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Sample records for antibody variants produced

  1. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    PubMed

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

  2. Characterization and identification of alanine to serine sequence variants in an IgG4 monoclonal antibody produced in mammalian cell lines.

    PubMed

    Fu, Jinmei; Bongers, Jacob; Tao, Li; Huang, Dan; Ludwig, Richard; Huang, Yunping; Qian, Yueming; Basch, Jonathan; Goldstein, Joel; Krishnan, Ramji; You, Li; Li, Zheng Jian; Russell, Reb J

    2012-11-01

    Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Heterologous expressed toxic and non-toxic peptide variants of toxin CssII are capable to produce neutralizing antibodies against the venom of the scorpion Centruroides suffusus suffusus.

    PubMed

    Hernández-Salgado, Kenya; Estrada, Georgina; Olvera, Alejandro; Coronas, Fredy I; Possani, Lourival D; Corzo, Gerardo

    2009-08-15

    Two toxic and one non-toxic recombinant peptide variants of the mammalian neurotoxin CssII was cloned into the expression vector pQE30 containing a 6His-tag and a Factor Xa proteolytic cleavage site. The toxic recombinant peptides rCssII, HisrCssII and the non-toxic rCssIIE15R were expressed under induction with isopropyl thiogalactoside (IPTG), isolated using chromatographic techniques and folded correctly in vitro. The three recombinant variants showed similar secondary structures as the native CssII, but only the rCssIIE15R was not toxic to mice at concentrations up to 30microg/20g mouse body weight when injected intraperitoneally. All three recombinant peptides were capable of displacing the native CssII from their receptor sites in rat brain synaptosomes, suggesting that they had similar structural and functional characteristics of the native peptides. The three recombinant variants of CssII and the native one were used as antigens for immunization of New Zealand rabbits. The antibodies present in the rabbit antisera were able to recognize the native CssII. Additionally and more importantly, the sera of the immunized rabbits were able to neutralize both the native toxin CssII and the whole soluble venom of the scorpion Centruroides suffusus suffusus. These results indicate that the recombinant peptides can be used to produce antidotes against the venom of this species of scorpion.

  4. Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

    PubMed

    Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

    2016-07-01

    Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars.

  5. Equine monoclonal antibodies recognize common epitopes on variants of equine infectious anaemia virus.

    PubMed Central

    Perryman, L E; O'Rourke, K I; Mason, P H; McGuire, T C

    1990-01-01

    Equine-murine xenohybridoma cells were produced using SP2/0 murine myeloma cells and splenic lymph node cells obtained from horses infected with 10(6) TCID50 of single cloned variants of equine infectious anaemia virus (EIAV). The xenohybridomas secreted equine IgG monoclonal antibodies reactive with EIAV in enzyme immunoassays employing purified virus. Seven antibodies were studied in detail. They bound to viral glycoproteins (gp90 or gp45) in radioimmunoprecipitation assays, and reacted with homologous EIAV as well as five other cloned variants of EIAV. When evaluated against a single cloned variant of EIAV (EIAV-WSU5), two antibodies bound to different epitopes on gp90. The five remaining antibodies reacted with the same or overlapping epitopes on gp45. None of the antibodies exhibited viral neutralizing activity. Images Figure 2 PMID:1703988

  6. Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies.

    PubMed

    Leon, Paul E; Wohlbold, Teddy John; He, Wenqian; Bailey, Mark J; Henry, Carole J; Wilson, Patrick C; Krammer, Florian; Tan, Gene S

    2017-08-29

    Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

  7. Glycosylation of plant produced human antibodies.

    PubMed

    Kallolimath, Somanath; Steinkellner, Herta

    2015-12-23

    Human immunoglobulins circulate as highly heterogeneously glycosylated mixture of otherwise homogeneous protein backbones. A series of studies, mainly on IgG, have unequivocally proven that antibodies modulate their effector function through sugars present in the Fc domain. However, our limited technology in producing complex proteins such as antibodies, with defined glycan structures hamper in depths studies. This review introduces a plant based expression platform enabling engineering of antibody glycans. The procedure is based on the simultaneous delivery of appropriate constructs, carrying cDNAs of target proteins (e.g. heavy and light chain of antibodies) in combination with human glycosylation enzymes into plant leaves. Harvesting of recombinant proteins one week post construct delivery allows high speed and flexibility. Major achievements include the production of functional active slialylated pentameric IgMs in tobacco leaves. The system provides a viable approach to the generation of antibodies with defined glycoforms on demand, contributing to studies on antibody glycans and the development of novel antibody based drugs.

  8. Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

    PubMed Central

    Hintersteiner, Beate; Lingg, Nico; Zhang, Peiqing; Woen, Susanto; Hoi, Kong Meng; Stranner, Stefan; Wiederkum, Susanne; Mutschlechner, Oliver; Schuster, Manfred; Loibner, Hans; Jungbauer, Alois

    2016-01-01

    ABSTRACT We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.1,2,3 We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted. PMID:27559765

  9. Functional antibodies produced by oncolytic clostridia.

    PubMed

    Groot, Arjan J; Mengesha, Asferd; van der Wall, Elsken; van Diest, Paul J; Theys, Jan; Vooijs, Marc

    2007-12-28

    Hypoxia is a hallmark of solid cancer and characterized by regions of low oxygen and necrosis due to insufficient blood perfusion. Intratumoral hypoxia triggers the transcription of genes responsible for cell survival. The transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) is a key regulator of this response. HIF activation is associated with resistance to radio- and chemotherapy and poor clinical outcome, and may therefore provide an attractive therapeutic target. Clostridium-based oncolysis is a promising therapeutic strategy for the treatment of hypoxic tumors where these microorganisms naturally home. Here, we report for the first time the isolation of transconjugants of two excellent tumor colonizing Clostridium strains, C. novyi-NT and C. sporogenes, expressing single chain antibodies specific for human HIF-1alpha. This is a first step towards Clostridium-directed antibody therapy (CDAT) that holds promise as a carrier of cancer therapeutics targeting the most resistant regions in human solid cancer.

  10. Isolation and Characterization of Monoclonal Antibody Charge Variants by Free Flow Isoelectric Focusing.

    PubMed

    Hosken, Brian D; Li, Charlene; Mullappally, Berny; Co, Carl; Zhang, Boyan

    2016-06-07

    Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.

  11. Considerations in producing preferentially reduced half-antibody fragments.

    PubMed

    Makaraviciute, Asta; Jackson, Carolyn D; Millner, Paul A; Ramanaviciene, Almira

    2016-02-01

    Half-antibody fragments are a promising reagent for biosensing, drug-delivery and labeling applications, since exposure of the free thiol group in the Fc hinge region allows oriented reaction. Despite the structural variations among the molecules of different IgG subclasses and those obtained from different hosts, only generalized preferential antibody reduction protocols are currently available. Preferential reduction of polyclonal sheep anti-digoxin, rabbit anti-Escherichia coli and anti-myoglobin class IgG antibodies to half-antibody fragments has been investigated. A mild reductant 2-mercaptoethylamine (2-MEA) and a slightly stronger reductant tris(2-carboxyethyl)phosphine (TCEP) were used and the fragments obtained were quantitatively determined by SDS-PAGE analysis. It has been shown that the yields of half-antibody fragments could be increased by lowering the pH of the reduction mixtures. However, antibody susceptibility to the reductants varied. At pH4.5 the highest yield of sheep anti-digoxin IgG half-antibody fragments was obtained with 1M 2-MEA. Conversely, rabbit IgG half-antibody fragments could only be obtained with the stronger reductant TCEP. Preferential reduction of rabbit anti-myoglobin IgG antibodies was optimized and the highest half-antibody yield was obtained with 35 mM TCEP. Finally, it has been demonstrated that produced anti-myoglobin half-IgG fragments retained their binding activity.

  12. Studies on the Transfer of Antibody Producing Capacity

    PubMed Central

    Nossal, G. J. V.

    1959-01-01

    Normal adult spleen cells were antigenically stimulated by incubation with isolated flagella from Salmonella bacteria, and after neutralization of free antigen and washing, were transferred to normal and sublethally X-irradiated mice and rats of various ages by intraperitoneal injection. Neonatal rats and Hall Institute mice could not produce antibody following such injections, but more mature rats and Hall Institute mice, and neonatal C3H mice, could produce antibody. Cells from immunized spleens were secondarily stimulated in vitro and transferred to neonatal and more mature recipients. Regardless of the age of the recipients, antibody production typical of a secondary response ensued. Cells from neonatal rat spleens were stimulated in vitro and transferred to more mature recipient rats. Some antibody production resulted. The theoretical implications of these findings are discussed. PMID:13653734

  13. Vaccination with peptide mimotopes produces antibodies recognizing bacterial capsular polysaccharides.

    PubMed

    Wu, Yang; Zhang, Qibo; Sales, Debra; Bianco, Albert Edward; Craig, Alister

    2010-09-07

    A phage display peptide library was screened using a panel of antibodies to the capsular polysaccharides of Streptococcus agalactiae and Neisseria meningitidis. Mimotopes NPDHPRVPTFMA (2-8), LIPFHKHPHHRG (3-2) and EQEIFTNITDRV (G3) showing the highest binding capacity and strongest ELISA reaction were selected for immunization experiments. These mimotopes were either synthesised as oligodeoxynucleotides for DNA immunization or MAP (multiple antigen peptide) for peptide immunization. Mimotope-DNA vaccination, particularly for G3, induced antibodies recognizing a number of target bacteria. This response was seen after the second boost injection and was significantly enhanced by the 3rd boost injection with a Th1-associated profile, which was dominated by IgG2a, followed by IgG1. Mimotope-MAP immunization also produced strong humoral immune responses to the bacteria. Antibodies from G3 DNA immunization reacted with the surface molecules of S. agalactiae, N. meningitidis and Escherichia coli K5 shown by indirect immunofluorescence staining, indicating a possible localization to the bacterial capsule. Antibodies produced both from DNA/MAP immunization reacted with purified bacterial capsular polysaccharides by ELISA and were of high avidity. We have further characterized peptide G3 by a 'tiling path' study to examine the effect of changing individual residues in the peptide in raising antibodies, which showed that the EIFTN motif in G3 was important in generating antibodies to several capsulated bacteria. We conclude that mimotope immunization with DNA or MAP potentially induces strong antibody responses against encapsulated bacteria. It is suggested that the antibody targets are polysaccharides, and these antibodies may cross react at least among closely related species of bacteria.

  14. NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana.

    PubMed

    Yagi, Hirokazu; Fukuzawa, Noriho; Tasaka, Yasushi; Matsuo, Kouki; Zhang, Ying; Yamaguchi, Takumi; Kondo, Sachiko; Nakazawa, Shiori; Hashii, Noritaka; Kawasaki, Nana; Matsumura, Takeshi; Kato, Koichi

    2015-06-01

    We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a (15)N-enriched plant growth medium. The Fc fragment derived from the (15)N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

  15. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    PubMed

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  16. Detection of Food Allergens by Phage-Displayed Produced Antibodies.

    PubMed

    Madrid, Raquel; de la Cruz, Silvia; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

    2017-01-01

    Phage display is a powerful tool to produce recombinant antibodies against a given antigen without animal immunization. This technology employs libraries of recombinant bacteriophages that display billions of different functional antibody fragments on their surface. They are selected by panning in vitro against the target antigen in search for specific binders. In this chapter, we describe the selection of single chain variable fragment (scFv) antibodies to be used for detection of allergenic proteins from nuts in food products. The artificial libraries TomLinson I+J (MRC Laboratory of Molecular Biology and MRC Centre for Protein Engineering) were employed that resulted in successful phage-ELISA systems for detection of almond and walnut proteins in commercial food products.

  17. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  18. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

    PubMed Central

    Yin, Gang; Garces, Eudean D; Yang, Junhao; Zhang, Juan; Tran, Cuong; Steiner, Alexander R; Roos, Christine; Bajad, Sunil; Hudak, Susan; Penta, Kalyani; Zawada, James; Pollitt, Sonia

    2012-01-01

    We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics. PMID:22377750

  19. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  20. Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

    PubMed Central

    Li, Yantao; Fu, Tuo; Liu, Tao; Guo, Huaizu; Guo, Qingcheng; Xu, Jin; Zhang, Dapeng; Qian, Weizhu; Dai, Jianxin; Li, Bohua; Guo, Yajun; Hou, Sheng; Wang, Hao

    2016-01-01

    ABSTRACT Nivolumab is a therapeutic fully human IgG4 antibody to programmed death 1 (PD-1). In this study, a nivolumab biosimilar, which was produced in our laboratory, was analyzed and characterized. Sequence variants that contain undesired amino acid sequences may cause concern during biosimilar bioprocess development. We found that low levels of sequence variants were detected in the heavy chain of the nivolumab biosimilar by ultra performance liquid chromatography (UPLC) and tandem mass spectrometry. It was further identified with UPLC-MS/MS by IdeS or trypsin digestion. The sequence variant was confirmed through addition of synthetic mutant peptide. Subsequently, the mixing base signal of normal and mutant sequence was detected through DNA sequencing. The relative levels of mutant A424V in the Fc region of the heavy chain have been detected and demonstrated to be 12.25% and 13.54%, via base peak intensity (BPI) and UV chromatography of the tryptic peptide mapping, respectively. A424V variant was also quantified by real-time PCR (RT-PCR) at the DNA and RNA level, which was 19.2% and 16.8%, respectively. The relative content of the mutant was consistent at the DNA, RNA and protein level, indicating that the A424V mutation may have little influence at transcriptional or translational levels. These results demonstrate that orthogonal state-of-the-art techniques such as LC- UV- MS and RT-PCR should be implemented to characterize recombinant proteins and cell lines for development of biosimilars. Our study suggests that it is important to establish an integrated and effective analytical method to monitor and characterize sequence variants during antibody drug development, especially for antibody biosimilar products. PMID:27050807

  1. Simulation model for overloaded monoclonal antibody variants separations in ion-exchange chromatography.

    PubMed

    Guélat, Bertrand; Ströhlein, Guido; Lattuada, Marco; Delegrange, Lydia; Valax, Pascal; Morbidelli, Massimo

    2012-08-31

    A model was developed for the design of a monoclonal antibody charge variants separation process based on ion-exchange chromatography. In order to account for a broad range of operating conditions in the simulations, an explicit pH and salt concentration dependence has been included in the Langmuir adsorption isotherm. The reliability of this model was tested using experimental chromatographic retention times as well as information about the structural characteristics of the different charge variants, e.g. C-terminal lysine groups and deamidated groups. Next, overloaded isocratic elutions at various pH and salt concentrations have been performed to determine the saturation capacity of the ion-exchanger. Furthermore, the column simulation model was applied for the prediction of monoclonal antibody variants separations with both pH and salt gradient elutions. A good prediction of the elution times and peak shapes was observed, even though none of the model parameters was adjusted to fit the experimental data. The trends in the separation performance obtained through the simulations were generally sufficient to identify the most promising operating conditions. The predictive column simulation model thus developed in this work, including a set of parameters determined through specific independent experiments, was experimentally validated and offers a useful basis for a rational optimization of monoclonal antibody variants separation processes on ion-exchange chromatography.

  2. Development of a stable phosphoarginine analog for producing phosphoarginine antibodies.

    PubMed

    Ouyang, Han; Fu, Chuan; Fu, Songsen; Ji, Zhe; Sun, Ying; Deng, Peiran; Zhao, Yufen

    2016-02-14

    Protein phosphorylation is one of the most common and extensively studied post-translational modifications (PTMs). Compared to the O-phosphorylation on Ser, Thr and Tyr residues, our understanding of arginine phosphorylation is relatively limited, both in prokaryotes and eukaryotes, due to the intrinsic instability of phosphoarginine (pArg) and the lack of a feasible method to produce anti-pArg antibodies. We report the design and synthesis of a stable pArg analog, in which the labile N-P bond is replaced with a non-hydrolyzable C-P bond. Significantly, this analog was successfully used as a hapten to raise an immune response and the first mouse polyclonal antibody that specifically recognizes pArg-containing peptides and proteins was produced using analog-KLH conjugated as the immunogen. The generated antibody shows excellent specificity towards pArg-containing peptides and proteins, and could be used for a variety of biological detection methods. This provides us an invaluable tool to unravel the mystery of the biological function of pArg.

  3. Producing and recognizing words with two pronunciation variants: evidence from novel schwa words.

    PubMed

    Bürki, Audrey; Frauenfelder, Ulrich H

    2012-01-01

    This study examined the lexical representations and psycholinguistic mechanisms underlying the production and recognition of novel words with two pronunciation variants in French. Participants first learned novel schwa words (e.g., /ʃənyk/), which varied in their alternating status (i.e., whether these words were learned with one or two variants) and, for alternating words, in the frequency of their variants. They were then tested in picture-naming (free or induced) and recognition memory tasks (i.e., deciding whether spoken items were learned during the experiment or not). Results for free naming show an influence of variant frequency on responses, more frequent variants being produced more often. Moreover, our data show an effect of the alternating status of the novel words on naming latencies, with longer latencies for alternating than for nonalternating novel words. These induced naming results suggest that both variants are stored as lexical entries and compete during the lexeme selection process. Results for recognition show an effect of variant frequency on reaction times and no effect of variant type (i.e., schwa versus reduced variant). Taken together, our findings suggest that participants both comprehend and produce novel French schwa words using two lexical representations, one for each variant.

  4. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    PubMed

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection.

  5. [Vaccination against Newcastle disease with variants and differentiation between post-vaccinal and post-infectious antibodies].

    PubMed

    Jestin, V; Cherbonnel, M; Bennejan, G

    1991-01-01

    Three monoclonal antibody (anti-HN Mab 3115) resistant variants of the Newcastle disease virus (NDV) La Sota strain, were selected (a25, b23, a16); once cloned and shown by haemagglutination inhibition, ELISA and Western blot, not to bind to Mab 3115 they were used as experimental vaccines for chicken. The intracerebral pathogenicity index (ICPI) of a25 and b23 variants was low (0.2 and 0.0 respectively). Three to 4 weeks post-administration of alive variants or inactivated b23, respectively administered via eye drop and subcutaneously, the protection against a challenge was not different from that following La Sota vaccination. Antibody titers induced by a25 and b23, as measured by 2 ELISA blocking tests (the first employing a NDV specific Mab 2114, the second employing Mab 3115), were significantly lower (P less than 0.001) than post-challenge antibody titers. On the contrary, the difference between post-La Sota vaccination antibodies and post challenge antibodies was weak (P less than 0.02). Following 3 successive exposures by contact of chickens to live b23 variant, no variation in antibody titers was observed as measured by ELISA employing Mab 3115. This constituted a necessary criterion, but insufficient to test the stability of the b23 variant. At the same time, the latter exhibited poor ability to diffuse. Vaccination with these variants should be considered in differentiating post-vaccinal from post-infectious antibodies.

  6. Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

    PubMed

    Edgeworth, Matthew J; Phillips, Jonathan J; Lowe, David C; Kippen, Alistair D; Higazi, Daniel R; Scrivens, James H

    2015-12-07

    Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

  7. C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes.

    PubMed

    Dick, Lawrence W; Qiu, Difei; Mahon, David; Adamo, Michael; Cheng, Kuang-Chuan

    2008-08-15

    The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation. 2008 Wiley Periodicals, Inc.

  8. Chromatographic separation of three monoclonal antibody variants using multicolumn countercurrent solvent gradient purification (MCSGP).

    PubMed

    Müller-Späth, Thomas; Aumann, Lars; Melter, Lena; Ströhlein, Guido; Morbidelli, Massimo

    2008-08-15

    Multicolumn countercurrent solvent gradient purification (MCSGP) is a continuous chromatographic process developed in recent years (Aumann and Morbidelli, 2007a; Aumann et al., 2007) that is particularly suited for applications in the field of bioseparations. Like batch chromatography, MCSGP is suitable for three-fraction chromatographic separations and able to perform solvent gradients but it is superior in terms of solvent consumption, yield, purity, and productivity due to the countercurrent movement of the liquid and the solid phases. In this work, the MCSGP process is applied to the separation of three monoclonal antibody variants on a conventional preparative cation exchange resin. The experimental process performance was compared to simulations based on a lumped kinetic model. Yield and purity values of the target variant of 93%, respectively were obtained experimentally. The batch reference process was clearly outperformed by the MCSGP process.

  9. Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors.

    PubMed

    Alam, Muntasir; Kuwata, Takeo; Shimura, Kazuya; Yokoyama, Masaru; Ramirez Valdez, Kristel Paola; Tanaka, Kazuki; Maruta, Yasuhiro; Oishi, Shinya; Fujii, Nobutaka; Sato, Hironori; Matsuoka, Masao; Matsushita, Shuzo

    2016-09-27

    HIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors. Mutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the "conformational unmasking" of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant

  10. Anti-D Antibodies in Pregnant D Variant Antigen Carriers Initially Typed as RhD+

    PubMed Central

    Lukacevic Krstic, Jelena; Dajak, Slavica; Bingulac-Popovic, Jasna; Dogic, Vesna; Mratinovic-Mikulandra, Jela

    2016-01-01

    Background To evaluate the incidence, the consequences, and the prevention strategy of anti-D alloimmunizations of D variant carriers in the obstetric population of Split-Dalmatia County, Croatia. Methods RhD immunization events were evaluated retrospectively for the period between 1993 and 2012. Women were tested for RhD antigen and irregular antibodies. Those with anti-D antibody who were not serologically D- were genotyped for RHD. They were evaluated for their obstetric and transfusion history and their titer of anti-D. The neonates were evaluated for RhD status, direct antiglobulin test (DAT), hemoglobin and bilirubin levels, transfusion therapy as well as phototherapy and outcome. Results Out of 104,884 live births 102,982 women were tested for RhD antigen. Anti-D immunization occurred in 184 women which accounts for 0.9% of individuals at risk of anti-D formation. 181 cases occurred in women serologically typed as D-. Three women were partial D carriers (DVa n = 2, DNB n = 1), initially typed RhD+, and recognized as D variant carriers after the immunization occurred. Anti-D titer varied from 1:1 to 1:16. Six children were RhD+, four had positive DAT, and two underwent phototherapy. Conclusion Anti-D immunization occurred in pregnant partial D carriers (DVa, DNB). RhD+ children had serologic markers of hemolytic disease of the fetus and newborn (HDFN), with no cases of severe HDFN. PMID:27994529

  11. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens.

    PubMed

    Tan, Joshua; Pieper, Kathrin; Piccoli, Luca; Abdi, Abdirahman; Foglierini, Mathilde; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Ndungu, Francis Maina; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro; Marsh, Kevin; Thathy, Vandana; Corti, Davide; Sallusto, Federica; Bull, Peter; Lanzavecchia, Antonio

    2016-01-07

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

  12. A LAIR-1 insertion generates broadly reactive antibodies against malaria variant antigens

    PubMed Central

    Abdi, Abdirahman; Perez, Mathilde Foglierini; Geiger, Roger; Tully, Claire Maria; Jarrossay, David; Maina Ndungu, Francis; Wambua, Juliana; Bejon, Philip; Fregni, Chiara Silacci; Fernandez-Rodriguez, Blanca; Barbieri, Sonia; Bianchi, Siro; Marsh, Kevin; Thathy, Vandana; Corti, Davide; Sallusto, Federica

    2016-01-01

    Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies1–4. Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here, we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 100 amino acid collagen-binding domain of LAIR-1, an Ig superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B cell clone and carry distinct somatic mutations in the LAIR-1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. PMID:26700814

  13. An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

    PubMed Central

    Irving, James A.; Miranda, Elena; Haq, Imran; Perez, Juan; Kotov, Vadim R.; Faull, Sarah V.; Motamedi-Shad, Neda; Lomas, David A.

    2015-01-01

    A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen–whose native state is susceptible to the formation of a proto-oligomeric intermediate–we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states. PMID:25738741

  14. Mechanistic modeling of ion-exchange process chromatography of charge variants of monoclonal antibody products.

    PubMed

    Kumar, Vijesh; Leweke, Samuel; von Lieres, Eric; Rathore, Anurag S

    2015-12-24

    Ion-exchange chromatography (IEX) is universally accepted as the optimal method for achieving process scale separation of charge variants of a monoclonal antibody (mAb) therapeutic. These variants are closely related to the product and a baseline separation is rarely achieved. The general practice is to fractionate the eluate from the IEX column, analyze the fractions and then pool the desired fractions to obtain the targeted composition of variants. This is, however, a very cumbersome and time consuming exercise. A mechanistic model that is capable of simulating the peak profile will be a much more elegant and effective way to make a decision on the pooling strategy. This paper proposes a mechanistic model, based on the general rate model, to predict elution peak profile for separation of the main product from its variants. The proposed approach uses inverse fit of process scale chromatogram for estimation of model parameters using the initial values that are obtained from theoretical correlations. The packed bed column has been modeled along with the chromatographic system consisting of the mixer, tubing and detectors as a series of dispersed plug flow and continuous stirred tank reactors. The model uses loading ranges starting at 25% to a maximum of 70% of the loading capacity and hence is applicable to process scale separations. Langmuir model has been extended to include the effects of salt concentration and temperature on the model parameters. The extended Langmuir model that has been proposed uses one less parameter than the SMA model and this results in a significant ease of estimating the model parameters from inverse fitting. The proposed model has been validated with experimental data and has been shown to successfully predict peak profile for a range of load capacities (15-28mg/mL), gradient lengths (10-30CV), bed heights (6-20cm), and for three different resins with good accuracy (as measured by estimation of residuals). The model has been also

  15. Biological properties of two distinct pilus types produced by isogenic variants of Neisseria gonorrhoeae P9.

    PubMed Central

    Lambden, P R; Robertson, J N; Watt, P J

    1980-01-01

    Isogenic variants from a single strain of Neisseria gonorrhoeae were shown to produce two distinct types of pili. These pili, designated alpha and beta, differed in both subunit molecular weight and in ability to attach to buccal epithelial cells. Images PMID:6101593

  16. Genomic copy number variants: evidence for association with antibody response to anthrax vaccine adsorbed.

    PubMed

    Falola, Michael I; Wiener, Howard W; Wineinger, Nathan E; Cutter, Gary R; Kimberly, Robert P; Edberg, Jeffrey C; Arnett, Donna K; Kaslow, Richard A; Tang, Jianming; Shrestha, Sadeep

    2013-01-01

    Anthrax and its etiologic agent remain a biological threat. Anthrax vaccine is highly effective, but vaccine-induced IgG antibody responses vary widely following required doses of vaccinations. Such variation can be related to genetic factors, especially genomic copy number variants (CNVs) that are known to be enriched among genes with immunologic function. We have tested this hypothesis in two study populations from a clinical trial of anthrax vaccination. We performed CNV-based genome-wide association analyses separately on 794 European Americans and 200 African-Americans. Antibodies to protective antigen were measured at week 8 (early response) and week 30 (peak response) using an enzyme-linked immunosorbent assay. We used DNA microarray data (Affymetrix 6.0) and two CNV detection algorithms, hidden markov model (PennCNV) and circular binary segmentation (GeneSpring) to determine CNVs in all individuals. Multivariable regression analyses were used to identify CNV-specific associations after adjusting for relevant non-genetic covariates. Within the 22 autosomal chromosomes, 2,943 non-overlapping CNV regions were detected by both algorithms. Genomic insertions containing HLA-DRB5, DRB1 and DQA1/DRA genes in the major histocompatibility complex (MHC) region (chromosome 6p21.3) were moderately associated with elevated early antibody response (β = 0.14, p = 1.78×10(-3)) among European Americans, and the strongest association was observed between peak antibody response and a segmental insertion on chromosome 1, containing NBPF4, NBPF5, STXMP3, CLCC1, and GPSM2 genes (β = 1.66, p = 6.06×10(-5)). For African-Americans, segmental deletions spanning PRR20, PCDH17 and PCH68 genes on chromosome 13 were associated with elevated early antibody production (β = 0.18, p = 4.47×10(-5)). Population-specific findings aside, one genomic insertion on chromosome 17 (containing NSF, ARL17 and LRRC37A genes) was associated with elevated peak antibody

  17. Quality assurance of monoclonal antibody pharmaceuticals based on their charge variants using microchip isoelectric focusing method.

    PubMed

    Kinoshita, Mitsuhiro; Nakatsuji, Yuki; Suzuki, Shigeo; Hayakawa, Takao; Kakehi, Kazuaki

    2013-09-27

    Monoclonal antibody (mAb) pharmaceuticals are much more complex than small-molecule drugs. Such complex characteristics raise challenging questions for regulatory evaluation. Although heterogeneity in mAbs based on their charge variants has been mainly evaluated using gel-based isoelectric focusing (IEF) method, recent development in capillary electrophoresis and microchip electrophoresis has made it possible to assure their heterogeneities in more easy and rapid manner. In the present paper, we customized the imaged microchip isoelectric focusing (mIEF) for the analysis of mAbs, and compared the customized version with the conventional capillary isoelectric focusing (cIEF) method, and found that mIEF has much higher performance in operations, and its resolving powers are comparable with those obtained by cIEF. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer?

    PubMed

    Chusainow, Janet; Yang, Yuan Sheng; Yeo, Jessna H M; Toh, Poh Choo; Asvadi, Parisa; Wong, Niki S C; Yap, Miranda G S

    2009-03-01

    Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize

  19. Recognition of influenza H3N2 variant virus by human neutralizing antibodies

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.

    2016-01-01

    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  20. Genetic variants are major determinants of CSF antibody levels in multiple sclerosis.

    PubMed

    Goris, An; Pauwels, Ine; Gustavsen, Marte W; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D'Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G; Gourraud, Pierre-Antoine; Sawcer, Stephen J; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F

    2015-03-01

    Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

  1. Genetic variants are major determinants of CSF antibody levels in multiple sclerosis

    PubMed Central

    Pauwels, Ine; Gustavsen, Marte W.; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D.; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D’Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A.; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H.; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G.; Gourraud, Pierre-Antoine; Sawcer, Stephen J.; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F.

    2015-01-01

    Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index—the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10−16). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10−7). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10−37). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10−22), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10−6). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

  2. Is there a difference among human populations in the rate with which mutation produces electrophoretic variants?

    PubMed Central

    Neel, J V; Rothman, E

    1981-01-01

    Data are summarized that suggest that tropical-zone/tribal/nonindustrialized populations have higher frequencies of certain types of protein variants than temperate-zone/civilized/industrial populations, and it is demonstrated that these differences are not an artifact produced by the contagious type of sampling used with respect to tribal populations. Evidence is reviewed that suggests that a possible explanation of this difference is higher mutation rates in the tribal populations studied. PMID:6942419

  3. Effects of carboxypeptidase B treatment and elevated temperature on recombinant monoclonal antibody charge variants in cation-exchange chromatography analysis.

    PubMed

    Kim, Do Gyun; Kim, Hyoung Jin; Kim, Hong-Jin

    2016-10-01

    Charge variants (acidic and basic) of recombinant monoclonal antibodies (Mabs) have received much attention due to their potential biological effects. C-terminal lysine variants are common in Mabs and their proportion is affected by the manufacturing process. In the present study, changes of trastuzumab charge variants brought about by carboxypeptidase B treatment and subsequent storage at 8 or 37 °C for up to 24 h were monitored by cation-exchange chromatography analysis to investigate the effects of C-terminal lysine cleavage and its subsequent reaction at 8 or 37 °C. C-terminal lysine cleavage at 8 °C reduced the fraction of basic species and had little effect on the fraction of acidic species. Analysis of individual peaks demonstrated that C-terminal lysine cleavage induced both increases and decreases in individual acidic variants, with the result that there was little overall change in the overall proportion of acidic species. It appeared that most of the basic variant Mab molecules but only a fraction of the acidic variant molecules had C-terminal lysines. Increasing the temperature to 37 °C appeared to increase the fraction of acidic species and decrease main species significantly, without a similar change in basic species. These results indicate that length of exposure to elevated temperature is a critical consideration in charge variant analysis.

  4. Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Funk, J.; Biber, N.; Schneider, M.; Hauser, E.; Enzenmüller, S.; Förtsch, C.

    2015-01-01

    In this study, the cytotoxicity of the recently described subtilase variant SubAB2-2 of Shiga toxin-producing Escherichia coli was determined and compared to the plasmid-encoded SubAB1 and the chromosome-encoded SubAB2-1 variant. The genes for the respective enzymatic active (A) subunits and binding (B) subunits of the subtilase toxins were amplified and cloned. The recombinant toxin subunits were expressed and purified. Their cytotoxicity on Vero cells was measured for the single A and B subunits, as well as for mixtures of both, to analyze whether hybrids with toxic activity can be identified. The results demonstrated that all three SubAB variants are toxic for Vero cells. However, the values for the 50% cytotoxic dose (CD50) differ for the individual variants. Highest cytotoxicity was shown for SubAB1. Moreover, hybrids of subunits from different subtilase toxins can be obtained which cause substantial cytotoxicity to Vero cells after mixing the A and B subunits prior to application to the cells, which is characteristic for binary toxins. Furthermore, higher concentrations of the enzymatic subunit SubA1 exhibited cytotoxic effects in the absence of the respective B1 subunit. A more detailed investigation in the human HeLa cell line revealed that SubA1 alone induced apoptosis, while the B1 subunit alone did not induce cell death. PMID:25824835

  5. Use of antigen mimics to produce specific antibodies to anti-coccidial drugs.

    PubMed

    Fodey, Terence L; Delahaut, Philippe; Elliott, Christopher T

    2007-05-31

    A range of polyclonal antibodies was successfully produced to the coccidiostatic drugs diclazuril and robenidine. Initial attempts to make immunogenic complexes of both drugs were ineffective due to difficulties encountered while trying to couple the compounds to large carrier proteins. Structural mimics, which could act as haptens for each drug, were sought and identified. The compounds identified were more open to chemical manipulation and were conjugated to carrier proteins to produce effective immunogens. The most sensitive antisera produced displayed IC50s of 1.5 ng/ml and 13 ng/ml for diclazuril and robenidine respectively. The antibody for diclazuril was shown to be specific, cross-reacting only with clazuril by 15%. The robenidine antibody displayed a low cross-reactivity of 1.2% to the compound used to produce the antibody.

  6. Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes.

    PubMed

    Juárez, Paloma; Presa, Silvia; Espí, Joaquín; Pineda, Benito; Antón, María T; Moreno, Vicente; Buesa, Javier; Granell, Antonio; Orzaez, Diego

    2012-04-01

    Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.

  7. Antiviral antibody-producing cells in parenchymatous organs during persistent virus infection

    PubMed Central

    1987-01-01

    In mice persistently infected with lymphocytic choriomeningitis virus (LCMV), the parenchymatous organs contain infiltrates of mononuclear cells, the sizes and numbers of which vary between strains and become more numerous and extensive when the animals grow older. Histologically, these were found to possess a tissue-like structure, and by use of immunohistologic procedures they were shown to contain plasma cells secreting IgM and IgG. Cells of kidneys, livers, brains, and spleens of LCMV carrier mice were dispersed by digestion with trypsin, leukocytes were separated by density gradient centrifugation, and numbers of cells producing antibodies against LCMV were determined by use of a solid-phase immunoenzymatic technique. In all these organs, cells producing LCMV-specific IgM and IgG antibodies were demonstrated, the latter more numerous than the former. Their numbers correlated with numbers and extent of the lymphoid cell infiltrates. The blood of the same mice was essentially free of antiviral antibody-forming cell. The proportion of cells producing LCMV-specific antibodies to all cells producing Ig of any specificity varied between organs, being lowest in spleen, intermediate in liver and kidney, and highest in the brain, where in individual mice up to 90% of all active cells produced virus- specific antibodies. The LCMV carrier mouse should prove to be a useful animal model to investigate antibody production in parenchymatous organs during persistent virus infections. PMID:3546579

  8. A human-mouse hybridoma producing monoclonal antibody against human sperm coating antigen.

    PubMed Central

    Kyurkchiev, S D; Shigeta, M; Koyama, K; Isojima, S

    1986-01-01

    Since anti-sperm antibodies were first discovered in the sera of women, the relationship of these antibodies to sterility has been studied by many investigators. In order to determine the antigens of spermatozoa responsible for raising antibodies to spermatozoa in humans, many studies have been carried out by purifying human spermatozoa cell membrane and seminal plasma components. Since it was found that the purification was difficult by physiochemical procedures, the immunoaffinity chromatography bound monoclonal antibody (Mab) to spermatozoa antigens was attempted for this purpose. The establishment of hybridomas producing Mabs to human seminal plasma and human spermatozoa was reported by Shigeta et al. (1980), Isojima, Koyoma & Fujiwara (1982), Lee et al. (1982) and Isahakia & Alexander (1984). The ordinary approaches to obtain the Mabs consisted of xenogenic immunization with human semen and cell fusion of immunized spleen cells with mouse myeloma cells. However, the antigenic epitopes of human spermatozoa, which induced antibody production, are xenogenic for the mouse, and therefore there is a possibility that there is a difference in recognized antigenic epitopes in humans as isotypic and in mice as xenogenic. In order to study these antigenic epitopes, which correspond to antibodies against spermatozoa in women, the establishment of human-mouse hybridomas, which produced anti-semen antibodies as produced in sterile women, became essential. In these studies, we used recently developed cell fusion techniques to fuse immunized human peripheral lymphocytes with mouse myeloma cells. PMID:3456978

  9. Generation of polyclonal antibodies against recombinant human glucocerebrosidase produced in Escherichia coli.

    PubMed

    Novo, Juliana Branco; Oliveira, Maria Leonor Sarno; Magalhães, Geraldo Santana; Morganti, Ligia; Raw, Isaías; Ho, Paulo Lee

    2010-11-01

    Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

  10. Elucidating the effects of pH shift on IgG1 monoclonal antibody acidic charge variant levels in Chinese hamster ovary cell cultures.

    PubMed

    Xie, Panpan; Niu, Huijie; Chen, Xinning; Zhang, Xintao; Miao, Shiwei; Deng, Xiancun; Liu, Xuping; Tan, Wen-Song; Zhou, Yan; Fan, Li

    2016-12-01

    Charge variants, especially acidic charge variants, in recombinant monoclonal antibodies are critical quality attributes, which can affect antibodies' properties in vitro and in vivo. Meanwhile, charge variants are cumulative effects of various post-translational modifications and chemical degradations on antibody. In this work, to investigate the effect of lowering culture pH in the stationary phase on acidic charge variant contents in fed-batch cultures and its mechanism, cell culture experiments in 2-L bioreactors were firstly performed to explore the changes in the charge distribution under the pH downshift condition using weak cation exchange chromatography. It is found that acidic charge variant contents were significantly decreased by pH downshift. Then, to reveal the mechanism by which the content of acidic charge variants is reduced under pH downshift condition, the variation of post-translational modifications and chemical degradations under the pH downshift condition was explored. Meanwhile, the structure of the acidic charge variants was characterized. Several analysis experiments including size exclusion chromatography, capillary electrophoresis-sodium dodecyl sulfate under non-reducing conditions, tryptic peptide map, and reduced antibody mass were applied in this study. The results show that the mechanism by which the content of acidic charge variants is reduced is that the contents of disulfide bond reduction, galactosylation, and asparagine deamination of the HC-N388 in the Fc domain were reduced by pH downshift.

  11. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  12. Envelope variants circulating as initial neutralization breadth developed in two HIV-infected subjects stimulate multiclade neutralizing antibodies in rabbits.

    PubMed

    Malherbe, Delphine C; Pissani, Franco; Sather, D Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F; Stuart, Andrew B; Robins, Harlan; Park, Byung; Krebs, Shelly J; Schuman, Jason T; Kalams, Spyros; Hessell, Ann J; Haigwood, Nancy L

    2014-11-01

    Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable of generating

  13. Effects of different amino acids in culture media on surfactin variants produced by Bacillus subtilis TD7.

    PubMed

    Liu, Jin-Feng; Yang, Juan; Yang, Shi-Zhong; Ye, Ru-Qiang; Mu, Bo-Zhong

    2012-04-01

    Surfactin produced by Bacillus subtilis has different variants, which are affected by the composition of substrate available. To demonstrate the effects of amino acids on surfactin variants, B. subtilis TD7 was cultivated under the same conditions but with different amino acids supplied in media, respectively, and the type as well as the proportion of surfactin variants produced was analyzed with electrospray ionization mass spectrometry and gas chromatography-mass spectrometry. The result shows that the addition of different amino acids significantly influences the proportion of surfactin variants with different fatty acids. When Arg, Gln, or Val was added to the culture medium of B. subtilis TD7, the proportion of produced surfactin variants with even β-hydroxy fatty acids significantly increased, while the addition of Cys, His, Ile, Leu, Met, Ser, or Thr enhanced the proportion of surfactin variants with odd β-hydroxy fatty acids markedly. This result may be of some reference value in enhancing the production of specific surfactin variants as well as in the research on the relationship between culture media and the corresponding products of a certain bacterium.

  14. Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency

    PubMed Central

    Chen, Long; Cohen, Justin; Song, Xiaoda; Zhao, Aishan; Ye, Zi; Feulner, Christine J.; Doonan, Patrick; Somers, Will; Lin, Laura; Chen, Peng R.

    2016-01-01

    Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme’s poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation. PMID:27534437

  15. Decreased SMG7 expression associates with lupus-risk variants and elevated antinuclear antibody production.

    PubMed

    Deng, Yun; Zhao, Jian; Sakurai, Daisuke; Sestak, Andrea L; Osadchiy, Vadim; Langefeld, Carl D; Kaufman, Kenneth M; Kelly, Jennifer A; James, Judith A; Petri, Michelle A; Bae, Sang-Cheol; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Criswell, Lindsey A; Freedman, Barry I; Kamen, Diane L; Gilkeson, Gary S; Jacob, Chaim O; Merrill, Joan T; Gaffney, Patrick M; Sivils, Kathy Moser; Niewold, Timothy B; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Boackle, Susan A; Vilá, Luis M; Sohn, I I Woong; Lee, Seung; Chang, Deh-Ming; Song, Yeong Wook; Vyse, Timothy J; Harley, John B; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Cantor, Rita M; Hahn, Bevra H; Grossman, Jennifer M; Tsao, Betty P

    2016-11-01

    Following up the systemic lupus erythematosus (SLE) genome-wide association studies (GWAS) identification of NMNAT2 at rs2022013, we fine-mapped its 150 kb flanking regions containing NMNAT2 and SMG7 in a 15 292 case-control multi-ancestry population and tested functions of identified variants. We performed genotyping using custom array, imputation by IMPUTE 2.1.2 and allele specific functions using quantitative real-time PCR and luciferase reporter transfections. SLE peripheral blood mononuclear cells (PBMCs) were cultured with small interfering RNAs to measure antinuclear antibody (ANA) and cyto/chemokine levels in supernatants using ELISA. We confirmed association at NMNAT2 in European American (EA) and Amerindian/Hispanic ancestries, and identified independent signal at SMG7 tagged by rs2702178 in EA only (p=2.4×10(-8), OR=1.23 (95% CI 1.14 to 1.32)). In complete linkage disequilibrium with rs2702178, rs2275675 in the promoter region robustly associated with SMG7 mRNA levels in multiple expression quantitative trait locus (eQTL) datasets. Its risk allele was dose-dependently associated with decreased SMG7 mRNA levels in PBMCs of 86 patients with SLE and 119 controls (p=1.1×10(-3) and 6.8×10(-8), respectively) and conferred reduced transcription activity in transfected HEK-293 (human embryonic kidney cell line) and Raji cells (p=0.0035 and 0.0037, respectively). As a critical component in the nonsense-mediated mRNA decay pathway, SMG7 could regulate autoantigens including ribonucleoprotein (RNP) and Smith (Sm). We showed SMG7 mRNA levels in PBMCs correlated inversely with ANA titres of patients with SLE (r=-0.31, p=0.01), and SMG7 knockdown increased levels of ANA IgG and chemokine (C-C motif) ligand 19 in SLE PBMCs (p=2.0×10(-5) and 2.0×10(-4), respectively). We confirmed NMNAT2 and identified independent SMG7 association with SLE. The inverse relationship between levels of the risk allele-associated SMG7 mRNAs and ANA suggested the novel contribution

  16. Protein adsorption on ion exchange resins and monoclonal antibody charge variant modulation.

    PubMed

    Guélat, Bertrand; Khalaf, Rushd; Lattuada, Marco; Costioli, Matteo; Morbidelli, Massimo

    2016-05-20

    A novel multicomponent adsorption equilibrium model for proteins on ion-exchange resins is developed on a statistical thermodynamic basis including surface coverage effects and protein-resin and protein-protein interactions. The resulting model exhibits a general competitive Langmuirian behavior and was applied to the study and optimization of the separation of monoclonal antibody charge variants on two strong cation exchangers. The model accounts explicitly for the effect of both pH and salt concentration, and its parameters can be determined in diluted conditions, that is, through physically sound assumptions, all model parameters can be obtained using solely experiments in diluted conditions, and be used to make predictions in overloaded conditions. The parameterization of the model and optimization of the separation is based on a two-step approach. First, gradient experiments in diluted conditions are undertaken in order to determine the model parameters. Based on these experiments and on information about the proteins of interest and the stationary phase used, all the model parameters can be estimated. Second, using the parameterized model, an initial Pareto optimization is undertaken where overloaded operating conditions are investigated. Experiments from this Pareto set are then used to refine the estimation of the model parameters. A second Pareto optimization can then be undertaken, this time with the refined parameters. This can be repeated until a satisfactory set of model parameters is found. This iterative approach is shown to be extremely efficient and to provide large amounts of knowledge based on only a few experiments. It is shown that due to the strong physical foundation of the model and the very low number of adjustable parameters, the number of iterations is expected to be at most two or three. Furthermore, the model based tool is improved as more experimental knowledge is provided, allowing for better estimations of the chromatographic

  17. Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas.

    PubMed

    Sado, Yoshikazu; Inoue, Satoko; Tomono, Yasuko; Matsuyama, Makoto; Fukushima, Masaki; Oohashi, Toshitaka; Jogahara, Takamichi; Oda, Sen-Ichi

    2017-04-27

    We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

  18. Medaka tert produces multiple variants with differential expression during differentiation in vitro and in vivo

    PubMed Central

    Rao, Feng; Wang, Tiansu; Li, Mingyou; Li, Zhendong; Hong, Ni; Zhao, Haobin; Yan, Yan; Lu, Wenqing; Chen, Tiansheng; Wang, Weijia; Lim, Menghuat; Yuan, Yongming; Liu, Ling; Zeng, Lingbing; Wei, Qiwei; Guan, Guijun; Li, Changming; Hong, Yunhan

    2011-01-01

    Embryonic stem (ES) cells have immortality for self-renewal and pluripotency. Differentiated human cells undergo replicative senescence. In human, the telomerase reverse transcriptase (Tert), namely the catalytic subunit of telomerase, exhibits differential expression to regulate telomerase activity governing cellular immortality or senescence, and telomerase activity or tert expression is a routine marker of pluripotent ES cells. Here we have identified the medaka tert gene and determined its expression and telomerase activity in vivo and in vitro. We found that the medaka tert locus produces five variants called terta to terte encoding isoforms TertA to TertE. The longest TertA consists of 1090 amino acid residues and displays a maximum of 34% identity to the human TERT and all the signature motifs of the Tert family. TertB to TertE are novel isoforms and have considerable truncation due to alternative splicing. The terta RNA is ubiquitous in embryos, adult tissues and cell lines, and accompanies ubiquitous telomerase activity in vivo and in vitro as revealed by TRAP assays. The tertb RNA was restricted to the testis, absent in embryos before gastrulation and barely detectable in various cell lines The tertc transcript was absent in undifferentiated ES cells but became evident upon ES cell differentiation, in vivo it was barely detectable in early embryos and became evident when embryogenesis proceeds. Therefore, ubiquitous terta expression correlates with ubiquitous telomerase activity in medaka, and expression of other tert variants appears to delineate cell differentiation in vitro and in vivo. PMID:21547060

  19. Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

    PubMed Central

    Schütte, Mark; Thullier, Philippe; Pelat, Thibaut; Wezler, Xenia; Rosenstock, Philip; Hinz, Dominik; Kirsch, Martina Inga; Hasenberg, Mike; Frank, Ronald; Schirrmann, Thomas; Gunzer, Matthias

    2009-01-01

    Background Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. Results The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. Conclusion Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus. PMID:19675673

  20. Evidence for the genetic control of antibody affinity from breeding studies with inbred mouse strains producing high and low affinity antibody.

