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Sample records for camelid anti-prp antibody

  1. Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

    PubMed

    Rouet, Romain; Dudgeon, Kip; Christie, Mary; Langley, David; Christ, Daniel

    2015-05-01

    Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.

  2. Potential of mean force for human lysozyme camelid vhh hl6 antibody interaction studies

    NASA Astrophysics Data System (ADS)

    Wang, Yeng-Tseng; Liao, Jun-Min; Chen, Cheng-Lung; Su, Zhi-Yuan; Chen, Chang-Hung; Hu, Jeu-Jiun

    2008-04-01

    Calculating antigen-antibody interaction energies is crucial for understanding antigen-antibody associations in immunology. To shed further light into this equation, we study a separation of human lysozyme-camelid vhh hl6 antibody (cAb-HuL6) complex. The c-terminal end-to-end stretching of the lysozyme-antibody complex structures have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and explicit water model. For the lysozyme-antibody complex, there are six important intermediates in the c-terminal extensions process. Inclusion of our simulations may help to understand the binding mechanics of lysozyme-cAb-HuL6 antibody complex.

  3. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

    PubMed

    Henry, Kevin A; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2016-01-01

    Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. PMID:27631624

  4. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

    PubMed Central

    Henry, Kevin A.; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O.; MacKenzie, C. Roger; Arbabi-Ghahroudi, Mehdi

    2016-01-01

    Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species. PMID:27631624

  5. Inhibition of biofilm formation by Camelid single-domain antibodies against the flagellum of Pseudomonas aeruginosa.

    PubMed

    Adams, Hendrik; Horrevoets, Wannie M; Adema, Simon M; Carr, Hannah E V; van Woerden, Richard E; Koster, Margot; Tommassen, Jan

    2014-09-30

    Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell-cell and cell-surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.

  6. Heterologous Antigen Selection of Camelid Heavy Chain Single Domain Antibodies against Tetrabromobisphenol A

    PubMed Central

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous flame retardant. A high-throughput immunoassay would allow for monitoring of human and environmental exposures as a part of risk assessment. Naturally occurring antibodies in camelids that are devoid of light chain, show great promise as an efficient tool in monitoring environmental contaminants, but they have been rarely used for small molecules. An alpaca was immunized with a TBBPA hapten coupled to thyroglobulin and a variable domain of heavy chain antibody (VHH) T3–15 highly selective for TBBPA was isolated from a phage displayed VHH library using heterologous coating antigens. Compared to the VHHs isolated using homologous antigens, VHH T3–15 had about a 10-fold improvement in sensitivity in an immunoassay. This assay, under the optimized conditions of 10% methanol in the assay buffer (pH 7.4), had an IC50 for TBBPA of 0.40 ng mL–1 and negligible cross reactivity (<0.1%) with other tested analogues. After heating the VHH at 90 °C for 90 min about 20% of the affinity for coating antigen T3-BSA remained. The recoveries of TBBPA from spiked soil and fetal bovine serum samples ranged from 90.3% to 110.7% by ELISA and agreed well with a liquid chromatography–tandem mass spectrometry method. We conclude the many advantages of VHH make them attractive for the development of immunoassays to small molecules. PMID:25068372

  7. Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

    PubMed

    Alturki, Norah A; Henry, Kevin A; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.

  8. A camelid anti-PrP antibody abrogates PrP replication in prion-permissive neuroblastoma cell lines.

    PubMed

    Jones, Daryl Rhys; Taylor, William Alexander; Bate, Clive; David, Monique; Tayebi, Mourad

    2010-01-01

    The development of antibodies effective in crossing the blood brain barrier (BBB), capable of accessing the cytosol of affected cells and with higher affinity for PrP(Sc) would be of paramount importance in arresting disease progression in its late stage and treating individuals with prion diseases. Antibody-based therapy appears to be the most promising approach following the exciting report from White and colleagues, establishing the "proof-of-principle" for prion-immunotherapy. After passive transfer, anti-prion antibodies were shown to be very effective in curing peripheral but not central rodent prion disease, due to the fact that these anti-prion antibodies are relatively large molecules and cannot therefore cross the BBB. Here, we show that an anti-prion antibody derived from camel immunised with murine scrapie material adsorbed to immunomagnetic beads is able to prevent infection of susceptible N2a cells and cure chronically scrapie-infected neuroblastoma cultures. This antibody was also shown to transmigrate across the BBB and cross the plasma membrane of neurons to target cytosolic PrP(C). In contrast, treatment with a conventional anti-prion antibody derived from mouse immunised with recombinant PrP protein was unable to prevent recurrence of PrP(Sc) replication. Furthermore, our camelid antibody did not display any neurotoxic effects following treatment of susceptible N2a cells as evidenced by TUNEL staining. These findings demonstrate the potential use of anti-prion camelid antibodies for the treatment of prion and other related diseases via non-invasive means.

  9. Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

    PubMed Central

    Klarenbeek, Alex; Mazouari, Khalil El; Desmyter, Aline; Blanchetot, Christophe; Hultberg, Anna; de Jonge, Natalie; Roovers, Rob C; Cambillau, Christian; Spinelli, Sylvia; Del-Favero, Jurgen; Verrips, Theo; de Haard, Hans J; Achour, Ikbel

    2015-01-01

    Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1β and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelid-derived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform. PMID:26018625

  10. Development and utilization of camelid VHH antibodies from alpaca for 2,2',4,4'-tetrabrominated diphenyl ether detection.

    PubMed

    Bever, Candace R S; Majkova, Zuzana; Radhakrishnan, Rajeswaran; Suni, Ian; McCoy, Mark; Wang, Yanru; Dechant, Julie; Gee, Shirley; Hammock, Bruce D

    2014-08-01

    An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies.

  11. Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

    PubMed Central

    Akazawa-Ogawa, Yoko; Takashima, Mizuki; Lee, Young-Ho; Ikegami, Takahisa; Goto, Yuji; Uegaki, Koichi; Hagihara, Yoshihisa

    2014-01-01

    The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation. PMID:24739391

  12. Novel camelid antibody fragments targeting recombinant nucleoprotein of Araucaria hantavirus: a prototype for an early diagnosis of Hantavirus Pulmonary Syndrome.

    PubMed

    Pereira, Soraya S; Moreira-Dill, Leandro S; Morais, Michelle S S; Prado, Nidiane D R; Barros, Marcos L; Koishi, Andrea C; Mazarrotto, Giovanny A C A; Gonçalves, Giselle M; Zuliani, Juliana P; Calderon, Leonardo A; Soares, Andreimar M; Pereira da Silva, Luiz H; Duarte dos Santos, Claudia N; Fernandes, Carla F C; Stabeli, Rodrigo G

    2014-01-01

    In addition to conventional antibodies, camelids produce immunoglobulins G composed exclusively of heavy chains in which the antigen binding site is formed only by single domains called VHH. Their particular characteristics make VHHs interesting tools for drug-delivery, passive immunotherapy and high-throughput diagnosis. Hantaviruses are rodent-borne viruses of the Bunyaviridae family. Two clinical forms of the infection are known. Hemorrhagic Fever with Renal Syndrome (HFRS) is present in the Old World, while Hantavirus Pulmonary Syndrome (HPS) is found on the American continent. There is no specific treatment for HPS and its diagnosis is carried out by molecular or serological techniques, using mainly monoclonal antibodies or hantavirus nucleoprotein (N) to detect IgM and IgG in patient serum. This study proposes the use of camelid VHHs to develop alternative methods for diagnosing and confirming HPS. Phage display technology was employed to obtain VHHs. After immunizing one Lama glama against the recombinant N protein (prNΔ₈₅) of a Brazilian hantavirus strain, VHH regions were isolated to construct an immune library. VHHs were displayed fused to the M13KO7 phage coat protein III and the selection steps were performed on immobilized prNΔ₈₅. After selection, eighty clones recognized specifically the N protein. These were sequenced, grouped based mainly on the CDRs, and five clones were analyzed by western blot (WB), surface plasmon resonance (SPR) device, and ELISA. Besides the ability to recognize prNΔ85 by WB, all selected clones showed affinity constants in the nanomolar range. Additionaly, the clone KC329705 is able to detect prNΔ₈₅ in solution, as well as the native viral antigen. Findings support the hypothesis that selected VHHs could be a powerful tool in the development of rapid and accurate HPS diagnostic assays, which are essential to provide supportive care to patients and reduce the high mortality rate associated with hantavirus

  13. Analysis and modeling of the variable region of camelid single-domain antibodies.

    PubMed

    Sircar, Aroop; Sanni, Kayode A; Shi, Jiye; Gray, Jeffrey J

    2011-06-01

    Camelids have a special type of Ab, known as heavy chain Abs, which are devoid of classical Ab light chains. Relative to classical Abs, camelid heavy chain Abs (cAbs) have comparable immunogenicity, Ag recognition diversity and binding affinities, higher stability and solubility, and better manufacturability, making them promising candidates for alternate therapeutic scaffolds. Rational engineering of cAbs to improve therapeutic function requires knowledge of the differences of sequence and structural features between cAbs and classical Abs. In this study, amino acid sequences of 27 cAb variable regions (V(H)H) were aligned with the respective regions of 54 classical Abs to detect amino acid differences, enabling automatic identification of cAb V(H)H CDRs. CDR analysis revealed that the H1 often (and sometimes the H2) adopts diverse conformations not classifiable by established canonical rules. Also, although the cAb H3 is much longer than classical H3 loops, it often contains common structural motifs and sometimes a disulfide bond to the H1. Leveraging these observations, we created a Monte Carlo-based cAb V(H)H structural modeling tool, where the CDR H1 and H2 loops exhibited a median root-mean-square deviation to natives of 3.1 and 1.5 Å, respectively. The protocol generated 8-12, 14-16, and 16-24 residue H3 loops with a median root-mean-square deviation to natives of 5.7, 4.5, and 6.8 Å, respectively. The large deviation of the predicted loops underscores the challenge in modeling such long loops. cAb V(H)H homology models can provide structural insights into interaction mechanisms to enable development of novel Abs for therapeutic and biotechnological use. PMID:21525384

  14. Analysis of camelid antibodies for antivenom development: Neutralisation of venom-induced pathology.

    PubMed

    Cook, Darren A N; Owen, Timothy; Wagstaff, Simon C; Kinne, Joerg; Wernery, Ulrich; Harrison, Robert A

    2010-09-01

    Camelid IgG has been reported to be less immunogenic, less able to activate the complement cascade and more thermostable than IgG from other mammals, and has the ability to bind antigens that are unreactive with other mammalian IgGs. We are investigating whether these attributes of camelid IgG translate into antivenom with immunological and venom-neutralising efficacy advantages over conventional equine and ovine antivenoms. The objective of this study was to determine the preclinical venom-neutralising effectiveness of IgG from camels immunised with venoms, individually or in combination, of the saw-scaled viper, Echis ocellatus, the puff adder, Bitis arietans and the spitting cobra, Naja nigricollis - the most medically-important snake species in West Africa. Neutralisation of the pathological effects of venoms from E. ocellatus, B. arietans and N. nigricollis by IgG from the venom-immunised camels, or commercial antivenom, was compared using assays of venom lethality (ED(50)), haemorrhage (MHD) and coagulopathy (MCD). The E. ocellatus venom ED(50), MHD and MCD results of the E. ocellatus monospecific camel IgG antivenom were broadly equivalent to comparable ovine (EchiTAbG, MicroPharm Ltd, Wales) and equine (SAIMR Echis, South African Vaccine Producer, South Africa) antivenoms, although the equine antivenom required half the amount of IgG. The B. arietans monospecific camel IgG neutralised the lethal effects of B. arietans venom at one fourth the concentration of the SAIMR polyspecific antivenom (a monospecific B. arietans antivenom is not available). The N. nigricollis camel IgG antivenom was ineffective (at the maximum permitted dose, 100 mul) against the lethal effects of N. nigricollis venom. All the equine polyspecific antivenoms required more than 100 microl to be effective against this venom. The polyspecific camel IgG antivenom, prepared from five camels, was effective against the venom-induced effects of E. ocellatus but not against that of B. arietans

  15. The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.

    PubMed

    Akazawa-Ogawa, Yoko; Uegaki, Koichi; Hagihara, Yoshihisa

    2016-01-01

    Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.

  16. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

    SciTech Connect

    Fanning, Sean W.; Horn, James R.

    2014-03-05

    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

  17. One-step immunoassay for tetrabromobisphenol a using a camelid single domain antibody-alkaline phosphatase fusion protein.

    PubMed

    Wang, Jia; Majkova, Zuzana; Bever, Candace R S; Yang, Jun; Gee, Shirley J; Li, Ji; Xu, Ting; Hammock, Bruce D

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environmental and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. The ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL(-1). Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogues was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP-based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples via this one-step assay ranged from 96.7% to 109.9% and correlated well with a high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS) method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring.

  18. One-step Immunoassay for Tetrabromobisphenol A Using a Camelid Single Domain Antibody-Alkaline Phosphatase Fusion Protein

    PubMed Central

    Wang, Jia; Majkova, Zuzana; Bever, Candace R. S.; Yang, Jun; Gee, Shirley J.; Li, Ji; Xu, Ting; Hammock, Bruce D.

    2015-01-01

    Tetrabromobisphenol A (TBBPA) is a ubiquitous brominated flame retardant, showing widespread environment and human exposures. A variable domain of the heavy chain antibody (VHH), naturally occurring in camelids, approaches the lower size limit of functional antigen-binding entities. Ease of genetic manipulation makes such VHHs a superior choice to use as an immunoreagent. In this study, a highly selective anti-TBBPA VHH T3-15 fused with alkaline phosphatase (AP) from E. coli was expressed, showing both an integrated TBBPA-binding capacity and enzymatic activity. A one-step immunoassay based on the fusion protein T3-15-AP was developed for TBBPA in 5% dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS, pH 7.4), with a half-maximum signal inhibition concentration (IC50) of 0.20 ng mL−1. Compared to the parental VHH T3-15, T3-15-AP was able to bind to a wider variety of coating antigens and the assay sensitivity was slightly improved. Cross-reactivity of T3-15-AP with a set of important brominated analogs was negligible (<0.1%). Although T3-15-AP was susceptible to extreme heat (90 °C), much higher binding stability at ambient temperature was observed in the T3-15-AP based assay for at least 70 days. A simple pretreatment method of diluting urine samples with DMSO was developed for a one-step assay. The recoveries of TBBPA from urine samples by this one-step assay ranged from 96.7–109.9% and correlated well with an HPLC-MS/MS method. It is expected that the dimerized fusion protein, VHH-AP, will show promising applications in human exposure and environmental monitoring. PMID:25849972

  19. Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin*

    PubMed Central

    Vance, David J.; Tremblay, Jacqueline M.; Mantis, Nicholas J.; Shoemaker, Charles B.

    2013-01-01

    In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody VH domains (VHHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific VHHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific VHHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics. PMID:24202178

  20. A camelid antibody candidate for development of a therapeutic agent against Hemiscorpius lepturus envenomation.

    PubMed

    Yardehnavi, Najmeh; Behdani, Mahdi; Bagheri, Kamran Pooshang; Mahmoodzadeh, Amir; Khanahmad, Hossein; Shahbazzadeh, Delavar; Habibi-Anbouhi, Mahdi; Hassanzadeh Ghassabeh, Gholamreza; Muyldermans, Serge

    2014-09-01

    Hemiscorpius lepturus scorpionism poses one of the most dangerous health problems in many parts of the world. The common therapy consists of using antivenom antibody fragments derived from a polyclonal immune response raised in horses. However, this immunotherapy creates serious side effects, including anaphylactic shock sometimes even leading to death. Thus, many efforts have been made to introduce new replacement therapeutics that cause less adverse reactions. One of the most attractive approaches to replacing the available therapy is offered by single-domain antibody fragments, or nanobodies (Nbs). We immunized dromedaries with H. lepturus toxin and identified a functional recombinant Nb (referred to as F7Nb) against heminecrolysin (HNc), the major known hemolytic and dermonecrotic fraction of H. lepturus venom. This Nb was retrieved from the immune library by phage display selection. The in vitro neutralization tests indicated that 17.5 nmol of the F7Nb can inhibit 45% of the hemolytic activity of 1 EC100 (7.5 μg/ml) of HNc. The in vivo neutralization tests demonstrated that F7Nb had good antihemolytic and antidermonecrotic effects against HNc in all tested mice. Surprisingly, F7Nb (8.75 nmol) neutralized 1 LD100 of HNc (10 μg) via an intracerebroventricular route or 1 LD100 (80 μg) via a subcutaneous route. All of the control mice died. Hence, this Nb is a potential leading novel candidate for treating H. lepturus scorpionism in the near future. PMID:24891523

  1. Development of protective agent against Hottentotta saulcyi venom using camelid single-domain antibody.

    PubMed

    Darvish, Maryam; Behdani, Mahdi; Shokrgozar, Mohammad Ali; Pooshang-Bagheri, Kamran; Shahbazzadeh, Delavar

    2015-12-01

    Hottentotta saulcyi, medically important scorpion species, causes some of harmful toxic exposure in Iran. Administrated, conventional antivenom-based immunotherapy is still limited and hardly meet ideal characteristic of effective treatment for scorpion envenomation. In this study we aimed to develop a neutralizing agent directed against scorpion venom based on VHH, variable domain of the Camelidae heavy chain antibody or Nanobody. This promising biomolecule is well-established as an advantageous tool for therapeutic purposes due to its small size, stability, monomeric performance and less immunogenicity. In this study, a large Nb library was constructed and phage displayed after successful camel immunization using H. saulcyi scorpion crude venom. After a series of biopanning rounds on Sephadex G50 purified venom fraction and screening by monoclonal phage ELISA, the best reactive Nb was retrieved and designated Nb12. The selected Nb was then expressed as soluble protein in Escherichia coli, purified and confirmed by SDS-PAGE analysis and western blotting. The lead candidate Nb12 bound scorpion venom with Kaff value of 5×10(7)M(-1). Nb12 was shown to be capable of neutralizing 2 LD50 of whole venom of scorpion toxin when injected in the ratio of the Nb/toxin of 1.4:1 into C57BL/6 mice. In challenge experiment, Nb succeeded to rescue all i.p. lethal dose injected mice even when administrated i.v., 20min after envenoming. These results with ease of production and superior neutralizing activity make Nb a suitable anti-toxin candidate for treatment of scorpion envenoming. PMID:26468036

  2. Development and Utilization of Camelid VHH Antibodies from Alpaca for 2,2′,4,4′-Tetrabrominated Diphenyl Ether Detection

    PubMed Central

    2015-01-01

    An antibody-based analytical method for the detection of a chemical flame retardant using antibody fragments isolated from an alpaca has been developed. One specific chemical flame retardant congener, 2,2′,4,4′-tetrabrominated diphenyl ether (BDE-47), is often the major poly-BDE (PBDE) congener present in human and environmental samples and that which is the most frequently detected. An alpaca was immunized with a surrogate of BDE-47 covalently attached to a carrier protein. The resulting mRNA coding for the variable domain of heavy-chain antibodies (VHH) were isolated, transcribed to cDNA, and cloned into a phagemid vector for phage display library construction. Selection of VHHs recognizing BDE-47 was achieved by panning under carefully modified conditions. The assay sensitivity for detecting BDE-47 was down to the part-per-billion (microgram per liter) level. Cross-reactivity analyses confirmed that this method was highly selective for BDE-47 and selected hydroxylated metabolites. When exposed to elevated temperatures, the camelid VHH antibodies retained more reactivity than a polyclonal antibody developed to the same target analyte. The use of this VHH antibody reagent immobilized onto a Au electrode for impedance biosensing demonstrates the increased versatility of VHH antibodies. PMID:25005746

  3. Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays.

    PubMed

    Friedrich, Adrián; Ledesma, Martín; Landone, Ignacio; Ferrari, Alejandro; Leoni, Juliana

    2014-09-01

    A monoclonal antibody (mAb) was produced against immunoglobulin M (IgM) of South American camelids. A single radial immunodiffusion (SRID) assay and a competitive enzyme-linked immunosorbent assay (ELISA) were developed to measure IgM in serum samples. Isotype and specificity of the mAb were assessed. The performance of the SRID assay was preliminarily evaluated in terms of working range, plate stability over a 4-week period, and initial intra- and interassay variation. The concentration of IgM was determined in 55 samples by SRID assay and ELISA, and results were not significantly different by t-test (0.64 ± 0.19 mg/ml for the SRID assay, and 0.58 ± 0.24 mg/ml for ELISA; P = 0.1489). The mAb was shown to be stable over the 4-week evaluation period, and the SRID assay was reproducible when tested in triplicate for intra-assay variability and in quadruplicate for interassay variability, with a percentage coefficient of variation of less than or equal to 5%. Also, the SRID assay proved to be sensitive enough to measure IgM levels in undiluted serum samples, and had a good correlation with ELISA. The current study is intended to submit a preliminary report of a mAb against IgM of South American camelids, and suggest the future potential of the mAb developed for diagnostic application, including use in the SRID assay.

  4. Hematology of camelids.

    PubMed

    Vap, Linda; Bohn, Andrea A

    2015-01-01

    Interpretation of camelid hematology results is similar to that of other mammals. Obtaining accurate results and using appropriate reference intervals can be a bit problematic, particularly when evaluating the erythron. Camelid erythrocytes vary from other mammals in that they are small, flat, and elliptical. This variation makes data obtained from samples collected from these species prone to error when using some automated instruments. Normal and abnormal findings in camelid blood are reviewed as well as how to ensure accurate results.

  5. Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays.

    PubMed

    Friedrich, Adrián; Ledesma, Martín; Landone, Ignacio; Ferrari, Alejandro; Leoni, Juliana

    2014-09-01

    A monoclonal antibody (mAb) was produced against immunoglobulin M (IgM) of South American camelids. A single radial immunodiffusion (SRID) assay and a competitive enzyme-linked immunosorbent assay (ELISA) were developed to measure IgM in serum samples. Isotype and specificity of the mAb were assessed. The performance of the SRID assay was preliminarily evaluated in terms of working range, plate stability over a 4-week period, and initial intra- and interassay variation. The concentration of IgM was determined in 55 samples by SRID assay and ELISA, and results were not significantly different by t-test (0.64 ± 0.19 mg/ml for the SRID assay, and 0.58 ± 0.24 mg/ml for ELISA; P = 0.1489). The mAb was shown to be stable over the 4-week evaluation period, and the SRID assay was reproducible when tested in triplicate for intra-assay variability and in quadruplicate for interassay variability, with a percentage coefficient of variation of less than or equal to 5%. Also, the SRID assay proved to be sensitive enough to measure IgM levels in undiluted serum samples, and had a good correlation with ELISA. The current study is intended to submit a preliminary report of a mAb against IgM of South American camelids, and suggest the future potential of the mAb developed for diagnostic application, including use in the SRID assay. PMID:25057160

  6. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments

    PubMed Central

    Prado, Nidiane D. R.; Pereira, Soraya S.; da Silva, Michele P.; Morais, Michelle S. S.; Kayano, Anderson M.; Moreira-Dill, Leandro S.; Luiz, Marcos B.; Zanchi, Fernando B.; Fuly, André L.; E. F. Huacca, Maribel; Fernandes, Cleberson F.; Calderon, Leonardo A.; Zuliani, Juliana P.; Soares, Andreimar M.; Stabeli, Rodrigo G.; F. C. Fernandes, Carla

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  7. Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

    PubMed

    Prado, Nidiane D R; Pereira, Soraya S; da Silva, Michele P; Morais, Michelle S S; Kayano, Anderson M; Moreira-Dill, Leandro S; Luiz, Marcos B; Zanchi, Fernando B; Fuly, André L; Huacca, Maribel E F; Fernandes, Cleberson F; Calderon, Leonardo A; Zuliani, Juliana P; Pereira da Silva, Luiz H; Soares, Andreimar M; Stabeli, Rodrigo G; Fernandes, Carla F C

    2016-01-01

    Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem. PMID:27028872

  8. Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS).

    PubMed

    Mejías, Maria P; Hiriart, Yanina; Lauché, Constanza; Fernández-Brando, Romina J; Pardo, Romina; Bruballa, Andrea; Ramos, María V; Goldbaum, Fernando A; Palermo, Marina S; Zylberman, Vanesa

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome. PMID:27118524

  9. Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)

    PubMed Central

    Mejías, Maria P.; Hiriart, Yanina; Lauché, Constanza; Fernández-Brando, Romina J.; Pardo, Romina; Bruballa, Andrea; Ramos, María V.; Goldbaum, Fernando A.; Palermo, Marina S.; Zylberman, Vanesa

    2016-01-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome. PMID:27118524

  10. Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (VHH) scaffold

    PubMed Central

    Tereshko, Valentina; Uysal, Serdar; Koide, Akiko; Margalef, Katrina; Koide, Shohei; Kossiakoff, Anthony A.

