwise specified (NOS) category in the 2016 WHO edition of myeloid neoplasm classification. While recent data proposes that FAB classification does not provide additional prognostic information for patients for whom NPM1 status is available, it is unknown whether FAB still retains a current prognostic role in predicting outcome of AML patients undergoing allogeneic stem cell transplantation. Using the European Society of Blood and Bone Marrow Transplantation registry we analyzed outcome of 1690 patients transplanted in CR1 to determine if FAB classification provides additional prognostic value. Multivariate analysis revealed that M6/M7 patients had decreased leukemia free survival (hazard ratio (HR) of 1.41, 95% confidence interval (CI), 1.01-1.99; P = .046) in addition to increased nonrelapse mortality (NRM) rates (HR, 1.79; 95% CI, 1.06-3.01; P = .028) compared with other FAB types. In the NPM1 wt AML, NOS cohort, FAB M6/M7 was also associated with increased NRM (HR, 2.17; 95% CI, 1.14-4.16; P = .019). Finally, in FLT3-ITD + patients, multivariate analyses revealed that specific FAB types were tightly associated with adverse outcome. In conclusion, FAB classification may predict outcome following transplantation in AML, NOS patients. © 2017 Wiley Periodicals, Inc.

Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

PubMed

Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

2014-01-15

Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. Copyright © 2013 Elsevier B.V. All rights reserved.

Type II Fatty Acid Synthesis Is Essential for the Replication of Chlamydia trachomatis*

PubMed Central

Yao, Jiangwei; Abdelrahman, Yasser M.; Robertson, Rosanna M.; Cox, John V.; Belland, Robert J.; White, Stephen W.; Rock, Charles O.

2014-01-01

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μm at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis. PMID:24958721

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

NASA Technical Reports Server (NTRS)

Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

1990-01-01

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

CroFab reconstitution in various media: an in vitro solubility study.

PubMed

Vohra, Rais; Clark, Rick; Kelner, Michael

2008-11-01

We investigated the solubility of Crotalidae Polyvalent Ovine Immune Fab antivenom (CroFab, Savage Labs and Protherics Inc., Brentwood, TN, USA) in solutions not listed in the Food and Drug Administration (FDA)-approved product package insert. We also assessed whether adsorption to plastic tubing occurs with CroFab preparations. Nine vials of expired CroFab were divided into three groups according to the solution used for reconstitution. Assignment to the solution groups of normal saline, lactated Ringer's solution, or half-normal saline (NS, LR, 1/2NS) was blinded. The antivenom was diluted to a final volume of 75 mL of test solution. Protein concentration was measured after reconstitution, after storage at 4-6 degrees C for 4 h, and after passage through plastic intravenous (IV) tubing. Higher measured protein yields were noted when half-normal saline was used in comparison with normal saline at each step of the study. Lactated Ringer's solution yielded higher protein concentrations than normal saline only at one out of the three measurement steps. There was no adsorption effect when CroFab was infused through plastic IV tubing. These data suggest that CroFab is slightly more soluble in the hypotonic solution we tested, and the amounts of measured antivenom did not diminish after 4 h of refrigeration or passage through plastic tubing. Our study may be of relevance when clinicians or pharmacists mix CroFab into non-standard solutions.

'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

PubMed

Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

2016-04-01

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A human Fab exclusively binding to the extracellular domain of LMP2A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Qing; Zhang, Dawei; Mao, Yuan

In the areas of North Africa, Southeast Asia as well as South China, Nasopharyngeal carcinoma (NPC) is among the most widespread cancers. Plenty of research findings confirmed that Epstein-Barr virus (EBV) played a crucial role in NPC. EBV-encoded Latent membrane protein 2A (LMP2A) which continuously expressed in cell membrane protein induced an epithelial-mesenchymal transition and increased the number of side population stem-like cancer cells in NPC. This reveals that LMP2A could contribute to the development and recurrence in NPC. Above evidences suggest that LMP2A could be the potential target molecule in the treatment of NPC. In the current study, amore » novel human antibody Fab (Fab29) against the extracellular domain of LMP2A was produced with success. Through immunofluorescence experiment it was proved that human antibody Fab29 exclusively combined the surface of SUNE cells (LMP2A-positive). Then flow cytometry result exhibited that the fluorescent intensities of SUNE cells and CNE cells were distinct (96.89% and 0.02% respectively). After that, it was shown by affinity test that the Fab29 fragment had high affinity (KD (M) 1.79E-09) with LMP2A. It was also revealed by immunohistochemical analysis that the Fab29 fragment could combine with LMP2A-positive human NPC tissues in comparison with the control group. Finally, the MTT result indicated that the Fab29 fragment could inhibit the proliferation of LMP2A-positive NPC cells. The inhibiting rate to SUNE cell proliferation reached a peak by Fab29 (19.67%) compared with unrelated Fab and CNE with Fab29 at a concentration of 500 μg/L in first 24 h and in the next 24 h the inhibition rate grew to 22.54%. In brief, it was shown that Fab29, a characteristic human antibody, could recognize LMP2A protein and inhibit the proliferation of LMP2A-expressing NPC cells in vitro.« less

Late hematologic toxicity following treatment of rattlesnake envenomation with crotalidae polyvalent immune Fab antivenom.

PubMed

Ruha, Anne-Michelle; Curry, Steven C; Albrecht, Clay; Riley, Brad; Pizon, Anthony

2011-01-01

North American rattlesnake envenomations commonly produce defibrination, coagulopathy and/or thrombocytopenia, which may be reversed following treatment with Crotalidae Polyvalent Immune Fab Ovine (FabAV). Despite initial resolution with FabAV, late onset or recurrence of venom-induced hematologic effects may occur. Time at which onset of late hematotoxicity may first be detected is unknown. The purpose of this study was to identify the incidence and time of onset of recurrent or new late hypofibrinogenemia, coagulopathy, or thrombocytopenia in a cohort of rattlesnake envenomation patients seen in outpatient follow-up after treatment with FabAV, and to report hematologic outcomes in these patients. Review of 66 charts of patients with rattlesnake envenomation who were treated with FabAV, and subsequently had outpatient follow-up evaluation at least 48 h after last FabAV, was performed. Demographic information, rattlesnake and bite characteristics, dose and timing of antivenom administration, adverse events, in-patient laboratory values, length of hospital stay, and follow-up laboratory values were collected. The primary outcome parameters were recurrent or delayed onset coagulopathy, hypofibrinogenemia, or thrombocytopenia identified no sooner than 48 h after last dose of FabAV. Prior to control of the envenomation with FabAV, 42 patients (63.6%) experienced hematologic toxicity. At follow-up, 21 patients (32%) were found to have late coagulopathy, hypofibrinogenemia, or thrombocytopenia. Of twenty-three patients (35%) with more than one follow-up visit, fifteen had normal laboratory findings at the first follow-up visit. Five of these 15 patients (8% of total study group; 33% of this subgroup) with normal hematologic studies at first follow-up exhibited late hematologic toxicity at second follow-up. Severe late hematologic toxicity developed in five of 66 (8%) patients. One patient was retreated with FabAV for late severe thrombocytopenia. Recurrent and delayed onset of hematologic toxicity in rattlesnake envenomation victims treated with FabAV is common. Follow-up more than three days after treatment is necessary to detect all cases of late hematologic toxicity. Copyright © 2010 Elsevier Ltd. All rights reserved.

Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

PubMed

Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

2015-01-01

D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.

Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

PubMed

Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

2017-05-01

Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy for Pompe disease.

78 FR 49771 - Proposed Extension of Information Collection Request Submitted for Public Comment

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-15

... (FAB) 2013-01 concerning the new disclosure requirements mandated the MAP-21. The FAB addresses a need...), as amended by the MAP-21. The FAB sets forth technical questions and answers and provides a model...

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

PubMed

Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

2015-06-01

Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

PubMed

Duan, Dongyu; Wang, Aiping; Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

PubMed Central

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Karch, Francois

2015-01-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. PMID:26303531

Research and Development Advances Impacting Diminishing Manufacturing Sources and Material Shortages Management

DTIC Science & Technology

2016-06-01

commercially available in 2 to 3 years. The fabs that fabricate today’s ICs service high-volume customers. Manufacturing the small volumes needed by DOD...is simply not cost effective and is disruptive to the process flow. There are smaller specialty fabs that focus on smaller orders and process smaller...process wafers with leading-edge technology, these fabs would have to invest in the same tools as the high-volume fabs —a prohibitive expense. Multi

Protein crystallization aboard the Space Shuttle and the Mir space station

NASA Technical Reports Server (NTRS)

Delbaere, Louis T. J.; Vandonselaar, Margaret; Prasad, Lata; Quail, J. W.; Birnbaum, George I.; Delucas, Lawrence J.; Moore, Karen; Bugg, Charles E.

1993-01-01

Two different protein crystallizations, namely ,the free Fab fragment of the Je142 monoclonal antibody and the complex of Fab fragment/HPr with antigen, were performed aboard the Discovery Space Shuttle flights and the Mir space station, respectively. Medium sized crystals of the Je142 Fab fragment were obtained. The Je142 Fab fragment/Hpr complex produced two medium-sized crystals after two months aboard the Mir space station. Microgravity was found to eliminate the tendency of these crystals to form clusters.

An Improved Thermal Blooming Model for the Laser Performance Code Anchor

DTIC Science & Technology

2016-06-01

target, ANCHOR uses the following equations: Pmelt = FABS ⋅PBKT − Ploss (4) . (5) In this case, FABS is the fractional target absorption; is...for hard kill of a given target, several material properties for the target are used to calculate Qmelt, FABS , and Ploss, discussed previously in...is approximately 50 kJ, Ploss is approximately 2.5 kJ, and FABS is 15%. Using these values ANCHOR produces the dwell time plots shown in Figures 9

The Work Disability Functional Assessment Battery (WD-FAB): Feasibility and Psychometric Properties

PubMed Central

Meterko, Mark; Marfeo, Elizabeth E.; McDonough, Christine M.; Jette, Alan M.; Ni, Pengsheng; Bogusz, Kara; Rasch, Elizabeth K; Brandt, Diane E.; Chan, Leighton

2015-01-01

Objectives To assess the feasibility and psychometric properties of eight scales covering two domains of the newly developed Work Disability Functional Assessment Battery (WD-FAB): physical function (PF) and behavioral health (BH) function. Design Cross-sectional. Setting Community. Participants Adults unable to work due to a physical (n=497) or mental (n=476) disability. Interventions None. Main Outcome Measures Each disability group responded to a survey consisting of the relevant WD-FAB scales and existing measures of established validity. The WD-FAB scales were evaluated with regard to data quality (score distribution; percent “I don’t know” responses), efficiency of administration (number of items required to achieve reliability criterion; time required to complete the scale) by computerized adaptive testing (CAT), and measurement accuracy as tested by person fit. Construct validity was assessed by examining both convergent and discriminant correlations between the WD-FAB scales and scores on same-domain and cross-domain established measures. Results Data quality was good and CAT efficiency was high across both WD-FAB domains. Measurement accuracy was very good for the PF scales; BH scales demonstrated more variability. Construct validity correlations, both convergent and divergent, between all WD-FAB scales and established measures were in the expected direction and range of magnitude. Conclusions The data quality, CAT efficacy, person fit and construct validity of the WD-FAB scales were well supported and suggest that the WD-FAB could be used to assess physical and behavioral health function related to work disability. Variation in scale performance suggests the need for future work on item replenishment and refinement, particularly regarding the Self-Efficacy scale. PMID:25528263

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

PubMed

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

PubMed Central

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

PubMed

Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

2016-11-01

Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

PubMed

Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

2016-07-08

Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016. © 2016 American Institute of Chemical Engineers.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

PubMed Central

Organtini, Lindsey J.; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.

2016-01-01

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. PMID:27535057

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

PubMed Central

Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

2012-01-01

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

A Case Study of a High School Fab Lab

NASA Astrophysics Data System (ADS)

Lacy, Jennifer E.

This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

Type II fatty acid synthesis is essential for the replication of Chlamydia trachomatis.

PubMed

Yao, Jiangwei; Abdelrahman, Yasser M; Robertson, Rosanna M; Cox, John V; Belland, Robert J; White, Stephen W; Rock, Charles O

2014-08-08

The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of CroFab antivenin in the management of a very young pediatric copperhead envenomation.

PubMed

Trinh, Hai H; Hack, Jason B

2005-08-01

The use of crotalid Fab antivenin (CroFab) in the treatment of snake envenomations in the pediatric population is still an underexplored area. There are very limited data to confirm the efficacy and safety of dosing children the same as adults and even less information available to evaluate this antivenin use in copperhead snake bites in children. We report the first use of crotalid Fab antivenin in an adult dose for a copperhead snake envenomation in a 2-year-old child. She had rapid resolution of symptoms with no adverse effects. The report serves to increase the literature supporting the current dosing recommendations of crotalid Fab antivenin in very young pediatric patients evidenced by its effectiveness in this patient.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.

PubMed

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-03-24

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

PubMed

Yu, Min; Kumar, T R Santha; Nkrumah, Louis J; Coppi, Alida; Retzlaff, Silke; Li, Celeste D; Kelly, Brendan J; Moura, Pedro A; Lakshmanan, Viswanathan; Freundlich, Joel S; Valderramos, Juan-Carlos; Vilcheze, Catherine; Siedner, Mark; Tsai, Jennifer H-C; Falkard, Brie; Sidhu, Amar Bir Singh; Purcell, Lisa A; Gratraud, Paul; Kremer, Laurent; Waters, Andrew P; Schiehser, Guy; Jacobus, David P; Janse, Chris J; Ager, Arba; Jacobs, William R; Sacchettini, James C; Heussler, Volker; Sinnis, Photini; Fidock, David A

2008-12-11

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Crystallization and preliminary X-ray diffraction analysis of FabG from Yersinia pestis.

PubMed

Nanson, Jeffrey David; Forwood, Jade Kenneth

2014-01-01

The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported. Recombinant FabG was expressed, purified to homogeneity and crystallized via the hanging-drop vapour-diffusion technique. Diffraction data were collected at the Australian Synchrotron to 2.30 Å resolution. The crystal displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 68.22, b = 98.68, c = 169.84 Å, and four ypFabG molecules in the asymmetric unit.

Teratogenicity induced by targeting a placental immunoglobulin transporter

PubMed Central

Kolonin, Mikhail G.; Pasqualini, Renata; Arap, Wadih

2002-01-01

Approximately 3% of children in developed countries are born with nongenetic birth defects. However, the nature and mechanisms of teratogenesis are poorly understood. We investigated mechanisms of teratogen-mediated blockade of maternofetal transport by screening a combinatorial library for peptides that bind nonendothelial placental vasculature in pregnant mice. Here, we identified a peptide motif, TPKTSVT, that homes to the yolk sac, induces placental necrosis, and disrupts embryo development. We show that TPKTSVT promotes transcytosis of phage into the embryo and blocks the transplacental transport of immunoglobulins. Based on these data, we propose a model in which TPKTSVT targets a placental Fc receptor. Absence of TPKTSVT placental homing in mice lacking β2-microglobulin (β2m) suggests FcRn/β2m as a target for the TPKTSVT, which is unexpected, given the normal development of FcRn/β2m-deficient progeny. High-throughput screening for embryotoxins that target placental receptors could be developed to systematically identify and avoid exposure to teratogenic drugs. PMID:12242328

The community FabLab platform: applications and implications in biomedical engineering.

PubMed

Stephenson, Makeda K; Dow, Douglas E

2014-01-01

Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

LC-MS/MS quantification of free and Fab-bound colchicine in plasma, urine and organs following colchicine administration and colchicine-specific Fab fragments treatment in Göttingen minipigs.

PubMed

Fabresse, Nicolas; Allard, Julien; Sardaby, Marine; Thompson, Adrian; Clutton, R Eddie; Eddleston, Michael; Alvarez, Jean-Claude

2017-08-15

Clinical evaluation of a colchicine specific antigen-binding fragment (Fab) in order to treat colchicine poisoning required the development of an accurate method allowing quantification of free and Fab-bound colchicine in plasma and urine, and free colchicine in tissues, to measure colchicine redistribution after Fab administration. Three methods have been developed for this purpose, and validated in plasma, urine and liver: total colchicine was determined after denaturation of Fab by dilution in water and heating; free colchicine was separated from Fab-bound colchicine by filtration with 30KDa micro-filters; tissues were homogenized in a tissue mixer. Deuterated colchicine was used as internal standard. Samples were extracted by liquid-liquid extraction and analyzed with a LC-MS/MS. LOQ were 0.5ng/mL in plasma and urine for free and total colchicine and 5pg/mg in tissues. The methods were linear in the 0.5-100ng/mL range in plasma and urine, and 5-300pg/mg in tissues with determination coefficients>0.99. Precision and accuracy of QC samples presented a CV<9.4%. The methods require only 200μL of sample and allow a high throughput due to short analytical run (2min). These methods were successfully applied to a pig intoxicated with colchicine and treated with colchicine specific Fab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

PubMed

Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

2011-07-18

The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

20 CFR 30.314 - How is a hearing conducted?

Code of Federal Regulations, 2010 CFR

2010-04-01

... Decisions on Claims § 30.314 How is a hearing conducted? (a) The FAB reviewer retains complete discretion to... telephone or teleconference. As part of the hearing process, the FAB reviewer will consider the written... claimant. The reviewer may also conduct whatever investigation is deemed necessary. (1) The FAB reviewer...

20 CFR 30.300 - What process will OWCP use to decide claims for entitlement and to provide for administrative...

Code of Federal Regulations, 2010 CFR

2010-04-01

.../or Part E of the Act will be forwarded to the Final Adjudication Branch (FAB). Claimants will be given an opportunity to object to all or part of the recommended decision before the FAB. The FAB will...

3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

DTIC Science & Technology

2013-07-01

FabI, but share low sequence identity and are poorly inhibited by triclosan.25,26 S. pneumoniae and P. aeruginosa contain FabK,24 and Vibrio cholerae,27...with 0.2 mM IPTG. The cells were harvested after an overnight induction period at 17 °C. The cells were lysed and sonicated and loaded onto a nickel...of enoyl- (acyl-carrier protein) reductase, FabV, from Vibrio fischeri. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 78−80. (27

Production and Characterization of F(Ab')2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies.

PubMed

de Araujo, Anna Erika Vieira; de Souza, Natalia Plinio; de Sousa, Alvaro Paiva Braga; Lara, Flavio Alves; Senna, Jose Procopio Moreno

2018-05-01

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab') 2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab') 2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab') 2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab') 2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab') 2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab') 2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

PubMed

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

2015-11-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Glycoprotein CD98 as a receptor for colitis-targeted delivery of nanoparticle.

PubMed

Xiao, Bo; Yang, Yang; Viennois, Emilie; Zhang, Yuchen; Ayyadurai, Saravanan; Baker, Mark; Laroui, Hamed; Merlin, Didier

2014-03-21

Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, ex vivo studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

PubMed

Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

2018-05-01

Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

FabAV antivenin use after copperhead snakebite: clinically indicated or knee-jerk reaction?