    PubMed Central

    Steward, M W; Petty, R E

    1976-01-01

    The amount (Abt) and relative affinity (KR) of antibody produced in response to protein antigens injected in saline has been measured in the parents, F1 hybrids and backcross offspring of inbred mice which produce high and low KR antibody to these antigens. The results obtained support the view that antibody affinity is under polygenic control. Furthermore, strain related variation in Abt is independent of KR and the breeding experiments indicate that these two parameters are under independent genetic control. PMID:1027713

  1. Characterization of antibody variants during process development: the tale of incomplete processing of N-terminal secretion peptide.

    PubMed

    Ambrogelly, Alexandre; Liu, Yan-Hui; Li, Hong; Mengisen, Selina; Yao, Bingyi; Xu, Wei; Cannon-Carlson, Susan

    2012-01-01

    Monoclonal antibodies (mAbs) have emerged as one of the most important classes of biotherapeutics, although development of these molecules is long and arduous. A production cell line must be established, and growth conditions for the cells and purification processes for the product must be optimized. Integration of the appropriate analytical strategies in these activities is the cornerstone of Quality by Design and in-process control approaches are encouraged by the Food and Drug Administration. We report here the development of a reversed phase-high performance liquid chromatography (RP-HPLC) method to follow the presence of a mAb product-related variant observed during the purification process development. The variant eluted as a later peak on RP-HPLC, compared with the mAb control (3.25 min and 2.85 min, respectively). We isolated this hydrophobic variant and further analyzed it by mass spectrometry. We identified the variant as a mAb with an incompletely processed leader sequence attached to the N-terminus of one of the two heavy chains.

  2. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    PubMed

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  3. Osteo-cementum Producing Odontogenic Myxomas. A Literature Review of a Distinctive Variant.

    PubMed

    Rallis, George; Dais, Panagiotis; Kostakis, George; Stathopoulos, Panagiotis

    2015-06-01

    Odontogenic myxoma (OM) is a benign neoplasm of mesenchymal origin with growth characteristics, clinical behaviour and radiographic presentation similar to those of ameloblastoma. It is an intraosseous neoplasm characterized by stellate and spindleshaped cells embedded in loose myxoid or mucoid extracellular matrix. Although sometimes bony islands that represent residual trabeculae are found throughout the lesion, the formation of osteocement-like calcified spherules within the tumour is an extremely rare phenomenon. We report a very rare case of an OM of the left maxilla exhibiting osteo-cementous metaplasia within the substance of the tumour and beyond the facial skeleton, in the nasopharynx. A review of all four similar cases previously reported in the literature is also presented. Whether or not this property to produce significant amounts of bone can be associated with a different biological behavior for this specific variant of OM remains to be proved with the study of more similar cases.

  4. Specificity of antibodies produced against native or desialylated human immunodeficiency virus type 1 recombinant gp160.

    PubMed Central

    Benjouad, A; Gluckman, J C; Montagnier, L; Bahraoui, E

    1993-01-01

    In a previous report we have shown that, in contrast to antibodies produced against native or fully deglycosylated human immunodeficiency virus type 1 (HIV-1) gp160 in rabbits, antibodies raised against desialylated HIV-1 gp160 also recognize gp140 from HIV-2 at high titers. Here, we characterize the fine specificity of these cross-reactive antibodies. Inhibition assays with a panel of synthetic peptides as competitors showed that cross-reactivity to gp140 was due to antibodies that were specific for the region encompassing HIV-1 gp41 immunodominant epitope, mimicked by peptide P39 (residues 583 to 609), the latter being able to totally inhibit the formation of complexes between radiolabeled HIV-2 gp140 and antibodies elicited by desialylated HIV-1 gp160. In addition, anti-desialylated gp160 antibodies retained on a P39 affinity column still bound HIV-2 gp140. Fine mapping has enabled us to localize the cross-reactive epitope within the N-terminal extremity of the gp41 immunodominant region. Interestingly, this cross-reactive antibody population did not recognize glycosylated or totally deglycosylated simian immunodeficiency virus gp140 despite an amino acid homology with HIV-1 within this region that is comparable to that of HIV-2. This cross-reactivity between HIV-1 and HIV-2 did not correlate with cross-neutralization. These results illustrate the influence of carbohydrate moieties on the specificity of the antibodies produced and clearly indicate that such procedures may be an efficient way to raise specific immune responses that are not type specific. Moreover, this cross-reactivity might explain the double-positive reactivity observed, in some human sera, against both HIV-1 and HIV-2 envelope antigens. PMID:7679751

  5. Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission

    PubMed Central

    Gregory, James A.; Li, Fengwu; Tomosada, Lauren M.; Cox, Chesa J.; Topol, Aaron B.; Vinetz, Joseph M.; Mayfield, Stephen

    2012-01-01

    Subunit vaccines are significantly more expensive to produce than traditional vaccines because they are based primarily on recombinant proteins that must be purified from the expression system. Despite the increased cost, subunit vaccines are being developed because they are safe, effective, and can elicit antibodies that confer protection against diseases that are not currently vaccine-preventable. Algae are an attractive platform for producing subunit vaccines because they are relatively inexpensive to grow, genetically tractable, easily scaled to large volumes, have a short generation time, and are devoid of inflammatory, viral, or prion contaminants often present in other systems. We tested whether algal chloroplasts can produce malaria transmission blocking vaccine candidates, Plasmodium falciparum surface protein 25 (Pfs25) and 28 (Pfs28). Antibodies that recognize Pfs25 and Pfs28 disrupt the sexual development of parasites within the mosquito midgut, thus preventing transmission of malaria from one human host to the next. These proteins have been difficult to produce in traditional recombinant systems because they contain tandem repeats of structurally complex epidermal growth factor-like domains, which cannot be produced in bacterial systems, and because they are not glycosylated, so they must be modified for production in eukaryotic systems. Production in algal chloroplasts avoids these issues because chloroplasts can fold complex eukaryotic proteins and do not glycosylate proteins. Here we demonstrate that algae are the first recombinant system to successfully produce an unmodified and aglycosylated version of Pfs25 or Pfs28. These antigens are structurally similar to the native proteins and antibodies raised to these recombinant proteins recognize Pfs25 and Pfs28 from P. falciparum. Furthermore, antibodies to algae-produced Pfs25 bind the surface of in-vitro cultured P. falciparum sexual stage parasites and exhibit transmission blocking activity. Thus

  6. Surface display vectors for selective detection and isolation of high level antibody producing cells.

    PubMed

    Lang, Sabine; Drewello, Delia; Wichter, Johannes; Nommay, Audrey; Wilms, Burkhard; Knopf, Hans-Peter; Jostock, Thomas

    2016-11-01

    Cell line generation for production of biopharmaceuticals in mammalian cells usually involves intensive screening of clones to identify the rare high producers. In order to facilitate efficient and selective fluorescence activated cell sorting (FACS) based enrichment and cloning of antibody producing CHO cells, we developed a special vector setup by inserting a leaky translation termination signal between the heavy chain of an IgG antibody and an IgG transmembrane domain. Partial read-through during translation of the antibody heavy chain leads to display of a subset of the produced antibody on the surface of the expressing cell. We could show that the level of surface expression correlates well with the productivity. By applying FACS, high producing cells can be selectively enriched and cloned. Two sequential FACS enrichment cycles were performed which led to more than eightfold increased productivities of transfected and selected cell populations without cloning. The combination of selective FACS enrichment and FACS cloning with the new vector setup led to a sevenfold higher average productivity of the resulting clones as compared to a reference vector. Productivity and production stability assessment of clones generated with the new vector showed no negative impact of the co-expression of transmembrane antibody. Clone productivities of 4 g/L in a generic shake flask fed-batch model were achieved. Thus, this new vector setup facilitates fast and selective isolation of high producing production cell lines and allows significant reduction of clone screening efforts during cell line development for production cell lines. Additionally, the high productivity of FACS-enriched but non-clonal cell populations supports rapid, high yield, and cost efficient material production in early project phases. Biotechnol. Bioeng. 2016;113: 2386-2393. © 2016 Wiley Periodicals, Inc.

  7. Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells.

    PubMed

    Prieto, Y; Rojas, L; Hinojosa, L; González, I; Aguiar, D; de la Luz, K; Castillo, A; Pérez, R

    2011-08-01

    The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

  8. Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

    PubMed

    Jooß, Kevin; Hühner, Jens; Kiessig, Steffen; Moritz, Bernd; Neusüß, Christian

    2017-08-12

    Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

  9. Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies

    PubMed Central

    Parma, Y. R.; Chacana, P. A.; Lucchesi, P. M. A.; Rogé, A.; Granobles Velandia, C. V.; Krüger, A.; Parma, A. E.; Fernández-Miyakawa, M. E.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx2EDL933, stx2vha, stx2vhb, stx2g, stx1EDL933, and stx1d were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli. PMID:22919675

  10. DIFFERENTIATION AND FUNCTIONAL EXPRESSION OF POTENTIAL ANTIBODY-PRODUCING CELLS IN THE PRESENCE OF CHLORAMPHENICOL

    PubMed Central

    Schoenberg, Melvin D.; Moore, Richard D.; Weisberger, Austin S.

    1967-01-01

    Rabbits were immunized with diphtheria toxoid combined with complete Freund's adjuvant. Half of the animals were started on intramuscular injections of chloramphenicol 24 hr before the injection of the antigens. There was a general depression of protein synthesis in the immune system in the presence of chloramphenicol, but a greater effect on the synthesis of antibody than on the synthesis of proteins necessary for reproduction and maturation. In contrast to the finding of antibody in cells of the spleen and in the circulation of the control animals, those animals receiving chloramphenicol did not have measurable circulating antibody, and their spleens contained only a few cells with intracytoplasmic antibody late in the course of the experiment. Cytologically there was maturation of potential antibody-producing cells in the red pulp and nonfollicular white pulp of the spleen while the animals were receiving chloramphenicol. These cells developed more slowly, and were fewer and smaller than those of the control animals. They had numerous small, electron-opaque particles in their cytoplasm early in development. Ribosomes were synthesized, though fewer in number. The endoplasmic reticulum formed more slowly. PMID:10976231

  11. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA

    PubMed Central

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Objectives Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Methods Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. Results The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10−4, OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10−7, OR 0.71; case-only pmeta=1.9×10−4, OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. Conclusions These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. PMID:25180293

  12. [An efficient method for producing monoclonal antibodies against multi-pass membrane proteins].

    PubMed

    Yagi, Hideki; Masuko, Takashi

    2013-01-01

    Antibodies have greatly contributed to the development of medical science and pharmacology, because of their high specificity. The cell fusion method has developed monoclonal antibodies (mAb) technology, such that massive amounts of mAb with a uniform structure can be produced. Although mAb have been produced against many proteins so far, the production of mAb against multi-pass transmembrane proteins, such as G-protein coupled receptor (GPCR) and various transporter proteins has been extremely difficult. The complicated structures, poorly extracellular regions, and high hydrophobicity of multiple-transmembrane proteins make it difficult to produce mAb against them. Production of mAb that recognize the extracellular region of living cells is thought to be important in determining the ability of a protein. Based on these findings, we tried to produce mAb against a multi-pass transmembrane transporter using green fluorescent protein (GFP)-fused full-length target proteins as immunogens. Furthermore, the immunizing method has proved to be important in generating functional mAb. We succeeded in producing functional mAb that react against the extracellular region of a 12-pass transmembrane transporter in a living cell. Based on this success, we began to produce mAb against seven-transmembrane GPCR. In this symposium, we report on the results of producing mAb against S1P receptors, a type of GPCR.

  13. Activation of clones producing self-reactive antibodies by foreign antigen and antiidiotype antibody carrying the internal image of the antigen.

    PubMed Central

    Bailey, N C; Fidanza, V; Mayer, R; Mazza, G; Fougereau, M; Bona, C

    1989-01-01

    Because we found in previous work that a high fraction of antibodies exhibiting various specificities bound to glutamic acid 50-tyrosine50 homopolymer (GT) and expressed pGAT cross-reactive idiotype (IdX), we studied the activation of clones producing multireactive antibodies in 1-mo-old MRL/lpr and C3H/HeJ mice bearing VHJ haplotype. The activation of such clones was studied after mice were immunized with GT in CFA, HP20 (an anti-Id MAb carrying the internal image of GT in the D region), and a synthetic peptide corresponding to the D segment of HP20. Our results indicate that immunized mice produced both GT- and self-reactive antibodies. Study of the immunochemical properties of MAb showed that they exhibit multispecific properties and bind with similar-affinity constants to GT or self-antigens such as DNA, Smith antigen (Sm), and IgG2a. An important fraction of antibodies obtained from MRL/lpr mice immunized with HP20 expressed pGAT IdX and some of these antibodies share IdX expressed on anti-DNA, Sm, and rheumatoid factor (RFs) antibodies. The hybridomas producing multispecific autoantibodies use heavy-chain- (VH) and light-chain-variable region (VK) genes from various V gene families, suggesting that they do not derive from the pool of GAT precursors. Sequencing of VH and VK genes of two antibodies show that they can use closely related VHJ558, unmutated VK1, or different VK genes than those used by anti-GT antibodies. Our data demonstrate that clones producing antibodies binding to GT and self-antigens with similar-affinity constants can be activated by foreign or anti-Id antibodies carrying the internal image of the antigen or even by a synthetic peptide corresponding to the D segment of anti-Id antibodies. Images PMID:2760212

  14. Rapid characterization of hybridomas producing monoclonal antibodies against platelet β3 integrin using ELIspot.

    PubMed

    Maenner, Denis; Werth, Silke; Bein, Gregor; Santoso, Sentot

    2016-12-01

    Generally, B-cell responses against human platelet antigens are assessed by the serological detection of specific platelet antibodies, mostly against β3 integrin. However, this approach seems to be of low sensitivity, since platelet autoantibodies against αIIbβ3 are detected in only 50% of all patients with immune thrombocytopenia (ITP). In this study, a novel B-cell ELIspot method was established to characterize the specificity of mouse monoclonal antibodies (moabs) against human β3 integrin. Moabs produced by hybridomas were immobilized on membrane and bound antibodies were visualized as spots using biotinylated recombinant proteins αIIbβ3 or αvβ3 and the enzyme labeled streptavidin-substrate system. Three hybridomas, Gi5, Gi16 and AP3, designated previously as anti-αIIbβ3, anti-αIIb and anti-β3, respectively, were investigated. Hybridoma producing moab against CD177 was used as the negative control. Whereas AP3 reacted with αIIbβ3 and αvβ3, Gi5 only formed spots with αIIbβ3. Titration analysis showed that the number of spots correlated significantly with the number of seeded cells. Approximately 15 antibody producing hybridoma cells could be identified among 10(3) nonproducing B-cells. Furthermore, superior correlation with the total number of IgG producing cells was obtained. Analysis of the third hybridoma, Gi16 (anti-αIIb), showed only few spots with αIIbβ3, indicating that this hybridoma contained different clones (producer and non-producer). Significant increased number of spots could be identified after re-cloning of these clones by limiting dilution method. Our results demonstrate that this B-cell ELIspot assay can be used for the identification of a small number of hybridoma cells producing moabs against β3 integrin, verification of their monoclonality, productivity and for determining their specificity in the early state of workup steps. In the future, this approach may be useful to define B-cell clones in patients who developed

  15. Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

    PubMed Central

    Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

    2016-01-01

    anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines. PMID:26819303

  16. Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing

    PubMed Central

    Grosse-Hovest, Ludger; Müller, Sigrid; Minoia, Rosa; Wolf, Eckhard; Zakhartchenko, Valeri; Wenigerkind, Hendrik; Lassnig, Caroline; Besenfelder, Urban; Müller, Mathias; Lytton, Simon D.; Jung, Gundram; Brem, Gottfried

    2004-01-01

    Complex recombinant antibody fragments for modulation of immune function such as tumor cell destruction have emerged at a rapid pace and diverse anticancer strategies are being developed to benefit patients. Despite improvements in molecule design and expression systems, the quantity and stability, e.g., of single-chain antibodies produced in cell culture, is often insufficient for treatment of human disease, and the costs of scale-up, labor, and fermentation facilities are prohibitive. The ability to yield mg/ml levels of recombinant antibodies and the scale-up flexibility make transgenic production in plants and livestock an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. Here, we report on the efficient production of a bispecific single-chain antibody in the serum of transgenic rabbits and a herd of nine cloned, transgenic cattle. The bispecific protein, designated r28M, is directed to a melanoma-associated proteoglycan and the human CD28 molecule on T cells. Purified from the serum of transgenic animals, the protein is stable and fully active in mediating target cell-restricted T cell stimulation and tumor cell killing. PMID:15105446

  17. Trans-splicing as a novel method to rapidly produce antibody fusion proteins

    SciTech Connect

    Iwasaki, Ryohei; Kiuchi, Hiroki; Ihara, Masaki; Mori, Toshihiro; Kawakami, Masayuki; Ueda, Hiroshi

    2009-07-03

    To cultivate the use of trans-splicing as a novel means to rapidly express various antibody fusion proteins, we tried to express antibody-reporter enzyme fusions in a COS-1 co-transfection model. When a vector designed to induce trans-splicing with IgH pre-mRNA was co-transfected with a vector encoding the mouse IgM locus, the expression of V{sub H}-secreted human placental alkaline phosphatase (SEAP) as well as Fab-SEAP were successfully expressed both in mRNA and protein levels. Especially, the vectors encoding complementary sequence to S{mu} as a binding domain was accurate and efficient, producing trans-spliced mRNA of up to 2% of cis-spliced one. Since S{mu} sequence should exist in every IgH pre-mRNA, our finding will lead to the rapid production and analysis of various antibody-enzyme fusions suitable for enzyme-linked immunosorbent assay (ELISA) or antibody-dependent enzyme prodrug therapy (ADEPT).

  18. Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

    PubMed Central

    Muellenbeck, Matthias F.; Ueberheide, Beatrix; Amulic, Borko; Epp, Alexandra; Fenyo, David; Busse, Christian E.; Esen, Meral; Theisen, Michael; Mordmüller, Benjamin

    2013-01-01

    Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection. PMID:23319701

  19. Crystal structure of a 3B3 variant - A broadly neutralizing HIV-1 scFv antibody

    SciTech Connect

    Clark, K. Reed; Walsh, Scott T.R.

    2009-12-10

    We present the crystal structure determination of an anti-HIV-1 gp120 single-chain variable fragment antibody variant, 3B3, at 2.5 {angstrom} resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site-directed mutagenesis of the variable heavy chain (V{sub H}) complementary-determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross-clade primary isolates of HIV-1 by interaction with the recessed CD4-binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR-H3 of the V{sub H} domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR-H3 of 3B3, in light of the b12-gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR-L3 of the variable light (VL) domain triggered by a point mutation in CDR-H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the VL and VH domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3-gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the VL and VH domains possibly through more favorable entropic effect through the expulsion of water.

  20. Directed evolution of chemotaxis inhibitory protein of Staphylococcus aureus generates biologically functional variants with reduced interaction with human antibodies.

    PubMed

    Gustafsson, Erika; Rosén, Anna; Barchan, Karin; van Kessel, Kok P M; Haraldsson, Karin; Lindman, Stina; Forsberg, Cecilia; Ljung, Lill; Bryder, Karin; Walse, Björn; Haas, Pieter-Jan; van Strijp, Jos A G; Furebring, Christina

    2010-02-01

    Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a protein that binds and blocks the C5a receptor (C5aR) and formylated peptide receptor, thereby inhibiting the immune cell recruitment associated with inflammation. If CHIPS was less reactive with existing human antibodies, it would be a promising anti-inflammatory drug candidate. Therefore, we applied directed evolution and computational/rational design to the CHIPS gene in order to generate new CHIPS variants displaying lower interaction with human IgG, yet retaining biological function. The optimization was performed in four rounds: one round of random mutagenesis to add diversity into the CHIPS gene and three rounds of DNA recombination by Fragment INduced Diversity (FIND). Every round was screened by phage selection and/or ELISA for decreased interaction with human IgG and retained C5aR binding. The mean binding of human anti-CHIPS IgG decreased with every round of evolution. For further optimization, new amino acid substitutions were introduced by rational design, based on the mutations identified during directed evolution. Finally, seven CHIPS variants with low interaction with human IgG and retained C5aR blocking capacity could be identified.

  1. Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

    PubMed Central

    Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

    2017-01-01

    Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

  2. A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA

    PubMed Central

    Esposito, Maria Valeria; Nunziato, Marcella; Starnone, Flavio; Telese, Antonella; Calabrese, Alessandra; D’Aiuto, Giuseppe; Pucci, Pietro; D’Aiuto, Massimiliano; Baralle, Francisco; D’Argenio, Valeria; Salvatore, Francesco

    2016-01-01

    About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient’s RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient’s transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset. PMID:28009814

  3. Distinct reactivity of the commercially available monoclonal antibodies of D-dimer and plasma FDP testing to the molecular variants of fibrin degradation products.

    PubMed

    Madoiwa, Seiji; Kitajima, Isao; Ohmori, Tsukasa; Sakata, Yoichi; Mimuro, Jun

    2013-10-01

    Fibrin degradation products (FDP) are an important marker of coagulopathy. We assessed the reactivity of the monoclonal antibodies used in clinical laboratory testing (6 D-dimer reagents, D-dimer-1-6; 4 plasma FDP reagents, plasma FDP-1-4) to quantify FDP using in vitro-generated FDP as well as FDP in clinical samples. The monoclonal antibodies used in D-dimer-1, -2, -5, and -6 reacted poorly to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in D-dimer-3 and -4 had better reactivity to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in plasma FDP-2, -3, and -4 reacted well to the high and low molecular weight FDP forms, while the monoclonal antibody in plasma FDP-1 reacted poorly to the low molecular weight FDP forms. Analysis of clinical samples revealed deviations in FDP molecular weight forms in DIC samples. The reactivity of the monoclonal antibodies of laboratory FDP testing to FDP variants in clinical samples was similar to that of in vitro-generated FDP. In conclusion, the monoclonal antibodies used in clinical laboratories to detect FDP have distinct reactivity to the molecular variants of FDP generated in vitro as well as those present in clinical samples. Our findings support the consensus for the standardization of D-dimer and plasma FDP testing.

  4. Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation.

    PubMed

    Dada, Oluwatosin O; Jaya, Nomalie; Valliere-Douglass, John; Salas-Solano, Oscar

    2015-08-20

    Characterization of both the acidic and basic regions of imaged capillary isoelectric focusing (icIEF) profile of an IgG1 antibody was achieved through preparative immobilized pH gradient isoelectric focusing (IPG-IEF) fractionation. Recent attempts at using this method to fractionate charge variants of monoclonal antibodies (mAbs) have shown promising results, but identification of the chemical modifications in the variants was limited to the basic species. We have optimized the method to achieve enrichment of each variant across the icIEF profile of an IgG1 mAb. The fractionation was followed by extended characterization to elucidate the composition of the acidic, main, and basic species observed in the icIEF profile. Deamidation, sialylation, glycation, and fragmentation were identified as the main modifications contributing to acidic variants of the mAb while C-terminal lysine, C-terminal proline amidation, and uncyclized N-terminal glutamine were the major species contributing to the basic variants. This characterization allows a better understanding of the modifications that contribute to the charge variants observed by icIEF, facilitating the evaluation of impacts on product safety and efficacy.

  5. Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

    PubMed

    Zhao, Jian; Giles, Brendan M; Taylor, Rhonda L; Yette, Gabriel A; Lough, Kara M; Ng, Han Leng; Abraham, Lawrence J; Wu, Hui; Kelly, Jennifer A; Glenn, Stuart B; Adler, Adam J; Williams, Adrienne H; Comeau, Mary E; Ziegler, Julie T; Marion, Miranda; Alarcón-Riquelme, Marta E; Alarcón, Graciela S; Anaya, Juan-Manuel; Bae, Sang-Cheol; Kim, Dam; Lee, Hye-Soon; Criswell, Lindsey A; Freedman, Barry I; Gilkeson, Gary S; Guthridge, Joel M; Jacob, Chaim O; James, Judith A; Kamen, Diane L; Merrill, Joan T; Sivils, Kathy Moser; Niewold, Timothy B; Petri, Michelle A; Ramsey-Goldman, Rosalind; Reveille, John D; Scofield, R Hal; Stevens, Anne M; Vilá, Luis M; Vyse, Timothy J; Kaufman, Kenneth M; Harley, John B; Langefeld, Carl D; Gaffney, Patrick M; Brown, Elizabeth E; Edberg, Jeffrey C; Kimberly, Robert P; Ulgiati, Daniela; Tsao, Betty P; Boackle, Susan A

    2016-01-01

    Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  6. Clinical Associations of Biallelic and Monoallelic TNFRSF13B Variants in Italian Primary Antibody Deficiency Syndromes

    PubMed Central

    Pulvirenti, Federica; Zuntini, Roberta; Milito, Cinzia; Specchia, Fernando; Spadaro, Giuseppe; Danieli, Maria Giovanna; Pession, Andrea; Quinti, Isabella; Ferrari, Simona

    2016-01-01

    We assessed the prevalence of TNFRSF13B mutations and the clinical correlates in an Italian cohort of 189 CVID, 67 IgAD patients, and 330 healthy controls to substantiate the role of TACI genetic testing in diagnostic workup. We found that 11% of CVID and 13% of IgAD carried at least one mutated TNFRSF13B allele. Seven per cent of CVID had monoallelic-mutations and 4% had biallelic-mutations. The frequency of C104R monoallelic-mutations was not higher than that found in healthy controls. Biallelic-mutations were exclusively found in CVID. CVID patients carrying monoallelic-mutations had an increased prevalence of lymphadenopathy, granulomata, and autoimmune cytopenias. CVID carrying biallelic-mutations had a low prevalence of autoimmunity in comparison with TACI wild-type CVID. Moreover, biallelic-mutated CVID had higher frequency of switched memory B-cells and higher IgM and IgA antibodies to polysaccharide antigens than TACI wild-type and monoallelic-mutated CVID. TACI-mutated IgAD patients had only monoallelic-mutations and did not display clinical difference from IgAD wild-type patients. In conclusion, TNFRSF13B genetic screening of antibody deficiencies may allow the identification of mutational patterns. However, as with counseling for risk assessment, geneticists should be aware that the interpretation of genetic testing for TACI mutations is difficult and the potential impact on clinical management is still limited. PMID:27123465

  7. A region of the N-terminal domain of meningococcal factor H-binding protein that elicits bactericidal antibody across antigenic variant groups.

    PubMed

    Beernink, Peter T; LoPasso, Carla; Angiolillo, Antonella; Felici, Franco; Granoff, Dan

    2009-05-01

    Meningococcal factor H-binding protein (fHbp) is a promising vaccine antigen. Previous studies described three fHbp antigenic variant groups and identified amino acid residues between 100 and 255 as important targets of variant-specific bactericidal antibodies. We investigated residues affecting expression of an epitope recognized by a murine IgG2a anti-fHbp mAb, designated JAR 4, which cross-reacted with fHbps in variant group 1 or 2 (95% of strains), and elicited human complement-mediated, cooperative bactericidal activity with other non-bactericidal anti-fHbp mAbs with epitopes involving residues between 121 and 216. From filamentous bacteriophage libraries containing random peptides that were recognized by JAR 4, we identified a consensus tripeptide, DHK that matched residues 25-27 in the N-terminal domain of fHbp. Since DHK was present in both JAR 4-reactive and non-reactive fHbps, the tripeptide was necessary but not sufficient for reactivity. Based on site-directed mutagenesis studies, the JAR 4 epitope could either be knocked out of a reactive variant 1 fHbp, or introduced into a non-reactive variant 3 protein. Collectively, the data indicated that the JAR 4 epitope was discontinuous and involved DHK residues beginning at position 25; YGN residues beginning at position 57; and a KDN tripeptide that was present in variant 3 proteins beginning at position 67 that negatively affected expression of the epitope. Thus, the region of fHbp encompassing residues 25-59 in the N-terminal domain is important for eliciting antibodies that can cooperate with other anti-fHbp antibodies for cross-reactive bactericidal activity against strains expressing fHbp from different antigenic variant groups.

  8. Feasibility studies of using the Catfish Immune System to produce monoclonal antibodies

    SciTech Connect

    Poston, T.M.

    1987-03-01

    The objective of these studies was to determine the feasibility of using a teleost cell line to produce monoclonal antibodies. Studies were undertaken to demonstrate the production of a polyclonal response of channel catfish (Icatalurus punctatus) challenged with mycotoxins coupled to a protein carrier. Companion studies were also performed to induce a permanent cell line with catfish lymphocytes. Attempts to demonstrate a polyclonal response to haptenized mycotoxins were inconclusive. Tests to induce an immortal, permanent cell line with benzene and x-ray irradiated cells were also inconclusive. 3 refs., 13 tabs.

  9. Mutation Detection in an Antibody-Producing Chinese Hamster Ovary Cell Line by Targeted RNA Sequencing

    PubMed Central

    Zhang, Siyan; Hughes, Jason D.; Murgolo, Nicholas; Levitan, Diane; Chen, Janice; Liu, Zhong

    2016-01-01

    Chinese hamster ovary (CHO) cells have been used widely in the pharmaceutical industry for production of biological therapeutics including monoclonal antibodies (mAb). The integrity of the gene of interest and the accuracy of the relay of genetic information impact product quality and patient safety. Here we employed next-generation sequencing, particularly RNA-seq, and developed a method to systematically analyze the mutation rate of the mRNA of CHO cell lines producing a mAb. The effect of an extended culturing period to mimic the scale of cell expansion in a manufacturing process and varying selection pressure in the cell culture were also closely examined. PMID:27088091

  10. IgE antibodies produced in mice instrumental in analyses of antigenicity of cephalothin preparation.

    PubMed

    Muranaka, M; Tadokoro, K; Hirai, K; Koizumi, K; Fukuba, S; Suzuki, S

    1980-01-01

    IgE antibodies to a cephalothin preparation were produced in mice which had been immunized with cephalothin-Ascaris extract conjugate mixed with Al(OH)3 gel plus Bordetella pertussis. Mice which had been irradiated just before receiving the booster injection wtih the conjugate showed higher IgE antibody levels against the preparation in comparison with unirradiated mice. The specificities of IgE antibodies against the preparation were examined with an inhibition test of passive cutaneous anaphylactic (PCA) reaction. The intensity of the PCA reaction evoked by the cephalothin preparation in the rats was substantially or partially reduced by a pretreatment of the animals with an injection of a cephalothin preparation from another source or a cephaloridine preparation. Meanwhile, a prior injection of a cefazolin, a 7-aminocephalosporanic acid, a benzylpenicillin (PcG) preparation, or an aminobenzylpenicillin preparation into the animals showed only weak or no influence on the PCA reaction. These results suggest that the acyl side chain of cephalothin plays an important role as eliciting antigenic determinant in the PCA reaction. The difference in the antigenic specificity between the cephalothin and PcG preparation is also discussed.

  11. Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits.

    PubMed

    Acero, G; Manoutcharian, K; Vasilevko, V; Munguia, M E; Govezensky, T; Coronas, G; Luz-Madrigal, A; Cribbs, D H; Gevorkian, G

    2009-08-18

    N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.

  12. IMMUNODOMINANT EPITOPE AND PROPERTIES OF PYROGLUTAMATE-MODIFIED Aβ-SPECIFIC ANTIBODIES PRODUCED IN RABBITS

    PubMed Central

    Acero, G.; Manoutcharian, K.; Vasilevko, V.; Munguia, M.E.; Govezensky, T.; Coronas, G.; Luz-Madrigal, A.; Cribbs, DH.; Gevorkian, G.

    2009-01-01

    N-truncated and N-modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer’s disease (AD) and Down’s syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Aβ is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full-length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 3 (AβN3(pE)). We demonstrated that AβN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AβN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AβN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AβN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:19545911

  13. Semiautomated pH gradient ion-exchange chromatography of monoclonal antibody charge variants.

    PubMed

    Talebi, Mohammad; Shellie, Robert A; Hilder, Emily F; Lacher, Nathan A; Haddad, Paul R

    2014-10-07

    A new approach using a chromatography system equipped with isocratic pumps and an electrolytic eluent generator (EG) is introduced, replacing external pH gradient delivery using conventional gradient systems, in which bottled buffers with preadjusted pH are mixed using a gradient pump. The EG is capable of generating high purity base or acid required for online preparation of the buffer at the point of use, utilizing deionized water as the only carrier stream. Typically, the buffer was generated from online titration of a reagent composed of low molecular weight amines. The reagent was delivered isocratically into a static mixing tee, where it was titrated to the required pH with electrolytically generated base or acid. The required pH gradient was thus conveniently generated by electrically controlling the concentration of titrant. Also, since the pH was adjusted at the point of use, this approach offered enhanced throughput in terms of eluent preparation time and labor, and with a more reproducible pH profile. The performance of the system was demonstrated by running pH gradients ranging from pH 8.2 to 10.9 on a polymer monolith cation-exchange column for high throughput profiling of charge heterogeneity of intact, basic therapeutic monoclonal antibodies. A high degree of flexibility in modulating the key parameters of the pH gradient, including the buffer concentration, the pH gradient slope and the operating pH range was demonstrated. This enabled fine-tuning of the separation conditions for each individual antibody in order to enhance the chromatographic resolution.

  14. Bioengineering of a Nisin A‐producing Lactococcus lactis to create isogenic strains producing the natural variants Nisin F, Q and Z

    PubMed Central

    Piper, Clare; Hill, Colin; Cotter, Paul D.; Ross, R. Paul

    2011-01-01

    Summary Nisin is the prototypical example of the lantibiotic family of antimicrobial peptides and has been employed as a food preservative for over half a century. It has also attracted attention due to its potency against a number of multidrug‐resistant clinical pathogens. Nisin A is the originally isolated form of Nisin and a further five natural variants have been described which differ by up to 10 amino acids (of 34 in total in Nisin A). Nisins A, Z, F and Q are produced by Lactococcus lactis, while Nisins U and U2 are produced by Streptococcus sp. In this study we bioengineered the nisA gene of a Nisin A producer to generate genes encoding Nisins Z, F, Q, U and U2. We determined that while active Nisin Z, F and Q can be produced against this genetic background, active forms of Nisin U and U2 are not generated. Minimum inhibitory concentration studies with Nisin A, Z, F and Q variants against a series of different clinically significant pathogens establish differences in specific activities against selected targets. Nisin F was most impressive, being the most active, or one of the most active, against the MRSA strain ST 525, EC 676, EC 725, VISA 22900, VISA 22781, hVISA 35197, Staphylococcus aureus 8325‐4 and L. lactis HP. Nisin Z was most active against ST 299, hVISA 32683 and, together with Nisin F, HP but had contrastingly poor activity against ST 525, EC 676 and 8325‐4. Nisin F, Q and A exhibited similar potency against VISA 22900. This was the only target against which Nisin Q and Nisin A were among the most active variants. PMID:21375711

  15. Bioengineering of a Nisin A-producing Lactococcus lactis to create isogenic strains producing the natural variants Nisin F, Q and Z.

    PubMed

    Piper, Clare; Hill, Colin; Cotter, Paul D; Ross, R Paul

    2011-05-01

    Nisin is the prototypical example of the lantibiotic family of antimicrobial peptides and has been employed as a food preservative for over half a century. It has also attracted attention due to its potency against a number of multidrug-resistant clinical pathogens. Nisin A is the originally isolated form of Nisin and a further five natural variants have been described which differ by up to 10 amino acids (of 34 in total in Nisin A). Nisins A, Z, F and Q are produced by Lactococcus lactis, while Nisins U and U2 are produced by Streptococcus sp. In this study we bioengineered the nisA gene of a Nisin A producer to generate genes encoding Nisins Z, F, Q, U and U2. We determined that while active Nisin Z, F and Q can be produced against this genetic background, active forms of Nisin U and U2 are not generated. Minimum inhibitory concentration studies with Nisin A, Z, F and Q variants against a series of different clinically significant pathogens establish differences in specific activities against selected targets. Nisin F was most impressive, being the most active, or one of the most active, against the MRSA strain ST 525, EC 676, EC 725, VISA 22900, VISA 22781, hVISA 35197, Staphylococcus aureus 8325-4 and L. lactis HP. Nisin Z was most active against ST 299, hVISA 32683 and, together with Nisin F, HP but had contrastingly poor activity against ST 525, EC 676 and 8325-4. Nisin F, Q and A exhibited similar potency against VISA 22900. This was the only target against which Nisin Q and Nisin A were among the most active variants. © 2010 The Authors. Journal compilation © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  16. A ribonucleic acid-specific antibody produced during autoimmune disease: evidence for nucleotide sequence specificity.

    PubMed

    Eilat, D; Ben Sasson, S A; Laskov, R

    1980-11-01

    A hybridoma secreting RNA-binding autoantibody has been produced by fusion of spleen cells from autoimmune NZB/NZWF1 mice with drug-resistant IgG2b-producing myeloma cells from BALB/c mice. Studies on the specificity of the purified monoclonal autoantibody revealed: (a) absolute specificity for ribopolynucleotides as compared with deoxyribopolynucleotides; (b) specificity for single-stranded RNA as compared with double-stranded RNA; (c) high affinity for the random copolymer poly(G, C); and (d) preference for the random heteropolymer poly(G, C, U). These studies were complemented by stoichiometric measurements of the antibody-RNA complex and computer analysis of the abundance of various di-, tri- and tetranucleotide sequences in native RNA. Taken together, these data suggest that the antigenic determinant recognized by the monoclonal autoantibody is largely composed of a trinucleotide sequence of single-stranded RNA containing, G, C and U residues.

  17. Anti-human AFP variant monoclonal antibody in radioimmunodetection of primary hepatocellular carcinoma

    PubMed Central

    Liu, Yang; Wu, Meng-Chao; Chen, Han; Zhang, Bai-He; Qian, Guang-Xiang; Pan, Wen-Zhou; Qiang, Mei-Yu

    1997-01-01

    AIM: To investigate the affinity of AFP-R-LCA monoclonal antibody (AFP-R-LCA McAb) for AFP-positive primary hepatocellular carcinoma (HCC) cells. METHODS: AFP-R-LCA McAb was labeled by 131I. Eleven cases of HCC with AFP positivity, 6 with AFP negativity, and 4 with hepatitis B-related cirrhosis were investigated by radioimmunodetection. RESULTS: The 131I-AFP-R-LCA McAb immunoreacted with 9 of the HCC AFP-positive cases (9/11), but with none of the 6 AFP negative HCC cases or of the 4 cirrhosis patients. 131I-AFP-R-LCA McAb at a small dose (7.4 × 107 Bq/300 μg) was associated with no side effects as determined by the liver function test, prothrombin time (Pt) test and thyroid gland function test (P > 0.05). Two cases of AFP-positive HCC were not imaged because of large tumor size (diameter > 10 cm) and higher AFP concentration in serum (20000 μg/L). CONCLUSION: AFP-R-LCA McAb has a strong and special affinity to AFP-positive HCC cells and may be useful as a carrier for radioimmunodetection and radioimmunotherapy. PMID:27053873

  18. Combined Linkage and Association Studies Show that HLA Class II Variants Control Levels of Antibodies against Epstein-Barr Virus Antigens

    PubMed Central

    Cobat, Aurélie; Guergnon, Julien; Brice, Pauline; Fermé, Christophe; Carde, Patrice; Hermine, Olivier; Pendeven, Catherine Le-; Amiel, Corinne; Taoufik, Yassine; Alcaïs, Alexandre; Theodorou, Ioannis; Besson, Caroline; Abel, Laurent

    2014-01-01

    Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10–5 for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL. PMID:25025336

  19. Combined linkage and association studies show that HLA class II variants control levels of antibodies against Epstein-Barr virus antigens.

    PubMed

    Pedergnana, Vincent; Syx, Laurène; Cobat, Aurélie; Guergnon, Julien; Brice, Pauline; Fermé, Christophe; Carde, Patrice; Hermine, Olivier; Le-Pendeven, Catherine; Amiel, Corinne; Taoufik, Yassine; Alcaïs, Alexandre; Theodorou, Ioannis; Besson, Caroline; Abel, Laurent

    2014-01-01

    Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10(-5) for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL.

  20. New Monoclonal Antibodies against a Novel Subtype of Shiga Toxin 1 Produced by Enterobacter cloacae and Their Use in Analysis of Human Serum.

    PubMed

    Skinner, Craig; Patfield, Stephanie; Khalil, Rowaida; Kong, Qiulian; He, Xiaohua

    2016-01-01

    Shiga toxin (Stx) is a major virulence factor of several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges for the clinical diagnosis of infections caused by Stx-producing organisms. Here, we report the development of four new monoclonal antibodies (MAbs) against Stx1e, a novel subtype of Stx1 that was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these MAbs were Stx1 specific, bound to the A subunit, and had distinct preferences for subtypes of Stx1. Of the four MAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from Stx1e in Vero cells. Enzyme-linked immunosorbent assays assembled with these high-affinity MAbs detected Stx1e at concentrations as low as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity, were investigated with the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms. IMPORTANCE Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently discovered in

  1. New Monoclonal Antibodies against a Novel Subtype of Shiga Toxin 1 Produced by Enterobacter cloacae and Their Use in Analysis of Human Serum

    PubMed Central

    Skinner, Craig; Patfield, Stephanie; Khalil, Rowaida; Kong, Qiulian

    2016-01-01

    ABSTRACT Shiga toxin (Stx) is a major virulence factor of several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges for the clinical diagnosis of infections caused by Stx-producing organisms. Here, we report the development of four new monoclonal antibodies (MAbs) against Stx1e, a novel subtype of Stx1 that was produced by an Enterobacter cloacae strain and had limited reactivity with existing anti-Stx1 antibodies. Western blot analysis indicates that these MAbs were Stx1 specific, bound to the A subunit, and had distinct preferences for subtypes of Stx1. Of the four MAbs, Stx1e-2 was capable of partially neutralizing cytotoxicities derived from Stx1e in Vero cells. Enzyme-linked immunosorbent assays assembled with these high-affinity MAbs detected Stx1e at concentrations as low as 4.8 pg/ml in phosphate-buffered saline and 53.6 pg/ml in spiked human serum samples and were also capable of distinguishing Stx1e-producing strains in enriched cultures. These assays may therefore have clinical value in diagnosing Stx1e-producing bacterial infection. Additionally, characteristics of Stx1e, such as the origin of stx1e genes, conditions for toxin expression, receptor binding, and cytotoxicity, were investigated with the new antibodies developed in this study. This information should be useful for further understanding the clinical significance and prevalence of Stx1e-harboring E. cloacae and other organisms. IMPORTANCE Stxs are among the most clinically important virulence factors of Shigella and enterohemorrhagic Escherichia coli. There are many varieties of Stx, and although Stx1a and Stx2a are the most common and widely distributed types of Stx, new variants of Stx are continually emerging. These new variants of Stx can be challenging to detect, since most Stx detection kits are optimized for the detection of Stx1a and Stx2a. Stx1e, recently

  2. Verotoxins in Bovine and Meat Verotoxin-Producing Escherichia coli Isolates: Type, Number of Variants, and Relationship to Cytotoxicity▿

    PubMed Central

    Krüger, Alejandra; Lucchesi, Paula M. A.; Parma, Alberto E.

    2011-01-01

    In this study, we determined vt subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing Escherichia coli (VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a vt2 gene. Moreover, the vt2 subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other vt subtypes detected were vt1, vt1d, vt2vha, vt2vhb, vt2O118, vt2d (mucus activatable), and vt2g. A total of 41 (22%) of the isolates possessed more than one vt subtype in its genome, and among them the most frequent combination was vt1/vt2, but we also observed multiple combinations among vt2 subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single vt2 variant, those carrying the vt2 subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying vt2vha or vt2hb variants. Notably, the isolates carrying the vt1 subtype showed a lesser increase than that of most of the vt2-positive VTEC strains. Furthermore, the presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of vt variants more than with the serotype or origin of the isolate. PMID:21037301

  3. [Importance of the conjugated antibody for the induction of selective effect of adriamycin conjugated with anti AFP monoclonal antibody and entrapped in liposomes against AFP producing tumors].

    PubMed

    Konno, H; Kumai, K; Tsubouchi, T; Ishibiki, K; Abe, O; Tadakuma, T; Yasuda, T; Nagaike, K; Hosokawa, S; Sakaguchi, S

    1989-06-01

    We investigated experimentally the effect of adriamycin (ADM) conjugated with anti alpha-fetoprotein (AFP) monoclonal antibodies and entrapped in liposomes (Lip-ADM = AbAFP) in vitro or in vivo. In the present study, we examined the importance of the conjugated antibody for the induction of selective therapeutic effect of Lip-ADM = AbAFP against AFP producing tumors. As the target tumors, AFP producing human hepatoma strain, Li-7, and AFP non-producing human breast cancer strain, MX-1 maintained in BALB/c nu/nu male mice were used. In order to evaluate the importance of the conjugated antibody, we prepared also ADM conjugated with normal mouse IgG, and entrapped in liposomes Lip-ADM = NIgG, of which therapeutic effects were compared with that of Lip-ADM = AbAFP. Judging from the tumor growth curve and the tumor weight, the therapeutic effect of Lip-ADM = AbAFP was greater against Li-7 than that of Lip-ADM = NIgG. On the other hand, both conjugates showed similar effects against MX-1. As the results it is suggested that the antibody which recognizes the antigen expressed on the target tumor cells can solely increase the therapeutic effect of ADM entrapped in liposomes (Lip-ADM) and that the main factors which contribute to the efficient therapeutic effect of the conjugate were the sensitibility to ADM, the affinity of the tumor cells to liposomes and the superiority of the conjugated antibody.

  4. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

    PubMed

    Tada, Minoru; Tatematsu, Ken-ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

  5. Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

    PubMed Central

    Tada, Minoru; Tatematsu, Ken-Ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

    2015-01-01

    In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. PMID:26261057

  6. Antigen identification and characterization of lung cancer specific monoclonal antibodies produced by mAb proteomics.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Guergova-Kuras, Mariana; Kadas, Janos; Takacs, Laszlo; Karger, Barry L

    2010-04-05

    A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.

  7. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    DOE PAGES

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; ...

    2015-07-13

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In thismore » paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.« less

  8. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    SciTech Connect

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; Sapparapu, Gopal; Crowe, James. E.; Spiller, Benjamin W.

    2015-07-13

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. In this paper, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. Finally, H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility.

  9. Vaccine-elicited antibody that neutralizes H5N1 influenza and variants binds the receptor site and polymorphic sites

    PubMed Central

    Winarski, Katie L.; Thornburg, Natalie J.; Yu, Yingchun; Sapparapu, Gopal; Crowe, James. E.; Spiller, Benjamin W.

    2015-01-01

    Antigenic drift of circulating seasonal influenza viruses necessitates an international vaccine effort to reduce the impact on human health. A critical feature of the seasonal vaccine is that it stimulates an already primed immune system to diversify memory B cells to recognize closely related, but antigenically distinct, influenza glycoproteins (hemagglutinins). Influenza pandemics arise when hemagglutinins to which no preexisting adaptive immunity exists acquire the capacity to infect humans. Hemagglutinin 5 is one subtype to which little preexisting immunity exists and is only a few acquired mutations away from the ability to transmit efficiently between ferrets, and possibly humans. Here, we describe the structure and molecular mechanism of neutralization by H5.3, a vaccine-elicited antibody that neutralizes hemagglutinin 5 viruses and variants with expanded host range. H5.3 binds in the receptor-binding site, forming contacts that recapitulate many of the sialic acid interactions, as well as multiple peripheral interactions, yet is not sensitive to mutations that alter sialic acid binding. H5.3 is highly specific for a subset of H5 strains, and this specificity arises from interactions to the periphery of the receptor-binding site. H5.3 is also extremely potent, despite retaining germ line-like conformational flexibility. PMID:26170302

  10. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. CHO-gmt5, a novel CHO glycosylation mutant for producing afucosylated and asialylated recombinant antibodies.