    2008-01-01

    A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (VHH) as a crystallization chaperone. By using a yeast surface display system for VHH, we successfully introduced additional Met residues in the core of the VHH scaffold. We identified a set of SeMet-labeled VHH variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched VHH chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and VHH model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for VHH, the enhanced VHH domain described here will be an excellent scaffold for producing effective crystallization chaperones. PMID:18445622

  11. Methane emission by camelids.

    PubMed

    Dittmann, Marie T; Runge, Ullrich; Lang, Richard A; Moser, Dario; Galeffi, Cordula; Kreuzer, Michael; Clauss, Marcus

    2014-01-01

    Methane emissions from ruminant livestock have been intensively studied in order to reduce contribution to the greenhouse effect. Ruminants were found to produce more enteric methane than other mammalian herbivores. As camelids share some features of their digestive anatomy and physiology with ruminants, it has been proposed that they produce similar amounts of methane per unit of body mass. This is of special relevance for countrywide greenhouse gas budgets of countries that harbor large populations of camelids like Australia. However, hardly any quantitative methane emission measurements have been performed in camelids. In order to fill this gap, we carried out respiration chamber measurements with three camelid species (Vicugna pacos, Lama glama, Camelus bactrianus; n = 16 in total), all kept on a diet consisting of food produced from alfalfa only. The camelids produced less methane expressed on the basis of body mass (0.32±0.11 L kg⁻¹ d⁻¹) when compared to literature data on domestic ruminants fed on roughage diets (0.58±0.16 L kg⁻¹ d⁻¹). However, there was no significant difference between the two suborders when methane emission was expressed on the basis of digestible neutral detergent fiber intake (92.7±33.9 L kg⁻¹ in camelids vs. 86.2±12.1 L kg⁻¹ in ruminants). This implies that the pathways of methanogenesis forming part of the microbial digestion of fiber in the foregut are similar between the groups, and that the lower methane emission of camelids can be explained by their generally lower relative food intake. Our results suggest that the methane emission of Australia's feral camels corresponds only to 1 to 2% of the methane amount produced by the countries' domestic ruminants and that calculations of greenhouse gas budgets of countries with large camelid populations based on equations developed for ruminants are generally overestimating the actual levels.

  12. Differential Neutralizing Activities of a Single Domain Camelid Antibody (VHH) Specific for Ricin Toxin’s Binding Subunit (RTB)

    PubMed Central

    Herrera, Cristina; Vance, David J.; Eisele, Leslie E.; Shoemaker, Charles B.; Mantis, Nicholas J.

    2014-01-01

    Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or “nanobody”) specific for ricin’s enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1∶10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined. PMID

  13. A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model.

    PubMed

    Moayeri, Mahtab; Leysath, Clinton E; Tremblay, Jacqueline M; Vrentas, Catherine; Crown, Devorah; Leppla, Stephen H; Shoemaker, Charles B

    2015-03-01

    Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from "pre-pore" to its SDS and heat-resistant "pore" conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.

  14. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

  15. Camelid herd health.

    PubMed

    Jones, Meredyth; Boileau, Melanie

    2009-07-01

    The area of herd health is particularly important when considering camelid operations because of the high frequency of travel for exhibition, breeding, and boarding. This article outlines the considerations for routine husbandry, facility and animal maintenance, and infectious disease control in the form of biosecurity, vaccination, and health testing that should be included in any farm's herd-health plan. Veterinary input into the design of programs for biosecurity and infectious disease prevention is critical and requires an active veterinary client-patient relationship with identification of the goals of the operation. Risk assessments should be made based on farm activities and should be the foundation for herd-health program design.

  16. Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

    PubMed

    Jiang, Wenzhi; Rosenberg, Julian N; Wauchope, Akelia D; Tremblay, Jacqueline M; Shoemaker, Charles B; Weeks, Donald P; Oyler, George A

    2013-11-01

    Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

  17. Toward chaperone-assisted crystallography: Protein engineering enhancement of crystal packing and X-ray phasing capabilities of a camelid single-domain antibody (V[subscript H]H) scaffold

    SciTech Connect

    Tereshko, Valentina; Uysal, Serdar; Koide, Akiko; Margalef, Katrina; Koide, Shohei; Kossiakoff, Anthony A.

    2008-07-28

    A crystallization chaperone is an auxiliary protein that binds to a target of interest, enhances and modulates crystal packing, and provides high-quality phasing information. We critically evaluated the effectiveness of a camelid single-domain antibody (V{sub H}H) as a crystallization chaperone. By using a yeast surface display system for V{sub H}H, we successfully introduced additional Met residues in the core of the V{sub H}H scaffold. We identified a set of SeMet-labeled V{sub H}H variants that collectively produced six new crystal forms as the complex with the model antigen, RNase A. The crystals exhibited monoclinic, orthorhombic, triclinic, and tetragonal symmetry and have one or two complexes in the asymmetric unit, some of which diffracted to an atomic resolution. The phasing power of the Met-enriched V{sub H}H chaperone allowed for auto-building the entire complex using single-anomalous dispersion technique (SAD) without the need for introducing SeMet into the target protein. We show that phases produced by combining SAD and VHH model-based phases are accurate enough to easily solve structures of the size reported here, eliminating the need to collect multiple wavelength multiple-anomalous dispersion (MAD) data. Together with the presence of high-throughput selection systems (e.g., phage display libraries) for V{sub H}H, the enhanced V{sub H}H domain described here will be an excellent scaffold for producing effective crystallization chaperones.

  18. Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

    PubMed Central

    Barrera, Daniel J.; Rosenberg, Julian N.; Chiu, Joanna G.; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T.; Shoemaker, Charles B.; Oyler, George A.; Mayfield, Stephen P.

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VHH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen binding proteins and the heterodimer fusion protein containing two VHH domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism. PMID:25229405

  19. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

    PubMed

    Barrera, Daniel J; Rosenberg, Julian N; Chiu, Joanna G; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T; Shoemaker, Charles B; Oyler, George A; Mayfield, Stephen P

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

  20. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

    PubMed

    Barrera, Daniel J; Rosenberg, Julian N; Chiu, Joanna G; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T; Shoemaker, Charles B; Oyler, George A; Mayfield, Stephen P

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism. PMID:25229405

  1. Complement component 3 binding to Haemophilus influenzae type b in the presence of anticapsular and anti-outer membrane antibodies.

    PubMed Central

    Hetherington, S V; Patrick, C C

    1992-01-01

    Antibodies directed against the capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) or the outer membrane proteins (OMP) of Haemophilus influenzae type b (Hib) promote bactericidal activity, complement 3 (C3) binding, and ingestion by phagocytic cells. To assess the relative contribution of anti-OMP to host defense against Hib, we compared the opsonic activities of anti-PRP and anti-OMP as reflected by the amounts of C3 bound to the bacterial surface. Immunoglobulin G (IgG) fractions containing either anti-PRP or anti-OMP were incubated with Hib in the presence of a C5-deficient complement source. C3, total IgG, and IgG subclasses bound to the bacteria were quantified by enzyme-linked immunosorbent assay. The maximum amount of C3 which could be bound to Hib was greater in the presence of anti-PRP than in the presence of anti-OMP. Also, except at low IgG concentrations, the rate of increase in bound C3 as a function of increasing IgG concentration was greater for anti-PRP than for anti-OMP. Hib-bound anti-OMP consisted primarily of IgG1 and IgG3, whereas bound anti-PRP was primarily IgG1 and IgG2. Thus, the potential for C3 binding to Hib is greater in the presence of anti-PRP than in the presence of anti-OMP, probably because of the larger number of binding sites available to the former. Nonetheless, OMP appear to provide important targets for opsonic antibody and would be logical components of a PRP-conjugate vaccine or may be efficacious as vaccines against nontypeable H. influenzae. Images PMID:1729183

  2. Fluid therapy in small ruminants and camelids.

    PubMed

    Jones, Meredyth; Navarre, Christine

    2014-07-01

    Body water, electrolytes, and acid-base balance are important considerations in the evaluation and treatment of small ruminants and camelids with any disease process, with restoration of these a priority as adjunctive therapy. The goals of fluid therapy should be to maintain cardiac output and tissue perfusion, and to correct acid-base and electrolyte abnormalities. Hypoglycemia, hyperkalemia, and acidosis are the most life-threatening abnormalities, and require most immediate correction.

  3. [Endo- and ectoparasites of South American camelids and their control].

    PubMed

    Schmäschke, R

    2015-01-01

    In a literature review, common endo- and ectoparasites of South American camelids are described, presenting morphological details and clinical signs important for diagnosis. Based on the life cycle of the parasites, possibilities for prophylaxis and therapy are indicated. The review should aid the veterinarian to diagnose and control common parasitic infections in South American camelids.

  4. [Testing for BTV, BVDV and BHV-1 in blood samples of new world camelids kept in middle Germany].

    PubMed

    Locher, Lena; Nieper, Hermann; Volkery, Janine; Fürll, Manfred; Wittek, Thomas

    2010-01-01

    The susceptibility of camelids for infectious agents which may result in severe economic losses or which are strictly regulated for epidemiological reasons in farm animals potentially causes a mutual risk of transmission. This study aimed to investigate the presence of antibodies against bovine herpesvirus 1 (BHV-1), bluetongue virus (BTV) and bovine viral diarrhoea virus (BVDV) as well as the presence of pestivirus antigen in new world camelids in Central Germany. Therefore 107 serum samples from 93 alpacas and lamas from this region which had been obtained from 2007 to 2009 were examined using ELISA, serum neutralisation test, RT-PCR and a pestivirus specific gene probe. All sample were negative for BHV-1 antibodies. Antibodies against BVDV-1 could be detected in four animals, titres reaching from 1:64 to > 1:256. One animal was positive for BTV antibodies in the year 2008. This animal had been tested negative for BTV antibodies in 2007. It can be concluded that up to now, these viruses seem to be of minor importance as pathogens in new world camelids in Central Germany. Therefore the risk of infection originating from new world camelids for production animals could be considered to be rather low in this region at the moment. However, it must be taken into consideration that these animals due to lack of antibodies are fully susceptible in case of occurrence of one of these viruses. For maintenance and improvement of the present status, general hygienic precautions should be applied; direct and indirect contact between animals from different herds must be avoided and virological diagnostic and quarantine should be required trading these animals. PMID:21141278

  5. [Testing for BTV, BVDV and BHV-1 in blood samples of new world camelids kept in middle Germany].

    PubMed

    Locher, Lena; Nieper, Hermann; Volkery, Janine; Fürll, Manfred; Wittek, Thomas

    2010-01-01

    The susceptibility of camelids for infectious agents which may result in severe economic losses or which are strictly regulated for epidemiological reasons in farm animals potentially causes a mutual risk of transmission. This study aimed to investigate the presence of antibodies against bovine herpesvirus 1 (BHV-1), bluetongue virus (BTV) and bovine viral diarrhoea virus (BVDV) as well as the presence of pestivirus antigen in new world camelids in Central Germany. Therefore 107 serum samples from 93 alpacas and lamas from this region which had been obtained from 2007 to 2009 were examined using ELISA, serum neutralisation test, RT-PCR and a pestivirus specific gene probe. All sample were negative for BHV-1 antibodies. Antibodies against BVDV-1 could be detected in four animals, titres reaching from 1:64 to > 1:256. One animal was positive for BTV antibodies in the year 2008. This animal had been tested negative for BTV antibodies in 2007. It can be concluded that up to now, these viruses seem to be of minor importance as pathogens in new world camelids in Central Germany. Therefore the risk of infection originating from new world camelids for production animals could be considered to be rather low in this region at the moment. However, it must be taken into consideration that these animals due to lack of antibodies are fully susceptible in case of occurrence of one of these viruses. For maintenance and improvement of the present status, general hygienic precautions should be applied; direct and indirect contact between animals from different herds must be avoided and virological diagnostic and quarantine should be required trading these animals.

  6. Seminal plasma components in camelids and comparisons with other species.

    PubMed

    Kershaw-Young, C M; Maxwell, W M C

    2012-08-01

    Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility. PMID:22827394

  7. Camelid nanobodies with high affinity for broad bean mottle virus: a possible promising tool to immunomodulate plant resistance against viruses.

    PubMed

    Ghannam, Ahmed; Kumari, Safa; Muyldermans, Serge; Abbady, Abdul Qader

    2015-03-01

    Worldwide, plant viral infections decrease seriously the crop production yield, boosting the demand to develop new strategies to control viral diseases. One of these strategies to prevent viral infections, based on the immunomodulation faces many problems related to the ectopic expression of specific antibodies in planta. Camelid nanobodies, expressed in plants, may offer a solution as they are an attractive tool to bind efficiently to viral epitopes, cryptic or not accessible to conventional antibodies. Here, we report a novel, generic approach that might lead to virus resistance based on the expression of camelid specific nanobodies against Broad bean mottle virus (BBMV). Eight nanobodies, recognizing BBMV with high specificity and affinity, were retrieved after phage display from a large 'immune' library constructed from an immunized Arabic camel. By an in vitro assay we demonstrate how three nanobodies attenuate the BBMV spreading in inoculated Vicia faba plants. Furthermore, the in planta transient expression of these three selected nanobodies confirms their virus neutralizing capacity. In conclusion, this report supports that plant resistance against viral infections can be achieved by the in vivo expression of camelid nanobodies. PMID:25648551

  8. Camelid antivenom development and potential in vivo neutralization of Hottentotta saulcyi scorpion venom.

    PubMed

    Darvish, Maryam; Ebrahimi, Soltan Ahmad; Shahbazzadeh, Delavar; Bagheri, Kamran-Pooshang; Behdani, Mahdi; Shokrgozar, Mohammad Ali

    2016-04-01

    Scorpion envenoming is a serious health problem which can cause a variety of clinical toxic effects. Of the many scorpion species native to Iran, Hottentotta saulcyi is important because its venom can produce toxic effects in man. Nowadays, antivenom derived from hyper immune horses is the only effective treatment for sever scorpion stings. Current limitations of immunotherapy urgently require an efficient alternative with high safety, target affinity and more promising venom neutralizing capability. Recently, heavy chain-only antibodies (HC-Abs) found naturally in camelid serum met the above mentioned advantages. In this study, immuno-reactivities of polyclonal antibodies were tested after successful immunization of camel using H. saulcyi scorpion crude venom. The lethal potency of scorpion venom in C57BL/6 mice injected intraperitoneally was determined to be 2.7 mg/kg. These results were followed by the efficient neutralization of lethal activity of H. saulcyi scorpion venom by injection of antivenom and purified IgG fractions into mice intraperitonelly or intravenously, respectively. HC-Ab camelid antivenom could be considered as a useful serotherapeutics instead of present treatment for scorpion envenomation. PMID:26809016

  9. Parasitic diversity found in coprolites of camelids during the Holocene.

    PubMed

    Taglioretti, Verónica; Fugassa, Martín Horacio; Sardella, Norma Haydée

    2015-07-01

    Knowledge of parasitic infections to which fauna was exposed in the past provides information on the geographical origin of some parasites, on the possible dispersal routes and for archaeological fauna on the potential zoonotic risk that human and animal populations could be exposed. The aim of the present study was to examine the gastrointestinal parasite present in camelid coprolites collected from the archaeological site Cerro Casa de Piedra, cave 7 (CCP7), Patagonia, Argentina. Coprolites were collected from different stratified sequences dating from the Pleistocene-Holocene transition to the late Holocene. Paleoparasitological examination revealed the presence of eggs of Trichostrongylidae attributed to Lamanema chavezi or Nematodirus lamae, eggs of three unidentified capillariids, Strongylus-type eggs and oocysts of Eimeria macusaniensis. These parasites affected camelids living in the studied area since the Pleistocene-Holocene transition, about 10,000 years ago. Gastrointestinal parasite fauna of patagonian camelids did not vary significatively from Pleistocene-Holocene transition to late Holocene, although environmental conditions fluctuated greatly throughout this period, as indicative of the strength and the stability of these associations over time. In this study, the zoonotic and biogeography importance of parasites of camelids are also discussed.

  10. Diagnostic sampling and gross pathology of New World camelids.

    PubMed

    Bildfell, Robert J; Löhr, Christiane V; Tornquist, Susan J

    2012-11-01

    This article provides an overview of tests and appropriate samples to send to a Veterinary Diagnostic Laboratory for the diagnosis of common diseases of New World Camelids (NWC) such as abortions, congenital anomalies, anemia, enteritis, endoparasitism, gastric ulcer, hepatic lipidosis, encephalitis, pneumonia, dermatosis, neoplasia and cryptococcosis. Unique anatomic features of NWC and common findings encountered during gross necropsy examination are briefly reviewed.

  11. Synthetic trimer and tetramer of 3-beta-D-ribose-(1-1)-D-ribitol-5-phosphate conjugated to protein induce antibody responses to Haemophilus influenzae type b capsular polysaccharide in mice and monkeys.

    PubMed Central

    Peeters, C C; Evenberg, D; Hoogerhout, P; Käyhty, H; Saarinen, L; van Boeckel, C A; van der Marel, G A; van Boom, J H; Poolman, J T

    1992-01-01

    Synthetic oligosaccharides derived from the capsular polysaccharide (PRP) of Haemophilus influenzae type b were conjugated to carrier proteins via a thioether linkage. Conjugates were made of trimeric and tetrameric ribose-ribitol-phosphate and tetanus toxoid or diphtheria toxin. All conjugates elicited anti-PRP antibody responses with an increasing immunoglobulin G/immunoglobulin M ratio in adult mice and monkeys. Trimer conjugates elicited lower anti-PRP antibody responses compared with tetramer conjugates. Adult monkeys responded equally well to the tetrameric oligosaccharide-tetanus toxoid conjugate as to the oligosaccharide-CRM197 conjugate (HbOC), which elicits protective levels of serum antibodies in human infants after two or three injections. PMID:1563770

  12. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    PubMed

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. PMID:24673401

  13. Elevated levels of maternal anti-tetanus toxin antibodies do not suppress the immune response to a Haemophilus influenzae type b polyribosylphosphate-tetanus toxoid conjugate vaccine.

    PubMed Central

    Panpitpat, C.; Thisyakorn, U.; Chotpitayasunondh, T.; Fürer, E.; Que, J. U.; Hasler, T.; Cryz, S. J.

    2000-01-01

    Reported are the effects of elevated levels of anti-tetanus antibodies on the safety and immune response to a Haemophilus influenzae type b polyribosylphosphate (PRP)-tetanus toxoid conjugate (PRP-T) vaccine. A group of Thai infants (n = 177) born to women immunized against tetanus during pregnancy were vaccinated with either a combined diphtheria-tetanus-pertussis (DTP) PRP-T vaccine or DTP and a PRP-conjugate vaccine using Neisseria meningitidis group B outer-membrane proteins as a carrier (PedVax HIB). Although most infants possessed high titres (> 1 IU/ml) of anti-tetanus antibodies, the DTP-PRP-T combined vaccine engendered an excellent antibody response to all vaccine components. In both vaccine groups > 98% of infants attained anti-PRP antibody titres > or = 0.15 microgram/ml. The geometric mean anti-PRP antibody titres were 5.41 micrograms/ml and 2.1 micrograms/ml for infants immunized with three doses of PRP-T versus two doses of PedVax HIB vaccines, respectively (P < 0.005). Similarly, the proportion of infants who achieved titres > or = 1 microgram/ml was higher in the PRP-T group (87.8%) than in the group immunized with PedVax HIB (74.2%) (P = 0.036). A subgroup analysis showed that there was no significant difference in the anti-PRP antibody response for infants exhibiting either < 1 IU of anti-tetanus antibody per millilitre or > or = 1 IU/ml at baseline. These finding indicate that pre-existing anti-carrier antibody does not diminish the immune response to the PRP moiety. All infants possessed protective levels of anti-D and anti-T antibody levels after immunization. PMID:10812736

  14. 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 ...

  15. Camelid genomes reveal evolution and adaptation to desert environments.

    PubMed

    Wu, Huiguang; Guang, Xuanmin; Al-Fageeh, Mohamed B; Cao, Junwei; Pan, Shengkai; Zhou, Huanmin; Zhang, Li; Abutarboush, Mohammed H; Xing, Yanping; Xie, Zhiyuan; Alshanqeeti, Ali S; Zhang, Yanru; Yao, Qiulin; Al-Shomrani, Badr M; Zhang, Dong; Li, Jiang; Manee, Manee M; Yang, Zili; Yang, Linfeng; Liu, Yiyi; Zhang, Jilin; Altammami, Musaad A; Wang, Shenyuan; Yu, Lili; Zhang, Wenbin; Liu, Sanyang; Ba, La; Liu, Chunxia; Yang, Xukui; Meng, Fanhua; Wang, Shaowei; Li, Lu; Li, Erli; Li, Xueqiong; Wu, Kaifeng; Zhang, Shu; Wang, Junyi; Yin, Ye; Yang, Huanming; Al-Swailem, Abdulaziz M; Wang, Jun

    2014-10-21

    Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedarius) and alpaca (Vicugna pacos) are economically important livestock. Although the Bactrian camel and dromedary are large, typically arid-desert-adapted mammals, alpacas are adapted to plateaus. Here we present high-quality genome sequences of these three species. Our analysis reveals the demographic history of these species since the Tortonian Stage of the Miocene and uncovers a striking correlation between large fluctuations in population size and geological time boundaries. Comparative genomic analysis reveals complex features related to desert adaptations, including fat and water metabolism, stress responses to heat, aridity, intense ultraviolet radiation and choking dust. Transcriptomic analysis of Bactrian camels further reveals unique osmoregulation, osmoprotection and compensatory mechanisms for water reservation underpinned by high blood glucose levels. We hypothesize that these physiological mechanisms represent kidney evolutionary adaptations to the desert environment. This study advances our understanding of camelid evolution and the adaptation of camels to arid-desert environments.

  16. [The occurrence of "Candidatus Mycoplasma haemolamae" infections in clinically asymptomatic South American Camelids in Austria].

    PubMed

    Franz, Sonja; Spergser, Joachim; Schwendenwein, Ilse; Stanitznig, Anna; Lambacher, Bianca; Tichy, Alexander; Wittek, Thomas

    2016-01-01

    Reports of CMhl infections in South American Camelids in Europe are only available from the United Kingdom and Switzerland. Knowing that CMhl infections can lead to severe disease resulting in death if combined with other diseases or stress, it was the aim of this study to assess prevalence data from camelids in Austria. In comparison to the previous studies a representative number of camelids was investigated nationwide. Data were assessed due to differences in geographical region, age, sex, species, and origin. A relatively high prevalence of 25.8% was recorded. CMhl was detected significantly more often in alpacas (Vicunja pacos) than in llamas (Lama glama) and more frequently in animals younger than 2 years. Additionally regional differences have been observed, which might be due to climatic differences and/or variations in insect vectors. In this study apperantly clinical healthy animals were shown to be infected with CMhl. Camelids infected with CMhl are a pathogen reservoir. The results of this study indicate different risk levels of infection between llamas and alpacas and between younger and older animals. The data presented underline the necessity of further studies on CMhlI infections in South American Camelids.

  17. [The occurrence of "Candidatus Mycoplasma haemolamae" infections in clinically asymptomatic South American Camelids in Austria].

    PubMed

    Franz, Sonja; Spergser, Joachim; Schwendenwein, Ilse; Stanitznig, Anna; Lambacher, Bianca; Tichy, Alexander; Wittek, Thomas

    2016-01-01

    Reports of CMhl infections in South American Camelids in Europe are only available from the United Kingdom and Switzerland. Knowing that CMhl infections can lead to severe disease resulting in death if combined with other diseases or stress, it was the aim of this study to assess prevalence data from camelids in Austria. In comparison to the previous studies a representative number of camelids was investigated nationwide. Data were assessed due to differences in geographical region, age, sex, species, and origin. A relatively high prevalence of 25.8% was recorded. CMhl was detected significantly more often in alpacas (Vicunja pacos) than in llamas (Lama glama) and more frequently in animals younger than 2 years. Additionally regional differences have been observed, which might be due to climatic differences and/or variations in insect vectors. In this study apperantly clinical healthy animals were shown to be infected with CMhl. Camelids infected with CMhl are a pathogen reservoir. The results of this study indicate different risk levels of infection between llamas and alpacas and between younger and older animals. The data presented underline the necessity of further studies on CMhlI infections in South American Camelids. PMID:27529994

  18. Reprint of “Inhibition of biofilm formation by Camelid single-domainantibodies against the flagellum of Pseudomonas aeruginosa”.