PubMed

Gale, Stephen C; Peters, Jo Ann; Allen, LaDonna; Creath, Robert; Dombrovskiy, Viktor Y

2016-01-01

Crotalidae Polyvalent Immune Fab (Ovine) (FabAV) antivenin is commonly recommended after pit viper snakebites. Because copperhead envenomations are usually self-limited, some physicians are reluctant to use this costly treatment routinely, while others follow a more liberal approach. We hypothesized that, in practice, only patients with evidence of significant (moderate or severe) copperhead envenomation [those with snakebite severity score (SSS) > 3] receive FabAV and examined a large cohort to determine the relationship between clinical findings and FabAV administration. All data from patients evaluated for copperhead snakebite at a rural tertiary referral center from 5/2002 to 10/2013 were compiled. Demographics, transfer status, antivenin use, and clinical findings were collected; SSS was calculated. The relationships among FabAV use, clinical findings, and SSS were analyzed using t-test, chi-square, and Pearson's coefficient (p < 0.05 was significant). During the study period, 318 patients were treated for copperhead snakebite; 44 (13.8 %) received antivenin. Median dose was four vials (range: 1-10; IQR: 4,6). There were no deaths. Most patients receiving FabAV (63.6 %) were admitted. With regard to demographics and symptoms, only the degree of swelling (moderate vs. none/mild; p < 0.01) and bite location (hand/arm vs. leg: p < 0.0001) were associated with FabAV use. A SSS > 3, indicating moderate or severe envenomation, was only very weakly correlated with antivenin use (r = 0.217; p < 0.0001). The majority of patients with SSS > 3 (65.8 %) did not receive antivenin while most patients who did receive antivenin (70.5 %) had SSS ≤ 3 (indicating mild envenomation). Considerable variation occurs in antivenin administration after copperhead snakebite. Use of FabAV appears poorly correlated with patients' symptoms. This practice may expose patients to the risks of antivenin and increasing costs of medical care without improving outcomes. Guidelines used for treating other pit viper strikes, such as rattlesnake or cottonmouth snakebite may be too liberal for copperhead envenomations. Our data suggests that most patients with mild or moderate envenomation appear to do well independent of FabAV use. We suggest, for patients with copperhead snakebite, that consideration be given to withholding FabAV for those without clinical evidence of severe envenomation until prospective randomized data are available.

The Efficacy of Crotalidae Polyvalent Immune Fab (Ovine) Antivenom Versus Placebo Plus Optional Rescue Therapy on Recovery From Copperhead Snake Envenomation: A Randomized, Double-Blind, Placebo-Controlled, Clinical Trial.

PubMed

Gerardo, Charles J; Quackenbush, Eugenia; Lewis, Brandon; Rose, S Rutherfoord; Greene, Spencer; Toschlog, Eric A; Charlton, Nathan P; Mullins, Michael E; Schwartz, Richard; Denning, David; Sharma, Kapil; Kleinschmidt, Kurt; Bush, Sean P; Ryan, Samantha; Gasior, Maria; Anderson, Victoria E; Lavonas, Eric J

2017-08-01

Copperhead snake (Agkistrodon contortrix) envenomation causes limb injury resulting in pain and disability. It is not known whether antivenom administration improves limb function. We determine whether administration of antivenom improves recovery from limb injury in patients envenomated by copperhead snakes. From August 2013 through November 2015, we performed a multicenter, randomized, double-blind, placebo-controlled, clinical trial to evaluate the effect of ovine Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) antivenom therapy on recovery of limb function in patients with copperhead snake envenomation at 14 days postenvenomation. The study setting was 18 emergency departments in regions of the United States where copperhead snakes are endemic. Consecutive patients aged 12 years or older with mild- to moderate-severity envenomation received either FabAV or placebo. The primary outcome was limb function 14 days after envenomation, measured by the Patient-Specific Functional Scale. Additional outcomes included the Patient-Specific Functional Scale at other points; the Disorders of the Arm, Shoulder, and Hand, Lower Extremity Functional Scale, and Patient's Global Impression of Change instruments; grip strength; walking speed; quality of life (Patient-Reported Outcomes Measurement Information System Physical Fucntion-10); pain; and analgesic use. Seventy-four patients received study drug (45 FabAV, 29 placebo). Mean age was 43 years (range 12 to 86 years). Fifty-three percent were men, 62% had lower extremity envenomation, and 88% had mild initial severity. The primary outcome, the least square mean Patient-Specific Functional Scale score at 14 days postenvenomation, was 8.6 for FabAV-treated subjects and 7.4 for placebo recipients (difference 1.2; 95% confidence interval 0.1 to 2.3; P=.04). Additional outcome assessments generally favored FabAV. More FabAV-treated subjects experienced treatment-emergent adverse events (56% versus 28%), but few were serious (1 in each group). Treatment with FabAV reduces limb disability measured by the Patient-Specific Functional Scale 14 days after copperhead envenomation. Copyright © 2017 American College of Emergency Physicians. Published by Elsevier Inc. All rights reserved.

Elderly patients with suspected chronic digoxin toxicity: A comparison of clinical characteristics of patients receiving and not receiving digoxin-Fab.

PubMed

Arbabian, Hooman; Lee, Hwee Min; Graudins, Andis

2018-04-01

The aim of the present study was to compare clinical features of patients with elevated serum digoxin concentrations who were treated with digoxin-Fab with those where the immunotherapy was not given by a tertiary hospital toxicology service. This was a retrospective series of patients with supratherapeutic serum digoxin concentrations referred to the toxicology service from August 2013 to October 2015. Data collected included demographics, presenting complaint, digoxin dose, other medications taken, serum digoxin, potassium and creatinine concentration on presentation and initial and post-digoxin-Fab heart rate. There were 47 referrals. Digoxin-Fab was administered in 21 cases. It was given more commonly when the heart rate was <51/min or serum potassium was >5.0 mmol/L. Patients receiving digoxin-Fab were more likely to be on maintenance therapy with beta-blockers or calcium channel blockers (95% vs 61%; OR 13.1; 95% CI 1.5-113) and/or potassium-sparing medications (95% vs 54%; OR 17.1; 95% CI 2.0-147). They had elevated serum creatinine (76% vs 42%; OR 8.2; 95% CI 1.9-34), higher serum potassium (median: 5.1 mmol/L vs 4.2 mmol/L, P = 0.02), higher serum digoxin concentration (median: 3.5 nmol/L vs 2.3 nmol/L, P = 0.02) and pretreatment heart rate <51/min (66% vs 31%; OR 4.5; 95% CI 1.3-15). There were no patients with ventricular arrhythmias or hypotension. Median heart rate increased by 10/min 1 and 4 h after digoxin-Fab. However, individual heart rate response to digoxin-Fab was variable. Digoxin-Fab was more commonly administered when heart rate was <51/min. It had a small effect on increasing heart rate; however, individual response to digoxin-Fab was variable as patients were using other negative chronotropic medications. In symptomatic bradycardic patients on multiple heart failure medications, positive chronotropic and potassium-lowering therapies should be considered in concert with digoxin-Fab. © 2018 Australasian College for Emergency Medicine and Australasian Society for Emergency Medicine.

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

PubMed Central

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits. PMID:29326789

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

PubMed

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab') 2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab') 2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab') 2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab') 2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab') 2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab') 2 and polyclonal anti-IgG F(ab') 2 are useful tools in biomedical and biochemical researches and diagnostic kits.

Safety and efficacy of Crotalidae Polyvalent Immune Fab in pediatric crotaline envenomations.

PubMed

Pizon, Anthony F; Riley, Bradley D; LoVecchio, Frank; Gill, Ruqayya

2007-04-01

Since it was approved by the Food and Drug Administration in October 2000, Crotalidae Polyvalent Immune Fab (CroFab) has largely replaced previously used crotaline antivenom. CroFab is more specifically tailored for crotalids of North America and is less allergenic than whole immunoglobulin antivenoms. However, premarketing and postmarketing studies have excluded children. To describe the safety and efficacy of CroFab in pediatric crotaline envenomations. Using admission and billing records, the authors identified all children 13 years of age and younger treated with CroFab at a pediatric hospital between October 2000 and September 2005. Charts were reviewed by two trained, blinded extractors. Data regarding age, signs of envenomation, laboratory values, total antivenom vials used, total vials used to gain control, transfused blood products, signs of acute allergy to antivenom, and any surgical procedures were abstracted. Data were analyzed using descriptive statistics. Twenty-four patients were identified, and their mean age was 7.3 (range, 1.9-13) years. At presentation, all had swelling, 14 (58%) had a prothrombin time >13 seconds, two (8.3%) had a fibrinogen level <150 mg/dL, and three (12.5%) had platelet counts <150,000/mL. The mean number of total antivenom vials used was 12.3 (range, 4-24). Five patients had resolution of swelling, but platelet counts continued to fall despite antivenom treatment. No patient required blood products, debridement of skin, or fasciotomy. There was only one (4.2%) possible acute allergy to CroFab, and there were no deaths. In this pediatric series, CroFab appears safe and effective, despite occasional resistant thrombocytopenia.

Brand-specific consumption of flavored alcoholic beverages among underage youth in the United States.

PubMed

Fortunato, Erin K; Siegel, Michael; Ramirez, Rebecca L; Ross, Craig; DeJong, William; Albers, Alison B; Jernigan, David H

2014-01-01

Although several studies have identified flavored alcoholic beverages (FABs) as being popular among underage drinkers, no previous study has ascertained the prevalence of brand-specific FAB consumption among a national sample of underage youth. To ascertain the brand-specific consumption prevalence and consumption share of FABs among a national sample of underage drinkers in the United States. In 2012, we conducted an online, self-administered survey of a national sample of 1031 underage drinkers, ages 13-20 years, to determine the prevalence of past 30-day consumption for each of 898 alcoholic beverage brands, including 62 FABs, and each brand's youth consumption share, based on the estimated total number of standard drinks consumed. There were three brand-specific outcome measures: prevalence of consumption, prevalence of consumption during heavy episodic drinking, and consumption share, defined as the percentage of the total drinks consumed by all respondents combined that was attributable to a particular brand. The FAB brands with the highest prevalence of past 30-day consumption were Smirnoff malt beverages, 17.7%; Mike's, 10.8%; Bacardi malt beverages, 8.0%; and Four Loko/Four MaXed, 6.1%. Just five brands accounted for almost half (49.1%) of the total consumption share by volume within the FAB category. Flavored alcoholic beverages are highly popular among underage drinkers, and the FAB brand preferences of this group are highly concentrated among a small number of brands. To decrease the consumption of FABs by underage youth, all states should reclassify these beverages as distilled spirits rather than beer.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

PubMed

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

PubMed

Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

2016-01-01

Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

Acute adverse events associated with the administration of Crotalidae polyvalent immune Fab antivenom within the North American Snakebite Registry.

PubMed

Kleinschmidt, Kurt; Ruha, Anne-Michelle; Campleman, Sharan; Brent, Jeffrey; Wax, Paul

2018-04-24

Crotalidae Polyvalent Immune Fab (Fab Antivenom) is the primary Viperid antivenom used in the United States since 2000. Adverse event data associated with its use are limited. The purpose of this study is to describe the prevalence of acute adverse events associated with the use of Fab antivenom. The American College of Medical Toxicology's Toxicology Investigators Consortium maintains a prospective case registry of poisoned and envenomated patients managed by medical toxicologists at the bedside. This registry includes the North American Snakebite sub-registry. We performed a review of 438 cases entered into the Snakebite sub-registry. A total of 373 (85.2%) received at least one vial of Fab Antivenom. Forty percent were children. Adverse events occurred in 10 patients (2.7%) of whom six were adults. Rash was the most common adverse event. More severe adverse events (hypotension, bronchospasm, and/or angioedema) occurred in four (1.1%) patients. Prophylaxis was administered prior to Fab antivenom in 4.0%. Eight patients received various treatments for their adverse events. Neither the initial number of Fab antivenom vials, atopic history, nor prior envenomation correlated with the prevalence of adverse events. This prevalence of adverse events was lower than in previous studies and in a meta-analysis of 11 studies. The types of adverse events and treatments used are consistent with those in previous reports. There were no prior reports of prophylaxis use with which to compare. The prevalence of Fab antivenom adverse events in the North American Snakebite Registry was 2.7%.

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

75 FR 9438 - Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-02

... Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation, Including On-Site Leased... Semiconductor, LLC, a subsidiary of Samsung Electronics Corporation, DRAM Fab 1, including on-site leased.... The workers are engaged in activities related to the production of DRAM chips for use in electronics...

78 FR 23472 - Amendments to Existing Validated End-User Authorizations: CSMC Technologies Corporation in the...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-19

... of Wuxi CR Semiconductor Wafers & Chips Co., Ltd. and CSMC Technologies Fab 1 Co., Ltd., which is... Validated End-User: CSMC Technologies Corporation. Eligible Destinations: CSMC Technologies Fab 1 Co., Ltd., 14 Liangxi Road, Wuxi, Jiangsu 214061, China. CSMC Technologies Fab 2 Co., Ltd., 8 Xinzhou Rd., Wuxi...

Functionally Approached Body (FAB) Strategies for Young Children Who Have Behavioral and Sensory Processing Challenges

ERIC Educational Resources Information Center

Pagano, John

2005-01-01

Functionally Approached Body (FAB) Strategies offer a clinical approach to help parents of young children with behavioral and sensory processing strategies. This article introduces the FAB Strategies, clinical strategies developed by the author for understanding and addressing young children's behavioral and sensory processing challenges. The FAB…

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix.

PubMed

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-02-17

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn't showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody-drug conjugates (ADC) or immunotoxins.

Structure determination of an Fab fragment that neutralizes human rhinovirus 14 and analysis of the Fab-virus complex.

PubMed

Liu, H; Smith, T J; Lee, W M; Mosser, A G; Rueckert, R R; Olson, N H; Cheng, R H; Baker, T S

1994-07-08

The crystal structure of Fab17-IA, an antigen-binding fragment from a murine immunoglobulin that neutralizes human rhinovirus 14 (HRV14), has been solved to 2.7 A resolution. Fab17-IA crystallized into three different space groups depending upon the method used to purify the intact antibody. The structure was determined by use of molecular and isomorphous replacement methods. The current model has a crystallographic R-factor of approximately 19% for 10,192 independent reflections between 8 and 2.7 A. Correlation coefficient calculations showed that the Fab17-IA structure can be fit into the Fab17-IA/HRV14 image reconstruction density to within 5 A positional accuracy and to within a few degrees of rotation. The resulting interface of the docked antibody was examined and showed extensive charge and shape complementarity with the virus surface that was supported by site-directed mutagenesis experiments. The success of this approach validates the utility of combining X-ray crystallography with cryo-electron microscopy of complex macromolecular assemblies.

Two decades of pharmacovigilance and clinical experience with highly purified rabies immunoglobulin F(ab')2 fragments.

PubMed

Reveneau, Elisa; Cottin, Pascale; Rasuli, Anvar

2017-03-01

Rabies is a worldwide zoonotic viral disease with no specific treatment once symptoms occur; manifest disease is almost always fatal. WHO recommendations for exposed individuals include immediate attention to the wound and use of rabies immunoglobulin and/or vaccine for post-exposure prophylaxis (PEP). Here, we provide an overview of the clinical experience with a highly purified preparation of F(ab') 2 fragments from equine rabies immunoglobulin (F(ab') 2 pERIG; Favirab TM ) in rabies PEP. Areas covered: Our review comprises a retrospective analysis of adverse event reports in the Sanofi Pasteur global pharmacovigilance database for F(ab') 2 pERIG, including adverse event reports from eight Sanofi Pasteur-sponsored clinical trials and post-market surveillance data collected between 1995 and 2014. The general safety profile of F(ab') 2 pERIG is discussed, as are the occurrence of rare anaphylactic reactions, and suspected intervention failure. Expert commentary: Over 20 years of clinical development and post-licensure experience has established the safety and effectiveness of F(ab') 2 pERIG (Favirab TM ) in rabies PEP.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

PubMed Central

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-01-01

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma. PMID:27009516

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

PubMed

Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

2015-11-01

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

A collagen-binding EGFR antibody fragment targeting tumors with a collagen-rich extracellular matrix

PubMed Central

Liang, Hui; Li, Xiaoran; Wang, Bin; Chen, Bing; Zhao, Yannan; Sun, Jie; Zhuang, Yan; Shi, Jiajia; Shen, He; Zhang, Zhijun; Dai, Jianwu

2016-01-01

Many tumors over-express collagen, which constitutes the physical scaffold of tumor microenvironment. Collagen has been considered to be a target for cancer therapy. The collagen-binding domain (CBD) is a short peptide, which could bind to collagen and achieve the sustained release of CBD-fused proteins in collagen scaffold. Here, a collagen-binding EGFR antibody fragment was designed and expressed for targeting the collagen-rich extracellular matrix in tumors. The antibody fragment (Fab) of cetuximab was fused with CBD (CBD-Fab) and expressed in Pichia pastoris. CBD-Fab maintained antigen binding and anti-tumor activity of cetuximab and obtained a collagen-binding ability in vitro. The results also showed CBD-Fab was mainly enriched in tumors and had longer retention time in tumors in A431 s.c. xenografts. Furthermore, CBD-Fab showed a similar therapeutic efficacy as cetuximab in A431 xenografts. Although CBD-Fab hasn’t showed better therapeutic effects than cetuximab, its smaller molecular and special target may be applicable as antibody–drug conjugates (ADC) or immunotoxins. PMID:26883295

ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

PubMed

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

2015-07-01

To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida.

PubMed

Kobayashi, H; Oyamada, H; Suzuki, A; Shibata, N; Suzuki, S; Okawa, Y

1996-10-21

We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme-linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1-mnn 4 and mnn 2. Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, latron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively. To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs. The results indicated that FAb 34 preferentially recognizes linear side chains containing a non-reducing terminal alpha-1,3-linked mannose residue, Man(alpha)1 --> 3Man(alpha)1 --> (2Man(alpha)1 --> )n(2Man) (n > or = 0), and that one of the recognition sites of FAb 9 is linear alpha-1,6-linked oligomannosyl series, Man(alpha)1 --> (6Man(alpha)1 --> )n(6Man) (n > or = 2). On the other hand, the recognition site of FAb 8 apparently consisted of two alpha-1,2-linked oligomannosyl side chains and an alpha-1,6-linked mannose residue that originated from the mannan backbone, Man(alpha)1 --> 2Man(alpha)1 --> 2(Man(alpha)1 -->2Man(alpha)1 --> 6)Man.

A novel series of enoyl reductase inhibitors targeting the ESKAPE pathogens, Staphylococcus aureus and Acinetobacter baumannii.

PubMed

Kwon, Jieun; Mistry, Tina; Ren, Jinhong; Johnson, Michael E; Mehboob, Shahila

2018-01-01

S. aureus and A. baumannii are among the ESKAPE pathogens that are increasingly difficult to treat due to the rise in the number of drug resistant strains. Novel therapeutics targeting these pathogens are much needed. The bacterial enoyl reductase (FabI) is as potentially significant drug target for developing pathogen-specific antibiotics due to the presence of alternate FabI isoforms in many other bacterial species. We report the identification and development of a novel N-carboxy pyrrolidine scaffold targeting FabI in S. aureus and A. baumannii, two pathogens for which FabI essentiality has been established. This scaffold is unrelated to other known antibiotic families, and FabI is not targeted by any currently approved antibiotic. Our data shows that this scaffold displays promising enzyme inhibitory activity against FabI from both S. aureus and A. baumannii, as well as encouraging antibacterial activity in S. aureus. Compounds also display excellent synergy when combined with colistin and tested against A. baumannii. In this combination the MIC of colistin is reduced by 10-fold. Our first generation compound displays promising enzyme inhibition, targets FabI in S. aureus with a favorable selectivity index (ratio of cytotoxicity to MIC), and has excellent synergy with colistin against A. baumannii, including a multidrug resistant strain. Copyright © 2017 Elsevier Ltd. All rights reserved.