    PubMed

    Haryadi, Ryan; Zhang, Peiqing; Chan, Kah Fai; Song, Zhiwei

    2013-01-01

    Engineered zinc-finger nucleases (ZFNs) are powerful tools for creating double-stranded-breaks (DSBs) in genomic DNA in a site-specific manner. These DSBs generated by ZFNs can be repaired by homology-directed repair or nonhomologous end joining, in which the latter can be exploited to generate insertion or deletion mutants. Based on published literature, we designed a pair of zinc-finger nucleases and inactivated the GDP-fucose transporter gene (Slc35c1) in a previously reported CHO mutant that has a dysfunctional CMP-sialic acid transporter gene (Slc35a1). The resulting mutant cell line, CHO-gmt5, lacks functional GDP-fucose transporter and CMP-sialic acid transporter. As a result, these cells can only produce asialylated and afucosylated glycoproteins. It is now widely recognized that removal of the core fucose from the N-glycans attached to Asn(297) of human IgG1 significantly enhances its binding to its receptor, FcγRIIIa, and thereby dramatically improves antibody-dependent cellular cytotoxicity (ADCC). Recent reports showed that removal of sialic acid from IgG1 also enhances ADCC. Therefore, CHO-gmt5 may represent a more advantageous cell line for the production of recombinant antibodies with enhanced ADCC. These cells show comparable growth rate to wild type CHO-K1 cells and uncompromised transfection efficiency, which make them desirable for use as a production line.

  12. Hepatitis C Virus Hypervariable Region 1 Variants Presented on Hepatitis B Virus Capsid-Like Particles Induce Cross-Neutralizing Antibodies

    PubMed Central

    Bankwitz, Dorothea; Osburn, William; Viazov, Sergei; Brovko, Olena; Zekri, Abdel-Rahman; Khudyakov, Yury; Nassal, Michael; Pumpens, Paul; Pietschmann, Thomas; Timm, Jörg; Roggendorf, Michael; Walker, Andreas

    2014-01-01

    Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses. PMID:25014219

  13. Effect of cultivation conditions on the activity of the beromycin producer Streptomyces glomeratus 3980 and its spontaneous variants.

    PubMed

    Blumauerová, M; Podojil, M; Gauze, G F; Maksimova, T S; Panos, J; Vanĕk, Z

    1980-01-01

    Optimal conditions for the submerged cultivation of Streptomyces glomeratus 3980, producer of the anthracycline antibiotics beromycins, and its variants were sought in media with glucose, soybean meal and salts differing in the content of ammonium sulphate. As compared with the original activity of the strain the antibiotic titre of some variants increased about 12 times on increasing the glucose concenration from 3 to 5%, or on omitting CaCO3 from the medium (i.e. under conditions leading to an increased production of propionic acid and suppression of production of the melanin-like pigment). In melanin-less variants accumulating propionate even under standard conditions the activity increased about 18-40 times in the medium with 3% glucose and 0.2% CaCO3 under conditions of more intensive aeration (i.e. under conditions when no propionic acid accumulated). Individual strains also differed in the requirement for (NH4)2SO4 in the medium, their response to changes of volume of the vegetative inoculum and semsitivity to MgSO4 x 7H2O. The biosynthetic activity of all strains was inhibited by the addition of ZnSO4 x 7H2O or CaCl2 and substitution of glucose with starch, lactose or sucrose.

  14. Identification and characterization of a truncated variant of the 5-hydroxytryptamine(2A) receptor produced by alternative splicing.

    PubMed

    Guest, P C; Salim, K; Skynner, H A; George, S E; Bresnick, J N; McAllister, G

    2000-09-08

    We have identified an alternatively spliced 5-hydroxytryptamine 2A receptor (5-HT(2A)-R) transcript by PCR of human brain cDNA using degenerate oligonucleotide primers to transmembrane (TM) domains 3 and 7 of the 5-HT(2)-R subfamily. The variant contains a 118-bp insertion at the exon II/III boundary of the 5-HT(2A)-R, which produces a frame shift in the coding sequence and a premature stop codon. PCR analysis showed that the truncated receptor (5-HT(2A-tr)) and native 5-HT(2A)-R were co-expressed in most brain tissues, with the highest levels being found in hippocampus, corpus collosum, amygdala and caudate nucleus. Western blot analysis of HEK-293 cells transfected transiently with a 5-HT(2A-tr) construct showed that a 30-kDa protein was expressed on cell membranes. Co-transfection studies showed no effect of the 5-HT(2A-tr) variant on 3H-ketanserin binding to the native 5-HT(2A)-R or on functional coupling of the 5-HT(2A)-R to 5-HT-stimulated Ca(2+) mobilization. The functional significance of the 5-HT(2A-tr) variant and other truncated receptors remains to be established.

  15. Specificity and isotype of Rh specific antibodies produced by human B-cell lines established from alloimmunized Rh negative women.

    PubMed

    Pasha, Roya Payam Khaja; Bahrami, Zahra Samadi; Niroomanesh, Shirin; Ramzi, Fereshteh; Razavi, Ali Reza; Shokri, Fazel

    2005-10-01

    Despite the successful outcome of anti-D prophylaxis program, alloimmunization still occurs. The aim of this study was to examine the specificity and isotype of anti-Rh antibodies in plasma samples of Rh negative alloimmunized individuals and to study the same parameters in lymphoblastoid cell lines (LCLs) generated from the same donors. Specificity of anti-Rh antibodies was determined in plasma of nine alloimmunized subjects by direct hemagglutination using a panel of known RBC genotypes and isotype of specific antibodies were identified by an antigen specific ELISA. Similar methods were employed to determine specificity and isotype of antibodies produced by Rh specific LCLs established from four donors. LCLs were generated by Epstein-Barr virus transformation of peripheral blood mononuclear cells isolated from each donor followed by their culture over a feeder of human fetal fibroblasts. Upon emergence of lymphoblastoid cells, culture supernatants were assayed for presence of Rh specific antibody by hemagglutination assay. Anti-D was the predominant antibody in both plasma samples and among the 128 established LCLs; however, antibodies to other Rh specificities namely C and E were also produced. The isotype of anti-Rh antibody in all plasma samples was found to be IgG, predominantly IgG1, combined in 7 samples with IgM. Similarly 76%, 9.2% and 14.8% of LCLs were determined to produce antibody of IgG, IgM and of both isotypes, respectively. The data supported that the D antigen is the immunodominant component of the Rh system as indicated by the in vitro and in vivo profiles of Rh specificities in our alloimmunized subjects.

  16. Clinical Significance of Heparanase Splice Variant (T5) in Renal Cell Carcinoma: Evaluation by a Novel T5-Specific Monoclonal Antibody

    PubMed Central

    Barash, Uri; Arvatz, Gil; Farfara, Roy; Naroditsky, Inna; Doweck, Ilana; Feld, Sari; Ben-Izhak, Ofer; Ilan, Neta; Nativ, Ofer; Vlodavsky, Israel

    2012-01-01

    T5 is a novel splice variant of heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites. T5 splice variant is endowed with pro-tumorigenic properties, enhancing cell proliferation, anchorage independent growth and tumor xenograft development despite lack of heparan sulfate-degrading activity typical of heparanase. T5 is over expressed in the majority of human renal cell carcinoma biopsies examined, suggesting that this splice variant is clinically relevant. T5 is thought to assume a distinct three-dimensional conformation compared with the wild type heparanase protein. We sought to exploit this presumed feature by generating monoclonal antibodies that will recognize the unique structure of T5 without, or with minimal recognition of heparanase, thus enabling more accurate assessment of the clinical relevance of T5. We provide evidence that such a monoclonal antibody, 9c9, preferentially recognizes T5 compared with heparanase by ELISA, immunoblotting and immunohistochemistry. In order to uncover the clinical significance of T5, a cohort of renal cell carcinoma specimens was subjected to immunostaining applying the 9c9 antibody. Notably, T5 staining intensity was significantly associated with tumor size (p = 0.004) and tumor grade (p = 0.02). Our results suggest that T5 is a functional, pro-tumorigenic entity. PMID:23251556

  17. Repair of 254 nm ultraviolet-induced (6-4) photoproducts: monoclonal antibody recognition and differential defects in xeroderma pigmentosum complementation groups A, D, and variant

    SciTech Connect

    Hiramoto, T.; Matsunaga, T.; Ichihashi, M.; Nikaido, O.; Fujiwara, Y.; Mishima, Y. )

    1989-11-01

    Repair kinetics of ultraviolet (UV) light-induced (6-4) photoproducts in xeroderma pigmentosum complementation group A, D, and variant cells were studied by the enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody raised against (6-4) photoproducts, together with unscheduled DNA synthesis (UDS) and loss of T4 endonuclease V-susceptible sites (ESS). Group AXP35KO cells completely failed to repair both ESS (cyclobutane pyrimidine dimers) and antibody-recognizing (6-4) photoproducts until tested 24 h after irradiation, and had 2% early-time UDS. Group DXP43KO cells showed about 10% removal of both (6-4) photoproducts and ESS in 24 h, despite showing a residually higher level of 40% early-time and cumulative UDS. Thus, the results substantiated the extreme UV hypersensitivity of XP group A and D cells. However, XP52KO variant cells exhibited the normal level of UDS and ESS loss, but a slightly reduced repair of antibody-recognizing (6-4) photoproducts at 6 and 12 h after irradiation, which may account for a small UV hypersensitivity of the XP variant cells.

  18. Risk of squamous cell skin cancer after organ transplant associated with antibodies to cutaneous papillomaviruses, polyomaviruses, and TMC6/8 (EVER1/2) variants.

    PubMed

    Madeleine, Margaret M; Carter, Joseph J; Johnson, Lisa G; Wipf, Gregory C; Davis, Connie; Berg, Daniel; Nelson, Karen; Daling, Janet R; Schwartz, Stephen M; Galloway, Denise A

    2014-10-01

    Squamous cell skin cancer (SCSC) disproportionately affects organ transplant recipients, and may be related to increased viral replication in the setting of immune suppression. We conducted a nested case-control study among transplant recipients to determine whether SCSC is associated with antibodies to cutaneous human papillomaviruses (HPV), to genes associated with a rare genetic susceptibility to HPV (TMC6/TMC8), or to human polyomaviruses (HPyV). Cases (n = 149) had histologically confirmed SCSC, and controls (n = 290) were individually matched to cases on time since transplant, type of transplant, gender, and race. All subjects had serum drawn immediately prior to transplant surgery. Antibodies to 25 cutaneous HPVs and six HPyVs were assayed by detection of binding to virus-like particles, and 11 TMC6/8 variants were genotyped. After correction for multiple comparisons, only antibodies to HPV37 were associated with SCSC (OR 2.0, 95% CI 1.2-3.4). Common genetic variants of TMC6/8 were not associated with SCSC, but three variants in TMC8 (rs12452890, rs412611, and rs7208422) were associated with greater seropositivity for species 2 betapapillomaviruses among controls. This study suggests that some betaHPVs, but not polyomaviruses, may play a role in the excess risk of SCSC among transplant recipients.

  19. Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing

    PubMed Central

    Vollmers, Christopher; Penland, Lolita; Kanbar, Jad N.; Quake, Stephen R.

    2015-01-01

    Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain. PMID:25611855

  20. Neonatal antibody responses are attenuated by interferon-γ produced by NK and T cells during RSV infection.

    PubMed

    Tregoning, John S; Wang, Belinda Lei; McDonald, Jacqueline U; Yamaguchi, Yuko; Harker, James A; Goritzka, Michelle; Johansson, Cecilia; Bukreyev, Alexander; Collins, Peter L; Openshaw, Peter J

    2013-04-02

    Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4(+) and CD8(+) cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-γ, and blocking IFN-γ also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-γ reduced antibody titer, confirming that IFN-γ plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-γ-secreting cells might, in some situations, be less protective than those that do not.

  1. Neonatal antibody responses are attenuated by interferon-γ produced by NK and T cells during RSV infection

    PubMed Central

    Tregoning, John S.; Wang, Belinda Lei; McDonald, Jacqueline U.; Yamaguchi, Yuko; Harker, James A.; Goritzka, Michelle; Johansson, Cecilia; Bukreyev, Alexander; Collins, Peter L.; Openshaw, Peter J.

    2013-01-01

    Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4+ and CD8+ cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-γ, and blocking IFN-γ also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-γ reduced antibody titer, confirming that IFN-γ plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-γ–secreting cells might, in some situations, be less protective than those that do not. PMID:23509276

  2. Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

    PubMed Central

    Wong-Baeza, Carlos; Reséndiz-Mora, Albany; Donis-Maturano, Luis; Wong-Baeza, Isabel; Zárate-Neira, Luz; Yam-Puc, Juan Carlos; Calderón-Amador, Juana; Medina, Yolanda; Wong, Carlos; Baeza, Isabel; Flores-Romo, Leopoldo

    2016-01-01

    Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers. PMID:27746783

  3. Antibodies to capsular polysaccharide and clumping factor A prevent mastitis and the emergence of unencapsulated and small-colony variants of Staphylococcus aureus in mice.

    PubMed

    Tuchscherr, Lorena P N; Buzzola, Fernanda R; Alvarez, Lucía P; Lee, Jean C; Sordelli, Daniel O

    2008-12-01

    The pathogenesis of Staphylococcus aureus infections is influenced by multiple virulence factors that are expressed under variable conditions, and this has complicated the design of an effective vaccine. Clinical trials that targeted the capsule or clumping factor A (ClfA) failed to protect the recipients against staphylococcal infections. We passively immunized lactating mice with rabbit antibodies to S. aureus capsular polysaccharide (CP) serotype 5 (CP5) or CP8 or with monoclonal antibodies to ClfA. Mice immunized with antibodies to CP5 or CP8 or with ClfA had significantly reduced tissue bacterial burdens 4 days after intramammary challenge with encapsulated S. aureus strains. After several passages in mice passively immunized with CP-specific antiserum, increasing numbers of stable unencapsulated variants of S. aureus were cultured from the infected mammary glands. Greater numbers of these unencapsulated S. aureus variants than of the corresponding encapsulated parental strains were internalized in vitro in MAC-T bovine cells. Furthermore, small-colony variants (SCVs) were recovered from the infected mammary glands after several passages in mice passively immunized with CP-specific antiserum. A combination of antibodies effectively sterilized mammary glands in a significant number of passively immunized mice. More importantly, passive immunization with antibodies to both CP and ClfA fully inhibited the emergence of unencapsulated "escape mutants" and significantly reduced the appearance of SCVs. A vaccine formulation comprising CP conjugates plus a surface-associated protein adhesin may be more effective than either antigen alone for prevention of S. aureus infections.

  4. Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

    NASA Astrophysics Data System (ADS)

    Nicoletti, Sarah E.

    With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further

  5. Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges.

    PubMed

    Dhanapala, Pathum; Withanage-Dona, Dulashi; Tang, Mimi L K; Doran, Tim; Suphioglu, Cenk

    2017-02-21

    Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients' sera. A pool of non-allergic patients' sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis.

  6. Hypoallergenic Variant of the Major Egg White Allergen Gal d 1 Produced by Disruption of Cysteine Bridges

    PubMed Central

    Dhanapala, Pathum; Withanage-Dona, Dulashi; Tang, Mimi L. K.; Doran, Tim; Suphioglu, Cenk

    2017-01-01

    Background: Gal d 1 (ovomucoid) is the dominant allergen in the chicken egg white. Hypoallergenic variants of this allergen can be used in immunotherapy as an egg allergy treatment approach. We hypothesised that disruption of two of the nine cysteine-cysteine bridges by site-directed mutagenesis will allow the production of a hypoallergenic variant of the protein; Methods: Two cysteine residues at C192 and C210 in domain III of the protein were mutated to alanine using site-directed mutagenesis, to disrupt two separate cysteine-cysteine bridges. The mutated and non-mutated proteins were expressed in Escherichia coli (E. coli) by induction with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed proteins were analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting to confirm expression. Immunoglobulin E (IgE) reactivity of the two proteins was analysed, by immunoblotting, against a pool of egg-allergic patients’ sera. A pool of non-allergic patients’ sera was also used in a separate blot as a negative control; Results: Mutant Gal d 1 showed diminished IgE reactivity in the immunoblot by showing lighter bands when compared to the non-mutated version, although there was more of the mutant protein immobilised on the membrane when compared to the wild-type protein. The non-allergic negative control showed no bands, indicating an absence of non-specific binding of secondary antibody to the proteins; Conclusion: Disruption of two cysteine bridges in domain III of Gal d 1 reduces IgE reactivity. Following downstream laboratory and clinical testing, this mutant protein can be used in immunotherapy to induce tolerance to Gal d 1 and in egg allergy diagnosis. PMID:28230769

  7. Molecular modeling assisted hapten design to produce broad selectivity antibodies for fluoroquinolone antibiotics.

    PubMed

    Pinacho, Daniel G; Sánchez-Baeza, Francisco; Marco, M-Pilar

    2012-05-15

    Antibodies with a wide recognition profile of fluoroquinolone antibiotics have been produced based on chemical criteria, theoretical studies, and molecular modeling assisted hapten design. The immunizing hapten preserves the most important and characteristic epitopes of this antibiotic family. The studies have taken into consideration the zwitterionic character of most of the fluoroquinolones and the relative concentration of the different species in equilibrium at physiologic pH. The hapten is prepared in the form of a stable prehapten through a 5 step synthetic pathway. Immediately before conjugation, the immunizing hapten is obtained by removing the diphenylmethane protecting group. The specificity of the antibodies obtained is directed toward the common area defined by the fluorine atom at position 6 and the β-ketoacid moiety. The ELISA developed is able to recognize with very good detectability important fluoroquinolones used in the veterinary field such as ciprofloxacin (CPFX, IC(50), 0.35 μg L(-1)), enrofloxacin (ERFX, IC(50), 0.65 μg L(-1)), danofloxacin (DNFX, IC(50), 7.31 μg L(-1)), difloxacin (DFX, IC(50), 0.91 μg L(-1)), sarafloxacin (SRFX, IC(50), 0.96 μg L(-1)), norfloxacin (NRFX, IC(50), 0.78 μg L(-1)), ofloxacin (OFX, IC(50), 1.84 μg L(-1)), flumequine (Flume, IC(50), 3.91 μ gL(-1)), marbofloxacin (MBFX, IC(50), 4.30 μ gL(-1)), and oxolinic acid (OXO, IC(50), 23.53 μg L(-1)). The results presented here demonstrate that the antibody affinity is strongly affected by the presence of divalent cations, owing to their complexation with the fluoroquinolone molecules. Moreover, the outcome from the effect of the pH on the immunochemical assays suggests that the selectivity could be modulated with the pH due to the zwitterionic character of the fluoroquinolones and as a function of their different pK(a) values.

  8. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

    PubMed Central

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G.; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans. PMID:26437211

  9. Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum.

    PubMed

    Vanier, Gaëtan; Hempel, Franziska; Chan, Philippe; Rodamer, Michael; Vaudry, David; Maier, Uwe G; Lerouge, Patrice; Bardor, Muriel

    2015-01-01

    Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.

  10. Campylobacter hyointestinalis Isolated from Pigs Produces Multiple Variants of Biologically Active Cytolethal Distending Toxin

    PubMed Central

    Kamei, Kazumasa; Hatanaka, Noritoshi; Asakura, Masahiro; Somroop, Srinuan; Samosornsuk, Worada; Hinenoya, Atsushi; Misawa, Naoaki; Nakagawa, Shinsaku

    2015-01-01

    Campylobacter hyointestinalis isolated from swine with proliferative enteritis often is considered to be pathogenic. While the precise virulence mechanisms of this species remain unclear, we have recently identified a cytolethal distending toxin (cdt) gene cluster in C. hyointestinalis isolated from a patient with diarrhea (W. Samosornsuk et al., J Med Microbiol, 27 July 2015, http://dx.doi.org/10.1099/jmm.0.000145). However, the sequences of the cdt genes in C. hyointestinalis were found to be significantly different and the gene products are immunologically distinct from those of other Campylobacter species. In this study, we demonstrate the presence of a second variant of the cdt gene cluster in C. hyointestinalis, designated cdt-II, while the former is named cdt-I. Sequencing of the cdt-II gene cluster and deduced amino acid sequences revealed that homologies between the subunits CdtA, CdtB, and CdtC of ChCDT-I and ChCDT-II are 25.0, 56.0, and 24.8%, respectively. Furthermore, the CdtB subunit of ChCDT-II was found to be immunologically unrelated to that of ChCDT-I by Ouchterlony double gel diffusion test. Recombinant ChCDT-II also induced cell distention and death of HeLa cells by blocking the cell cycle at G2/M phase. Interestingly, the cdt-II genes were detected in all 23 animal isolates and in 1 human isolate of C. hyointestinalis, and 21 of these strains carried both cdt-I and cdt-II gene clusters. Altogether, our results indicate that ChCDT-II is an important virulence factor of C. hyointestinalis in animals. PMID:26283337

  11. Human polyclonal antibodies produced in transchromosomal cattle prevent lethal Zika virus infection and testicular atrophy in mice.

    PubMed

    Stein, Derek R; Golden, Joseph W; Griffin, Bryan D; Warner, Bryce M; Ranadheera, Charlene; Scharikow, Leanne; Sloan, Angela; Frost, Kathy L; Kobasa, Darwyn; Booth, Stephanie A; Josleyn, Matthew; Ballantyne, John; Sullivan, Eddie; Jiao, Jin-An; Wu, Hua; Wang, Zhongde; Hooper, Jay W; Safronetz, David

    2017-09-08

    Zika virus (ZIKV) is rapidly spreading throughout the Americas and is associated with significant fetal complications, most notably microcephaly. Treatment with polyclonal antibodies for pregnant women at risk of ZIKV-related complications could be a safe alternative to vaccination. We found that large quantities of human polyclonal antibodies could be rapidly produced in transchromosomal bovines (TcB) and successfully used to protect mice from lethal infection. Additionally, antibody treatment eliminated ZIKV induced tissue damage in immunologically privileged sites such as the brain and testes and protected against testicular atrophy. These data indicate that rapid development and deployment of human polyclonal antibodies could be a viable countermeasure against ZIKV. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  12. Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.

    PubMed

    Lombardi, Raffaele; Donini, Marcello; Villani, Maria Elena; Brunetti, Patrizia; Fujiyama, Kazuhito; Kajiura, Hiroyuki; Paul, Matthew; Ma, Julian K-C; Benvenuto, Eugenio

    2012-10-01

    We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

  13. Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum.

    PubMed

    Carey, Anne-Marie; Hacking, Kirsty; Picken, Nichola; Honkanen, Suvi; Kelly, Sharon; Niedzwiedzki, Dariusz M; Blankenship, Robert E; Shimizu, Yuuki; Wang-Otomo, Zheng-Yu; Cogdell, Richard J

    2014-11-01

    This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.

  14. Recombinant proteinase 3 produced in different expression systems: recognition by anti-PR3 antibodies.

    PubMed

    van der Geld, Y M; Oost-Kort, W; Limburg, P C; Specks, U; Kallenberg, C G

    2000-10-20

    Anti-neutrophil cytoplasm autoantibodies (ANCA) directed against proteinase 3 (PR3) are highly sensitive and specific markers for Wegener's granulomatosis (WG). Consequently, antigen-specific assays for detection of PR3-ANCA are helpful for the diagnosis and follow-up of patients with WG. Purification of PR3 is laborious and requires large amounts of granulocytes. Therefore, several attempts have been made to produce recombinant PR3 that is recognized by PR3-ANCA. The purpose of this study was to compare the recognition of different recombinant forms of PR3 (rPR3) by anti-PR3 antibodies. Recombinant PR3 produced in E. coli (rcPR3), P. pastoris (rpPR3), insect cells using the baculovirus system (rbPR3), the human mast cell line, HMC-1 (HMC-1/PR3-S176A), or the human epithelial cell line, 293 (Delta-rPR3-S176A) as well as purified neutrophil PR3 (nPR3) were used. Recognition of these rPR3s by anti-PR3 antibodies was determined by direct and capture ELISA with 19 PR3-ANCA sera, 13 anti-PR3 mAbs and a rabbit serum raised against human PR3. In the capture ELISA rabbit anti-PR3 strongly bound nPR3 and all rPR3 products. By capture ELISA rcPR3 and rpPR3 were recognized by 11 (57%) and 13 (68%) of the 19 PR3-ANCA sera, respectively, whereas rbPR3, HMC-1/PR3-S176A, Delta-rPR3-S176A and nPR3 were recognized by all PR3-ANCA sera. By direct ELISA rabbit anti-PR3 strongly bound nPR3 and all tested rPR3 products. Using the direct ELISA none of the PR3-ANCA sera recognized rcPR3, whereas rpPR3 and rbPR3 were recognized by two (11%) and 17 (89%) of the 19 PR3-ANCA sera, respectively. All 13 anti-PR3 mAbs recognized nPR3 in the direct as well as in the capture ELISA. The rcPR3 was recognized by two mAbs in the capture ELISA but by none of the mAbs in the direct ELISA. The rpPR3 was recognized by seven mAbs in the capture ELISA and only by two mAbs in the direct ELISA. All but one of the anti-PR3 mAbs recognized rbPR3, whereas HMC-1/PR3-S176A and Delta-rPR3-S176A were recognized by

  15. Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484

    PubMed Central

    O'Connor, Paula M.; O'Shea, Eileen F.; Guinane, Caitriona M.; O'Sullivan, Orla; Cotter, Paul D.; Hill, Colin

    2015-01-01

    Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions—Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys—-and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin. PMID:25841003

  16. Marburg virus-like particles produced in insect cells induce neutralizing antibodies in rhesus macaques.

    PubMed

    Gai, Weiwei; Zheng, Xuexing; Wang, Chong; Zhao, Yongkun; Wang, Qi; Wang, Hualei; Wong, Gary; Xie, Ying; Wang, Haijun; Cao, Zengguo; Feng, Na; Chi, Hang; Wang, Tiecheng; Gao, Yuwei; Shan, Junjie; Yang, Songtao; Xia, Xianzhu

    2017-04-12

    Marburg virus (MARV), which is one of the most virulent agents in the world, causes lethal haemorrhagic fever in humans and nonhuman primates (NHPs) with a mortality rate of up to 90%. Currently, there is no effective treatment or approved vaccine for MARV for human use to control disease outbreak and spread. Virus-like particles (VLPs), which are morphologically identical to the native infectious virus particle, are efficacious as vaccines against many viruses, including human papilloma virus (HPV), porcine circovirus (PCV) type 2 and hepatitis B virus (HBV). In this study, we generated MARV virus-like particles (VLPs) by co-expressing a glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. Rhesus macaques vaccinated with MARV VLPs mixed with adjuvant Poria cocos polysaccharides (PCP-II) produced a GP-specific IgG titer of up to 1:1280 and virus-neutralizing antibody titers that reached 1:320. MARV VLPs also elicited interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion associated with T-helper 1 cell (Th1)- and T-helper 2 cell (Th2)-mediated immunity, as detected using enzyme-linked immunospot (ELISpot) assays. These data indicate that MARV VLPs mixed with adjuvant PCP-II have excellent immunogenicity in rhesus macaques and may be a promising candidate vaccine against MARV.This article is protected by copyright. All rights reserved.

  17. Application of dielectric spectroscopy for monitoring high cell density in monoclonal antibody producing CHO cell cultivations.

    PubMed

    Párta, László; Zalai, Dénes; Borbély, Sándor; Putics, Akos

    2014-02-01

    The application of dielectric spectroscopy was frequently investigated as an on-line cell culture monitoring tool; however, it still requires supportive data and experience in order to become a robust technique. In this study, dielectric spectroscopy was used to predict viable cell density (VCD) at industrially relevant high levels in concentrated fed-batch culture of Chinese hamster ovary cells producing a monoclonal antibody for pharmaceutical purposes. For on-line dielectric spectroscopy measurements, capacitance was scanned within a wide range of frequency values (100-19,490 kHz) in six parallel cell cultivation batches. Prior to detailed mathematical analysis of the collected data, principal component analysis (PCA) was applied to compare dielectric behavior of the cultivations. PCA analysis resulted in detecting measurement disturbances. By using the measured spectroscopic data, partial least squares regression (PLS), Cole-Cole, and linear modeling were applied and compared in order to predict VCD. The Cole-Cole and the PLS model provided reliable prediction over the entire cultivation including both the early and decline phases of cell growth, while the linear model failed to estimate VCD in the later, declining cultivation phase. In regards to the measurement error sensitivity, remarkable differences were shown among PLS, Cole-Cole, and linear modeling. VCD prediction accuracy could be improved in the runs with measurement disturbances by first derivative pre-treatment in PLS and by parameter optimization of the Cole-Cole modeling.

  18. Antibodies to plant-produced Plasmodium falciparum sexual stage protein Pfs25 exhibit transmission blocking activity.

    PubMed

    Farrance, Christine E; Chichester, Jessica A; Musiychuk, Konstantin; Shamloul, Moneim; Rhee, Amy; Manceva, Slobodanka D; Jones, R Mark; Mamedov, Tarlan; Sharma, Satish; Mett, Vadim; Streatfield, Stephen J; Roeffen, Will; van de Vegte-Bolmer, Marga; Sauerwein, Robert W; Wu, Yimin; Muratova, Olga; Miller, Louis; Duffy, Patrick; Sinden, Robert; Yusibov, Vidadi

    2011-01-01

    Malaria is a serious and sometimes fatal mosquito-borne disease caused by a protozoan parasite. Each year, it is estimated that over one million people are killed by malaria, yet the disease is preventable and treatable. Developing vaccines against the parasite is a critical component in the fight against malaria and these vaccines can target different stages of the pathogen's life cycle. We are targeting sexual stage proteins of P. falciparum which are found on the surface of the parasite reproductive cells present in the mosquito gut. Antibodies against these proteins block the progression of the parasite's life cycle in the mosquito, and thus block transmission to the next human host. Transmission blocking vaccines are essential to the malaria eradication program to ease the disease burden at the population level. We have successfully produced multiple versions of the Pfs25 antigen in a plant virus-based transient expression system and have evaluated these vaccine candidates in an animal model. The targets are expressed in plants at a high level, are soluble and most importantly, generate strong transmission blocking activity as determined by a standard membrane feeding assay. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economic production of a transmission blocking vaccine against malaria.

  19. [A case of pharyngeal-cervical-brachial variant of Guillain-Barré syndrome with positive anti-galactocerebroside (Gal-C) IgM antibody].

    PubMed

    Kasuya, J; Miyazono, T; Takenaga, S; Arimura, K; Osame, M; Kusunoki, S

    1999-05-01

    A 49-year-old man presented with hoarseness, dysphagia, muscle atrophy and weakness of deltoid, trapezius, sternocleidomastoid, rhomboid, anterior serratus, infraspinatus and supraspinatus. Anti-Gal-C IgM antibody was positive in the serum. The other antiganglioside antibodies (GM1, GM2, GM3, GD1a, GD1b, GD3, GT1a, GT1b, GQ1b, GA1, GalNAc-GD1a, GM1b) were negative. Patient contracted pneumonia but whether it was due to mycoplasma was not evident. Plasmapheresis improved his clinical state including a decrease of the antibody. This case was diagnosed pharyngeal-cervical-brachial variant of Guillain-Barré syndrome, and anti-Gal-C antibody seemed to be correlated with the pathogenesis of this syndrome. Gal-C is a major glycolipid of myelin and the cell membrane of the myelin-forming cell (oligodendrocytes and Schwann cells) and is free of specific localization and distribution. The mechanism how the anti-Gal-C IgM antibody induced bulbar paralysis and the symptoms localizing neck and upper limbs remains to be known.

  20. Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

    PubMed Central

    Lian, Zhirui; Wu, Qindong; Wang, Tongtong

    2016-01-01

    ABSTRACT Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins. PMID:26652198

  1. Myeloperoxidase-antineutrophil Cytoplasmic Antibodies with Cytoplasmic Fluorescence Pattern.

    PubMed

    Chhabra, Seema; Minz, Ranjana Walker; Goyal, Lekha; Sharma, Nidhi

    2010-01-01

    We report here two rare cases of myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-positive Wegener's granulomatosis (limited variant) which deceptively produced a cytoplasmic (C-ANCA) pattern on indirect immunofluorescence.

  2. Definition of glomerular antigens by monoclonal antibodies produced against a human glomerular membrane fraction.

    PubMed

    Neale, T J; Callus, M S; Donovan, L C; Baird, H

    1990-10-01

    Experimental animal models of glomerulonephritis (GN) produced by direct antibody binding to non-basement membrane glomerular capillary wall antigens do not to date have human parallels. To examine the potential for this form of humoral glomerular injury in man, we sought to define discrete human non-GBM glomerular antigenic targets using hybridoma technology. Mice were immunised intraperitoneally with 20-100 micrograms of a human glomerular membrane fraction (HGMF). Six fusions have yielded 12 stable reagents defined by positive glomerular indirect immunofluorescence (IF) and microELISA using HGMF as the screening antigen. Subclass analysis of ascitic McAbs indicated several IgG1, one IgG2b, and three IgM reagents. Distinctive IF patterns of reactivity with epithelial, endothelial or mesangial structures have been observed, with or without peritubular capillary, tubular basement membrane and vessel wall reactivity. Seven normal non-renal human organs and the kidneys of rat, rabbit and sheep have shown patterns characteristic of each individual McAb, restricted to human or with species cross reactivity. To partially characterise McAb-reactive antigens, detergent-solubilised renal cortex and collagenase-solubilised GBM (CS-GBM) extracts have been probed by immunoblot. A unique McAb 7-5Q, reactive with glomerular and tubular epithelial structures, binds major bands of approximately 107 KD and 93 KD in detergent solubilised cortex and a single band of similar size by immunoprecipitation (110 KD). 5-3A (a human-restricted linear-reacting McAb) binds bands of 20-200 KD (major band 58 KD) in CS-GBM. In conclusion, distinct species-restricted and more broadly disposed glomerular epitopes are definable in man by McAbs and are potential targets for humoral injury. Purification of these antigens will allow assay for circulating putative nephritogenic auto-antibody and potentially, McAbs may be useful in screening urine for evidence of occult structural renal disease.

  3. Distinct short-lived and long-lived antibody-producing cell populations.

    PubMed

    Ho, F; Lortan, J E; MacLennan, I C; Khan, M

    1986-10-01

    This report analyzes the life span of Ig-containing cells (IgCC) in different sites of antibody production. The experimental approach was based upon the observations that most IgCC are derived from proliferating precursors while IgCC themselves are mainly nondividing end cells. Rats were given a continuous infusion of [3H] thymidine via an osmotic pump inserted in the peritoneal cavity. At intervals of 1, 3, 5 or 10 days after starting infusions, tissues were taken and analyzed by a combination of immunohistology and autoradiography to identify the proportions of IgCC which had gone through S phase of the cell cycle during the period of infusion. After 3 days infusion the median and (range) percent-labeled IgCC in the medullary cords of mesenteric and cervical lymph nodes and the red pulp of the spleen were, respectively, 88 (81-90), 75 (66-77) and 88 (82-93). Conversely that for IgCC in bone marrow was only 13 (11-17) and that in the lamina propria of the jejunum 47 (33-68). The rate of increase in labeling of bone marrow IgCC with length of infusion was approximately linear. Extrapolation of this slope suggests that bone marrow IgCC have a life span in excess of 3 weeks. The slopes of increase in IgCC labeled with time for lymph nodes and spleen were clearly biphasic suggesting that while most IgCC in these tissues have a life span of less than 3 days, there is also a minor population of long-lived IgCC. The lamina propria appears to have approximately equal proportions of long and short-lived IgCC. The life span of IgCC, with the exception of IgMCC, appears to be a feature of the site of antibody production rather than the Ig class produced. Almost all IgM-containing cells were found to be short lived.

  4. Directed selection of influenza virus produces antigenic variants that match circulating human virus isolates and escape from vaccine-mediated immune protection.

    PubMed

    DeDiego, Marta L; Anderson, Christopher S; Yang, Hongmei; Holden-Wiltse, Jeanne; Fitzgerald, Theresa; Treanor, John J; Topham, David J

    2016-06-01

    Influenza vaccination does not provide 100% protection from infection, partly due to antigenic drift of the haemagglutinin (HA) protein. Low serum antibody titres increase the risk of infection. To determine whether there were additional correlates of risk, we examined the relationship between human serum immunity and antigenic variation in seasonal H3N2 influenza viruses. Seasonal H3N2 vaccine strains grown in the presence of heterogeneous human or mono-specific ferret antisera selected variants with mutations in the HA antigenic sites. Surprisingly, circulating strains infecting human subjects in the same seasons displayed mutations in the same positions, although only in one case did the change correspond to the same amino acid. Serum antibody titres were lower against both the in vitro selected and clinical isolates compared with the vaccine strains, suggesting that the mutations are relevant to vaccine failure. Antibody titres were also significantly lower in sera from infected subjects than in non-infected subjects, suggesting relatively poor responses to vaccination in the infected subjects. Collectively, the data suggest that risk from influenza infection is a result of poor response to vaccination, as well as encounter with drifted seasonal influenza virus antigenic variants. The results also show that directed selection under human immune pressure could reveal antigenic variants relevant to real-world drifted viruses, helping in annual vaccine re-formulation. © 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.

  5. Rapid O serogroup identification of the six clinically relevant Shiga toxin-producing Escherichia coli by antibody microarray

    USDA-ARS?s Scientific Manuscript database

    Antibody array was developed for the detection of the top six non-O157 Shiga toxin-producing Escherichia coli O serogroups. Sensitivity of the array was 10**5 CFU, and the limit of detection of serogroups in ground beef was 1-10 CFU following 12 h of enrichment. The array utilized a minimal amount...

  6. Effect of temperature shift on levels of acidic charge variants in IgG monoclonal antibodies in Chinese hamster ovary cell culture.

    PubMed

    Kishishita, Shohei; Nishikawa, Tomoko; Shinoda, Yasuharu; Nagashima, Hiroaki; Okamoto, Hiroshi; Takuma, Shinya; Aoyagi, Hideki

    2015-06-01

    During the production of therapeutic monoclonal antibodies (mAbs), not only enhancement of mAb productivity but also control of quality attributes is critical. Charge variants, which are among the most important quality attributes, can substantially affect the in vitro and in vivo properties of mAbs. During process development for the production of mAbs in a Chinese hamster ovary cell line, we have observed that an improvement in mAb titer is accompanied by an increase in the content of acidic charge variants. Here, to help maintain comparability among mAbs, we aimed to identify the process parameters that controlled the content of acidic charge variants. First, we used a Plackett-Burman design to identify the effect of selected process parameters on the acidic charge variant content. Eight process parameters were selected by using a failure modes and effects analysis. Among these, temperature shift was identified from the Plackett-Burman design as the factor most influencing the acidic charge variant content. We then investigated in more detail the effects of shift temperature and temperature shift timing on this content. The content decreased with a shift to a lower temperature and with earlier timing of this temperature shift. Our observations suggest that Plackett-Burman designs are advantageous for preliminary screening of bioprocess parameters. We report here for the first time that temperature downshift is beneficial for effective control of the acidic peak variant content. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Effect of the Anti-C1s Humanized Antibody TNT009 and its Parental Mouse Variant TNT003 on HLA Antibody-induced Complement Activation - A Preclinical in Vitro Study.

    PubMed

    Wahrmann, M; Mühlbacher, J; Marinova, L; Regele, H; Huttary, N; Eskandary, F; Cohen, G; Fischer, G F; Parry, Graham C; Gilbert, J C; Panicker, S; Böhmig, G A

    2017-03-01

    The classical pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 μg/ml, both in solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients. This article is protected by copyright. All rights reserved.

  8. Draft Genome Sequences of Five Shiga Toxin-Producing Escherichia coli Isolates Harboring the New and Recently Described Subtilase Cytotoxin Allelic Variant subAB2-3

    PubMed Central

    Tasara, Taurai; Fierz, Lisa; Schmidt, Herbert

    2017-01-01

    ABSTRACT We present here the draft genome sequences of five Shiga toxin-producing Escherichia coli (STEC) strains which tested positive in a primary subAB screening. Assembly and annotation of the draft genomes revealed that all strains harbored the recently described allelic variant subAB2-3. Based on the sequence data, primers were designed to identify and differentiate this variant. PMID:28232433

  9. 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

  10. Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides.

    PubMed

    Kirkham, Lea-Ann S; Wiertsema, Selma P; Corscadden, Karli J; Mateus, Tulia; Mullaney, Gemma L; Zhang, Guicheng; Richmond, Peter C; Thornton, Ruth B

    2017-03-01

    The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. Copyright © 2017 Kirkham et al.

  11. Otitis-Prone Children Produce Functional Antibodies to Pneumolysin and Pneumococcal Polysaccharides

    PubMed Central

    Wiertsema, Selma P.; Corscadden, Karli J.; Mateus, Tulia; Mullaney, Gemma L.; Zhang, Guicheng; Richmond, Peter C.; Thornton, Ruth B.

    2016-01-01

    ABSTRACT The pneumococcus is a major otitis media (OM) pathogen, but data are conflicting regarding whether otitis-prone children have impaired humoral immunity to pneumococcal antigens. We and others have shown that otitis-prone and healthy children have similar antibody titers to pneumococcal proteins and polysaccharides (vaccine and nonvaccine types); however, the quality of antibodies from otitis-prone children has not been investigated. Antibody function, rather than titer, is considered to be a better correlate of protection from pneumococcal disease. Therefore, we compared the capacities of antibodies from otitis-prone (cases) and healthy (controls) children to neutralize pneumolysin, the pneumococcal toxin currently in development as a vaccine antigen, and to opsonize pneumococcal vaccine and nonvaccine serotypes. A pneumolysin neutralization assay was conducted on cholesterol-depleted complement-inactivated sera from 165 cases and 61 controls. A multiplex opsonophagocytosis assay (MOPA) was conducted on sera from 20 cases and 20 controls. Neutralizing and opsonizing titers were calculated with antigen-specific IgG titers to determine antibody potency for pneumolysin, pneumococcal conjugate vaccine (PCV) polysaccharides, and non-PCV polysaccharides. There was no significant difference in antibody potencies between cases and controls for the antigens tested. Antipneumolysin neutralizing titers increased with the number of episodes of acute OM, but antibody potency did not. Pneumolysin antibody potency was lower in children colonized with pneumococci than in noncarriers, and this was significant for the otitis-prone group (P < 0.05). The production of functional antipneumococcal antibodies in otitis-prone children demonstrates that they respond to the current PCV and are likely to respond to pneumolysin-based vaccines as effectively as healthy children. PMID:28031178

  12. Characterization of Alpha-Toxin hla Gene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

    PubMed Central

    Wu, Yuling; Tabor, David E.; Mok, Hoyin; Sellman, Bret R.; Jenkins, Amy; Yu, Li; Jafri, Hasan S.; Rude, Thomas H.; Ruffin, Felicia; Schell, Wiley A.; Park, Lawrence P.; Yan, Qin; Thaden, Joshua T.; Messina, Julia A.; Esser, Mark T.

    2014-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study. PMID:25392350

  13. Characterization of cysteine related variants in an IgG2 antibody by LC-MS with an automated data analysis approach.

    PubMed

    Zhang, Yuling; Bailey, Robert; Nightlinger, Nancy; Gillespie, Alison; Balland, Alain; Rogers, Richard

    2015-08-01

    In this communication, a high-throughput method for automated data analysis of cysteine-related product quality attributes (PQAs) in IgG2 antibodies is reported. This method leverages recent advances in the relative quantification of PQAs to facilitate the characterization of disulfide variants and free sulfhydryls (SHs) in IgG2 antibodies. The method uses samples labeled with a mass tag (N-ethyl maleimide [NEM]) followed by enzymatic digestion under non-reducing conditions to maintain the cysteine connectivity. The digested IgG2 samples are separated and detected by mass spectrometry (MS) and the resulting peptide map is analyzed in an automated fashion using Pinpoint software (Thermo Scientific). Previous knowledge of IgG2 disulfide structures can be fed into the Pinpoint software to create workbooks for various disulfide linkages and hinge disulfide variants. In addition, the NEM mass tag can be added to the workbooks for targeted analysis of labeled cysteine-containing peptides. The established Pinpoint workbooks are a high-throughput approach to quantify relative abundances of unpaired cysteines and disulfide linkages, including complicated hinge disulfide variants. This approach is especially efficient for comparing large sets of similar samples such as those created in comparability and stability studies or chromatographic fractions. Here, the high throughput method is applied to quantify the relative abundance of hinge disulfide variants and unpaired cysteines in the IgG2 fractions from non-reduced reversed-phase high-performance liquid chromatography (nrRP-HPLC). The LC-MS data analyzed by the Pinpoint workbook suggests that the nrRP-HPLC separated peaks contain hinge disulfide isoforms and free cysteine pairs for each major disulfide isoform structure.

  14. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

    SciTech Connect

    Ye Ling; Lin Jianguo; Sun Yuliang; Bennouna, Soumaya; Lo, Michael; Wu Qingyang; Bu Zhigao; Pulendran, Bali; Compans, Richard W. . E-mail: compans@microbio.emory.edu; Yang Chinglai . E-mail: chyang@emory.edu

    2006-08-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity of Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.

  15. Production of monoclonal antibody recognizing a subpopulation of human B lymphocytes producing B cell growth factor (BCGF)

    SciTech Connect

    Witzel, N.; Ambrus, J.L. Jr.; Jurgensen, C.H.; Mostowski, H.; Fauci, A.S.

    1986-03-05

    Several laboratories have recently reported production of B cell growth factors (BCGF) by a variety of human B cell lines. The authors have described production of BCGF by clones of B cell lymphoma lines and by normal Staphylococcus aureus Cowan I-activated B cells. To further evaluate whether BCGF production was the function of particular subpopulations of B cells, they immunized mice with the BCGF producing line Namalva and then developed a panel of hybridomas. Monoclonal antibodies were screened for binding to Namalva and the absence of binding to a non-BCGF producing B cell line. One monoclonal antibody, NN4, which met these criteria was also noted by fluorescence-activated cell sorter analysis to stain clones from a B cell lymphoma line producing BCGF but not clones from the same line which do not produce BCGF. The monoclonal antibody did not stain T cells or monocytes but stained 1-5% of normal human B lymphocytes from peripheral blood or tonsils. Binding of /sup 125/I-labeled NN4 to NN4/sup +/ B cells was not affected by the presence of BCGF. Therefore, NN4 recognizes a unique antigen present on B cell lines which produces BCGF; studies are in progress to determine the significance of this antigen on normal B cells.

  16. Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients.

    PubMed Central

    Scarselli, E; Cerino, A; Esposito, G; Silini, E; Mondelli, M U; Traboni, C

    1995-01-01

    The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed. PMID:7539508

  17. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody.

    PubMed Central

    Gorny, M K; Conley, A J; Karwowska, S; Buchbinder, A; Xu, J Y; Emini, E A; Koenig, S; Zolla-Pazner, S

    1992-01-01

    The third variable region (V3) of the HIV-1 gp120 envelope glycoprotein is thought to induce potent neutralizing antibodies which are generally defined as type specific and reactive with individual viral isolates. In contrast, the CD4-binding domain is thought to induce neutralizing antibodies that are group specific and capable of neutralizing all isolates of HIV-1. However, in this study, we used a panel of human monoclonal antibodies to these regions of gp120 which displays specificities and neutralizing activities that challenge these tenets. In particular, we used a human monoclonal antibody to the V3 domain with exceptionally potent and broad neutralizing activity against many diverse HIV-1 isolates. The anti-CD4-binding domain antibodies, on the other hand, showed a more restricted pattern of activity. PMID:1433529

  18. DARPA ANTIBODY TECHNOLOGY PROGRAM STANDARDIZED TEST BED FOR ANTIBODY CHARACTERIZATION: CHARACTERIZATION OF TWO MS2 SCFV ANTIBODIES PRODUCED BY THE UNIVERSITY OF TEXAS

    DTIC Science & Technology

    2017-05-01

    extinction coefficient was used in conjunction with the A280 value to determine an accurate concentration. The MS2 scFv concentrations were determined...by dividing the average A280 value by 1.77, which is the extinction coefficient for a scFv. Each reading required a 2 µL sample, which was placed on...For each antibody, these three numbers were averaged and divided by the extinction coefficient of 1.77. The final concentrations were determined to

  19. DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

    DTIC Science & Technology

    2016-08-01

    For this reason, the extinction coefficient is used in conjunction with the A280 value to determine an accurate concentration. The MS2 scFv...concentrations were determined by dividing the average A280 value by 1.77, which is the extinction coefficient for a scFv. Each reading required a 2 µL sample...4.056 AFX-719 1 4.423 2 4.961 3 4.497 6 For each antibody, these three numbers were averaged and divided by the extinction coefficient of

  20. Macrophages from chickens selected for high antibody response produced more nitric oxide and have greater phagocytic capacity.