    PubMed

    Adams, Hendrik; Horrevoets, Wannie M; Adema, Simon M; Carr, Hannah E V; van Woerden, Richard E; Koster, Margot; Tommassen, Jan

    2014-12-10

    Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in patients with compromised host defense mechanisms, including burn wound victims. In addition to its intrinsic resistance against most antibiotics, P. aeruginosa has the ability to form biofilms adhering to biotic or abiotic surfaces. These factors make treatment of P. aeruginosa infections complicated and demand new therapies and drugs. The flagellum of P. aeruginosa plays an important role in cell–cell and cell–surface interactions during the first stage of biofilm formation. In this study, we describe the selection of monoclonal anti-flagellin single-domain antibodies (VHHs) derived from the Camelid heavy-chain antibody repertoire of a llama immunized with P. aeruginosa antigens. The anti-flagellin VHHs could be produced efficiently in Saccharomyces cerevisiae, and surface plasmon resonance experiments demonstrated that they have apparent affinities in the nanomolar range. Functional screens showed that the anti-flagellin VHHs are capable of inhibiting P. aeruginosa from swimming and that they prevent biofilm formation in an in vitro assay. These data open doors for the development of novel methods for the prevention of P. aeruginosa-related infections.

  19. Rotavirus strains in neglected animal species including lambs, goats and camelids.

    PubMed

    Papp, Hajnalka; Malik, Yashpal S; Farkas, Szilvia L; Jakab, Ferenc; Martella, Vito; Bányai, Krisztián

    2014-01-01

    Surveillance of rotavirus infections and circulating strains in small ruminants (i.e. lambs, goats and camelids) has been a neglected research area in the past. However, recent years that have seen an intensification of surveillance in humans and livestock animals, where vaccines to reduce disease burden caused by Rotavirus A (RVA) are available, led to the efforts to better understand the epidemiology, ecology and evolution of RVA strains in other hosts, including lambs, goats and camelids. The aim of this review is to provide an update of the epidemiology and strain diversity of RV strains in these species through searching for relevant information in public data bases. PMID:25674588

  20. Digesta retention patterns of solute and different-sized particles in camelids compared with ruminants and other foregut fermenters.

    PubMed

    Dittmann, Marie T; Runge, Ullrich; Ortmann, Sylvia; Lang, Richard A; Moser, Dario; Galeffi, Cordula; Schwarm, Angela; Kreuzer, Michael; Clauss, Marcus

    2015-07-01

    The mean retention times (MRT) of solute or particles in the gastrointestinal tract and the forestomach (FS) are crucial determinants of digestive physiology in herbivores. Besides ruminants, camelids are the only herbivores that have evolved rumination as an obligatory physiological process consisting of repeated mastication of large food particles, which requires a particle sorting mechanism in the FS. Differences between camelids and ruminants have hardly been investigated so far. In this study we measured MRTs of solute and differently sized particles (2, 10, and 20 mm) and the ratio of large-to-small particle MRT, i.e. the selectivity factors (SF(10/2mm), SF(20/2mm), SF(20/10mm)), in three camelid species: alpacas (Vicugna pacos), llamas (Llama glama), and Bactrian camels (Camelus bactrianus). The camelid data were compared with literature data from ruminants and non-ruminant foregut fermenters (NRFF). Camelids and ruminants both had higher SF(10/2mm)FS than NRFF, suggesting convergence in the function of the FS sorting mechanism in contrast to NRFF, in which such a sorting mechanism is absent. The SF(20/10mm)FS did not differ between ruminants and camelids, indicating that there is a particle size threshold of about 1 cm in both suborders above which particle retention is not increased. Camelids did not differ from ruminants in MRT(2mm)FS, MRTsoluteFS, and the ratio MRT(2mm)FS/MRTsoluteFS, but they were more similar to 'cattle-' than to 'moose-type' ruminants. Camelids had higher SF(10/2mm)FS and higher SF(20/2mm)FS than ruminants, indicating a potentially slower particle sorting in camelids than in ruminants, with larger particles being retained longer in relation to small particles.

  1. [Physiology and pathology of reproduction in domesticated New World camelids with special emphasis on ultrasonography].

    PubMed

    Hoops, M; Kauffold, J

    2013-01-01

    The number of New World camelids in Germany is increasing. Owners and breeders are usually well educated regarding their animals. For practitioners, this means being up-to-date with respect to their veterinary knowledge. This includes the physiology and pathology of reproduction. Specifics of reproduction in domesticated New World camelids are an induced ovulation, the absence of cyclic sexual activity, a relatively long gestation of 336-349 days and a predominantly left-horn gestation. Ultrasonography plays an important role as part of the gynecological examination. Generally, the ultrasonographic examination can be performed transrectally and transcutaneously in the left or right flanks. Transrectal ultrasonography has to be carried out with particular caution to avoid rectal injuries. An accurate pregnancy diagnosis by transrectal scanning is possible starting from day 20 of pregnancy; using transcutaneous scanning, diagnosis is accurate starting on days 50-60 (left flank) or from day 90 (right flank) of pregnancy, respectively. Ultrasonography is also appropriate to examine the non-gravid uterus and the ovaries. Based on 5 years of experience working with farmed New World camelids, the article describes the physiology and pathology of reproduction in domesticated New World camelids. Particular consideration is given to the ultrasonographical examination of the genital organs.

  2. Low habitat overlap at landscape scale between wild camelids and feral donkeys in the Chilean desert

    NASA Astrophysics Data System (ADS)

    Malo, Juan E.; González, Benito A.; Mata, Cristina; Vielma, André; Donoso, Denise S.; Fuentes, Nicolás; Estades, Cristián F.

    2016-01-01

    Feral domestic ungulates may compete with the populations of wild herbivores with which they coexist, particularly so in arid regions. The potential competition between wild camelids and feral donkeys at the eastern sector of the Atacama Desert is evaluated in terms of their coincidence or segregation in habitat use and complemented with a comparison of reproductive output (yearling/adult ratio) of vicuña family groups in the proximity vs. distant from donkey observations. Habitat use of wild camelids and donkeys was sampled driving some 1250 km of roads and tracks at the dry and wet seasons. There were 221 vicuñas (Vicugna vicugna) sightings, 77 for donkeys (Equus asinus), 25 for guanacos (Lama guanicoe) and 8 for hybrids between guanacos and domestic llamas (Lama glama), as well as 174 randomly selected control locations. By means of Generalised Discriminant Analysis and Analysis of Variance we show that all ungulates actively select their habitat, with significant differences between use and availability in the area. Donkeys are relatively abundant in comparison with camelids and coincide broadly with both of them across the altitudinal gradient, but they fall between them in local scale habitat selection and do not seem to force their displacement from their preferred habitats. Thus donkeys occur preferentially on slopes with a high cover of tall shrubs, whereas vicuñas use valley bottoms with grass and guanacos the upper slope zones with grass. The potential for competition between donkeys and wild camelids is thus limited and it does not affect the reproductive output of vicuña in this region. Therefore, with the present knowledge we suggest that population control is not currently merited for feral donkeys.

  3. Pathology of Haemonchus contortus in New World camelids in the southeastern United States: a retrospective review.

    PubMed

    Edwards, Erin E; Garner, Bridget C; Williamson, Lisa H; Storey, Bob E; Sakamoto, Kaori

    2016-03-01

    Most small ruminant farms in tropical climates are plagued by Haemonchus contortus, a hematophagous, abomasal parasite. Heavy burdens of this parasite can cause anemia, hypoproteinemia, weight loss, and mortality in susceptible animals. Haemonchus contortus is becoming a major health concern in New World camelids as well, namely llamas (Llama glama) and alpacas (Vicugna pacos), yet little research has been conducted regarding its prevalence or pathology in these species. Herein, we present a retrospective review of llamas and alpacas that were admitted to The University of Georgia Veterinary Teaching Hospital and Athens Diagnostic Laboratory between the years 2002 and 2013. Antemortem fecal egg count (FEC) estimates performed on 30 alpacas were negatively correlated with hematocrit, hemoglobin, and red blood cell count. Total protein was not significantly correlated with FEC. On postmortem examination, 55 of 198 camelids, including 2 from the aforementioned antemortem review, were infected with H. contortus, with llamas (42.6%) having a significantly higher infection rate than alpacas (22.2%). In 15.7% of the total cases, the parasite was the major cause of death. Common gross lesions included peritoneal, thoracic, and pericardial effusions, visceral pallor, subcutaneous edema, and serous atrophy of fat. Histologic lesions included centrilobular hepatic necrosis, hepatic atrophy, lymphoplasmacytic inflammation of the mucosa of the third gastric compartment (C3), extramedullary hematopoiesis in both the liver and spleen, and the presence of nematodes in C3. Our study emphasizes the importance of H. contortus diagnosis and herd monitoring in New World camelids, particularly llamas.

  4. Antithyroglobulin antibody

    MedlinePlus

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

  5. Natural and man-made V-gene repertoires for antibody discovery

    PubMed Central

    Finlay, William J. J.; Almagro, Juan C.

    2012-01-01

    Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process. PMID:23162556

  6. Bluetongue disease and seroprevalence in South American camelids from the northwestern region of the United States.

    PubMed

    Allen, Andrew J; Stanton, James B; Evermann, James F; Fry, Lindsay M; Ackerman, Melissa G; Barrington, George M

    2015-03-01

    In late summer/early fall of 2013, 2 South American camelids from central Washington were diagnosed with fatal bluetongue viral disease, an event which is rarely reported. A 9-year-old intact male llama (Lama glama), with a 1-day history of anorexia, recumbency, and dyspnea before death. Abundant foam discharged from the mouth and nostrils, and the lungs were severely edematous on postmortem examination. Histologically, there was abundant intra-alveolar edema with fibrin. Hemorrhage and edema disrupted several other organs. Bluetongue viral RNA was detected by reverse transcription polymerase chain reaction (RT-PCR), and serotype 11 was identified by sequencing a segment of the VP2 outer capsid gene. Approximately 1 month later, at a site 150 miles north of the index case, a 2-year-old female alpaca with similar, acutely progressive clinical signs was reported. A postmortem examination was performed, and histologic lesions from the alpaca were similar to those of the llama, and again serotype 11 was detected by PCR. The occurrence of bluetongue viral infection and disease is described in the context of seasonal Bluetongue virus activity within the northwestern United States and southwestern Canada.

  7. Review of laboratory submissions from New World camelids in England and Wales (2000-2011).

    PubMed

    Twomey, D F; Wu, G; Nicholson, R; Watson, E N; Foster, A P

    2014-04-01

    Sample submissions to the Animal Health and Veterinary Laboratories Agency's (AHVLA's) diagnostic laboratory network in England and Wales were reviewed for diseases affecting New World camelids (NWCs). In the years 2000-2011, 6757 submissions were analysed, including 5154/6757 (76.3%) for diagnosing a disease problem and 1603/6757 (23.7%) for monitoring (no clinical disease). Wasting (weight loss, ill-thrift) was the most commonly reported clinical sign across all age groups. A diagnosis was reached for 1765/5154 (34.2%) diagnostic submissions. The proportion of submissions with diagnoses was higher for carcasses than non-carcass samples and multiple diagnoses were more likely to be reached from carcasses. Parasitic diseases were collectively the most common problem, including parasitic gastroenteritis (319/1765, 18.2%), coccidiosis (187/1765, 10.6%), fascioliasis (151/1765, 8.6%), ectoparasitic infestations (86/1765, 4.9%) and cryptosporidiosis (24/1765, 1.4%). The most frequently diagnosed non-parasitic problems included nutritional diseases (182/1765, 10.3%), septicaemia (104/1765, 5.9%, including 45 cases of colisepticaemia), gastric ulceration (79/1765, 4.5%), tumours/neoplastic diseases (65/1765, 3.7%), tuberculosis (57/1765, 3.2%), clostridial diseases (44/1765, 2.5%), congenital anomalies (41/1765, 2.3%), peritonitis (39/1765, 2.2%) and Johne's disease (20/1765, 1.1%).

  8. The history of Old World camelids in the light of molecular genetics.

    PubMed

    Burger, Pamela Anna

    2016-06-01

    Old World camels have come into the focus as sustainable livestock species, unique in their morphological and physiological characteristics and capable of providing vital products even under extreme environmental conditions. The evolutionary history of dromedary and Bactrian camels traces back to the middle Eocene (around 40 million years ago, mya), when the ancestors of Camelus emerged on the North American continent. While the genetic status of the two domestic species has long been established, the wild two-humped camel has only recently been recognized as a separate species, Camelus ferus, based on molecular genetic data. The demographic history established from genome drafts of Old World camels shows the independent development of the three species over the last 100,000 years with severe bottlenecks occurring during the last glacial period and in the recent past. Ongoing studies involve the immune system, relevant production traits, and the global population structure and domestication of Old World camels. Based on the now available whole genome drafts, specific metabolic pathways have been described shedding new light on the camels' ability to adapt to desert environments. These new data will also be at the origin for genome-wide association studies to link economically relevant phenotypes to genotypes and to conserve the diverse genetic resources in Old World camelids.

  9. Hybridizing Old and New World camelids: Camelus dromedarius x Lama guanicoe.

    PubMed

    Skidmore, J A; Billah, M; Binns, M; Short, R V; Allen, W R

    1999-04-01

    Thirty female dromedary camels were inseminated on a total of 50 occasions with 2-4 ml of fresh guanaco semen diluted with an equal volume of commercially available camel semen extender. Similarly, nine female guanacos were inseminated on 34 occasions with 4-6 ml of fresh, diluted camel semen. Only two of the dromedary females conceived; one aborted a female foetus on day 260 of gestation and the other gave birth to a stillborn female calf on day 365. Six conceptions occurred in the female guanacos. Two of these conceptuses, diagnosed by ultrasound, were resorbed between days 25 and 40 of gestation, one female foetus was aborted on day 291, another female foetus was aborted on day 302, and one female calf was stillborn on day 365 of gestation. The sixth foetus, a male, was born prematurely but alive after a 328-day gestation. It had a phenotypic appearance intermediate between that of a camel and a guanaco and its hybrid parentage was confirmed by the DNA fingerprinting of eight llama microsatellites. To our knowledge, this is the first viable hybrid ever to be produced between Old World and New World camelids, which have been reproductively isolated from one another for at least 11 million years. The preponderance of female hybrids is in accordance with Haldane's law. Histological examination of their ovaries revealed a failure of meiosis, with only an occasional abnormal oocyte surrounded by follicle cells. Although the diploid chromosone number of camels and guanacos is the same (2n = 74), sufficient genetic change has taken place to make the pairing of homologous chromosomes no longer possible. PMID:10331286

  10. [Dry matter intake of South American camelids and its effects on the composition of feed rations].

    PubMed

    Stölzl, Anna Maria; Lambertz, Christian; Moors, Eva; Stiehl, Jennifer; Gauly, Matthias

    2014-01-01

    The number of South American camelids (SAC) is increasing in Germany since decades. Due to a lack of scientifically based publications the knowledge about feeding SACs is still poor. Therefore, the aim of this study was to estimate the dry matter intake (DMI) of SACs as a basis for calculations of feed rations. Previous studies proposed a DMI of up to 3% of the body weight (BW) (Vaughan und Gauly, 2011). In the present study, eight llamas (Llama glama) were allocated to two groups of four animals each. The two groups were fed with hay of different qualities over a total period often weeks, which was divided into two runs of five weeks each. During the first run, group 1 was fed with hay 1 (15.1% crude protein; 8.5% crude ash; 3.1% crude fat; 52.6% NDF per kg DM) and group 2 with hay 2 (6.6% crude protein; 6.2% crude ash; 2.1% crude fat; 64.3% NDF per kg DM). After five weeks the groups were changed and group 1 received hay 2 and group 2 received hay 1. BW was measured at the start and end of each run (week zero, five and ten). The hay quality affected the DMI, but the animals did not compensate a lower feed quality with an increased DMI. The total DMI was 1.26% and 0.89% of the BW for hay 1 and hay 2, respectively, which was lower than expected in both groups. In conclusion, calculations of feed rations for SACs should be adjusted to the present findings of a lower DMI capability.

  11. Schmallenberg virus infection in South American camelids: Field and experimental investigations.

    PubMed

    Schulz, Claudia; Beer, Martin; Hoffmann, Bernd

    2015-11-18

    During the first epizootic wave of the novel, teratogenic Schmallenberg virus (SBV, Orthobunyavirus) in ruminants in Northern Europe, serological evidence of a previous SBV-infection demonstrated that South American camelids (SAC) are also susceptible to SBV. However, their potential role in SBV spread remains unknown. To investigate the prevalence and course of SBV-infection in SAC, a German field study and an animal trial with three llamas and three alpacas were conducted. From September 2012 to December 2013, 313 of 502 SAC (62.35%) were found SBV seropositive, but negative for SBV-RNA. The estimated between-district (94.23% of 52) and median within-district (71.43%) and herd (73.13%) SBV seroprevalence in German SAC was similar to the seroprevalence reported in cattle herds and sheep flocks at the time. An age of >1 year was found a statistically significant risk factor for SBV-infection, which could be explained by the spatio-temporal spread of SBV in Germany during the study period. No clinical signs or an increase of abortion and congenital malformation associated with SBV-infection in SAC were reported by the study participants. Similar to SBV-infected ruminants, SBV-RNAemia in experimentally SBV-infected SAC was detected for a short time between days 3 and 7 after infection (dpi), and seroconversion occurred between 9 and 21 dpi. Despite the similar virological and serological results, the lack of clinical signs and congenital malformation associated with SBV-infection suggests that SBV causes subclinical infection in SAC. However, their role as reservoirs in the spread of SBV has to be further investigated.

  12. Molecular characterization and antibiotic resistance of Enterococcus species from gut microbiota of Chilean Altiplano camelids

    PubMed Central

    Guerrero-Olmos, Katheryne; Báez, John; Valenzuela, Nicomédes; Gahona, Joselyne; del Campo, Rosa; Silva, Juan

    2014-01-01

    Background Enterococcus is one of the major human pathogens able to acquire multiple antibiotic-resistant markers as well as virulence factors which also colonize remote ecosystems, including wild animals. In this work, we characterized the Enterococcus population colonizing the gut of Chilean Altiplano camelids without foreign human contact. Material and methods Rectal swabs from 40 llamas and 10 alpacas were seeded in M-Enterococcus agar, and we selected a total of 57 isolates. Species identification was performed by biochemical classical tests, semi-automated WIDER system, mass spectrometry analysis by MALDI-TOF (matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer), and, finally, nucleotide sequence of internal fragments of the 16S rRNA, rpoB, pheS, and aac(6)-I genes. Genetic diversity was measured by pulsed field gel electrophoresis (PFGE)-SmaI, whereas the antibiotic susceptibility was determined by the WIDER system. Carriage of virulence factors was explored by polymerase chain reaction (PCR). Results Our results demonstrated that the most prevalent specie was Enterococcus hirae (82%), followed by other non–Enterococcus faecalis and non–Enterococcus faecium species. Some discrepancies were detected among the identification methods used, and the most reliable were the rpoB, pheS, and aac(6)-I nucleotide sequencing. Selected isolates exhibited susceptibility to almost all studied antibiotics, and virulence factors were not detected by PCR. Finally, some predominant clones were characterized by PFGE into a diverse genetic background. Conclusion Enterococcus species from the Chilean camelids’ gut microbiota were different from those adapted to humans, and they remained free of antibiotic resistance mechanisms as well as virulence factors. PMID:25405007

  13. Pulmonary Arterial Lesions in New World Camelids in Association With Dicrocoelium dendriticum and Fasciola hepatica Infection.

    PubMed

    Hilbe, M; Robert, N; Pospischil, A; Gerspach, C

    2015-11-01

    In Switzerland, dicrocoeliasis is regarded as the most significant parasitic infection of llamas and alpacas. Fasciola hepatica infestation is also a problem but less common. The aim of the present retrospective study was to evaluate the lungs of New World camelids (NWCs) for evidence of arterial hypertension in association with liver changes due to liver fluke infestation. The lungs of 20 llamas and 20 alpacas with liver fluke infestation were histologically evaluated. The hematoxylin and eosin and van Gieson (VG)-elastica stains as well as immunohistology for the expression of α-smooth muscle actin (α-SMA) were used to visualize the structures of arterial walls. Parasitology of fecal matter (11 llamas and 17 alpacas) confirmed that most of these animals were infested with both Dicrocoelium dendriticum and other gastrointestinal parasites. In most cases (10/12 llamas, 4/6 alpacas), liver enzyme activity in serum was elevated. Histologically, arteries in the lungs of 9 of 20 llamas (45%) and 3 of 20 alpacas (15%) showed severe intimal and adventitial and slight to moderate medial thickening, which was confirmed with α-SMA and VG-elastica staining. All animals exhibited typical liver changes, such as fibrosis and biliary hyperplasia, in association with the presence of liver flukes. This study shows that liver flukes can induce proliferative changes in lung arteries in NWCs that resemble those seen with pulmonary arterial hypertension due to liver parasites in humans. However, the degree of liver fluke infestation was not correlated with the extent of liver damage, or with the amount of thoracic or abdominal effusion or pulmonary arterial changes.

  14. Efficacy of anthelmintics on South American camelid (llama and alpaca) farms in Georgia.

    PubMed

    Gillespie, Rose-Ann M; Williamson, Lisa H; Terrill, Thomas H; Kaplan, Ray M

    2010-08-27

    The number of South American camelid (SAC; llama and alpaca) farms is growing in the southeastern United States, and infection with gastrointestinal nematodes (GIN) is a major health concern in this region. There is widespread resistance to anthelmintic remedies in small ruminants (sheep and goats), but a paucity of information on llamas and alpacas. Anthelmintic resistance was evaluated on three SAC farms (two llama; one alpaca) in Georgia in the southern United States using fecal egg count reduction (FECR) tests. For each farm, animals were randomly assigned to 1 of 5 treatment groups based on initial fecal egg count (FEC) and number of animals available (2-5 groups, n=9-11 per treatment). Ivermectin (IVM, subcutaneous injection; 0.3mg/kg body weight (BW)) and a control group were tested on an alpaca farm, and fenbendazole (FBZ, oral; 10mg/kg BW; two farms), moxidectin (MOX oral; 0.2mg/kg BW; two farms), and levamisole (LEV, oral; 8 mg/kg BW; one farm) were added for the llama farms. Anthelmintic efficacy was determined by comparing FEC of treatment and control animals 14 days post-treatment, with resistance evaluated using the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Based upon these guidelines, there was GIN resistance to IVM in both llamas and alpacas in Georgia and to FBZ on both llama farms where this drug was tested. There was MOX resistance on one llama farm using the FECR test, while there was no resistance to LEV detected in this study. These data demonstrate a serious emerging problem in the United States of llama and alpaca GIN resistant to drugs from two of the three major anthelmintic classes.

  15. The Structure of Natural and Recombinant Antibodies.

    PubMed

    Ma, Hui; O'Kennedy, Richard

    2015-01-01

    Immunoglobulins (Ig) isotypes A, D, E, G, and M are glycoproteins which are mainly composed of a "Y"-shaped Ig monomer (~150 kDa), consisting of two light and two heavy chains. Both light and heavy chains contain variable (N-terminal) and constant regions (C-terminal). Each light chain consists of one variable domain and one constant domain, whereas each heavy chain has one variable domain and three constant domains. However, heavy-chain antibodies consisting of only heavy chains and lacking the light chains are found in camelids and cartilaginous fishes. Unlike other immunoglobulins, the heavy chain of avian antibody IgY (~180 kDa) consists of four constant domains. The single-chain variable fragment (scFv; ~25 kDa) of an antibody contains variable regions of antibody heavy and light chains. The fragment antigen-binding (Fab; ~50 kDa) region has the full antibody light chain but the heavy chain is composed of a variable region and one constant domain.

  16. Palynological analysis of camelid coprolites: seasonality in the use of the site Cerro Casa de Piedra 7 (Santa Cruz, Argentina)

    NASA Astrophysics Data System (ADS)

    Velázquez, Nadia Jimena; Burry, Lidia Susana; Fugassa, Martín Horacio; Civalero, María Teresa; Aschero, Carlos Alberto

    2014-01-01

    Palynological, palaeoparasitological and paleobotanical studies of coprolites found in archaeological sites from Perito Moreno National Park (47°57‧S72°05‧W) yielded information on diet, palaeoenvironment and health. These studies allowed adding evidence to the reconstruction of life history of the hunter-gatherers that inhabited Patagonia during the Holocene. We examined the season of the year when camelid Lama guanicoe coprolites (5400 ± 64 yr 14C BP to 9640 ± 190 yr 14C BP) were deposited at Cerro Casa de Piedra 7 (site CCP7). The study used palynological evidence and comparison with pollen spectra of modern feces collected during summer, fall, winter and spring of 2010. The dominant types were: pollen of Nothofagus, Empetrum rubrum, Asteraceae subfam. Asteroideae, Nassauvia, Caryophyllaceae and Poaceae; fern spores; remains of Eimeria macusaniensis; and plant remains of Poaceae, Festuca pallescens, Stipa speciosa, Armeria maritima, Gaultheria mucronata and E. rubrum. Pollen spectra of modern and fossil feces were used for multivariate analysis. Coprolites associated to fall and winter modern feces. These results and those obtained from pollen concentration values and the presence of pollen types indicators of seasonality, allowed the determination of summer, fall and winter coprolites. However, caution must be taken with the seasonality results of coprolites dated earlier than 9000 years BP since the environmental conditions differed from now. The site was probably a camelid shelter during the unfavorable seasons.