Production and characterization of anti-human IgG F(ab')2 antibody fragment.

PubMed

Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

2018-04-10

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

Improving Assessment of Work Related Mental Health Function Using the Work Disability Functional Assessment Battery (WD-FAB).

PubMed

Marfeo, Elizabeth E; Ni, Pengsheng; McDonough, Christine; Peterik, Kara; Marino, Molly; Meterko, Mark; Rasch, Elizabeth K; Chan, Leighton; Brandt, Diane; Jette, Alan M

2018-03-01

Purpose To improve the mental health component of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Specifically our goal was to expand the WD-FAB scales of mood & emotions, resilience, social interactions, and behavioral control to improve the depth and breadth of the current scales and expand the content coverage to include aspects of cognition & communication function. Methods Data were collected from a random, stratified sample of 1695 claimants applying for the SSA work disability benefits, and a general population sample of 2025 working age adults. 169 new items were developed to replenish the WD-FAB scales and analyzed using factor analysis and item response theory (IRT) analysis to construct unidimensional scales. We conducted computer adaptive test (CAT) simulations to examine the psychometric properties of the WD-FAB. Results Analyses supported the inclusion of four mental health subdomains: Cognition & Communication (68 items), Self-Regulation (34 items), Resilience & Sociability (29 items) and Mood & Emotions (34 items). All scales yielded acceptable psychometric properties. Conclusions IRT methods were effective in expanding the WD-FAB to assess mental health function. The WD-FAB has the potential to enhance work disability assessment both within the context of the SSA disability programs as well as other clinical and vocational rehabilitation settings.

Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

PubMed

Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

2015-05-01

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

77 FR 37920 - Proposed Extension of Information Collection Requests Submitted for Public Comment: National...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-25

... Bulletin (FAB) 2009-1 to provide interim guidance under section 101(f) of ERISA in order to assist plan... notices plan administrators may use to satisfy the annual funding notice content requirements. The FAB... the guidance contained in the FAB and has acted in accordance with a good faith, reasonable...

20 CFR 30.315 - May a claimant postpone a hearing?

Code of Federal Regulations, 2010 CFR

2010-04-01

... and Final Decisions on Claims § 30.315 May a claimant postpone a hearing? (a) The FAB will entertain... made at the time that the hearing is requested. Scheduling is at the discretion of the FAB, and is not... (b) of this section, unless the FAB reviewer can reschedule the hearing on the same docket (that is...

78 FR 70070 - Agency Information Collection Activities; Submission for OMB Review; Comment Request; Annual...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-22

... DOL released EBSA Field Assistance Bulletin (FAB) 2013-01 concerning the new disclosure requirements mandated by MAP-21 provisions. The FAB addresses a need for interim guidance pending the adoption of regulations or other guidance under ERISA section 101(f), as amended by the MAP-21. The FAB sets forth...

Phage Wrapping with Cationic Polymers Eliminates Non-specific Binding between M13 Phage and High pI Target Proteins

PubMed Central

Lamboy, Jorge A.; Arter, Jessica A.; Knopp, Kristeene A.; Der, Denise; Overstreet, Cathie M.; Palermo, Edmund; Urakami, Hiromitsu; Yu, Ting-Bin; Tezgel, Ozgul; Tew, Gregory; Guan, Zhibin; Kuroda, Kenichi; Weiss, Gregory A.

2011-01-01

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO2, and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient “phage wrapping” strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking non-specific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials, and solve a major problem in phage display – non-specific binding by the phage to high pI target proteins. PMID:19856910

Phage wrapping with cationic polymers eliminates nonspecific binding between M13 phage and high pI target proteins.

PubMed

Lamboy, Jorge A; Arter, Jessica A; Knopp, Kristeene A; Der, Denise; Overstreet, Cathie M; Palermo, Edmund F; Urakami, Hiromitsu; Yu, Ting-Bin; Tezgel, Ozgul; Tew, Gregory N; Guan, Zhibin; Kuroda, Kenichi; Weiss, Gregory A

2009-11-18

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Fear-avoidance beliefs-a moderator of treatment efficacy in patients with low back pain: a systematic review.

PubMed

Wertli, Maria M; Rasmussen-Barr, Eva; Held, Ulrike; Weiser, Sherri; Bachmann, Lucas M; Brunner, Florian

2014-11-01

Psychological factors are believed to influence the development of chronic low back pain. To date, it is not known how fear-avoidance beliefs (FABs) influence the treatment efficacy in low back pain. To summarize the evidence examining the influence of FABs measured with the Fear-Avoidance Belief Questionnaire or the Tampa Scale of Kinesiophobia on treatment outcomes in patients with low back pain. This is a systematic review. Patients with low back pain. Work-related outcomes and perceived measures including return to work, pain, and disability. In January 2013, the following databases were searched: BIOSIS, CINAHL, Cochrane Library, Embase, OTSeeker, PeDRO, PsycInfo, PubMed/Medline, Scopus, and Web of Science. A hand search of the six most often retrieved journals and a bibliography search completed the search. research studies that included patients with low back pain who participated in randomized controlled trials (RCTs) investigating nonoperative treatment efficacy. Out of 646 records, 78 articles were assessed in full text and 17 RCTs were included. Study quality was high in five studies and moderate in 12 studies. In patients with low back pain of up to 6 months duration, high FABs were associated with more pain and/or disability (4 RCTs) and less return to work (3 RCTs) (GRADE high-quality evidence, 831 patients vs. 322 in nonpredictive studies). A decrease in FAB values during treatment was associated with less pain and disability at follow-up (GRADE moderate evidence, 2 RCTs with moderate quality, 242 patients). Interventions that addressed FABs were more effective than control groups based on biomedical concepts (GRADE moderate evidence, 1,051 vs. 227 patients in studies without moderating effects). In chronic patients with LBP, the findings were less consistent. Two studies found baseline FABs to be associated with more pain and disability and less return to work (339 patients), whereas 3 others (832 patients) found none (GRADE low evidence). Heterogeneity of the studies impeded a pooling of the results. Evidence suggests that FABs are associated with poor treatment outcome in patients with LBP of less than 6 months, and thus early treatment, including interventions to reduce FABs, may avoid delayed recovery and chronicity. Patients with high FABs are more likely to improve when FABs are addressed in treatments than when these beliefs are ignored, and treatment strategies should be modified if FABs are present. Copyright © 2014 Elsevier Inc. All rights reserved.

Evidence-based, multidisciplinary approach to the development of a crotalidae polyvalent antivenin (CroFab) protocol at a university hospital.

PubMed

Weant, Kyle A; Johnson, Peter N; Bowers, Rebecca C; Armitstead, John A

2010-03-01

Several thousand people are bitten annually by venomous snakes in the US. While the development of ovine Crotalidae polyvalent immune Fab antivenin (FabAV) for Crotalinae snakebite envenomations has greatly changed the way this clinical presentation is treated, multiple issues complicate its use. From patient assessment and evaluation, to medication preparation and administration, to the management of adverse drug reactions, the use of this antidote carries with it multiple points of possible medication variances. The inappropriate use of this agent can result in adverse patient consequences and a significant financial burden for both the hospital and the patient. To describe an evidence-based, multidisciplinary approach that was taken to ensure optimal, safe, and cost-effective treatment of patients with FabAV. Following an analysis of the available literature, a multidisciplinary committee was formed to construct a protocol for use of FabAV. This group included clinical pharmacists, pharmacy administrators, emergency medicine physicians who specialized in wilderness medicine and pharmacy residents. A multidisciplinary FabAV usage protocol was constructed and implemented to ensure appropriate patient evaluation, FabAV use and preparation, monitoring, and follow-up. This protocol was based on the available literature and the expert opinion of the committee. Through the use of a 24-hour in-house pharmacy resident on-call system, clinical pharmacy services were provided to ensure a multidisciplinary approach to the care of these patients emergently. Although limited, initial data show that this approach is effective and may result in substantial cost savings. Initial results from implementation of a protocol for use of FabAV have limited inappropriate use, reduced medication wastage, and decreased costs.

Investigation of degradation processes in IgG1 monoclonal antibodies by limited proteolysis coupled with weak cation-exchange HPLC.

PubMed

Lau, Hollis; Pace, Danielle; Yan, Boxu; McGrath, Theresa; Smallwood, Scott; Patel, Ketaki; Park, Jihea; Park, Sungae S; Latypov, Ramil F

2010-04-01

A new cation-exchange high-performance liquid chromatography (HPLC) method that separates fragment antigen-binding (Fab) and fragment crystallizable (Fc) domains generated by the limited proteolysis of monoclonal antibodies (mAbs) was developed. This assay has proven to be suitable for studying complex degradation processes involving various immunoglobulin G1 (IgG1) molecules. Assignment of covalent degradations to specific regions of mAbs was facilitated by using Lys-C and papain to generate Fab and Fc fragments with unique, protease-dependent elution times. In particular, this method was useful for characterizing protein variants formed in the presence of salt under accelerated storage conditions. Two isoforms that accumulated during storage were readily identified as Fab-related species prior to mass-spectrometric analysis. Both showed reduced biological activity likely resulting from modifications within or in proximity of the complementarity-determining regions (CDRs). Utility of this assay was further illustrated in the work to characterize light-induced degradations in mAb formulations. In this case, a previously unknown Fab-related species which populated upon light exposure was observed. This species was well resolved from unmodified Fab, allowing for direct and high-purity fractionation. Mass-spectrometric analysis subsequently identified a histidine-related degradation product associated with the CDR2 of the heavy chain. In addition, the method was applied to assess the structural organization of a noncovalent IgG1 dimer. A new species corresponding to a Fab-Fab complex was found, implying that interactions between Fab domains were responsible for dimerization. Overall, the data presented demonstrate the suitability of this cation-exchange HPLC method for studying a wide range of covalent and noncovalent degradations in IgG1 mAbs. 2010 Elsevier B.V. All rights reserved.

Dysexecutive performance of healthy oldest old subjects on the Frontal Assessment Battery.

PubMed

Iavarone, Alessandro; Lorè, Elisa; De Falco, Caterina; Milan, Graziella; Mosca, Raffaela; Pappatà, Sabina; Galeone, Filomena; Sorrentino, Paolo; Scognamiglio, Mario; Postiglione, Alfredo

2011-01-01

Frontal lobes and executive functions appear to be more vulnerable to normal aging than other cerebral regions and domains. The aim of the study was to evaluate executive functions by the Frontal Assessment Battery (FAB) in healthy oldest old subjects free of dementia. Thirty-two healthy oldest old subjects (age range 85-97 yrs) and 32 young old subjects (aged 61-74 yrs) were studied. All subjects were living with their families or alone and were considered normal, since they were fully independent in their activities of daily living and without signs or symptoms characteristic of any type of dementia. Mental status was assessed by the Mini- Mental State Examination (MMSE) and executive functions by the FAB. Mean MMSE scores were 23.12 ± 4.68 in oldest old and 26.78 ± 2.60 in young old subjects (p<0.005). Delayed recall was the most impaired domain, followed by executive (Serial 7). Mean FAB scores were 9.37 ± 4.14 in the oldest old and 13.53 ± 2.12 in the young old (p<0.0001). Among the FAB subtests, conceptualization was the most impaired in both groups, with sensitivity to interference and inhibitory control exhibiting higher discrimination between the oldest old and young old. Education influenced performance on MMSE and FAB in both groups. On the FAB test, healthy oldest old subjects showed executive impairment with respect to the young olds, due to the involvement of functions depending on activities of different regions of the frontal lobes. FAB results were consistent with the hypothesis that frontal lobes have a high vulnerability to normal aging. Short composite batteries like the FAB are suitable for rapid and reliable description of patterns of executive functioning in the oldest old.

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

PubMed

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

PubMed

Amirzargar, M A; Amirzargar, A; Basiri, A; Hajilooi, M; Roshanaei, G; Rajabi, G; Solgi, G

2015-05-01

Immunologic factors are reliable markers for allograft monitoring, because of their seminal role in rejection process. One of these factors is the immunoglobulin (Ig)A anti-Fab of the IgG antibody. This study aimed to evaluate the predictive value of pre- and posttransplant levels of this marker for kidney allograft function and survival. Sera samples of 59 living unrelated donor kidney recipients were collected before and after transplantation (days 7, 14, and 30) and investigated for IgA anti-Fab of IgG antibody levels using enzyme-linked immunosorbent assay in relation with allograft outcome. Among 59 patients, 15 cases (25%) including 10 with acute rejection and 5 with chronic rejection episodes showed graft failure during a mean of 5 years of follow-up. High posttransplant levels of IgA anti-Fab antibodies were observed more frequently in patients with stable graft function (SGF) compared with patients with graft failure (P = 2 × 10(-6)). None of patients with acute or chronic rejection episodes had high levels of IgA anti-Fab antibodies at day 30 posttransplant compared with the SGF group (P = 10(-6) and P = .01, respectively). In addition, high levels of IgA anti-Fab antibody correlated with lesser concentration of serum creatinine at 1 month posttransplantation (P = .01). Five-year graft survival was associated with high levels of pre- and posttransplant IgA anti-Fab antibodies (P = .02 and P = .003, respectively). Our findings indicate the protective effect of higher levels of IgA anti-Fab antibodies regarding to kidney allograft outcomes and long-term graft survival. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

PubMed

Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

2017-05-08

The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

Comparison of childhood myelodysplastic syndrome, AML FAB M6 or M7, CCG 2891: report from the Children's Oncology Group.

PubMed

Barnard, Dorothy R; Alonzo, Todd A; Gerbing, Robert B; Lange, Beverly; Woods, William G

2007-07-01

Myelodysplastic syndromes (MDS), acute erythroleukemia (FAB M6), and acute megakaryocytic leukemia (FAB M7) have overlapping features. Children without Down syndrome or acute promyelocytic leukemia who were newly diagnosed with primary myelodysplastic syndrome or acute myeloid leukemia (AML) M6 or M7 were compared to children with de novo AML M0-M5. All children were entered on the Children's Cancer Group therapeutic research study CCG 2891. The presentation and outcomes of the 132 children diagnosed with MDS (60 children), AML FAB M6 (19 children), or AML FAB M7 (53 children) were similar. Children with AML FAB M7 were diagnosed at a significantly younger age (P = 0.001). Children with MDS, M6, or M7 had significantly lower white blood cell (WBC) counts (P = 0.001), lower peripheral blast counts (P < 0.001), and an increased frequency of -7/7q- (P = 0.003) at presentation. All three groups had significantly inferior overall survival (OS) (P < 0.001) and event free survival (P < 0.001) compared with the 748 children diagnosed with AML FAB M0-M5 when assessed from entry on study. This poor survival was largely attributable to induction death and failure. However, when assessed from successful completion of induction therapy, the 5-year OS (P = 0.090)(49.1 vs. 56.9%) and disease-free survival (DFS) (P = 0.113)(38.0 vs. 46.3%) therapy were not significantly different from other children with AML. Childhood AML FAB M6 and AML M7 resemble MDS in presentation, poor induction success rates, and outcomes.

Basic Phage Mathematics.

PubMed

Abedon, Stephen T; Katsaounis, Tena I

2018-01-01

Basic mathematical descriptions are useful in phage ecology, applied phage ecology such as in the course of phage therapy, and also toward keeping track of expected phage-bacterial interactions as seen during laboratory manipulation of phages. The most basic mathematical descriptor of phages is their titer, that is, their concentration within stocks, experimental vessels, or other environments. Various phenomena can serve to modify phage titers, and indeed phage titers can vary as a function of how they are measured. An important aspect of how changes in titers can occur results from phage interactions with bacteria. These changes tend to vary in degree as a function of bacterial densities within environments, and particularly densities of those bacteria that are susceptible to or at least adsorbable by a given phage type. Using simple mathematical models one can describe phage-bacterial interactions that give rise particularly to phage adsorption events. With elaboration one can consider changes in both phage and bacterial densities as a function of both time and these interactions. In addition, phages along with their impact on bacteria can be considered as spatially constrained processes. In this chapter we consider the simpler of these concepts, providing in particular detailed verbal explanations toward facile mathematical insight. The primary goal is to stimulate a more informed use and manipulation of phages and phage populations within the laboratory as well as toward more effective phage application outside of the laboratory, such as during phage therapy. More generally, numerous issues and approaches to the quantification of phages are considered along with the quantification of individual, ecological, and applied properties of phages.

Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus).

PubMed

Uchiyama, Jumpei; Suzuki, Masato; Nishifuji, Koji; Kato, Shin-Ichiro; Miyata, Reina; Nasukawa, Tadahiro; Yamaguchi, Kotoe; Takemura-Uchiyama, Iyo; Ujihara, Takako; Shimakura, Hidekatsu; Murakami, Hironobu; Okamoto, Noriaki; Sakaguchi, Yoshihiko; Shibayama, Keigo; Sakaguchi, Masahiro; Matsuzaki, Shigenobu

2016-08-01

Pseudomonas aeruginosa causes serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treat P. aeruginosa infections, and phages belonging to the PB1-like virus genus in the Myoviridae family have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones of P. aeruginosa strain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in the Myoviridae family. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets of in vitro experiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in the wzy gene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading frames orf060, orf065, and orf086 The information obtained in this study should be useful for further development of safe and efficient phage therapy. Pseudomonas aeruginosa causes serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treat P. aeruginosa infections, and phages that belong to the PB1-like virus genus in the family Myoviridae have been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, the in vitro short-term antagonistic evolution was studied using P. aeruginosa strain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Flavored alcoholic beverages: an international marketing campaign that targets youth.

PubMed

Mosher, James F; Johnsson, Diane

2005-09-01

Flavored alcoholic beverages (FABs) were first introduced into the alcohol market in the early I980s in the form of wine coolers. FABs are sweet, relatively low alcohol content beverages that are designed for "entry-level" drinkers. The alcohol industry has introduced new products and production methods to expand the category's popularity. Research suggests that they are popular with underage drinkers, particularly teenage girls, and that the industry uses marketing practices that appear to target youth. FABs are now marketed globally, and their production and marketing vary by country based on national regulatory restraints. In the United States, industry representations that the products are malt beverages for regulatory purposes appears to violate many state laws because the alcohol in the FABs is derived from distilled spirits. Recommendations for regulatory reform, including new legal definitions of FABs, increased taxes, and restrictions on availability, are applicable at both national and state levels.

[Isolation and characterization of siphovirus phages infecting bovine Streptococcus agalactiae].