    PubMed

    Guimarães, Marco Cesar Cunegundes; Guillermo, Landi Veivi Costilla; Matta, Marcos Fernando de Rezende; Soares, Sandro Gomes; DaMatta, Renato Augusto

    2011-04-15

    Macrophages are fundamental cells of the innate immune system, which, through phagocytosis and nitric oxide production, eliminate pathogens. The aim of the present study was to determine if macrophages from chicken families divergently selected to high and low antibodies response differ in nitric oxide production and phagocytic capacity. Blood monocytes derived macrophages were activated with lipopolysaccharide and supernatant from chicken spleen lymphocytes cultured with Concanavalin A (containing chicken interferon). Nitric oxide production was evaluated in culture supernatants. Phagocytic capacity of activated and non-activated macrophages was assayed using yeasts and IgY opsonized sheep red blood cells. Activated and non-activated macrophages from the high antibodies response family produced higher nitric oxide levels, internalized more yeast and significantly more opsonized sheep red blood cells than macrophages from the low antibodies response family. Moreover, activated macrophages became more elongated and widely spread. These findings indicate that macrophages from the high antibodies response family were more active suggesting that the differences in antibody response also depend on macrophage function.

  1. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

    PubMed Central

    Devey, M E; Major, P J; Bleasdale-Barr, K M; Holland, G P; Dal Canto, M C; Paterson, P Y

    1990-01-01

    Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease. Images Figure 2 PMID:2335373

  2. Mountain Yellow-legged Frogs (Rana muscosa) did not Produce Detectable Antibodies in Immunization Experiments with Batrachochytrium dendrobatidis.

    PubMed

    Poorten, Thomas J; Stice-Kishiyama, Mary J; Briggs, Cheryl J; Rosenblum, Erica Bree

    2016-01-01

    Chytridiomycosis is a devastating infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). A growing number of studies have examined the role of amphibian adaptive immunity in response to this pathogen, with varying degrees of immune activation reported. Here we present immunologic data for the mountain yellow-legged frog, Rana muscosa, and the Sierra Nevada yellow-legged frog, Rana sierrae, which are two endangered and ecologically important species experiencing Bd-inflicted declines. Previous studies on these species that examined transcriptional response during Bd infection, and the effective of immunization, provided little evidence of immune activation to Bd. However, the studies did not directly assay immune effectors in the frog hosts. We performed experiments to examine antibody production, which is a hallmark of systemic adaptive immune activation. We used controlled laboratory experiments and enzyme-linked immunosorbent assays to examine the antibody response to Bd immunization and live Bd exposure. Rana muscosa and R. sierrae individuals did not produce detectable antibodies with the capacity to bind to denatured Bd antigens under our experimental conditions. While we cannot rule out antibody response to Bd in these species, our results suggest weak, poor, or inefficient production of antibodies to denatured Bd antigens. Our findings are consistent with susceptibility to chytridiomycosis in these species and suggest additional work is needed to characterize the potential for adaptive immunity.

  3. Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

    PubMed

    Love, Tracey E; Redmond, Caroline; Mayers, Carl N

    2008-05-20

    We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

  4. Vaccination of cattle with a methanogen protein produces specific antibodies in the saliva which are stable in the rumen.

    PubMed

    Subharat, Supatsak; Shu, Dairu; Zheng, Tao; Buddle, Bryce M; Janssen, Peter H; Luo, Dongwen; Wedlock, D Neil

    2015-04-15

    Methane is produced in the rumen of cattle by a group of archaea (single-celled organisms forming a domain distinct from bacteria and eucarya) called methanogens. Vaccination against methanogens has the potential to reduce methane emissions by inducing antibodies in saliva which are transferred to the rumen and diminish the ability of methanogens to produce methane. Since it is likely that an effective vaccination strategy will need to produce high levels of methanogen-specific antibody in the saliva; the choice of adjuvant, route of vaccination and stability of saliva-derived antibody in the rumen all need to be considered. In this study, stability of IgA and IgG in rumen fluid was determined using an in vitro assay. IgA levels in cattle saliva were reduced by only 40% after 8h exposure to rumen contents while IgG levels were reduced by 80%. These results indicated that antibody is relatively stable in the bovine rumen. A trial was conducted in cattle to investigate induction of immune responses to a methanogen protein, recombinant glycosyl transferase protein (rGT2) from Methanobrevibacter ruminantium M1. Groups of cattle (n=6) were vaccinated subcutaneously with rGT2, formulated with Montanide ISA61 with or without the TLR4 agonist, monophosphoryl lipid A (MPL). A control group (n=6) was not vaccinated. Strong antigen-specific IgG and moderate IgA responses were measured in the serum and saliva of the vaccinated animals and antibody was also detected in the rumen.

  5. Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever

    PubMed Central

    Golden, Joseph W.; Maes, Piet; Kwilas, Steven A.; Ballantyne, John

    2016-01-01

    ABSTRACT Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT50), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can

  6. Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

    PubMed

    Golden, Joseph W; Maes, Piet; Kwilas, Steven A; Ballantyne, John; Hooper, Jay W

    2016-01-20

    Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT(50)), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the

  7. Subtilase cytotoxin-encoding subAB2 variants in verotoxin-producing Escherichia coli strains isolated from goats and sheep.

    PubMed

    Orden, José A; Domínguez-Bernal, Gustavo; de la Fuente, Ricardo; Carrión, Javier

    2016-04-01

    Subtilase cytotoxin (SubAB) is a cytotoxin which might contribute to the virulence of verotoxin-producing Escherichia coli (VTEC) strains in humans. Three variants of SubAB encoding genes have been described (subAB1, subAB2-1, and subAB2-2) and it has been suggested that the strains positive for two variants of subAB may be more pathogenic for humans. In this study, 188 subAB2-positive VTEC strains isolated from goats and sheep were investigated for the presence of the subAB2-1 and subAB2-2 variants by PCR. Eighty-one of the 132 (61.4%) caprine strains and 36 of the 56 (64.3%) ovine strains possessed the subAB2-1 variant and all ovine and caprine strains, except one, were positive for the subAB2-2 variant. The results of this study show for first time that the subAB2-1 and subAB2-2 variants are found in caprine subAB2-positive VTEC strains and confirm that both subAB2 variants are detected in ovine subAB2-positive VTEC strains. Since no significant difference in the presence of both subAB2 variants was found among strains belonging to serotypes associated with severe illness in humans and strains not belonging to these serotypes, the occurrence of two subAB2 variants seems not to be associated with a higher risk of severe disease in humans.

  8. Human genetic variants of homologous recombination repair genes first found to be associated with Epstein-Barr virus antibody titers in healthy Cantonese.

    PubMed

    Shen, Guo-Ping; Pan, Qing-Hua; Hong, Ming-Huang; Qin, Hai-De; Xu, Ya-Fei; Chen, Li-Zhen; Feng, Qi-Sheng; Jorgensen, Timothy J; Shugart, Yin Yao; Zeng, Yi-Xin; Jia, Wei-Hua

    2011-09-15

    Epstein-Barr virus (EBV) infection is a major risk factor for nasopharyngeal carcinoma (NPC). Despite high prevalence of infection among the general population worldwide, only a small proportion of infected individuals presents with seropositivity for EBV-specific IgA antibodies. This seropositive subgroup of EBV carriers has an elevated cumulative risk for NPC during their lifetime. Previous studies reported that the host homologous recombination repair (HRR) system participates in EBV lytic replication, suggesting a potential mechanism to influence EBV reactivation status and thus seropositivity. To investigate whether genetic variants of HRR genes are associated with the serostatus in a healthy population, we investigated the association between seropositivity for anti-VCA-IgA and 156 tagging SNPs in 35 genes connected with HRR in an observational study among 755 healthy Cantonese speakers in southern China. Six variant alleles of MDC1, RAD54L, TP53BP1, RPA1, LIG3 and RFC1 exhibited associations with seropositivity (p(trend) from 0.0085 to 0.00027). Our study provides evidence that genetic variation within the HRR might affect an individual's propensity for EBV seropositive status of anti-VCA IgA antibody.

  9. Antibodies to variant surface antigens of Plasmodium falciparum-infected erythrocytes are associated with protection from treatment failure and the development of anemia in pregnancy.

    PubMed

    Feng, Gaoqian; Aitken, Elizabeth; Yosaatmadja, Francisca; Kalilani, Linda; Meshnick, Steven R; Jaworowski, Anthony; Simpson, Julie A; Rogerson, Stephen J

    2009-07-15

    In pregnancy-associated malaria (PAM), Plasmodium falciparum-infected erythrocytes (IEs) express variant surface antigens (VSA-PAM) that evade existing immunity and mediate placental sequestration. Antibodies to VSA-PAM develop with gravidity and block placental adhesion or opsonize IEs for phagocytic clearance, helping to prevent maternal anemia and low birth weight in infants. Using serum samples from 141 pregnant Malawian women with parasitemia enrolled in a randomized trial of antimalarials and VSA-PAM-expressing CS2 IEs, we quantified levels of immunoglobulin (Ig) G to VSA-PAM by flow cytometry and levels of opsonizing antibodies by measuring uptake of IEs by THP1 promonocytes. After controlling for gravidity and antimalarial treatment, higher levels of IgG to VSA-PAM were associated with decreased anemia at delivery (odds ratio [OR], 0.66 [95% confidence interval {CI}, 0.46-0.93]; P = .018) and were weakly associated with decreased parasitological failure (OR, 0.78 [95% CI, 0.60-1.03]; P = .075), especially reinfection (OR, 0.73 [95% CI, 0.53-1.01]; P = .057). Higher levels of opsonizing antibodies to CS2 IEs were associated with less maternal anemia (OR, 0.31 [95% CI, 0.13-0.74]; P = .008) and treatment failure (OR, 0.48 [95% CI, 0.25-0.90]; P = .023), primarily because of recrudescent infection (OR, 0.49 [95% CI, 0.21-1.12]; P = .089). Higher levels of both IgG antibodies to VSA-PAM and opsonizing antibodies, a functional measure of immunity, correlate with parasite clearance and less anemia in pregnancy malaria.

  10. Antibodies with specificities against a dispase-produced 15-kilodalton hexon fragment neutralize adenovirus type 2 infectivity.

    PubMed Central

    Varga, M J; Bergman, T; Everitt, E

    1990-01-01

    During the entrance of adenovirus type 2 into cells, it has been suggested that the virion undergoes a conformational change. In this investigation, we have further characterized the hypothetical conformational change, which the structural protein hexon undergoes in response to low pH. From pH 5.0 to pH 6.0, the proteolytic enzyme dispase cleaved the hexon into a few distinct fragments with a dominating low-molecular-weight fragment with a molecular weight of 15,000 (15K peptide), whereas between pH 6.5 and pH 8.0, the cleavage of the hexon was negligible. The degradation of the hexon with dispase at low pH was not due to an increased activity or alteration of the active site of dispase at low pH. The 15K fragment was identified as a segment of the N-terminal part of the hexon polypeptide beginning at amino acid residue 5. An immune serum produced in response to acid-treated and glutaraldehyde-fixed hexons contained a small amount of antibodies directed towards the 15K fragment, as judged by Western immunoblotting. An anti-15K antibody fraction was isolated by affinity chromatography by removing antibodies recognizing the hexon in the alkaline configuration. Such antibodies displayed a higher relative titer at pH 5.0 than at pH 7.5 in an enzyme-linked immunosorbent assay. The isolated antibodies showed a specific neutralizing capacity five times higher than that of the corresponding unfractionated polyclonal anti-hexon serum; however, the neutralizing ability was independent of pH. The neutralization of adenovirus type 2 infection by the isolated anti-15K antibodies implies that the N-terminal end of the hexon may play a critical role in the early steps of the virion-cell interaction. Images PMID:1696636

  11. Rabbits immunized with thyroid-stimulating hormone produce autoantiidiotypic thyroid-stimulating antibodies.

    PubMed

    Beall, G N; Rapoport, B; Chopra, I J; Kruger, S R

    1985-05-01

    We immunized rabbits with thyroid-stimulating hormone (TSH) to investigate the hypothesis that such immunization could result in production of thyroid-stimulating autoantiidiotypic antibodies to anti-TSH. Thyroid-stimulating immunoglobulin (TSI) appeared in the serum of several rabbits after immunization. At 160 d, TSI equivalent to 6-18 microU TSH/1.5 mg IgG was present in two of six human (h)TSH-, two of six hTSH beta chain-, and two of the four surviving bovine (b)TSH-immunized animals. Control (human serum albumin-immunized rabbits) serum TSI was 4.3 +/- 0.4 (mean +/- SD) at this time. Antiidiotypic antibodies that could bind to monoclonal anti-hTSH were found in the sera of the bTSH-immunized rabbits. The peak TSI activity occurred 3 mo after a TSH booster immunization and declined gradually during subsequent weeks. Evidence that antiidiotypic antibodies to anti-TSH can cause thyroid stimulation strengthens the notion that such antibodies may be the cause of Graves' hyperthyroidism.

  12. Fusion of Mouse-Mouse Cells to Produce Hybridomas Secreting Monoclonal Antibody,

    DTIC Science & Technology

    the plasmacytoma cells for fusion, the fusion method, and the method of taking samples for identification of colonies secreting monoclonal antibody...The plasmacytoma cell line used is designated SP2/0 ag14. This continuous cell line is a nonimmunoglobulin secreting mouse hybridoma cell line

  13. Harnessing the immune system's arsenal: producing human monoclonal antibodies for therapeutics and investigating immune responses

    PubMed Central

    Sullivan, Meghan; Kaur, Kaval; Pauli, Noel

    2011-01-01

    Monoclonal antibody technology has undergone rapid and innovative reinvention over the last 30 years. Application of these technologies to human samples revealed valuable therapeutic and experimental insights. These technologies, each with their own benefits and flaws, have proven indispensable for immunological research and in our fight to provide new treatments and improved vaccines for infectious disease. PMID:21876728

  14. Rotavirus A-specific single-domain antibodies produced in baculovirus-infected insect larvae are protective in vivo

    PubMed Central

    2012-01-01

    Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in insect larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after insect processing (1 L extract represents about 125 g of insect biomass or about 375 insect larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that insects have been used as living biofactories to produce a VHH molecule. PMID:22953695

  15. Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation*

    PubMed Central

    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

    2014-01-01

    Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. PMID:24958729

  16. Multiple length peptide-pheromone variants produced by Streptococcus pyogenes directly bind Rgg proteins to confer transcriptional regulation.

    PubMed

    Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J

    2014-08-08

    Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Cell surface expression of MR1B, a splice variant of the MHC class I-related molecule MR1, revealed with antibodies.

    PubMed

    Yamaguchi, Hisateru; Tsukamoto, Kentaro; Hashimoto, Keiichiro

    2014-01-10

    The major histocompatibility complex (MHC) class I-related molecule, MR1, is highly conserved in mammals and can present bacteria-derived vitamin B metabolites to mucosal-associated invariant T (MAIT) cells, possibly having important defense function in the microbial infection. MR1B is a splice variant of MR1 and possesses an intriguing domain structure with only two extracellular domains resembling some NKG2D ligand molecules. Thus far, cell surface expression of MR1B could not be analyzed with flow cytometry due to a lack of appropriate antibodies reactive with MR1B. Here we clarified the expression of MR1B recombinant protein on the cell surface of the transfected cells by flow cytometry analyses using the antiserum against MR1. Consistently, MR1B tagged with FLAG peptide at the N-terminus also could be detected with anti-FLAG monoclonal antibodies. Our result showed that MR1B can be recognized on the cell surface by macromolecules such as antibodies, indicating its potential of interaction with certain receptor(s). We discuss possibility of interaction of MR1B and/or the full-length MR1 with some receptor(s) other than αβ T cell receptor (TCR) of MAIT cells based on the highly conserved characteristic residues of the ligand-binding domains of MR1 and its MAIT cells αβTCR footprints.

  18. Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

    PubMed

    Zhao, Qinjian; Modis, Yorgo; High, Katrina; Towne, Victoria; Meng, Yuan; Wang, Yang; Alexandroff, Jaime; Brown, Martha; Carragher, Bridget; Potter, Clinton S; Abraham, Dicky; Wohlpart, Dave; Kosinski, Mike; Washabaugh, Mike W; Sitrin, Robert D

    2012-02-22

    Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes

  19. Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins.

    PubMed

    Arnaoty, Ahmed; Gouilleux-Gruart, Valérie; Casteret, Sophie; Pitard, Bruno; Bigot, Yves; Lecomte, Thierry

    2013-08-01

    The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

  20. Production of specific IgY antibody to the recombinant FanC protein produced in Escherichia coli

    PubMed Central

    Nasiri, Khadijeh; Zibaee, Saeed; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza

    2016-01-01

    Objective(s): Enterotoxigenic Escherichia coli (ETEC) strains are one of the primary causes of diarrhea in newborn calves and in humans, pigs, and sheep. IgY technology has been identified as a promising alternative to generating a mass amount of specific antibody for use in immunotherapy and immunodiagnostics. The purpose of this study was to produce specific antibody by egg yolk antibody (IgY) to recombinant FanC protein from ETEC. Materials and Methods: FanC (K99) gene was amplified from ETEC by specific primers and polymerase chain reaction. The gene was cloned and subcloned into pTZ57R/T and pET32a (+) vectors, respectively. Recombinant vector was transferred into E. coli BL21 CodonPlus (DE3). Protein expression was investigated by 1 mM IPTG induction. Hens were immunized by the purified recombinant FanC protein. The activity and specificity of the IgY antibody were detected by dot-blotting, Western blotting, and indirect ELISA. Results: We obtained FanC specific IgYs by immunizing the hens with the recombinant FanC protein. The anti-FanC IgY showed binding specifically to the FanC protein of ETEC. Conclusion: The results emphasize that specific IgY against the recombinant FanC protein could be recommended as a candidate for passive immunization against ETEC infection in animals and humans. PMID:27746871

  1. The CEA/CD3-Bispecific Antibody MEDI-565 (MT111) Binds a Nonlinear Epitope in the Full-Length but Not a Short Splice Variant of CEA

    PubMed Central

    Huang, Jiaqi; Brohawn, Philip; Morehouse, Chris; Lekstrom, Kristen; Baeuerle, Patrick A.; Wu, Herren; Yao, Yihong; Coats, Steven R.; Dall’Acqua, William; Damschroder, Melissa; Hammond, Scott A.

    2012-01-01

    MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE®) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326–349 and 388–410, with critical residues F326, T328, N333, V388, G389, P390, E392, I408, and N410. Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank® database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. PMID:22574157

  2. Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail.

    PubMed

    Kugelman, Jeffrey R; Kugelman-Tonos, Johanny; Ladner, Jason T; Pettit, James; Keeton, Carolyn M; Nagle, Elyse R; Garcia, Karla Y; Froude, Jeffrey W; Kuehne, Ana I; Kuhn, Jens H; Bavari, Sina; Zeitlin, Larry; Dye, John M; Olinger, Gene G; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

    2015-09-29

    MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.

  3. Charge variants characterization of a monoclonal antibody by ion exchange chromatography coupled on-line to native mass spectrometry: Case study after a long-term storage at +5°C.

    PubMed

    Leblanc, Y; Ramon, C; Bihoreau, N; Chevreux, G

    2017-03-24

    Numerous putative post-translational modifications may induce variations of monoclonal antibodies charge distribution that can potentially affect their biological activity. The characterization and the monitoring of these charge variants are critical quality requirements to ensure stability and process consistency. Charge variants are usually characterized by preparative ion exchange chromatography, collection of fractions and subsequent reverse-phase liquid chromatography with mass spectrometry analysis. While this process can be automatized by on-line two-dimensional chromatography, it remains often complex and time consuming. For this reason, a straightforward on-line charge variant analysis method is highly desirable and analytical laboratories are actively pursuing efforts to overcome this challenge. In this study, a mixed mode ion exchange chromatographic method using volatile salts and coupled on-line to native mass spectrometry was developed in association with a middle-up approach for a detailed characterization of monoclonal antibodies charge variants. An aged monoclonal antibody, presenting a complex charge variant profile was successfully investigated by this methodology as a case study. Results demonstrate that deamidation of the heavy chain was the major degradation pathway after long-term storage at 5°C while oxidation was rather low. The method was also very useful to identify all the clipped forms of the antibody. Copyright © 2017 LFB Biotechnologies. Published by Elsevier B.V. All rights reserved.

  4. Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India, after causing a cholera outbreak.

    PubMed

    Kumar, P; Mishra, D K; Deshmukh, D G; Jain, M; Zade, A M; Ingole, K V; Yadava, P K

    2014-05-01

    Vibrio cholerae O1 biotype El Tor producing Haitian variant Cholera Toxin (HCT) and showing reduced susceptibility to ciprofloxacin caused a cholera outbreak associated with a high case fatality rate (4.5) in India. HCT-secreting strains responsible for severe cholera epidemics in Orissa (India), Western Africa and Haiti were associated with increased mortality. There is a pressing need for an integrated multidisciplinary approach to combat further spread of newly emerging variant strains. The therapeutic effect of ciprofloxacin was diminished whereas use of doxycycline in moderate to severe cholera patients was found to be effective in outbreak management.

  5. Chromosome-Based blaOXA-48-Like Variants in Shewanella Species Isolates from Food-Producing Animals, Fish, and the Aquatic Environment.

    PubMed

    Ceccarelli, Daniela; van Essen-Zandbergen, Alieda; Veldman, Kees T; Tafro, Nedzib; Haenen, Olga; Mevius, Dik J

    2017-02-01

    Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based blaOXA-48-like variants in Shewanella spp., including two new alleles, blaOXA-514 and blaOXA-515 Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

  6. Specific recognition pattern of IgM and IgG antibodies produced in the course of experimental paracoccidioidomycosis.

    PubMed Central

    Vaz, C A; Mackenzie, D W; Hearn, V M; Camargo, Z P; Singer-Vermes, L M; Burger, E; Calich, V L

    1992-01-01

    Specific IgM and IgG responses to Paracoccidioides brasiliensis produced in resistant and susceptible mice during experimental paracoccidioidomycosis were examined by the immunoblotting procedure. Sera from infected mice recognized 51 antigen bands with apparent molecular masses from 8 to 86 kD. Sixteen of these were defined as major antigen bands because of almost universal presence of antibodies to them, and their intense staining. All sera, including those from normal control mice, tested for both IgM and IgG antibody reacted with the major E antigen which appeared as a large diffuse band from 43 to 47 kD. Comparisons between resistant and susceptible mice showed some significant differences in IgM responses to many antigen bands. While IgG responses were quite similar for both strains, differences were apparent in the response to the antigens at 62 and 68 kD. Images Fig. 1 Fig. 2 Fig. 3 PMID:1563097

  7. Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III.

    PubMed

    Jiang, Hua; Wang, Huamao; Tan, Zhonghua; Hu, Suwen; Wang, Hai; Shi, Bizhi; Yang, Lin; Li, Peiyong; Gu, Jianren; Wang, Hongyang; Li, Zonghai

    2011-02-18

    Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.

  8. Growth Suppression of Human Hepatocellular Carcinoma Xenografts by a Monoclonal Antibody CH12 Directed to Epidermal Growth Factor Receptor Variant III*

    PubMed Central

    Jiang, Hua; Wang, Huamao; Tan, Zhonghua; Hu, Suwen; Wang, Hai; Shi, Bizhi; Yang, Lin; Li, Peiyong; Gu, Jianren; Wang, Hongyang; Li, Zonghai

    2011-01-01

    Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (Kd) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-xL, Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression. PMID:21163950

  9. Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies.

    PubMed

    Cheung, Jackie L Y; Wand, Nadina V; Ooi, Cher-Pheng; Ridewood, Sophie; Wheeler, Richard J; Rudenko, Gloria

    2016-11-01

    The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a

  10. Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies

    PubMed Central

    Cheung, Jackie L. Y.; Wand, Nadina V.; Ooi, Cher-Pheng; Ridewood, Sophie

    2016-01-01

    The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a

  11. Detailed functional characterization of glycosylated and nonglycosylated variants of malaria vaccine candidate PfAMA1 produced in Nicotiana benthamiana and analysis of growth inhibitory responses in rabbits.

    PubMed

    Boes, Alexander; Spiegel, Holger; Edgue, Gueven; Kapelski, Stephanie; Scheuermayer, Matthias; Fendel, Rolf; Remarque, Edmond; Altmann, Friedrich; Maresch, Daniel; Reimann, Andreas; Pradel, Gabriele; Schillberg, Stefan; Fischer, Rainer

    2015-02-01

    One of the most promising malaria vaccine candidate antigens is the Plasmodium falciparum apical membrane antigen 1 (PfAMA1). Several studies have shown that this blood-stage antigen can induce strong parasite growth inhibitory antibody responses. PfAMA1 contains up to six recognition sites for N-linked glycosylation, a post-translational modification that is absent in P. falciparum. To prevent any potential negative impact of N-glycosylation, the recognition sites have been knocked out in most PfAMA1 variants expressed in eukaryotic hosts. However, N-linked glycosylation may increase efficacy by improving immunogenicity and/or focusing the response towards relevant epitopes by glycan masking. We describe the production of glycosylated and nonglycosylated PfAMA1 in Nicotiana benthamiana and its detailed characterization in terms of yield, integrity and protective efficacy. Both PfAMA1 variants accumulated to high levels (>510 μg/g fresh leaf weight) after transient expression, and high-mannose-type N-glycans were confirmed for the glycosylated variant. No significant differences between the N. benthamiana and Pichia pastoris PfAMA1 variants were detected in conformation-sensitive ligand-binding studies. Specific titres of >2 × 10(6) were induced in rabbits, and strong reactivity with P. falciparum schizonts was observed in immunofluorescence assays, as well as up to 100% parasite growth inhibition for both variants, with IC₅₀ values of ~35 μg/mL. Competition assays indicated that a number of epitopes were shielded from immune recognition by N-glycans, warranting further studies to determine how glycosylation can be used for the directed targeting of immune responses. These results highlight the potential of plant transient expression systems as a production platform for vaccine candidates.

  12. CD27− B-Cells Produce Class Switched and Somatically Hyper-Mutated Antibodies during Chronic HIV-1 Infection

    PubMed Central

    Cagigi, Alberto; Du, Likun; Dang, Linh Vu Phuong; Grutzmeier, Sven; Atlas, Ann; Chiodi, Francesca

    2009-01-01

    Class switch recombination and somatic hypermutation occur in mature B-cells in response to antigen stimulation. These processes are crucial for the generation of functional antibodies. During HIV-1 infection, loss of memory B-cells, together with an altered differentiation of naïve B-cells result in production of low quality antibodies, which may be due to impaired immunoglobulin affinity maturation. In the current study, we evaluated the effect of HIV-1 infection on class switch recombination and somatic hypermutation by studying the expression of activation-induced cytidine deaminase (AID) in peripheral B-cells from a cohort of chronically HIV-1 infected patients as compared to a group of healthy controls. In parallel, we also characterized the phenotype of B-cells and their ability to produce immunoglobulins in vitro. Cells from HIV-1 infected patients showed higher baseline levels of AID expression and increased IgA production measured ex-vivo and upon CD40 and TLR9 stimulation in vitro. Moreover, the percentage of CD27−IgA+ and CD27−IgG+ B-cells in blood was significantly increased in HIV-1 infected patients as compared to controls. Interestingly, our results showed a significantly increased number of somatic hypermutations in the VH genes in CD27− cells from patients. Taken together, these results show that during HIV-1 infection, CD27− B-cells can also produce class switched and somatically hypermutated antibodies. Our data add important information for the understanding of the mechanisms underlying the loss of specific antibody production observed during HIV-1 infection. PMID:19412542

  13. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry*

    PubMed Central

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D.

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG–FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG–FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG–FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG–FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. PMID:25378534

  14. Investigating the interaction between the neonatal Fc receptor and monoclonal antibody variants by hydrogen/deuterium exchange mass spectrometry.

    PubMed

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman; Kettenberger, Hubert; Hilger, Maximiliane; Rand, Kasper D

    2015-01-01

    The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG(1) and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found that several regions in the IgG Fab region also showed reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc and Fab regions upon FcRn binding. Further, we investigated the role of IgG glycosylation in the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increased the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding did not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild-type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of hydrogen/deuterium exchange mass spectrometry to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The localization of antigen in relation to specific antibody-producing cells

    PubMed Central

    McDevitt, H. O.; Askonas, Brigitte A.; Humphrey, J. H.; Schechter, I.; Sela, M.

    1966-01-01

    A synthetic multichain polypeptide, (T,G,)-A--L 509, was trace labelled with 125I in the tyrosine end groups, composing the main antigenic determinants, and used to study the distribution of antigen in the draining lymph nodes after injection into the footpads of mice. The polypeptide was administered: (a) in saline solution into unprimed mice—in which form it is not demonstrably immunogenic; (b) in Freund's adjuvant into unprimed mice—in which form it is immunogenic; and (c) in saline solution to primed mice, so as to give a booster response. Experiments comparable to (c) were also done with [125I]haemocyanin. At various time intervals from 12 hours to 21 days later sections of the draining nodes were examined by radioautography and methylgreen—pyronine staining, or by a combination of immunofluorescent staining for antibody containing cells with radioautography. In unprimed mice, irrespective of whether they made antibody, labelling was found predominantly in the phagocytic cells of the medulla and the cortical sinus, but definite weaker labelling was also seen in the germinal centres. The label persisted throughout the period of observation, but tended to become more prominent with time in the germinal centres of mice making antibody in response to antigen in Freund's adjuvant. In primed mice the label was rapidly and predominantly concentrated in germinal centres, where it persisted throughout the period of observation while its intensity gradually diminished in other sites. The distribution of label in germinal centres was in a lace-like or dendritic pattern, similar to that described by Nossal and co-workers, and did not correspond to the lymphocytes or pyroninophilic cells in the centres. Grain counts were made over specific antibody containing cells in both primary and booster responses to (T,G)-A--L. Such cells sometimes lay close to and sometimes many cell diameters distant from phagocytic cells containing concentrations of the antigen, but the

  16. The Presence, Persistence and Functional Properties of Plasmodium vivax Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants

    PubMed Central

    Torres, Leticia M.; Lima, Barbara A. S.; Sousa, Taís N.; Alves, Jéssica R. S.; Rocha, Roberto S.; Fontes, Cor J. F.; Sanchez, Bruno A. M.; Adams, John H.; Brito, Cristiana F. A.; Pires, Douglas E. V.; Ascher, David B.; Sell, Ana Maria; Carvalho, Luzia H.

    2016-01-01

    Background The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. Methodology/Principal Findings A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. Conclusions/Significance The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase

  17. The Presence, Persistence and Functional Properties of Plasmodium vivax Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants.

    PubMed

    Kano, Flora S; Souza-Silva, Flávia A; Torres, Leticia M; Lima, Barbara A S; Sousa, Taís N; Alves, Jéssica R S; Rocha, Roberto S; Fontes, Cor J F; Sanchez, Bruno A M; Adams, John H; Brito, Cristiana F A; Pires, Douglas E V; Ascher, David B; Sell, Ana Maria; Carvalho, Luzia H

    2016-12-01

    The human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked. A community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII-erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses. The current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II

  18. Coexistence of hepatitis B surface antigen (HBs Ag) and anti-HBs antibodies in chronic hepatitis B virus carriers: influence of "a" determinant variants.

    PubMed

    Lada, Olivier; Benhamou, Yves; Poynard, Thierry; Thibault, Vincent

    2006-03-01

    In chronic hepatitis B (CHB), the persistence of hepatitis B surface antigen (HBs Ag) is sometimes associated with antibodies (Ab) to HBs (anti-HBs). To assess the hypothesis of the selection of HBs Ag immune escape variants in CHB patients, the variability of the HBV S gene was determined for patients persistently carrying both HBs Ag and anti-HBs antibodies and patients solely positive for HBs Ag. We selected 14 patients who presented both markers (group I) in several consecutive samples and 12 patients positive for HBs Ag only (group II). The HBs Ag-encoding gene was amplified and cloned, and at least 15 clones per patient were sequenced and analyzed. The number of residue changes within the S protein was 2.7 times more frequent for group I than for group II patients and occurred mostly in the "a" determinant of the major hydrophilic region (MHR), with 9.52 versus 2.43 changes per 100 residues (P = 0.009), respectively. Ten patients (71%) from group I, but only three (25%) from group II, presented at least two residue changes in the MHR. The most frequent changes in group I patients were located at positions s145, s129, s126, s144, and s123, as described for immune escape variants. In CHB patients, the coexistence of HBs Ag and anti-HBs Ab is associated with an increase of "a" determinant variability, suggesting a selection of HBV immune escape mutants during chronic carriage. The consequences of this selection process with regard to vaccine efficacy, diagnosis, and clinical evolution remain partially unknown.

  19. Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles.

    PubMed

    Lin, Tao; Xianyu, Lingzhi; Lyu, Songya

    2015-06-01

    Periodic outbreaks of hand, foot and mouth disease (HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71 (EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice (6-8 weeks old) were immunized with virus-like particles (VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing mAbs (D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect >95% cells from 100 TCID50 EV71 infection at 25 µg/mL solution (lowest concentration). Those two neutralizing mAbs identified in the study may be promising candidates in development for mAbs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.

  20. Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

    PubMed Central

    Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

    2016-01-01

    In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders. PMID:27006517

  1. Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes

    PubMed Central

    Salzer, Ulrich; Bacchelli, Chiara; Buckridge, Sylvie; Pan-Hammarström, Qiang; Jennings, Stephanie; Lougaris, Vassilis; Bergbreiter, Astrid; Hagena, Tina; Birmelin, Jennifer; Plebani, Alessandro; Webster, A. David B.; Peter, Hans-Hartmut; Suez, Daniel; Chapel, Helen; McLean-Tooke, Andrew; Spickett, Gavin P.; Anover-Sombke, Stephanie; Ochs, Hans D.; Urschel, Simon; Belohradsky, Bernd H.; Ugrinovic, Sanja; Kumararatne, Dinakantha S.; Lawrence, Tatiana C.; Holm, Are M.; Franco, Jose L.; Schulze, Ilka; Schneider, Pascal; Gertz, E. Michael; Schäffer, Alejandro A.; Hammarström, Lennart; Thrasher, Adrian J.; Gaspar, H. Bobby

    2009-01-01

    TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell– specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n = 39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n = 41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P <.001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P < .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD−CD27+ B cells (P = .019), benign lymphoproliferation (P < .001), and autoimmune complications (P = .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation. PMID:18981294

  2. A CLEC16A variant confers risk for juvenile idiopathic arthritis and anti-cyclic citrullinated peptide antibody negative rheumatoid arthritis.

    PubMed

    Skinningsrud, Beate; Lie, Benedicte A; Husebye, Eystein S; Kvien, Tore K; Førre, Øystein; Flatø, Berit; Stormyr, Alice; Joner, Geir; Njølstad, Pål R; Egeland, Thore; Undlien, Dag E

    2010-08-01

    Variants in CLEC16A have conferred susceptibility to autoimmune diseases in genome-wide association studies. The present work aimed to investigate the locus' involvements in juvenile idiopathic arthritis (JIA) and further explore the association with rheumatoid arthritis (RA), type 1 diabetes (T1D) and Addison's disease (AD) in the Norwegian population. Three single nucleotide polymorphisms (SNPs) were genotyped in patients with RA (n=809), JIA (n=509), T1D (n=1211) and AD (n=414) and in healthy controls (n=2149). All diseases were associated with CLEC16A, but with different SNPs. The intron 22 SNP, rs6498169, was associated with RA (p=0.006) and JIA (p=0.016) and the intron 19 SNPs, rs12708716/rs12917716, with T1D (p=1x10-5) and AD (p=2x10-4). The RA association was confined to the anti-cyclic citrullinated peptide antibody (anti-CCP) negative subgroup (p=2x10-4). This is the first report of a CLEC16A association with JIA and a split of the RA association according to anti-CCP status. Different causative variants underlie the rheumatic versus the organ specific diseases.

  3. Multiproduct high-resolution monoclonal antibody charge variant separations by pH gradient ion-exchange chromatography.

    PubMed

    Farnan, Dell; Moreno, G Tony

    2009-11-01

    In the biotechnology industry, ion-exchange chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Ionic strength based ion exchange separations, while having excellent resolving power and robustness, are product specific and time-consuming to develop. In the present work, a pH gradient based separation using a cation exchange column is described and shown to be a multiproduct charge sensitive separation method for monoclonal antibodies. Simple mixtures of defined buffer components were used to generate the pH-gradients that separate closely related antibody species. The form of the pH gradient was controlled and optimized by the pump as well as the buffer composition if necessary. During this work, the buffer compositions for the separation were optimized in parallel for several MAbs. The data shows that the multiproduct method is optimal for all of the MAbs studied. Operational aspects of the separation such as column chemistry, column length, and sample matrix indicate a very robust method. The pH gradient ion-exchange method is demonstrated to have significant resolving power and peak capacities far in excess of what we would expect for ionic strength elution ion-exchange. Data obtained demonstrates that the separation is relatively insensitive to column length. Direct analysis (no buffer exchange) of samples in matrixes consistent with in-process manufacturing pools is demonstrated. Such a capability is extremely useful for the high throughput evaluation of in-process and final product samples.

  4. Characterization of drug-product-related impurities and variants of a therapeutic monoclonal antibody by higher energy C-trap dissociation mass spectrometry.

    PubMed

    Wang, Deyun; Wynne, Colin; Gu, Flora; Becker, Chris; Zhao, Jia; Mueller, Hans-Martin; Li, Huijuan; Shameem, Mohammed; Liu, Yan-Hui

    2015-01-20

    Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.

  5. Antibody-producing cells correlated to body weight in juvenile chinook salmon (Oncorhynchus tshawytscha) acclimated to optimal and elevated temperatures

    USGS Publications Warehouse

    Harrahy, L.N.M.; Schreck, C.B.; Maule, A.G.

    2001-01-01

    The immune response of juvenile chinook salmon (Oncorhynchus tshawytscha) ranging in weight from approximately 10 to 55 g was compared when the fish were acclimated to either 13 or 21?? C. A haemolytic plaque assay was conducted to determine differences in the number of antibody-producing cells (APC) among fish of a similar age but different body weights. Regression analyses revealed significant increases in the number of APC with increasing body weight when fish were acclimated to either water temperature. These results emphasise the importance of standardising fish weight in immunological studies of salmonids before exploring the possible effects of acclimation temperatures. ?? 2001 Academic Press.

  6. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-02-24

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding.

  7. Antibody-integrated and functionalized graphite-encapsulated magnetic beads, produced using ammonia gas plasma technology, for capturing Salmonella.

    PubMed

    Sakudo, Akikazu; Chou, Han; Nagatsu, Masaaki

    2015-03-01

    Salmonella spp. is the single and most important causative agent of foodborne infections, especially involving foods such as eggs, milk and meat. To prevent infection, a reliable surveillance system is required that can quickly and sensitively detect Salmonella. Here, we describe the development of antibody-integrated magnetic beads that are functionalized by a novel strategy using ammonia gas plasma. Ammonia plasma, produced by a radio frequency (RF) power supply, was allowed to react with the surface of graphite-encapsulated magnetic beads, resulting in the introduction of amino groups. An anti-Salmonella antibody was then anchored by sulfide groups present on the protein surface to the amino groups of the magnetic beads via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The potential usefulness of these magnetic beads for capturing Salmonella was examined as follows. The beads were incubated with Salmonella in liquid medium and then separated from the supernatant by applying a magnetic field. After thorough washing, adsorption of Salmonella to the beads was confirmed by immunochromatography, polymerase chain reaction and a direct culture assay. Our findings indicate that the capture and concentration of Salmonella using the antibody-integrated magnetic beads was more efficient than commercial Dynabeads® anti-Salmonella, which are conventionally used for concentrating Salmonella from liquid cultures. We believe this novel bead technology will contribute to the enhanced detection of Salmonella.

  8. An In Vitro Combined Antibiotic-Antibody Treatment Eliminates Toxicity from Shiga Toxin-Producing Escherichia coli

    PubMed Central

    Skinner, Craig; Zhang, Guodong; Patfield, Stephanie

    2015-01-01

    Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the “big six” serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments. PMID:26100707

  9. An in vitro combined antibiotic-antibody treatment eliminates toxicity from Shiga toxin-producing Escherichia coli.

    PubMed

    Skinner, Craig; Zhang, Guodong; Patfield, Stephanie; He, Xiaohua

    2015-09-01

    Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is difficult. The utility of antibiotics for STEC treatment is controversial, since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase the expression of Shiga toxins (Stxs), which are some of the most important virulence factors in STEC. Stxs contribute to life-threatening hemolytic uremic syndrome (HUS), which develops in considerable proportions of patients with STEC infections. Understanding the antibiotic resistance profiles of STEC isolates and the Stx induction potential of promising antibiotics is essential for evaluating any antibiotic treatment of STEC. In this study, 42 O157:H7 or non-O157 STEC isolates (including the "big six" serotypes) were evaluated for their resistance against 22 antibiotics by using an antibiotic array. Tigecycline inhibited the growth of all of the tested STEC isolates and also inhibited the production of Stxs (Stx2 in particular). In combination with neutralizing antibodies to Stx1 and Stx2, the tigecycline-antibody treatment fully protected Vero cells from Stx toxicity, even when the STEC bacteria and the Vero cells were cultured together. The combination of an antibiotic such as tigecycline with neutralizing antibodies presents a promising strategy for future STEC treatments. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Variant RH alleles and Rh immunisation in patients with sickle cell disease

    PubMed Central

    Sippert, Emilia; Fujita, Claudia R.; Machado, Debora; Guelsin, Glaucia; Gaspardi, Ane C.; Pellegrino, Jordão; Gilli, Simone; Saad, Sara S.T.O.; Castilho, Lilian

    2015-01-01

    Background Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. Materials and methods RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. Results We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. Discussion In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered. PMID:24960646

  11. Variant RH alleles and Rh immunisation in patients with sickle cell disease.

    PubMed

    Sippert, Emilia; Fujita, Claudia R; Machado, Debora; Guelsin, Glaucia; Gaspardi, Ane C; Pellegrino, Jordão; Gilli, Simone; Saad, Sara S T O; Castilho, Lilian

    2015-01-01

    Alloimmunisation is a major complication in patients with sickle cell disease (SCD) receiving red blood cell (RBC) transfusions and despite provision of Rh phenotyped RBC units, Rh antibodies still occur. These antibodies in patients positive for the corresponding Rh antigen are considered autoantibodies in many cases but variant RH alleles found in SCD patients can also contribute to Rh alloimmunisation. In this study, we characterised variant RH alleles in 31 SCD patients who made antibodies to Rh antigens despite antigen-positive status and evaluated the clinical significance of the antibodies produced. RHD and RHCE BeadChip™ from BioArray Solutions and/or amplification and sequencing of exons were used to identify the RH variants. The serological features of all Rh antibodies in antigen-positive patients were analysed and the clinical significance of the antibodies was evaluated by retrospective analysis of the haemoglobin (Hb) levels before and after transfusion; the change from baseline pre-transfusion Hb and the percentage of HbS were also determined. We identified variant RH alleles in 31/48 (65%) of SCD patients with Rh antibodies. Molecular analyses revealed the presence of partial RHD alleles and variant RHCE alleles associated with altered C and e antigens. Five patients were compound heterozygotes for RHD and RHCE variants. Retrospective analysis showed that 42% of antibodies produced by the patients with RH variants were involved in delayed haemolytic transfusion reactions or decreased survival of transfused RBC. In this study, we found that Rh antibodies in SCD patients with RH variants can be clinically significant and, therefore, matching patients based on RH variants should be considered.

  12. [Transverse myelitis associated with toxocariasis and the importance of locally produced antibodies for diagnosis].

    PubMed

    Ural, Serap; Özer, Behiye; Gelal, Fazıl; Dirim Erdoğan, Derya; Sezak, Nurbanu; Balık, Recep; Demirdal, Tuna; Korkmaz, Metin

    2016-07-01

    Toxocariasis caused by Toxocara canis or less frequently by T.catis is a common parasitic infection worldwide. Clinical spectrum in humans can vary from asymptomatic infection to serious organ disfunction depending on the load of parasite, migration target of the larva and the inflammatory response of the host. Transverse myelitis (TM) due to toxocariasis is an uncommon illness identified mainly as case reports in literature. In this report, a case of TM who was diagnosed as neurotoxocariasis by serological findings has been presented. A 44-year-old male patient complained with backache was diagnosed as TM in a medical center in which he has admitted two years ago, and treated with pregabalin and nonsteroidal drugs for six months. Because of the progression of the lesions he readmitted to another center and treated with high dose steroid therapy for three months. After six months of follow up, improvement has been achieved, however, since his symptoms reccurred in the following year he was admitted to our hospital. Magnetic resonance imaging (MRI) examination revealed a TM in a lower segment of spinal cord. He was suffering with weakness and numbness in the left lower extremity. There was no history of rural life or contact with cats or dogs in his anamnesis. Physical examination revealed normal cranial nerve functions, sensory and motor functions. There has been no pathological reflexes, and deep tendon reflexes were also normal. Laboratory findings yielded normal hemogram and biochemical tests, negative PPD and parasitological examination of stool were negative for cysts and ova. Viral hepatitis markers, anti-HIV, toxoplasma-IgM, CMV-IgM, rubella-IgM, EBV-VCA-IgM, VDRL, Brucella tube agglutination, echinococcus antibody, autoantibody tests and neuromyelitis optica test were negative. Examination of CSF showed 20 cells/mm3 (mononuclear cells), 45 mg/dl protein and normal levels of glucose and chlorine. In both serum and CSF samples of the patient Toxocara

  13. Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

    PubMed Central

    Benschop, K. S. M.; Koen, G.; Claassen, Y. B.; Wagner, K.; Bakker, A. Q.; Wolthers, K. C.

    2015-01-01

    ABSTRACT The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPe

  14. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

    PubMed Central

    Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart

    2015-01-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  15. Natural killer cells produce T cell-recruiting chemokines in response to antibody-coated tumor cells.

    PubMed

    Roda, Julie M; Parihar, Robin; Magro, Cynthia; Nuovo, Gerard J; Tridandapani, Susheela; Carson, William E

    2006-01-01

    In the current report, we have examined the ability of natural killer (NK) cells to produce T cell-recruiting chemokines following dual stimulation with interleukin (IL)-2 or IL-12 and human breast cancer cells coated with an antitumor antibody (trastuzumab). NK cells stimulated in this manner secreted an array of T cell-recruiting chemotactic factors, including IL-8, macrophage-derived chemokine, macrophage inflammatory protein 1alpha (MIP-1alpha), monocyte chemoattractant protein 1, and regulated on activation, normal T-cell expressed and secreted (RANTES), whereas stimulation of NK cells with either agent alone had minimal effect. Furthermore, these factors were functional for T-cell chemotaxis as culture supernatants derived from costimulated NK cells induced migration of both naïve and activated T cells in an in vitro chemotaxis assay. T-cell migration was significantly reduced when neutralizing antibodies to IL-8, MIP-1alpha, or RANTES were added to culture supernatants before their use in the chemotaxis assay. In addition, coadministration of trastuzumab-coated tumor cells and IL-12 to mice led to enhanced serum MIP-1alpha. As a clinical correlate, we examined the chemokine content of serum samples from breast cancer patients enrolled on a phase I trial of trastuzumab and IL-12, and found elevated levels of IL-8, RANTES, IFN-gamma inducible protein 10, monokine induced by IFN-gamma, and MIP-1alpha, specifically in those patients that experienced a clinical benefit. Sera from these patients exhibited the ability to direct T-cell migration in a chemotaxis assay, and neutralization of chemokines abrogated this effect. These data are the first to show chemokine production by NK cells, specifically in response to stimulation with antibody-coated tumor cells, and suggest a potential role for NK cell-derived chemokines in patients receiving therapeutic monoclonal antibodies.