  17. Morphofunctional structure of the lingual papillae in three species of South American Camelids: Alpaca, guanaco, and llama.

    PubMed

    Erdoğan, Serkan; Villar Arias, Silvia; Pérez, William

    2016-02-01

    The aim of this study was to compare the anatomical and functional characteristics of the lingual papilla among the Camelidae. For this purpose, tongues of alpaca, guanaco, and llama were used. Numerous long and thin filiform papillae were located in the median groove and none were detected on the rest of the dorsal surface of the lingual apex in alpaca. Secondary papillae originated from the base of some filiform papillae on the ventral surface of alpaca tongue. The bases of some filiform papillae of the lateral surface of the lingual apex were inserted into conspicuous grooves in guanaco and tips of filiform papillae on the dorsal surface of the lingual body were ended by bifurcated apex. On the dorsal surface of the lingual apex of llama, there were no filiform papillae but there were numerous filiform papillae on both the lateral margins of the ventral surface of the lingual apex. Fungiform papillae were distributed randomly on dorsal lingual surface and ventral margins of the tongues of all camelid species. Lenticular papillae were located on the lingual torus and varied in size and topographical distribution for each species. Circumvallate papillae had irregular surfaces in llama and alpaca, and smooth surface in guanaco. In conclusion, llama and alpaca tongues were more similar to each other, and tongues of all camelid species displayed more similarities to those of Bactrian and dromedary camels in comparison with other herbivores and ruminants.

  18. Morphofunctional structure of the lingual papillae in three species of South American Camelids: Alpaca, guanaco, and llama.

    PubMed

    Erdoğan, Serkan; Villar Arias, Silvia; Pérez, William

    2016-02-01

    The aim of this study was to compare the anatomical and functional characteristics of the lingual papilla among the Camelidae. For this purpose, tongues of alpaca, guanaco, and llama were used. Numerous long and thin filiform papillae were located in the median groove and none were detected on the rest of the dorsal surface of the lingual apex in alpaca. Secondary papillae originated from the base of some filiform papillae on the ventral surface of alpaca tongue. The bases of some filiform papillae of the lateral surface of the lingual apex were inserted into conspicuous grooves in guanaco and tips of filiform papillae on the dorsal surface of the lingual body were ended by bifurcated apex. On the dorsal surface of the lingual apex of llama, there were no filiform papillae but there were numerous filiform papillae on both the lateral margins of the ventral surface of the lingual apex. Fungiform papillae were distributed randomly on dorsal lingual surface and ventral margins of the tongues of all camelid species. Lenticular papillae were located on the lingual torus and varied in size and topographical distribution for each species. Circumvallate papillae had irregular surfaces in llama and alpaca, and smooth surface in guanaco. In conclusion, llama and alpaca tongues were more similar to each other, and tongues of all camelid species displayed more similarities to those of Bactrian and dromedary camels in comparison with other herbivores and ruminants. PMID:26572928

  19. Optimizing selection of large animals for antibody production by screening immune response to standard vaccines.

    PubMed

    Thompson, Mary K; Fridy, Peter C; Keegan, Sarah; Chait, Brian T; Fenyö, David; Rout, Michael P

    2016-03-01

    Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals.

  20. Optimizing selection of large animals for antibody production by screening immune response to standard vaccines.

    PubMed

    Thompson, Mary K; Fridy, Peter C; Keegan, Sarah; Chait, Brian T; Fenyö, David; Rout, Michael P

    2016-03-01

    Antibodies made in large animals are integral to many biomedical research endeavors. Domesticated herd animals like goats, sheep, donkeys, horses and camelids all offer distinct advantages in antibody production. However, their cost of use is often prohibitive, especially where poor antigen response is commonplace; choosing a non-responsive animal can set a research program back or even prevent experiments from moving forward entirely. Over the course of production of antibodies from llamas, we found that some animals consistently produced a higher humoral antibody response than others, even to highly divergent antigens, as well as to their standard vaccines. Based on our initial data, we propose that these "high level responders" could be pre-selected by checking antibody titers against common vaccines given to domestic farm animals. Thus, time and money can be saved by reducing the chances of getting poor responding animals and minimizing the use of superfluous animals. PMID:26775851

  1. Pastoralism in northern Peru during pre-Hispanic times: insights from the Mochica Period (100-800 AD) based on stable isotopic analysis of domestic camelids.

    PubMed

    Dufour, Elise; Goepfert, Nicolas; Gutiérrez Léon, Belkys; Chauchat, Claude; Jordán, Régulo Franco; Vásquez Sánchez, Segundo

    2014-01-01

    Llama (Lama glama) and alpaca (Vicugna pacos) are the only large domesticated animals indigenous to the Americas. Pastoralism occupies a fundamental economic, social and religious role in Andean life. Today, camelid livestock are confined to the ecozone of the puna (above 3,500 masl), while their presence on the Pacific coast during pre-Hispanic times is attested by archaeological skeletal remains. This study aims to document herding practices on the northern Peruvian coast during the Early Intermediate Period (200 BC-600 AD) by gaining insights into diet, location of breeding and mobility of archaeological camelids from the funerary and ritual contexts of two Mochica sites, Uhle Platform in Huacas de Moche and El Brujo. The three first early years and the long-term life histories of the animals were documented by the combined bulk analysis of bone collagen (δ(13)C col and δ(15)N col) and bone structural carbonate (δ(13)C bone and δ(18)O bone) and the serial analysis of structural carbonate of molar tooth enamel (δ(13)C enamel and δ(18)O enamel). Mochica camelids were bred in the low and/or middle valleys, unlike their modern counterparts, who are restricted to highland puna C3 pastures. Archaeological camelids had diverse and complex life histories, usually with substantial maize foddering. An ontogenetic switch in diet and possible residential mobility during the course of life were identified for some specimens. Although the inference of geographic origin from δ(18)O bone and δ(18)O enamel values was limited because of the lack of understanding of the influence of environmental and biological factors, tooth enamel analysis has great potential for exploring camelid herding practices and Andean pastoralism. Our study suggested that Mochica herders adapted their practices to the difficult lowland environment and that herding practices were varied and not restricted to breeding at higher altitudes. The role of maize in different aspects of the economic life

  2. Pastoralism in Northern Peru during Pre-Hispanic Times: Insights from the Mochica Period (100–800 AD) Based on Stable Isotopic Analysis of Domestic Camelids

    PubMed Central

    Dufour, Elise; Goepfert, Nicolas; Gutiérrez Léon, Belkys; Chauchat, Claude; Franco Jordán, Régulo; Sánchez, Segundo Vásquez

    2014-01-01

    Llama (Lama glama) and alpaca (Vicugna pacos) are the only large domesticated animals indigenous to the Americas. Pastoralism occupies a fundamental economic, social and religious role in Andean life. Today, camelid livestock are confined to the ecozone of the puna (above 3,500 masl), while their presence on the Pacific coast during pre-Hispanic times is attested by archaeological skeletal remains. This study aims to document herding practices on the northern Peruvian coast during the Early Intermediate Period (200 BC-600 AD) by gaining insights into diet, location of breeding and mobility of archaeological camelids from the funerary and ritual contexts of two Mochica sites, Uhle Platform in Huacas de Moche and El Brujo. The three first early years and the long-term life histories of the animals were documented by the combined bulk analysis of bone collagen (δ13Ccol and δ15Ncol) and bone structural carbonate (δ13Cbone and δ18Obone) and the serial analysis of structural carbonate of molar tooth enamel (δ13Cenamel and δ18Oenamel). Mochica camelids were bred in the low and/or middle valleys, unlike their modern counterparts, who are restricted to highland puna C3 pastures. Archaeological camelids had diverse and complex life histories, usually with substantial maize foddering. An ontogenetic switch in diet and possible residential mobility during the course of life were identified for some specimens. Although the inference of geographic origin from δ18Obone and δ18Oenamel values was limited because of the lack of understanding of the influence of environmental and biological factors, tooth enamel analysis has great potential for exploring camelid herding practices and Andean pastoralism. Our study suggested that Mochica herders adapted their practices to the difficult lowland environment and that herding practices were varied and not restricted to breeding at higher altitudes. The role of maize in different aspects of the economic life of the Mochicas is also

  3. Persistent Bovine Viral Diarrhea Virus Infection in Domestic and Wild Small Ruminants and Camelids Including the Mountain Goat (Oreamnos americanus)

    PubMed Central

    Nelson, Danielle D.; Duprau, Jennifer L.; Wolff, Peregrine L.; Evermann, James F.

    2016-01-01

    Bovine viral diarrhea virus (BVDV) is a pestivirus best known for causing a variety of disease syndromes in cattle, including gastrointestinal disease, reproductive insufficiency, immunosuppression, mucosal disease, and hemorrhagic syndrome. The virus can be spread by transiently infected individuals and by persistently infected animals that may be asymptomatic while shedding large amounts of virus throughout their lifetime. BVDV has been reported in over 40 domestic and free-ranging species, and persistent infection has been described in eight of those species: white-tailed deer, mule deer, eland, mousedeer, mountain goats, alpacas, sheep, and domestic swine. This paper reviews the various aspects of BVDV transmission, disease syndromes, diagnosis, control, and prevention, as well as examines BVDV infection in domestic and wild small ruminants and camelids including mountain goats (Oreamnos americanus). PMID:26779126

  4. Persistent Bovine Viral Diarrhea Virus Infection in Domestic and Wild Small Ruminants and Camelids Including the Mountain Goat (Oreamnos americanus).

    PubMed

    Nelson, Danielle D; Duprau, Jennifer L; Wolff, Peregrine L; Evermann, James F

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is a pestivirus best known for causing a variety of disease syndromes in cattle, including gastrointestinal disease, reproductive insufficiency, immunosuppression, mucosal disease, and hemorrhagic syndrome. The virus can be spread by transiently infected individuals and by persistently infected animals that may be asymptomatic while shedding large amounts of virus throughout their lifetime. BVDV has been reported in over 40 domestic and free-ranging species, and persistent infection has been described in eight of those species: white-tailed deer, mule deer, eland, mousedeer, mountain goats, alpacas, sheep, and domestic swine. This paper reviews the various aspects of BVDV transmission, disease syndromes, diagnosis, control, and prevention, as well as examines BVDV infection in domestic and wild small ruminants and camelids including mountain goats (Oreamnos americanus).

  5. Persistent Bovine Viral Diarrhea Virus Infection in Domestic and Wild Small Ruminants and Camelids Including the Mountain Goat (Oreamnos americanus).

    PubMed

    Nelson, Danielle D; Duprau, Jennifer L; Wolff, Peregrine L; Evermann, James F

    2015-01-01

    Bovine viral diarrhea virus (BVDV) is a pestivirus best known for causing a variety of disease syndromes in cattle, including gastrointestinal disease, reproductive insufficiency, immunosuppression, mucosal disease, and hemorrhagic syndrome. The virus can be spread by transiently infected individuals and by persistently infected animals that may be asymptomatic while shedding large amounts of virus throughout their lifetime. BVDV has been reported in over 40 domestic and free-ranging species, and persistent infection has been described in eight of those species: white-tailed deer, mule deer, eland, mousedeer, mountain goats, alpacas, sheep, and domestic swine. This paper reviews the various aspects of BVDV transmission, disease syndromes, diagnosis, control, and prevention, as well as examines BVDV infection in domestic and wild small ruminants and camelids including mountain goats (Oreamnos americanus). PMID:26779126

  6. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines.

    PubMed

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela; Van Der Wielen, Marie

    2016-07-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  7. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines

    PubMed Central

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela

    2016-01-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  8. Antibody Persistence in Young Children 5 Years after Vaccination with a Combined Haemophilus influenzae Type b-Neisseria meningitidis Serogroup C Conjugate Vaccine Coadministered with Diphtheria-Tetanus-Acellular Pertussis-Based and Pneumococcal Conjugate Vaccines.

    PubMed

    Tejedor, Juan Carlos; Brzostek, Jerzy; Konior, Ryszard; Grunert, Detlef; Kolhe, Devayani; Baine, Yaela; Van Der Wielen, Marie

    2016-07-01

    We evaluated antibody persistence in children up to 5 years after administration of a combined Haemophilus influenzae type b (Hib)-Neisseria meningitidis serogroup C (MenC)-tetanus toxoid (TT) conjugate vaccine coadministered with a pneumococcal conjugate vaccine. This is the follow-up study of a randomized trial (ClinicalTrials.gov registration no. NCT00334334/00463437) in which healthy children were vaccinated (primary vaccinations at 2, 4, and 6 months of age and booster vaccination at 11 to 18 months of age) with Hib-MenC-TT or a control MenC conjugate vaccine, coadministered with diphtheria-tetanus-acellular pertussis (DTPa)-based combination vaccines (DTPa/Hib for control groups) and a pneumococcal conjugate vaccine (10-valent pneumococcal nontypeable H. influenzae protein D conjugate vaccine [PHiD-CV] or 7-valent cross-reacting material 197 [CRM197] conjugate vaccine [7vCRM]). MenC antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (i.e., rabbit SBA [rSBA]), and antibodies against Hib polyribosylribitol phosphate (PRP) were measured with an enzyme-linked immunosorbent assay. Antibody persistence up to 5 years after booster vaccination is reported for 530 children ∼6 years of age. The percentages of children with seroprotective rSBA-MenC titers were between 24.2% and 40.1% in all groups approximately 5 years after booster vaccination. More than 98.5% of children in each group retained seroprotective anti-PRP concentrations. No vaccine-related serious adverse events and no events related to a lack of vaccine efficacy were reported. Approximately 5 years after booster vaccination, the majority of children retained seroprotective anti-PRP antibody concentrations. The percentage of children retaining seroprotective rSBA-MenC titers was low (≤40%), suggesting that a significant proportion of children may be unprotected against MenC disease. (This study has been registered at ClinicalTrials.gov under

  9. Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

    PubMed Central

    Even-Desrumeaux, Klervi; Baty, Daniel; Chames, Patrick

    2010-01-01

    Antibodies microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnosis and proteome analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecule for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs produced in crude bacterial lysates to generate proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of “multi-tagged” sdAbs via anti-tag antibodies and direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers. PMID:20859568

  10. Single Domain Antibodies as a Powerful Tool for High Quality Surface Plasmon Resonance Studies

    PubMed Central

    Della Pia, Eduardo Antonio; Martinez, Karen L.

    2015-01-01

    Single domain antibodies are recombinantly expressed functional antibodies devoid of light chains. These binding elements are derived from heavy chain antibodies found in camelids and offer several distinctive properties for applications in biotechnology such as small size, stability, solubility, and expression in high yields. In this study we demonstrated the potential of using single domain antibodies as capturing molecules in biosensing applications. Single domain antibodies raised against green fluorescent protein were anchored onto biosensor surfaces by using several immobilization strategies based on Ni2+:nitrilotriacetic acid-polyhistidine tag, antibody-antigen, biotin-streptavidin interactions and amine-coupling chemistry. The interaction with the specific target of the single domain antibodies was characterized by surface plasmon resonance. The immobilized single domain antibodies show high affinities for their antigens with KD = 3–6 nM and outperform other antibody partners as capturing molecules facilitating also the data analysis. Furthermore they offer high resistance and stability to a wide range of denaturing agents. These unique biophysical properties and the production of novel single domain antibodies against affinity tags make them particularly attractive for use in biosensing and diagnostic assays. PMID:25822527

  11. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein

    PubMed Central

    Anderson, George P.; Teichler, Daniel D.; Zabetakis, Dan; Shriver-Lake, Lisa C.; Liu, Jinny L.; Lonsdale, Stephen G.; Goodchild, Sarah A.; Goldman, Ellen R.

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  12. Importance of Hypervariable Region 2 for Stability and Affinity of a Shark Single-Domain Antibody Specific for Ebola Virus Nucleoprotein.

    PubMed

    Anderson, George P; Teichler, Daniel D; Zabetakis, Dan; Shriver-Lake, Lisa C; Liu, Jinny L; Lonsdale, Stephen G; Goodchild, Sarah A; Goldman, Ellen R

    2016-01-01

    Single-domain antibodies derived from the unique New Antigen Receptor found in sharks have numerous potential applications, ranging from diagnostic reagents to therapeutics. Shark-derived single-domain antibodies possess the same characteristic ability to refold after heat denaturation found in single-domain antibodies derived from camelid heavy-chain-only antibodies. Recently, two shark derived single-domain antibodies specific for the nucleoprotein of Ebola virus were described. Our evaluation confirmed their high affinity for the nucleoprotein, but found their melting temperatures to be low relative to most single-domain antibodies. Our first approach towards improving their stability was grafting antigen-binding regions (complementarity determining regions) of one of these single-domain antibodies onto a high melting temperature shark single-domain antibody. This resulted in two variants: one that displayed excellent affinity with a low melting temperature, while the other had poor affinity but a higher melting temperature. These new proteins, however, differed in only 3 amino acids within the complementarity determining region 2 sequence. In shark single-domain antibodies, the complementarity determining region 2 is often referred to as hypervariable region 2, as this segment of the antibody domain is truncated compared to the sequence in camelid single-domain antibodies and conventional heavy chain variable domains. To elucidate which of the three amino acids or combinations thereof were responsible for the affinity and stability we made the 6 double and single point mutants that covered the intermediates between these two clones. We found a single amino acid change that achieved a 10°C higher melting temperature while maintaining sub nM affinity. This research gives insights into the impact of the shark sdAb hypervariable 2 region on both stability and affinity. PMID:27494523

  13. Phenotypic lentivirus screens to identify functional single domain antibodies.

    PubMed

    Schmidt, Florian I; Hanke, Leo; Morin, Benjamin; Brewer, Rebeccah; Brusic, Vesna; Whelan, Sean P J; Ploegh, Hidde L

    2016-01-01

    Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway. PMID:27573105

  14. Successful use of camelid (alpaca) antivenom to treat a potentially lethal tiger snake (Notechis scutatus) envenomation in a dog.

    PubMed

    Padula, Andrew M; Winkel, Kenneth D

    2016-05-01

    This report describes a confirmed clinical case of tiger snake (Notechis scutatus) envenomation in a domestic dog that was successfully treated with a novel polyvalent camelid (alpaca; Llama pacos) antivenom. Samples collected from the dog were assayed for tiger snake venom (TSV) using a highly sensitive and specific ELISA. The TSV concentration in serum and urine at initial presentation was 365 ng/mL and 11,640 ng/mL respectively. At the time of initial presentation whole blood collected from the dog did not clot and the Prothrombin Time was abnormally increased (>300 s). Serum was also visibly hemolysed. The dog was administered antihistamine, dexamethasone and 4000 Units (sufficient to neutralise 40 mg of TSV) of a novel polyvalent alpaca antivenom diluted in 0.9% NaCl. At 4 h post-antivenom treatment the dog's clinical condition had improved markedly with serum TSV concentrations below the limit of detection (<0.015 ng/mL), consistent with complete binding of venom antigens by the alpaca antivenom. Coagulation parameters had begun to improve by 4 h and had fully normalised by 16 h post-antivenom. Venom concentrations in both serum and urine remained undetectable at 16 h post-antivenom. The dog made a complete recovery, without complications, suggesting that the alpaca-based antivenom is both clinically safe and effective.

  15. Expression of orexin A and its receptor 1 in the epididymis of the South American camelid alpaca (Vicugna pacos).

    PubMed

    Liguori, G; Paino, S; Mirabella, N; Squillacioti, C; De Luca, A; Vittoria, A

    2014-02-01

    Orexins A (ox A) and B are two peptides originally discovered in neurons of rat hypothalamus, and later found in different cellular types of the gastrointestinal and genital tracts. They arise from the proteolytic cleavage of a common precursor molecule, prepro-orexin, and bind to two receptors, namely receptor 1 (ox1r) and receptor 2 for orexins, that show different binding affinity. The central role of the two peptides has been extensively studied, whereas their activity in the periphery is still poorly known. Here, we investigated the presence of ox A and ox1r in the epididymis of a South American camelid species, the alpaca, by immunohistochemistry, and we also assessed the expression of prepro-orexin and ox1r in tissue extracts by Western blotting analysis. Ox A- and ox1r-immunoreactivity was found in the cytoplasm of principal cells of the caput epididymis. A prevalent supranuclear localization of granular-shaped positive material was observed. No positivity was present in the other cytotypes of epididymis. The expression of two peptides with molecular weight corresponding to those of prepro-orexin and ox1r, respectively, was detected in the tissue extracts from the organ.

  16. Successful use of camelid (alpaca) antivenom to treat a potentially lethal tiger snake (Notechis scutatus) envenomation in a dog.

    PubMed

    Padula, Andrew M; Winkel, Kenneth D

    2016-05-01

    This report describes a confirmed clinical case of tiger snake (Notechis scutatus) envenomation in a domestic dog that was successfully treated with a novel polyvalent camelid (alpaca; Llama pacos) antivenom. Samples collected from the dog were assayed for tiger snake venom (TSV) using a highly sensitive and specific ELISA. The TSV concentration in serum and urine at initial presentation was 365 ng/mL and 11,640 ng/mL respectively. At the time of initial presentation whole blood collected from the dog did not clot and the Prothrombin Time was abnormally increased (>300 s). Serum was also visibly hemolysed. The dog was administered antihistamine, dexamethasone and 4000 Units (sufficient to neutralise 40 mg of TSV) of a novel polyvalent alpaca antivenom diluted in 0.9% NaCl. At 4 h post-antivenom treatment the dog's clinical condition had improved markedly with serum TSV concentrations below the limit of detection (<0.015 ng/mL), consistent with complete binding of venom antigens by the alpaca antivenom. Coagulation parameters had begun to improve by 4 h and had fully normalised by 16 h post-antivenom. Venom concentrations in both serum and urine remained undetectable at 16 h post-antivenom. The dog made a complete recovery, without complications, suggesting that the alpaca-based antivenom is both clinically safe and effective. PMID:26930223

  17. Analysis of mitochondrial DNA in Bolivian llama, alpaca and vicuna populations: a contribution to the phylogeny of the South American camelids.

    PubMed

    Barreta, J; Gutiérrez-Gil, B; Iñiguez, V; Saavedra, V; Chiri, R; Latorre, E; Arranz, J J

    2013-04-01

    The objectives of this work were to assess the mtDNA diversity of Bolivian South American camelid (SAC) populations and to shed light on the evolutionary relationships between the Bolivian camelids and other populations of SACs. We have analysed two different mtDNA regions: the complete coding region of the MT-CYB gene and 513 bp of the D-loop region. The populations sampled included Bolivian llamas, alpacas and vicunas, and Chilean guanacos. High levels of genetic diversity were observed in the studied populations. In general, MT-CYB was more variable than D-loop. On a species level, the vicunas showed the lowest genetic variability, followed by the guanacos, alpacas and llamas. Phylogenetic analyses performed by including additional available mtDNA sequences from the studied species confirmed the existence of the two monophyletic clades previously described by other authors for guanacos (G) and vicunas (V). Significant levels of mtDNA hybridization were found in the domestic species. Our sequence analyses revealed significant sequence divergence within clade G, and some of the Bolivian llamas grouped with the majority of the southern guanacos. This finding supports the existence of more than the one llama domestication centre in South America previously suggested on the basis of archaeozoological evidence. Additionally, analysis of D-loop sequences revealed two new matrilineal lineages that are distinct from the previously reported G and V clades. The results presented here represent the first report on the population structure and genetic variability of Bolivian camelids and may help to elucidate the complex and dynamic domestication process of SAC populations.

  18. Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies*

    PubMed Central

    Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

    2016-01-01

    Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229. PMID:27129274

  19. Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies.

    PubMed

    Blanchetot, Christophe; De Jonge, Natalie; Desmyter, Aline; Ongenae, Nico; Hofman, Erik; Klarenbeek, Alex; Sadi, Ava; Hultberg, Anna; Kretz-Rommel, Anke; Spinelli, Silvia; Loris, Remy; Cambillau, Christian; de Haard, Hans

    2016-06-24

    Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).

  20. 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 ...

  1. 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)

  2. Antimitochondrial antibody

    MedlinePlus

    ... antibodies (AMA) are substances ( antibodies ) that form against mitochondria. The mitochondria are an important part of cells. They are ... often, in people with other kinds of liver disease and some autoimmune diseases. Risks Risks for having ...

  3. Culicoides vector species on three South American camelid farms seropositive for bluetongue virus serotype 8 in Germany 2008/2009.