PubMed

Bai, Qinqin; Yang, Yongchun; Lu, Chengping

2016-02-04

To isolate and identify Streptococcus agalactiae phages and screen candidate phages to control infection caused by bovine S. agalactiae. We used two methods for isolation of S. agalactiae phages, namely (1) isolation of phages from milk and environmental samples, and (2) isolation of phages via induction of lysogens with Mitomycin C. Double-layer agar culture method was used to purify phages. Then the newly obtained phages, with S. agalactiae phage JX01 isolated from mastitis milk, were comparatively analyzed in the following aspects: morphology of phages by transmission electron microscopy, host range of phages to 55 S. agalactiae strains and other Streptococcus strains, phages DNA using EcoR I, Xba I, Pst I and Sal I, the optical multiplicity of infection, absorption curve and one step growth curve, and the stability of phages at different storage conditions. The comparative analysis of the 3 novel phages LYGO9, HZ04 and pA11 (induced from S. agalctiae bovine clinical isolate HAJL2011070601) with JX01 showed that the 4 phages were classified as the member of Siphovirdae family. EcoR I, Sal I, Xba I and Pst I separately digested the 4 phages DNA provided 4, 3, 3 and 2 profiles, respectively. This suggested that they were different strains. All the 4 phages specifically infected bovine S. agalactiae isolates. LYGO9, pA11, JX01 and HZ04 could lyse 12, 13, 20 and 23 of 42 tested bovine S. agalctiae isolates, respectively. This clearly indicated that these 4 phages are closely related. The 3 new phages which specifically lyse bovine S. agalactiae isolates are siphovirus phages. Phage LYGO9 was shown having a short latent period and a larger burst size.

Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

NASA Astrophysics Data System (ADS)

Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad

2015-12-01

A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.

Development of tools to study personal weight control strategies: OxFAB taxonomy

PubMed Central

Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A.

2016-01-01

Objective To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. Methods The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think‐aloud interviews were conducted to test the face/concept validity of the questionnaire, and test–retest reliability was assessed in a sample of 138 participants. Results The OxFAB taxonomy consists of 117 strategies grouped into 23 domains. Compared to taxonomies used to describe interventions, around half of the domains and strategies identified are unique to the OxFAB taxonomy. The OxFAB questionnaire consists of 117 questions, one for each strategy from the taxonomy. Test–retest resulted in a mean PABAK score of 0.61 (SD 0.15). Questions were revised where appropriate. Conclusions The OxFAB taxonomy and questionnaire provide a conceptual framework to identify the cognitive and behavioral strategies used by individuals during attempts at weight control. PMID:26748902

Development of tools to study personal weight control strategies: OxFAB taxonomy.

PubMed

Hartmann-Boyce, Jamie; Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A

2016-02-01

To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think-aloud interviews were conducted to test the face/concept validity of the questionnaire, and test-retest reliability was assessed in a sample of 138 participants. The OxFAB taxonomy consists of 117 strategies grouped into 23 domains. Compared to taxonomies used to describe interventions, around half of the domains and strategies identified are unique to the OxFAB taxonomy. The OxFAB questionnaire consists of 117 questions, one for each strategy from the taxonomy. Test-retest resulted in a mean PABAK score of 0.61 (SD 0.15). Questions were revised where appropriate. The OxFAB taxonomy and questionnaire provide a conceptual framework to identify the cognitive and behavioral strategies used by individuals during attempts at weight control. © 2016 The Authors. Obesity published by Wiley Periodicals, Inc. on behalf of The Obesity Society (TOS).

Frontal Assessment Battery (FAB) is a simple tool for detecting executive deficits in chronic cannabis users.

PubMed

Fontes, Maria Alice; Bolla, Karen I; Cunha, Paulo Jannuzzi; Almeida, Priscila Previato; Jungerman, Flávia; Laranjeira, Ronaldo Ramos; Bressan, Rodrigo A; Lacerda, Acioly L T

2011-06-01

Cannabis is the most used illicit drug in the world, and its use has been associated with prefrontal cortex (PFC) dysfunction, including deficits in executive functions (EF). Considering that EF may influence treatment outcome, it would be interesting to have a brief neuropsychological battery to assess EF in chronic cannabis users (CCU). In the present study, the Frontal Assessment Battery (FAB), a brief, easy to use neuropsychological instrument aimed to evaluate EF, was used to evaluate cognitive functioning of CCU. We evaluated 107 abstinent CCU with the FAB and compared with 44 controls matched for age, estimated IQ, and years of education. CCU performed poorly as compared to controls (FAB total score = 16.53 vs. 17.09, p < .05). CCU had also a poor performance in the Motor Programming subtest (2.47 vs. 2.73, p < .05). This study examined effects of cannabis in executive functioning and showed evidence that the FAB is sensitive to detect EF deficits in early abstinent chronic cannabis users. Clinical significance of these findings remains to be investigated in further longitudinal studies. FAB may be useful as a screening instrument to evaluate the necessity for a complete neuropsychological assessment in this population.

Functional Neuroanatomical Correlates of The Frontal Assessment Battery Performance in Alzheimer Disease: A FDG-PET Study.

PubMed

Lee, Jun Ho; Byun, Min Soo; Sohn, Bo Kyung; Choe, Young Min; Yi, Dahyun; Han, Ji Young; Choi, Hyo Jung; Baek, Hyewon; Woo, Jong Inn; Lee, Dong Young

2015-09-01

We aimed to elucidate the functional neuroanatomical correlates of Frontal Assessment Battery (FAB) performances by applying [(18)F]fluorodeoxyglucose positron emission tomography (FDG-PET) to a large population of patients with Alzheimer disease (AD). The FAB was administered to 177 patients with AD, and regional cerebral glucose metabolism (rCMglc) was measured by FDG-PET scan. Correlations between FAB scores and rCMglc were explored using both region-of-interest-based (ROI-based) and voxel-based approaches. The ROI-based analysis showed that FAB scores correlated with the rCMglc of the dorsolateral prefrontal cortices. Voxel-based approach revealed significant positive correlations between FAB scores and rCMglc which were in various cortical regions including the temporal and parietal cortices as well as frontal regions, independent of age, gender, and education. After controlling the effect of global disease severity with Mini-Mental State Examination score, significant positive correlation was found only in the bilateral prefrontal regions. Although FAB scores are influenced by temporoparietal dysfunction due to the overall progression of AD, it likely reflects prefrontal dysfunction specifically regardless of global cognitive state or disease severity in patients with AD. © The Author(s) 2015.

PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

PubMed

Freches, Danielle; Patil, Harshad P; Machado Franco, Maria; Uyttenhove, Catherine; Heywood, Sam; Vanbever, Rita

2017-04-15

The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

(67/68)Ga-labeling agent that liberates (67/68)Ga-NOTA-methionine by lysosomal proteolysis of parental low molecular weight polypeptides to reduce renal radioactivity levels.

PubMed

Uehara, Tomoya; Rokugawa, Takemi; Kinoshita, Mai; Nemoto, Souki; Fransisco Lazaro, Guerra Gomez; Hanaoka, Hirofumi; Arano, Yasushi

2014-11-19

The renal localization of gallium-67 or gallium-68 ((67/68)Ga)-labeled low molecular weight (LMW) probes such as peptides and antibody fragments constitutes a problem in targeted imaging. Wu et al. previously showed that (67)Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bz-NOTA)-conjugated methionine ((67)Ga-NOTA-Met) was rapidly excreted from the kidney in urine following lysosomal proteolysis of the parental (67)Ga-NOTA-Bz-SCN-disulfide-stabilized Fv fragment (Bioconjugate Chem., (1997) 8, 365-369). In the present study, a new (67/68)Ga-labeling reagent for LMW probes that liberates (67/68)Ga-NOTA-Met was designed, synthesized, and evaluated using longer-lived (67)Ga in order to reduce renal radioactivity levels. We employed a methionine-isoleucine (MI) dipeptide bond as the cleavable linkage. The amine residue of MI was coupled with SCN-Bz-NOTA for (67)Ga-labeling, while the carboxylic acid residue of MI was derivatized to maleimide for antibody conjugation in order to synthesize NOTA-MI-Mal. A Fab fragment of the anti-Her2 antibody was thiolated with iminothiolane, and NOTA-MI-Mal was conjugated with the antibody fragment by maleimide-thiol chemistry. The Fab fragment was also conjugated with SCN-Bz-NOTA (NOTA-Fab) for comparison. (67)Ga-NOTA-MI-Fab was obtained at radiochemical yields of over 95% and was stable in murine serum for 24 h. In the biodistribution study using normal mice, (67)Ga-NOTA-MI-Fab registered significantly lower renal radioactivity levels from 1 to 6 h postinjection than those of (67)Ga-NOTA-Fab. An analysis of urine samples obtained 6 h after the injection of (67)Ga-NOTA-MI-Fab showed that the majority of radioactivity was excreted as (67)Ga-NOTA-Met. In the biodistribution study using tumor-bearing mice, the tumor to kidney ratios of (67)Ga-NOTA-MI-Fab were 4 times higher (6 h postinjection) than those of (67)Ga-NOTA-Fab. Although further studies including the structure of radiometabolites and/or cleavable linkages are required, the results of the present study indicate that the current chemical design is applicable to the development of (67)Ga-labeled Fabs for low renal radioactivity levels.

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

PubMed

Lu, Danfeng; Li, Zhuoran; Li, Lingling; Yang, Liping; Chen, Guijun; Yang, Deying; Zhang, Yue; Singh, Vikrant; Smith, Sheryl; Xiao, Yu; Wang, Erlin; Ye, Yunshuang; Zhang, Wei; Zhou, Lei; Rong, Yikang; Zhou, Jumin

2018-01-01

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains.

PubMed

Rohde, Christine; Resch, Grégory; Pirnay, Jean-Paul; Blasdel, Bob G; Debarbieux, Laurent; Gelman, Daniel; Górski, Andrzej; Hazan, Ronen; Huys, Isabelle; Kakabadze, Elene; Łobocka, Małgorzata; Maestri, Alice; Almeida, Gabriel Magno de Freitas; Makalatia, Khatuna; Malik, Danish J; Mašlaňová, Ivana; Merabishvili, Maia; Pantucek, Roman; Rose, Thomas; Štveráková, Dana; Van Raemdonck, Hilde; Verbeken, Gilbert; Chanishvili, Nina

2018-04-05

Phage therapy is increasingly put forward as a "new" potential tool in the fight against antibiotic resistant infections. During the "Centennial Celebration of Bacteriophage Research" conference in Tbilisi, Georgia on 26-29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.

Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains

PubMed Central

Rohde, Christine; Resch, Grégory; Blasdel, Bob G.; Gelman, Daniel; Górski, Andrzej; Hazan, Ronen; Huys, Isabelle; Kakabadze, Elene; Łobocka, Małgorzata; Maestri, Alice; Makalatia, Khatuna; Malik, Danish J.; Mašlaňová, Ivana; Merabishvili, Maia; Rose, Thomas; Štveráková, Dana; Van Raemdonck, Hilde; Verbeken, Gilbert; Chanishvili, Nina

2018-01-01

Phage therapy is increasingly put forward as a “new” potential tool in the fight against antibiotic resistant infections. During the “Centennial Celebration of Bacteriophage Research” conference in Tbilisi, Georgia on 26–29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application. PMID:29621199

Generation of Affibody ligands binding interleukin-2 receptor alpha/CD25.

PubMed

Grönwall, Caroline; Snelders, Eveline; Palm, Anna Jarelöv; Eriksson, Fredrik; Herne, Nina; Ståhl, Stefan

2008-06-01

Affibody molecules specific for human IL-2Ralpha, the IL-2 (interleukin-2) receptor alpha subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody molecules bound to CD4+ CD25+ PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody molecules as targeting agents for medical imaging and for therapeutic applications is discussed.

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

PubMed

Duska, L R; Hamblin, M R; Bamberg, M P; Hasan, T

1997-01-01

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okamura, Hitomi; Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501; Nio, Yasunori, E-mail: yasunori.nio@takeda.com

Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturatedmore » fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.« less

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

PubMed

Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia.

PubMed

Farag, Mina; Badowski, Dominika; Koschny, Ronald; Skopp, Gisela; Brcic, Andreas; Szabo, Gabor B

2017-12-01

Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options. We report the case of a suicidal yew ingestion admitted to our hospital under prolonged cardiopulmonary resuscitation due to pulseless electrical activity. Extra-corporeal life support (ECLS) was established to maintain adequate organ perfusion. Repeated administration of digoxin-specific Fab antibody fragments, which cross-react with taxine, was associated with an immediate conversion from asystole to broad-complex bradycardia and a gradual normalization of the electrocardiogram (ECG). This was paralleled by a recovery of the cardiac function and weaning from the ECLS. The taxine metabolite 3,5-dimethoxyphenol could be detected by mass spectrometry before but not after the first Fab-fragment treatment. In contrast, the total amount of taxine (including the neutralized, Fab fragment-bound fraction) was increased after each Fab fragment administration, suggesting an accumulation of neutralized, since antibody-bound taxine in the blood by anti-digoxin Fab fragments. In conclusion, the successful clinical course of this case suggests a benefit of an early anti-digoxin Fab-fragment administration for the treatment of yew intoxication. Copyright © 2017 Elsevier Inc. All rights reserved.

Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus.

PubMed

Pereira, Jose H; Goh, Ee-Been; Keasling, Jay D; Beller, Harry R; Adams, Paul D

2012-10-01

Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

Characterization of Two Virulent Phages of Lactobacillus plantarum

PubMed Central

Briggiler Marcó, Mariángeles; Garneau, Josiane E.; Tremblay, Denise; Quiberoni, Andrea

2012-01-01

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria. PMID:23042172

Toward Understanding Phage:Host Interactions in the Rumen; Complete Genome Sequences of Lytic Phages Infecting Rumen Bacteria

PubMed Central

Gilbert, Rosalind A.; Kelly, William J.; Altermann, Eric; Leahy, Sinead C.; Minchin, Catherine; Ouwerkerk, Diane; Klieve, Athol V.

2017-01-01

The rumen is known to harbor dense populations of bacteriophages (phages) predicted to be capable of infecting a diverse range of rumen bacteria. While bacterial genome sequencing projects are revealing the presence of phages which can integrate their DNA into the genome of their host to form stable, lysogenic associations, little is known of the genetics of phages which utilize lytic replication. These phages infect and replicate within the host, culminating in host lysis, and the release of progeny phage particles. While lytic phages for rumen bacteria have been previously isolated, their genomes have remained largely uncharacterized. Here we report the first complete genome sequences of lytic phage isolates specifically infecting three genera of rumen bacteria: Bacteroides, Ruminococcus, and Streptococcus. All phages were classified within the viral order Caudovirales and include two phage morphotypes, representative of the Siphoviridae and Podoviridae families. The phage genomes displayed modular organization and conserved viral genes were identified which enabled further classification and determination of closest phage relatives. Co-examination of bacterial host genomes led to the identification of several genes responsible for modulating phage:host interactions, including CRISPR/Cas elements and restriction-modification phage defense systems. These findings provide new genetic information and insights into how lytic phages may interact with bacteria of the rumen microbiome. PMID:29259581

Advance in phage display technology for bioanalysis.

PubMed

Tan, Yuyu; Tian, Tian; Liu, Wenli; Zhu, Zhi; J Yang, Chaoyong

2016-06-01

Phage display technology has emerged as a powerful tool for target gene expression and target-specific ligand selection. It is widely used to screen peptides, proteins and antibodies with the advantages of simplicity, high efficiency and low cost. A variety of targets, including ions, small molecules, inorganic materials, natural and biological polymers, nanostructures, cells, bacteria, and even tissues, have been demonstrated to generate specific binding ligands by phage display. Phages and target-specific ligands screened by phage display have been widely used as affinity reagents in therapeutics, diagnostics and biosensors. In this review, comparisons of different types of phage display systems are first presented. Particularly, microfluidic-based phage display, which enables screening with high throughput, high efficiency and integration, is highlighted. More importantly, we emphasize the advances in biosensors based on phages or phage-derived probes, including nonlytic phages, lytic phages, peptides or proteins screened by phage display, phage assemblies and phage-nanomaterial complexes. However, more efficient and higher throughput phage display methods are still needed to meet an explosion in demand for bioanalysis. Furthermore, screening of cyclic peptides and functional peptides will be the hotspot in bioanalysis. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Validating a faster method for reconstitution of Crotalidae Polyvalent Immune Fab (ovine).

PubMed

Gerring, David; King, Thomas R; Branton, Richard

2013-07-01

Reconstitution of CroFab(®) (Crotalidae Polyvalent Immune Fab [ovine]) lyophilized drug product was previously performed using 10 mL sterile water for injection followed by up to 36 min of gentle swirling of the vial. CroFab has been clinically demonstrated to be most effective when administered within 6 h of snake envenomation, and improved clinical outcomes are correlated with quicker timing of administration. An alternate reconstitution method was devised, using 18 mL 0.9% saline with manual inversion, with the goal of shortening reconstitution time while maintaining a high quality, efficacious product. An analytical study was designed to compare the physicochemical properties of 3 separate batches of CroFab when reconstituted using the standard procedure (10 mL WFI with gentle swirling) and a modified rapid procedure using 18 mL 0.9% saline and manual inversion. The physical and chemical characteristics of the same 3 batches were assessed using various analytic methodologies associated with routine quality control release testing. In addition further analytical methodologies were applied in order to elucidate possible structural changes that may be induced by the changed reconstitution procedure. Batches A, B, and C required mean reconstitution times of 25 min 51 s using the label method and 3 min 07 s (a 88.0% mean decrease) using the modified method. Physicochemical characteristics (color and clarity, pH, purity, protein content, potency) were found to be highly comparable. Characterization assays (dynamic light scattering, analytical ultracentrifugation, LC-MS, SDS-PAGE and circular dichroism spectroscopy were also all found to be comparable between methods. When comparing CroFab batches that were reconstituted using the labeled and modified methods, the physicochemical and biological (potency) characteristics of CroFab were not significantly changed when challenged by the various standard analytical methodologies applied in routine quality control analysis. Additionally, no changes in the CroFab molecule regarding degradation, aggregation, purity, structure, or mass were observed. The analyses performed validated the use of the more rapid reconstitution method using 18 mL 0.9% saline in order to allow a significantly reduced time to administration of CroFab to patients in need. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impaired frontal lobe functions in patients with Parkinson's disease and psychosis.

PubMed

Thota, Naveen; Lenka, Abhishek; George, Lija; Hegde, Shantala; Arumugham, Shyam Sundar; Prasad, Shweta; Stezin, Albert; Kamble, Nitish; Yadav, Ravi; Pal, Pramod Kumar

2017-12-01

Patients with Parkinson's disease (PD) may develop several non-motor symptoms (NMS). Psychosis is one of the debilitating NMS of PD. The neurobiology of psychosis is not fully understood. This study aims to compare the frontal lobe functions of PD patients with and without psychosis using the Frontal Assessment Battery (FAB). This study included 69 patients with PD; 34 with psychosis (PD-P) and 35 without psychosis (PD-NP). Mini Mental Status Examination (MMSE) was used to screen for cognitive impairment. Unified Parkinson's disease Rating scale part-III (UPDRS-III) was used to measure the severity and Hoehn and Yahr score (H&Y) was used to measure the stage of PD. Frontal lobe functions were assessed by FAB. The PD-P and PD-NP groups were comparable for age (58.7±8.4 vs 55.7±8.2, p=0.14), age at onset of symptoms (51.4±8.1 vs 50.0±8.8, p=0.48), gender distribution (men: 88%vs 80%, p=0.51), MMSE (28.2±1.9 vs 28.7±1.2 p=0.12), levodopa equivalent dose/day (736.0±376.3 vs 625.2±332.2, p=0.19), UPDRS-III OFF-score (36.7±8.8 vs 35.4±13.2, p=0.64), UPDRS-III ON-score (13.2±5.4 vs 12.4±6.6, p=0.44) and H&Y stage (2.3±0.3 vs 2.3±0.3, p=0.07). PD-P group had lower total FAB score compared to PD-NP group (13.9±2.2 vs 16.5±1.8, p<0.01). On the FAB, PD-P group had lower scores compared to PD-NP in lexical fluency (FAB-2), programming (FAB-3), sensitivity to interference (FAB-4) and inhibitory control (FAB-5). Patients with PD-P had significant frontal lobe dysfunction compared to PD-NP. FAB may be a simple and useful bedside tool to assess frontal dysfunction in patients with PD in a busy neurological set up. Copyright © 2017 Elsevier B.V. All rights reserved.

PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

PubMed

Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

2015-06-01

The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Engineered phages for electronics.

PubMed

Cui, Yue

2016-11-15

Phages are traditionally widely studied in biology and chemistry. In recent years, engineered phages have attracted significant attentions for functionalization or construction of electronic devices, due to their specific binding, catalytic, nucleating or electronic properties. To apply the engineered phages in electronics, these are a number of interesting questions: how to engineer phages for electronics? How are the engineered phages characterized? How to assemble materials with engineered phages? How are the engineered phages micro or nanopatterned? What are the strategies to construct electronics devices with engineered phages? This review will highlight the early attempts to address these questions and explore the fundamental and practical aspects of engineered phages in electronics, including the approaches for selection or expression of specific peptides on phage coat proteins, characterization of engineered phages in electronics, assembly of electronic materials, patterning of engineered phages, and construction of electronic devices. It provides the methodologies and opens up ex-cit-ing op-por-tu-ni-ties for the development of a variety of new electronic materials and devices based on engineered phages for future applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

PubMed

Gadermaier, E; Marth, K; Lupinek, C; Campana, R; Hofer, G; Blatt, K; Smiljkovic, D; Roder, U; Focke-Tejkl, M; Vrtala, S; Keller, W; Valent, P; Valenta, R; Flicker, S

2018-01-09

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. © 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

PubMed

Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

2012-07-01

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirkpatrick, Andrew S.; Yokoyama, Takeshi; Choi, Kyoung-Jae

Fatty acid biosynthesis is crucial for all living cells. In contrast to higher organisms, bacteria use a type II fatty acid synthase (FAS II) composed of a series of individual proteins, making FAS II enzymes excellent targets for antibiotics discovery. The {beta}-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. Here, we report the structure of Campylobacter jejuni FabZ (CjFabZ), showing a hexamer both in crystals and solution, with each protomer adopting the characteristic hot dog fold. Together with biochemical analysis of CjFabZ, we define the first functional FAS II enzyme from this pathogen, and provide amore » framework for investigation on roles of FAS II in C. jejuni virulence« less

Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

NASA Astrophysics Data System (ADS)

Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2016-07-01

Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

PubMed

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

2016-10-01

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators

PubMed Central

Scheer, Justin M.; Sandoval, Wendy; Elliott, J. Michael; Shao, Lily; Luis, Elizabeth; Lewin-Koh, Sock-Cheng; Schaefer, Gabriele; Vandlen, Richard

2012-01-01

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules. PMID:23284778

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

PubMed Central

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

2016-01-01

ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

A pancultural perspective on the fading affect bias in autobiographical memory.

PubMed

Ritchie, Timothy D; Batteson, Tamzin J; Bohn, Annette; Crawford, Matthew T; Ferguson, Georgie V; Schrauf, Robert W; Vogl, Rodney J; Walker, W Richard

2015-01-01

The fading affect bias (FAB) refers to the negative affect associated with autobiographical events fading faster than the positive affect associated with such events, a reliable and valid valence effect established by researchers in the USA. The present study examined the idea that the FAB is a ubiquitous emotion regulating phenomenon in autobiographical memory that is present in people from a variety of cultures. We tested for evidence of the FAB by sampling more than 2400 autobiographical event descriptions from 562 participants in 10 cultures around the world. Using variations on a common method, each sample evidenced a FAB: positive affect faded slower than negative affect did. Results suggest that in tandem with local norms and customs, the FAB may foster recovery from negative life events and promote the retention of the positive emotions, within and outside of the USA. We discuss these findings in the context of Keltner and Haidt's levels of analysis theory of emotion and culture.

20 CFR 30.301 - May subpoenas be issued for witnesses and documents in connection with a claim under Part B of...

Code of Federal Regulations, 2010 CFR

2010-04-01

... adjudication of a claim under Part B of EEOICPA, an OWCP district office and/or a FAB reviewer may, at their... a FAB reviewer. No subpoenas will be issued at the request of the claimant under any other portion of the claims process. The decision to grant or deny such request is within the discretion of the FAB...

Anti-fouling properties of Fab' fragments immobilized on silane-based adlayers

NASA Astrophysics Data System (ADS)

Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

2015-12-01

Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab') in biosensors. One Fab' linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab' fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab' fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab'-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element.

PubMed

Canudas, Silvia; Pérez, Silvia; Fanti, Laura; Pimpinelli, Sergio; Singh, Navjot; Hanes, Steven D; Azorín, Fernando; Espinás, M Lluïsa

2005-01-01

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Immediate and delayed allergic reactions to Crotalidae polyvalent immune Fab (ovine) antivenom.

PubMed

Clark, Richard F; McKinney, Patrick E; Chase, Peter B; Walter, Frank G

2002-06-01

Allergic reactions are the most commonly reported adverse events after administration of antivenoms. Conventional horse serum-based crotalid antivenom used in the United States (Antivenin [Crotalidae] polyvalent) can lead to both immediate and delayed hypersensitivity reactions. Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) has recently been approved for use in the United States. Experience from premarketing trials of this product and in the administration of other types of Fab, such as in digoxin poisoning, has demonstrated these fragments to be safe and effective, with a low incidence of sequella; however, allergic reactions can occur when any animal-protein derivatives are administered to human subjects. We report in detail the nature and course of allergic reactions that occurred in 4 patients treated with FabAV. Cases of anaphylaxis, acute urticaria, angioedema, and delayed serum sickness are described. All reactions were easily treated with some combination of antihistamines, epinephrine, and steroids, with prompt resolution of signs and symptoms enabling further dosing of antivenom as required. Several of these cases may have resulted from batches of antivenom contaminated with Fc fragments. The overall incidence of immediate and delayed allergic reactions to this product appears so far to be lower than that reported with conventional whole-immunoglobulin G (IgG) antivenom, but postmarketing surveillance is warranted.

Identification of a new binding site in E. coli FabH using Molecular dynamics simulations: validation by computational alanine mutagenesis and docking studies.

PubMed

Ramamoorthy, Divya; Turos, Edward; Guida, Wayne C

2013-05-24

FabH (Fatty acid biosynthesis, enzyme H, also referred to as β-ketoacyl-ACP-synthase III) is a key condensing enzyme in the type II fatty acid synthesis (FAS) system. The FAS pathway in bacteria is essential for growth and survival and vastly differs from the human FAS pathway. Enzymes involved in this pathway have arisen as promising biomolecular targets for discovery of new antibacterial drugs. However, currently there are no clinical drugs that selectively target FabH, and known inhibitors of FabH all act within the active site. FabH exerts its catalytic function as a dimer, which could potentially be exploited in developing new strategies for inhibitor design. The aim of this study was to elucidate structural details of the dimer interface region by means of computational modeling, including molecular dynamics (MD) simulations, in order to derive information for the structure-based design of new FabH inhibitors. The dimer interface region was analyzed by MD simulations, trajectory snapshots were collected for further analyses, and docking studies were performed with potential small molecule disruptors. Alanine mutation and docking studies strongly suggest that the dimer interface could be a potential target for anti-infection drug discovery.

Structural Comparison of Different Antibodies Interacting with Parvovirus Capsids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hafenstein, Susan; Bowman, Valorie D.; Sun, Tao

2009-05-13

The structures of canine parvovirus (CPV) and feline parvovirus (FPV) complexed with antibody fragments from eight different neutralizing monoclonal antibodies were determined by cryo-electron microscopy (cryoEM) reconstruction to resolutions varying from 8.5 to 18 {angstrom}. The crystal structure of one of the Fab molecules and the sequence of the variable domain for each of the Fab molecules have been determined. The structures of Fab fragments not determined crystallographically were predicted by homology modeling according to the amino acid sequence. Fitting of the Fab and virus structures into the cryoEM densities identified the footprints of each antibody on the viral surface.more » As anticipated from earlier analyses, the Fab binding sites are directed to two epitopes, A and B. The A site is on an exposed part of the surface near an icosahedral threefold axis, whereas the B site is about equidistant from the surrounding five-, three-, and twofold axes. One antibody directed to the A site binds CPV but not FPV. Two of the antibodies directed to the B site neutralize the virus as Fab fragments. The differences in antibody properties have been linked to the amino acids within the antibody footprints, the position of the binding site relative to the icosahedral symmetry elements, and the orientation of the Fab structure relative to the surface of the virus. Most of the exposed surface area was antigenic, although each of the antibodies had a common area of overlap that coincided with the positions of the previously mapped escape mutations.« less

Acute hypersensitivity reactions associated with administration of crotalidae polyvalent immune Fab antivenom.

PubMed

Cannon, Robert; Ruha, Anne-Michelle; Kashani, John

2008-04-01

Acute hypersensitivity reactions are well known to occur with the administration of the Antivenin (Crotalidae) Polyvalent (Wyeth Laboratories, Marietta, PA). Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV, Protherics, Inc., Brentwood, TN) was introduced in 2001, and early studies reported a hypersensitivity reaction rate up to 19%. We describe the incidence of acute hypersensitivity reactions to FabAV in patients bitten by rattlesnakes. This was a nonconcurrent observational cohort study, with data obtained by chart review of all patients admitted to our service for rattlesnake bites from July 2000 to June 2004. The study was conducted at an urban Level I trauma center and urban children's hospital. All patients treated with FabAV were included. Those who received no antivenom or who were treated with Antivenin (Crotalidae) Polyvalent were excluded. The main outcome variable was whether an acute hypersensitivity reaction developed. Ninety-three patients were included in the review (72 male and 21 female patients). The mean age was 34.5 years (range 16 months to 91 years), and the mean dose of antivenom was 12 vials (range 4 to 32 vials). The incidence of acute hypersensitivity reactions was 5 of 93, or 5.4%. Four patients developed a mild reaction that was easily treated and were able to finish the full course of antivenom. Only 1 patient developed a reaction that prevented further antivenom administration. FabAV appears to be associated with a lower incidence of acute hypersensitivity than initially reported. Most reactions are mild and easily treated and do not preclude further dosing of antivenom.

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

PubMed Central

Duska, L. R.; Hamblin, M. R.; Bamberg, M. P.; Hasan, T.

1997-01-01

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer. PMID:9062404

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

PubMed

Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

2016-11-25

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Genomic, proteomic and bioinformatic analysis of two temperate phages in Roseobacter clade bacteria isolated from the deep-sea water.

PubMed

Tang, Kai; Lin, Dan; Zheng, Qiang; Liu, Keshao; Yang, Yujie; Han, Yu; Jiao, Nianzhi

2017-06-27

Marine phages are spectacularly diverse in nature. Dozens of roseophages infecting members of Roseobacter clade bacteria were isolated and characterized, exhibiting a very high degree of genetic diversity. In the present study, the induction of two temperate bacteriophages, namely, vB_ThpS-P1 and vB_PeaS-P1, was performed in Roseobacter clade bacteria isolated from the deep-sea water, Thiobacimonas profunda JLT2016 and Pelagibaca abyssi JLT2014, respectively. Two novel phages in morphological, genomic and proteomic features were presented, and their phylogeny and evolutionary relationships were explored by bioinformatic analysis. Electron microscopy showed that the morphology of the two phages were similar to that of siphoviruses. Genome sequencing indicated that the two phages were similar in size, organization, and content, thereby suggesting that these shared a common ancestor. Despite the presence of Mu-like phage head genes, the phages are more closely related to Rhodobacter phage RC1 than Mu phages in terms of gene content and sequence similarity. Based on comparative genomic and phylogenetic analysis, we propose a Mu-like head phage group to allow for the inclusion of Mu-like phages and two newly phages. The sequences of the Mu-like head phage group were widespread, occurring in each investigated metagenomes. Furthermore, the horizontal exchange of genetic material within the Mu-like head phage group might have involved a gene that was associated with phage phenotypic characteristics. This study is the first report on the complete genome sequences of temperate phages that infect deep-sea roseobacters, belonging to the Mu-like head phage group. The Mu-like head phage group might represent a small but ubiquitous fraction of marine viral diversity.

Molecular Basis for Lytic Bacteriophage Resistance in Enterococci.

PubMed

Duerkop, Breck A; Huo, Wenwen; Bhardwaj, Pooja; Palmer, Kelli L; Hooper, Lora V

2016-08-30

The human intestine harbors diverse communities of bacteria and bacteriophages. Given the specificity of phages for their bacterial hosts, there is growing interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. A significant barrier to such therapies is the rapid development of phage-resistant bacteria, highlighting the need to understand how bacteria acquire phage resistance in vivo Here we identify novel lytic phages in municipal raw sewage that kill Enterococcus faecalis, a Gram-positive opportunistic pathogen that resides in the human intestine. We show that phage infection of E. faecalis requires a predicted integral membrane protein that we have named PIPEF (for phage infection protein from E. faecalis). We find that PIPEF is conserved in E. faecalis and harbors a 160-amino-acid hypervariable region that determines phage tropism for distinct enterococcal strains. Finally, we use a gnotobiotic mouse model of in vivo phage predation to show that the sewage phages temporarily reduce E. faecalis colonization of the intestine but that E. faecalis acquires phage resistance through mutations in PIPEF Our findings define the molecular basis for an evolutionary arms race between E. faecalis and the lytic phages that prey on them. They also suggest approaches for engineering E. faecalis phages that have altered host specificity and that can subvert phage resistance in the host bacteria. Bacteriophage therapy has received renewed attention as a potential solution to the rise in antibiotic-resistant bacterial infections. However, bacteria can acquire phage resistance, posing a major barrier to phage therapy. To overcome this problem, it is necessary to understand phage resistance mechanisms in bacteria. We have unraveled one such resistance mechanism in Enterococcus faecalis, a Gram-positive natural resident of the human intestine that has acquired antibiotic resistance and can cause opportunistic infections. We have identified a cell wall protein hypervariable region that specifies phage tropism in E. faecalis Using a gnotobiotic mouse model of in vivo phage predation, we show that E. faecalis acquires phage resistance through mutations in this cell wall protein. Our findings define the molecular basis for lytic phage resistance in E. faecalis They also suggest opportunities for engineering E. faecalis phages that circumvent the problem of bacterial phage resistance. Copyright © 2016 Duerkop et al.

Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

PubMed Central

Koetter, Ina; Schwab, Matthias; Fritz, Peter; Kimmel, Martin; Alscher, M. Dominik; Braun, Niko

2013-01-01

Background Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. Methods Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. Results Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. Conclusion This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy. PMID:24244376

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

PubMed Central

Essono, Sosthène; Mondielli, Grégoire; Lamourette, Patricia; Boquet, Didier; Grassi, Jacques; Marchot, Pascale

2013-01-01

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis. PMID:24146971

Incidence of immediate hypersensitivity reaction and serum sickness following administration of Crotalidae polyvalent immune Fab antivenom: a meta-analysis.

PubMed

Schaeffer, Tammi H; Khatri, Vaishali; Reifler, Liza M; Lavonas, Eric J

2012-02-01

  Crotalidae polyvalent immune Fab (ovine) (FabAV) is commonly used in the treatment of symptomatic North American crotaline snake envenomation. When approved by the U.S. Food and Drug Administration in 2000, the incidences of immediate hypersensitivity reactions and serum sickness were reported as 0.14 and 0.18, respectively. The objective of this meta-analysis was to evaluate the incidence of immediate hypersensitivity reactions and serum sickness reported in studies of patients treated with FabAV therapy after North American crotaline envenomation.   The authors searched PubMed, Ovid MEDLINE, and EMBASE from January 1, 1997, to September 20, 2010, for English-language medical literature and cross-referenced bibliographies of reviewed articles. The published abstracts of the major toxicology conferences were also searched. All prospective and retrospective cohort studies with patients receiving FabAV therapy for North American crotaline envenomations were eligible for data abstraction. Two content experts reviewed full-text articles and extracted relevant study design and outcome data. Proportions of immediate hypersensitivity and serum sickness for each study were analyzed in a random-effects model to produce an overall estimate of immediate hypersensitivity and serum sickness incidence associated with FabAV administration.   The literature search revealed 11 unique studies of patients who received FabAV that contained information on immediate hypersensitivity reactions and serum sickness. The meta-analysis produced a combined estimate of the incidence of immediate hypersensitivity of 0.08 (95% confidence interval [CI] = 0.05 to 0.11) and a combined estimate of the incidence of serum sickness of 0.13 (95% CI = 0.07 to 0.21).   In this systematic literature review and meta-analysis, the combined estimates of the incidence of immediate hypersensitivity reactions and serum sickness from FabAV in the treatment of symptomatic North American crotaline envenomations appear to be lower than previously reported, at 0.08 and 0.13, respectively. © 2012 by the Society for Academic Emergency Medicine.

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro gastrointestinal cancers.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

PubMed

Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-07-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex

PubMed Central

Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-01-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors. PMID:27428541

Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

PubMed

Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

2015-12-01

Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

PubMed Central

Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

2015-01-01

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

PubMed Central

Gillis, Annika; Mahillon, Jacques

2014-01-01

Many bacteriophages (phages) have been widely studied due to their major role in virulence evolution of bacterial pathogens. However, less attention has been paid to phages preying on bacteria from the Bacillus cereus group and their contribution to the bacterial genetic pool has been disregarded. Therefore, this review brings together the main information for the B. cereus group phages, from their discovery to their modern biotechnological applications. A special focus is given to phages infecting Bacillus anthracis, B. cereus and Bacillus thuringiensis. These phages belong to the Myoviridae, Siphoviridae, Podoviridae and Tectiviridae families. For the sake of clarity, several phage categories have been made according to significant characteristics such as lifestyles and lysogenic states. The main categories comprise the transducing phages, phages with a chromosomal or plasmidial prophage state, γ-like phages and jumbo-phages. The current genomic characterization of some of these phages is also addressed throughout this work and some promising applications are discussed here. PMID:25010767

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

PubMed Central

Ikehara, Yukio; Pitot, Henry C.

1973-01-01

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [125I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [125I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [125I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[125I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [125I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[125I]Fab to free polysomes was also obtained. The [125I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[125I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase. PMID:4201708

The Use of a Selenium-Peptide to Specifically Inactivate Yersinia pestis

DTIC Science & Technology

2005-10-01

Control 2 (F1 Antibody+Anti-F1 Antibody) 2ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab 1ug/ mlF1 Ab+1011 Phage+Anti-F1 Antibody .5ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab...25ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .125ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0625ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .03125ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab...0156ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0078ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .0039ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab .00195ug/ mlF1 Ab+1011 Phage+Anti-F1 Ab

Landscape Phage: Evolution from Phage Display to Nanobiotechnology.