  16. High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis.

    PubMed

    Orellana, Camila A; Marcellin, Esteban; Schulz, Benjamin L; Nouwens, Amanda S; Gray, Peter P; Nielsen, Lars K

    2015-02-06

    Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.

  17. Taking advantage of unspecific interactions to produce highly active magnetic nanoparticle-antibody conjugates.

    PubMed

    Puertas, Sara; Batalla, Pilar; Moros, María; Polo, Ester; Del Pino, Pablo; Guisan, José M; Grazú, Valeria; de la Fuente, Jesús M

    2011-06-28

    Several strategies for linking antibodies (Abs) through their Fc region in an oriented manner have been proposed at the present time. By using these strategies, the Fab region of the Ab is available for antigen molecular recognition, leading to a more efficient interaction. Most of these strategies are complex processes optimized mainly for the functionalization of surfaces or microbeads. These methodologies imply though the Ab modification through several steps of purification or the use of expensive immobilized proteins. Besides, the functionalization of magnetic nanoparticles (MNPs) turned out to be much more complex than expected due to the lack of stability of most MNPs at high ionic strength and non-neutral pH values. Therefore, there is still missing an efficient, easy and universal methodology for the immobilization of nonmodified Abs onto MNPs without involving their Fab regions during the immobilization process. Herein, we propose the functionalization of MNPs via a two-steps strategy that takes advantage of the ionic reversible interactions between the Ab and the MNP. These interactions make possible the orientation of the Ab on the MNP surface before being attached in an irreversible way via covalent bonds. Three Abs (Immunoglobulin G class) with very different isoelectric points (against peroxidase, carcinoembryonic antigen, and human chorionic gonadotropin hormone) were used to prove the general applicability of the strategy here proposed and its utility for the development of more bioactive NPs.

  18. An investigation into the relationship between metabolic responses and energy regulation in antibody-producing cell.

    PubMed

    Sun, Ya-Ting; Zhao, Liang; Ye, Zhao-Yang; Fan, Li; Liu, Xu-Ping; Tan, Wen-Song

    2013-11-28

    Energy-efficient metabolic responses were often noted in high-productive cultures. To better understand these metabolic responses, an investigation into the relationship between metabolic responses and energy regulation was conducted via a comparative analysis among cultures with different energy source supplies. Both glycolysis and glutaminolysis were studied through the kinetic analyses of major extracellular metabolites concerning the fast and slow cell growth stages, respectively, as well as the time-course profiles of intracellular metabolites. In three cultures showing distinct antibody productivities, the amino acid metabolism and energy state were further examined. Both the transition of lactate from production to consumption and steady intracellular pools of pyruvate and lactate were observed to be correlated with efficient energy regulation. In addition, an efficient utilization of amino acids as the replenishment for the TCA cycle was also found in the cultures with upregulated energy metabolism. It was further revealed that the inefficient energy regulation would cause low cell productivity based on the comparative analysis of cell growth and productivity in cultures having distinct energy regulation.

  19. IgA and IgG antineutrophil cytoplasmic antibody engagement of Fc receptor genetic variants influences granulomatosis with polyangiitis.

    PubMed

    Kelley, James M; Monach, Paul A; Ji, Chuanyi; Zhou, Yebin; Wu, Jianming; Tanaka, Sumiaki; Mahr, Alfred D; Johnson, Sharleen; McAlear, Carol; Cuthbertson, David; Carette, Simon; Davis, John C; Dellaripa, Paul F; Hoffman, Gary S; Khalidi, Nader; Langford, Carol A; Seo, Phillip; St Clair, E William; Specks, Ulrich; Stone, John H; Spiera, Robert F; Ytterberg, Steven R; Merkel, Peter A; Edberg, Jeffrey C; Kimberly, Robert P

    2011-12-20

    Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR (FCAR) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ∼30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced (P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).

  20. Monoclonal antibodies of three different immunoglobulin G isotypes produced by immunization with a synthetic peptide or native protein protect mice against challenge with Plasmodium yoelii sporozoites.

    PubMed

    Ak, M; Bower, J H; Hoffman, S L; Sedegah, M; Lees, A; Carter, M; Beaudoin, R L; Charoenvit, Y

    1993-06-01

    Passive transfer of monoclonal antibodies (MAbs) against malaria circumsporozoite (CS) proteins protects animals against malaria. Active immunization with synthetic or recombinant peptides induces a level of polyclonal antibodies to sporozoites comparable to those found after passive immunization but does not provide comparable protection. In the Plasmodium yoelii system, synthetic or recombinant peptide-induced antibodies have never been shown to protect. The current studies were designed to determine whether immunogen structure (native protein versus synthetic peptide) or immunoglobulin G (IgG) subclass of antibodies was responsible for the absolute differences between protective, passively transferred MAbs and nonprotective, actively induced polyclonal antibodies. In this study we produced two MAbs, QGP-S1 (IgG1) and QGP-S2 (IgG2b), by immunization with a synthetic peptide based on the P. yoelii CS major repeat, (QGPGAP)4, conjugated to keyhole limpet hemocyanin. These MAbs were compared tp NYS1 (IgG3), an anti-CS protein MAb previously produced by immunization with irradiated P. yoelii sporozoites, which recognizes (QGP GAP)2. QGP-S1 and QGP-S2 passively transferred protection. However, when compared with NYS1, there was a hierarchy of protection, NYS1 > QGP-S1 > QGP-S2. There was no correlation between antibody level at challenge as determined by immunofluorescent antibody test against sporozoites or enzyme-linked immunosorbent assay against (QGPGAP)2 or apparent antibody avidity for (QGPGAP)2 by sodium thiocyanate elution assay. The data demonstrate that a synthetic peptide can induce protective antibodies and that a specific antibody subclass is not required for protection. Work to determine whether antibody affinity or fine specificity can explain the hierarchy of protection among the MAbs is under way.

  1. Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.

    PubMed

    Raven, Nicole; Rasche, Stefan; Kuehn, Christoph; Anderlei, Tibor; Klöckner, Wolf; Schuster, Flora; Henquet, Maurice; Bosch, Dirk; Büchs, Jochen; Fischer, Rainer; Schillberg, Stefan

    2015-02-01

    Tobacco BY-2 cells have emerged as a promising platform for the manufacture of biopharmaceutical proteins, offering efficient protein secretion, favourable growth characteristics and cultivation in containment under a controlled environment. The cultivation of BY-2 cells in disposable bioreactors is a useful alternative to conventional stainless steel stirred-tank reactors, and orbitally-shaken bioreactors could provide further advantages such as simple bag geometry, scalability and predictable process settings. We carried out a scale-up study, using a 200-L orbitally-shaken bioreactor holding disposable bags, and BY-2 cells producing the human monoclonal antibody M12. We found that cell growth and recombinant protein accumulation were comparable to standard shake flask cultivation, despite a 200-fold difference in cultivation volume. Final cell fresh weights of 300-387 g/L and M12 yields of ∼20 mg/L were achieved with both cultivation methods. Furthermore, we established an efficient downstream process for the recovery of M12 from the culture broth. The viscous spent medium prevented clarification using filtration devices, but we used expanded bed adsorption (EBA) chromatography with SP Sepharose as an alternative for the efficient capture of the M12 antibody. EBA was introduced as an initial purification step prior to protein A affinity chromatography, resulting in an overall M12 recovery of 75-85% and a purity of >95%. Our results demonstrate the suitability of orbitally-shaken bioreactors for the scaled-up cultivation of plant cell suspension cultures and provide a strategy for the efficient purification of antibodies from the BY-2 culture medium.

  2. Hemoglobin Abraham Lincoln, β32 (B14) Leucine → Proline AN UNSTABLE VARIANT PRODUCING SEVERE HEMOLYTIC DISEASE

    PubMed Central

    Honig, George R.; Green, David; Shamsuddin, Mir; Vida, Loyda N.; Mason, R. George; Gnarra, David J.; Maurer, Helen S.

    1973-01-01

    An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t½ using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[14C]leucine indicated a t½ of 7.2 days. When stroma-free hemolysates were heated at 50°C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37°C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant β-chain by precipitation with p-hydroxymercuribenzoate. Purification of the β-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for β32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at β31 to impair heme stabilization. Images PMID:4352462

  3. Changes of the immune reactivities of antibodies produced against gamma-irradiated antigen

    NASA Astrophysics Data System (ADS)

    Byun, Myung-Woo; Lee, Ju-Woon; Seo, Ji-Hyun; Kim, Jae-Hun; Jo, Cheorun; Kim, Dong-Ho; Chung, Hyung-Wook

    2004-09-01

    To observe the changes of immunogenicity and antigenicity of gamma-irradiated ovalbumin (OVA), an antigen (Ag) solution (2.0 mg/ml) was prepared and irradiated with the absorbed doses of 3 and 10 kGy. Immunoglobulin G (IgG) was produced for each Ag. 0, 3 and 10 kGy-IgG were individually reacted against 3 Ags in an ELISA cross reactivity test. Cross reactivity of each IgG was significantly different for each Ag. Especially the 10 kGy-irradiated OVA lost most antigenicity compared to the 0 kGy-IgG.

  4. A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours.

    PubMed

    Francis, R J; Sharma, S K; Springer, C; Green, A J; Hope-Stone, L D; Sena, L; Martin, J; Adamson, K L; Robbins, A; Gumbrell, L; O'Malley, D; Tsiompanou, E; Shahbakhti, H; Webley, S; Hochhauser, D; Hilson, A J; Blakey, D; Begent, R H J

    2002-09-09

    Antibody-directed enzyme prodrug therapy is a targeted therapy in which a prodrug is activated selectively at the tumour site by an enzyme, which has been targeted to the tumour by an antibody (antibody-enzyme conjugate). Previous clinical trials have shown evidence of tumour response, however, the activated drug had a long half-life, which resulted in dose-limiting myelosuppression. Also, the targeting system, although giving high tumour to blood ratios of antibody-enzyme conjugate (10 000 : 1) required administration of a clearing antibody in addition to the antibody-enzyme conjugate. The purpose of this current study therefore was to attempt tumour targeting of the antibody-enzyme conjugate without the clearing antibody, and to investigate a new prodrug (bis-iodo phenol mustard, ZD2767P) whose activated form is highly potent and has a short half-life. Twenty-seven patients were treated with antibody-directed enzyme prodrug therapy using A5CP antibody-enzyme conjugate and ZD2767P prodrug, in a dose-escalating phase I trial. The maximum tolerated dose of ZD2767P was reached at 15.5 mg m(-2)x three administrations with a serum carboxypeptidase G2 level of 0.05 U ml(-1). Myelosuppression limited dose escalation. Other toxicities were mild. Patients' quality of life was not adversely affected during the trial as assessed by the measures used. There were no clinical or radiological responses seen in the study, but three patients had stable disease at day 56. Human anti-mouse antibody and human anti-carboxypeptidase G2 antibody were produced in response to the antibody enzyme conjugate (A5CP). The antibody-enzyme conjugate localisation data (carboxypeptidase G2 enzyme levels by HPLC on tumour and normal tissue samples, and gamma camera analysis of I-131 radiolabelled conjugate) are consistent with inadequate tumour localisation (median tumour: normal tissue ratios of antibody-enzyme conjugate of less than 1). A clearance system is therefore desirable with this antibody

  5. Theoretical study of catalytic efficiency of a Diels-Alderase catalytic antibody: an indirect effect produced during the maturation process.

    PubMed

    Martí, Sergio; Andrés, Juan; Moliner, Vicent; Silla, Estanislao; Tuñón, Iñaki; Bertrán, Juan

    2008-01-01

    The Diels-Alder reaction is one of the most important and versatile transformations available to organic chemists for the construction of complex natural products, therapeutics agents, and synthetic materials. Given the lack of efficient enzymes capable of catalyzing this kind of reaction, it is of interest to ask whether a biological catalyst could be designed from an antibody-combining site. In the present work, a theoretical study of the different behavior of a germline catalytic antibody (CA) and its matured form, 39 A-11, that catalyze a Diels-Alder reaction has been carried out. A free-energy perturbation technique based on a hybrid quantum-mechanics/molecular-mechanics scheme, together with internal energy minimizations, has allowed free-energy profiles to be obtained for both CAs. The profiles show a smaller barrier for the matured form, which is in agreement with the experimental observation. Free-energy profiles were obtained with this methodology, thereby avoiding the much more demanding two-dimensional calculations of the energy surfaces that are normally required to study this kind of reaction. Structural analysis and energy evaluations of substrate-protein interactions have been performed from averaged structures, which allows understanding of how the single mutations carried out during the maturation process can be responsible for the observed fourfold enhancement of the catalytic rate constant. The conclusion is that the mutation effect in this studied germline CA produces a complex indirect effect through coupled movements of the backbone of the protein and the substrate.

  6. Association of the interleukin-12 polymorphic variants with the development of antibodies to surface antigen of hepatitis B virus in hemodialysis patients in response to vaccination or infection.

    PubMed

    Grzegorzewska, Alicja E; Wobszal, Piotr M; Sowińska, Anna; Mostowska, Adrianna; Jagodziński, Paweł P

    2013-12-01

    Cytokines, involved in the T-helper 1 system, play a role in the regulation of hepatitis B virus (HBV) clearance and the immune response to HBV antigens during natural infection or planned vaccination. Our aim was to examine whether the polymorphic variants of IL-12 are equally associated with development of antibodies to HBV surface antigen (anti-HBs) in hemodialysis (HD) patients in the case of HBV vaccination or HBV infection. The IL-12A rs568408 and IL-12B rs3212227 polymorphisms were analyzed in relation to anti-HBs development in 602 HD patients with negative antibodies to HBV core antigen (anti-HBc) who were hepatitis B vaccinated (group I) as well as in 237 anti-HBc positive HD patients who were infected with HBV in the past (group II). In group I, 199 patients did not develop an anti-HBs titre >10 IU/L (subgroup Ia), whereas in group II, 55 patients did not develop an anti-HBs titre >10 IU/L (subgroup IIa). Patients of groups I and II that developed an anti-HBs >10 IU/L were included into subgroups Ib and IIb, respectively. In hepatitis B vaccinated HD patients, development of a protective anti-HBs titre was positively associated with vintage of renal replacement therapy (RRT), chronic glomerulonephritis as a cause of RRT, and GA rs 568408 IL-12A (OR 1.6, 95 % CI 1.0-2.5, P = 0.035), but a frequency distribution of this genotype between responders and non-responders was not significant when the Bonferroni correction was applied. In HBV infected HD patients, anti-HBs development was positively associated with AC rs3212227 IL-12B (OR 8.0, 95 % CI 2.6-24.9, P < 0.001), whereas HBsAg positivity, AA rs3212227 IL-12B (OR 0.3, 95 % CI 0.1-0.7, P = 0.007), and CC rs3212227 IL-12B (OR 0.1, 95 % CI 0.03-0.6, P = 0.011) were negative predictors of positive anti-HBs phenotype. When the Bonferroni correction was applied, if appropriate, these associations remained significant. In HD patients, the studied IL-12 polymorphic variants seem to be associated

  7. Identification with monoclonal antibody 140.240 of a structural variant of melanotransferrin shed by human melanoma cell lines in vitro.

    PubMed

    Liao, S K

    1996-01-01

    Shedding by cultured human melanoma cells of a well-characterised cell- surface glycoprotein antigen known as "melanotransferrin" was studied with two monoclonal antibodies, 140.240 and 96.5. By means of [35S]-cysteine metabolically-labelled melanoma cells and immunoprecipitation studies, identification was made, by 140.240 in the spent media of two of six melanoma cell lines, of a new molecule of 100-kDa, aside from the 88-kDa molecule. Only the 88-kDa shed molecule was detected in the remaining four melanoma cell lines with both antibodies. None of nine clonal sublines derived from the two melanoma cell lines were found to shed the 100-kDa or 88-kDa molecule exclusively. Both shed antigens were released spontaneously to the medium from the live melanoma cells rather than as a result of cell death and lysis, since there was no obvious cell death or debris in the spent medium nor in the monolayer cells detected at the time of spent medium collection. Digestion of the isolated 100-kDa and 88-kDa shed molecules with N-glycanase followed by SDS-polyacrylamide gel electrophoresis resulted in the appearance of a single band of the 77-kDa molecule, which is deduced to be the polypeptide precursor of the cell-associated 87-kDa antigen. It is concluded that some melanoma lines shed the variant 100-kDa molecule, in addition to the 88-kDa molecule, and that both shed molecules and their cellular counterpart 87-kDa differ in their degrees of glycosylation.

  8. Antibody response against plasmid-encoded toxin (Pet) and the protein involved in intestinal colonization (Pic) in children with diarrhea produced by enteroaggregative Escherichia coli.

    PubMed

    Bellini, Estela M; Elias, Waldir P; Gomes, Tânia A T; Tanaka, Tânia L; Taddei, Carla R; Huerta, Rocio; Navarro-Garcia, Fernando; Martinez, Marina B

    2005-02-01

    Enteroaggregative Escherichia coli (EAEC) is an emerging cause of pediatric and adult travellers diarrhea. The mechanism by which EAEC induce diarrhea is not completely known. Two serine protease autotransporter proteins, named Pet and Pic have been identified in EAEC strains. Pet has enterotoxic and cytotoxic activities, while the role of Pic in pathogenesis may lie on its mucinolytic activity. Little is known about Pet and Pic biological activities in vivo. In this study the antibody responses against these autotransporter proteins in convalescent children is investigated. Fifteen (83%) children showed specific antibodies against Pet or Pic in their sera. IgG and IgM antibodies were the main isotype found. Specific antibodies against Pic, but not against Pet, were detected in sera from age-matched control group. These data show that specific anti-Pet and anti-Pic antibodies are produced during the course of a natural EAEC infection in children.

  9. Effect of a conjugate of daunomycin and antibodies to rat alpha-fetoprotein on the growth of alpha-fetoprotein-producing tumor cells.

    PubMed Central

    Tsukada, Y; Bischof, W K; Hibi, N; Hirai, H; Hurwitz, E; Sela, M

    1982-01-01

    Daunomycin was covalently attached via a dextran bridge to specific antibodies against rat alpha-fetoprotein produced in a horse. The effect of this conjugate on an alpha-fetoprotein-producing tumor was investigated in terms of cytotoxicity and inhibition or retardation of tumor development. Under the experimental conditions used, the covalent conjugate was by both criteria more efficient than either daunomycin alone or a mixture of daunomycin and specific antibodies or a conjugate of daunomycin with horse immunoglobulin. These results show that the conjugate may be useful as a specific cytotoxic agent against alpha-fetoprotein-producing tumors. PMID:6176999

  10. Efficient generation in vitro, from human peripheral blood cells, of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization.

    PubMed Central

    Winger, L; Winger, C; Shastry, P; Russell, A; Longenecker, M

    1983-01-01

    This paper describes a simple protocol for the efficient generation of large numbers of human monoclonal antibody-producing cells. This system is based on initial limiting-dilution culture after Epstein-Barr virus exposure of highly enriched precursors selected from peripheral blood mononuclear cells. Precursors can be enriched by using rosetting or panning approaches. Antibodies to erythrocytes, a mouse mammary carcinoma, DNA, and sperm antigens, produced without any deliberate immunization, are described. Large-scale human monoclonal antibody production may be facilitated by a combination of this protocol with a human cellular fusion system. For efficient precursor analysis and short-term (2 months or more) monoclonal antibody production, however, the system described here may be sufficient. Images PMID:6308627

  11. Effector properties and glycosylation patterns of recombinant human anti-D-IgG1 antibodies produced by human PER.C6(®) cells.

    PubMed

    Olovnikova, N I; Grigorieva, O V; Petrov, A V

    2012-12-01

    Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

  12. Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia.

    PubMed

    Kawahara, T; Ichikawa, A; Katakura, Y; Teruya, K; Yoshida, T; Kikuchi, M; Kamei, M; Hashizume, S; Shirahata, S

    2001-07-01

    Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4(+) T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells.

  13. Analyzing Clonal Variation of Monoclonal Antibody-Producing CHO Cell Lines Using an In Silico Metabolomic Platform

    PubMed Central

    Ghorbaniaghdam, Atefeh; Chen, Jingkui; Henry, Olivier; Jolicoeur, Mario

    2014-01-01

    Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones. PMID

  14. AHNP-Streptavidin: A Tetrameric Bacterially Produced Antibody Surrogate Fusion Protein Against p185her2/neu

    SciTech Connect

    Masuda,K.; Richter, M.; Song, X.; Berezov, A.; Masuda, K.; Murali, R.; Greene, M.; Zhang, H.

    2006-01-01

    The anti-p185her2/neu peptidomimetic (AHNP) is a small exo-cyclic peptide derived from the anti-p185her2/neu rhumAb 4D5 (h4D5). AHNP mimics many but not all of the antitumor characteristics exhibited by h4D5. However, the pharmacokinetic profiles of AHNP are less than optimal for therapeutic or diagnostic purposes. To improve the binding affinity to p185her2/neu and the antitumor efficacy, we have engineered a fusion protein containing AHNP and a nonimmunoglobulin protein scaffold, streptavidin (SA). The recombinant protein, AHNP-SA (ASA) bound to p185her2/neu with high affinity, inhibited the proliferation of p185her2/neu-overexpressing cells, and reduced tumor growth induced by p185her2/neu-transformed cells. These data suggest that the bacterially produced tetrameric ASA can be used as an antibody-surrogate molecule. This class of molecule will play a role in the diagnosis and treatment of p185her2/neu-related tumors. Our studies establish a general principle by which a small biologically active synthetic exo-cyclic peptide can be engineered to enhance functional aspects by structured oligomerization and can be produced recombinantly using bacterial expression.

  15. Hospital sewage water - a reservoir for variants of new delhi metallo-β-lactamase (blaNDM) and ESBL-producing enterobacteriaceae.

    PubMed

    Parvez, Shadab; Khan, Asad U

    2017-09-05

    New Delhi metallo-β-lactamase (NDM)-producing Enterobacteriaceae has become a threat to public health. Hospital sewage is generally unexplored, having a potential for harbouring bacteria causing healthcare-associated infections. Hence, we initiated this study to monitor NDM-producing Enterobacteriaceae in hospital sewage water. We detected 32 isolates with blaNDM variants, 17 Escherichia coli, 8 Citrobacter freundii, 4 Shigella boydii, 2 Citrobacter braakii, and 1 Citrobacter farmeri in hospital sewage water, showing resistance to all antibiotics except colistin. All isolates carried blaNDM (of which nine were blaNDM-1, eleven blaNDM-4, ten blaNDM-5, and two blaNDM-7), blaCMY variants (one blaCMY-2, three blaCMY-4, five blaCMY-6, eleven blaCMY-42, two blaCMY-86, and two blaCMY-139) in 24 isolates, blaOXA-type (blaOXA-1 in seventeen isolates, and blaOXA-48 in three isolates), blaCTX-M in 19 isolates and/or ampC in 9 isolates on conjugative plasmid of IncFIA, IncFIB, IncFIC, IncP, IncY, IncHI1 and IncI1 types. In Escherichia coli, coexistence of blaNDM-1 with blaCMY-6 and blaCMY-139, blaNDM-4 with blaCMY-6, blaCMY-42, and blaCMY-86, blaNDM-5 with blaCMY-6 and blaCMY-42, and blaNDM-7 with blaCMY-6, were observed. Identification of NDM-5-producing Shigella boydii and NDM-7-producing Citrobacter freundii and detection of association of blaNDM-4 and blaOXA-48 in Citrobacter braakii and Citrobacter farmeri. Class 1 integron was also found on the plasmid. ISAba125 and bleomycin genes were found surrounding all blaNDM variants. The emergence and dissemination of blaNDM variants in the hospital sewage water is a matter of concern, creating an endemic scenario, leading to the level of an outbreak. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  16. Identification of a T cell surface molecule using a monoclonal antibody produced by TCR/CD3 complex immunization.

    PubMed

    Mahasongkram, Kodchakorn; Pata, Supansa; Chruewkamlow, Nuttapol; Kasinrerk, Watchara

    2015-06-01

    Several molecules are known to be involved in T-cell activation via the TCR/CD3 complex and while the mechanisms of late T cell signaling have been well characterized, the very early events are still not fully understood. The aim of this study was to identify yet unknown molecules associated with the TCR/CD3 complex. To identify new molecules associated with the TCR/CD3 complex, a monoclonal antibody termed MT3 was produced by immunoprecipitated beads immunization. Colocalization of the MT3 mAb recognizing molecules with the TCR/CD3 complexes was verified by confocal microscopic analysis. The surface antigen recognized by MT3 antibody was expressed on a subpopulation of CD3+ T cells, and on both CD4+ and CD8+ lymphocytes. The antigen was also expressed on na?ve CD4+ T cells and on a subset of memory CD4+ T cells. In contrast, in the CD8 population, the majority of MT3+ cells were found in the na?ve population. The MT3 mAb recognizing molecules were also expressed on red blood cells but only in particular subjects. Similar to peripheral blood leukocytes, MT3 mAb recognizing molecules are exclusively expressed on T cell lines. Based on the cellular distribution patterns and confocal microscopic analysis, the MT3 mAb recognizing molecule that we investigated is proposed to be a TCR/CD3 associated molecule and might be involved in the antigen recognition of T cells.

  17. Characterization of new hybridoma clones producing monoclonal antibodies reactive against both live and heat-killed Listeria monocytogenes.

    PubMed

    Heo, Seok A; Nannapaneni, Ramakrishna; Story, Robert P; Johnson, Michael G

    2007-01-01

    The objective of this study is to develop high affinity monoclonal antibody (MAb) probes recognizing all major serotypes of Listeria monocytogenes cells. From 500 candidate hybridoma clones, 2 new monoclonal antibody-producing hybridomas were selected and evaluated. MAbs 22D10 and 24F6 reacted strongly with live cells of most serotypes of L. monocytogenes except 4c and 4e and with some L. innocua strains; MAb 22D10 reacted strongly with both live and heat-killed cells (100 degrees C for 20 min) of Listeria. Both MAbs 22D10 and 24F6 did not show any cross-reactions with the other non-Listeria G(+) bacteria tested in ELISA. The mixture of EM-7G1 and 22D10 or 24F6 reacted with all 13 major serotypes of live L. monocytogenes except serotype 4c, while none of these 3 MAbs when tested alone did so. MAb 22D10 mixed with 7G1 reacted with all heat-killed L. monocytogenes serotypes except 4c and 4e. In Western blots, MAbs 22D10 and 24F6 reacted with 1 major protein band of 66 kDa in extracts from L. monocytogenes, but with 2 major protein bands of 66 kDa and 76 kDa in extracts from L. innocua. These results suggest that MAbs 22D10 and 24F6 have high affinity for 11 of 13 serotypes of L. monocytogenes, both live and heat-killed cells. MAbs 22D10 and 24F6--in combination with species-specific MAb EM-7G1--should be useful candidates for use in an ELISA sandwich assays for detecting L. monocytogenes in RTE meat and poultry products.

  18. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    PubMed

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross

  19. Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology

    PubMed Central

    Al-Homsi, Lamis; Al-Okla, Souad; Abbady, Abdul Q.

    2015-01-01

    Mutacin I, a bacteriocin produced by streptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology. The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of soluble sfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carrier sfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin. Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide. PMID:26668664

  20. Antibody-producing cell responses to an isolated outer membrane protein and to complexes of this antigen with lipopolysaccharide or with vesicles of phospholipids from Proteus mirabilis.

    PubMed Central

    Karch, H; Nixdorff, K

    1981-01-01

    Antibody-producing cell responses of mice to a protein isolated from the outer membrane of Proteus mirabilis were typical of the responses to a thymus-dependent antigen. The immunoglobulin G antibody-producing cell responses to the protein were increased after administration of the antigen complexed with either lipopolysaccharide or with vesicles of phospholipids extracted from P. mirabilis. The protein in turn significantly increased the immune response to lipopolysaccharide and also converted this response from predominantly immunoglobulin M to predominantly immunoglobulin G. PMID:6164651

  1. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  2. Binding specificities of eight monoclonal antibodies to human glycophorin A - studies with M/sup c/M, and M/sub k/En(UK) variant human erythrocytes and M- and MN/sup V/-type chimpanzee erythrocytes

    SciTech Connect

    Bigbee, W.L.; Langlois, R.G.; Vanderlaan, M.; Jensen, R.H.

    1984-12-01

    Four newly derived mouse monoclonal antibodies to human glycophorin A are described. Three of these antibodies bind preferentially to the N form of glycophorin A; the fourth recognizes a shared determinant of the M and N forms. All four antibodies are directed toward the 39 amino acid, amino-terminal portion of the protein, and the N-specific antibodies require for binding the presence of N-acetyl-neuraminic acid on the glycosidically linked oligosaccharides. Cross-reaction of the N-specific antibodies to homozygous MM erythrocytes appears to result from binding to glycophorin B. In addition, these antibodies together with four previously reported glycophorin monoclonal antibodies, including two that specifically recognize the M form of glycophorin A, were tested for binding to M/sup c/M and M/sup k/En(UK) variant human erythrocytes. Results obtained for five of the six M- or N-specific monoclonal antibodies point to the general immunodominance of the amino-terminal serine-leucine polymorphism and the requirement for sialic acid. The epitopes for all three N-specific monoclonal antibodies include the amino terminal leucine that occurs in the N form of glycophorin A and may also include the glutamic acid that occurs at position five. Their studies support the proposed Lepore-type glycophorin A-B hybrid gene rearrangement for the En(UK) allele found in the English En(a-) family. The data also confirm the expression of the M-like glycoprotein on chimpanzee erythrocytes and the presence of a human glycophorin B-like antigen on the MN/sup V/-type cells.

  3. A kinetic study of antibody producing cells in the spleen of mice immunized intravenously with sheep erythrocytes

    PubMed Central

    Biozzi, G.; Stiffel, C.; Mouton, D.; Bouthillier, Y.; Decreusefond, C.

    1968-01-01

    The number of antibody producing cells, i.e. rosette forming cells (RFC) has been studied in the spleen of mice injected intravenously with a full immunizing dose of sheep RBC. The spleen of a non-immunized mouse contains a background of about 70,000 RFC (normal RFC), which do not appear to be the `target cells' for antigens of sheep RBC. Our findings suggest that the spleen of a mouse contains about 4000 `target cells' which initiate the immune response to sheep RBC. In the primary response, the rise of RFC is exponential for about 96 hours with a doubling time of 13 hours involving seven to eight consecutive doubling periods. The peak value of RFC is 1·6 × 106 per spleen. In the secondary response, the doubling time of RFC is 6–7 hours. The exponential rise lasts 72 hours and includes nine to eleven doubling periods leading to a peak of 3·5 × 106 RFC/spleen. Adjuvant in the primary response leaves the doubling time of RFC unaltered but prolongs the exponential rise until the 120th hour leading to a peak of 6·3 × 106 RFC/spleen after ten doubling periods. The effects of priming and adjuvant are not fully additive. PMID:5635755

  4. Human macrophages produce dimeric forms of IL-18 which can be detected with monoclonal antibodies specific for inactive IL-18.

    PubMed

    Kikkawa, S; Matsumoto, M; Shida, K; Fukumori, Y; Toyoshima, K; Seya, T

    2001-02-23

    We established two monoclonal antibodies (mAbs) which specifically recognize human 'functionally inactive' recombinant IL-18, and IL-18 protein polymorphism was examined using human monocytes and macrophages (M phi). In 6 day GM-CSF-treated M phi, an 'inactive' IL-18-recognizing mAb 21 detected the IL-18 proform (24 kDa) and a 48-kDa protein, which were gradually increased concomitant with maturation stage. Majority of the 24- and 48-kDa forms were barely detectable with other mAbs recognizing 'active' IL-18. No reagents including Toll stimulators up-regulated these IL-18 populations in M phi. The 21-recognizable IL-18 species were separated using an anion-exchanger column and their IFN gamma-inducing activity was assessed with human lymphocytes plus IL-12. Virtually no as yet known activity was detected with these IL-18 species. After processed with M phi proteases, an 18-kDa form was generated to express the IFN gamma-inducing activity, although the activity was far weaker than that of control 'active' IL-18. These observations suggested that large amounts of various IL-18 species are produced with monocyte-M phi differentiation and most of these IL-18 species are functionally 'inactive' in terms of the reported IL-18 function even after proteolytic 18-kDa conversion.

  5. ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice.

    PubMed

    Phan, Hoang T; Pohl, Julia; Floss, Doreen M; Rabenstein, Frank; Veits, Jutta; Le, Binh T; Chu, Ha H; Hause, Gerd; Mettenleiter, Thomas; Conrad, Udo

    2013-06-01

    Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5-ELP. ELPylated trimers could be purified by a membrane-based inverse transition cycling procedure with the potential of successful scale-up. The trimeric form of AIV HA was found to enhance the HA-specific immune response compared with the monomeric form. Plant-derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus-like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale-up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.

  6. Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody.

    PubMed

    Carrillo-Cocom, L M; Genel-Rey, T; Araíz-Hernández, D; López-Pacheco, F; López-Meza, J; Rocha-Pizaña, M R; Ramírez-Medrano, A; Alvarez, M M

    2015-10-01

    Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.

  7. Cell lines for the production of monoclonal antibodies to human glycophorin A

    SciTech Connect

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.; Langlois, R.G.

    1988-08-30

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  8. Cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, William L.; Fong, Stella S. N.; Jensen, Ronald H.; Vanderlaan, Martin; Langlois, Richard G.

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  9. Method and cell lines for the production of monoclonal antibodies to human glycophorin A

    DOEpatents

    Bigbee, W.L.; Fong, S.S.N.; Jensen, R.H.; Vanderlaan, M.

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that differentiate between the M and N forms of human glycophorin A. These antibodies have potential application as human blood group reagents, as markers for terminally differentiated erythroid cells and as immunofluorescent labels of somatically variant human erythrocytes.

  10. Application of an enzyme-labeled antigen method for visualizing plasma cells producing antibodies against Strep A, a carbohydrate antigen of Streptococcus pyogenes, in recurrent tonsillitis.

    PubMed

    Onouchi, Takanori; Mizutani, Yasuyoshi; Shiogama, Kazuya; Inada, Ken-ichi; Okada, Tatsuyoshi; Naito, Kensei; Tsutsumi, Yutaka

    2015-01-01

    Streptococcus pyogenes is the main causative pathogen of recurrent tonsillitis. Histologically, lesions of recurrent tonsillitis contain numerous plasma cells. Strep A is an antigenic carbohydrate molecule on the cell wall of S. pyogenes. As expected, plasma cells in subjects with recurrent tonsillitis secrete antibodies against Strep A. The enzyme-labeled antigen method is a novel histochemical technique that visualizes specific antibody-producing cells in tissue sections by employing a biotin-labeled antigen as a probe. The purpose of the present study was to visualize plasma cells producing antibodies reactive with Strep A in recurrent tonsillitis. Firstly, the lymph nodes of rats immunized with boiled S. pyogenes were paraformaldehyde-fixed and specific plasma cells localized in frozen sections with biotinylated Strep A. Secondly, an enzyme-labeled antigen method was used on human tonsil surgically removed from 12 patients with recurrent tonsillitis. S. pyogenes genomes were PCR-detected in all 12 specimens. The emm genotypes belonged to emm12 in nine specimens and emm1 in three. Plasma cells producing anti-Strep A antibodies were demonstrated in prefixed frozen sections of rat lymph nodes, 8/12 human specimens from patients with recurrent tonsillitis but not in two control tonsils. In human tonsils, Strep A-reactive plasma cells were observed within the reticular squamous mucosa and just below the mucosa, and the specific antibodies belonged to either IgA or IgG classes. Our technique is effective in visualizing immunocytes producing specific antibodies against the bacterial carbohydrate antigen, and is thus a novel histochemical tool for analyzing immune reactions in infectious disorders. © 2014 The Authors. Microbiology and Immunology Published by The Societies and Wiley Publishing Asia Pty Ltd.

  11. [Influence of estradiol on the capacity to produce antibodies during experimental amebiasis of the female golden hamster].

    PubMed

    Duong, T H; Barrabes, A

    1976-01-01

    During experimental tissular amibiasis, the level of circulating antibodies, as detected by indirect immunofluorescence, is lower in female castrated golden hamsters who have been implanted with a pellet of oestradiol than in castrated control animals. Antibodies are first detected in the serum twenty four hours after infestation and their level becomes maximal near the fourteenth day.

  12. Automated in situ measurement of cell-specific antibody secretion and laser-mediated purification for rapid cloning of highly-secreting producers.

    PubMed

    Hanania, Elie G; Fieck, Annabeth; Stevens, Janine; Bodzin, Leon J; Palsson, Bernhard Ø; Koller, Manfred R

    2005-09-30

    Cloning of highly-secreting recombinant cells is critical for biopharmaceutical manufacturing, but faces numerous challenges including the fact that secreted protein does not remain associated with the producing cell. A fundamentally new approach was developed combining in situ capture and measurement of individual cell protein secretion followed by laser-mediated elimination of all non- and poorly-secreting cells, leaving only the highest-secreting cell in a well. Recombinant cells producing humanized antibody were cultured serum-free on a capture matrix, followed by staining with fluorescently-labeled anti-human antibody fragment. A novel, automated, high-throughput instrument (called LEAP) was used to image and locate every cell, quantify the cell-associated and secreted antibody (surrounding each cell), eliminate all undesired cells from a well via targeted laser irradiation, and then track clone outgrowth and stability. Temporarily sparing an island of helper cells around the clone of interest improved cloning efficiency (particularly when using serum-free medium), and helper cells were easily eliminated with the laser after several days. The in situ nature of this process allowed several serial sub-cloning steps to be performed within days of one another, resulting in rapid generation of clonal populations with significantly increased and more stable, homogeneous antibody secretion. Cell lines with specific antibody secretion rates of > 50 pg/cell per day (in static batch culture) were routinely obtained as a result of this cloning approach, often times representing up to 20% of the clones screened.

  13. Association of high levels of serum antibody to staphylococcal toxic shock antigen with nasal carriage of toxic shock antigen-producing strains of Staphylococcus aureus.

    PubMed Central

    Ritz, H L; Kirkland, J J; Bond, G G; Warner, E K; Petty, G P

    1984-01-01

    Forty-four asymptomatic male subjects were examined for their nasal carriage of strains of Staphylococcus aureus capable of producing staphylococcal toxic shock antigen (TSA), an exotoxin implicated in the pathogenesis of toxic shock syndrome. In addition, the levels of antibody to TSA in sera from these subjects were determined by an enzyme-linked immunosorbent assay. S. aureus was isolated from the anterior nares of 23 subjects. Of those 23 isolates of S. aureus, 9 were found to produce TSA. All individuals carrying strains of S. aureus capable of producing TSA had high to moderate levels of antibody to TSA. In contrast, those individuals carrying strains not producing TSA had levels of antibody to TSA ranging from high to nondetectable. A second examination of nasal samples from 42 of these subjects revealed that 86% of those carrying S. aureus initially still carried S. aureus after a period of 3 months; all subjects found to carry TSA-producing strains initially and that were examined a second time yielded TSA-producing strains once again. PMID:6698614

  14. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation

    PubMed Central

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-01-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies. PMID:26674415

  15. Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice

    PubMed Central

    Brightbill, Hans D.; Jeet, Surinder; Lin, Zhonghua; Yan, Donghong; Zhou, Meijuan; Tan, Martha; Nguyen, Allen; Yeh, Sherry; Delarosa, Donnie; Leong, Steven R.; Wong, Terence; Chen, Yvonne; Ultsch, Mark; Luis, Elizabeth; Ramani, Sree Ranjani; Jackman, Janet; Gonzalez, Lino; Dennis, Mark S.; Chuntharapai, Anan; DeForge, Laura; Meng, Y. Gloria; Xu, Min; Eigenbrot, Charles; Lee, Wyne P.; Refino, Canio J.; Balazs, Mercedesz; Wu, Lawren C.

    2010-01-01

    IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1′ domain, and using genetically modified mice that contain the human M1′ domain inserted into the mouse IgE locus, we demonstrated that M1′-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1′-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1′-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1′-specific antibodies may be a novel treatment for asthma and allergy. PMID:20458139

  16. A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

    PubMed Central

    Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

    2017-01-01

    Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

  17. Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors.

    PubMed

    van der Geest, Kornelis S M; Lorencetti, Pedro G; Abdulahad, Wayel H; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

    2016-03-01

    Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So far, few studies have directly assessed the effect of aging on the latter B cell function. Here, we determined if and how human aging influences the production of cytokines by B cells. In a cross-sectional study, we found that absolute numbers of circulating B cells were similar in 31 young (ages 19-39) and 73 old (age ≥ 60) individuals. Numbers of transitional B cells (CD19(+)CD27(-)CD38(High)CD24(High)) were decreased in old individuals, whereas numbers of naive and memory B cell subsets were comparable in young and old individuals. Short-term in vitro stimulation of whole blood samples revealed that numbers of B cells capable of producing TNF-α were similar in young and old individuals. In contrast, B cells capable of IL-10 production were decreased in old subjects. This decline of IL-10(+) B cells was observed in old individuals that were ANA positive, and in those that were negative for both ANAs and RFs. However, IL-10(+) B cells were remarkably well retained in the circulation of old subjects that were RF positive. Thus, pro-inflammatory TNF-α(+) B cells are retained in the elderly, whereas IL-10(+) B cells generally decline. In addition, our findings indicate that IL-10(+) B cells may differentially impact the development of ANAs and RFs in the elderly.

  18. Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus

    PubMed Central

    1990-01-01

    We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus. These B cells expressed the surface CD5 molecule. The antibodies they produced were polyreactive IgM that displayed a relatively low affinity for the virus components (Kd, 1.0- 2.4 x 10(-6) g/microliters). After immunization, different anti-virus (IgG and IgA) antibody-producing cells consistently appeared in the circulation and increased from less than 0.005% to greater than 10% of the total B cells committed to the production of IgG and IgA, respectively. Most of such B cells do not express CD5 and produce monoreactive antibodies of high affinity for rabies virus (Kd, 6.5 x 10(-9) to 1.2 x 10(-10) g/microliters). One of these IgG mAbs efficiently neutralized rabies virus in vitro and in vivo, as detailed elsewhere (Dietzschold, B., P. Casali, Y. Ueki, M. Gore, C. E. Rupprecht, A. L. Notkins, and H. Koprowski, manuscript submitted for publication). Hybridization experiments using probes specific for the different human V gene segment families revealed that cell precursors producing low affinity IgM binding to rabies virus utilized a restricted number of VH gene segments (i.e., only members of the VHIIIb subfamily), whereas cell precursors producing high affinity IgG and IgA to rabies virus utilized an assortment of different VH gene segments (i.e., members of the VHI, VHIII, VHIV, and VHVI families and VHIIIb subfamily). In conclusion, our studies show that EBV transformation in conjunction with limiting dilution technology and somatic cell hybridization techniques are useful methods for quantitating, at the B cell clonal level, the human

  19. Genetic Variants in Toll-Like Receptor 2 (TLR2), TLR4, TLR9, and FCγ Receptor II Are Associated with Antibody Response to Quadrivalent Meningococcal Conjugate Vaccine in HIV-Infected Youth

    PubMed Central

    Qin, Min; Lujan-Zilbermann, Jorge; Singh, Kumud K.; Warshaw, Meredith G.; Williams, Paige L.; Jean-Philippe, Patrick; Fenton, Terence; Siberry, George K.

    2013-01-01

    This study examined the association of host genetic variants with the antibody response to the quadrivalent meningococcal conjugate vaccine (MCV4) in HIV-infected youth. Genetic variants associated with severity of meningococcal disease, including the IgG Fc receptor (FCγRII)-A484T, interleukin-10 (IL-10)-A1082G, -C819T, and -C627A, IL-4-C589T, mannose binding lectin-2 (MBL2)-A/O, -H/L, -P/Q, and -X/Y, toll-like receptor 2 (TLR2)-G2408A, TLR4-A12874G and -C13174T, and TLR9-T1237C and -T1486C were determined by real-time PCR (RT-PCR) for 271 HIV-infected subjects (median, 17 years). Response was defined as a ≥4-fold increase from entry in bactericidal antibody titers to each serogroup. Generalized estimating equation (GEE) models were used to evaluate the association of allelic variants with the immunologic response to all serogroups within each subject with and without adjusting for CD4 percentage and HIV viral load. At week 4, but not after, subjects with TLR2-2408-G/A versus -G/G genotypes and the TLR4-12874-A/A genotype were more likely to achieve a ≥4-fold increase overall in the four serogroups (unadjusted P of 0.006 and adjusted P of 0.008 and unadjusted P of 0.008 and adjusted P of 0.019, respectively). At week 28, the TLR9-1237 T allele was associated with enhanced antibody response (T allele versus C/C, unadjusted P of 0.014 and adjusted P of 0.009), which was maintained at week 72 (unadjusted and adjusted P of 0.008). At week 72, the FcγRII-131Arg allotype was associated with a ≥4-fold increase in antibody titer versus those with His/His (unadjusted P of 0.009; adjusted P of <0.001). These findings suggest that for HIV-infected youth, the initial antibody response to MCV4 is associated with variants in TLR2 and TLR4 while the long-term response is associated with genetic polymorphisms in TLR9 and FcγRIIa. PMID:23595505

  20. Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.

    PubMed

    Shymanets, Aliaksei; Prajwal; Vadas, Oscar; Czupalla, Cornelia; LoPiccolo, Jaclyn; Brenowitz, Michael; Ghigo, Alessandra; Hirsch, Emilio; Krause, Eberhard; Wetzker, Reinhard; Williams, Roger L; Harteneck, Christian; Nürnberg, Bernd

    2015-07-01

    Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

  1. In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method

    PubMed Central

    Mizutani, Y; Tsuge, S; Takeda, H; Hasegawa, Y; Shiogama, K; Onouchi, T; Inada, K; Sawasaki, T; Tsutsumi, Y

    2014-01-01

    Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis. PMID:24698402

  2. Outbreak Caused by an Ertapenem-Resistant, CTX-M-15-Producing Klebsiella pneumoniae Sequence Type 101 Clone Carrying an OmpK36 Porin Variant

    PubMed Central

    Poulou, Aggeliki; Voulgari, Evangelia; Vrioni, Georgia; Koumaki, Vasiliki; Xidopoulos, Grigorios; Chatzipantazi, Vasiliki; Markou, Fani

    2013-01-01

    Although numerous studies have documented outbreaks of carbapenem-resistant Klebsiella pneumoniae (CRKP) possessing various carbapenemases, reports on outbreaks due to CRKP possessing extended-spectrum β-lactamases (ESBLs) and/or AmpCs with porin lesions have been limited. Here, we describe an outbreak caused by an ertapenem-resistant, CTX-M-15-producing clonal K. pneumoniae strain expressing an OmpK36 porin variant. From May 2012 to November 2012, 37 ertapenem-resistant K. pneumoniae isolates phenotypically negative for carbapenemase production were recovered from 19 patients hospitalized in the intensive care unit of a Greek hospital. The isolates were either susceptible or intermediate to other carbapenems and resistant to all remaining β-lactams but cefotetan. Phenotypic and molecular analysis revealed the presence in all isolates of the blaCTX-M-15 gene on a conjugative 100-kb plasmid, disruption in the expression of the ompK35 gene, and the production of an Ompk36 porin variant. The index case was a patient admitted from another hospital. Active surveillance upon admission and on a weekly basis was immediately initiated; environmental samples were also periodically tested. Molecular typing showed that all clinical isolates as well as two ertapenem-resistant environmental K. pneumoniae isolates belonged to the same clonal type and were assigned to multilocus sequence typing (MLST) sequence type 101 (ST101). As all colonized/infected patients were hospitalized during overlapping periods, cross-infection was considered the main route for the dissemination of the outbreak strain. Despite reinforcement of infection control measures and active surveillance, the outbreak lasted approximately 7 months. Identification of hidden carriers upon admission and by screening on a weekly basis was found valuable for early recognition and subsequent successful management of the outbreak. PMID:23850951

  3. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

    PubMed

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  4. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    PubMed

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. © 2015 Society for Experimental Biology, Association of

  5. Prevalence of Toxic Shock Syndrome Toxin 1-Producing Staphylococcus aureus and the Presence of Antibodies to This Superantigen in Menstruating Women

    PubMed Central

    Parsonnet, Jeffrey; Hansmann, Melanie A.; Delaney, Mary L.; Modern, Paul A.; DuBois, Andrea M.; Wieland-Alter, Wendy; Wissemann, Kimberly W.; Wild, John E.; Jones, Michaelle B.; Seymour, Jon L.; Onderdonk, Andrew B.