    PubMed

    Schulz, Claudia; Ziller, Mario; Kampen, Helge; Gauly, Matthias; Beer, Martin; Grevelding, Christoph G; Hoffmann, Bernd; Bauer, Christian; Werner, Doreen

    2015-12-15

    Palearctic species of Culicoides (Diptera, Ceratopogonidae), in particular of the Obsoletus and Pulicaris complexes, were identified as putative vectors of bluetongue virus serotype 8 (BTV-8) on ruminant farms during the epizootic in Germany from 2006 to 2009. BTV may cause severe morbidity and mortality in ruminants and sporadically in South American camelids (SAC). However, the fauna of Culicoides spp. on SAC farms has not been investigated. Therefore, the ceratopogonid fauna was monitored on three farms with BTV-seropositive SAC in Germany. Black-light traps were set up on pastures and in stables from summer 2008 to autumn 2009. Additionally, ceratopogonids were caught in emergence traps mounted on llama dung and dung-free pasture from spring to autumn 2009. After morphological identification, selected Culicoides samples were analysed for BTV-RNA by real-time RT-PCR. The effects of the variables 'location', 'temperature' and 'humidity' on the number of Culicoides caught in black-light traps were modelled using multivariable Poisson regression. In total, 26 species of Culicoides and six other genera of biting midges were identified. The most abundant Culicoides spp. collected both outdoors and indoors with black-light traps belonged to the Obsoletus (77.4%) and Pulicaris (16.0%) complexes. The number of Culicoides peaked in summer, while no biting midges were caught during the winter months. Daily collections of Culicoides were mainly influenced by the location and depended on the interaction of temperature and humidity. In the emergence traps, species of the Obsoletus complex predominated the collections. In summary, the absence of BTV-RNA in any of the analysed Culicoides midges and in the BTV-seropositive SAC on the three farms together with the differences in the pathogenesis of BTV-8 in SAC compared to ruminants suggests a negligible role of SAC in the spread of the virus. Although SAC farms may provide similar suitable habitats for putative Culicoides

  4. Culicoides vector species on three South American camelid farms seropositive for bluetongue virus serotype 8 in Germany 2008/2009.

    PubMed

    Schulz, Claudia; Ziller, Mario; Kampen, Helge; Gauly, Matthias; Beer, Martin; Grevelding, Christoph G; Hoffmann, Bernd; Bauer, Christian; Werner, Doreen

    2015-12-15

    Palearctic species of Culicoides (Diptera, Ceratopogonidae), in particular of the Obsoletus and Pulicaris complexes, were identified as putative vectors of bluetongue virus serotype 8 (BTV-8) on ruminant farms during the epizootic in Germany from 2006 to 2009. BTV may cause severe morbidity and mortality in ruminants and sporadically in South American camelids (SAC). However, the fauna of Culicoides spp. on SAC farms has not been investigated. Therefore, the ceratopogonid fauna was monitored on three farms with BTV-seropositive SAC in Germany. Black-light traps were set up on pastures and in stables from summer 2008 to autumn 2009. Additionally, ceratopogonids were caught in emergence traps mounted on llama dung and dung-free pasture from spring to autumn 2009. After morphological identification, selected Culicoides samples were analysed for BTV-RNA by real-time RT-PCR. The effects of the variables 'location', 'temperature' and 'humidity' on the number of Culicoides caught in black-light traps were modelled using multivariable Poisson regression. In total, 26 species of Culicoides and six other genera of biting midges were identified. The most abundant Culicoides spp. collected both outdoors and indoors with black-light traps belonged to the Obsoletus (77.4%) and Pulicaris (16.0%) complexes. The number of Culicoides peaked in summer, while no biting midges were caught during the winter months. Daily collections of Culicoides were mainly influenced by the location and depended on the interaction of temperature and humidity. In the emergence traps, species of the Obsoletus complex predominated the collections. In summary, the absence of BTV-RNA in any of the analysed Culicoides midges and in the BTV-seropositive SAC on the three farms together with the differences in the pathogenesis of BTV-8 in SAC compared to ruminants suggests a negligible role of SAC in the spread of the virus. Although SAC farms may provide similar suitable habitats for putative Culicoides

  5. Antibody validation

    PubMed Central

    Bordeaux, Jennifer; Welsh, Allison W.; Agarwal, Seema; Killiam, Elizabeth; Baquero, Maria T.; Hanna, Jason A.; Anagnostou, Valsamo K.; Rimm, David L.

    2013-01-01

    Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence. PMID:20359301

  6. Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy.

    PubMed

    Godar, Marie; Morello, Virginia; Sadi, Ava; Hultberg, Anna; De Jonge, Natalie; Basilico, Cristina; Hanssens, Valérie; Saunders, Michael; Lambrecht, Bart N; El Khattabi, Mohamed; de Haard, Hans; Michieli, Paolo; Blanchetot, Christophe

    2016-08-22

    Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

  7. Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

    PubMed Central

    Godar, Marie; Morello, Virginia; Sadi, Ava; Hultberg, Anna; De Jonge, Natalie; Basilico, Cristina; Hanssens, Valérie; Saunders, Michael; Lambrecht, Bart N.; El Khattabi, Mohamed; de Haard, Hans; Michieli, Paolo; Blanchetot, Christophe

    2016-01-01

    Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo. PMID:27546726

  8. Retrospective study of central nervous system lesions and association with Parelaphostrongylus species by histology and specific nested polymerase chain reaction in domestic camelids and wild ungulates.

    PubMed

    Dobey, Carrie L; Grunenwald, Caroline; Newman, Shelley J; Muller, Lisa; Gerhold, Richard W

    2014-11-01

    Formalin-fixed, paraffin-embedded tissues from elk (Cervus elaphus), goats, and camelids with case histories and lesions suggestive of Parelaphostrongylus tenuis were examined by histology to characterize lesions that could aid in definitively diagnosing P. tenuis infection. Additionally, sections of paraffin-embedded tissue were used in a nested polymerase chain reaction (nPCR) using Parelaphostrongylus-specific primers to determine how PCR results corresponded with histological findings. Histological changes in brain and spinal cord consisted of linear tracks of hemorrhage; tracks or perivascular accumulations of hemosiderin-laden macrophages; acute foci of axonal degeneration and/or linear glial scars; and perivascular, parenchymal, or meningeal accumulations of eosinophils and/or lymphocytes and plasma cells. Of the 43 samples with histologic lesions consistent with neural larval migrans, 19 were PCR positive; however, only 8 were confirmed Parelaphostrongylus by DNA sequencing. Additionally, 1 goat was identified with a protostrongylid that had a 97% identity to both Parelaphostrongylus odocoilei and a protostrongylid nematode from pampas deer (Ozotoceros bezoarticus celer) from Argentina. None of the histologic lesions individually or in combination correlated statistically to positive molecular tests for the nematode. The results indicate that it is possible to extract Parelaphostrongylus DNA from formalin-fixed, paraffin-embedded tissue, but extended fixation presumably can cause DNA crosslinking. Nested PCR provides another diagnostic tool to identify the cause of neurologic disease in camelids and elk with histologic lesions consistent with neural larval migrans. Furthermore, potential novel protostrongylid DNA was detected from a goat with lesions consistent with P. tenuis infection, suggesting that other neurotropic Parelaphostrongylus species may occur locally.

  9. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers

    PubMed Central

    Dahms, Sven O.; Creemers, John W. M.; Schaub, Yvonne; Bourenkov, Gleb P.; Zögg, Thomas; Brandstetter, Hans; Than, Manuel E.

    2016-01-01

    Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors. PMID:27670069

  10. Antiparietal cell antibody test

    MedlinePlus

    APCA; Anti-gastric parietal cell antibody; Atrophic gastritis - anti-gastric parietal cell antibody; Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ...

  11. Platelet antibodies.

    PubMed

    Pulkrabek, S M

    1996-12-01

    The proper diagnosis of patients with immune-mediated thrombocytopenias can be accomplished by using the advances made in the field of platelet serology. These techniques range from solid phase red cell adherence to sequencing platelet antigen amino acids by polymerase chain reaction. This article describes platelet antigens, the clinical tests available to detect platelet antigens and antibodies, and the value of these tests in supporting clinical diagnoses.

  12. Antibody Engineering and Therapeutics

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul WHI; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates. PMID:24589717

  13. Presence, distribution and steroidogenic effect of the peptides orexin A and receptor 1 for orexins in the testis of the South American camelid alpaca (Vicugna pacos).

    PubMed

    Liguori, Giovanna; Assisi, Loredana; Squillacioti, Caterina; Paino, Salvatore; Mirabella, Nicola; Vittoria, Alfredo

    2012-10-01

    The orexins A (oxA) and B are peptides discovered in the rat hypothalamus and successively found in some peripheral organs of the mammalian body. They binds two protein G-coupled receptors defined receptor 1 (ox1r) and 2 for orexins, the first of which is highly specific for oxA while the second binds both the peptides with equal affinity. This work aimed to detect the presence of oxA and ox1r in the testis of the South American camelid alpaca (Vicugna pacos) and investigate the role played by them on Leydig cell steroidogenesis. The species alpaca acquired, in the last years, increasing zootechnical interest for the quality of the wool produced and its breeding spread from the country of origin to USA, Australia and Europe. Immunohistochemistry allowed us to detect oxA in Leydig and Sertoli cells, spermatogonia, resting spermatocytes, round and oval spermatids. Ox1r-immunoreactivity was found in Leydig cells and round, oval and elongated spermatids. The expression of the two peptides in tissue extracts was established by using Western blotting technique. Such results demonstrated that in the alpaca testis exists in a cellular complex able to produce and/or internalize oxA. Finally, the effect of oxA on steroidogenesis was investigated by means of in vitro cultured thin testis slices which were added with oxA or/and Müllerian Inhibiting Substance (MIS), a steroidolitic agent basally produced by the Sertoli cell. OxA evoked increase of testosterone production while MIS a decrease. The consecutive addition of oxA and MIS, or vice versa, highlighted an antagonistic interplay between the two substances which has been thought to be the main molecular event at the basis of the oxA-stimulated steroidogenesis mechanism.

  14. Monoclonal antibodies.

    PubMed

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  15. Linking Single Domain Antibodies that Recognize Different Epitopes on the Same Target

    PubMed Central

    Glaven, Richard H.; Anderson, George P.; Zabetakis, Dan; Liu, Jinny L.; Long, Nina C.; Goldman, Ellen R.

    2012-01-01

    Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb. PMID:25585631

  16. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

    PubMed

    Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J; Alvarez-Vallina, Luis

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

  17. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains

    PubMed Central

    Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M.; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet, Jaume; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva, Baldo; Sanz, Laura; Blanco, Francisco J.; Alvarez-Vallina, Luis

    2016-01-01

    Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas. PMID:27345490

  18. [Antinuclear antibodies].

    PubMed

    Cabiedes, Javier; Núñez-Álvarez, Carlos A

    2010-01-01

    Anti-nuclear antibodies (ANA) are immunoglobulin directed against autologous cell nuclear and cytoplasmic components. Besides the autoimmune ANA there are other ANA that can be detected in circulation, like natural and infectious ANA. Because of its high sensibility, detection of the ANA must be done by indirect immunofluorescence (IIF) as screening test and all of those positive samples are convenient to confirm its specificity by ELISA, western blot or other techniques. Positive ANA detected by IIF must be evaluated taking in to account the pattern and titer. The following recommended step is the specificity characterization (reactivity against extractable nuclear antigens [ENA], dsDNA, etc.) which is useful for the diagnosis and follow up of patients with autoimmune diseases, and by such reasoning, its detection must be performed in an orderly and reasonable way using guides or strategies focused to the good use and interpretation of the autoantibodies. The objective of this review is to present a compilation of the literature and our experience in the detection and study of the ANA.

  19. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.

  20. Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

    PubMed Central

    2011-01-01

    Background Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive candidates for diagnostic assays. Results Here we describe the isolation of an sdAb against Staphyloccocus aureus enterotoxin B (SEB). The clone, A3, was found to have high affinity (Kd = 75 pM) and good specificity for SEB, showing no cross reactivity to related molecules such as Staphylococcal enterotoxin A (SEA), Staphylococcal enterotoxin D (SED), and Shiga toxin. Most remarkably, this anti-SEB sdAb had an extremely high Tm of 85°C and an ability to refold after heating to 95°C. The sharp Tm determined by circular dichroism, was found to contrast with the gradual decrease observed in intrinsic fluorescence. We demonstrated the utility of this sdAb as a capture and detector molecule in Luminex based assays providing limits of detection (LODs) of at least 64 pg/mL. Conclusion The anti-SEB sdAb A3 was found to have a high affinity and an extraordinarily high Tm and could still refold to recover activity after heat denaturation. This combination of heat resilience and strong, specific binding make this sdAb a good candidate for use in antibody-based toxin detection technologies. PMID:21933444

  1. Antibodies and antibody-derived analytical biosensors

    PubMed Central

    Sharma, Shikha; Byrne, Hannah

    2016-01-01

    The rapid diagnosis of many diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Biosensors are now being applied for rapid diagnostics due to their capacity for point-of-care use with minimum need for operator input. Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection of a plethora of analytes such as disease markers, food and environmental contaminants, biological warfare agents and illicit drugs. Antibodies are ideal biorecognition elements that provide sensors with high specificity and sensitivity. This review describes monoclonal and recombinant antibodies and different immobilization approaches crucial for antibody utilization in biosensors. Examples of applications of a variety of antibody-based sensor formats are also described. PMID:27365031

  2. Antibodies and antibody-derived analytical biosensors.

    PubMed

    Sharma, Shikha; Byrne, Hannah; O'Kennedy, Richard J

    2016-06-30

    The rapid diagnosis of many diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Biosensors are now being applied for rapid diagnostics due to their capacity for point-of-care use with minimum need for operator input. Antibody-based biosensors or immunosensors have revolutionized diagnostics for the detection of a plethora of analytes such as disease markers, food and environmental contaminants, biological warfare agents and illicit drugs. Antibodies are ideal biorecognition elements that provide sensors with high specificity and sensitivity. This review describes monoclonal and recombinant antibodies and different immobilization approaches crucial for antibody utilization in biosensors. Examples of applications of a variety of antibody-based sensor formats are also described. PMID:27365031

  3. Structural basis for antibody recognition in the receptor-binding domains of toxins A and B from Clostridium difficile.

    PubMed

    Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N; Klassen, John S; Ng, Kenneth K S

    2014-01-24

    Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.

  4. Isolation and characterization of antigen-specific alpaca (Lama pacos) VHH antibodies by biopanning followed by high-throughput sequencing.

    PubMed

    Miyazaki, Nobuo; Kiyose, Norihiko; Akazawa, Yoko; Takashima, Mizuki; Hagihara, Yosihisa; Inoue, Naokazu; Matsuda, Tomonari; Ogawa, Ryu; Inoue, Seiya; Ito, Yuji

    2015-09-01

    The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 × 10(7) from cDNA of peripheral blood mononuclear cells of an alpaca (Lama pacos) immunized with a fragment of IZUMO1 (IZUMO1PFF) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (>93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

  5. Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile*

    PubMed Central

    Murase, Tomohiko; Eugenio, Luiz; Schorr, Melissa; Hussack, Greg; Tanha, Jamshid; Kitova, Elena N.; Klassen, John S.; Ng, Kenneth K. S.

    2014-01-01

    Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents. PMID:24311789

  6. Antibody Blood Tests

    MedlinePlus

    ... discovered that people with celiac disease who eat gluten have higher than normal levels of certain antibodies ... rye and barley that are generically known as “gluten.” Antibody Testing: Only A First Step To help ...

  7. RBC Antibody Screen

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Screen Share this page: Was this page ... Screen Related tests: Direct Antiglobulin Test ; Blood Typing ; RBC Antibody Identification ; Type and Screen; Crossmatch All content ...

  8. Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

    PubMed

    Maffey, Lucia; Vega, Celina G; Parreño, Viviana; Garaicoechea, Lorena

    2015-01-01

    Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects. PMID:26654700

  9. Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

    PubMed Central

    Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Gabriel; González-Sapienza, Gualberto

    2015-01-01

    BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. PMID:25819371

  10. Modeling Antibody Diversity.

    ERIC Educational Resources Information Center

    Baker, William P.; Moore, Cathy Ronstadt

    1998-01-01

    Understanding antibody structure and function is difficult for many students. The rearrangement of constant and variable regions during antibody differentiation can be effectively simulated using a paper model. Describes a hands-on laboratory exercise which allows students to model antibody diversity using readily available resources. (PVD)

  11. Anti-insulin antibody test

    MedlinePlus

    Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... Normally, there are no antibodies against insulin in your blood. ... different laboratories. Some labs use different measurements or ...

  12. Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

    PubMed Central

    Rosa, Sofia Tabares-da; Rossotti, Martin; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Last, Jerold A.; Hammock, Bruce D; González-Sapienza, Gualberto

    2011-01-01

    Single-domain antibodies (sdAbs) found in camelids, lack a light chain and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. While this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC50 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, KD 0.98–1.37 nM (SPR) which allowed development of a competitive assay for TCC with an IC50 = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC50 attained with other anti-hapten VHHs reported thus far. Despite the modest overall anti-hapten sdAbs response in llamas, a small subpopulation of high affinity VHHs are generated that can be isolated by carefully design of the selection process. PMID:21827167

  13. [VGKC-complex antibodies].

    PubMed

    Watanabe, Osamu

    2013-04-01

    Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).

  14. Advances in Antibody Design.

    PubMed

    Tiller, Kathryn E; Tessier, Peter M

    2015-01-01

    The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody-drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

  15. Antibody Therapeutics in Oncology

    PubMed Central

    Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

    2016-01-01

    One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment. PMID:27081677

  16. Antibodies to prothrombin.

    PubMed

    Bertolaccini, M L

    2012-06-01

    Research on antiphospholipid antibodies (aPL) and the thrombotic manifestations associated with these antibodies has grown since the description of anticardiolipin antibodies (aCL) by Harris and colleagues in the early 1980s. Antiprothrombin (aPT) antibodies are commonly detected by ELISA, using irradiated plates (aPT) or prothrombin in complex with phosphatidylserine (aPS/PT). Although aPT and/or aPS/PT are associated with antiphospholipid syndrome (APS) -related clinical features and these antibodies correlate with each other, aPT and aPS/PT belong to different populations of autoantibodies even though they can both be present in the same patient. Early studies suggested that these antibodies might be the antigenic target of lupus anticoagulant (LA) and their correlation and clinical significance is being investigated. PMID:22635215

  17. Engineering antibody therapeutics.

    PubMed

    Chiu, Mark L; Gilliland, Gary L

    2016-06-01

    The successful introduction of antibody-based protein therapeutics into the arsenal of treatments for patients has within a few decades fostered intense innovation in the production and engineering of antibodies. Reviewed here are the methods currently used to produce antibodies along with how our knowledge of the structural and functional characterization of immunoglobulins has resulted in the engineering of antibodies to produce protein therapeutics with unique properties, both biological and biophysical, that are leading to novel therapeutic approaches. Antibody engineering includes the introduction of the antibody combining site (variable regions) into a host of architectures including bi and multi-specific formats that further impact the therapeutic properties leading to further advantages and successes in patient treatment. PMID:27525816

  18. Antibodies in Transplantation

    PubMed Central

    Platt, Jeffrey L.

    2011-01-01

    Transplantation of cells, tissues, and organs from one individual to another can incite the production of antibodies specific for foreign antigens, especially major histocompatibility antigens, in the graft. Antibodies specific for a graft provide an index of immunity and a potential trigger for injury and rejection. However, the index of immunity can sometimes miss antibody-mediated rejection and besides causing injury the antibodies against a graft can also protect a graft from injury by blocking immune recognition, called enhancement, regulating activation of complement, and inducing changes in the graft that resist damage. Reviewed here are potential limitations in the use of antibodies as an index of immunity and the ways antibodies cause and/or prevent injury. PMID:20807473

  19. Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

    PubMed

    Doyle, Patrick J; Saeed, Hanaa; Hermans, Anne; Gleddie, Steve C; Hussack, Greg; Arbabi-Ghahroudi, Mehdi; Seguin, Charles; Savard, Marc E; Mackenzie, C Roger; Hall, J Christopher

    2009-12-11

    15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops. PMID:19783651

  20. Antibodies to cholesterol.

    PubMed Central

    Swartz, G M; Gentry, M K; Amende, L M; Blanchette-Mackie, E J; Alving, C R

    1988-01-01

    Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained. Images PMID:3162316

  1. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  2. Antibodies as natural adjuvants.

    PubMed

    Heyman, Birgitta

    2014-01-01

    Antibodies in complex with specific antigen can dramatically change the antibody response to this antigen. Depending on antibody class and type of antigen, >99 % suppression or >100-fold enhancement of the response can take place. IgM and IgG3 are efficient enhancers and operate via the complement system. In contrast, IgG1, IgG2a, and IgG2b enhance antibody and CD4(+) T cell responses to protein antigens via activating Fcγ-receptors. IgE also enhances antibody and CD4(+) T cell responses to small proteins but uses the low-affinity receptor for IgE, CD23. Most likely, IgM and IgG3 work by increasing the effective concentration of antigen on follicular dendritic cells in splenic follicles. IgG1, IgG2a, IgG2b, and IgE probably enhance antibody responses by increasing antigen presentation by dendritic cells to T helper cells. IgG antibodies of all subclasses have a dual effect, and suppress antibody responses to particulate antigens such as erythrocytes. This capacity is used in the clinic to prevent immunization of Rhesus-negative women to Rhesus-positive fetal erythrocytes acquired via transplacental hemorrage. IgG-mediated suppression in mouse models can take place in the absence of Fcγ-receptors and complement and to date no knock-out mouse strain has been found where suppression is abrogated.

  3. 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

  4. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  5. Production Of Human Antibodies

    NASA Technical Reports Server (NTRS)

    Sammons, David W.; Neil, Garry A.

    1993-01-01

    Process for making human monoclonal antibodies based on combination of techniques. Antibodies made active against specific antigen. Process involves in vivo immunization of human B lymphocyte cells in mice. B cells of interest enriched in vitro before fusion. Method potentially applicable to any antigen. Does not rely on use of Epstein-Barr virus at any step. Human lymphocytes taken from any source.

  6. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Antibodies are provided in a variety of formats that includes antiserum, hybridoma culture supernatant or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facil...

  7. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  8. NMDA receptor antibodies

    PubMed Central

    Ramberger, Melanie; Bsteh, Gabriel; Schanda, Kathrin; Höftberger, Romana; Rostásy, Kevin; Baumann, Matthias; Aboulenein-Djamshidian, Fahmy; Lutterotti, Andreas; Deisenhammer, Florian; Berger, Thomas

    2015-01-01

    Objectives: To analyze the frequency of NMDA receptor (NMDAR) antibodies in patients with various inflammatory demyelinating diseases of the CNS and to determine their clinical correlates. Methods: Retrospective case-control study from 2005 to 2014 with the detection of serum IgG antibodies to NMDAR, aquaporin-4, and myelin oligodendrocyte glycoprotein by recombinant live cell-based immunofluorescence assays. Fifty-one patients with acute disseminated encephalomyelitis, 41 with neuromyelitis optica spectrum disorders, 34 with clinically isolated syndrome, and 89 with multiple sclerosis (MS) were included. Due to a known association of NMDAR antibodies with seizures and behavioral symptoms, patients with those clinical manifestations were preferentially included and are therefore overrepresented in our cohort. Nine patients with NMDAR encephalitis, 94 patients with other neurologic diseases, and 48 healthy individuals were used as controls. Results: NMDAR antibodies were found in all 9 patients with NMDAR encephalitis but in only 1 of 215 (0.5%) patients with inflammatory demyelination and in none of the controls. This patient had relapsing-remitting MS with NMDAR antibodies present at disease onset, with an increase in NMDAR antibody titer with the onset of psychiatric symptoms and cognitive deficits. Conclusion: In demyelinating disorders, NMDAR antibodies are uncommon, even in those with symptoms seen in NMDAR encephalitis. PMID:26309901

  9. Mining human antibody repertoires

    PubMed Central

    2010-01-01

    Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties. PMID:20505349

  10. Anti-cartilage antibody.

    PubMed

    Greenbury, C L; Skingle, J

    1979-08-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change.

  11. Anti-cartilage antibody.

    PubMed Central

    Greenbury, C L; Skingle, J

    1979-01-01

    Antibody to cartilage has been demonstrated by indirect immunofluorescence on rat trachea in the serum of about 3% of 1126 patients with rheumatoid arthritis. Titres ranged from 1:20 to 1:640. The antibody was not found in 284 patients with primary or secondary osteoarthritis or in 1825 blood donors, nor, with the exception of two weak reactors, in 1314 paraplegic patients. In most cases the antibody appears to be specific for native type II collagen. Using this as an antigen in a haemagglutination test 94% of anti-cartilage sera were positive, whereas among 100 rheumatoid control sera there were only three weak positives. More than 80% of patients with antibody had some erosion of articular cartilage, but there was no correlation with age, sex, duration of disease, nor any recognisable clinical event or change. Images Fig. 1 PMID:389957

  12. HIV Antibody Test

    MedlinePlus

    ... despite the fact that the person is infected ( false negative ). If an HIV antibody test is negative ... infection (around 28 days) and may give a false-negative result. ^ Back to top Is there anything ...