PubMed

Petrenko, Valery A

2018-06-07

The development of phage engineering technology has led to the construction of a novel type of phage display library-a collection of nanofiber materials with diverse molecular landscapes accommodated on the surface of phage particles. These new nanomaterials, called the "landscape phage", serve as a huge resource of diagnostic/detection probes and versatile construction materials for the preparation of phage-functionalized biosensors and phage-targeted nanomedicines. Landscape-phage-derived probes interact with biological threat agents and generate detectable signals as a part of robust and inexpensive molecular recognition interfaces introduced in mobile detection devices. The use of landscape-phage-based interfaces may greatly improve the sensitivity, selectivity, robustness, and longevity of these devices. In another area of bioengineering, landscape-phage technology has facilitated the development and testing of targeted nanomedicines. The development of high-throughput phage selection methods resulted in the discovery of a variety of cancer cell-associated phages and phage proteins demonstrating natural proficiency to self-assemble into various drug- and gene-targeting nanovehicles. The application of this new "phage-programmed-nanomedicines" concept led to the development of a number of cancer cell-targeting nanomedicine platforms, which demonstrated anticancer efficacy in both in vitro and in vivo experiments. This review was prepared to attract the attention of chemical scientists and bioengineers seeking to develop functionalized nanomaterials and use them in different areas of bioscience, medicine, and engineering.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

PubMed Central

Forsman, Päivi; Alatossava, Tapani

1991-01-01

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages. Images PMID:16348513

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

PubMed Central

Szymczak, Paula; Neves, Ana Rute; Kot, Witold; Hansen, Lars H.; Lametsch, René; Neve, Horst; Franz, Charles M. A. P.

2016-01-01

ABSTRACT Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. PMID:28039135

Bacteriophages of Gordonia spp. Display a Spectrum of Diversity and Genetic Relationships.

PubMed

Pope, Welkin H; Mavrich, Travis N; Garlena, Rebecca A; Guerrero-Bustamante, Carlos A; Jacobs-Sera, Deborah; Montgomery, Matthew T; Russell, Daniel A; Warner, Marcie H; Hatfull, Graham F

2017-08-15

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution. Copyright © 2017 Pope et al.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

PubMed

Szymczak, Paula; Janzen, Thomas; Neves, Ana Rute; Kot, Witold; Hansen, Lars H; Lametsch, René; Neve, Horst; Franz, Charles M A P; Vogensen, Finn K

2017-03-01

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos - or pac -type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos - or pac -type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos - or pac -type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis , extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed. IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations. Copyright © 2017 Szymczak et al.

Receptor Diversity and Host Interaction of Bacteriophages Infecting Salmonella enterica Serovar Typhimurium

PubMed Central

Kim, Hyeryen; Choi, Younho; Heu, Sunggi; Ryu, Sangryeol

2012-01-01

Background Salmonella enterica subspecies enterica serovar Typhimurium is a Gram-negative pathogen causing salmonellosis. Salmonella Typhimurium-targeting bacteriophages have been proposed as an alternative biocontrol agent to antibiotics. To further understand infection and interaction mechanisms between the host strains and the bacteriophages, the receptor diversity of these phages needs to be elucidated. Methodology/Principal Findings Twenty-five Salmonella phages were isolated and their receptors were identified by screening a Tn5 random mutant library of S. Typhimurium SL1344. Among them, three types of receptors were identified flagella (11 phages), vitamin B12 uptake outer membrane protein, BtuB (7 phages) and lipopolysaccharide-related O-antigen (7 phages). TEM observation revealed that the phages using flagella (group F) or BtuB (group B) as a receptor belong to Siphoviridae family, and the phages using O-antigen of LPS as a receptor (group L) belong to Podoviridae family. Interestingly, while some of group F phages (F-I) target FliC host receptor, others (F-II) target both FliC and FljB receptors, suggesting that two subgroups are present in group F phages. Cross-resistance assay of group B and L revealed that group L phages could not infect group B phage-resistant strains and reversely group B phages could not infect group L SPN9TCW-resistant strain. Conclusions/Significance In this report, three receptor groups of 25 newly isolated S. Typhimurium-targeting phages were determined. Among them, two subgroups of group F phages interact with their host receptors in different manner. In addition, the host receptors of group B or group L SPN9TCW phages hinder other group phage infection, probably due to interaction between receptors of their groups. This study provides novel insights into phage-host receptor interaction for Salmonella phages and will inform development of optimal phage therapy for protection against Salmonella. PMID:22927964

Therapeutic and prophylactic applications of bacteriophage components in modern medicine.

PubMed

Adhya, Sankar; Merril, Carl R; Biswas, Biswajit

2014-01-01

As the interactions of phage with mammalian innate and adaptive immune systems are better delineated and with our ability to recognize and eliminate toxins and other potentially harmful phage gene products, the potential of phage therapies is now being realized. Early efforts to use phage therapeutically were hampered by inadequate phage purification and limited knowledge of phage-bacterial and phage-human relations. However, although use of phage as an antibacterial therapy in countries that require controlled clinical studies has been hampered by the high costs of patient trials, their use as vaccines and the use of phage components such as lysolytic enzymes or lysozymes has progressed to the point of commercial applications. Recent studies concerning the intimate associations between mammalian hosts and bacterial and phage microbiomes should hasten this progress.

Current insights into phage biodiversity and biogeography.

PubMed

Thurber, Rebecca Vega

2009-10-01

Phages exert tremendous ecological and evolutionary forces directly on their bacterial hosts. Phage induced cell lysis also indirectly contributes to organic and inorganic nutrient recycling. Phage abundance, diversity, and distribution are therefore important parameters in ecosystem function. The assumption that phage consortia are ubiquitous and homogenous across habitats (everything is everywhere) is currently being re-evaluated. New studies on phage biogeography have found that some phages are globally distributed while others are unique and perhaps endemic to specific environments. Furthermore, advances in technology have allowed scientists to conduct experiments aimed at analyzing phage consortia over temporal scales, and surprisingly have found reoccurring patterns. This review discusses currents in the field of phage ecology with particular focus on efforts to characterize phage diversity and biogeography across various spatial and temporal scales.

20 CFR 30.310 - What must the claimant do if he or she objects to the recommended decision or wants to request a...

Code of Federal Regulations, 2010 CFR

2010-04-01

... exposed (if any), and whether a hearing is desired. This written statement should be filed with the FAB at... with the FAB, the statement will be considered to be “filed” on the date that the claimant mails it to the FAB, as determined by postmark, or on the date that such written statement is actually received by...

Regioselective chemical modification of monoclonal antibodies

DOEpatents

Ranadive, Girish; Rosenzweig, Howard S.; Epperly, Michael; Bloomer, William

1993-01-01

A method of selectively modifying an immunoglobulin having at least one Fab region and at least one Fc region, each region having an isoelectric point wherein said isoelectric point of the Fab fragment of said immunoglobulin is different than the isoelectric point of the Fc fragment of the immunoglobulin, said method comprising modification of the immunoglobulin at a pH between the respective isoelectric points of the Fab and Fc fragments of the immunoglobulin.

Fires Readiness: The State of US Army Fires in Support of Combined Arms Maneuver at the Division Level

DTIC Science & Technology

2016-06-10

proficiency in their core competencies. 15. SUBJECT TERMS Fires, Fire Support, DIVARTY, FAB , Combined Arms Maneuver, DOTMLPF 16. SECURITY...FAA Functional Area Analysis FAB Field Artillery Brigade FER Final Exercise Report FFA HQ Force Field Artillery Headquarters FM Field Manual FNA...function. This provided me the opportunity to work with several Division Artillerys (DIVARTYs) and Field Artillery Brigades ( FABs ). During this time

An Assessment of Early Competitive Prototyping for Major Defense Acquisition Programs

DTIC Science & Technology

2016-04-30

with 20/80 share ratio for EMD; CPFF for test execution. o Percent change in PAUC from development baseline. -2.3%. 3. FAB -T–FET. The Air Force’s...Family of Advanced Beyond Line-of-Sight Terminals ( FAB -T) provides for survivable terminals for communicating strategic nuclear execution orders via...jam-resistant, low probability of intercept waveforms through the Milstar and Advanced Extremely High Frequency (AEHF) satellite constellations. FAB

3D printing in social education: Eki-Fab and student PBL

NASA Astrophysics Data System (ADS)

Makino, Masato; Saito, Azusa; Kodama, Mai; Takamatsu, Kyuuichiro; Tamate, Hideaki; Sakai, Kazuyuki; Wada, Masato; Khosla, Ajit; Kawakami, Masaru; Furukawa, Hidemitsu

2017-04-01

Additive manufacturing or 3D printer is one of the most innovative material processing methods. We are considering that human resources for 3D printing would be needed in the future. To educate the abilities of the digital fabrication, we have the public digital fabrication space "Eki-Fab" for junior and high school students and Project Based Learning (PBL) class for undergraduate students. Eki-Fab is held on every Saturday at the Yonezawa train station. In the "Eki-Fab", anybody can study the utilizing of 3D printer and modeling technics under the instruction of staff in Yamagata University. In the PBL class, we have the class every Thursday. The students get the techniques of the digital fabrication through the PBL.

Surface antigen in early differentiation.

PubMed Central

Kemler, R; Babinet, C; Eisen, H; Jacob, F

1977-01-01

Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A. Images PMID:270688

Allosteric Modulation of protein oligomerization: an emerging approach to drug design

NASA Astrophysics Data System (ADS)

Gabizon, Ronen; Friedler, Assaf

2014-03-01

Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the "shiftides" approach developed in our lab.

Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

PubMed

Welschof, M; Little, M; Dörsam, H

1998-01-01

Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

Domain repertoires as a tool to derive protein recognition rules.

PubMed

Zucconi, A; Panni, S; Paoluzi, S; Castagnoli, L; Dente, L; Cesareni, G

2000-08-25

Several approaches, some of which are described in this issue, have been proposed to assemble a complete protein interaction map. These are often based on high throughput methods that explore the ability of each gene product to bind any other element of the proteome of the organism. Here we propose that a large number of interactions can be inferred by revealing the rules underlying recognition specificity of a small number (a few hundreds) of families of protein recognition modules. This can be achieved through the construction and characterization of domain repertoires. A domain repertoire is assembled in a combinatorial fashion by allowing each amino acid position in the binding site of a given protein recognition domain to vary to include all the residues allowed at that position in the domain family. The repertoire is then searched by phage display techniques with any target of interest and from the primary structure of the binding site of the selected domains one derives rules that are used to infer the formation of complexes between natural proteins in the cell.

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

PubMed Central

Krueger, A. P.; Scribner, E. J.; Brown, B. B.

1946-01-01

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]plaq., [extracellular phage]act., and [extracellular phage]plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück's objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out. PMID:19873475

Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

PubMed Central

Khan Mirzaei, Mohammadali; Nilsson, Anders S.

2015-01-01

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection. PMID:25761060

Diversity and geographical distribution of Flavobacterium psychrophilum isolates and their phages: patterns of susceptibility to phage infection and phage host range.

PubMed

Castillo, Daniel; Christiansen, Rói Hammershaimb; Espejo, Romilio; Middelboe, Mathias

2014-05-01

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that "enhanced infection" is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli.

PubMed

Weiss, Marietta; Denou, Emmanuel; Bruttin, Anne; Serra-Moreno, Ruth; Dillmann, Marie-Lise; Brüssow, Harald

2009-10-10

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

Whey powders are a rich source and excellent storage matrix for dairy bacteriophages.

PubMed

Wagner, Natalia; Brinks, Erik; Samtlebe, Meike; Hinrichs, Jörg; Atamer, Zeynep; Kot, Witold; Franz, Charles M A P; Neve, Horst; Heller, Knut J

2017-01-16

Thirteen whey powders and 5 whey powder formulations were screened for the presence of dairy bacteriophages using a representative set of 8 acid-producing Lactococcus lactis and 5 Streptococcus thermophilus, and 8 flavour-producing Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides strains. Lytic L. lactis phages were detected in all samples, while S. thermophilus and Leuconostoc phages were present in 50% or 40% of the samples, respectively. Maximal phage titers were 6×10 7 plaque-forming units (pfu)/g of whey powder for L. lactis phages, 1×10 7 pfu/g for Leuconostoc phages and 1×10 5 pfu/g for S. thermophilus phages. In total, 55 phages were isolated and characterized. Thirty one of the 33 lactococcal phages tested belonged to the wide-spread 936 phage group. In the course of this study, a PCR detection method for Leuconostoc phages (Ali et al., 2013) was adapted to new phage isolates. Furthermore, a remarkably high stability of phages in whey powder samples was documented during a long-term storage period of 4 years. Copyright © 2016 Elsevier B.V. All rights reserved.

In vivo growth rates are poorly correlated with phage therapy success in a mouse infection model.

PubMed

Bull, J J; Otto, G; Molineux, I J

2012-02-01

Two classes of phages yield profoundly different levels of recovery in mice experimentally infected with an Escherichia coli O18:K1:H7 strain. Phages requiring the K1 capsule for infection (K1-dep) rescue virtually all infected mice, whereas phages not requiring the capsule (K1-ind) rescue modest numbers (∼30%). To rescue infected mice, K1-ind phages require at least a 10(6)-fold-higher inoculum than K1-dep phages. Yet their in vivo growth dynamics are only modestly inferior to those of K1-dep phages, and competition between the two phage types in the same mouse reveals only a slight growth advantage for the K1-dep phage. The in vivo growth rate seems unlikely to be the primary determinant of phage therapy success. An alternative explanation is that the success of K1-dep phages is due substantially to their proteomic composition. They encode an enzyme that degrades the K1 capsule, which has been shown in other work to be sufficient to cure infection in the complete absence of phages.

Filamentous Phage: Structure and Biology.

PubMed

Rakonjac, Jasna; Russel, Marjorie; Khanum, Sofia; Brooke, Sam J; Rajič, Marina

2017-01-01

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE). This chapter will give an overview of the Ff filamentous phage structure and biology, emphasizing those properties of the Ff phage life cycle and virion that are pertinent to phage display applications.

Characterisation of a novel enterobacteria phage, CAjan, isolated from rat faeces.

PubMed

Carstens, Alexander B; Kot, Witold; Lametsch, Rene; Neve, Horst; Hansen, Lars H

2016-08-01

In this study, we describe the isolation and characterisation of the novel enterobacteria phage CAjan. This phage belongs to the order Caudovirales and the family Siphoviridae. The phage possesses a linear, double-stranded DNA genome consisting of 59,670 bp with a G+C content of 44.7 % and 91 predicted open reading frames (ORFs). Putative functions were assigned to 39 of the ORFs (37.4 %). The phage structural genes were furthermore functionally characterised by LC MS/MS. CAjan, together with Escherichia phage Seurat and Escherichia phage slur01, represent a novel and genetically distinct clade of Siphoviridae phages that could be considered to constitute a new phage genus. Despite limited sequence similarity, the phages in this group share a number of other common features, including genome structure and the presence of queuosine biosynthesis genes.

Bacteriophage Ecology in a Commercial Cucumber Fermentation

PubMed Central

Pérez-Díaz, I. M.; Hayes, J. S.; Breidt, F.

2012-01-01

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations. PMID:23023756

Bacteriophage of Haemophilus influenzae III. Morphology, DNA Homology, and Immunity Properties of HP1c1, S2, and the Defective Bacteriophage from Strain Rd

PubMed Central

Boling, Maxon E.; Allison, David P.; Setlow, Jane K.

1973-01-01

The phages HP1c1 and S2 and a defective phage of Haemophilus influenzae have been compared. The morphology of the phages and the mol wt of their DNAs are similar, although the defective phage appears to have a different tail plate region. Electron microscope observation indicates that the defective phage does not attach to the cell surface, and its DNA appears to lack cohesive ends. The homology of the DNAs of the phages has been measured by hydridization. DNA from the defective phage shows little or no homology with the other phage DNAs. HP1c1 and S2 DNAs show a high level of homology. Each of these phages can form plaques on lawns of the lysogen of the other phage but at reduced plating efficiencies, suggesting that the two phages have related but not identical immunity systems. Images PMID:4540713

Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection

PubMed Central

Carmody, Lisa A.; Gill, Jason J.; Summer, Elizabeth J.; Sajjan, Uma S.; Gonzalez, Carlos F.; Young, Ryland F.; LiPuma, John J.

2009-01-01

The therapeutic potential of bacteriophage (phage) in a mouse model of acute B. cenocepacia pulmonary infection was assessed. Phage were administered by either intranasal (i.n.) inhalation or intraperitoneal (i.p.) injection. Bacterial density, macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-α (TNFα) levels were significantly reduced in lungs of mice treated with i.p. phage. No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with i.n. phage, i.p. UV-inactivated phage, or i.p. λ phage controls. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNFα levels compared to mock-infected / mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic administration of phage was more effective than inhalational administration, suggesting that circulating phage have better access to bacteria in lung compared to topical phage. PMID:20001604

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries

PubMed Central

Noppe, Wim; Plieva, Fatima; Galaev, Igor Yu; Pottel, Hans; Deckmyn, Hans; Mattiasson, Bo

2009-01-01

Background Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. Results Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. Conclusion Chromato-panning allows combining several steps of the panning procedure resulting in 4–8 fold decrease of total time needed for phage selection. PMID:19292898

Structural Insights into Antibody Sequestering and Neutralizing of Na+ Channel α-Type Modulator from Old World Scorpion Venom

PubMed Central

Fabrichny, Igor P.; Mondielli, Grégoire; Conrod, Sandrine; Martin-Eauclaire, Marie-France; Bourne, Yves; Marchot, Pascale

2012-01-01

The Old World scorpion Androctonus australis hector (Aah) produces one of the most lethal venoms for humans. Peptidic α-toxins AahI to AahIV are responsible for its potency, with AahII accounting for half of it. All four toxins are high affinity blockers of the fast inactivation phase of mammalian voltage-activated Na+ channels. However, the high antigenic polymorphism of α-toxins prevents production of a polyvalent neutralizing antiserum, whereas the determinants dictating their trapping by neutralizing antibodies remain elusive. From an anti-AahII mAb, we generated an antigen binding fragment (Fab) with high affinity and selectivity for AahII and solved a 2.3 Å-resolution crystal structure of the complex. Sequestering of the C-terminal region of the bound toxin within a groove formed by the Fab combining loops is associated with a toxin orientation and main and side chain conformations that dictate the AahII antigenic specificity and efficient neutralization. From an anti-AahI mAb, we also preformed and crystallized a high affinity AahI-Fab complex. The 1.6 Å-resolution structure solved revealed a Fab molecule devoid of a bound AahI and with combining loops involved in packing interactions, denoting expulsion of the bound antigen upon crystal formation. Comparative analysis of the groove-like combining site of the toxin-bound anti-AahII Fab and planar combining surface of the unbound anti-AahI Fab along with complementary data from a flexible docking approach suggests occurrence of distinctive trapping orientations for the two toxins relative to their respective Fab. This study provides complementary templates for designing new molecules aimed at capturing Aah α-toxins and suitable for immunotherapy. PMID:22371498

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41.

PubMed

Hartono, Yossa Dwi; Lazim, Raudah; Yip, Yew Mun; Zhang, Dawei

2012-02-15

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Relevance of spontaneous fabT mutations to a streptococcal toxic shock syndrome to non-streptococcal toxic shock syndrome transition in the novel-type Streptococcus pyogenes isolates that lost a salRK.