    2005-01-01

    Menstrual toxic shock syndrome (mTSS) is thought to be associated with colonization with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus aureus in women with insufficient antibody titers. mTSS has been associated with menstruation and tampon use, and although it is rare, the effects can be life threatening. It remains of interest because of the widespread use of tampons, reported to be about 70% of women in the United States, Canada, and much of Western Europe. This comprehensive study was designed to determine S. aureus colonization and TSST-1 serum antibody titers in 3,012 menstruating women in North America between the ages of 13 and 40, particularly among age and racial groups that could not be assessed reliably in previous small studies. One out of every four subjects was found to be colonized with S. aureus in at least one of three body sites (nose, vagina, or anus), with approximately 9% colonized vaginally. Eighty-five percent of subjects had antibody titers (≥1:32) to TSST-1, and the vast majority (81%) of teenaged subjects (13 to 18 years) had already developed antibody titers. Among carriers of toxigenic S. aureus, a significantly lower percentage of black women than of white or Hispanic women were found to have antibody titers (≥1:32) to TSST-1 (89% versus 98% and 100%). These findings demonstrate that the majority of teenagers have antibody titers (≥1:32) to TSST-1 and are presumed to be protected from mTSS. These findings also suggest that black women may be more susceptible to mTSS than previously thought. PMID:16145118

  6. Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3

    PubMed Central

    Tsekoa, Tsepo Lebiletsa; Lotter-Stark, Therese; Buthelezi, Sindisiwe; Chakauya, Ereck; Stoychev, Stoyan H.; Sabeta, Claude; Shumba, Wonderful; Phahladira, Baby; Hume, Steve; Morton, Josh; Rupprecht, Charles E.; Steinkellner, Herta; Pauly, Michael; Zeitlin, Larry; Whaley, Kevin; Chikwamba, Rachel

    2016-01-01

    Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG. PMID:27427976

  7. Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

    PubMed

    Tsekoa, Tsepo Lebiletsa; Lotter-Stark, Therese; Buthelezi, Sindisiwe; Chakauya, Ereck; Stoychev, Stoyan H; Sabeta, Claude; Shumba, Wonderful; Phahladira, Baby; Hume, Steve; Morton, Josh; Rupprecht, Charles E; Steinkellner, Herta; Pauly, Michael; Zeitlin, Larry; Whaley, Kevin; Chikwamba, Rachel

    2016-01-01

    Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

  8. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology

    PubMed Central

    SAKUDO, AKIKAZU; VISWAN, ANCHU; CHOU, HAN; SASAKI, TADAHIRO; IKUTA, KAZUYOSHI; NAGATSU, MASAAKI

    2016-01-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito-borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave-excited inductively-coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody-integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV-infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1–4 onto the beads was confirmed using reverse transcription-polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody-integration, represents a significant improvement in the detection of DENV. PMID:27221214

  9. Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

    PubMed

    Rodríguez, Meilyn; Pérez, Lincidio; Gavilondo, Jorge V; Garrido, Greta; Bequet-Romero, Mónica; Hernández, Ignacio; Huerta, Vivian; Cabrera, Gleysin; Pérez, Marlene; Ramos, Osmani; Leyva, René; León, Mariela; Ramos, Pedro Luis; Triguero, Ada; Hernández, Abel; Sánchez, Belinda; Ayala, Marta; Soto, Jeny; González, Ernesto; Mendoza, Osmani; Tiel, Kenia; Pujol, Merardo

    2013-01-01

    A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins. © 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  10. Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate.

    PubMed

    Wetzel, Hanna N; Webster, Rose P; Saeed, Fatima O; Kirley, Terence L; Ball, William J; Norman, Andrew B

    2017-06-03

    We have generated a humanized anti-cocaine monoclonal antibody (mAb), which is at an advanced stage of pre-clinical development. We report here in vitro binding affinity studies, and in vivo pharmacokinetic and efficacy studies of the recombinant mAb. The overall aim was to characterize the recombinant antibody from each of the three highest producing transfected clones and to select one to establish a master cell bank. In mAb pharmacokinetic studies, after injection with h2E2 (120 mg/kg iv) blood was collected from the tail tip of mice over 28 days. Antibody concentrations were quantified using ELISA. The h2E2 concentration as a function of time was fit using a two-compartment pharmacokinetic model. To test in vivo efficacy, mice were injected with h2E2 (120 mg/kg iv), then one hour later injected with an equimolar dose of cocaine. Blood and brain were collected 5 min after cocaine administration. Cocaine concentrations were quantified using LC/MS. The affinity of the antibody for cocaine was determined using a [(3)H] cocaine binding assay. All three antibodies had long elimination half-lives, 2-5 nM Kd for cocaine, and prevented cocaine's entry into the brain by sequestering it in the plasma. Pharmacokinetic and radioligand binding assays supported designation of the highest producing clone 85 as the master cell bank candidate. Overall, the recombinant h2E2 showed favorable binding properties, pharmacokinetics, and in vivo efficacy. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Prevalence of Toxic Shock Syndrome Toxin 1 (TSST-1)-Producing Strains of Staphylococcus aureus and Antibody to TSST-1 among Healthy Japanese Women▿

    PubMed Central

    Parsonnet, Jeffrey; Goering, Richard V.; Hansmann, Melanie A.; Jones, Michaelle B.; Ohtagaki, Kumiko; Davis, Catherine C.; Totsuka, Kyoichi

    2008-01-01

    Many cases of neonatal toxic shock syndrome (TSS)-like exanthematous disease but few cases of menstrual TSS (mTSS) have been reported in Japan. We determined the prevalence of mucosal colonization with Staphylococcus aureus and of positive antibodies to TSS toxin 1 (TSST-1) among 209 healthy Japanese women in Tokyo. S. aureus isolates from mucosal sites were characterized with respect to TSST-1 production and resistance genotype. Antibody titers were determined for test subjects and for 133 Japanese and 137 Caucasian control women living in the United States. S. aureus was isolated from at least one site in 108 of 209 women (52%) in Tokyo. Of the 159 S. aureus isolates recovered, 14 (9%) were TSST-1 positive (12 unique strains). Twelve of 209 women (6%) were colonized with a TSST-1-producing strain; two (<1%) had vaginal colonization. Only 2 of 12 unique toxigenic strains (14%) were methicillin resistant. Of the 12 TSST-1-positive strains isolated, 6 (50%) were pulsed-field gel electrophoresis type USA200, multilocus sequence type clonal complex 30. Fewer Japanese women in Tokyo (47%) than Caucasian and Japanese women in the United States (89% and 75%, respectively) had TSST-1 antibodies. The prevalences of colonization with TSST-1-producing S. aureus were comparable in Japan and the United States, despite low seropositivity to TSST-1 in Japan. Environmental factors appear to be important in promoting the development of anti-TSST-1 antibodies, as there was a significant difference in titers between Japanese women living in Tokyo and those living in the United States. Most colonizing TSST-1-producing S. aureus strains in Japan were genotypically similar to mTSS strains found in the United States. PMID:18550735

  12. High-level carbapenem-resistant OXA-48-producing Klebsiella pneumoniae with a novel OmpK36 variant and low-level, carbapenem-resistant, non-porin-deficient, OXA-181-producing Escherichia coli from Thailand.

    PubMed

    Lunha, Kamonwan; Chanawong, Aroonwadee; Lulitanond, Aroonlug; Wilailuckana, Chotechana; Charoensri, Nicha; Wonglakorn, Lumyai; Saenjamla, Pimjai; Chaimanee, Prajuab; Angkititrakul, Sunpetch; Chetchotisakd, Ploenchan

    2016-06-01

    Five blaOXA-48-like-carrying Enterobacteriaceae isolates collected from two Thai patients in December 2012 were characterized. Three Klebsiella pneumoniae isolates giving two different pulsed-field gel electrophoresis patterns and sequence types (ST11 and ST37) from patient 1 harbored blaOXA-48 locating on Tn1999.2, whereas two Escherichia coli isolates with the same pulsotype and ST5 from Patient 2 carried ISEcp1-associated blaOXA-181. One K. pneumoniae strain had blaSHV-12, blaDHA-1, qnrB, and qnrS, while another strain harbored blaCTX-M-15, qnrS and aac(6')-Ib-cr. The E. coli strain contained blaCTX-M-15, blaCMY-2, qnrS, and aac(6')-Ib-cr. Interestingly, the OXA-48 producers with a novel OmpK36 variant by a substitution of Gly to Asp in the L3 loop-borne PEFXG motif exhibited high-level resistance to ertapenem, imipenem, and meropenem. In contrast, the OXA-181 producer with non-porin-deficient background showed low-level resistance to ertapenem only. Both patients died because of either septic shock or pneumonia. This study showed the impact of OXA-48-like carbapenemases in porin-defective clinical isolate background, which may lead to serious therapeutic problems in the near future.

  13. T cells produce an antigen-binding factor with in vivo activity analogous to IgE antibody

    PubMed Central

    1983-01-01

    T cell-dependent activation of resident tissue mast cells is required for the elicitation of delayed-type hypersensitivity skin reactions in mice. A T cell-derived antigen-binding factor that transfers the ability to elicit an immediate hypersensitivity-like skin reaction is described and compared with a hybridoma IgE antibody. Both the T cell factor and IgE mediate reactions with increased vascular permeability and both are mast cell dependent, as they are inactive in two different types of mast cell deficient mice (W/Wv and Sl/Sld). The T cell factor was distinguished from IgE by affinity chromatography using specific anti-IgE and anti-factor antibodies and by a shorter duration of passive sensitization. The T cell factor is a suitable candidate for participation in the mechanism by which T cells activate mast cells in delayed-type hypersensitivity. PMID:6187880

  14. Human Polyclonal Antibodies Produced in Transchromosomal Bovine Prevent Lethal Zika Virus Infection and Testicular Atrophy in Mice

    DTIC Science & Technology

    2017-03-21

    reproductive tract leading to documented cases of sexual transmission (8,   9). Animal models of ZIKV infection have rapidly been established and...targets immune privileged sites for replication, which include the brain and reproductive organs. Recently, an epidemiological study showed that up...treated on days -1/+1 and +1/+3 with ZIKV-TcB antibody had no detectable virus in the brain, testis, or epididymis. Sexual transmission and

  15. An Antibody to the Lutheran Glycoprotein (Lu) Recognizing the LU4 Blood Type Variant Inhibits Cell Adhesion to Laminin α5

    PubMed Central

    Kikkawa, Yamato; Miwa, Takahiro; Tohara, Yukiko; Hamakubo, Takayuki; Nomizu, Motoyoshi

    2011-01-01

    Background The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM. Methodology/Principal Findings In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg175, the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5. Conclusion/Significance This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion. PMID:21858073

  16. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

    PubMed Central

    Spiesberger, Katrin; Paulfranz, Florian; Egger, Anton; Reiser, Judith; Vogl, Claus; Rudolf-Scholik, Judith; Mayrhofer, Corina; Grosse-Hovest, Ludger; Brem, Gottfried

    2015-01-01

    Background 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4). Results With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M. Conclusion Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma. PMID:26469402

  17. RNA interference-mediated phosphodiesterase 4D splice variants knock-down in the prefrontal cortex produces antidepressant-like and cognition-enhancing effects

    PubMed Central

    Wang, Zhen-Zhen; Zhang, Yi; Liu, Yan-Qin; Zhao, Nan; Zhang, You-Zhi; Yuan, Li; An, Lei; Li, Jing; Wang, Xiao-Yun; Qin, Juan-Juan; Wilson, Steven P; O'Donnell, James M; Zhang, Han-Ting; Li, Yun-Feng

    2013-01-01

    Background and Purpose Phosphodiesterase 4 (PDE4) inhibitors produce potent antidepressant-like and cognition-enhancing effects. However, their clinical utility is limited by the major side effect of emesis, which appears to be PDE4 isoform-specific. Although PDE4D subtype plays the pivotal role in these therapeutic profiles, it is also the primary subtype responsible for emesis. Therefore, the aim of present research was to investigate whether long-form PDE4D variants mediate antidepressant-like and cognition-enhancing effects, but are irrespective with emesis. Experimental Approach In mice microinfused with lentiviral vectors that contained shRNA-mir hairpin structure targeting long-form PDE4Ds into bilateral prefrontal cortices, the tail-suspension and forced-swim tests were used to measure antidepressant-like effects; novel object recognition and Morris water-maze tasks were used to determine cognition-enhancing effects. The emetic potential was assessed by alpha2 adrenergic receptor-mediated anaesthesia, a surrogate measure of emesis. Intracellular cAMP signalling was analysed by time-resolved FRET immunoassay and Western-blot. Dendritic complexity was assessed by Golgi staining. Key Results Microinfusions of lentiviral PDE4D-shRNA down-regulated PDE4D4 and PDE4D5, and imitated the antidepressant-like and cognition-enhancing effects of the prototypical PDE4 inhibitor rolipram. The behavioural effects were related to dendritic complexity and mediated by the increased cAMP signalling. In addition, these effects were not enhanced in the presence of rolipram. Finally, while rolipram shortened the duration of combined anaesthesia, RNA interference-mediated PDE4D knock-down in the prefrontal cortex did not. Conclusion and Implications These data suggest that long-form PDE4Ds, at least PDE4D4 and PDE4D5, may be the promising targets for the development of PDE4 variant-selective inhibitors as the new pharmacotherapies for depressive disorders and neurodegenerative

  18. Cross-Neutralizing Antibodies in HIV-1 Individuals Infected by Subtypes B, F1, C or the B/Bbr Variant in Relation to the Genetics and Biochemical Characteristics of the env Gene

    PubMed Central

    de Almeida, Dalziza Victalina; Macieira, Karine Venegas; Grinsztejn, Beatriz Gilda Jegerhorn; Veloso dos Santos, Valdiléa Gonçalves; Guimarães, Monick Lindenmeyer

    2016-01-01

    Various HIV-1 env genetic and biochemical features impact the elicitation of cross-reactive neutralizing antibodies in natural infections. Thus, we aimed to investigate cross-neutralizing antibodies in individuals infected with HIV-1 env subtypes B, F1, C or the B/Bbr variant as well as env characteristics. Therefore, plasma samples from Brazilian chronically HIV-1 infected individuals were submitted to the TZM-bl neutralization assay. We also analyzed putative N-glycosylation sites (PNGLs) and the size of gp120 variable domains in the context of HIV-1 subtypes prevalent in Brazil. We observed a greater breadth and potency of the anti-Env neutralizing response in individuals infected with the F1 or B HIV-1 subtypes compared with the C subtype and the variant B/Bbr. We observed greater V1 B/Bbr and smaller V4 F1 than those of other subtypes (p<0.005), however neither was there a correlation verified between the variable region length and neutralization potency, nor between PNLG and HIV-1 subtypes. The enrichment of W at top of V3 loop in weak neutralizing response viruses and the P in viruses with higher neutralization susceptibility was statistically significant (p = 0.013). Some other signatures sites were associated to HIV-1 subtype-specific F1 and B/Bbr samples might influence in the distinct neutralizing response. These results indicate that a single amino acid substitution may lead to a distinct conformational exposure or load in the association domain of the trimer of gp120 and interfere with the induction power of the neutralizing response, which affects the sensitivity of the neutralizing antibody and has significant implications for vaccine design. PMID:27936047

  19. Polymorphisms in the F8 Gene and MHC-II Variants as Risk Factors for the Development of Inhibitory Anti-Factor VIII Antibodies during the Treatment of Hemophilia A: A Computational Assessment

    PubMed Central

    Pandey, Gouri Shankar; Yanover, Chen; Howard, Tom E.; Sauna, Zuben E.

    2013-01-01

    The development of neutralizing anti-drug-antibodies to the Factor VIII protein-therapeutic is currently the most significant impediment to the effective management of hemophilia A. Common non-synonymous single nucleotide polymorphisms (ns-SNPs) in the F8 gene occur as six haplotypes in the human population (denoted H1 to H6) of which H3 and H4 have been associated with an increased risk of developing anti-drug antibodies. There is evidence that CD4+ T-cell response is essential for the development of anti-drug antibodies and such a response requires the presentation of the peptides by the MHC-class-II (MHC-II) molecules of the patient. We measured the binding and half-life of peptide-MHC-II complexes using synthetic peptides from regions of the Factor VIII protein where ns-SNPs occur and showed that these wild type peptides form stable complexes with six common MHC-II alleles, representing 46.5% of the North American population. Next, we compared the affinities computed by NetMHCIIpan, a neural network-based algorithm for MHC-II peptide binding prediction, to the experimentally measured values and concluded that these are in good agreement (area under the ROC-curve of 0.778 to 0.972 for the six MHC-II variants). Using a computational binding predictor, we were able to expand our analysis to (a) include all wild type peptides spanning each polymorphic position; and (b) consider more MHC-II variants, thus allowing for a better estimation of the risk for clinical manifestation of anti-drug antibodies in the entire population (or a specific sub-population). Analysis of these computational data confirmed that peptides which have the wild type sequence at positions where the polymorphisms associated with haplotypes H3, H4 and H5 occur bind MHC-II proteins significantly more than a negative control. Taken together, the experimental and computational results suggest that wild type peptides from polymorphic regions of FVIII constitute potential T-cell epitopes and thus

  20. [Immunization experiments for producing antibody-like substances in caterpillars of Mamestra brassicae L. (Insecta, Lepid., Noct.)].

    PubMed

    Luther, P; Otto, D; Köhler, W; Fischer, G

    1975-01-01

    The agglutinins against human blood cells described in caterpillars of Mamestra brassicae L. were not demonstrable when feeding the animals with a semisynthetic food. After injection or oral intake of certain bacteria (E. coli or streptococci of group C) or even Pope's broth the "antibody-like substances" known from feeding with natural food are being formed, and they agglutinated all human blood cells. The individual animals showed differences regarding the strength of agglutinin formation. The immune reactions observed possibly indicate the existence of a primitive immune system in these species (arthropods).

  1. Maternal autism-associated IgG antibodies delay development and produce anxiety in a mouse gestational transfer model.

    PubMed

    Braunschweig, Daniel; Golub, Mari S; Koenig, Claire M; Qi, Lihong; Pessah, Isaac N; Van de Water, Judy; Berman, Robert F

    2012-11-15

    A murine passive transfer model system was employed to ascertain the effects of gestational exposure to a single, intravenous dose of purified, brain-reactive IgG antibodies from individual mothers of children with autism (MAU) or mothers with typically developing children (MTD). Growth and behavioral outcomes in offspring were measured from postnatal days 8 to 65 in each group. Comparisons revealed alterations in early growth trajectories, significantly impaired motor and sensory development, and increased anxiety. This report demonstrates for the first time the effects of a single, low dose gestational exposure of IgG derived from individual MAU on their offspring's physical and social development.

  2. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

    PubMed

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-02-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

  3. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

    PubMed Central

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-01-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

  4. An ultra scale-down approach identifies host cell protein differences across a panel of mAb producing CHO cell line variants.

    PubMed

    Hogwood, Catherine E M; Ahmad, Shahina S; Tarrant, Richard D; Bracewell, Daniel G; Smales, C Mark

    2016-03-01

    During the manufacture of biopharmaceutical products, the final product must lie within strict pre-set specifications, for example the host cell protein (HCP) content. A number of specific HCPs have been identified in particular products and the interactions between product/HCPs have also been recently investigated; however, a comparison of the HCP dynamics between related cell lines and their response to early downstream processing to aid process development and cell line selection has not been published. We have utilised a proteomic approach coupled with an ultra scale-down study to determine the HCP profile dynamics, at harvest and during early downstream processing, across a panel of recombinant GS-CHOK1SV antibody producing cell lines. The results reveal that cell culture viability upon harvest has the greatest impact upon shear sensitivity and HCP concentration. Whilst the general HCP population/profile was broadly similar across the cell lines, the actual amounts of some specific HCPs in the supernatant differed and a number of cell line specific differences in the response to early downstream processing were observed. We anticipate that such knowledge can now be applied to cell line selection and downstream processing development to target reduction/removal of general and specific problematic HCPs before and during downstream processing.

  5. Capture of dengue viruses using antibody-integrated graphite-encapsulated magnetic beads produced using gas plasma technology.

    PubMed

    Sakudo, Akikazu; Viswan, Anchu; Chou, Han; Sasaki, Tadahiro; Ikuta, Kazuyoshi; Nagatsu, Masaaki

    2016-07-01

    Despite significant advances in medicine, global health is threatened by emerging infectious diseases caused by a number of viruses. Dengue virus (DENV) is a mosquito‑borne virus, which can be transmitted to humans via mosquito vectors. Previously, the Ministry of Health, Labour and Welfare in Japan reported the country's first domestically acquired case of dengue fever for almost 70 years. To address this issue, it is important to develop novel technologies for the sensitive detection of DENV. The present study reported on the development of plasma-functionalized, graphite-encapsulated magnetic nanoparticles (GrMNPs) conjugated with anti-DENV antibody for DENV capture. Radiofrequency wave‑excited inductively‑coupled Ar and ammonia gas plasmas were used to introduce amino groups onto the surface of the GrMNPs. The GrMNPs were then conjugated with an antibody against DENV, and the antibody‑integrated magnetic beads were assessed for their ability to capture DENV. Beads incubated in a cell culture medium of DENV‑infected mosquito cells were separated from the supernatant by applying a magnetic field and were then washed. The adsorption of DENV serotypes 1‑4 onto the beads was confirmed using reverse transcription‑polymerase chain reaction, which detected the presence of DENV genomic RNA on the GrMNPs. The methodology described in the present study, which employed the plasma-functionalization of GrMNPs to enable antibody‑integration, represents a significant improvement in the detection of DENV.

  6. Are children's vitamin D levels and BMI associated with antibody titers produced in response to 2014-2015 influenza vaccine?

    PubMed

    Lin, Chyongchiou J; Martin, Judith M; Cole, Kelly Stefano; Zimmerman, Richard K; Susick, Michael; Moehling, Krissy K; Levine, Min Z; Spencer, Sarah; Flannery, Brendan; Nowalk, Mary Patricia

    2017-07-03

    Vitamin D is an immunomodulating hormone, which has been associated with susceptibility to infectious diseases. Serum vitamin D levels in 135 children ages 3-17 y were measured at baseline and hemagglutinin influenza antibody titers were measured pre- and 21 d post influenza vaccination with live attenuated influenza vaccine (LAIV) or inactivated influenza vaccine (IIV). Height and weight were derived from the electronic medical record and were used to calculate body mass index (BMI). Thirty-nine percent of children were ages 3-8 years; 75% were black, 34% were obese (BMI ≥ 95(th) percentile); vitamin D levels were >20 ng/ml in 55%. In linear regression analyses, post vaccination antibody titers for LAIV B lineages (B Brisbane and B Massachusetts) were significantly higher among those with lower vitamin D levels and among younger participants (P < 0.05). No associations between vitamin D levels and responses to LAIV A strains (A/H1N1 and A/H3N2) or to any IIV strains or lineages were found. Low vitamin D levels were associated with higher response to LAIV B lineages in the 2014-2015 LAIV, but not related to LAIV A or any IIV strains.

  7. Anti-malaria antibody-producing B cell frequencies in adults after a Plasmodium falciparum outbreak in Madagascar.

    PubMed Central

    Migot, F; Chougnet, C; Henzel, D; Dubois, B; Jambou, R; Fievet, N; Deloron, P

    1995-01-01

    The central highlands of Madagascar offer a unique opportunity to explore the malaria immune memory, as the last murderous epidemic in the study area occurred 8 years ago. Quantification of the circulating memory B lymphocytes reacting to Plasmodium falciparum was assessed among 14 Madagascans by using a limiting dilution assay, applied to the EL4 culture system, which leads to activation, proliferation and differentiation into antibody-secreting cells (ASC) of most peripheral B cells. This system allowed us to observe, without any malaria-specific restimulation, a geometric mean frequency of one anti-P. falciparum ASC among 2992 circulating B cells, except for one Madagascan who did not have any detectable ASC. A geometric mean frequency of one anti-P. falciparum ASC among 1403 was obtained for six malaria hyperimmune Cameroonians, but conversely, no anti-malaria ASC was detected in the blood of six malaria non-immune French control subjects. Anti-P. falciparum ASC frequencies and serum specific antibodies were strongly related. Our results indicate that anti-malaria ASC are still present in peripheral blood of Madagascan subjects, who have not been exposed to P. falciparum for several years. These responder B cells reflect the malaria B cell memory acquired during the last epidemic. PMID:8536368

  8. Enhanced transport of plant-produced rabies single chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.

    PubMed

    Phoolcharoen, Waranyoo; Prehaud, Christophe; van Dolleweerd, Craig J; Both, Leonard; da Costa, Anaelle; Lafon, Monique; Ma, Julian K-C

    2017-03-08

    The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In the present study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system (CNS). This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralising single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognises the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralisation activity against RABV in cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVG(P) fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVG(P) fusion protein was able to cross the in cellulo BBB and neutralise RABV. This article is protected by copyright. All rights reserved.

  9. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512

    PubMed Central

    Di Pilato, Vincenzo; Arena, Fabio; Tascini, Carlo; Cannatelli, Antonio; Henrici De Angelis, Lucia; Fortunato, Simona; Giani, Tommaso; Menichetti, Francesco

    2016-01-01

    A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa). PMID:27401575

  10. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512.

    PubMed

    Di Pilato, Vincenzo; Arena, Fabio; Tascini, Carlo; Cannatelli, Antonio; Henrici De Angelis, Lucia; Fortunato, Simona; Giani, Tommaso; Menichetti, Francesco; Rossolini, Gian Maria

    2016-09-01

    A novel mcr variant, named mcr-1.2, encoding a Gln3-to-Leu functional variant of MCR-1, was detected in a KPC-3-producing ST512 Klebsiella pneumoniae isolate collected in Italy from a surveillance rectal swab from a leukemic child. The mcr-1.2 gene was carried on a transferable IncX4 plasmid whose structure was very similar to that of mcr-1-bearing plasmids previously found in Escherichia coli and K. pneumoniae strains from geographically distant sites (Estonia, China, and South Africa).

  11. Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody.

    PubMed

    Yuki, Yoshikazu; Kurokawa, Shiho; Kozuka-Hata, Hiroko; Tokuhara, Daisuke; Mejima, Mio; Kuroda, Masaharu; Oyama, Masaaki; Nishimaki-Mogami, Tomoko; Teshima, Reiko; Kiyono, Hiroshi

    2016-04-01

    To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor-like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor-like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

  12. A Phase Variant of Azospirillum lipoferum Lacks a Polar Flagellum and Constitutively Expresses Mechanosensing Lateral Flagella

    PubMed Central

    Alexandre, Gladys; Rohr, René; Bally, René

    1999-01-01

    Flagellation of a nonswimming variant of the mixed flagellated bacterium Azospirillum lipoferum 4B was characterized by electron microscopy, and polyclonal antibodies were raised against polar and lateral flagellins. The variant cells lacked a polar flagellum due to a defect in flagellin synthesis and constitutively expressed lateral flagella. The variant cells were able to respond to conditions that restricted the rotation of lateral flagella by producing more lateral flagella, suggesting that the lateral flagella, as well as the polar flagellum, are mechanosensing. PMID:10508112

  13. De Novo Assembly of the Complete Genome of an Enhanced Electricity-Producing Variant of Geobacter sulfurreducens Using Only Short Reads

    PubMed Central

    Nagarajan, Harish; Butler, Jessica E.; Klimes, Anna; Qiu, Yu; Zengler, Karsten; Ward, Joy; Young, Nelson D.; Methé, Barbara A.; Palsson, Bernhard Ø.; Lovley, Derek R.; Barrett, Christian L.

    2010-01-01

    State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated. We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm. The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another. PMID:20544019

  14. Intrahippocampal administration of a domain antibody that binds aggregated amyloid-β reverses cognitive deficits produced by diet-induced obesity

    PubMed Central

    Osborne, Danielle M.; Fitzgerald, Dennis P.; O’Leary, Kelsey E.; Anderson, Brian M.; Lee, Christine C.; Tessier, Peter M.; McNay, Ewan C.

    2016-01-01

    Background The prevalence of high fat diets (HFD), diet-induced obesity (DIO) and Type 2 diabetes continues to increase, associated with cognitive impairment in both humans and rodent models. Mechanisms transducing these impairments remain largely unknown: one possibility is that a common mechanism may be involved in the cognitive impairment seen in obese and/or diabetic states and in dementia, specifically Alzheimer’s disease (AD). DIO is well established as a risk factor for development of AD. Oligomeric amyloid-β (Aβ) is neurotoxic, and we showed that intrahippocampal oligomeric Aβ produces cognitive and metabolic dysfunction similar to that seen in DIO or diabetes. Moreover, animal models of DIO show elevated brain Aβ, a hallmark of AD, suggesting that this may be one source of cognitive impairment in both conditions. Methods Intrahippocampal administration of a novel anti-Aβ domain antibody for aggregated Aβ, or a control domain antibody, to control or HFD-induced DIO rats. Spatial learning measured in a conditioned contextual fear (CCF) task after domain antibody treatment; postmortem, hippocampal NMDAR and AMPAR were measured. Results DIO caused impairment in CCF, and this impairment was eliminated by intrahippocampal administration of the active domain antibody. Measurement of hippocampal proteins suggests that DIO causes dysregulation of hippocampal AMPA receptors, which is also reversed by acute domain antibody administration. Conclusions Our findings support the concept that oligomeric Aβ within the hippocampus of DIO animals may not only be a risk factor for development of AD but may also cause cognitive impairment before the development of dementia. General Significance and Interest Our work integrates the engineering of domain antibodies with conformational-and sequence-specificity for oligomeric amyloid beta with a clinically relevant model of diet-induced obesity in order to demonstrate not only the pervasive effects of obesity on several

  15. Intrahippocampal administration of a domain antibody that binds aggregated amyloid-β reverses cognitive deficits produced by diet-induced obesity.

    PubMed

    Osborne, Danielle M; Fitzgerald, Dennis P; O'Leary, Kelsey E; Anderson, Brian M; Lee, Christine C; Tessier, Peter M; McNay, Ewan C

    2016-06-01

    The prevalence of high fat diets (HFD), diet-induced obesity (DIO) and Type 2 diabetes continues to increase, associated with cognitive impairment in both humans and rodent models. Mechanisms transducing these impairments remain largely unknown: one possibility is that a common mechanism may be involved in the cognitive impairment seen in obese and/or diabetic states and in dementia, specifically Alzheimer's disease (AD). DIO is well established as a risk factor for development of AD. Oligomeric amyloid-β (Aβ) is neurotoxic, and we showed that intrahippocampal oligomeric Aβ produces cognitive and metabolic dysfunction similar to that seen in DIO or diabetes. Moreover, animal models of DIO show elevated brain Aβ, a hallmark of AD, suggesting that this may be one source of cognitive impairment in both conditions. Intrahippocampal administration of a novel anti-Aβ domain antibody for aggregated Aβ, or a control domain antibody, to control or HFD-induced DIO rats. Spatial learning measured in a conditioned contextual fear (CCF) task after domain antibody treatment; postmortem, hippocampal NMDAR and AMPAR were measured. DIO caused impairment in CCF, and this impairment was eliminated by intrahippocampal administration of the active domain antibody. Measurement of hippocampal proteins suggests that DIO causes dysregulation of hippocampal AMPA receptors, which is also reversed by acute domain antibody administration. Our findings support the concept that oligomeric Aβ within the hippocampus of DIO animals may not only be a risk factor for development of AD but may also cause cognitive impairment before the development of dementia. Our work integrates the engineering of domain antibodies with conformational- and sequence-specificity for oligomeric amyloid beta with a clinically relevant model of diet-induced obesity in order to demonstrate not only the pervasive effects of obesity on several aspects of brain biochemistry and behavior, but also the bioengineering of

  16. Persistence survey of Toxic Shock Syndrome toxin-1 producing Staphylococcus aureus and serum antibodies to this superantigen in five groups of menstruating women

    PubMed Central

    2010-01-01

    Background Menstrual Toxic Shock Syndrome (mTSS) is thought to be associated with the vaginal colonization with specific strains of Staphylococcus aureus TSST-1 in women who lack sufficient antibody titers to this toxin. There are no published studies that examine the seroconversion in women with various colonization patterns of this organism. Thus, the aim of this study was to evaluate the persistence of Staphylococcus aureus colonization at three body sites (vagina, nares, and anus) and serum antibody to toxic shock syndrome toxin-producing Staphylococcus aureus among a small group of healthy, menstruating women evaluated previously in a larger study. Methods One year after the completion of that study, 311 subjects were recalled into 5 groups. Four samples were obtained from each participant at several visits over an additional 6-11 month period: 1) an anterior nares swab; 2) an anal swab; 3) a vagina swab; and 4) a blood sample. Gram stain, a catalase test, and a rapid S. aureus-specific latex agglutination test were performed to phenotypically identify S. aureus from sample swabs. A competitive ELISA was used to quantify TSST-1 production. Human TSST-1 IgG antibodies were determined from the blood samples using a sandwich ELISA method. Results We found only 41% of toxigenic S. aureus and 35.5% of non-toxigenic nasal carriage could be classified as persistent. None of the toxigenic S. aureus vaginal or anal carriage could be classified as persistent. Despite the low persistence of S. aureus colonization, subjects colonized with a toxigenic strain were found to display distributions of antibody titers skewed toward higher titers than other subjects. Seven percent (5/75) of subjects became seropositive during recall, but none experienced toxic shock syndrome-like symptoms. Conclusions Nasal carriage of S. aureus appears to be persistent and the best predicator of subsequent colonization, whereas vaginal and anal carriage appear to be more transient. From these

  17. Human Polyclonal Antibodies Produced through DNA Vaccination of Transchromosomal Cattle Provide Mice with Post-Exposure Protection against Lethal Zaire and Sudan Ebolaviruses

    PubMed Central

    Bounds, Callie E.; Kwilas, Steven A.; Kuehne, Ana I.; Brannan, Jennifer M.; Bakken, Russell R.; Dye, John M.; Hooper, Jay W.; Dupuy, Lesley C.; Ellefsen, Barry; Hannaman, Drew; Wu, Hua; Jiao, Jin-an; Sullivan, Eddie J.; Schmaljohn, Connie S.

    2015-01-01

    DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR -/-) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe

  18. Human Polyclonal Antibodies Produced through DNA Vaccination of Transchromosomal Cattle Provide Mice with Post-Exposure Protection against Lethal Zaire and Sudan Ebolaviruses.

    PubMed

    Bounds, Callie E; Kwilas, Steven A; Kuehne, Ana I; Brannan, Jennifer M; Bakken, Russell R; Dye, John M; Hooper, Jay W; Dupuy, Lesley C; Ellefsen, Barry; Hannaman, Drew; Wu, Hua; Jiao, Jin-an; Sullivan, Eddie J; Schmaljohn, Connie S

    2015-01-01

    DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR(-/-)) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe

  19. Human anti-varicella-zoster virus (VZV) recombinant monoclonal antibody produced after Zostavax immunization recognizes the gH/gL complex and neutralizes VZV infection.

    PubMed

    Birlea, Marius; Owens, Gregory P; Eshleman, Emily M; Ritchie, Alanna; Traktinskiy, Igor; Bos, Nathan; Seitz, Scott; Azarkh, Yevgeniy; Mahalingam, Ravi; Gilden, Don; Cohrs, Randall J

    2013-01-01

    Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.

  20. A trial with IgY chicken antibodies to eradicate faecal carriage of Klebsiella pneumoniae and Escherichia coli producing extended-spectrum beta-lactamases

    PubMed Central

    Jonsson, Anna-Karin; Larsson, Anders; Tängdén, Thomas; Melhus, Åsa; Lannergård, Anders

    2015-01-01

    Background Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is an emerging therapeutic challenge, especially in the treatment of urinary tract infections. Following an outbreak of CTX-M-15 Klebsiella pneumoniae in Uppsala, Sweden, an orphan drug trial on IgY chicken antibodies was undertaken in an attempt to eradicate faecal carriage of ESBL-producing K. pneumoniae and Escherichia coli. Methods Hens were immunised with epitopes from freeze-dried, whole-cell bacteria (ESBL-producing K. pneumoniae and E. coli) and recombinant proteins of two K. pneumoniae fimbriae subunits (fimH and mrkD). The egg yolks were processed according to good manufacturing practice and the product was stored at−20°C until used. Using an internal database from the outbreak and the regular laboratory database, faecal carriers were identified and recruited from May 2005 to December 2013. The participants were randomised in a placebo-controlled 1:1 manner. Results From 749 eligible patients, 327 (44%) had deceased, and only 91 (12%) were recruited and signed the informed consent. In the initial screening performed using the polymerase chain reaction, 24 participants were ESBL positive and subsequently randomised and treated with either the study drug or a placebo. The study was powered for 124 participants. Because of a very high dropout rate, the study was prematurely terminated. From the outbreak cohort (n=247), only eight patients were screened, and only one was positive with the outbreak strain in faeces. Conclusions The present study design, using IgY chicken antibodies for the eradication of ESBL-producing K. pneumonia and E. coli, was ineffective in reaching its goal due to high mortality and other factors resulting in a low inclusion rate. Spontaneous eradication of ESBL-producing bacteria was frequently observed in recruited participants, which is consistent with previous reports. PMID:26560861

  1. Validation of a pH gradient-based ion-exchange chromatography method for high-resolution monoclonal antibody charge variant separations.

    PubMed

    Rea, Jennifer C; Moreno, G Tony; Lou, Yun; Farnan, Dell

    2011-01-25

    Ion-exchange chromatography is widely used for profiling the charge heterogeneity of proteins, including monoclonal antibodies. Despite good resolving power and robustness, ionic strength-based ion-exchange separations are product-specific and time-consuming to develop. We have previously reported a novel pH-based separation of proteins by cation exchange chromatography that was multi-product, high-resolution, and robust against variations in sample matrix salt concentration and pH. In this study, a pH gradient-based separation method using cation exchange chromatography was evaluated in a mock validation. This method was shown to be robust for monoclonal antibodies and suitable for its intended purpose of charge heterogeneity analysis. Simple mixtures of defined buffer components were used to generate the pH gradients that separated closely related antibody species. Validation characteristics, such as precision and linearity, were evaluated. Robustness to changes in protein load, buffer pH and column oven temperature was demonstrated. The stability-indicating capability of this method was determined using thermally stressed antibody samples. In addition, intermediate precision was demonstrated using multiple instruments, multiple analysts, multiple column lots, and different column manufacturers. Finally, the precision for this method was compared to conventional ion-exchange chromatography and imaged capillary isoelectric focusing. These results demonstrate the superior precision and robustness of this multi-product method, which can be used for the high-throughput evaluation of in-process and final product samples.

  2. An in vitro combined antibiotic/antibody treatment eliminates toxicity from Shiga toxin-producing E. coli

    USDA-ARS?s Scientific Manuscript database

    Background: Treating Shiga toxin-producing Escherichia coli (STEC) gastrointestinal infections is a difficult endeavor. The utility of antibiotics as an STEC treatment is controversial since antibiotic resistance among STEC isolates is widespread and certain antibiotics dramatically increase express...

  3. Interaction between PLA2R1 and HLA-DQA1 Variants Associates with Anti-PLA2R Antibodies and Membranous Nephropathy

    PubMed Central

    Lv, Jicheng; Hou, Wanyin; Zhou, Xujie; Liu, Gang; Zhou, Fude; Zhao, Na; Hou, Ping; Zhao, Minghui

    2013-01-01

    Risk alleles at genome loci containing phospholipase A2 receptor 1 (PLA2R1) and HLA-DQA1 closely associate with idiopathic membranous nephropathy (IMN) in the European population, but it is unknown whether a similar association exists in the Chinese population and whether high-risk alleles promote the development of anti-PLA2R antibodies. Here, we genotyped 2132 Chinese individuals, including 1112 patients with IMN and 1020 healthy controls, for three single nucleotide polymorphisms (SNPs) within PLA2R1 and three SNPs within HLA genes. We also selected 71 patients, with varying genotypes, to assess for circulating anti-PLA2R antibody and for PLA2R expression in glomeruli. Three SNPs within PLA2R1 and one SNP within HLA-DQA1 strongly associated with IMN, and we noted gene–gene interactions involving these SNPs. Furthermore, these risk alleles strongly associated with the presence of anti-PLA2R antibodies and glomerular PLA2R expression. Among individuals who carried risk alleles for both genes, 73% had anti-PLA2R antibodies and 75% expressed PLA2R in glomeruli. In contrast, among individuals who carried protective genotypes of both genes, none had anti-PLA2R antibodies and glomerular expression of PLA2R was weak or absent. In conclusion, the interaction between PLA2R1 and HLA-DQA1 risk alleles associates with the development of IMN in the Chinese population. Individuals carrying risk alleles are predisposed to the generation of circulating anti-PLA2R autoantibodies, which may contribute to the development of IMN. PMID:23813219

  4. Versuche zur Gewinnung von katalytischen Antikörpern zur Hydrolyse von Arylcarbamaten und Arylharnstoffen. (English Title: Attempts to produce catalytic antibodies for hydrolysis of arylcarbamates and arylureas)

    NASA Astrophysics Data System (ADS)

    Werner, Deljana

    2002-05-01

    Umsatzgeschwindigkeit eine lineare Abhängigkeit festgestellt. Die thermodynamische Gleichtgewichtsdissoziationskonstante KD des Abzyms von 2,6 nM zeugt von einer sehr guten Affinität zum ÜZA. Hydrolytisch aktiv waren nur Antikörper, die gegen das Übergangszustandsanalogon Hei3 hergestellt worden waren. Es wird vermutet, dass die Hydrolyse der Benzylphenylcarbamate über einen Additions-Eliminierungsmechanismus unter Ausbildung eines tetraedrischen Übergangszustandes verläuft, dessen analoge Verbindung Hei3 ist. Im Rahmen der Generierung von Nachweisantikörpern zur Detektion der Substratabnahme bei der Hydrolyse wurden Anti-Diuron-Antikörper hergestellt. Einer der Antikörper (B91-CG5) ist spezifisch für das Herbizid Diuron und hat einen IC50-Wert von 0,19 µg/l und eine untere Nachweisgrenze von 0,04 µg/l. Ein anderer Antikörper (B91-KF5) reagiert kreuz mit einer Palette ähnlicher Herbizide. Mit diesen Antikörpern wurde ein empfindlicher Labortest, der ein Monitoring von Diuron auf Grundlage des durch die Trinkwasserverordnung festgeschriebenen Wertes für Pflanzenschutzmittel von 0,1 µg/l erlaubt, aufgebaut. Der Effekt der Anti-Diuron-Antikörper auf die Diuron-inhibierte Photosynthese wurde in vitro und in vivo untersucht. Es wurde nachgewiesen, dass sowohl in isolierten Thylakoiden, als auch in intakten Algen eine Vorinkubation der Anti-Diuron-Antikörper mit Diuron zur Inaktivierung seiner Photosynthese-hemmenden Wirkung führt. Wurde der Elektronentransport in den isolierten Thylakoiden oder in Algen durch Diuron unterbrochen, so führte die Zugabe der Anti-Diuron-Antikörper zur Reaktivierung der Elektronenübertragung. Attempts to produce catalytic antibodies for hydrolysis of arylcarbamates and arylureas: The aim of the investigations was to produce antibodies which are able to cleave herbicides resistant to naturally occuring enzymes. Structurally similar carbamate and urea derivatives were chosen for the experiments. Phosphonate derivatives were synthesized

  5. Optimization of hapten-peptide labeling for pretargeted immunoPET of bispecific antibody using generator-produced 68Ga.

    PubMed

    Karacay, Habibe; Sharkey, Robert M; McBride, William J; Rossi, Edmund A; Chang, Chien-Hsing; Goldenberg, David M

    2011-04-01

    Bispecific antibody pretargeting is highly sensitive and specific for cancer detection by PET. In this study, the preparation of a high-specific-activity (68)Ga-labeled hapten-peptide, IMP288, was evaluated. IMP288 (DOTA-D-Tyr-D-Lys(histamine-succinyl-glycine [HSG])-D-glu-D-Lys(HSG)-NH(2)) was added to buffered (68)Ga and then heated in boiling water and purified on a reversed-phase cartridge. Tumor-bearing nude mice were used for biodistribution and tumor localization studies. (68)Ga-IMP288 was prepared at a starting specific activity up to 1.78 GBq/nmol, with final yields of 0.74 GBq/nmol (decay-corrected) and less than 1% unbound (68)Ga. Purification was essential to remove unbound (68)Ga and (68)Ge breakthrough. Pretargeted animals showed a high (68)Ga-IMP288 uptake (27.5 ± 5.8 percentage injected dose per gram), with ratios of 13.6 ± 4.8, 66.8 ± 14.5, and 325.9 ± 61.9 for the kidneys, liver, and blood, respectively, at 1.5 h after peptide injection. High-specific-activity labeling of DOTA-hapten-peptide was obtained from the (68)Ga/(68)Ge generator for approximately 1 y, yielding products suitable for immunoPET.

  6. Application of a risk analysis method to different technologies for producing a monoclonal antibody employed in hepatitis B vaccine manufacturing.

    PubMed

    Milá, Lorely; Valdés, Rodolfo; Tamayo, Andrés; Padilla, Sigifredo; Ferro, Williams

    2012-03-01

    CB.Hep-1 monoclonal antibody (mAb) is used for a recombinant Hepatitis B vaccine manufacturing, which is included in a worldwide vaccination program against Hepatitis B disease. The use of this mAb as immunoligand has been addressed into one of the most efficient steps of active pharmaceutical ingredient purification process. Regarding this, Quality Risk Management (QRM) provides an excellent framework for the risk management use in pharmaceutical manufacturing and quality decision-making applications. Consequently, this study sought applying a prospective risk analysis methodology Failure Mode Effects Analysis (FMEA) as QRM tool for analyzing different CB.Hep-1 mAb manufacturing technologies. As main conclusions FMEA was successfully used to assess risks associated with potential problems in CB.Hep-1 mAb manufacturing processes. The severity and occurrence of risks analysis evidenced that the percentage of very high severe risks ranged 31.0-38.7% of all risks and the huge majority of risks have a very low occurrence level (61.9-83.3%) in all assessed technologies. Finally, additive Risk Priority Number, was descending ordered as follow: transgenic plants (2636), ascites (2577), transgenic animals (2046) and hollow fiber bioreactors (1654), which also corroborated that in vitro technology, should be the technology of choice for CB.Hep-1 mAb manufacturing in terms of risks and mAb molecule quality. Copyright © 2011 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  7. Top-down and middle-down approach by fraction collection enrichment using off-line capillary electrophoresis - mass spectrometry coupling: Application to monoclonal antibody Fc/2 charge variants.

    PubMed

    Biacchi, Michael; Said, Nassur; Beck, Alain; Leize-Wagner, Emmanuelle; François, Yannis-Nicolas

    2017-02-27

    The characterization of complex protein mixtures represents one of the biggest challenge in many research fields such as biological or biopharmaceutical sciences. Out of all categories, monoclonal antibodies (mAbs) and related products drawn the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need for analytical methods to provide comprehensive in-depth characterization of these proteins. In this work, we developed a methodology using CE-UV/MALDI-MS to perform top-down or middle-down characterization after fraction collection enrichment applied to intact protein and mAbs samples. The performance of the method was evaluated with the rapid separation of three intact protein mixture. Good robustness of CZE separation and quality of MALDI-MS spectra and MALDI-ISD spectra of each protein confirms the usefulness of sample enrichment to obtain adequate quantity of deposed protein for top-down analysis and the proof of principle of the method. In a second step, the method was applied to the middle-down characterization of Fc/2 cetuximab variants. Identification of around 9% sequence coverage of Fc/2 cetuximab fragments allows to conclude on the feasibility of the strategy for middle-down characterization of Fc/2 cetuximab variants using CE-UV/MALDI-MS. Moreover, MALDI-ISD fragmentation of Fc/2 cetuximab variants confirm separation phenomenon based on the formation of Fc/2 dimers with and without C-terminal truncation.