  13. Platelet associated antibodies

    MedlinePlus

    ... of the following: For unknown reasons (idiopathic thrombocytopenic purpura, or ITP ) Side effect of certain drugs such ... 2012:chap 134. Read More Antibody Idiopathic thrombocytopenic purpura (ITP) Platelet count Serum globulin electrophoresis Thrombocytopenia Update ...

  14. Anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2009-09-15

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  15. Engineering antibodies for therapy.

    PubMed

    Adair, J R

    1992-12-01

    Success in the generation of an antibody-based therapeutic requires careful consideration of the binding site, to achieve specificity and high affinity; of the effector, to produce the desired therapeutic effect; of the means of attachment of the effector to the binding site; production of the end product; and the response made by the patient to the administered compound. Each of these areas is receiving attention by antibody-engineering techniques. The number of potentially useful monoclonal antibodies developed over the last 10 years, and currently in clinical trials or preregistration, is now being increased by these engineered newcomers. It will be interesting to see over the next few years how many of these antibodies, and of which kind, emerge as products.

  16. Antibody tumor penetration

    PubMed Central

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    Antibodies have proven to be effective agents in cancer imaging and therapy. One of the major challenges still facing the field is the heterogeneous distribution of these agents in tumors when administered systemically. Large regions of untargeted cells can therefore escape therapy and potentially select for more resistant cells. We present here a summary of theoretical and experimental approaches to analyze and improve antibody penetration in tumor tissue. PMID:18541331

  17. Monoclonal antibodies and cancer therapy

    SciTech Connect

    Reisfeld, R.A.; Sell, S.

    1985-01-01

    These proceedings collect papers on the subject of monoclonal antibodies. Topics include: Monoclonal antibody, biochemical effects and cancer therapeutic potential of tunicamycin, use of monoclonal antibodies for detection of lymph node metastases, active specific immunotherapy, and applications of monoclonal antibodies to investigations of growth factors.

  18. Engineering antibodies for cancer therapy.

    PubMed

    Boder, Eric T; Jiang, Wei

    2011-01-01

    The advent of modern antibody engineering has led to numerous successes in the application of these proteins for cancer therapy in the 13 years since the first Food and Drug Administration approval, which has stimulated active interest in developing more and better drugs based on these molecules. A wide range of tools for discovering and engineering antibodies has been brought to bear on this challenge in the past two decades. Here, we summarize mechanisms of monoclonal antibody therapeutic activity, challenges to effective antibody-based treatment, existing technologies for antibody engineering, and current concepts for engineering new antibody formats and antibody alternatives as next generation biopharmaceuticals for cancer treatment.

  19. Human antibody technology and the development of antibodies against cytomegalovirus.

    PubMed

    Ohlin, Mats; Söderberg-Nauclér, Cecilia

    2015-10-01

    Cytomegalovirus (CMV) is a virus that causes chronic infections in a large set of the population. It may cause severe disease in immunocompromised individuals, is linked to immunosenescence and implied to play an important role in the pathogenesis of cardiovascular diseases and cancer. Modulation of the immune system's abilities to manage the virus represent a highly viable therapeutic option and passive immunotherapy with polyclonal antibody preparations is already in clinical use. Defined monoclonal antibodies offer many advantages over polyclonal antibodies purified from serum. Human CMV-specific monoclonal antibodies have consequently been thoroughly investigated with respect to their potential in the treatment of diseases caused by CMV. Recent advances in human antibody technology have substantially expanded the breadth of antibodies for such applications. This review summarizes the fundamental basis for treating CMV disease by use of antibodies, the basic technologies to be used to develop such antibodies, and relevant human antibody specificities available to target this virus.

  20. A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking

    PubMed Central

    Herrera, Cristina; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten

    2016-01-01

    JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin’s enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin’s bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells. PMID:27300140

  1. A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking.

    PubMed

    Herrera, Cristina; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

    2016-01-01

    JJX12 is an engineered bispecific antibody against ricin, a member of the medically important A-B family of toxins that exploits retrograde transport as means to gain entry into the cytosol of target cells. JJX12 consists of RTA-D10, a camelid single variable domain (VHH) antibody directed against an epitope on ricin's enzymatic subunit (RTA), linked via a 15-mer peptide to RTB-B7, a VHH against ricin's bivalent galactose binding subunit (RTB). We previously reported that JJX12, but not an equimolar mixture of RTA-D10 and RTB-B7 monomers, was able to passively protect mice against a lethal dose ricin challenge, demonstrating that physically linking RTB-B7 and RTA-D10 is critical for toxin-neutralizing activity in vivo. We also reported that JJX12 promotes aggregation of ricin in solution, presumably through the formation of intermolecular crosslinking. In the current study, we now present evidence that JJX12 affects the dynamics of ricin uptake and trafficking in human epithelial cells. Confocal microscopy, as well as live cell imaging coupled with endocytosis pathway-specific inhibitors, revealed that JJX12-toxin complexes are formed on the surfaces of mammalian cells and internalized via a pathway sensitive to amiloride, a known inhibitor of macropinocytosis. Moreover, in the presence of JJX12, retrograde transport of ricin to the trans-Golgi network was significantly reduced, while accumulation of the toxin in late endosomes was significantly enhanced. In summary, we propose that JJX12, by virtue of its ability to crosslink ricin toxin, alters the route of toxin uptake and trafficking within cells. PMID:27300140

  2. Polyreactive Antibodies: Function and Quantification.

    PubMed

    Gunti, Sreenivasulu; Notkins, Abner Louis

    2015-07-15

    Polyreactive antibodies, a major component of the natural antibody repertoire, bind with low affinity to a variety of structurally unrelated antigens. Many of these antibodies are germline or near germline in sequence. Little is known, however, about the function of these antibodies. In the present mini-review we show: (1) that the broad antibacterial activity of the natural antibody repertoire is largely due to polyreactive antibodies, which in the presence of complement lyse bacteria and enhance phagocytosis; (2) that polyreactive antibodies bind to UV- or human immunodeficiency virus-induced apoptotic cells and with complement enhance the phagocytosis of these cells by macrophages; and (3) that dinitrophenol can be used as a surrogate for quantitating the level of polyreactive antibodies in serum. We conclude that polyreactive antibodies protect the host against both foreign invaders and its own damaged/apoptotic cells.

  3. ANTIBODY FORMATION IN VITRO

    PubMed Central

    Fishman, M.

    1961-01-01

    Neutralizing activity against T2 bacteriophage appeared in cultures of lymph node cells from normal rats in response to their in vitro stimulation with a cell-free filtrate derived from homogenized rat macrophages which had been incubated with T2 bacteriophage. This activity was specifically directed against T2 bacteriophage. It resided in a fraction of the culture fluid which had the salting-out properties of serum globulin. Phage neutralization was inhibited by antibody specific for rat serum gamma globulin. Antibody production against T2 bacteriophage in cultures of lymph node cells from normal animals failed to occur if (a) T2 bacteriophage alone was added, (b) if the incubation period of macrophages and T2 phage was unduly shortened, (c) if the cell-free filtrate was heated at 80–100°C for 15 minutes, (d) if more than an optimal amount of T2 bacteriophage was added to the macrophages. Additional factors which prevented the formation of antibody were the heat inactivation of the lymph node cells or the addition to the culture medium of either streptomycin or ribonuclease. Finally, it was found that macrophages and lymph node cells had to be obtained from animals of one and the same species. All essential findings on the production of antibody to T2 bacteriophage in vitro could be confirmed by substitution of the chick embryo for the tissue culture medium. The results are discussed in terms of a possible mechanism of antibody production in which an RNAse-sensitive substance resulting from the interaction of macrophages and antigen is capable of stimulating antibody synthesis in lymphocytic cells. PMID:13893304

  4. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

    Willis, Jordan R; Briney, Bryan S; DeLuca, Samuel L; Crowe, James E; Meiler, Jens

    2013-04-01

    Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.

  5. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation.

    PubMed

    Weider, Elodie; Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Nimesh, Surendra; Ashraf, Yahya; Wycoff, Keith L; Zhang, Jianbing; Prat, Annik; Seidah, Nabil G

    2016-08-01

    Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes. PMID:27284008

  6. Therapeutic antibodies in ophthalmology

    PubMed Central

    Magdelaine-Beuzelin, Charlotte; Pinault, Coralie; Paintaud, Gilles

    2010-01-01

    More than a century after the first successful use of serotherapy, antibody-based therapy has been renewed by the availability of recombinant monoclonal antibodies. As in the past, current clinical experience has prompted new pharmacological questions and induced much debate among practitioners, notably in the field of ophthalmology. An examination of the history of antibodies as treatments for ocular disorders reveals interesting parallels to the modern era. The fact that a treatment administered by a systemic route could be efficacious in a local disease was not widely accepted and the “chemical” nature of antibodies was not clearly understood in the late 19th century. Clinical studies by Henry Coppez, a Belgian ophthalmologist, established in 1894 that antidiphtheric antitoxins could be used to treat conjunctival diphtheria. Nearly 20 years later, Coppez and Danis described age-related macular degeneration, a disorder which today benefits from ranibizumab therapy. The product, a locally-administered recombinant monoclonal Fab fragment, is directed against vascular endothelial growth factor A. Interestingly, its full-size counterpart, bevacizumab, which is approved for the treatment of solid tumors, has also demonstrated efficacy in age-related macular degeneration when administered either intravenously or locally, which raises new questions about antibody pharmacology and biodistribution. In order to shed some light on this debate, we recount the early history of serotherapy applied to ophthalmology, review the exact molecular differences between ranibizumab and bevacizumab, and discuss what is known about IgG and the blood-retina barrier and the possible role of FcRn, an IgG transporter. PMID:21358858

  7. Trifunctional antibody ertumaxomab

    PubMed Central

    Diermeier-Daucher, Simone; Ortmann, Olaf; Buchholz, Stefan; Brockhoff, Gero

    2012-01-01

    Background: The trifunctional antibody ertumaxomab bivalently targets the human epidermal growth factor receptor 2 (Her2) on epithelial (tumor) cells and the T cell specific CD3 antigen, and its Fc region is selectively recognized by Fcγ type I/III receptor-positive immune cells. As a trifunctional immunoglobulin, ertumaxomab therefore not only targets Her2 on cancer cells, but also triggers immunological effector mechanisms mediated by T and accessory cells (e.g., macrophages, dendritic cells, natural killer cells). Whether molecular effects, however, might contribute to the cellular antitumor efficiency of ertumaxomab are largely unknown. Methods: Potential molecular effects of ertumaxomab on Her2-overexpressing BT474 and SK-BR-3 breast cancer cells were evaluated. The dissociation constant Kd of ertumaxomab was calculated from titration curves that were recorded by flow cytometry. Treatment-induced changes in Her2 homodimerization were determined by flow cytometric fluorescence resonance energy transfer measurements on a cell-by-cell basis. Potential activation / deactivation of Her2, ERK1/2, AKT and STAT3 were analyzed by western blotting, Immunochemistry and immunofluorescent cell staining. Results: The Kd of ertumaxomab for Her2-binding was determined at 265 nM and the ertumaxomab binding epitope was found to not overlap with that of the therapeutic anti-Her2 monoclonal antibodies trastuzumab and pertuzumab. Ertumaxomab caused an increase in Her2 phosphorylation at higher antibody concentrations, but changed neither the rate of Her2-homodimerization /-phosphorylation nor the activation state of key downstream signaling proteins analyzed. Conclusions: The unique mode of action of ertumaxomab, which relies more on activation of immune-mediated mechanisms against tumor cells compared with currently available therapeutic antibodies for breast cancer treatment, suggests that modular or sequential treatment with the trifunctional bivalent antibody might complement

  8. The Art of Making Antibodies.

    ERIC Educational Resources Information Center

    Headon, Denis R.

    1986-01-01

    Provides background information for teachers on the nature and production of antibodies. Points out that the production of monoclonal antibodies blends the malignant with the beneficial to create a medical tool of exciting potential. (JN)

  9. Red Blood Cell Antibody Identification

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? RBC Antibody Identification Share this page: Was this page helpful? Also known as: Alloantibody Identification; Antibody ID, RBC; RBC Ab ID Formal name: Red Blood Cell ...

  10. 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.

  11. Anti-smooth muscle antibody

    MedlinePlus

    ... medlineplus.gov/ency/article/003531.htm Anti-smooth muscle antibody To use the sharing features on this page, please enable JavaScript. Anti-smooth muscle antibody is a blood test that detects the ...

  12. Natural antibodies in healthy adults.

    PubMed

    Veres, J

    1975-01-01

    The serum of 100 adults living in Budapest was examined for isohaemagglutinin titre with haemaglutination, for staphylococcal-antitoxin titre with haemolysis inhibition and for bacterial antibody titre against 17 different groups of bacteria with passive haemagglutination. Antibody levels in males, except for certain bacterial antibodies, were somewhat lower than in females. The antibody titres, especially in men, decreased gradually from 20 to 50 years of age and were usually lower in Rh negative than in Rh positive persons.

  13. Antibody-gold cluster conjugates

    DOEpatents

    Hainfeld, J.F.

    1988-06-28

    Antibody- or antibody fragment-gold cluster conjugates are shown wherein the conjugate size can be about 5.0 nm. Methods and reagents are disclosed in which antibodies or Fab' fragments thereof are covalently bound to a stable cluster of gold atoms. 2 figs.

  14. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    PubMed

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed.

  15. Antibody mimetics: promising complementary agents to animal-sourced antibodies.

    PubMed

    Baloch, Abdul Rasheed; Baloch, Abdul Wahid; Sutton, Brian J; Zhang, Xiaoying

    2016-01-01

    Despite their wide use as therapeutic, diagnostic and detection agents, the limitations of polyclonal and monoclonal antibodies have inspired scientists to design the next generation biomedical agents, so-called antibody mimetics that offer many advantages over conventional antibodies. Antibody mimetics can be constructed by protein-directed evolution or fusion of complementarity-determining regions through intervening framework regions. Substantial progress in exploiting human, butterfly (Pieris brassicae) and bacterial systems to design and select mimetics using display technologies has been made in the past 10 years, and one of these mimetics [Kalbitor® (Dyax)] has made its way to market. Many challenges lie ahead to develop mimetics for various biomedical applications, especially those for which conventional antibodies are ineffective, and this review describes the current characteristics, construction and applications of antibody mimetics compared to animal-sourced antibodies. The possible limitations of mimetics and future perspectives are also discussed. PMID:25264572

  16. Antibody Engineering and Therapeutics Conference

    PubMed Central

    Almagro, Juan Carlos; Gilliland, Gary L; Scott, Jamie; Larrick, James W; Plückthun, Andreas; Veldman, Trudi; Adams, Gregory P; Parren, Paul WHI; Chester, Kerry A; Bradbury, Andrew; Reichert, Janice M; Huston, James S

    2013-01-01

    The Antibody Engineering and Therapeutics conference, which serves as the annual meeting of The Antibody Society, will be held in Huntington Beach, CA from Sunday December 8 through Thursday December 12, 2013. The scientific program will cover the full spectrum of challenges in antibody research and development, and provide updates on recent progress in areas from basic science through approval of antibody therapeutics. Keynote presentations will be given by Leroy Hood (Institute of System Biology), who will discuss a systems approach for studying disease that is enabled by emerging technology; Douglas Lauffenburger (Massachusetts Institute of Technology), who will discuss systems analysis of cell communication network dynamics for therapeutic biologics design; David Baker (University of Washington), who will describe computer-based design of smart protein therapeutics; and William Schief (The Scripps Research Institute), who will discuss epitope-focused immunogen design.   In this preview of the conference, the workshop and session chairs share their thoughts on what conference participants may learn in sessions on: (1) three-dimensional structure antibody modeling; (2) identifying clonal lineages from next-generation data sets of expressed VH gene sequences; (3) antibodies in cardiometabolic medicine; (4) the effects of antibody gene variation and usage on the antibody response; (5) directed evolution; (6) antibody pharmacokinetics, distribution and off-target toxicity; (7) use of knowledge-based design to guide development of complementarity-determining regions and epitopes to engineer or elicit the desired antibody; (8) optimizing antibody formats for immunotherapy; (9) antibodies in a complex environment; (10) polyclonal, oligoclonal and bispecific antibodies; (11) antibodies to watch in 2014; and (12) polyreactive antibodies and polyspecificity.

  17. [Antibody therapy for Alzheimer's disease].

    PubMed

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially. PMID:22277519

  18. [Antibody therapy for Alzheimer's disease].

    PubMed

    Tabira, Takeshi; Matsumoto, Shin-Ei; Jin, Haifeng

    2011-11-01

    In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

  19. Targeting antibodies to the cytoplasm.

    PubMed

    Marschall, Andrea L J; Frenzel, André; Schirrmann, Thomas; Schüngel, Manuela; Dübel, Stefan

    2011-01-01

    A growing number of research consortia are now focused on generating antibodies and recombinant antibody fragments that target the human proteome. A particularly valuable application for these binding molecules would be their use inside a living cell, e.g., for imaging or functional intervention. Animal-derived antibodies must be brought into the cell through the membrane, whereas the availability of the antibody genes from phage display systems allows intracellular expression. Here, the various technologies to target intracellular proteins with antibodies are reviewed.

  20. Therapeutic antibodies against cancer

    PubMed Central

    Adler, Mark J.; Dimitrov, Dimiter S.

    2012-01-01

    Antibody-based therapeutics against cancer are highly successful in clinic and currently enjoy unprecedented recognition of their potential; 13 monoclonal antibodies (mAbs) have been approved for clinical use in the European Union and in the United States (one, mylotarg, was withdrawn from market in 2010). Three of the mAbs (bevacizumab, rituximab, trastuzumab) are in the top six selling protein therapeutics with sales in 2010 of more than $5 bln each. Hundreds of mAbs including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs and mAbs with optimized pharmacokinetics are in clinical trials. However, challenges remain and it appears that deeper understanding of mechanisms is needed to overcome major problems including resistance to therapy, access to targets, complexity of biological systems and individual variations. PMID:22520975

  1. Antibody Therapy for Histoplasmosis

    PubMed Central

    Nosanchuk, Joshua D.; Zancopé-Oliveira, Rosely M.; Hamilton, Andrew J.; Guimarães, Allan J.

    2012-01-01

    The endemic human pathogenic fungus Histoplasma capsulatum is a major fungal pathogen with a broad variety of clinical presentations, ranging from mild, focal pulmonary disease to life-threatening systemic infections. Although azoles, such as itraconazole and voriconazole, and amphotericin B have significant activity against H. capsulatum, about 1 in 10 patients hospitalized due to histoplasmosis die. Hence, new approaches for managing disease are being sought. Over the past 10 years, studies have demonstrated that monoclonal antibodies (mAbs) can modify the pathogenesis of histoplasmosis. Disease has been shown to be impacted by mAbs targeting either fungal cell surface proteins or host co-stimulatory molecules. This review will detail our current knowledge regarding the impact of antibody therapy on histoplasmosis. PMID:22347215

  2. Antibody-mediated radiotherapy

    SciTech Connect

    Bloomer, W.D.; Lipsztein, R.; Dalton, J.F.

    1985-05-01

    Antibodies that react with antigens on the surface of tumor cells but not normal cells have great potential for cancer detection and therapy. If radiolabeled without loss of immunologic specificity, such antibodies may be able to deliver cytoxic amounts of radiation. Target- cell specificity and a high extraction coefficient are necessary with any radionuclide in order to minimize normal tissue irradiation. Tumor- cell-retention time and the rate of catabolized radionuclide will also influence ultimate applicability. Among the unanswered questions for choosing a radionuclide is the choice of particle emitter. Although classic beta emitters have been used in a number of clinical situations, they have not had a major impact on disease outcome except in diseases of the thyroid. Unfortunately, Auger emitters such as iodine 125 are cytotoxic only when localized within close proximity to the genome. On the other hand, alpha emitters such as astatine 211 eliminate the need for subcellular sequestration but not cell-specific localization. 34 references.

  3. Prediction of Antibody Epitopes.

    PubMed

    Nielsen, Morten; Marcatili, Paolo

    2015-01-01

    Antibodies recognize their cognate antigens in a precise and effective way. In order to do so, they target regions of the antigenic molecules that have specific features such as large exposed areas, presence of charged or polar atoms, specific secondary structure elements, and lack of similarity to self-proteins. Given the sequence or the structure of a protein of interest, several methods exploit such features to predict the residues that are more likely to be recognized by an immunoglobulin. Here, we present two methods (BepiPred and DiscoTope) to predict linear and discontinuous antibody epitopes from the sequence and/or the three-dimensional structure of a target protein. PMID:26424260

  4. Commercial antibodies and their validation

    PubMed Central

    Voskuil, JLA

    2014-01-01

    Despite an impressive growth in the business of research antibodies a general lack of trust in commercial antibodies remains in place. A variety of issues, each one potentially causing an antibody to fail, underpin the frustrations that scientists endure. Lots of money goes to waste in buying and trying one failing antibody after the other without realizing all the pitfalls that come with the product: Antibodies can get inactivated, both the biological material and the assay itself can potentially be flawed, a single antibody featuring in many different catalogues can be deemed as a set of different products, and a bad choice of antibody type, wrong dilutions, and lack of proper validation can all jeopardize the intended experiments. Antibodies endorsed by scientific research papers do not always meet the scientist’s requirements either due to flawed specifications, or due to batch-to-batch variations. Antibodies can be found with Quality Control data obtained from previous batches that no longer represent the batch on sale. In addition, one cannot assume that every antibody is fit for every application. The best chance of success is to try an antibody that already was confirmed to perform correctly in the required platform. PMID:25324967

  5. Antibody Production with Synthetic Peptides.

    PubMed

    Lee, Bao-Shiang; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Jenny; Gupta, Shalini

    2016-01-01

    Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column. PMID:27515072

  6. Engineering antibody affinity and specificity.

    PubMed

    Webster, D M; Roberts, S; Cheetham, J C; Griest, R; Rees, A R

    1988-01-01

    A combination of ab initio calculations, "knowledge-based prediction", molecular graphics and site-directed mutagenesis has enabled us to probe the molecular details of antibody:antigen recognition and binding and to alter the affinity and specificity of an antibody for its antigen. The significance of electrostatic hydrogen bonding, hydrophilic/hydrophobic patch matching and van der Waals interactions as well as CDR:CDR interactions are discussed in relation to the results of site-directed mutagenesis experiments on the anti-lysozyme antibody Gloop2. The ability to generate reconstructed antibodies, chimeric antibodies, catalytic antibodies and the use of modelled antibodies for the design of drugs is discussed. PMID:3209295

  7. Engineering antibodies by yeast display.

    PubMed

    Boder, Eric T; Raeeszadeh-Sarmazdeh, Maryam; Price, J Vincent

    2012-10-15

    Since its first application to antibody engineering 15 years ago, yeast display technology has been developed into a highly potent tool for both affinity maturing lead molecules and isolating novel antibodies and antibody-like species. Robust approaches to the creation of diversity, construction of yeast libraries, and library screening or selection have been elaborated, improving the quality of engineered molecules and certainty of success in an antibody engineering campaign and positioning yeast display as one of the premier antibody engineering technologies currently in use. Here, we summarize the history of antibody engineering by yeast surface display, approaches used in its application, and a number of examples highlighting the utility of this method for antibody engineering.

  8. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Daugharty, Harry; Hopper, John E.; MacDonald, A. Bruce; Nisonoff, Alfred

    1969-01-01

    Specifically purified anti-p-azobenzoate antibodies of the IgG class from individual rabbits were used to elicit anti-idiotypic antibodies in recipient rabbits. Allotypes of each donor and recipient were matched. When polymerized antibodies were used for immunization, more than 80% of the recipients responded with the formation of antibodies that precipitated the monomeric donor antibody. Percentages of precipitable molecules in the donor antibody population (D) varied from 4 to 56. As little as 4% was readily detectable by the Ouchterlony method or precipitin test. Specificity of the reaction was tested by double diffusion in agar gel against a panel of purified antibenzoate antibodies from 14 heterologous rabbits and, quantitatively, in three systems by measurement of the extent of coprecipitation of heterologous, radiolabeled antibenzoate antibodies. No cross-reactions were observed. Reactions were shown to be attributable to antibenzoate antibodies in the donor serum, and contributions of allotypic reactions were excluded. In three systems investigated quantitatively, and in one studied qualitatively, two recipients of the same donor antibody produced anti-antibody that reacted with essentially the same subfraction of the donor antibody population. The findings that only a portion of the D population is immunogenic, and that the same subfraction is frequently immunogenic in different recipients, suggest that the immunogenic population comprises a limited number of homogeneous groups of antibody molecules. This is supported by the small number of bands usually observed by the Ouchterlony technique. Quantitative methods of analysis should provide an approach to the study of cell populations producing antibodies of a particular idiotype. PMID:5347693

  9. Contributions of the Complementarity Determining Regions to the Thermal Stability of a Single-Domain Antibody

    PubMed Central

    Zabetakis, Dan; Anderson, George P.; Bayya, Nikhil; Goldman, Ellen R.