PubMed

Tatsuno, Ichiro; Okada, Ryo; Matsumoto, Masakado; Hata, Nanako; Matsui, Hideyuki; Zhang, Yan; Isaka, Masanori; Hasegawa, Tadao

2016-05-01

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). Mutations in covR/S or rgg, negative regulators, can reportedly modulate the severity of infection in this pathogen. Recently, we showed that the regions encoding the SalR-SalK, a two-component regulatory system, were deleted in some emm 1-type isolates (named as 'novel-type'). In this study, the two novel 'STSS' isolates 10-85stss and 11-171stss were more virulent than the two novel 'non-STSS' isolates 11O-2non and 11T-3non when examined using a mouse model of invasive infection. Genome-sequencing experiments using the three strains 10-85stss , 11-171stss , and 11O-2non detected only one single nucleotide polymorphism that causes a non-synonymous mutation in fabT encoding a transcriptional regulator in strain 11O-2non . Loss of fabT reduced the high level of virulence observed in the STSS isolates to that in the non-STSS isolates, and introduction of an intact fabT compensated the lower virulence of 11O-2non , suggesting that the mutation in fabT, but not in covR/S or rgg, is involved in the differential virulence among the novel-type clinical isolates. This type of non-synonymous fabT mutation was also identified in 12 non-STSS isolates (including 11O-2non and 11T-3non ), and most of those 12 isolates showed impaired FabT function. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Reduced folate and serum vitamin metabolites in patients with rectal carcinoma: an open-label feasibility study of pemetrexed with folic acid and vitamin B12 supplementation

PubMed Central

Odin, Elisabeth A.; Carlsson, Göran U.; Kurlberg, Göran K.; Björkqvist, Hillevi G.; Tångefjord, Maria T.; Gustavsson, Bengt G.

2016-01-01

The objectives of this single-center, open-label, phase II study were to evaluate (a) the feasibility and safety of neoadjuvant administration of pemetrexed with oral folic acid and vitamin B12 (FA/B12) in newly diagnosed patients with resectable rectal cancer and (b) intracellular and systemic vitamin metabolism. Patients were treated with three cycles of pemetrexed (500 mg/m2, every 3 weeks) and FA/B12 before surgery. The reduced folates tetrahydrofolate, 5-methyltetrahydrofolate, and 5,10-methylenetetrahydrofolate were evaluated from biopsies in tumor tissue and in adjacent mucosa. Serum levels of homocysteine, cystathionine, and methylmalonic acid were also measured. All 37 patients received three cycles of pemetrexed; 89.2% completed their planned dosage within a 9-week feasibility time frame. Neither dose reductions nor study drug-related serious adverse events were reported. Reduced folate levels were significantly higher in tumor tissue compared with adjacent mucosa at baseline. After FA/B12 administration, tissue levels of reduced folates increased significantly and remained high during treatment in both tumor and mucosa until surgery. Serum levels of cystathionine increased significantly compared with baseline after FA/B12 administration, but then decreased, fluctuating cyclically during pemetrexed therapy. Homocysteine and methylmalonic acid levels decreased significantly after FA/B12 administration, and remained below baseline levels during the study. These results indicate that administration of three neoadjuvant cycles of single-agent pemetrexed, every 3 weeks, with FA/B12 in patients with resectable rectal cancer is feasible and tolerable. Tissue and serum vitamin metabolism results demonstrate the influence of pemetrexed and FA/B12 on vitamin metabolism and warrant further study. PMID:26825869

The Influence of Self-Efficacy, Fear-Avoidance Belief, and Coping Styles on Quality of Life for Chinese Patients with Chronic Nonspecific Low Back Pain: A Multisite Cross-Sectional Study.

PubMed

Du, Shizheng; Hu, Lingli; Bai, Yamei; Dong, Jianshu; Jin, Shengji; Zhang, Heng; Zhu, Ying

2017-11-23

As a bio-psycho-social issue, chronic low back pain (CLBP) has been a significant topic in health management, and patients' quality of life (QOL) is gaining extensive attention. Self-efficacy, pain fear-avoidance belief (FAB), and coping styles play important roles in the QOL of CLBP patients. However, it remains unclear how self-efficacy and FAB influence QOL through specific coping styles. This study aimed to explore the influencing paths of self-efficacy, FAB, and coping styles on the QOL of patients with CLBP. This study relies on a multisite, cross-sectional design involving 221 CLBP patients. Stepwise multiple regression and structural equation modeling were employed. CLBP patients lived with a poor global QOL. Self-efficacy played a direct, positive role in predicting QOL for patients with CLBP (β = 0.35), and it also played an indirect, positive role in predicting QOL (β = 0.19) through active coping styles (β = 0.31). FAB played a direct, negative role in predicting QOL (β = -0.33), and it also played an indirect, negative role in predicting QOL (β = -0.32) through passive coping styles (β = 0.32). Self-efficacy and FAB are both directly and indirectly related to global QOL, and coping styles are important mediating variables. Self-efficacy and active coping are protective factors for the QOL of CLBP patients, while FAB and passive coping are risk factors. Health education strategies are recommended by medical personnel to enhance CLBP patients' pain self-efficacy, decrease pain FAB, and modify pain coping styles, so that their global QOL can be improved. © 2017 World Institute of Pain.

QSAR models for prediction of chromatographic behavior of homologous Fab variants.

PubMed

Robinson, Julie R; Karkov, Hanne S; Woo, James A; Krogh, Berit O; Cramer, Steven M

2017-06-01

While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R 2  > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

A study on EUV reticle surface molecular contamination under different storage conditions in a HVM foundry fab

NASA Astrophysics Data System (ADS)

Singh, SherJang; Yatzor, Brett; Taylor, Ron; Wood, Obert; Mangat, Pawitter

2017-03-01

The prospect of EUVL (Extreme Ultraviolet Lithography) insertion into HVM (High Volume Manufacturing) has never been this promising. As technology is prepared for "lab to fab" transition, it becomes important to comprehend challenges associated with integrating EUVL infrastructure within existing high volume chip fabrication processes in a foundry fab. The existing 193nm optical lithography process flow for reticle handling and storage in a fab atmosphere is well established and in-fab reticle contamination concerns are mitigated with the reticle pellicle. However EUVL reticle pellicle is still under development and if available, may only provide protection against particles but not molecular contamination. HVM fab atmosphere is known to be contaminated with trace amounts of AMC's (Atmospheric Molecular Contamination). If such contaminants are organic in nature and get absorbed on the reticle surface, EUV photon cause photo-dissociation resulting into carbon generation which is known to reduce multilayer reflectivity and also degrades exposure uniformity. Chemical diffusion and aggregation of other ions is also reported under the e-beam exposure of a EUV reticle which is known to cause haze issues in optical lithography. Therefore it becomes paramount to mitigate absorbed molecular contaminant concerns on EUVL reticle surface. In this paper, we have studied types of molecular contaminants that are absorbed on an EUVL reticle surface under HVM fab storage and handling conditions. Effect of storage conditions (gas purged vs atmospheric) in different storage pods (Dual pods, Reticle Clamshells) is evaluated. Absorption analysis is done both on ruthenium capping layer as well as TaBN absorber. Ru surface chemistry change as a result of storage is also studied. The efficacy of different reticle cleaning processes to remove absorbed contaminant is evaluated as well.

Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

PubMed

Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

2014-02-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

PubMed Central

Kornberger, Petra; Skerra, Arne

2014-01-01

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

Characterization of Natronobacterium magadii phage phi Ch1, a unique archaeal phage containing DNA and RNA.

PubMed

Witte, A; Baranyi, U; Klein, R; Sulzner, M; Luo, C; Wanner, G; Krüger, D H; Lubitz, W

1997-02-01

A novel archaeal bacteriophage, phi Ch1, was isolated from a haloalkalophilic archaeon Natronobacterium magadii upon spontaneous lysis. The phage-cured strain N. magadii(L13) was used to demonstrate infectivity of phage phi Ch1. The turbid-plaque morphology and the fact that N. magadii cells isolated from plaques were able to produce phage indicated that phi Ch1 is a temperate phage. The phage morphology resembles other members of Myoviridae-infecting Halobacterium species. In solution below 2M NaCl, the phage lost its morphological stability and infectivity. One- and two-dimensional SDS-PAGE of phage particles revealed at least four major and five minor proteins with molecular masses ranging from 15 to 80 kDa and acidic isoelectric points. Southern blot analysis of chromosomal DNA of a lysogenic N. magadii strain showed that phi Ch1 exists as a chromosomally integrated prophage. The phage particles contain both double-stranded, linear DNA (approx. 55 kbp) as well as several RNA species (80-700 nucleotides). Hybridization of labelled RNA fragments to total DNA from N. magadii and phi Ch1 showed that the virion-associated RNA is host encoded. Part of the phage DNA population is modified and restriction analysis revealed evidence for adenine methylation. Phage phi Ch1 is the first virus described for the genus natronobacterium, and the first phage containing DNA and RNA in mature phage particles.

Citrulline-modified phage display: a novel high-throughput discovery approach for the identification of citrulline-containing ligands.

PubMed

Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2011-06-01

Phage display is a high-throughput technology used to identify ligands for a given target. A drawback of the approach is the absence of PTMs in phage-displayed peptides. The applicability of phage display could be broadened considerably by the implementation of PTMs in this system. The aim of this study was to investigate the possible application of citrullination, a PTM of an arginine into a citrulline amino acid, in filamentous (M13) and lytic (T7) phage display. After in vitro citrullination of T7 and M13 phages, citrullination was confirmed and the infectivity of both citrullinated and non-citrullinated phage was compared by titer determination. We demonstrated the successful in vitro citrullination of T7 and M13 phage-displayed peptides. This in vitro modification did not affect the viability or infectivity of the T7 virions, a necessary prerequisite for the implementation of this approach in T7 phage display. For M13 phage, however, the infecting phage titer decreased five-fold upon citrullination, limiting the use of this modification in M13 phage display. In conclusion, in vitro citrullination can be applied in T7 phage display giving rise to a high-throughput and sensitive approach to identify citrulline-containing ligands by the use of the strengths of phage display technology. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On The Influence Of Vector Design On Antibody Phage Display

PubMed Central

Soltes, Glenn; Hust, Michael; Ng, Kitty K.Y.; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I.H.; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-01

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection. PMID:16996161

On the influence of vector design on antibody phage display.

PubMed

Soltes, Glenn; Hust, Michael; Ng, Kitty K Y; Bansal, Aasthaa; Field, Johnathan; Stewart, Donald I H; Dübel, Stefan; Cha, Sanghoon; Wiersma, Erik J

2007-01-20

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.

Regioselective chemical modification of monoclonal antibodies

DOEpatents

Ranadive, G.; Rozenzweig, H.S.; Epperly, M.; Bloomer, W.

1993-05-04

A method is presented of selectively modifying an immunoglobulin having at least one Fab region and at least one Fc region. Each region has an isoelectric point where the isoelectric point of the Fab fragment of the immunoglobulin is different from the isoelectric point of the Fc fragment of the immunoglobulin. The method comprises of a modification of the immunoglobulin at a pH between the respective isoelectric points of the Fab and Fc fragments of the immunoglobulin.

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

PubMed Central

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; Ockenhouse, Christian F.; Emerling, Daniel; Glanville, Jacob; Volkmuth, Wayne; Flores-Garcia, Yevel; Zavala, Fidel; Ward, Andrew B.; King, C. Richter; Wilson, Ian A.

2017-01-01

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development in vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA)3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Together, these structural insights may aid in the design of a next-generation malaria vaccine. PMID:29138320

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE PAGES

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike; ...

2017-11-14

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oyen, David; Torres, Jonathan L.; Wille-Reece, Ulrike

Acquired resistance against antimalarial drugs has further increased the need for an effective malaria vaccine. The current leading candidate, RTS,S, is a recombinant circumsporozoite protein (CSP)-based vaccine against Plasmodium falciparum that contains 19 NANP repeats followed by a thrombospondin repeat domain. Although RTS,S has undergone extensive clinical testing and has progressed through phase III clinical trials, continued efforts are underway to enhance its efficacy and duration of protection. Here in this paper, we determined that two monoclonal antibodies (mAbs 311 and 317), isolated from a recent controlled human malaria infection trial exploring a delayed fractional dose, inhibit parasite development inmore » vivo by at least 97%. Crystal structures of antibody fragments (Fabs) 311 and 317 with an (NPNA) 3 peptide illustrate their different binding modes. Notwithstanding, one and three of the three NPNA repeats adopt similar well-defined type I β-turns with Fab311 and Fab317, respectively. Furthermore, to explore antibody binding in the context of P. falciparum CSP, we used negative-stain electron microscopy on a recombinant shortened CSP (rsCSP) construct saturated with Fabs. Both complexes display a compact rsCSP with multiple Fabs bound, with the rsCSP–Fab311 complex forming a highly organized helical structure. Lastly, together, these structural insights may aid in the design of a next-generation malaria vaccine.« less

Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography.

PubMed

Raweerith, Rutai; Ratanabanangkoon, Kavi

2003-11-01

A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

PubMed

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

PubMed

Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

2015-01-01

In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

Complex polar lipids of a hot spring cyanobacterial mat and its cultivated inhabitants

NASA Technical Reports Server (NTRS)

Ward, D. M.; Panke, S.; Kloppel, K. D.; Christ, R.; Fredrickson, H.

1994-01-01

The complex polar lipids of the hot spring cyanobacterial mat in the 50 to 55 degrees C region of Octopus Spring, Yellowstone National Park, and of thermophilic bacteria cultivated from this or similar habitats, were compared in an attempt to understand the microbial sources of the major lipid biomarkers in this community. Intact complex lipids were analyzed directly by fast atom bombardment mass spectrometry (FAB-MS), two-dimensional thin-layer chromatography (TLC), and combined TLC-FAB-MS. FAB-MS and TLC gave qualitatively similar results, suggesting that the mat contains major lipids most like those of the cyanobacterial isolate we studied, Synechococcus sp. strain Y-7c-s. These include monoglycosyl, diglycosyl, and sulfoquinosovyl diglycerides (MG, DG, and SQ, respectively) and phosphatidyl glycerol (PG). Though Chloroflexus aurantiacus also contains MG, DG, and PG, the fatty acid chain lengths of mat MGs, DGs, and PGs resemble more those of cyanobacterial than green nonsulfur bacterial lipids. FAB-MS spectra of the lipids of nonphototrophic bacterial isolates were distinctively different from those of the mat and phototrophic isolates. The lipids of these nonphototrophic isolates were not detected in the mat, but most could be detected when added to mat samples. The mat also contains major glycolipids and aminophospholipids of unknown structure and origin. FAB-MS and TLC did not always give quantitatively similar results. In particular, PG and SQ may give disproportionately high FAB-MS responses.

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

PubMed

Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

2015-09-01

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Improving 130nm node patterning using inverse lithography techniques for an analog process

NASA Astrophysics Data System (ADS)

Duan, Can; Jessen, Scott; Ziger, David; Watanabe, Mizuki; Prins, Steve; Ho, Chi-Chien; Shu, Jing

2018-03-01

Developing a new lithographic process routinely involves usage of lithographic toolsets and much engineering time to perform data analysis. Process transfers between fabs occur quite often. One of the key assumptions made is that lithographic settings are equivalent from one fab to another and that the transfer is fluid. In some cases, that is far from the truth. Differences in tools can change the proximity effect seen in low k1 imaging processes. If you use model based optical proximity correction (MBOPC), then a model built in one fab will not work under the same conditions at another fab. This results in many wafers being patterned to try and match a baseline response. Even if matching is achieved, there is no guarantee that optimal lithographic responses are met. In this paper, we discuss the approach used to transfer and develop new lithographic processes and define MBOPC builds for the new lithographic process in Fab B which was transferred from a similar lithographic process in Fab A. By using PROLITHTM simulations to match OPC models for each level, minimal downtime in wafer processing was observed. Source Mask Optimization (SMO) was also used to optimize lithographic processes using novel inverse lithography techniques (ILT) to simultaneously optimize mask bias, depth of focus (DOF), exposure latitude (EL) and mask error enhancement factor (MEEF) for critical designs for each level.

The MUT056399 Inhibitor of FabI Is a New Antistaphylococcal Compound▿

PubMed Central

Escaich, S.; Prouvensier, L.; Saccomani, M.; Durant, L.; Oxoby, M.; Gerusz, V.; Moreau, F.; Vongsouthi, V.; Maher, Kirsty; Morrissey, Ian; Soulama-Mouze, C.

2011-01-01

MUT056399 is a highly potent new inhibitor of the FabI enzyme of both Staphylococcus aureus and Escherichia coli. In vitro, MUT056399 was very active against S. aureus strains, including methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), linezolid-resistant, and multidrug-resistant strains, with MIC90s between 0.03 and 0.12 μg/ml. MUT056399 was also active against coagulase-negative staphylococci, with MIC90s between 0.12 and 4 μg/ml. The antibacterial spectrum is consistent with specific FabI inhibition with no activity against bacteria using FabK but activity against FabI-containing Gram-negative bacilli. In vitro, resistant clones of S. aureus were obtained at a low frequency. All of the resistant clones analyzed were found to contain mutations in the fabI gene. In vivo, MUT056399, administered subcutaneously, protected mice from a lethal systemic infection induced by MSSA, MRSA, and vancomycin-intermediate S. aureus strains (50% effective doses ranging from 19.3 mg/kg/day to 49.6 mg/kg/day). In the nonneutropenic murine thigh infection model, the same treatment with MUT056399 reduced the bacterial multiplication of MSSA and MRSA in the thighs of immunocompetent mice. These properties support MUT056399 as a very promising candidate for a novel drug to treat severe staphylococcal infections. PMID:21825292

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display.

PubMed

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-11-14

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.

A novel approach for separating bacteriophages from other bacteriophages using affinity chromatography and phage display

PubMed Central

Ceglarek, Izabela; Piotrowicz, Agnieszka; Lecion, Dorota; Miernikiewicz, Paulina; Owczarek, Barbara; Hodyra, Katarzyna; Harhala, Marek; Górski, Andrzej; Dąbrowska, Krystyna

2013-01-01

Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In ‘competitive phage display’ bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins. PMID:24225840

Marine phages as excellent tracers for reactive colloidal transport in porous media

NASA Astrophysics Data System (ADS)

Ghanem, Nawras; Chatzinotas, Antonis; Harms, Hauke; Wick, Lukas Y.

2016-04-01

Question: Here we evaluate marine phages as specific markers of hydrological flow and reactive transport of colloidal particles in the Earth's critical zone (CZ). Marine phages and their bacterial hosts are naturally absent in the CZ, and can be detected with extremely high sensitivity. In the framework of the DFG Collaborative Research Center AquaDiva, we asked the following questions: (1) Are marine phages useful specific markers of hydrological flow and reactive transport in porous media? and (2) Which phage properties are relevant drivers for the transport of marine phages in porous media? Methods: Seven marine phages from different families (as well two commonly used terrestrial phages) were selected based on their morphology, size and physico-chemical surface properties (surface charge and hydrophobicity). Phage properties were assessed by electron microscopy, dynamic light scattering and water contact angle analysis (CA). Sand-filled laboratory percolation columns were used to study transport. The breakthrough curves of the phages were analyzed using the clean bed filtration theory and the XDLVO theory of colloid stability, respectively. Phages were quantified by a modified high- throughput plaque assay and a culture-independent particle counting method approach. Results: Our data show that most marine tested phages exhibited highly variable transport rates and deposition efficiency, yet generally high colloidal stability and viability. We find that size, morphology and hydrophobicity are key factors shaping the transport efficiency of phages. Differing deposition efficiencies of the phages were also supported by calculated XDLVO interaction energy profile. Conclusion: Marine phages have a high potential for the use as sensitive tracers in terrestrial habitats with their surface properties playing a crucial role for their transport. Marine phages however, exhibit differences in their deposition efficiency depending on their morphology, hydrophobicity and availability.