  8. Anti-ICOS Monoclonal Antibody MEDI-570 in Treating Patients With Relapsed or Refractory Peripheral T-cell Lymphoma Follicular Variant or Angioimmunoblastic T-cell Lymphoma

    ClinicalTrials.gov

    2017-09-28

    Follicular Variant Peripheral T-Cell Lymphoma; Grade 1 Follicular Lymphoma; Grade 2 Follicular Lymphoma; Grade 3a Follicular Lymphoma; Recurrent Angioimmunoblastic T-cell Lymphoma; Recurrent Follicular Lymphoma; Recurrent Mature T- and NK-Cell Non-Hodgkin Lymphoma; Recurrent Mycosis Fungoides; Recurrent Primary Cutaneous T-Cell Non-Hodgkin Lymphoma; Refractory Angioimmunoblastic T-cell Lymphoma; Refractory Follicular Lymphoma; Refractory Mature T-Cell and NK-Cell Non-Hodgkin Lymphoma; Stage IB Mycosis Fungoides; Stage II Mycosis Fungoides; Stage III Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage III Mycosis Fungoides; Stage IV Cutaneous T-Cell Non-Hodgkin Lymphoma; Stage IV Mycosis Fungoides

  9. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells.

    PubMed

    Hussain, Musaddeq

    2015-11-10

    Chinese hamster ovary (CHO) cells are the host cell of choice for manufacturing of monoclonal antibody (mAb) drugs in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and must be controlled and monitored in order to ensure drug purity and safety. A conventional method for quantification of host residual DNA in drug requires extraction of DNA from the mAb drug substance with subsequent quantification of the extracted DNA using real-time PCR (qPCR). Here we report a method where the DNA extraction step is eliminated prior to qPCR. In this method, which we have named 'direct resDNA qPCR', the mAb drug substance is digested with a protease called KAPA in a 96-well PCR plate, the protease in the digest is then denatured at high temperature, qPCR reagents are added to the resultant reaction wells in the plate along with standards and controls in other wells of the same plate, and the plate subjected to qPCR for analysis of residual host DNA in the samples. This direct resDNA qPCR method for CHO is sensitive to 5.0fg of DNA with high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with four mAbs drug, two IgG1 and two IgG4. Both the purified drug substance as well as a number of process intermediate samples, e.g., bioreactor harvest, Protein A column eluate and ion-exchange column eluates were tested. This method simplifies the residual DNA quantification protocol, reduces time of analysis and leads to increased assay sensitivity and development of automated high-throughput methods.

  10. Blastomyces dermatitidis: Antibody Detection in Sera from Dogs with Blastomycosis with Yeast Lysate Antigens Produced from Human and Dog Isolates.

    PubMed

    Mondada, Katie; Fullmer, Jessie; Hungerford, Eric; Novack, Katrina; Vickers, Kristen; Scalarone, Gene

    2014-01-01

    Dogs are common hosts to the fungal organism Blastomyces dermatitidis, which causes the systemic disease blastomycosis. The goal of our study was to compare the reactivity of two B. dermatitidis yeast lysate antigens prepared from dog isolates (ERC-2, Wisconsin; T-58, Tennessee) and two lysate antigens prepared from human isolates (B5931 and B5896, Minnesota) against 48 serum specimens from dogs with confirmed blastomycosis using the indirect enzyme-linked immunosorbent assay (ELISA). Secondarily, we used three different ELISA substrates (Ultra TMB: A, SureBlue: B, and SureBlue Reserve: C) to compare the effectiveness of each substrate. Mean absorbance values ranged from 0.446 (B) to 0.651 (C) for the B5931 antigen and from 0.393 (B) to 0.540 (C) for the ERC-2 antigen in Trial 1. In Trial 2, the absorbance values ranged from 0.628 (B) to 0.909 (A) for the B5896 antigen and from 0.828 (B) to 1.375 (C) for the T-58 antigen. In Trial 1, the lysate antigen prepared from the human isolate B5931 exhibited the highest absorbance value and in Trial 2 the lysate prepared from the dog isolate T-58 was the most reactive. The overall results thus indicated that the T-58 lysate was the optimal reagent when used to detect antibody with the Sure-Blue Reserve substrate. Our laboratory is continuing to study B. dermatitidis antigen and substrate combinations for the reliable immunodiagnosis of blastomycosis in humans and animals.

  11. Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF.

    PubMed

    Weiser, W Y; Remold, H G; David, J R

    1985-01-01

    Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

  12. Exploring the capabilities of fluorometric online monitoring on chinese hamster ovary cell cultivations producing a monoclonal antibody.

    PubMed

    Schwab, Karen; Amann, Thomas; Schmid, Jakob; Handrick, René; Hesse, Friedemann

    2016-11-01

    Online monitoring of Chinese hamster ovary fed-batch cell cultures via two-dimensional fluorescence spectroscopy (2DFS) was evaluated in this work. Particular attention was directed toward different process strategies regarding the use of nutrient-rich feed media and temperature shifts. These intentionally performed process manipulations broadened the variances in the obtained fluorescence spectra and this was suspected to hamper the generation of reliable soft sensors. Principal component analysis of the obtained fluorescence data showed that temperature shift and feeding strategy had a considerable impact on the fluorescence signals. Partial least square regression models were calculated for the prediction of glucose, lactate, monoclonal antibody (mAb), and viable cell concentrations (VCC). It was aimed to integrate all 2DFS datasets in the respective calibration models regardless of the process-strategy-dependent diversity. Contrary to the expectations, it was feasible to calibrate soft sensors for the online prediction of glucose (7 latent variables (LVs), Rcal2 = 0.97, rout mean squared error of prediction (RMSEP) = 1.1 g L(-1) ), lactate (5 LV; Rcal2 = 0.96; RMSEP = 0.5 g L(-1) ) and mAb concentrations (4 LV; Rcal2 = 0.99; RMSEP = 11.4 mg L(-1) ). Feeding and temperature shifts had the highest impact on the VCC model (3 LV; Rcal2 = 0.94; RMSEP 3.8 × 10(5) mL(-1) ), nevertheless the prediction of VCC from the fed-batch 2DFS data was feasible. The results strongly indicate that variances in the datasets due to the process strategy can be tolerated to some extent by the respective soft sensors. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1592-1600, 2016.

  13. Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

    PubMed

    Revoltella, R P; Laricchia Robbio, L; Liberati, A M; Reato, G; Foa, R; Funaro, A; Vinante, F; Pizzolo, G

    2000-09-15

    We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing.

  14. A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.

    PubMed

    Mett, Vadim; Chichester, Jessica A; Stewart, Michelle L; Musiychuk, Konstantin; Bi, Hong; Reifsnyder, Carolyn J; Hull, Anna K; Albrecht, Mark T; Goldman, Stanley; Baillie, Les W J; Yusibov, Vidadi

    2011-01-01

    The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.

  15. Antibody titer has positive predictive value for vaccine protection against challenge with natural antigenic-drift variants of H5N1 high-pathogenicity avian influenza viruses from Indonesia.

    PubMed

    Swayne, David E; Suarez, David L; Spackman, Erica; Jadhao, Samadhan; Dauphin, Gwenaelle; Kim-Torchetti, Mia; McGrane, James; Weaver, John; Daniels, Peter; Wong, Frank; Selleck, Paul; Wiyono, Agus; Indriani, Risa; Yupiana, Yuni; Sawitri Siregar, Elly; Prajitno, Teguh; Smith, Derek; Fouchier, Ron

    2015-04-01

    antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses. H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Antibody Titer Has Positive Predictive Value for Vaccine Protection against Challenge with Natural Antigenic-Drift Variants of H5N1 High-Pathogenicity Avian Influenza Viruses from Indonesia

    PubMed Central

    Suarez, David L.; Spackman, Erica; Jadhao, Samadhan; Dauphin, Gwenaelle; Kim-Torchetti, Mia; McGrane, James; Weaver, John; Daniels, Peter; Wong, Frank; Selleck, Paul; Wiyono, Agus; Indriani, Risa; Yupiana, Yuni; Sawitri Siregar, Elly; Prajitno, Teguh; Smith, Derek; Fouchier, Ron

    2015-01-01

    antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses. IMPORTANCE H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear. PMID:25609805

  17. Characterization of N-Linked Glycosylation in a Monoclonal Antibody Produced in NS0 Cells Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection

    PubMed Central

    Hamm, Melissa; Wang, Yang; Rustandi, Richard R.

    2013-01-01

    The N-linked glycosylation in recombinant monoclonal antibodies (mAb) occurs at Asn297 on the Fc region in the CH2 domain. Glycosylation heterogeneities have been well documented to affect biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) through their interaction with Fc-receptors. Hence, it is critical to monitor and characterize the N-linked glycosylation profile in a therapeutic protein such as a mAb for product consistency. In one approach, the glycans are first released from the mAb using an enzyme specific digestion, such as Protein N-Glycosidase F (PNGase) and subsequently they are labeled using a fluorophore, for example, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) . Here we have applied this approach and used Capillary Electrophoresis with Laser-Induced Fluorescence detection (CE-LIF) to analyze a recombinant mAb produced in murine myeloma (NS0) cells. The technique provides short analysis times, efficient separations, and high sensitivity. CE-LIF peak identification was done by a combination of glycan standards and treatment with various exoglycosidases. Furthermore, the APTS-labeled glycans were also analyzed using hydrophilic interaction chromatography (HILIC) high performance liquid chromatography (HPLC) to aid identification of minor peaks by sample collection and off-line mass spectrometry (MS) analysis. PMID:24276024

  18. Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1.

    PubMed

    Matsuyama, Rima; Yamano, Noriko; Kawamura, Namiko; Omasa, Takeshi

    2017-03-01

    The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400 nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

    PubMed Central

    Sączyńska, Violetta; Romanik, Agnieszka; Florys, Katarzyna; Cecuda-Adamczewska, Violetta; Kęsik-Brodacka, Małgorzata; Śmietanka, Krzysztof; Olszewska, Monika; Domańska-Blicharz, Katarzyna; Minta, Zenon; Szewczyk, Bogusław; Płucienniczak, Grażyna; Płucienniczak, Andrzej

    2017-01-01

    The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of

  20. Effect of Corynebacterium parvum on the class and subclass of antibody produced in the response of different strains of mice to sheep erythrocytes.

    PubMed Central

    Warr, G W; James, K

    1975-01-01

    Several strains of mice were injected with sheep erythrocytes (SRBC) using C. parvum as adjuvant. The adjuvant effects on the amounts of class and subclass of antibody produced were ranked in the order IgG2b greater than IgG2a and IgM greater than IgG1. In addition, these effects were shown to vary depending on the time of administration of C. parvum relative to antigen. C parvum was shown to have no adjuvant effect on the response of congenitally athymic mice when given at the same time as the antigen, SRBC. On the basis of the reported observations it is suggested that certain of the adjuvant effects of C. parvum require T-cell function. PMID:1092609

  1. Multi-walled carbon nanotubes increase antibody-producing B cells in mice immunized with a tetravalent vaccine candidate for dengue virus.

    PubMed

    Calegari, Luan P; Dias, Roberto S; de Oliveira, Michelle D; Pessoa, Carine Ribeiro; de Oliveira, André S; Oliveira, Ana F C S; da Silva, Cynthia C; Fonseca, Flavio G; Versiani, Alice F; De Paula, Sérgio O

    2016-07-27

    In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile. Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC. The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

  2. Cellulase variants

    DOEpatents

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  3. Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-protonation-based charge variants.

    PubMed

    Luo, Haibin; Cao, Mingyan; Newell, Kelcy; Afdahl, Christopher; Wang, Jihong; Wang, William K; Li, Yuling

    2015-12-11

    We have systemically investigated unusual elution behaviors of an IgG4 (mAb A) in cation exchange chromatography (CEX). This mAb A exhibited two elution peaks under certain conditions when being purified by several strong CEX columns. When either of the two peaks was isolated and re-injected on the same column, the similar pattern was observed again during elution. The protein distribution between the two peaks could be altered by NaCl concentration in the feed, or NaCl concentration in wash buffer, or elution pH, suggesting two pH-associated strong-and-weak binding configurations. The protein distributions under different pH values showed good correlation with protonated/un-protonated fractions of a histidine residue. These results suggest that the double-peak elution profile associates with histidine-protonation-based charge variants. By conducting pepsin digestion, amino-acid specific chemical modifications, peptide mapping, and measuring the effects of elution residence time, a histidine in the variable fragment (Fab) was identified to be the root cause. Besides double-peak pattern, mAb A can also exhibit peak-shouldering or single elution peak on different CEX resins, reflecting different resins' resolving capability on protonated/un-protonated forms. This work characterizes a novel cause for unusual elution behaviors in CEX and also provides alternative avenues of purification development for mAbs with similar behaviors.

  4. Polymorphisms of the Hepatitis A Virus Cellular Receptor 1 in African Green Monkey Kidney Cells Result in Antigenic Variants That Do Not React with Protective Monoclonal Antibody 190/4

    PubMed Central

    Feigelstock, Dino; Thompson, Peter; Mattoo, Pravina; Kaplan, Gerardo G.

    1998-01-01

    Monoclonal antibody (MAb) 190/4 blocks binding of hepatitis A virus (HAV) to the HAV cellular receptor 1 (havcr-1) and protects African green monkey kidney (AGMK) clone GL37 cells (GL37 cells) against HAV infection. BS-C-1 and CV-1 cells, two widely used AGMK cell lines, did not react with MAb 190/4 but expressed havcr-1, as judged by Western blot analysis. The cDNA coding for havcr-1 was amplified from BS-C-1 and CV-1 total cellular RNA by reverse transcription-PCR. Alignment of the amino acid sequences inferred from the cDNA nucleotide sequences showed that BS-C-1 and CV-1 havcr-1 differed from GL37 havcr-1 by having two substitutions in the Cys-rich region, N48H and K108Q, and 10 to 11 additional substitutions plus the insertion of 18 to 22 amino acids in the mucin-like region. Studies with chimeras of GL37 havcr-1 and BS-C-1 havcr-1 showed that the K108Q substitution was responsible for the lack of reaction of MAb 190/4 with BS-C-1 and CV-1 cells. Binding studies indicated that HAV bound to dog cell transfectants expressing the BS-C-1 havcr-1 as well as the GL37/BS-C-1 havcr-1 chimeras. These results indicate that antigenic variants of havcr-1 are expressed in AGMK cells and that binding of HAV to these havcr-1 variants tolerates changes in protective epitope 190/4. PMID:9621093

  5. Polymorphisms of the hepatitis A virus cellular receptor 1 in African green monkey kidney cells result in antigenic variants that do not react with protective monoclonal antibody 190/4.

    PubMed

    Feigelstock, D; Thompson, P; Mattoo, P; Kaplan, G G

    1998-07-01

    Monoclonal antibody (MAb) 190/4 blocks binding of hepatitis A virus (HAV) to the HAV cellular receptor 1 (havcr-1) and protects African green monkey kidney (AGMK) clone GL37 cells (GL37 cells) against HAV infection. BS-C-1 and CV-1 cells, two widely used AGMK cell lines, did not react with MAb 190/4 but expressed havcr-1, as judged by Western blot analysis. The cDNA coding for havcr-1 was amplified from BS-C-1 and CV-1 total cellular RNA by reverse transcription-PCR. Alignment of the amino acid sequences inferred from the cDNA nucleotide sequences showed that BS-C-1 and CV-1 havcr-1 differed from GL37 havcr-1 by having two substitutions in the Cys-rich region, N48H and K108Q, and 10 to 11 additional substitutions plus the insertion of 18 to 22 amino acids in the mucin-like region. Studies with chimeras of GL37 havcr-1 and BS-C-1 havcr-1 showed that the K108Q substitution was responsible for the lack of reaction of MAb 190/4 with BS-C-1 and CV-1 cells. Binding studies indicated that HAV bound to dog cell transfectants expressing the BS-C-1 havcr-1 as well as the GL37/BS-C-1 havcr-1 chimeras. These results indicate that antigenic variants of havcr-1 are expressed in AGMK cells and that binding of HAV to these havcr-1 variants tolerates changes in protective epitope 190/4.

  6. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana

    PubMed Central

    Garcia, J.P.; Beingesser, J.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M.; Velasco, J.; Whaley, K.; Zeitlin, L.; Roy, C.J.; Uzal, F.A.

    2014-01-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 =1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 minutes when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 minutes post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. PMID:24950050

  7. A novel approach for the simultaneous quantification of a therapeutic monoclonal antibody in serum produced from two distinct host cell lines

    PubMed Central

    Geist, Brian J.; Davis, Darryl; McIntosh, Thomas; Yang, Tong-Yuan; Goldberg, Kenneth; Han, Chao; Pendley, Charles; Davis, Hugh M.

    2013-01-01

    Therapeutic monoclonal antibodies (mAbs) possess a high degree of heterogeneity associated with the cell expression system employed in manufacturing, most notably glycosylation. Traditional immunoassay formats used to quantify therapeutic mAbs are unable to discriminate between different glycosylation patterns that may exist on the same protein amino acid sequence. Mass spectrometry provides a technique to distinguish specific glycosylation patterns of the therapeutic antibody within the same sample, thereby allowing for simultaneous quantification of the same mAb with different glycosylation patterns. Here we demonstrate a two-step approach to successfully differentiate and quantify serum mixtures of a recombinant therapeutic mAb produced in two different host cell lines (CHO vs. Sp2/0) with distinct glycosylation profiles. Glycosylation analysis of the therapeutic mAb, CNTO 328 (siltuximab), was accomplished through sample pretreatment consisting of immunoaffinity purification (IAP) and enrichment, followed by liquid chromatography (LC) and mass spectrometry (MS). LC-MS analysis was used to determine the percentage of CNTO 328 in the sample derived from either cell line based on the N-linked G1F oligosaccharide on the mAb. The relative amount of G1F derived from each cell line was compared with ratios of CNTO 328 reference standards prepared in buffer. Glycoform ratios were converted to concentrations using an immunoassay measuring total CNTO 328 that does not distinguish between the different glycoforms. Validation of the IAP/LC-MS method included intra-run and inter-run variability, method sensitivity and freeze-thaw stability. The method was accurate (%bias range = -7.30–13.68%) and reproducible (%CV range = 1.49–10.81%) with a LOQ of 2.5 μg/mL. PMID:23182963

  8. Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation

    PubMed Central

    1992-01-01

    The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events. PMID:1370498

  9. A novel antibody engineering strategy for making monovalent bispecific heterodimeric IgG antibodies by electrostatic steering mechanism.

    PubMed

    Liu, Zhi; Leng, Esther C; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir; Liu, Hua; Fanslow, William; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio; Foltz, Ian N; Yan, Wei

    2015-03-20

    Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.

  10. Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination.

    PubMed

    Clarke, Jihong Liu; Paruch, Lisa; Dobrica, Mihaela-Olivia; Caras, Iuliana; Tucureanu, Catalin; Onu, Adrian; Ciulean, Sonya; Stavaru, Crina; Eerde, Andre; Wang, Yanliang; Steen, Hege; Haugslien, Sissel; Petrareanu, Catalina; Lazar, Catalin; Popescu, Costin-Ioan; Bock, Ralph; Dubuisson, Jean; Branza-Nichita, Norica

    2017-04-17

    The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium-mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and the Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Effect of Priming With Seasonal Influenza A(H3N2) Virus on the Prevalence of Cross-Reactive Hemagglutination-Inhibition Antibodies to Swine-Origin A(H3N2) Variants.

    PubMed

    Liu, Feng; Veguilla, Vic; Gross, F Liaini; Gillis, Eric; Rowe, Thomas; Xu, Xiyan; Tumpey, Terrence M; Katz, Jacqueline M; Levine, Min Z; Lu, Xiuhua

    2017-09-15

    Recent outbreaks of swine-origin influenza A(H3N2) variant (H3N2v) viruses have raised public health concerns. Previous studies indicated that older children and young adults had the highest levels of hemagglutination-inhibition (HI) antibodies to 2010-2011 H3N2v viruses. However, newly emerging 2013 H3N2v have acquired antigenic mutations in the hemagglutinin at amino acid position 145 (N145K/R). We estimated the levels of serologic cross-reactivity among humans primed with seasonal influenza A(H3N2) (sH3N2), using postinfection ferret antisera. We also explored age-related HI antibody responses to 2012-2013 H3N2v viruses. Human and ferret antisera were tested in HI assays against 1 representative 2012 H3N2v (145N) and 2 2013 H3N2v (145K/R) viruses, together with 9 sH3N2 viruses circulating since 1968. Low levels of cross-reactivity between the H3N2v and sH3N2 viruses from the 1970s-1990s were observed using postinfection ferret antisera. The overall seroprevalence among the sH3N2-primed population against 2012-2013 H3N2v viruses was >50%, and age-related seroprevalence was observed. Seroprevalence was significantly higher to 2013 H3N2v than to 2012 H3N2v viruses among some children likely to have been primed with A/Sydney/5/97-like (145K) or A/Wuhan/359/95-like viruses (145K). A single substitution (N145K/R) was sufficient to affect seropositivity to H3N2v viruses in some individuals. Insight into age-related antibody responses to newly emerging H3N2v viruses is critical for risk assessment and pandemic preparedness.

  12. Using simple models to describe the kinetics of growth, glucose consumption, and monoclonal antibody formation in naive and infliximab producer CHO cells.

    PubMed

    López-Meza, Julián; Araíz-Hernández, Diana; Carrillo-Cocom, Leydi Maribel; López-Pacheco, Felipe; Rocha-Pizaña, María Del Refugio; Alvarez, Mario Moisés

    2016-08-01

    Despite their practical and commercial relevance, there are few reports on the kinetics of growth and production of Chinese hamster ovary (CHO) cells-the most frequently used host for the industrial production of therapeutic proteins. We characterize the kinetics of cell growth, substrate consumption, and product formation in naive and monoclonal antibody (mAb) producing recombinant CHO cells. Culture experiments were performed in 125 mL shake flasks on commercial culture medium (CD Opti CHO™ Invitrogen, Carlsbad, CA, USA) diluted to different glucose concentrations (1.2-4.8 g/L). The time evolution of cell, glucose, lactic acid concentration and monoclonal antibody concentrations was monitored on a daily basis for mAb-producing cultures and their naive counterparts. The time series were differentiated to calculate the corresponding kinetic rates (rx = d[X]/dt; rs = d[S]/dt; rp = d[mAb]/dt). Results showed that these cell lines could be modeled by Monod-like kinetics if a threshold substrate concentration value of [S]t = 0.58 g/L (for recombinant cells) and [S]t = 0.96 g/L (for naïve cells), below which growth is not observed, was considered. A set of values for μmax, and Ks was determined for naive and recombinant cell cultures cultured at 33 and 37 °C. The yield coefficient (Yx/s) was observed to be a function of substrate concentration, with values in the range of 0.27-1.08 × 10(7) cell/mL and 0.72-2.79 × 10(6) cells/mL for naive and recombinant cultures, respectively. The kinetics of mAb production can be described by a Luedeking-Piret model (d[mAb]/dt = αd[X]/dt + β[X]) with values of α = 7.65 × 10(-7) µg/cell and β = 7.68 × 10(-8) µg/cell/h for cultures conducted in batch-agitated flasks and batch and instrumented bioreactors operated in batch and fed-batch mode.

  13. Antibody humanization by structure-based computational protein design.

    PubMed

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH ("Computationally-Driven Antibody Humanization") in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.

  14. The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells.

    PubMed

    Costa, Ana Rita; Withers, Joanne; Rodrigues, Maria Elisa; McLoughlin, Niaobh; Henriques, Mariana; Oliveira, Rosário; Rudd, Pauline M; Azeredo, Joana

    2013-06-25

    N-glycosylation is one of the most crucial parameters affecting the biological activity of therapeutic monoclonal antibodies (mAbs), and should therefore be closely monitored and controlled to guarantee a consistent and high-quality product in biopharmaceutical processes. In the present work, the effect of the time-consuming step of gradual cell adaptation to serum-free conditions on the glycosylation profile of a mAb produced by CHO-K1 cells was evaluated. High-performance liquid chromatography analysis revealed important changes in mAb glycosylation patterns in all steps of serum reduction. These changes could be grouped in two distinct phases of the process of adaptation: middle (2.5 to 0.15% serum) and final (0.075 and 0% serum). For intermediate levels of serum, a desirable increase of galactosylation and decrease of fucosylation, but an undesirable increase in sialylation were observed; while the inverse was obtained at the final stages of adaptation. These divergences may be related to the reduction of serum supplementation, to variations in the levels of cell density and viability achieved at these stages, and to the natural shift of the cell growth mode during adaptation from adherent to suspended. The divergent glycan profiles obtained in this study demonstrate a strong influence of the adaptation process on mAb glycosylation, suggesting that control and monitoring of product quality should be implemented at the early stages of process development.

  15. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

    PubMed Central

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B.; Fontana, Juan; Steven, Alasdair C.; King, Lisa R.; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K.; Golding, Hana

    2011-01-01

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified. PMID:21704111

  16. A novel modified vaccination technique produces IgG antibodies that cause complement-mediated lysis of multiple myeloma cells carrying CD38 antigen.

    PubMed

    Barabas, Arpad Z; Cole, Chad D; Graeff, Richard M; Morcol, Tulin; Lafreniere, Rene

    2016-01-01

    Objectives were to: 1) induce a lytic IgG antibody (ab) response (via the so called `third vaccination method') against CD38 antigen (ag) residing on the extra-cellular domain of multiple myeloma (MM) cells in recipient rabbits, by combining the CD38 ag with donor-derived anti-CD38 ag lytic IgG ab into an immune complex (IC); and 2) determine whether abs produced would cause complement-mediated lysis (in vitro) of human MM cells containing CD38 ag. The vaccine was created in a two-step process. First, ab (rabbit anti-CD38 ag IgG ab) was raised in donor rabbits by injections of low molecular weight soluble CD38 ag in Freund's complete adjuvant (FCA) and aqueous solution. Second, transfer of pathogenic lytic IgG ab response into recipient rabbits was achieved by injections of ICs composed of CD38 ag and homologous anti-CD38 ag IgG ab. Consequently, recipient rabbits produced the same ab with the same specificity against the target ag as was present in the inoculum, namely agglutinating, precipitating and lytic (as demonstrated in vitro). In an in vitro study, in the presence of complement, donor and recipient rabbits' immune sera caused lysis of CD38 ag associated human MM cells. The most effective lytic ab response causing sera were those from donor rabbits injected with CD38 ag in FCA and those from rabbits injected with ICs, especially when they were administered in adjuvants. These results provided proof of concept that the third vaccination method has good potential as a stand-alone and efficacious method of controlling cancer.

  17. The new allelic variant of the subtilase cytotoxin (subAB2) is common among Shiga toxin-producing Escherichia coli strains from large game animals and their meat and meat products.

    PubMed

    Sánchez, Sergio; Díaz-Sánchez, Sandra; Martínez, Remigio; Llorente, María Teresa; Herrera-León, Silvia; Vidal, Dolors

    2013-10-25

    Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Two allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, and subAB2, located on a pathogenicity island (PAI) together with tia gene. While subAB1 has been reported to be more frequent among bovine strains, subAB2 has been mainly associated with strains from small ruminants. We investigated the presence of the two variants of subAB among 59 eae-negative STEC from large game animals (deer and wild boar) and their meat and meat products in order to assess the role of other species in the epidemiology of subAB-positive, eae-negative STEC. For this approach, the strains were PCR-screened for the presence of subAB, including the specific detection of both allelic variants, for the presence of saa, tia and sab, and for stx subtyping. Overall, subAB genes were detected in 71.2% of the strains: 84.1% of the strains from deer and 33.3% of the strains from wild boar. Most of them (97.6%) possessed subAB2 and most of these subAB2-positive strains (92.7%) were also positive for tia and negative for saa, suggesting the presence of the subAB2-harbouring PAI. Subtype stx2b was present in most of the strains (67.8%) and a statistically significant association could be established between subAB2 and stx2b. Our results suggest that large game animals, mainly deer, may represent an important animal reservoir of subAB2-positive, eae-negative STEC, and also highlight the risk of human infection posed by the consumption of large game meat and meat products.

  18. Immunodetection of Triticum mosaic virus by DAS- and DAC-ELISA using antibodies produced against coat protein expressed in Escherichia coli: potential for high-throughput diagnostic methods.

    PubMed

    Tatineni, Satyanarayana; Sarath, Gautam; Seifers, Dallas; French, Roy

    2013-04-01

    Triticum mosaic virus (TriMV), an economically important virus infecting wheat in the Great Plains region of the USA, is the type species of the Poacevirus genus in the family Potyviridae. Sensitive and high-throughput serology-based detection methods are crucial for the management of TriMV and germplasm screening in wheat breeding programs. In this study, TriMV coat protein (CP) was expressed in Escherichia coli, and polyclonal antibodies were generated against purified soluble native form recombinant CP (rCP) in rabbits. Specificity and sensitivity of resulting antibodies were tested in Western immuno-blot and enzyme-linked immunosorbent assays (ELISA). In direct antigen coating (DAC)-ELISA, antibodies reacted specifically, beyond 1:20,000 dilution with TriMV in crude sap, but not with healthy extracts, and antiserum at a 1:10,000 dilution detected TriMV in crude sap up to 1:4860 dilution. Notably, rabbit anti-TriMV IgG and anti-TriMV IgG-alkaline phosphatase conjugate reacted positively with native virions in crude sap in a double antibody sandwich-ELISA, suggesting that these antibodies can be used as coating antibodies which is crucial for any 'sandwich' type of assays. Finally, the recombinant antibodies reacted positively in ELISA with representative TriMV isolates collected from fields, suggesting that antibodies generated against rCP can be used for sensitive, large-scale, and broad-spectrum detection of TriMV.

  19. A new SEMA7A variant found in Native Americans with alloantibody.

    PubMed

    Richard, M; St-Laurent, J; Perreault, J; Long, A; St-Louis, M

    2011-04-01

    John Milton Hagen (JMH) antigens are carried by Semaphorin 7A that plays important roles in the nervous system and the immune responses. Its role on the erythrocytes is unclear. Over the years, few samples were referred to our Immunohaematology Reference Laboratory to elucidate their JMH status. Seven blood samples with antibodies compatible with JMH1-negative red cells were studied at the molecular level to identify polymorphisms and explain the JMH diversity observed. Four samples were of Native American background and three were Caucasians. Molecular analyses of the SEMA7A were undertaken, and soluble form of recombinant Sema7A proteins was produced to characterize the antibodies. Sequencing of the cDNA showed a polymorphism in SEMA7A exon 9 at position 1040 (G>T) in the four Native American samples. Caucasians had a normal sequence. This polymorphism precludes a change at position 347 where an Arg is replaced by a Leu. Plasma was assayed in ELISA on wild-type Sema7AArg347 and variant Sema7ALeu347 proteins. Results clearly indicated a specific recognition of the antibody produced by the Native Americans for the wild-type Sema7AArg347 protein and not the variant one. A new SEMA7A variant was identified in this study. The antibody present in the Native American plasma samples should be considered as an alloantibody because it recognizes the wild-type protein. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

  20. Platelet transfusion refractoriness attributable to HLA antibodies produced by donor-derived cells after allogeneic bone marrow transplantation from one HLA-antigen-mismatched mother.

    PubMed

    Hatakeyama, Naoki; Hori, Tsukasa; Yamamoto, Masaki; Inazawa, Natsuko; Iesato, Kotoe; Miyazaki, Toru; Ikeda, Hisami; Tsutsumi, Hiroyuki; Suzuki, Nobuhiro

    2011-12-01

    PTR is a serious problem in patients being treated for hematologic disorders. Two patients with acute leukemia developed PTR after allogeneic BMT from one HLA-antigen-mismatched mother attributable to HLA antibodies, which could not be detected in their serum before BMT. HLA antibodies, whose specificity resembled that of each patient, were detected in each donor's serum. Each donor had probably been immunized during pregnancy by their partner's HLA antigens expressed by the fetus, consequently, transplanted donor-derived cells provoked HLA antibodies in each recipient early after BMT, and those HLA antibodies induced PTR. If the mothers are selected as donors for their children, they should be tested for the presence of HLA antibodies.

  1. Laboratory Scale Production and Purification of a Therapeutic Antibody.

    PubMed

    Elgundi, Zehra; Sifniotis, Vicki; Reslan, Mouhamad; Cruz, Esteban; Kayser, Veysel

    2017-01-24

    Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

  2. NNKTT120, an anti-iNKT cell monoclonal antibody, produces rapid and sustained iNKT cell depletion in adults with sickle cell disease

    PubMed Central

    Majerus, Elaine; Ataga, Kenneth I.; Vichinsky, Elliot P.; Schaub, Robert; Mashal, Robert; Nathan, David G.

    2017-01-01

    Invariant NKT (iNKT) cells can be activated to stimulate a broad inflammatory response. In murine models of sickle cell disease (SCD), interruption of iNKT cell activity prevents tissue injury from vaso-occlusion. NKTT120 is an anti-iNKT cell monoclonal antibody that has the potential to rapidly and specifically deplete iNKT cells and, potentially, prevent vaso-occlusion. We conducted an open-label, multi-center, single-ascending-dose study of NKTT120 to determine its pharmacokinetics, pharmacodynamics and safety in steady-state patients with SCD. Doses were escalated in a 3+3 study design over a range from 0.001 mg/kg to 1.0 mg/kg. Twenty-one adults with SCD were administered NKTT120 as part of 7 dose cohorts. Plasma levels of NKTT120 predictably increased with higher doses. Median half-life of NKTT120 was 263 hours. All subjects in the higher dose cohorts (0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg) demonstrated decreased iNKT cells below the lower limit of quantification within 6 hours after infusion, the earliest time point at which they were measured. In those subjects who received the two highest doses of NKTT120 (0.3, 1 mg/kg), iNKT cells were not detectable in the peripheral blood for a range of 2 to 5 months. There were no serious adverse events in the study deemed to be related to NKTT120. In adults with SCD, NKTT120 produced rapid, specific and sustained iNKT cell depletion without any infusional toxicity or attributed serious adverse events. The next step is a trial to determine NKTT120’s ability to decrease rate of vaso-occlusive pain episodes. Trial Registration: clinicaltrials.gov NCT01783691. PMID:28152086

  3. Characterization of N-glycan structures and biofunction of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus-insect cell expression system.

    PubMed

    Song, Mira; Park, Da-Young; Kim, Youngkwan; Lee, Kyung-Jin; Lu, Zhe; Ko, Kinarm; Choo, Young Kug; Han, Yeon Soo; Ahn, Mi-Hyun; Oh, Doo-Byoung; Ko, Kisung

    2010-08-01

    Advantages of the baculovirus insect cell expression system for production of recombinant proteins include high capacity, flexibility, and glycosylation capability. In this study, this expression system was exploited to produce anti-cancer monoclonal antibody (mAb) CO17-1A, which recognizes the antigen GA733. The heavy chain (HC) and light chain (LC) genes of mAb CO17-1A were cloned under the control of P(10) and Polyhedrin promoters in the pFastBac dual vector, respectively. Gene expression cassettes carrying the HC and LC genes were transposed into a bacmid in Escherichia coli (DH10Bac). The transposed bacmid was transfected to Sf9 insect cells to generate baculovirus expressing mAb CO17-1A. Confocal immunofluorescence and Western blot analyses confirmed expression of mAb CO17-1A in baculovirus-infected insect cells. The optimum conditions for mAb expression were evaluated at 24, 48, and 72 h after the virus infection at an optimum virus multiplicity of infection of 1. Expression of mAb CO17-1A in insect cells significantly increased at 72 h after infection. HPLC analysis of glycosylation status revealed that the insect-derived mAb (mAb(I)) CO17-1A had insect specific glycan structures. ELISA showed that the purified mAb(I) from cell culture supernatant specifically bound to SW948 human colorectal cancer cells. Fluorescence-activated cell sorting analysis showed that, although mAb(I) had insect specific glycan structures that differed from their mammalian counterparts, mAb(I) similarly interacted with CD64 (FcgammaRI) and Fc of IgG, compared to the interactions of mammalian-derived mAb. These results suggest that the baculovirus insect cell expression system is able to express, assemble, and secrete biofunctional full size mAb.

  4. Synthetic nanoparticle vaccines produced by layer-by-layer assembly of artificial biofilms induce potent protective T-cell and antibody responses in vivo.

    PubMed

    Powell, Thomas J; Palath, Naveen; DeRome, Mary E; Tang, Jie; Jacobs, Andrea; Boyd, James G

    2011-01-10

    Nanoparticle vaccines induce potent immune responses in the absence of conventional adjuvant due to the recognition by immune cells of the particle structures, which mimic natural pathogens such as viruses and bacteria. Nanoparticle vaccines were fabricated by constructing artificial biofilms using layer-by-layer (LbL) deposition of oppositely charged polypeptides and target designed peptides on CaCO(3) cores. LbL nanoparticles were efficiently internalized by dendritic cells in vitro by a mechanism that was at least partially phagocytic, and induced DC maturation without triggering secretion of inflammatory cytokines. LbL nanoparticle delivery of designed peptides to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. A single immunization of mice with LbL nanoparticles containing designed peptide induced vigorous T-cell responses characterized by a balanced effector (IFNγ) and Th2 (IL-4) ELISPOT profile and in vivo CTL activity. Mice immunized with LbL nanoparticles bearing ovalbumin-derived designed peptides were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin, confirming the relevance of the CTL/effector T-cell responses. LbL nanoparticles also elicited antibody responses to the target epitope but not to the matrix components of the nanoparticle, avoiding the vector or carrier affect that hampers utility of other vaccine platforms. The potency and efficacy of LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of immune responses to the matrix components, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.

  5. Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells

    PubMed Central

    Desin, Taseen S.; Townsend, Hugh G.; Potter, Andrew A.

    2015-01-01

    Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes. PMID:26451946

  6. Detection of Shiga toxin variants, virulence genes and the relationship to cytotoxicity in Shiga toxin-producing Escherichia coli (STEC) from domestic farm animals

    USDA-ARS?s Scientific Manuscript database

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to life-threating diseases such as hemorrhagic colitis, and hemolytic uremic syndrome in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in STEC stra...

  7. Characterization of shiga toxin-producing Escherichia coli recovered from domestic animals to determine stx variants, virulence genes, and cytotoxicity in mammalian cells

    USDA-ARS?s Scientific Manuscript database

    Shiga toxin-producing Escherichia coli (STEC) can cause foodborne illnesses ranging from diarrhea to severe diseases such as hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) in humans. In this study, we determined virulence genes, stx subtypes and we evaluated the cytotoxicity in mammal...

  8. Top-down proteomic identification of Shiga toxin 2 variants from Shiga toxin-producing Escherichia coli (STEC) using MALDI-TOF-TOF-MS/MS-PSD

    USDA-ARS?s Scientific Manuscript database

    Shiga toxin-producing Escherichia coli (STEC) are increasingly linked to severe outbreaks of foodborne illness throughout the world, e.g. Germany and France in 2011. STEC infections can result in bloody diarrhea, hemolytic uremic syndrome, kidney failure and death. New analytical techniques are ne...

  9. Developing recombinant antibodies for biomarker detection

    SciTech Connect

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    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 libraries 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.

  10. [Metabolic characteristics of GS-nS0 myeloma cells producing anti-CD25 monoclonal antibody in serum-free culture].

    PubMed

    Zhao, Liang; Fan, Li; Zhang, Xu; Tan, Wensong

    2009-07-01

    As an immunodepressant, anti-CD25 monoclonal antibody has a huge market with wide prospect and economic value. We developed a low protein serum-free medium for large-scale GS-NS0 myeloma cell culture and anti-CD25 monoclonal antibody production. Further study focused on the characteristics of GS-NSO cell growth, glucose and amino acid metabolism, and antibody production. In the serum-free medium, the maximal viable cell density and antibody concentration reached above 3x10(6) cells/mL and 300 mg/L in batch culture. Compared with the commercial serum-free medium (Excell 620 + 0.2% Primatone), the maximal viable cell density doubled and the maximal antibody concentration increased 46%. Results also showed the specific growth rate decreased when the glucose concentration was lower than 6 mmol/L. And the production of lactate increased when glucose concentration was excessively high (> 30 mmol/L). These results were important to provide technique and theory basis for developing optimized GS-NS0 cell culture and anti-CD25 monoclonal antibody production processes.

  11. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits [Vlaardingen, NL; Gualfetti, Peter [San Francisco, CA; Mitchinson, Colin [Half Moon Bay, CA; Larenas, Edmund [Moss Beach, CA

    2008-12-02

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  12. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits [Vlaardingen, NL; Gualfetti, Peter [San Francisco, CA; Mitchinson, Colin [Half Moon Bay, CA; Larenas, Edmund [Moss Beach, CA

    2011-05-31

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  13. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits [Vlaardingen, NL; Gualfetti, Peter [San Francisco, CA; Mitchinson, Colin [Half Moon Bay, CA; Larenas, Edmund [Moss Beach, CA

    2011-08-16

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  14. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits [Vlaardingen, NL; Gualfetti, Peter [San Francisco, CA; Mitchinson, Colin [Half Moon Bay, CA; Larenas, Edmund [Moss Beach, CA

    2012-08-07

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  15. Variant humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Edmund, Larenas

    2014-09-09

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  16. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegeburr, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2013-02-19

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  17. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2014-03-18

    Disclosed are variants of Humicola grisea Cel7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  18. Variant Humicola grisea CBH1.1

    DOEpatents

    Goedegebuur, Frits; Gualfetti, Peter; Mitchinson, Colin; Larenas, Edmund

    2017-05-09

    Disclosed are variants of Humicola grisea CeI7A (CBH1.1), H. jecorina CBH1 variant or S. thermophilium CBH1, nucleic acids encoding the same and methods for producing the same. The variant cellulases have the amino acid sequence of a glycosyl hydrolase of family 7A wherein one or more amino acid residues are substituted.

  19. DHAD variants and methods of screening

    DOEpatents

    Kelly, Kristen J.; Ye, Rick W.

    2017-02-28

    Methods of screening for dihydroxy-acid dehydratase (DHAD) variants that display increased DHAD activity are disclosed, along with DHAD variants identified by these methods. Such enzymes can result in increased production of compounds from DHAD requiring biosynthetic pathways. Also disclosed are isolated nucleic acids encoding the DHAD variants, recombinant host cells comprising the isolated nucleic acid molecules, and methods of producing butanol.

  20. A standard immunoglobulin preparation produced from bovine colostra shows antibody reactivity and neutralization activity against Shiga-like toxins and EHEC-hemolysin of Escherichia coli O157:H7.

    PubMed

    Lissner, R; Schmidit, H; Karch, H

    1996-01-01

    Enterohemorrhagic Escherichia coli (EHEC) causes a variety of clinical conditions, the most important being hemorrhagic colitis and hemolytic uremic syndrome. A curative therapy of EHEC diseases is not yet feasible. This study investigates the antibody reactivity of Lactobin, a standardized immunoglobulin (Ig) preparation, obtained from the colostra of non-immunized cows. Three different batches of Lactobin exhibited equally high titers of specific antibodies against Shiga-like toxins (SLTs, verocytotoxins) and EHEC hemolysin (EHEC-Hly) produced by E. coli O157. In addition, Lactobin blocked the cytotoxic effect of SLT-I and SLT-II on Vero cell monolayers and inhibited the cytolytic effects of EHEC-Hly on human erythrocytes. Since Lactobin contains high levels of antibodies and neutralizing activity against important virulence factors of EHEC O157, this drug has potential use in the treatment of diarrhea and the prevention of EHEC-associated hemolytic uremic syndrome.

  1. Invasive Stratified Mucin-producing Carcinoma and Stratified Mucin-producing Intraepithelial Lesion (SMILE): 15 Cases Presenting a Spectrum of Cervical Neoplasia With Description of a Distinctive Variant of Invasive Adenocarcinoma.

    PubMed

    Lastra, Ricardo R; Park, Kay J; Schoolmeester, J Kenneth

    2016-02-01

    Stratified mucin-producing intraepithelial lesion (SMILE) is a cervical intraepithelial lesion, distinct from conventional squamous or glandular counterparts, believed to arise from embryonic cells at the transformation zone by transdifferentiation during high-risk HPV-associated carcinogenesis. It is characterized by stratified, immature epithelial cells displaying varying quantities of intracytoplasmic mucin throughout the majority of the lesional epithelium. We identified a distinct form of invasive cervical carcinoma with morphologic features identical to those in SMILE, which we have termed "invasive stratified mucin-producing carcinoma." Fifteen cases from 15 patients (mean 36 y; range, 22 to 64 y) were retrieved from the pathology archives of multiple institutions with a diagnosis of either SMILE or invasive cervical carcinoma with a description or comment about the invasive tumor's resemblance to SMILE. Seven cases had solely intraepithelial disease with a component of SMILE (mean 29 y; range, 22 to 40 y). The 8 other cases had invasive stratified mucin-producing carcinoma (mean 44; range, 34 to 64 y) in which SMILE was identified in 7. All cases of invasive stratified mucin-producing carcinoma demonstrated stratified, immature nuclei with intracytoplasmic mucin, which morphologically varied between cases from "mucin-rich" to "mucin-poor" in a similar manner to SMILE. All cases had mitotic figures and apoptotic debris, and an intralesional neutrophilic infiltrate was seen in the majority of cases. In cases of invasive carcinoma, the depth of invasion ranged from <1 to 19 mm. Follow-up information was available in 8 cases and ranged from 1 to 36 months (mean 11 mo). Three cases of invasive stratified mucin-producing carcinoma had biopsy or resection-proven metastatic carcinoma on follow-up. These 15 cases of cervical stratified mucin-producing lesions show a combination of intraepithelial and invasive growth patterns. Given that SMILE is well rooted as a

  2. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  3. Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

    SciTech Connect

    Crowell, R.L.; Field, A.K.; Schleif, W.A.; Long, W.L.; Colonno, R.J.; Mapoles, J.E.; Emini, E. A.

    1986-02-01

    BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: (i) protection against CB3 on HeLa, (ii) protection against CB3-RD on rhabdomyosarcoma (RD) cells, and (iii) protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of (/sup 35/S)methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-Rd virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and /sup 125/I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

  4. Molecular Characterization of High-Level-Cholera-Toxin-Producing El Tor Variant Vibrio cholerae Strains in the Zanzibar Archipelago of Tanzania

    PubMed Central

    Naha, A.; Chowdhury, G.; Ghosh-Banerjee, J.; Senoh, M.; Takahashi, T.; Ley, B.; Thriemer, K.; Deen, J.; Seidlein, L. V.; Ali, S. M.; Khatib, A.; Ramamurthy, T.; Nandy, R. K.; Nair, G. B.; Takeda, Y.

    2013-01-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis. PMID:23325815

  5. Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

    PubMed

    Naha, A; Chowdhury, G; Ghosh-Banerjee, J; Senoh, M; Takahashi, T; Ley, B; Thriemer, K; Deen, J; Seidlein, L V; Ali, S M; Khatib, A; Ramamurthy, T; Nandy, R K; Nair, G B; Takeda, Y; Mukhopadhyay, A K

    2013-03-01

    Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

  6. Structural Characterization of New Peptide Variants Produced by Cyanobacteria from the Brazilian Atlantic Coastal Forest Using Liquid Chromatography Coupled to Quadrupole Time-of-Flight Tandem Mass Spectrometry

    PubMed Central

    Sanz, Miriam; Andreote, Ana Paula Dini; Fiore, Marli Fatima; Dörr, Felipe Augusto; Pinto, Ernani

    2015-01-01

    Cyanobacteria from underexplored and extreme habitats are attracting increasing attention in the search for new bioactive substances. However, cyanobacterial communities from tropical and subtropical regions are still largely unknown, especially with respect to metabolite production. Among the structurally diverse secondary metabolites produced by these organisms, peptides are by far the most frequently described structures. In this work, liquid chromatography/electrospray ionization coupled to high resolution quadrupole time-of-flight tandem mass spectrometry with positive ion detection was applied to study the peptide profile of a group of cyanobacteria isolated from the Southeastern Brazilian coastal forest. A total of 38 peptides belonging to three different families (anabaenopeptins, aeruginosins, and cyanopeptolins) were detected in the extracts. Of the 38 peptides, 37 were detected here for the first time. New structural features were proposed based on mass accuracy data and isotopic patterns derived from full scan and MS/MS spectra. Interestingly, of the 40 surveyed strains only nine were confirmed to be peptide producers; all of these strains belonged to the order Nostocales (three Nostoc sp., two Desmonostoc sp. and four Brasilonema sp.). PMID:26096276

  7. Tissue distribution of mucosal antibody-producing cells specific for respiratory syncytial virus in severe combined immune deficiency (SCID) mice engrafted with human tonsils.