    2013-01-01

    Single domain antibodies (sdAbs) are the recombinantly-expressed variable domain from camelid (or shark) heavy chain only antibodies and provide rugged recognition elements. Many sdAbs possess excellent affinity and specificity; most refold and are able to bind antigen after thermal denaturation. The sdAb A3, specific for the toxin Staphylococcal enterotoxin B (SEB), shows both sub-nanomolar affinity for its cognate antigen (0.14 nM) and an unusually high melting point of 85°C. Understanding the source of sdAb A3’s high melting temperature could provide a route for engineering improved melting temperatures into other sdAbs. The goal of this work was to determine how much of sdAb A3’s stability is derived from its complementarity determining regions (CDRs) versus its framework. Towards answering this question we constructed a series of CDR swap mutants in which the CDRs from unrelated sdAbs were integrated into A3’s framework and where A3’s CDRs were integrated into the framework of the other sdAbs. All three CDRs from A3 were moved to the frameworks of sdAb D1 (a ricin binder that melts at 50°C) and the anti-ricin sdAb C8 (melting point of 60°C). Similarly, the CDRs from sdAb D1 and sdAb C8 were moved to the sdAb A3 framework. In addition individual CDRs of sdAb A3 and sdAb D1 were swapped. Melting temperature and binding ability were assessed for each of the CDR-exchange mutants. This work showed that CDR2 plays a critical role in sdAb A3’s binding and stability. Overall, results from the CDR swaps indicate CDR interactions play a major role in the protein stability. PMID:24143255

  10. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    PubMed

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  11. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    PubMed

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  12. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  13. Induction of antihemagglutinin antibodies by polyclonal antiidiotype antibodies.

    PubMed

    Dinca, L; Neuwirth, S; Schulman, J; Bona, C

    1993-01-01

    Antiidiotypic antibodies can be envisioned as an alternative approach in the development of vaccines against influenza virus, which exhibits natural antigenic variations. In our work, we obtained two polyclonal cross-reactive anti-Id antibodies against PY102, VM113, and VM202 mAbs, which in turn are specific respectively for PR8 virus and laboratory-induced virus variants (PY102-V1 and VM113-V1). With these cross-reactive anti-Id antibodies, we were able to elicit anti-HA antibodies in mice. In comparing the anti-HA antibody response in animals injected with anti-Id antibodies to those immunized with PR8 influenza virus, we demonstrated that the HI titer was higher after virus immunization and that the PR8 virus boost was more efficient in this group. Our results showed that the polyclonal cross-reactive anti-Id antibodies were more efficient than the individual anti-Ids at eliciting responses. At the same time, we demonstrated that PR8-primed T cells, cultured with B cells from animals immunized with anti-Id antibodies, were able to produce anti-PR8 antibodies subsequent to stimulation with influenza virus. PMID:8476510

  14. Induction of antihemagglutinin antibodies by polyclonal antiidiotype antibodies.

    PubMed

    Dinca, L; Neuwirth, S; Schulman, J; Bona, C

    1993-01-01

    Antiidiotypic antibodies can be envisioned as an alternative approach in the development of vaccines against influenza virus, which exhibits natural antigenic variations. In our work, we obtained two polyclonal cross-reactive anti-Id antibodies against PY102, VM113, and VM202 mAbs, which in turn are specific respectively for PR8 virus and laboratory-induced virus variants (PY102-V1 and VM113-V1). With these cross-reactive anti-Id antibodies, we were able to elicit anti-HA antibodies in mice. In comparing the anti-HA antibody response in animals injected with anti-Id antibodies to those immunized with PR8 influenza virus, we demonstrated that the HI titer was higher after virus immunization and that the PR8 virus boost was more efficient in this group. Our results showed that the polyclonal cross-reactive anti-Id antibodies were more efficient than the individual anti-Ids at eliciting responses. At the same time, we demonstrated that PR8-primed T cells, cultured with B cells from animals immunized with anti-Id antibodies, were able to produce anti-PR8 antibodies subsequent to stimulation with influenza virus.

  15. Antibody Glossary —

    Cancer.gov

    The components of the immune system have diverse roles in the initial development of cancers, progression of early-stage malignancies to invasive tumors, establishment of metastatic lesions, tumor dormancy, and response or resistance to therapy. Characterizing the components of the immune system and their functional status in tissues and in tumors requires the use of highly specific reagents. Researchers employ antibodies in a variety of in vitro and in vivo applications to delineate, enrich, or deplete specific immune subsets in order to understand their role(s) in tumorigenesis. This is a glossary of validated reagents and protocols that are useful for functional phenotyping of the immune system in murine cancer models.

  16. Structural analysis of effector functions related motifs, complement activation and hemagglutinating activities in Lama glama heavy chain antibodies.

    PubMed

    Saccodossi, Natalia; De Simone, Emilio A; Leoni, Juliana

    2012-01-15

    Heavy chain antibodies (HCAbs), devoid of the light chains and the CH(1) domain, are present in the serum of camelids. IgG(2) and IgG(3) are HCAbs; whereas IgG(1) has the conventional structure. In order to study the immunological properties of llama HCAbs, from which to date little is known, llamas (Lama glama) HCAbs cDNA were cloned, sequenced and compared with other mammalian Igs. The sequence analysis showed that llama HCAbs cDNA organization is similar to other mammalian Igs and the presence of conserved binding motifs to Protein A, Protein G, FcγRI, FcγRIII and C1q in HCAbs were observed. In a previous work, different IgG isotypes purified by Protein A and Protein G chromatography, were assayed for their ability to fix complement. Both IgG(1) and the total serum were able to fix complement, whereas IgG(2) and IgG(3) fixed complement even in the absence of antigen (anti-complementary activity). Therefore, in this work we performed the complement activating activity of the different IgG isotypes purified under physiological conditions using Sephadex G-150 and their ability to induce hemagglutination. Llamas were immunized with sheep red blood cells (RBC) stroma and the different isotypes were purified from sera. Whole serum and IgG(1) could activate complement; however, HCAbs (IgG(2)+IgG(3)) could not, despite the presence of the C1q binding motif in their primary sequence. Unlike IgG(1), the fraction corresponding to IgG(2)+IgG(3) did not display hemagglutinating activity. Our findings suggest that HCAbs cannot crosslink efficiently with different antigens and that the C1q binding site might be hindered by the proximity of the variable domains. PMID:22197565

  17. Guinea-pig reaginic antibody

    PubMed Central

    Margni, R. A.; Hajos, Silvia E.

    1973-01-01

    The physicochemical and biological properties of purified guinea-pig reaginic antibody were studied. It is a labile protein different to γ1. Its antibody activity is completely destroyed by heating at 56° for 6 hours and by treatment with mercaptoethanol. The capacity to give PCA is decreased by repeated freezing and thawing. It is a bivalent antibody, haemagglutinating, does not fix complement and is capable of sensitizing guinea-pig skin for PCA reaction after a latent period of a week but not after 3 hours. Reaginic antibody appears on day 7–8 after the first inoculation and the higher levels (PCA reaction) are obtained at the eleventh to thirteenth days. After the fifteenth to seventeenth days the PCA is negative. The reaginic antibody does not pass the placenta. Higher levels of reaginic antibody were obtained when the guinea-pigs were inoculated with the antigen in saline with simultaneous inoculation, intraperitoneally, of killed Bordetella pertussis, phase I. PMID:4354828

  18. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies. PMID:20954327

  19. [Targeted therapy by monoclonal antibodies].

    PubMed

    Ohnuma, Kei; Morimoto, Chikao

    2010-10-01

    Human monoclonal antibodies are virtually indispensable for immunotherapy of cancer, infectious diseases, autoimmune diseases, or organ transplantation. The hybridoma technique, developed by Georges Köhler and César Milstein in 1975, has been shown to be most and highly producible method for generating murine monoclonal antibodies. However, poor results were obtained when it was administered in human bodies. With development of biotechnology, human monoclonal antibodies have been manufactured with higher efficiency. A major hindrance of producing therapeutic human monoclonal antibodies is the lack of an appropriate strategy for determining and selecting the antibodies that would be effective in vivo. In this review, we give an overview of the present techniques on therapeutic monoclonal antibodies.

  20. Monoclonal antibodies to Actinobacillus actinomycetemcomitans.

    PubMed Central

    Place, D A; Scidmore, N C; McArthur, W P

    1988-01-01

    Murine hybridoma cell lines were developed which synthesized monoclonal antibodies against Actinobacillus actinomycetemcomitans-associated antigens. Monoclonal antibodies specific for an antigen(s) common to all A. actinomycetemcomitans isolates tested but not detected on other gram-negative oral plaque microorganisms or other Actinobacillus species were identified. Monoclonal antibodies specific for each serotype group of A. actinomycetemcomitans which did not bind to other Actinobacillus species or oral plaque microorganisms were also identified. PMID:3356470

  1. A solid phase antibody screen.

    PubMed

    Plapp, F V; Sinor, L T; Rachel, J M; Beck, M L; Coenen, W M; Bayer, W L

    1984-12-01

    An automated solid phase antibody screen (SPAS) in microplates has been developed. Red blood cell (RBC) adherence was used as the end point instead of agglutination. Consequently, positive and negative reactions were readily distinguished by a microplate spectrophotometer. The SPAS performed as well as conventional antiglobulin methods for detecting IgG antibodies in donor sera and had increased sensitivity as determined by serial dilutions of antibodies.

  2. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Hopper, John E.; MacDonald, A. Bruce; Nisonoff, Alfred

    1970-01-01

    Idiotypic antibodies were investigated quantitatively by a method of indirect precipitation, which utilizes labeled F(ab')2 fragments of specifically purified antibenzoate antibody from the donor, anti-antibody, and an antiglobulin reagent. The contribution of allotypic and hidden determinants to these reactions was excluded. Greater fractions of an idiotypic antibody population are precipitated by this method, as compared to direct precipitation, and in two instances large proportions of idiotypic antibodies were detected in populations which failed to form precipitates by double diffusion in agar gel. The greater sensitivity of the indirect method was attributed to its capacity to detect molecules bearing a small number of antigenic determinants. Extensive studies of cross-reactions, carried out by an inhibition technique, failed to reveal any strong reactions of anti-idiotypic antibodies with heterologous antibenzoate antibody preparations, heterologous sera, or IgG, although a few weak cross-reactions were noted. One definite cross-reaction was observed by a direct binding measurement with heterologous antiserum. Antisera prepared in more than one recipient against a single donor preparation reacted with identical or overlapping subpopulations of the donor molecules. Instances in which two recipient antisera reacted with different proportions of the molecules of a single donor provided evidence for the existence of more than one idiotypic antibody population in the antibenzoate antibody of an individual rabbit. PMID:5308065

  3. Monoclonal antibodies and neuroblastoma

    SciTech Connect

    Miraldi, F. )

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.48 references.

  4. Monoclonal antibodies and neuroblastoma.

    PubMed

    Miraldi, F

    1989-10-01

    Several antineuroblastoma monoclonal antibodies (MoAbs) have been described and two have been used in radioimmunoimaging and radioimmunotherapy in patients. MoAb 3F8 is a murine IgG3 antibody specific for the ganglioside GD2. Radioiodine-labeled 3F8 has been shown to specifically target human neuroblastoma in patients, and radioimmunoimaging with this agent has provided consistently high uptakes with tumor-to-background ratios of greater than or equal to 10:1. Radioimmunotherapy has been attempted with both MoAb 3F8 and MoAb UJ13A, and although encouraging results have been obtained, dosimetry data and tissue dose response information for these agents is lacking, which impedes the development of such therapy. 124I, a positron emitter, can be used with 3F8 in positron emission tomography (PET) scanning to provide dosimetry information for radioimmunotherapy. The tumor radiation dose response from radiolabeled MoAb also can be followed with PET images with fluorodeoxyglucose (FDG) scanning of neuroblastoma tumors. Results to date indicate that radioimmunoimaging has clinical use in the diagnosis of neuroblastoma and the potential for radioimmunotherapy for this cancer remains high.

  5. Creating Ordered Antibody Arrays with Antibody-Polymer Conjugates

    NASA Astrophysics Data System (ADS)

    Dong, Xuehui; Obermeyer, Allie; Olsen, Bradley

    Antibodies are a category of functional proteins that play crucial roles in the immune system and have been widely applied in the area of cancer therapeutics, targeting delivery, signal detection, and sensors. Due to the extremely large size and lack of specific functional groups on the surface, it is challenging to functionalize antibodies and manipulate the ordered packing of antibodies in an array with high density and proper orientation, which is critical to achieve outstanding performance in materials. In this work, we demonstrate an efficient and facile approach for preparing antibody-polymer conjugates with two-step sequential ``click'' reaction to form antibody-polymer block copolymers. Highly ordered nanostructures are fabricated based on the principles of block copolymer self-assembly. The nanostructures are studied with both small angle X-ray scattering (SAXS) and transmission electron microscopy (TEM). Lamellae with alternating antibody domain and polymer domain are observed with an overall domain size of ~50 nm. The nanostructure not only increases the packing density and promotes proper orientation of the antibody, but also provides possible channel to facilitate substrate transportation and improves the stability of the antibody.

  6. Antibodies: Protective, destructive and regulatory role

    SciTech Connect

    Milgrom, F.; Abeyounis, C.J.; Albini, B.

    1985-01-01

    This book contains papers under 10 subject headings. The headings are: Production and Function of Antibodies, Protective Role of Antibodies, Antibodies to Foreign and Neoplastic Cells, Autoantibodies, Regulatory Mechanisms, Allergy, Immune Complexes, Antibodies in Pregnancy and Aging, Administration of Antibodies for Prevention and Therapy, and Abstracts of Poster Presentations.

  7. Production of monoclonal antibodies.

    PubMed

    Freysd'ottir, J

    2000-01-01

    The discovery of monoclonal antibodies (mAbs) produced by "hybridoma technology" by George Köhler and Cesar Milstein in 1975 has had a great impact both on basic biological research and on clinical medicine. However, this impact was not immediately recognized. It took around 10 years to appreciate the importance of using these mAbs in various fields of science other than immunology, such as cell biology, biochemistry, microbiology, virology, para-sitology, physiology, genetics, and molecular biology; and also in areas of clinical medicine, such as pathology, hematology, oncology, and infectious disease. The contribution of mAbs to science and clinical medicine was recognized in 1984 by the award of the Nobel Prize for Medicine to Köhler and Milstein.

  8. Micromechanical antibody sensor

    DOEpatents

    Thundat, Thomas G.; Jacobson, K. Bruce; Doktycz, Mitchel J.; Kennel, Stephen J.; Warmack, Robert J.

    2001-01-01

    A sensor apparatus is provided using a microcantilevered spring element having a coating of a detector molecule such as an antibody or antigen. A sample containing a target molecule or substrate is provided to the coating. The spring element bends in response to the stress induced by the binding which occurs between the detector and target molecules. Deflections of the cantilever are detected by a variety of detection techniques. The microcantilever may be approximately 1 to 200 .mu.m long, approximately 1 to 50 .mu.m wide, and approximately 0.3 to 3.0 .mu.m thick. A sensitivity for detection of deflections is in the range of 0.01 nanometers.

  9. Radiolabeled antibodies in gynecologic tumors

    SciTech Connect

    Hardy, J.G.; Perkins, A.C.; Symonds, E.M.; Wastie, M.L.; Pimm, M.V.

    1984-01-01

    A monoclonal antibody has been raised against an osteogenic sarcoma cell line and radiolabeled with iodine-131. The antibody was administered to 12 patients with suspected ovarian tumors, two with recurrent carcinoma of the cervix and one with carcinoma of the body of the uterus. Each patient received an intravenous dose of 70 MBq I-131-labeled antibody and was imaged either 24 or 48 hours later. Image enhancement was achieved by subtraction of background activity using Tc-99m-labeled red blood cells and pertechnetate. In eleven patients with ovarian malignancies antibody uptake was detected at the suspected tumor sites, and agreed with the operative findings in the eight patients who subsequently underwent surgery. The patient in whom the antibody failed to localize was found to have a benign lesion. Uptake of antibody was seen at the tumor sites in the patients with carcinoma of the cervix and body of the uterus. The localization of tumor sites using I-131-labeled antibodies is difficult due to background activity, particularly from radioiodine in the bladder. In only five cases could the abnormal antibody concentration be identified on the iodine images alone. This problem was overcome by the use of background subtraction techniques. Immunoscintigraphy is proving useful for the assessment of tumor recurrence and as an aid to radiotherapy treatment planning.

  10. 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

  11. Properties of protective malarial antibody

    PubMed Central

    Cohen, S.; Butcher, G. A.

    1970-01-01

    Properties of protective malarial antibody have been studied in cultures of P. knowlesi giving average parasite multiplication rates of sixfold in 24 hours. Parasite growth was assessed by incorporation of [3H]-leucine into protein. Immune serum has little effect upon the growth of intracellular parasites, but prevents reinvasion of red cells and inhibits the succeeding cycle of parasite development. Protective antibody is present in relatively low titre in immune sera even after long immunization and this may explain certain characteristic features of malarial immunity. Protective antibody in the sera studied is associated with IgG and IgM; its action is not complement dependent, but requires at least two combining sites per molecule. Anti-malarial antibody has several features in common with viral neutralizing antibody. PMID:4990404

  12. Endogenous Antibodies for Tumor Detection

    PubMed Central

    Rich, Barrie S.; Honeyman, Joshua N.; Darcy, David G.; Smith, Peter T.; Williams, Andrew R.; Lim, Irene Isabel P.; Johnson, Linda K.; Gönen, Mithat; Simon, Joel S.; LaQuaglia, Michael P.; Simon, Sanford M.

    2014-01-01

    The study of cancer immunology has provided diagnostic and therapeutic instruments through serum autoantibody biomarkers and exogenous monoclonal antibodies. While some endogenous antibodies are found within or surrounding transformed tissue, the extent to which this exists has not been entirely characterized. We find that in transgenic and xenograft mouse models of cancer, endogenous gamma immunoglobulin (IgG) is present at higher concentration in malignantly transformed organs compared to non-transformed organs in the same mouse or organs of cognate wild-type mice. The enrichment of endogenous antibodies within the malignant tissue provides a potential means of identifying and tracking malignant cells in vivo as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is possible through the use of agents that bind endogenous antibodies. PMID:24875800

  13. Metrics for antibody therapeutics development.

    PubMed

    Reichert, Janice M

    2010-01-01

    A wide variety of full-size monoclonal antibodies (mAbs) and therapeutics derived from alternative antibody formats can be produced through genetic and biological engineering techniques. These molecules are now filling the preclinical and clinical pipelines of every major pharmaceutical company and many biotechnology firms. Metrics for the development of antibody therapeutics, including averages for the number of candidates entering clinical study and development phase lengths for mAbs approved in the United States, were derived from analysis of a dataset of over 600 therapeutic mAbs that entered clinical study sponsored, at least in part, by commercial firms. The results presented provide an overview of the field and context for the evaluation of on-going and prospective mAb development programs. The expansion of therapeutic antibody use through supplemental marketing approvals and the increase in the study of therapeutics derived from alternative antibody formats are discussed.

  14. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Brient, Bruce W.; Nisonoff, Alfred

    1970-01-01

    Rabbit anti-idiotypic antibodies were prepared by injection of specifically purified anti-p-azobenzoate antibodies (D) from individual donor rabbits. Benzoate derivatives were found to be strong inhibitors of the reactions of D with anti-D antisera. There was a close correlation between the combining affinities of the benzoate derivatives used and their effectiveness as inhibitors. Compounds tested that are chemically unrelated to benzoate were ineffective. The results indicate either that the combining site of anti-benzoate antibody is part of an important idiotypic determinant, which is sterically blocked by hapten, or that the hapten induces a conformational change which alters idiotypic determinants not involving the active site. Such conformational changes, if they occur, must be restricted since hapten has little effect on the reactions of F(ab')2 fragments of anti-benzoate antibodies with antisera directed to rabbit fragment Fab and no detectable effect on reactions with antibodies directed to allotypic determinants. PMID:4097134

  15. RosettaAntibody: antibody variable region homology modeling server.

    PubMed

    Sircar, Aroop; Kim, Eric T; Gray, Jeffrey J

    2009-07-01

    The RosettaAntibody server (http://antibody.graylab.jhu.edu) predicts the structure of an antibody variable region given the amino-acid sequences of the respective light and heavy chains. In an initial stage, the server identifies and displays the most sequence homologous template structures for the light and heavy framework regions and each of the complementarity determining region (CDR) loops. Subsequently, the most homologous templates are assembled into a side-chain optimized crude model, and the server returns a picture and coordinate file. For users requesting a high-resolution model, the server executes the full RosettaAntibody protocol which additionally models the hyper-variable CDR H3 loop. The high-resolution protocol also relieves steric clashes by optimizing the CDR backbone torsion angles and by simultaneously perturbing the relative orientation of the light and heavy chains. RosettaAntibody generates 2000 independent structures, and the server returns pictures, coordinate files, and detailed scoring information for the 10 top-scoring models. The 10 models enable users to use rational judgment in choosing the best model or to use the set as an ensemble for further studies such as docking. The high-resolution models generated by RosettaAntibody have been used for the successful prediction of antibody-antigen complex structures.

  16. Epstein-Barr virus antibody test

    MedlinePlus

    EBV antibody test; EBV serology ... a lab, where a lab specialist looks for antibodies to the Epstein-Barr virus. In the first stages of an illness, little antibody may be detected. For this reason, the test ...

  17. Antibodies - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    NCI announces the release of monoclonal antipeptide antibodies from rabbit for distribution on the antibody portal. There are 60 recently added monoclonal antibodies, with 56 generated from mouse and 4 generated from rabbit.

  18. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Kuettner, Mirta Goffan; Wang, Ai-Lan; Nisonoff, Alfred

    1972-01-01

    Antisera were prepared in rabbits against anti-p-azobenzoate antibodies of an A/J and a BALB/c mouse and anti-p-azophenylarsonate antibodies of an A/J mouse. After appropriate absorption the antisera reacted with the anti-hapten antibody of the donor mouse but, by sensitive quantitative tests, not at all with other components of the hyperimmune serum or with preimmune serum of the donor mouse. The absorbed antiserum therefore appeared to be specific for idiotypic determinants. Nearly all idiotypic specificities identified in the serum of the donor were also present in the serum of other mice of the same strain, immunized against the same hapten group, but not in mice immunized with a different hapten. In each case the antibodies of the donor mouse reacted most effectively on a weight basis with antiidiotypic antiserum. Cross-reactions were observed among different strains of mice but homologous anti-bodies reacted most effectively with antiidiotypic antisera. C57/BL and DBA antisera contained very low concentrations of specificities present in the A/J and BALB/c antibody populations; antibodies of A/J and BALB/c antisera are more closely related to one another. The results indicate that idiotypic specificity may provide a genetic marker for the variable regions of immunoglobulin polypeptide chains. PMID:4110016

  19. The therapeutic monoclonal antibody market

    PubMed Central

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

  20. The therapeutic monoclonal antibody market.

    PubMed

    Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

    2015-01-01

    Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼ 70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion.

  1. Reducing heterophilic antibody interference in immunoassays using single chain antibodies

    SciTech Connect

    Baird, Cheryl L.; Tan, Ruimin; Fischer, Christopher J.; Victry, Kristin D.; Zangar, Richard C.; Rodland, Karin D.

    2011-12-15

    Sandwich ELISA microarrays have the potential to simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as seen with traditional ELISA diagnostics, heterophilic antibodies (HA) in patient sera have the potential to cause interference in these assays. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit, the variable heavy and light domains artificially connected with a short polypeptide linker (scFv), is an effective strategy for reducing the HA assay interference.

  2. Heterophile antibodies in human transplantation

    PubMed Central

    Rapaport, F. T.; Kano, K.; Milgrom, F.

    1968-01-01

    Sensitization of human recipients with transplantation antigens (leucocytes, skin, or kidney allografts) has resulted in the appearance of serum hemagglutinins directed against sheep, guinea pig, and rat erythrocytes. Such hemagglutinins have been identified as IgG and IgM antibodies. Their appearance was not related to AB0 erythrocyte group incompatibility between donors and recipients, and the antibodies were not of the Forssman or Paul-Bunnel type. The antibody responses appeared to be primarily directed against antigen(s) present on rat erythrocytes, but shared to varying extents by other species. The peak antibody titers occurred in association with allograft rejection. In this regard, they may be of interest as a possible early warning system for the diagnosis and prompt management of rejection crises in clinical organ transplantation. Images PMID:4866325

  3. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  4. Antiphospholipid antibodies and hypertension.