Generation and characterization of high affinity humanized fab against hepatitis B surface antigen.

PubMed

Tiwari, Ashutosh; Dutta, Durgashree; Khanna, Navin; Acharya, Subrat K; Sinha, Subrata

2009-09-01

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

Numerical analysis on temperature field in single-wire flux-aided backing-submerged arc welding

NASA Astrophysics Data System (ADS)

Pu, Juan; Wu, Ming Fang; Pan, Haichao

2017-07-01

Single-wire flux-aided backing-submerged arc welding (FAB-SAW) technology has been widely used to weld thick steel plate due to its easy assembly and high heat input. The microstructure and property of welded joint are closely related to the thermal field of FAB-SAW process. In this research, the feature of thermal field for single-wire FAB-SAW was investigated. Based on the heat transfer mechanism, a three-dimensional transient model for thermal field was developed based on the influence of steel thickness, groove angle and ceramic backing. The temperature profile in single-wire FAB-SAW of D36 steel under different welding conditions was simulated by ANSYS. The characteristic of thermal field was analyzed and the influences of groove angle on temperature field for different plate thicknesses were discussed. The calculated geometries and dimensions of weld cross-section under different conditions show a good agreement with the experimental results. This newly built model can describe the thermal field accurately, which would be helpful to understanding the thermophysical mechanism of FAB-SAW and optimizing the welding process.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esser, Lothar; Shukla, Suneet; Zhou, Fei

P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays tomore » 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.« less

Novel phage group infecting Lactobacillus delbrueckii subsp. lactis, as revealed by genomic and proteomic analysis of bacteriophage Ldl1.

PubMed

Casey, Eoghan; Mahony, Jennifer; Neve, Horst; Noben, Jean-Paul; Dal Bello, Fabio; van Sinderen, Douwe

2015-02-01

Ldl1 is a virulent phage infecting the dairy starter Lactobacillus delbrueckii subsp. lactis LdlS. Electron microscopy analysis revealed that this phage exhibits a large head and a long tail and bears little resemblance to other characterized phages infecting Lactobacillus delbrueckii. In vitro propagation of this phage revealed a latent period of 30 to 40 min and a burst size of 59.9 +/- 1.9 phage particles. Comparative genomic and proteomic analyses showed remarkable similarity between the genome of Ldl1 and that of Lactobacillus plantarum phage ATCC 8014-B2. The genomic and proteomic characteristics of Ldl1 demonstrate that this phage does not belong to any of the four previously recognized L. delbrueckii phage groups, necessitating the creation of a new group, called group e, thus adding to the knowledge on the diversity of phages targeting strains of this industrially important lactic acid bacterial species.

Phage Therapy Approaches to Reducing Pathogen Persistence and Transmission in Animal Production Environments: Opportunities and Challenges.

PubMed

Colavecchio, Anna; Goodridge, Lawrence D

2017-06-01

The era of genomics has allowed for characterization of phages for use as antimicrobials to treat animal infections with a level of precision never before realized. As more research in phage therapy has been conducted, several advantages of phage therapy have been realized, including the ubiquitous nature, specificity, prevalence in the biosphere, and low inherent toxicity of phages, which makes them a safe and sustainable technology for control of animal diseases. These unique qualities of phages have led to several opportunities with respect to emerging trends in infectious disease treatment. However, the opportunities are tempered by several challenges to the successful implementation of phage therapy, such as the fact that an individual phage can only infect one or a few bacterial strains, meaning that large numbers of different phages will likely be needed to treat infections caused by multiple species of bacteria. In addition, phages are only effective if enough of them can reach the site of bacterial colonization, but clearance by the immune system upon introduction to the animal is a reality that must be overcome. Finally, bacterial resistance to the phages may develop, resulting in treatment failure. Even a successful phage infection and lysis of its host has consequences, because large amounts of endotoxin are released upon lysis of Gram-negative bacteria, which can lead to local and systemic complications. Overcoming these challenges will require careful design and development of phage cocktails, including comprehensive characterization of phage host range and assessment of immunological risks associated with phage treatment.

Twelve previously unknown phage genera are ubiquitous in global oceans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh B

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in unknowns dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four wellknown viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria.more » Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage host systems for experimental hypothesis testing.« less

Somatostatin displayed on filamentous phage as a receptor-specific agonist

PubMed Central

Rousch, Mat; Lutgerink, Jan T; Coote, James; de Bruïne, Adriaan; Arends, Jan-Willem; Hoogenboom, Hennie R

1998-01-01

In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand. PMID:9776337

Twelve previously unknown phage genera are ubiquitous in global oceans.

PubMed

Holmfeldt, Karin; Solonenko, Natalie; Shah, Manesh; Corrier, Kristen; Riemann, Lasse; Verberkmoes, Nathan C; Sullivan, Matthew B

2013-07-30

Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in "unknowns" dominating culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage-host systems for experimental hypothesis testing.

64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

PubMed

Kwon, Luke Yongkyu; Scollard, Deborah A; Reilly, Raymond M

2017-02-06

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64 Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG 24 ) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFR mod /HER2 low ), MDA-MB-468 (EGFR high /HER2 neg ), MDA-MB-231-H2N (EGFR mod /HER2 mod ), and SKOV3 (EGFR low /HER2 high ) cells by competition and saturation cell binding assays to estimate the dissociation constant (K d ). The elimination of the 64 Cu-NOTA-trastuzumab Fab-PEG 24 -EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64 Cu-NOTA-trastuzumab Fab and 64 Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64 Cu-labeled bsRICs incorporating the PEG 24 spacer were eliminated more slowly from the blood than 64 Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64 Cu-NOTA-Fab or 64 Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly greater tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF (4.9 ± 0.4%ID/g) than 64 Cu-NOTA-Fab (1.9 ± 0.3%ID/g; P < 0.0001) and 64 Cu-NOTA-EGF (0.7 ± 0.2%ID/g; P < 0.0001). Furthermore, preadministration of an excess of trastuzumab Fab or trastuzumab Fab-PEG 24 -EGF significantly decreased the tumor uptake of 64 Cu-NOTA-Fab-PEG 24 -EGF in SK-OV-3 and MDA-MB-468 xenografts by 4.4-fold (P = 0.0012) and 1.8-fold (P = 0.0031), respectively. 64 Cu-labeled bsRICs bound HER2 or EGFR and were taken up specifically in vivo in tumor xenografts expressing one or both receptors. The PEG 24 linker prolonged the blood residence time contributing to the higher tumor uptake of the bsRICs than monospecific agents.

Safety analysis of a Russian phage cocktail: From MetaGenomic analysis to oral application in healthy human subjects

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCallin, Shawna, E-mail: semccallin@yahoo.com; Alam Sarker, Shafiqul, E-mail: sasarker@icddrb.org; Barretto, Caroline, E-mail: Caroline.Barretto@rdls.nestle.com

Phage therapy has a long tradition in Eastern Europe, where preparations are comprised of complex phage cocktails whose compositions have not been described. We investigated the composition of a phage cocktail from the Russian pharmaceutical company Microgen targeting Escherichia coli/Proteus infections. Electron microscopy identified six phage types, with numerically T7-like phages dominating over T4-like phages. A metagenomic approach using taxonomical classification, reference mapping and de novo assembly identified 18 distinct phage types, including 7 genera of Podoviridae, 2 established and 2 proposed genera of Myoviridae, and 2 genera of Siphoviridae. De novo assembly yielded 7 contigs greater than 30 kb,more » including a 147-kb Myovirus genome and a 42-kb genome of a potentially new phage. Bioinformatic analysis did not reveal undesired genes and a small human volunteer trial did not associate adverse effects with oral phage exposure. - Highlights: • We analyzed the composition of a commercial Russian phage cocktail. • The cocktail consists of at least 10 different phage genera. • No undesired genes were detected. • No adverse effects were seen upon oral application in a small human clinical trial.« less

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage.

PubMed

Brockmann, Eeva-Christine; Lamminmäki, Urpo; Saviranta, Petri

2005-06-20

Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.

A simple and rapid method to isolate purer M13 phage by isoelectric precipitation.

PubMed

Dong, Dexian; Sutaria, Sanjana; Hwangbo, Je Yeol; Chen, P

2013-09-01

M13 virus (phage) has been extensively used in phage display technology and nanomaterial templating. Our research aimed to use M13 phage to template sulfur nanoparticles for making lithium ion batteries. Traditional methods for harvesting M13 phage from Escherichia coli employ polyethylene glycol (PEG)-based precipitation, and the yield is usually measured by plaque counting. With this method, PEG residue is present in the M13 phage pellet and is difficult to eliminate. To resolve this issue, a method based on isoelectric precipitation was introduced and tested. The isoelectric method resulted in the production of purer phage with a higher yield, compared to the traditional PEG-based method. There is no significant variation in infectivity of the phage prepared using isoelectric precipitation, and the dynamic light scattering data indirectly prove that the phage structure is not damaged by pH adjustment. To maximize phage production, a dry-weight yield curve of M13 phage for various culture times was produced. The yield curve is proportional to the growth curve of E. coli. On a 200-mL culture scale, 0.2 g L(-1) M13 phage (dry-weight) was produced by the isoelectric precipitation method.

Lytic and inhibition responses to bacteriophages among marine bacteria, with special reference to the origin of phage-host systems

NASA Astrophysics Data System (ADS)

Moebus, K.

1983-12-01

The results of phage-host cross-reaction tests reported by Moebus & Nattkemper (1981) were re-examined using serially diluted bacteriophage suspensions to elicit the actual type of reaction between the bacteria and phage lysates tested. More than 1450 phage-host systems were studied at 25 °C incubation temperature. Among the nearly 300 phage strains used, 29 were identified as temperate ones. In about 65 % of the phage-host systems bacteriophage propagation was indicated by plaque formation. The remaining systems were characterized by the “inhibition” reaction of bacteria to phage lysates indicated by homogenously reduced bacterial growth within the test area without production of progeny phages. Since crude phage lysates had to be used, it remains obscure whether agents other than infective phage particles (defective ones or bacteriocins) caused this reaction. Among 269 systems of the inhibition type which were also tested at 5° and 15 °C, 54 were observed to propagate phages at one of or both the lower temperatures. Plaques produced at 15 °C with several phage-host systems were found to yield only few progeny phages which generally could not be propagated to produce high-titer phage stocks. With one system temperature-sensitive phage mutants were isolated. The probability of inhibition reactions occurring was found to be higher with phage-host systems isolated east of the Azores than with systems derived from the western Atlantic. With systems from the last mentioned area the proportion of inhibition versus lytic responses of bacteria to phages was observed to increase with the distance between the stations where both parts of the systems were derived. The latter findings are discussed in view of the assumption that bacterial and bacteriophage populations undergo genetic changes while being transported from west to east.

EFFECT OF SODIUM CHLORIDE ON STAPHYLOCOCCUS-PHAGE RELATIONSHIPS

PubMed Central

West, B.; Kelly, Florene C.; Shields, Doris A.

1963-01-01

West, B. (University of Oklahoma Medical Center, Oklahoma City), Florene C. Kelly, and Doris A. Shields. Effect of sodium chloride on staphylococcus-phage relationships. J. Bacteriol. 86:773–780. 1963.—Phage patterns of 21 phage-propagating strains of staphylococci on medium with high NaCl content appeared to be an expression of the staphylococcal cells, as well as of the salt tolerance of the phages. Serological group A phages, previously found to be NaCl-tolerant in the free state, were capable of lysing susceptible staphylococci on 3, 7.5, and 10% NaCl Trypticase Soy Agar. None of the other phages tested was active when the medium contained 7.5 and 10% NaCl. Increasing the NaCl content of the medium rarely resulted in nonspecific reactions; rather the effect was, generally, a narrowing of the phage spectrum of the cells, with persistence in the phage pattern of the phage, or phages, which were propagated on the cells being tested. Although NaCl tolerance of the phages was the chief limiting factor of phage activity in the presence of 7.5 and 10% NaCl, reactions on salt medium also depended on the degree of susceptibility of cells to phage on routine typing medium and to certain other unexplained factors. In some instances, under the influence of increased NaCl, significant lysis at 1000 RTD was replaced by thinning of growth (inhibition), with or without the presence of plaques. Conversely, certain phage-cell combinations, which gave inhibition at 1000 RTD on standard medium produced some degree of lysis when the NaCl concentration was increased. Studies of phage 81 and its propagating strain showed that replication of phage occurred in 10% NaCl medium, although adsorption diminished as salt concentration was increased, and the time required to reach maximal lytic activity was delayed. PMID:14066474

Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics

PubMed Central

Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Owczarek, Barbara; Międzybrodzki, Ryszard; Łusiak-Szelachowska, Marzanna; Łodej, Norbert; Górski, Andrzej

2017-01-01

Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes—contained in granules—that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy. The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy. PMID:28613272

Phage-Phagocyte Interactions and Their Implications for Phage Application as Therapeutics.

PubMed

Jończyk-Matysiak, Ewa; Weber-Dąbrowska, Beata; Owczarek, Barbara; Międzybrodzki, Ryszard; Łusiak-Szelachowska, Marzanna; Łodej, Norbert; Górski, Andrzej

2017-06-14

Phagocytes are the main component of innate immunity. They remove pathogens and particles from organisms using their bactericidal tools in the form of both reactive oxygen species and degrading enzymes-contained in granules-that are potentially toxic proteins. Therefore, it is important to investigate the possible interactions between phages and immune cells and avoid any phage side effects on them. Recent progress in knowledge concerning the influence of phages on phagocytes is also important as such interactions may shape the immune response. In this review we have summarized the current knowledge on phage interactions with phagocytes described so far and their potential implications for phage therapy . The data suggesting that phage do not downregulate important phagocyte functions are especially relevant for the concept of phage therapy.

Free Shiga toxin 1-encoding bacteriophages are less prevalent than Shiga toxin 2 phages in extraintestinal environments.

PubMed

Grau-Leal, Ferran; Quirós, Pablo; Martínez-Castillo, Alexandre; Muniesa, Maite

2015-11-01

Stx bacteriophages are involved in the pathogenicity of Stx-producing Escherichia coli. Induction of the Stx phage lytic cycle increases Stx expression and releases Stx phages that reach extracellular environments. Stx phage family comprises different phages that harbour any stx subtype. Stx2 is closely related with severe disease and therefore previous studies focused on free Stx2 phages in extraintestinal environments. To provide similar information regarding Stx1 phages, we evaluate free Stx1 phages in 357 samples of human and animal wastewater, faeces, river water, soil, sludge and food. Our method, based on quantification of stx1 in the DNA from the viral fraction, was validated using electron microscopy counting of phages and infectivity. The overall prevalence of Stx1 phages was very low: 7.6% of positive samples and values below 3 × 10(3) GC (gene copies) ml(-1) . These results contrast starkly with the abundance of Stx2 phages in the samples (68.4%). This environmental scarcity of free Stx1 phages is attributed to their lower rates of induction and the fact that Stx1 does not require phage induction to be expressed because it possesses an independent promoter. The implications of the low prevalence of free Stx1 phages for the emergence of new pathogenic strains in the environment are discussed. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine.

PubMed

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-06-21

Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (∼900 nm long and ∼8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic-inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials.

Phage as a Genetically Modifiable Supramacromolecule in Chemistry, Materials and Medicine

PubMed Central

Cao, Binrui; Yang, Mingying; Mao, Chuanbin

2016-01-01

CONSPECTUS Filamentous bacteriophage (phage) is a genetically modifiable supramacromolecule. It can be pictured as a semiflexible nanofiber (~900 nm long and ~8 nm wide) made of a DNA core and a protein shell with the former genetically encoding the latter. Although phage bioengineering and phage display techniques were developed before the 1990s, these techniques have not been widely used for chemistry, materials, and biomedical research from the perspective of supramolecular chemistry until recently. Powered by our expertise in displaying a foreign peptide on its surface through engineering phage DNA, we have employed phage to identify target-specific peptides, construct novel organic–inorganic nanohybrids, develop biomaterials for disease treatment, and generate bioanalytical methods for disease diagnosis. Compared with conventional biomimetic chemistry, phage-based supramolecular chemistry represents a new frontier in chemistry, materials science, and medicine. In this Account, we introduce our recent successful efforts in phage-based supramolecular chemistry, by integrating the unique nanofiber-like phage structure and powerful peptide display techniques into the fields of chemistry, materials science, and medicine: (1) successfully synthesized and assembled silica, hydroxyapatite, and gold nanoparticles using phage templates to form novel functional materials; (2) chemically introduced azo units onto the phage to form photoresponsive functional azo-phage nanofibers via a diazotization reaction between aromatic amino groups and the tyrosine residues genetically displayed on phage surfaces; (3) assembled phage into 2D films for studying the effects of both biochemical (the peptide sequences displayed on the phages) and biophysical (the topographies of the phage films) cues on the proliferation and differentiation of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) and identified peptides and topographies that can induce their osteogenic differentiation; (4) discovered that phage could induce angiogenesis and osteogenesis for MSC-based vascularized bone regeneration; (5) identified novel breast cancer cell-targeting and MSC-targeting peptides and used them to significantly improve the efficiency of targeted cancer therapy and MSC-based gene delivery, respectively; (6) employed engineered phage as a probe to achieve ultrasensitive detection of biomarkers from serum of human patients for disease diagnosis; and (7) constructed centimeter-scale 3D multilayered phage assemblies with the potential application as scaffolds for bone regeneration and functional device fabrication. Our findings demonstrated that phage is indeed a very powerful supramacromolecule suitable for not only developing novel nanostructures and biomaterials but also advancing important fields in biomedicine, including molecular targeting, cancer diagnosis and treatment, drug and gene delivery, stem cell fate direction, and tissue regeneration. Our successes in exploiting phage in chemistry, materials, and medicine suggest that phage itself is nontoxic at the cell level and can be safely used for detecting biomarkers in vitro. Moreover, although we have demonstrated successful in vivo tissue regeneration induced by phage, we believe future studies are needed to evaluate the in vivo biodistribution and potential risks of the phage-based biomaterials. PMID:27153341

Effectiveness of delayed use of crotalidae polyvalent immune Fab (ovine) antivenom.

PubMed

Bebarta, Vikhyat; Dart, Richard C

2004-01-01

Traditionally, horse-serum-based antivenom has been used in the United States for North American crotaline snake evenomation. Crotalidae polyvalent immune Fab (ovine) was approved in 2000 for use in mild to moderate envenomations. The manufacture recommends use within 6 h of envenomation. Published postmarketing retrospective reports describe its use up to 9 h after envenomation. We describe a case of effective use of FabAV 52 h after envenomation with resultant correction of coagulopathy and mild improvement of local symptoms.

Novel Therapeutic Approaches Targeting MDSC in Myelodysplastic Syndrome

DTIC Science & Technology

2017-09-01

anti-Fc Fab fragment antibody for half hour on ice before culture for 48 hours at which point total RNA was collected for gene expression analysis...MDS BMMNCs (Figure 3A). We then cross-linked BI 836858 or CD33Ab with a respective anti- Fab antibody on ice for 30 minutes followed by 24 hour...or CD33Ab with a respective anti- Fab antibody on ice for 30 min followed by 24 h culture to mimic ligand binding to the receptor. We found that cross