    PubMed Central

    Nadal, D; Albini, B; Schläpfer, E; Chen, C; Brodsky, L; Ogra, P L

    1991-01-01

    Groups of C.B-17 SCID mice were reconstituted intraperitoneally with human tonsillar mononuclear cells (hu-TMC) from children seropositive for antibody to respiratory syncytial virus (RSV) and subsequently challenged intraperitoneally with inactivated RSV or sham-immunized. The synthesis and the distribution characteristics of human antibody to RSV in various murine tissues were studied using an enzyme-linked immunospot assay (ELISPOT). No specific antibody was observed in sham-immunized animals. In contrast, mice engrafted with hu-TMC exhibited the appearance of specific human antibody secreting cells (hu-ASC) after i.p. immunization with inactivated RSV. RSV-specific hu-ASC were detected only in animals engrafted with cells from donors seropositive for antibodies to Epstein-Barr virus. Hu-TMC engrafted mice showed RSV-specific IgM and, in lower numbers, IgG hu-ASC in several tissues including the lungs. Numbers of RSV-specific IgA hu-ASC were low, however, and detected only in the lung. No RSV-specific hu-ASC were detected in the intestine. These data demonstrate for the first time that hu-TMC-SCID chimeras respond to immunization with viral antigen. Furthermore, the results suggest that hu-TMC engraft in lungs but not in the intestinal tissue. PMID:1893614

  8. Transcriptome and proteome analysis of antibody-producing mouse myeloma NS0 cells cultivated at different cell densities in perfusion culture.

    PubMed

    Krampe, Britta; Swiderek, Halina; Al-Rubeai, Mohamed

    2008-07-01

    A combined gene and protein expression profiling was performed to gain a deeper insight into the intracellular response of the antibody-producing GS-NS0 cell line in continuous perfusion culture. Growth rate, production rate, metabolic activity and viability declined with increasing cell density, dilution rate and time. Transcriptome and proteome analyses of cells at three different densities revealed 53 genes and 47 proteins as having significantly altered expression levels at HCD (high cell density). The results showed an increased up-regulation of genes/proteins involved in cellular energy production with increasing cell density. Furthermore, the intensified process triggered a cellular response to external stress stimuli, revealed by an overexpression of the genes/proteins implicated in cell-cycle arrest [e.g. Rb1 (retinoblastoma 1 gene) and Cdkn1b (cyclin-dependent kinase inhibitor 1B gene)] and in the induction of pro-apoptotic genes/proteins [e.g. Tnfrsf (tumour necrosis factor receptor superfamily gene), Nfkappa bia (gene coding for nuclear factor-kappaB inhibitor), HSP60 (heat-shock protein of molecular mass 60 kDa) and heterogeneous nuclear ribonucleoprotein K]. Interestingly, we observed a down-regulation of the transcription factor interferon regulatory factor 4 involved in the unfolded-protein-response process and protein disulfide-isomerase family members responsible for protein folding and assembly. Additionally, subunits of proteasome complex were highly expressed at HCD. Microarray, real-time quantitative reverse-transcription PCR and Western-blot analyses demonstrated a consistent trend of decrease in IgG heavy-chain level with increasing cell density. HSP60, which inhibits apoptosis by complexing with pro-apoptotic proteins such as Bax and Bak, was repressed at HCD. Overall, the data suggested that the balance among several factors involved in energy metabolism might be essential for fine-tuning the cell choice between survival and apoptosis

  9. Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

    PubMed Central

    Lu, Zhaohua; Perkins, Hillary M.

    2014-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. PMID:24351753

  10. Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

    PubMed

    Franz, A; Rehner, R; Kienle, A; Grammel, H

    2012-01-01

    The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics. © 2011 The Authors. Letters in Applied Microbiology ©2011 The Society for Applied Microbiology.

  11. [New antibodies in cancer treatment].

    PubMed

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  12. Rhesus monkeys protected against Plasmodium knowlesi malaria produce antibodies against a 65,000-MrP. knowlesi glycoprotein at the surface of infected erythrocytes.

    PubMed

    Schmidt-Ullrich, R; Miller, L H; Wallach, D F; Lightholder, J; Powers, K G; Gwadz, R W

    1981-11-01

    Sera from 27 rhesus monkeys immunized in various ways against the H strain of Plasmodium knowlesi were analyzed by quantitative crossed immunoelectrophoresis. The reaction of the sera was compared with a reference immune serum only reactive with P. knowlesi-specific 65,000-Mr glycoprotein-immune component 13 (gp65/ic13) in membranes of infected rhesus monkey erythrocytes. Triton X-100-solubilized, 125I-labeled membranes of schizont-infected erythrocytes were used as an antigen. Sera from 9 or 10 monkeys immunized by repeated infections with P. knowlesi reacted with gp65/ic13. In 6 of 10 sera, anti-gp65/ic13 was the only antibody reacting with host cell membrane proteins. In contrast, vaccination of 15 monkeys with predominantly sexual stages or trophozoites of P. knowlesi in Freund complete adjuvant resulted in protection against blood challenges in 7 monkeys, only 2 of which contained precipitating antibody against gp65/ic13. None of the sera from monkeys not protected by infections or vaccinations contained detectable levels of precipitating antibodies against gp65/ic13. Our data indicate that gp65/ic13 acts as a prominent immunogen in vivo during natural p. knowlesi infections of rhesus monkeys. There is a positive correlation suggested between anti-gp65/ic13 antibody and protection in the monkeys analyzed. This correlation does not apply to monkeys protected against P. knowlesi malaria by vaccination, pointing to other effective immune defense mechanisms.

  13. Vaccination of rhesus macaques with the anthrax vaccine adsorbed vaccine produces a serum antibody response that effectively neutralizes receptor-bound protective antigen in vitro.

    PubMed

    Clement, Kristin H; Rudge, Thomas L; Mayfield, Heather J; Carlton, Lena A; Hester, Arelis; Niemuth, Nancy A; Sabourin, Carol L; Brys, April M; Quinn, Conrad P

    2010-11-01

    Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

  14. Association of Kidney Graft Loss With De Novo Produced Donor-Specific and Non-Donor-Specific HLA Antibodies Detected by Single Antigen Testing.

    PubMed

    Süsal, Caner; Wettstein, Daniel; Döhler, Bernd; Morath, Christian; Ruhenstroth, Andrea; Scherer, Sabine; Tran, Thuong H; Gombos, Petra; Schemmer, Peter; Wagner, Eric; Fehr, Thomas; Živčić-Ćosić, Stela; Balen, Sanja; Weimer, Rolf; Slavcev, Antonij; Bösmüller, Claudia; Norman, Douglas J; Zeier, Martin; Opelz, Gerhard

    2015-09-01

    The association of donor-specific HLA antibodies (DSA) with kidney graft failure has been addressed previously; however, the majority of studies were based on small numbers of patients with graft failure. We investigated 83 patients with failed kidney transplants for a possible association of de novo development and persistence or loss of pre-existing DSA with graft failure. Single Antigen Bead assay-detected DSA and non-DSA antibodies were compared between patients with graft loss and matched controls with functioning grafts. The incidence of weak de novo DSA or non-DSA at a mean fluorescence intensity of 500 or higher was higher in the graft loss than in the nonrejector group (76% vs 40%, P < 0.001). Because of the low number of patients developing de novo DSA, the DSA results did not reach statistical significance (only 22% of patients with graft loss developed de novo DSA). However, at all cutoffs, there was a significantly higher rate of graft loss in patients with de novo non-DSA. The incidence of strong pretransplant DSA that persist after transplantation was higher in the graft loss group (10% vs 1%, P = 0.034). When C1q-binding ability in sera of rejectors and nonrejectors with posttransplant de novo or persistent DSA was compared, none of the nonrejectors demonstrated C1q positivity, whereas 43% of patients with graft loss showed C1q-positive antibodies, although not necessarily donor-specific (P < 0.001). Our data show that the posttransplant presence of persisting or de novo HLA antibodies, especially if C1q binding, is associated with graft loss, even if the antibodies are not specific for mismatched donor HLA.

  15. 21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's...

  16. Antimitochondrial antibody

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  17. Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

    PubMed Central

    Patra, Kailash P.; Li, Fengwu; Carter, Darrick; Gregory, James A.; Baga, Sheyenne; Reed, Steven G.; Mayfield, Stephen P.

    2015-01-01

    A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration. PMID:25690099

  18. Alga-produced malaria transmission-blocking vaccine candidate Pfs25 formulated with a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes.

    PubMed

    Patra, Kailash P; Li, Fengwu; Carter, Darrick; Gregory, James A; Baga, Sheyenne; Reed, Steven G; Mayfield, Stephen P; Vinetz, Joseph M

    2015-05-01

    A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene-oil-in-water emulsion, and GLA plus squalene-oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene-oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene-oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  20. Development and characterization of antibodies specific to caspase-3-produced alpha II-spectrin 120 kDa breakdown product: marker for neuronal apoptosis.

    PubMed

    Nath, R; Huggins, M; Glantz, S B; Morrow, J S; McGinnis, K; Nadimpalli, R; Wanga, K K

    2000-10-01

    Alpha II-spectrin (alpha-fodrin) is a demonstrated endogenous substrate for caspase-3 in neurons undergoing unscheduled apoptotic death. We have previously identified the caspase cleavage site that yields the distinctive 120 kDa spectrin breakdown product (SBDP120) as (DSLD(1478)*SVEAL). Here, by using a synthetic peptide (NH(2)-SVEALC) mimicking the neo-N-terminal of SBDP120 as antigen, we report the development of chicken antibodies that specifically recognize the SBDP120 generated by in vitro caspase-3 digestion of bovine alpha-spectrin on Western blot. These anti-SBDP120 antibodies recognize SBDP120 generated by two apoptotic challenges (staurosporine, EGTA) to human neuroblastoma SH-SY5Y cells. Yet they neither react with intact alpha-spectrin nor its other fragments on Western blots. These anti-SBDP120 work equally well in detecting SBDP120 generated in rat cerebellar granule neurons undergoing potassium withdrawal-induced apoptosis. In immunocytochemical studies, these antibodies also specifically stained apoptotic SH-SY5Y or CGN's undergoing apoptosis in a caspase- inhibitor-sensitive manner. These anti-SBDP120s might become powerful markers for apoptotic neurons in various neurological or neurodegenerative conditions in vivo.

  1. The germinal center antibody response in health and disease.

    PubMed

    DeFranco, Anthony L

    2016-01-01

    The germinal center response is the delayed but sustained phase of the antibody response that is responsible for producing high-affinity antibodies of the IgG, IgA and/or IgE isotypes. B cells in the germinal center undergo re-iterative cycles of somatic hypermutation of immunoglobulin gene variable regions, clonal expansion, and Darwinian selection for cells expressing higher-affinity antibody variants. Alternatively, selected B cells can terminally differentiate into long-lived plasma cells or into a broad diversity of mutated memory B cells; the former secrete the improved antibodies to fight an infection and to provide continuing protection from re-infection, whereas the latter may jumpstart immune responses to subsequent infections with related but distinct infecting agents. Our understanding of the molecules involved in the germinal center reaction has been informed by studies of human immunodeficiency patients with selective defects in the production of antibodies. Recent studies have begun to reveal how innate immune recognition via Toll-like receptors can enhance the magnitude and selective properties of the germinal center, leading to more effective control of infection by a subset of viruses. Just as early insights into the nature of the germinal center found application in the development of the highly successful conjugate vaccines, more recent insights may find application in the current efforts to develop new generations of vaccines, including vaccines that can induce broadly protective neutralizing antibodies against influenza virus or HIV-1.

  2. New engineered antibodies against prions

    PubMed Central

    Škrlj, Nives; Dolinar, Marko

    2014-01-01

    A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens. PMID:23941991

  3. AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

    PubMed

    van der Ploeg, Claudia A; Rogé, Ariel D; Bordagorria, Ximena L; de Urquiza, María T; Viñas, María R; Pichel, Mariana G; Bruno, Susana B

    2015-01-01

    Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

  4. Novel Epitopes Identified by Anti-PrP Monoclonal Antibodies Produced Following Immunization of Prnp0/0 Balb/cJ Mice with Purified Scrapie Prions

    PubMed Central

    Stanker, Larry H.; Scotcher, Miles C.; Lin, Alice; McGarvey, Jeffery; Prusiner, Stanley B.

    2012-01-01

    Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform (PrPSc) of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assays, as antibodies specific for the disease-causing PrPSc isoform have not been developed. Here we report the generation of three new monoclonal antibodies (MAbs) to PrP, which were isolated following immunization of Prnp0/0 Balb/cJ mice with highly purified PrPSc isolated from brain lipid rafts. Epitope mapping using synthetic PrP peptides revealed that the three MAbs bind different epitopes of PrP. The DRM1-31 MAb has a conformational epitope at the proposed binding site for the putative prion conversion co-factor “protein X.” The DRM1-60 MAb binds a single linear epitope localized to the β2–α2 loop region of PrP, whereas DRM2-118 binds an epitope that includes sequences within the octarepeat region and near the site of N-terminal truncation of PrPSc by proteinase K. Our novel anti-PrP MAbs with defined PrP epitopes may be useful in deciphering the conformational conversion of PrPC into PrPSc. PMID:23098297

  5. Swine-origin pandemic H1N1 influenza virus-like particles produced in insect cells induce hemagglutination inhibiting antibodies in BALB/c mice

    PubMed Central

    Krammer, Florian; Nakowitsch, Sabine; Messner, Paul; Palmberger, Dieter; Ferko, Boris; Grabherr, Reingard

    2015-01-01

    Recent outbreaks of influenza A highlight the importance of rapid and sufficient supply for pandemic and inter-pandemic vaccines. Classical manufacturing methods for influenza vaccines fail to satisfy this demand. Alternatively, cell culture-based production systems and virus-like particle (VLP)-based technologies have been established. We developed swine-origin pandemic H1N1 influenza VLPs consisting of hemagglutinin (A/California/04/2009) and matrix protein. Hemagglutinin and matrix protein were co-expressed in insect cells by the baculovirus expression system. VLPs were harvested from infection supernatants, purified and used for intraperitoneal immunization of BALB/c mice. Immunization induced high serum antibody titers against A/California/04/2009 as well as hemagglutination inhibiting antibodies. Additionally, we compared VLP production in two different insect cell lines, Sf9 and BTI-TN5B1-4 (High Five™). Taken together VLPs represent a potential strategy for the fight against new pandemic influenza viruses. PMID:20041443

  6. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    PubMed

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. [Progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases].

    PubMed

    Yao, Yuan; Yu, Chuan-xin

    2013-08-01

    Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

  8. 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.

  9. Variant Calling From Next Generation Sequence Data.

    PubMed

    Hansen, Nancy F

    2016-01-01

    The use of next generation nucleotide sequencing to discover and genotype small sequence variants has led to numerous insights into the molecular causes of various diseases. This chapter describes the use of freely available software to align next generation sequencing reads to a reference and then to use the resulting alignments to call, annotate, view, and filter small sequence variants. The suggested variant calling workflow includes read alignment with novoalign, the removal of polymerase chain reaction duplicate sequences with samtools or bamUtils, and the detection of variants with Freebayes or bam2mpg software. ANNOVAR is then used to annotate the predicted variants using gene models, population frequencies, and predicted mutation severity, producing variant files which can be viewed and filtered with the variant display tool VarSifter.

  10. Systematic comparison of single-chain Fv antibody-fusion toxin constructs containing Pseudomonas Exotoxin A or saporin produced in different microbial expression systems.

    PubMed

    Della Cristina, Pietro; Castagna, Monica; Lombardi, Alessio; Barison, Erika; Tagliabue, Giovanni; Ceriotti, Aldo; Koutris, Ilias; Di Leandro, Luana; Giansanti, Francesco; Vago, Riccardo; Ippoliti, Rodolfo; Flavell, Sopsamorn U; Flavell, David J; Colombatti, Marco; Fabbrini, Maria Serena

    2015-02-13

    Antibodies raised against selected antigens over-expressed at the cell surface of malignant cells have been chemically conjugated to protein toxin domains to obtain immunotoxins (ITs) able to selectively kill cancer cells. Since latest generation immunotoxins are composed of a toxic domain genetically fused to antibody fragment(s) which confer on the IT target selective specificity, we rescued from the hydridoma 4KB128, a recombinant single-chain variable fragment (scFv) targeting CD22, a marker antigen expressed by B-lineage leukaemias and lymphomas. We constructed several ITs using two enzymatic toxins both able to block protein translation, one of bacterial origin (a truncated version of Pseudomonas exotoxin A, PE40) endowed with EF-2 ADP-ribosylation activity, the other being the plant ribosome-inactivating protein saporin, able to specifically depurinate 23/26/28S ribosomal RNA. PE40 was selected because it has been widely used for the construction of recombinant ITs that have already undergone evaluation in clinical trials. Saporin has also been evaluated clinically and has recently been expressed successfully at high levels in a Pichia pastoris expression system. The aim of the present study was to evaluate optimal microbial expression of various IT formats. An anti-CD22 scFv termed 4KB was obtained which showed the expected binding activity which was also internalized by CD22+ target cells and was also competed for by the parental monoclonal CD22 antibody. Several fusion constructs were designed and expressed either in E. coli or in Pichia pastoris and the resulting fusion proteins affinity-purified. Protein synthesis inhibition assays were performed on CD22+ human Daudi cells and showed that the selected ITs were active, having IC50 values (concentration inhibiting protein synthesis by 50% relative to controls) in the nanomolar range. We undertook a systematic comparison between the performance of the different fusion constructs, with respect to yields in

  11. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  12. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  13. Gr1+ IL-4 producing innate cells are induced in response to Th2 stimuli and suppress Th1-dependent antibody responses.¶

    PubMed Central

    McKee, Amy; MacLeod, Megan; White, Janice; Crawford, Frances; Kappler, John; Marrack, Philippa

    2010-01-01

    Alum is used as a vaccine adjuvant and induces Th2 responses and Th2-driven antibody isotype production against co-injected antigens. Alum also promotes the appearance in the spleen of Gr1+, IL-4+ innate cells that, via IL-4 production, induce MHC II mediated signaling in B cells. To investigate whether these Gr1+ cells accumulate in the spleen in response to other Th2 inducing stimuli and to understand some of their functions, the effects of injection of alum and eggs from the helminth, Schistosoma mansoni, were compared. Like alum, schistosome eggs induced the appearance of Gr1+IL-4+ cells in spleen and promoted MHC II-mediated signaling in B cells. Unlike alum, however, schistosome eggs did not promote CD4 T cell responses against co-injected antigens, suggesting that the effects of alum or schistosome eggs on splenic B cells cannot by themselves explain the T cell adjuvant properties of alum. Accordingly, depletion of IL-4 or Gr1+ cells in alum injected mice had no effect on the ability of alum to improve expansion of primary CD4 T cells. However, Gr1+ cells and IL-4 played some role in the effects of alum, since depletion of either resulted in antibody responses to antigen that included not only the normal Th2-driven isotypes, like IgG1, but also a Th1-driven isotype, IgG2c. These data suggest that alum affects the immune response in at least two ways, one, independent of Gr1+ cells and IL-4, that promotes CD4 T cell proliferation and another, via Gr1+IL-4+ cells that participate in the polarization of the response. PMID:18343889

  14. Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

    PubMed

    Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

    2017-03-01

    Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10(-8) M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL(-1), respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL(-1)), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

  15. Single-cell antibody nanowells: a novel technology in detecting anti-SSA/Ro60- and anti-SSB/La autoantibody-producing cells in peripheral blood of rheumatic disease patients.

    PubMed

    Esfandiary, Lida; Gupta, Nirupama; Voigt, Alexandria; Wanchoo, Arun; Chan, Edward K L; Sukumaran, Sukesh; Nguyen, Cuong Q

    2016-05-17

    Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La. Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies. Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects. These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.

  16. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  17. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  18. Structural consequences of aglycosylated IgG Fc variants evolved for FcγRI binding.

    PubMed

    Ju, Man-Seok; Na, Jung-Hyun; Yu, Yeon Gyu; Kim, Jae-Yeol; Jeong, Cherlhyun; Jung, Sang Taek

    2015-10-01

    In contrast to the glycosylated IgG antibodies secreted by human plasma cells, the aglycosylated IgG antibodies produced by bacteria are unable to bind FcγRs expressed on the surface of immune effector cells and cannot trigger immune effector functions. To avoid glycan heterogeneity problems, elicit novel effector functions, and produce therapeutic antibodies with effector function using a simple bacterial expression system, FcγRI-specific Fc-engineered aglycosylated antibodies, Fc11 (E382V) and Fc (E382V/M428I), containing mutations in the CH3 region, were isolated in a previous study. To elucidate the relationship between FcγRI binding affinity and the structural dynamics of the upper CH2 region of Fc induced by the CH3 mutations, the conformational variation of Fc variants was observed by single-molecule Förster resonance energy transfer (FRET) analysis using alternating-laser excitation (ALEX). In sharp contrast to wild-type Fc, which exhibits a highly dynamic upper CH2 region, the mutations in the CH3 region significantly stabilized the upper CH2 region. The results indicate that conformational plasticity, as well as the openness of the upper CH2 region, is critical for FcγR binding and therapeutic effector functions of IgG antibodies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  20. Evaluation of options for harvest of a recombinant E. Coli fermentation producing a domain antibody using ultra scale‐down techniques and pilot‐scale verification

    PubMed Central

    Voulgaris, Ioannis; Chatel, Alex; Finka, Gary; Uden, Mark

    2016-01-01

    Ultra scale‐down (USD) methods operating at the millilitre scale were used to characterise full‐scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g., a centrifugation stage operating at 0.11 L/m2 equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m2/L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2016 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 32:382–392, 2016 PMID:26698375

  1. A polyclonal antibody based immunoassay detects seven subtypes of Shiga toxin 2 produced by escherichia coli in human and environmental samples

    USDA-ARS?s Scientific Manuscript database

    The increase of outbreaks and illnesses linked to Shiga toxin-producing Escherichia coli (STEC) has necessitated the development of effective detection methods for these pathogens in various matrices. The best way to determine if a bacterial strain is a STEC is to examine the production of Shiga tox...

  2. Comparison of antibody- versus pcr-based assays for serotyping shiga toxin-producing escherichia coli recovered from various cattle operations

    USDA-ARS?s Scientific Manuscript database

    Serotyping of Shiga toxin-producing Escherichia coli (STEC) strains is contingent upon the availability of quality antisera. In this study, the serogroup of 161 STEC strains typed by conventional antisera and isolated from the fecal samples of California cattle were compared to two newly developed ...

  3. New monoclonal antibodies against a novel subtype of Shiga toxin 1 produced by Enterobacter cloacae and their use in analysis of human serum

    USDA-ARS?s Scientific Manuscript database

    Shiga toxin (Stx) is a major virulence factor for several bacterial pathogens that cause potentially fatal illness, including Escherichia coli and Shigella spp. The continual emergence of new subtypes of Stxs presents challenges in clinical diagnosis of infections caused by Shiga toxin-producing org...

  4. Humanized Antibodies for Antiviral Therapy

    NASA Astrophysics Data System (ADS)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  5. Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

    PubMed

    Fauvelle, Catherine; Felmlee, Daniel J; Crouchet, Emilie; Lee, JiYoung; Heydmann, Laura; Lefèvre, Mathieu; Magri, Andrea; Hiet, Marie-Sophie; Fofana, Isabel; Habersetzer, François; Foung, Steven K H; Milne, Ross; Patel, Arvind H; Vercauteren, Koen; Meuleman, Philip; Zeisel, Mirjam B; Bartenschlager, Ralf; Schuster, Catherine; Baumert, Thomas F

    2016-01-01

    Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  6. Variant Interpretation: Functional Assays to the Rescue.

    PubMed

    Starita, Lea M; Ahituv, Nadav; Dunham, Maitreya J; Kitzman, Jacob O; Roth, Frederick P; Seelig, Georg; Shendure, Jay; Fowler, Douglas M

    2017-09-07

    Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  7. Effects of different molting procedures on incidence of Salmonella infection in flocks of naturally contaminated laying hens in a commercial egg-producing farm by detection of yolk antibodies to Salmonella in eggs.

    PubMed

    Murase, Toshiyuki; Chiba, Kaori; Sato, Tomoko; Otsuki, Koichi; Holt, Peter S

    2006-12-01

    Indirect enzyme-linked immunosorbent assays (ELISAs) have been applied to detect immunoglobulin Y antibodies to different serotypes of Salmonella in the yolks of chicken eggs with heat-extracted antigens of Salmonella enterica serotypes Agona (SA), Cerro (SC), Enteritidis (SE), Montevideo (SM), and Putten (SP). The egg yolk samples examined were classified as positive if their ELISA absorbance values exceeded the value for eggs from specific-pathogen-free flocks by more than two standard deviations. Of 30 egg yolk samples from three flocks vaccinated with a killed SE vaccine, 29 were antibody positive by the ELISA assay for the SE antigen. Four to 29 of the 29 yolk samples showed positive results for the other serovars, although the absorbance values for SE were higher than those obtained for the other serotypes in each of the yolk samples. All 30 yolks from three flocks that were not administered any SE vaccines were found to be antibody negative for SE, and two samples were determined to be positive for SC. Thirty-nine or 40 eggs were obtained from each of four layer flocks in a commercial egg production farm where the laying houses were naturally contaminated with SA, SC, SM, SP, Salmonella serovar Infantis (SI), and untypeable strains. The ELISA absorbance values for SM in the egg yolks obtained from the two flocks molted through feed withdrawal when the birds restarted laying were significantly (P < 0.05) higher than those observed in the yolks obtained before the molt. In egg yolks from the two other flocks that were molted through a wheat bran diet, there was no significant difference between the absorbance values before and after the molt. The observations in the present study provide further evidence to suggest that a molt initiated through the administration of a wheat bran diet can reduce the risk for Salmonella problems in a commercial egg-producing setting.

  8. Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. V. H-2 associativity of variant-specific antigens.

    PubMed

    Van Snick, J; Maryanski, J; Van Pel, A; Parmiani, G; Boon, T

    1982-11-01

    By in vitro mutagenesis of mastocytoma P815, it is possible to obtain tumor cell variants that are rejected by syngeneic mice (tum-). Most of these variants carry new individual antigens and stimulate a specific cytolytic T cell (CTL) response in mixed leukocyte tumor cell culture (MLTC). The H-2 associativity of this response was examined for six different variants by measuring the inhibition of cell-mediated cytolysis by antibodies directed against products of the K or the D end of the H-2d complex. The lysis was either not inhibited (variants P91 and P116) or inhibited selectively by anti-Kd (variants P21, P32 and P198) or anti-Dd antibodies (variant P35). All these tum- variants expressed Kd and Dd antigens as measured by absorption of H-2 alloantisera. Long-term CTL clones can be obtained that are specific for individual tum- antigens. The pattern of H-2 associativity obtained with MLTC-derived CTL against four tum- variants was verified with CTL clones directed against the specific antigens of these variants. Concordant results were observed in all cases. In addition to CTL clones specific for tum- antigens, it is possible to isolate clones against a P815 tumor-associated antigen found on all P815 tum- variants. For these clones no clear associativity with either Kd or Dd products was found.

  9. 2H-WS2 Quantum Dots Produced by Modulating the Dimension and Phase of 1T-Nanosheets for Antibody-Free Optical Sensing of Neurotransmitters.

    PubMed

    Kim, Man-Jin; Jeon, Su-Ji; Kang, Tae Woog; Ju, Jong-Min; Yim, DaBin; Kim, Hye-In; Park, Jung Hyun; Kim, Jong-Ho

    2017-03-28

    Modulating the dimensions and phases of transition metal dichalcogenides is of great interest to enhance their intrinsic properties or to create new physicochemical properties. Herein, we report an effective approach to synthesize 2H-WS2 quantum dots (QDs) via the dimension and phase engineering of 1T-WS2 nanosheets. The solvothermal reaction of chemically exfoliated 1T-WS2 nanosheets in N-methyl-2-pyrrolidone (NMP) under an N2 atmosphere induced their chopping and phase transition at lower temperature to produce 2H-WS2 QDs with a high quantum yield (5.5 ± 0.3%). Interestingly, this chopping and phase transition process showed strong dependency on solvent; WS2 QDs were not produced in other solvents such as 1,4-dioxane and dimethyl sulfoxide. Mechanistic investigations suggested that NMP radicals played a crucial role in the effective production of 2H-WS2 QDs from 1T-WS2 nanosheets. WS2 QDs were successfully applied for the selective, sensitive, and rapid detection of dopamine in human serum (4 min, as low as 23.8 nM). The intense fluorescence of WS2 QDs was selectively quenched upon the addition of dopamine and Au(3+) ions due to fluorescence resonance energy transfer between WS2 QDs and the quickly formed Au nanoparticles. This new sensing principle enabled us to discriminate dopamine from dopamine-derivative neurotransmitters including epinephrine and norepinephrine, as well as other interference compounds.

  10. A monoclonal antibody against leptin.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

  11. Glycan modulation and sulfoengineering of anti–HIV-1 monoclonal antibody PG9 in plants

    PubMed Central

    Loos, Andreas; Gach, Johannes S.; Hackl, Thomas; Maresch, Daniel; Henkel, Theresa; Porodko, Andreas; Bui-Minh, Duc; Sommeregger, Wolfgang; Wozniak-Knopp, Gordana; Forthal, Donald N.; Altmann, Friedrich; Steinkellner, Herta; Mach, Lukas

    2015-01-01

    Broadly neutralizing anti–HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells (CHOPG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for CHOPG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti–HIV-1 antibodies with effector functions superior to PG9 made in CHO cells. PMID:26417081

  12. From hybridomas to a robust microalgal-based production platform: molecular design of a diatom secreting monoclonal antibodies directed against the Marburg virus nucleoprotein.

    PubMed

    Hempel, Franziska; Maurer, Michael; Brockmann, Björn; Mayer, Christian; Biedenkopf, Nadine; Kelterbaum, Anne; Becker, Stephan; Maier, Uwe G

    2017-07-27

    The ideal protein expression system should provide recombinant proteins in high quality and quantity involving low production costs only. However, especially for complex therapeutic proteins like monoclonal antibodies many challenges remain to meet this goal and up to now production of monoclonal antibodies is very costly and delicate. Particularly, emerging disease outbreaks like Ebola virus in Western Africa in 2014-2016 make it necessary to reevaluate existing production platforms and develop robust and cheap alternatives that are easy to handle. In this study, we engineered the microalga Phaeodactylum tricornutum to produce monoclonal IgG antibodies against the nucleoprotein of Marburg virus, a close relative of Ebola virus causing severe hemorrhagic fever with high fatality rates in humans. Sequences for both chains of a mouse IgG antibody were retrieved from a murine hybridoma cell line and implemented in the microalgal system. Fully assembled antibodies were shown to be secreted by the alga and antibodies were proven to be functional in western blot, ELISA as well as IFA studies just like the original hybridoma produced IgG. Furthermore, synthetic variants with constant regions of a rabbit IgG and human IgG with optimized codon usage were produced and characterized. This study highlights the potential of microalgae as robust and low cost expression platform for monoclonal antibodies secreting IgG antibodies directly into the culture medium. Microalgae possess rapid growth rates, need basically only water, air and sunlight for cultivation and are very easy to handle.

  13. A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

    PubMed

    Gimenez, Alba Marina; Françoso, Katia S; Ersching, Jonatan; Icimoto, Marcelo Y; Oliveira, Vitor; Rodriguez, Anabel E; Schnittger, Leonhard; Florin-Christensen, Monica; Rodrigues, Mauricio M; Soares, Irene S

    2016-11-14

    Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4(+) T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

  14. Stereospecific antibodies to propranolol.

    PubMed

    Wang, L; Chorev, M; Feingers, J; Levitzki, A; Inbar, M

    1986-04-21

    The beta-adrenergic antagonist propranolol was activated through its side chain, coupled to bovine serum albumin, and injected into BALB/c mice. After fusion of the splenocytes from these immunized mice with the NS-1 myeloma cell line, two hybridomas, producing monoclonal anti-propranolol antibodies, were isolated. Clone P-49 was monospecific for propranolol, with a significant preference for the 1-stereoisomer, as compared to the d form. On the other hand, clone P-28 cross-reacted with alprenolol as well as some other beta-antagonists. Both classes of antibodies competed with A431 epidermoid carcinoma beta 2-adrenoceptors for the binding of [3H]propranolol. When ascites cells from clone P-28 were fixed with glutaraldehyde, the anti-propranolol monoclonal antibody became cell bound. These cell-bound P-28 antibodies bind propranolol and other beta-adrenergic ligands with a similar ranking order to the soluble monoclonal antibody. The cell-bound antibody displayed a 5-fold higher affinity towards 1-propranolol than the soluble monoclonal antibody. The practical implications of these findings are discussed.

  15. STUDIES ON ANTIBODY PRODUCTION

    PubMed Central

    White, Robert G.; Coons, Albert H.; Connolly, Jeanne M.

    1955-01-01

    After subcutaneous injection of hen's ovalbumin or diphtheria toxoid precipitated with aluminum phosphate, the production of antibody, as judged by the presence in the tissues of antibody-containing cells, proceeds partly within the regional lymphatic glands and partly in the granulation tissue surrounding the nodule which develops at the site of injection. The first production of antibody takes place in the regional lymphatic gland and antibody production in the local granuloma becomes apparent only from 14 days onwards (rabbit). Antibody-containing plasma cells were demonstrated in the local granuloma up to 7 weeks. Antibody-containing cells in the regional lymphatic glands reach maximum numbers at 2 weeks following injection and decrease thereafter to few cells at 5 weeks. The adjuvant effect of the aluminum phosphate is interpreted as due partly to the delay in absorption of antigen from the local site of its injection which results in prolongation of stimulation of cells within the regional lymphatic glands, and partly to the production of a local granuloma which contains antibody-producing plasma cells. PMID:14392242

  16. Monoclonal antibody heterogeneity analysis and deamidation monitoring with high-performance cation-exchange chromatofocusing using simple, two component buffer systems.

    PubMed

    Kang, Xuezhen; Kutzko, Joseph P; Hayes, Michael L; Frey, Douglas D

    2013-03-29

    The use of either a polyampholyte buffer or a simple buffer system for the high-performance cation-exchange chromatofocusing of monoclonal antibodies is demonstrated for the case where the pH gradient is produced entirely inside the column and with no external mixing of buffers. The simple buffer system used was composed of two buffering species, one which becomes adsorbed onto the column packing and one which does not adsorb, together with an adsorbed ion that does not participate in acid-base equilibrium. The method which employs the simple buffer system is capable of producing a gradual pH gradient in the neutral to acidic pH range that can be adjusted by proper selection of the starting and ending pH values for the gradient as well as the buffering species concentration, pKa, and molecular size. By using this approach, variants of representative monoclonal antibodies with isoelectric points of 7.0 or less were separated with high resolution so that the approach can serve as a complementary alternative to isoelectric focusing for characterizing a monoclonal antibody based on differences in the isoelectric points of the variants present. Because the simple buffer system used eliminates the use of polyampholytes, the method is suitable for antibody heterogeneity analysis coupled with mass spectrometry. The method can also be used at the preparative scale to collect highly purified isoelectric variants of an antibody for further study. To illustrate this, a single isoelectric point variant of a monoclonal antibody was collected and used for a stability study under forced deamidation conditions. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. Application of histone modification-specific interaction domains as an alternative to antibodies

    PubMed Central

    Kungulovski, Goran; Kycia, Ina; Tamas, Raluca; Jurkowska, Renata Z.; Kudithipudi, Srikanth; Henry, Chisato; Reinhardt, Richard; Labhart, Paul

    2014-01-01

    Post-translational modifications (PTMs) of histones constitute a major chromatin indexing mechanism, and their proper characterization is of highest biological importance. So far, PTM-specific antibodies have been the standard reagent for studying histone PTMs despite caveats such as lot-to-lot variability of specificity and binding affinity. Herein, we successfully employed naturally occurring and engineered histone modification interacting domains for detection and identification of histone PTMs and ChIP-like enrichment of different types of chromatin. Our results demonstrate that histone interacting domains are robust and highly specific reagents that can replace or complement histone modification antibodies. These domains can be produced recombinantly in Escherichia coli at low cost and constant quality. Protein design of reading domains allows for generation of novel specificities, addition of affinity tags, and preparation of PTM binding pocket variants as matching negative controls, which is not possible with antibodies. PMID:25301795

  18. Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

    PubMed

    Hadiji-Abbes, Nadia; Mihoubi, Wafa; Martin, Marta; Karakasyan-Dia, Carole; Frikha, Fakher; Gergely, Csilla; Jouenne, Thierry; Gargouri, Ali; Mokdad-Gargouri, Raja

    2015-10-01

    Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

  19. Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

    PubMed Central

    2012-01-01

    Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α). Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT

  20. Glyco-variant library of the versatile enzyme horseradish peroxidase

    PubMed Central

    Capone, Simona; Pletzenauer, Robert; Maresch, Daniel; Metzger, Karl; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2014-01-01

    When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. PMID:24859724

  1. Glyco-variant library of the versatile enzyme horseradish peroxidase.

    PubMed

    Capone, Simona; Pletzenauer, Robert; Maresch, Daniel; Metzger, Karl; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

    2014-09-01

    When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library. © The Author 2014. Published by Oxford University Press.

  2. Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

  3. Charge variant analysis of proposed biosimilar to Trastuzumab.

    PubMed

    Dakshinamurthy, Pravinkumar; Mukunda, Pavithra; Prasad Kodaganti, Bhargav; Shenoy, Bharath Ravindra; Natarajan, Bairavabalakumar; Maliwalave, Amol; Halan, Vivek; Murugesan, Sathyabalan; Maity, Sunit

    2017-03-01

    Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcγRIIIa) on an effector cell, such as natural killer (NK) cells. The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The ligand-independent HER2-HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival. An attempt has been made in this study to understand the impact of charge heterogeneity on the binding kinetics and potency of the monoclonal antibody. Trastuzumab has a pI range of 8.7-8.9 and is composed of mixture of acidic and basic variants beside the main peak. Ion exchange chromatography was used to isolate the acidic, basic, and main peak fractions from in-house proposed biosimilar to Trastuzumab and their activities were compared to the Innovator Trastuzumab Herclon(®). Data from the mass analysis confirmed the potential modifications in both acidic and basic variant. Binding activity studies performed using Surface Plasmon Resonance (SPR) revealed that acidic variants had lesser binding to HER2 in comparison to the basic variants. Both acidic and basic variant showed no significant changes in their binding to soluble CD16a receptors. In vitro assay studies using a breast cancer cell line (BT-474) confirmed the binding potency of acidic variant to be lesser than basic variant, along with reduced anti-proliferative activity for the acidic variant of Trastuzumab. Overall, these data has provided meaningful insights to the impact of antibody charge variants on in vitro potency and CD16 binding affinity of trastuzumab.

  4. Temporal analysis of HIV envelope sequence evolution and antibody escape in a subtype A-infected individual with a broad neutralizing antibody response

    PubMed Central

    Bosch, Katherine A.; Rainwater, Stephanie; Jaoko, Walter; Overbaugh, Julie

    2010-01-01

    The origin of broadly neutralizing HIV-specific antibodies and their relation to HIV evolution are not well defined. Here we examined virus evolution and neutralizing antibody escape in a subtype A infected individual with a broad, cross subtype, antibody response. The majority of envelope variants isolated over the first ~ 5 years post-infection were poorly neutralized by contemporaneous plasma that neutralized variants from earlier in infection, consistent with a dynamic process of escape. The majority of variants could be neutralized by later plasma, suggesting these evolving variants may have contributed to the elicitation of new antibody responses. However, some variants from later in infection were recognized by plasma from earlier in infection, including one notably neutralization-sensitive variant that was sensitive due to a proline at position 199 in V2. These studies suggest a complex pattern of virus evolution in this individual with a broad NAb response, including persistence of neutralization-sensitive viruses. PMID:20034648

  5. A novel multi-epitope vaccine based on Dipeptidyl Peptidase 4 prevents streptozotocin-induced diabetes by producing anti-DPP4 antibody and immunomodulatory effect in C57BL/6J mice.

    PubMed

    Li, Zhixin; Fang, Jinzhi; Jiao, Rui; Wei, Xiaomin; Ma, Yanjie; Liu, Xiaoran; Cheng, Peng; Li, Taiming

    2017-03-31

    Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing β-cells leads to impaired glucose metabolism and its attendant complications. A series of Dipeptidyl peptidase 4 (DPP4) inhibitors have been developed and granted approval in the treatment of type 2 diabetes mellitus by inhibiting the enzymatic degradation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). An increasing number of studies have shown the potential benefits of DPP4 inhibitors for type 1 diabetes. In this report, we describe a novel multi-epitope vaccine comprising a B cell epitope of DPP4, an anti-diabetic B cell epitope of Insulinoma antigen-2 (IA-2) and a Th2 epitope of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in streptozotocin (STZ) treated mice successfully induced specific anti-DPP4 antibody and increased serum GLP-1 level. Moreover, this antibody lasted for more than 7 weeks. Inoculation of this vaccine in C57BL/6J mice significantly reduced blood glucose level, improved glucose excursion and increased plasma insulin concentration. Consistent with a lower diabetic and insulitis incidence, induced splenic T cell proliferation and tolerance were observed. IFN-γ and IL-2 secretion reduced, but IL-10 and IL-4 increased significantly in the Dipeptidyl Peptidase 41-Insulinoma antigen-2(5)-P2-1 (D41-IP) treated mice compared to the Insulinoma antigen-2(5)-P2-1 (IA2(5)P2-1) and control group due to the potential immunomodulatory effect of the epitopes in the vaccine. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach against type 1 diabetes.

  6. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  7. Evaluation of selectivity in homologous multimodal chromatographic systems using in silico designed antibody fragment libraries.

    PubMed

    Karkov, Hanne Sophie; Woo, James; Krogh, Berit Olsen; Ahmadian, Haleh; Cramer, Steven M

    2015-12-24

    This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.

  8. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  9. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  10. HHF35, a muscle-actin-specific monoclonal antibody. I. Immunocytochemical and biochemical characterization.

    PubMed

    Tsukada, T; Tippens, D; Gordon, D; Ross, R; Gown, A M

    1987-01-01

    A monoclonal antibody to muscle cell actin isotypes was produced and characterized. Immunocytochemical analysis of methanol-Carnoy's-fixed, paraffin-embedded human tissue revealed that this antibody, termed HHF35, reacts with skeletal muscle cells, cardiac muscle cells, smooth muscle cells, pericytes, and myoepithelial cells, but is nonreactive with endothelial, epithelial, neural, or connective tissue cells. When assayed by indirect immunofluorescence, HHF35 reacts with microfilament bundles from various cultured mammalian smooth muscle cells, but does not react with cultured human dermal fibroblasts or various epithelial tumor cell lines. In one-dimensional gel electrophoresis immunoblot experiments this antibody detects a 42-kd polypeptide from tissue extracts of uterus, ileum, aorta, diaphragm, and heart and extract from smooth muscle cells. The antibody also reacts with a comigrating 42-kd band of highly purified rabbit skeletal muscle actin. HHF35 is nonreactive on immunoblots of extracts from all tested nonmuscle cell extracts. Immunoelectrophoresis followed by immunoblotting performed in the presence of urea and reducing agents reveals recognition of the alpha isoelectrophoretic variant of actin from skeletal, cardiac, and smooth muscle sources and of the gamma variant from smooth muscle sources. Because HHF35 reacts with virtually all muscle cells, it will be useful as a marker for muscle and muscle-derived cells.

  11. Development and Evaluation of an Ovine Antibody-Based Platform for Treatment of Clostridium difficile Infection

    PubMed Central

    Roberts, April; McGlashan, Joanna; Al-Abdulla, Ibrahim; Ling, Roger; Denton, Harriet; Green, Steve; Coxon, Ruth; Landon, John

    2012-01-01

    Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed. PMID:22144483

  12. Optimizing antibody expression by using the naturally occurring framework diversity in a live bacterial antibody display system.

    PubMed

    Lombana, T Noelle; Dillon, Michael; Bevers, Jack; Spiess, Christoph

    2015-12-03

    Rapid identification of residues that influence antibody expression and thermostability is often needed to move promising therapeutics into the clinic. To establish a method that can assess small expression differences, we developed a Bacterial Antibody Display (BAD) system that overcomes previous limitations, enabling the use of full-length formats for antibody and antigen in a live cell setting. We designed a unique library of individual framework variants using natural diversity introduced by somatic hypermutation, and screened half-antibodies for increased expression using BAD. We successfully identify variants that dramatically improve expression yields and in vitro thermostability of two therapeutically relevant antibodies in E. coli and mammalian cells. While we study antibody expression, bacterial display can now be expanded to examine the processes of protein folding and translocation. Additionally, our natural library design strategy could be applied during antibody humanization and library design for in vitro display methods to maintain expression and formulation stability.

  13. Charge variants in IgG1

    PubMed Central

    Goswami, Sirj; Hutchinson, Ryan; Kwong, Zephania W; Yang, Jihong; Wang, Xiangdan; Yao, Zhenling; Sreedhara, Alavattam; Cano, Tony; Tesar, Devin; Nijem, Ihsan; Allison, David E; Wong, Pin Yee; Kao, Yung-Hsiang; Quan, Cynthia; Joshi, Amita; Harris, Reed J; Motchnik, Paul

    2010-01-01

    Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7–9.1 and was composed of about 20% acidic variants, 12% basic variants and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity or the PK properties in rats. PMID:20818176

  14. FcR epsilon+ lymphocytes and regulation of the IgE antibody system. III. Suppressive factor of allergy (SFA) is produced during the in vitro FcR epsilon expression cascade and displays corollary physiologic activity in vivo.

    PubMed

    Marcelletti, J F; Katz, D H

    1984-12-01

    detectable SFA, selectively suppressed in vivo IgE synthesis after administration to intact mice. This indicates that EIRB can stimulate resident T cells of irradiated SJL mice to produce SFA. Finally, as shown previously with conventional ascites-derived SFA, the SFA produced in vitro after stimulation of lymphoid cells with IgE is devoid of IgE-binding properties, because its inhibitory effects on in vivo IgE antibody synthesis are not removed by passage over IgE affinity columns.

  15. Monoclonal Antibodies against Aβ42 Fibrils Distinguish Multiple Aggregation State Polymorphisms in Vitro and in Alzheimer Disease Brain*

    PubMed Central

    Hatami, Asa; Albay, Ricardo; Monjazeb, Sanaz; Milton, Saskia; Glabe, Charles

    2014-01-01

    Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process. PMID:25281743

  16. The Treponema denticola FhbB Protein Is a Dominant Early Antigen That Elicits FhbB Variant-Specific Antibodies That Block Factor H Binding and Cleavage by Dentilisin.

    PubMed

    Miller, Daniel P; Oliver, Lee D; Tegels, Brittney K; Reed, Lucas A; O'Bier, Nathaniel S; Kurniyati, Kurni; Faust, Lindsay A; Lawson, Christine K; Allard, Anna M; Caimano, Melissa J; Marconi, Richard T

    2016-07-01

    The Treponema denticola FhbB protein contributes to immune evasion by binding factor H (FH). Cleavage of FH by the T. denticola protease, dentilisin, may contribute to the local immune dysregulation that is characteristic of periodontal disease (PD). Although three FhbB phyletic types have been defined (FhbB1, FhbB2, and FhbB3), the in vivo expression patterns and antigenic heterogeneity of FhbB have not been assessed. Here, we demonstrate that FhbB is a dominant early antigen that elicits FhbB type-specific antibody (Ab) responses. Using the mu