    PubMed

    Rollino, C; Boero, R; Elia, F; Montaruli, B; Massara, C; Beltrame, G; Ferro, M; Quattrocchio, G; Quarello, F

    2004-01-01

    Hypertension is a common manifestation of antiphospholipid syndrome (APS). Antiphospholipid antibodies (aPL) have been described in patients with hypertension secondary to renal artery stenosis (RAS). Twenty-six patients with RAS and 25 patients with severe essential hypertension (diastolic blood pressure > 110 mmHg or > or = 3 hypertensive drugs) were studied and compared to 61 age- and sex-matched healthy subjects. Serum samples were tested for lupus anticoagulant (LA), anticardiolipin (aCL) IgG and IgM, antiprothrombin (aPT) IgG and IgM, anti-beta2glycoprotein 1 (abeta2GP1) IgG and IgM. aPL were negative in all patients with RAS. Two patients with essential hypertension had positive aPL (8%) (LA in one patient confirmed in a second assay and abeta2GP1-IgG in the other patient confirmed one year later together with aCL IgG positivity). Among healthy subjects, one case (1.6%) was found to be positive for LA, aCL IgM, abeta2GP1 IgM, aPT IgG, aPT IgM. In conclusion, the association between RAS and aPL seems to be casual rather than an expression of an elective thrombotic localization ofAPS. The positive finding of aPL in 8% of patients with essential hypertension, a frequency higher than that of the control population, deserves further studies in larger series to better explore the relationship between aPL and hypertension. PMID:15540508

  5. Antibodies to watch in 2015.

    PubMed

    Reichert, Janice M

    2015-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 7 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, blinatumomab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other 'antibodies to watch' are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are 'antibodies to watch' in 2015 because of their potential for entry into the US market and regulatory review, respectively.

  6. Novel antibodies as anticancer agents.

    PubMed

    Zafir-Lavie, I; Michaeli, Y; Reiter, Y

    2007-05-28

    In recent years antibodies, whether generated by traditional hybridoma technology or by recombinant DNA strategies, have evolved from Paul Ehrlich's 'magic bullets' to a modern age 'guided missile'. In the recent years of immunologic research, we are witnessing development in the fields of antigen screening and protein engineering in order to create specific anticancer remedies. The developments in the field of recombinant DNA, protein engineering and cancer biology have let us gain insight into many cancer-related mechanisms. Moreover, novel techniques have facilitated tools allowing unique distinction between malignantly transformed cells, and regular ones. This understanding has paved the way for the rational design of a new age of pharmaceuticals: monoclonal antibodies and their fragments. Antibodies can select antigens on both a specific and a high-affinity account, and further implementation of these qualities is used to target cancer cells by specifically identifying exogenous antigens of cancer cell populations. The structure of the antibody provides plasticity resonating from its functional sites. This review will screen some of the many novel antibodies and antibody-based approaches that are being currently developed for clinical applications as the new generation of anticancer agents. PMID:17530025

  7. Antibodies to watch in 2014.

    PubMed

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry's progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the "Antibodies to watch" series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration's Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914

  8. Antibodies to watch in 2015.

    PubMed

    Reichert, Janice M

    2015-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 7 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, blinatumomab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other 'antibodies to watch' are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are 'antibodies to watch' in 2015 because of their potential for entry into the US market and regulatory review, respectively. PMID:25484055

  9. Antibodies to watch in 2014

    PubMed Central

    Reichert, Janice M

    2014-01-01

    Since 2010, mAbs has documented the biopharmaceutical industry’s progress in transitioning antibody therapeutics to first Phase 3 clinical studies and regulatory review, and its success at gaining first marketing approvals for antibody-based products. This installment of the “Antibodies to watch” series outlines events anticipated to occur between December 2013 and the end of 2014, including first regulatory actions on marketing applications for vedolizumab, siltuximab, and ramucirumab, as well as the Fc fusion proteins Factor IX-Fc and Factor VIII-Fc; and the submission of first marketing applications for up to five therapeutics (secukinumab, ch14.18, onartuzumab, necitumumab, gevokizumab). Antibody therapeutics in Phase 3 studies are described, with an emphasis on those with study completion dates in 2014, including antibodies targeting interleukin-17a or the interleukin-17a receptor (secukinumab, ixekizumab, brodalumab), proprotein convertase subtilisin/kexin type 9 (alirocumab, evolocumab, bococizumab), and programmed death 1 receptor (lambrolizumab, nivolumab). Five antibodies with US Food and Drug Administration’s Breakthrough Therapy designation (obinutuzumab, ofatumumab, lambrolizumab, bimagrumab, daratumumab) are also discussed. PMID:24284914

  10. Natural monoclonal antibodies and cancer.

    PubMed

    Vollmers, Peter H; Brändlein, Stephanie

    2008-06-01

    Immunity is responsible for recognition and elimination of infectious particles and for removal of cellular waste, modified self structures and transformed cells. Innate or natural immunity acts as a first line defense and is also the link to acquired immunity and memory. By using the human hybridoma technology, a series of monoclonal antibodies and several new tumor-specific targets could be identified. A striking phenomenon of immunity against malignant cells is that all so far isolated tumor-specific antibodies were germ-line coded natural IgM antibodies. And neither in animals nor in humans affinity-maturated tumor-specific IgG antibodies have been detected so far. These IgM's preferentially bind to carbohydrate epitopes on post-transcriptionally modified surface receptors, which are recently patented and preferentially remove malignant cells by inducing apoptosis to avoid inflammatory processes. Our "biology-" or "function-driven" method represents a unique yet powerful approach compared to the typical approaches on screening compounds or antibodies against non-validated targets (mostly differentially expressed). Moreover, the approach creates a competitive patenting strategy of creating proprietary antibodies and validated targets at the same time, which has the potential of further streamlining the discovery of new cancer therapies. PMID:18537750

  11. Computer-aided antibody design

    PubMed Central

    Kuroda, Daisuke; Shirai, Hiroki; Jacobson, Matthew P.; Nakamura, Haruki

    2012-01-01

    Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (VH) and light (VL) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody–antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development. PMID:22661385

  12. Antibodies to watch in 2015

    PubMed Central

    Reichert, Janice M

    2015-01-01

    The commercial pipeline of recombinant antibody therapeutics is robust and dynamic. As of early December 2014, a total of 6 such products (vedolizumab, siltuximab, ramucirumab, pembrolizumab, nivolumab, blinatumomab) were granted first marketing approvals in 2014. As discussed in this perspective on antibodies in late-stage development, the outlook for additional approvals, potentially still in 2014 and certainly in 2015, is excellent as marketing applications for 6 antibody therapeutics (secukinumab, evolocumab, mepolizumab, dinutuximab, nivolumab, necitumumab) are undergoing a first regulatory review in the EU or US. Of the 39 novel mAbs currently in Phase 3 studies, a marketing application for one (alirocumab) may be submitted in late 2014, and marketing application submissions for at least 4 (reslizumab, ixekizumab, ocrelizumab, obiltoxaximab) are expected in 2015. Other ‘antibodies to watch’ are those in Phase 3 studies with estimated primary completion dates in late 2014 or 2015, which includes 13 for non-cancer indications (brodalumab, bimagrumab, bococizumab, MABp1, gevokizumab, dupilumab, sirukumab, sarilumab, tildrakizumab, guselkumab, epratuzumab, combination of actoxumab + bezlotoxumab, romosozumab) and 2 (racotumomab and clivatuzumab tetraxetan) undergoing evaluation as treatments for cancer. In addition to the novel antibody therapeutics mentioned, biosimilar infliximab and biosimilar trastuzumab are ‘antibodies to watch’ in 2015 because of their potential for entry into the US market and regulatory review, respectively. PMID:25484055

  13. Avian Diagnostic and Therapeutic Antibodies

    SciTech Connect

    Bradley, David Sherman

    2012-12-31

    A number of infectious agents have the potential of causing significant clinical symptomology and even death, but dispite this, the number of incidence remain below the level that supports producing a vaccine. Therapeutic antibodies provide a viable treatment option for many of these diseases. We proposed that antibodies derived from West Nile Virus (WNV) immunized geese would be able to treat WNV infection in mammals and potential humans. We demonstrated that WNV specific goose antibodies are indeed successful in treating WNV infection both prophylactically and therapeutically in a golden hamster model. We demonstrated that the goose derived antibodies are non-reactogenic, i.e. do not cause an inflammatory response with multiple exposures in mammals. We also developed both a specific pathogen free facility to house the geese during the antibody production phase and a patent-pending purification process to purify the antibodies to greater than 99% purity. Therefore, the success of these study will allow a cost effective rapidly producible therapeutic toward clinical testing with the necessary infrastructure and processes developed and in place.

  14. Antibody therapeutics - the evolving patent landscape.

    PubMed

    Petering, Jenny; McManamny, Patrick; Honeyman, Jane

    2011-09-01

    The antibody patent landscape has evolved dramatically over the past 30 years, particularly in areas of technology relating to antibody modification to reduce immunogenicity in humans or improve antibody function. In some cases antibody techniques that were developed in the 1980s are still the subject of patent protection in the United States or Canada.

  15. Alternative downstream processes for production of antibodies and antibody fragments.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke

    2014-11-01

    Protein-A or Protein-L affinity chromatography and virus inactivation are key processes for the manufacturing of therapeutic antibodies and antibody fragments. These two processes often involve exposure of therapeutic proteins to denaturing low pH conditions. Antibodies have been shown to undergo conformational changes at low pH, which can lead to irreversible damages on the final product. Here, we review alternative downstream approaches that can reduce the degree of low pH exposure and consequently damaged product. We and others have been developing technologies that minimize or eliminate such low pH processes. We here cover facilitated elution of antibodies using arginine in Protein-A and Protein-G affinity chromatography, a more positively charged amidated Protein-A, two Protein-A mimetics (MEP and Mabsorbent), mixed-mode and steric exclusion chromatography, and finally enhanced virus inactivation by solvents containing arginine. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. PMID:24859179

  16. Engineered antibodies take center stage.

    PubMed

    Huston, J S; George, A J

    2001-01-01

    The start of the post-genomic era provides a useful juncture for reflection on the state of antibody engineering, which will be a critical technology for relating function and pathology to genomic sequence in biology and medicine. The phenomenal progress in deciphering the human genome has given significant impetus to the application of engineered antibodies in proteomics. Thus, advances in phage display antibody libraries can now help to define novel gene function and the measurement of abnormal protein expression in pathological states. Furthermore, intrabody and antibody engineering provide vehicles for the development of molecular medicines of the future. In addition to these new directions, antibody engineering has begun to show concrete success in its long-term efforts to develop targeted immunotherapies for cancer and other diseases. The cornerstones of clinical development are the detailed academic clinical trials that continue to push the boundaries of engineered antibodies into the real world. The field displays a healthy impatience for practical results, as research accelerates with concerted efforts to transfer preclinical insights into clinical trials. Growing private and governmental expenditures will lead to the rapid expansion of life-saving immunotherapeutic agents. The present review developed from our effort to report on the 11th Annual International Conference on Antibody Engineering (3-6 December 2000). This annual meeting is a forum for discussions on the latest advances in antibody engineering groups from around the world, and now includes the broader agenda of engineering in molecular immunology. In bringing scientists together to exchange ideas at this open forum, new collaborations and the threads of new discoveries are woven. For example, Professors Gerhard Wagner (Harvard Medical School), Dennis Burton (Scripps Research Institute), and Peter Hudson (CSIRO, Melbourne, Australia) gave exciting insights on structural immunobiology that had

  17. Antibodies and immunoassays.

    PubMed

    Madersbacher, S; Berger, P

    2000-05-01

    As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a single molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation variants, such as its free beta (hCGbeta) and alpha (hCGalpha) subunits and clipped, cleaved, terminally differently sialylated, and overglycosylated forms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is highly dependent on the selection of target molecules and the natural variability of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immunoassays are still the state-of-the-art technique for both clinical and research applications but a major problem is the different extents of recognition of hCG variants by mAbs used in different immunoassays. On the whole, construction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, cross-reactivities, epitope locations and compatibilities, overlap and overhang in specificities (pairs of mAbs), and, finally, overspecificity. Consequences of overhang and overlap in antigen recognition of coating and detection mAb specificities are nondesirable assay cross-reactions and competitive interference by antigenic variants. The general agreement on the most favorable assay design is contrasted by the variety of isotopic and nonisotopic detection systems in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity. By measuring substrate absorption values off the absorption maximum, the measuring range of any IEMA can be extended significantly, as shown for 3,3',5, 5'-tetramethylbenzidine (TMB), without jeopardizing assay characteristics. Sensitivity of the IEMA can be enhanced by modifying the horseradish peroxidase (HRPO) labeling technique by using highly purified m

  18. [Correlation between measles-neutralizing antibody and HI antibody, between measles-neutralizing antibody and PA antibody among pregnant women, and protective levels of three titration types].

    PubMed

    Takayama, Naohide; Shoda, Akiko; Okazaki, Takayuki; Ichinohe, Sadato; Saika, Shizuko; Inaba, Noriyuki

    2007-11-01

    When measles antibody levels among pregnant women were measured with measles hemagglutinin inhibition (HI), 31% of subjects had negative HI antibody titers. When the same blood samples were tested with measles gelatin particle agglutination (PA) and neutralizing (NT), the percentages of those with negative antibody levels were 1% and 3%. We conducted the correlation between antibody titers measured by the three types of titration. Correlation between NT and HI antibody titers higher than 1:8 and that between NT and PA antibody titers were good, but 81% of subjects whose HI antibody titer was below 1:8 and all women with HI antibody of 1:8 were found to have NT antibody titer higher than 1:4. NT antibody titer higher than 1:4 was found in 95% of women having PA antibody titer of 1:256 and in 99% of those with PA antibody titer of 1:512. Based on the relationships to measles NT antibody level, the majority of subjects with HI antibody titer higher than 1:8 or PA antibody level higher than 1:512 was reasonably assumed to be protected against clinical measles. PA seemed superior to HI in finding subjects with insufficient immunity against measles, because the former detects weak immunity more efficiently than the latter.

  19. Antibodies: an alternative for antibiotics?

    PubMed

    Berghman, L R; Abi-Ghanem, D; Waghela, S D; Ricke, S C

    2005-04-01

    In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming" of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases. PMID:15844826

  20. Antibodies to watch in 2016.

    PubMed

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015. PMID:26651519

  1. Antibodies to watch in 2016.

    PubMed

    Reichert, Janice M

    2016-01-01

    The number of novel antibody therapeutics that received first marketing approvals in 2015 met expectations, with 6 (alirocumab (Praluent®), evolocumab (Repatha®), daratumumab (Darzalex®), dinutuximab (Unituxin®), idarucizumab (Praxbind®), mepolizumab (Nucala®)) granted first approvals as of mid-November*. Seven novel antibody therapeutics (begelomab, brodalumab, elotuzumab, ixekizumab, necitumumab, obiltoxaximab, reslizumab) are in regulatory review, and thus a similar number, if not more, are projected to gain first approvals in 2016. Commercial late-stage antibody therapeutics development exceeded expectations by increasing from 39 candidates in Phase 3 studies as of late 2014 to 53 as of late 2015. Of the 53 candidates, transitions to regulatory review by the end of 2016 are projected for 8 (atezolizumab, benralizumab, bimagrumab, durvalumab, inotuzumab ozogamicin, lebrikizumab, ocrelizumab, tremelimumab). Other "antibodies to watch" include 15 candidates (bavituximab, bococizumab, dupilumab, fasinumab, fulranumab, gevokizumab, guselkumab, ibalizumab, LY2951742, onartuzumab, REGN2222, roledumab, romosozumab, sirukumab, Xilonix) undergoing evaluation in Phase 3 studies that have estimated primary completion dates in 2016. As evidenced by the antibody therapeutics discussed in this perspective, the biopharmaceutical industry has a highly active late-stage clinical pipeline that may deliver numerous new products to the global market in the near future. *See Note added in proof for updates through December 31, 2015.

  2. Remembering antibodies coming of age.

    PubMed

    Melchers, Fritz

    2016-01-01

    Fifty years ago, Norbert Hilschmann discovered that antibodies have variable immunoglobulin domains to bind antigens, and constant domains to carry out effector functions in the immune system. Just as this happened, the author of this perspective entered the field of immunology. Ten years later, the genetic basis of antibody variability was discovered by Susumu Tonegawa and his colleagues at the Basel Institute for Immunology, where the author had become a scientific member. At the same time, Georges Köhler, a former graduate student of the author's at the Basel Institute, invented with Cesar Milstein at the Laboratory of Molecular Biology in Cambridge, England, the method to produce monoclonal antibodies. The author describes here his memories connected to these three monumental, paradigm-changing discoveries, which he observed in close proximity. PMID:27144253

  3. Remembering antibodies coming of age.

    PubMed

    Melchers, Fritz

    2016-01-01

    Fifty years ago, Norbert Hilschmann discovered that antibodies have variable immunoglobulin domains to bind antigens, and constant domains to carry out effector functions in the immune system. Just as this happened, the author of this perspective entered the field of immunology. Ten years later, the genetic basis of antibody variability was discovered by Susumu Tonegawa and his colleagues at the Basel Institute for Immunology, where the author had become a scientific member. At the same time, Georges Köhler, a former graduate student of the author's at the Basel Institute, invented with Cesar Milstein at the Laboratory of Molecular Biology in Cambridge, England, the method to produce monoclonal antibodies. The author describes here his memories connected to these three monumental, paradigm-changing discoveries, which he observed in close proximity.

  4. Antibodies to watch in 2013

    PubMed Central

    Reichert, Janice M

    2013-01-01

    The transitions of antibody therapeutics to late-stage clinical development, regulatory review and the market are proceeding at a rapid pace in 2013. Since late 2012, two monoclonal antibody (mAb) therapeutics (itolizumab, trastuzumab emtansine) received their first approvals, first marketing applications for three mAbs (vedolizumab, ramucirumab, obinutuzumab) were submitted to regulatory agencies, and five mAbs (brodalumab, MABp1, moxetumomab pasudotox, tildrakizumab, rilotumumab) entered their first Phase 3 studies. The current total of commercially-sponsored antibody therapeutics undergoing evaluation in late-stage studies is 30. Recently announced study results for farletuzumab, naptumomab estafenatox, and tabalumab indicate that clinical endpoints were not met in some Phase 3 studies of these product candidates. PMID:23727858

  5. Guinea-pig reaginic antibody

    PubMed Central

    Margni, R. A.; Hajos, Silvia E.

    1973-01-01

    The methods for isolation and purification of a guinea-pig serum protein with homocytotropic antibody activity and characteristics of IgE are described. By precipitation in the equivalence zone or immunoadsorption and chromatography on DEAE-cellulose, we isolated an homocytotropic antibody, that was not able to give a precipitin line when it was reacted directly with the antigen. It was capable of sensitizing guinea-pig skin for PCA after a latent period of 24–48 hours but not after 3 hours; it was sensitive to treatment with mercaptoethanol. It had antigenic determinants present in the other guinea-pig immunoglobulins and particular antigenic determinants. All these properties make us believe that this protein belongs to an immunoglobulin different from γ1 and similar to the reaginic antibody (IgE) described in other species. ImagesFIG. 3FIG. 4FIG. 5 PMID:4126261

  6. Molecular-specific urokinase antibodies

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

    2009-01-01

    Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

  7. Epigenetics of the antibody response.

    PubMed

    Li, Guideng; Zan, Hong; Xu, Zhenming; Casali, Paolo

    2013-09-01

    Epigenetic marks, such as DNA methylation, histone post-translational modifications and miRNAs, are induced in B cells by the same stimuli that drive the antibody response. They play major roles in regulating somatic hypermutation (SHM), class switch DNA recombination (CSR), and differentiation to plasma cells or long-lived memory B cells. Histone modifications target the CSR and, possibly, SHM machinery to the immunoglobulin locus; they together with DNA methylation and miRNAs modulate the expression of critical elements of that machinery, such as activation-induced cytidine deaminase (AID), as well as factors central to plasma cell differentiation, such as B lymphocyte-induced maturation protein-1 (Blimp-1). These inducible B cell-intrinsic epigenetic marks instruct the maturation of antibody responses. Their dysregulation plays an important role in aberrant antibody responses to foreign antigens, such as those of microbial pathogens, and self-antigens, such as those targeted in autoimmunity, and B cell neoplasia.

  8. Communication: Antibody stability and behavior on surfaces.

    PubMed

    Bush, Derek B; Knotts, Thomas A

    2015-08-14

    Antibody microarrays have the potential to revolutionize molecular detection in scientific, medical, and other biosensor applications, but their current use is limited because of poor reliability. It is hypothesized that one reason for their poor performance results from strong antibody-surface interactions that destabilize the antibody structure and create steric interference for antigen recognition. Using a recently developed coarse-grain protein-surface model that has been parameterized against experimental data, antibody-surface interactions for two antibody orientations on two types of surfaces have been investigated. The results show that regardless of attachment geometry, antibodies tend to collapse onto hydrophobic surfaces and exhibit lower overall stability compared to antibodies on hydrophilic surfaces or in bulk solution. The results provide an unprecedented view into the dynamics of antibodies on surfaces and offer new insights into the poor performance exhibited by current antibody microarrays. PMID:26277119

  9. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-22

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  10. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2013-04-09

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

  11. REGULATION OF CELLULAR ANTIBODY SYNTHESIS

    PubMed Central

    Möller, Göran

    1968-01-01

    Transfer of spleen cells from mice immunized against sheep red blood cells (SRBC) into irradiated (600 R) nonimmune, syngeneic mice in the presence of antigen resulted in excessive cellular 7S production 7 days later. The number of 7S plaque-forming cells usually exceeded 106 per spleen and the mean proportion varied between 1 and 70%. In occasional animals all spleen cells were producing antibodies to SRBC. Serum antibody synthesis was also excessively increased, the titers in agglutination after 2-ME treatment and in hemolysis varying between 215 and 225. The generation time of the 7S PFC was found to be 9.6 hr in the secondary hosts. It seemed possible that the excessive production of 7S PFC and antibodies in the irradiated nonimmune recipients was caused by the absence of feedback inhibition of the immune response by antibody, a mechanism which would normally function to restrict antibody synthesis. This conclusion was strengthened by the demonstration that transfer of antigen-stimulated immune cells into actively or passively immunized irradiated recipients resulted in a marked suppression of cellular 7S synthesis. Serial transfers of antigen-stimulated immune cell populations in irradiated hosts resulted in an equally high number of 7S PFC during the first four transfer generations. However, after the fifth to seventh transfer generation the number of 7S PFC rapidly declined and disappeared within one to three passages. Serum antibodies and 7S PFC declined in parallel during the last transfer generations. Further passages of antigen-stimulated spleen cells lacking 7S PFC did not lead to reappearance of PFC. Thus, antigen-sensitive cells have a limited lifespan and/or multiplication capacity. From the hypothesis that the 7S PFC developed by division from antigen-sensitive precursors it was calculated that 38–40 divisions occurred, Thus, one antigen-sensitive precursor has the potential to give rise to 1012 7S PFC. PMID:5635380

  12. Therapeutic Antibodies in Cancer Therapy.

    PubMed

    Gasser, Martin; Waaga-Gasser, Ana Maria

    2016-01-01

    The therapeutic arsenal in solid tumors comprises different anticancer strategies with diverse chemotherapeutic agents and a growing number of biological substances. Large clinical study-based chemotherapeutic protocols combined with biologicals have become an important component in (neo-) adjuvant therapy alongside surgery in solid cancers as well as radiation therapy in some instances. In recent years, monoclonal antibodies have entered the mainstream of cancer therapy. Their first use was as antagonists of oncogenic receptor tyrosine kinases, but today monoclonal antibodies have emerged as long-sought vehicles for the targeted delivery of potent chemotherapeutic agents and as powerful tools to manipulate anticancer immune responses. There is a growing number of FDA approved monoclonal antibodies and small molecules targeting specific types of cancer suggestive of the clinical relevance of this approach.Targeted cancer therapies , also referred to as personalized medicine, are being studied for use alone, in combination with other targeted therapies, and in combination with chemotherapy. The use of monoclonal antibodies in colorectal and gastric cancer for example have shown best outcome when combined with chemotherapy, even though single agent anti-EGFR antibodies seem to be active in particular setting of metastatic colorectal cancer patients. However, it is not well defined whether the addition of anti-VEGF - and anti-EGFR strategies to chemotherapy could improve outcome in those patients susceptible to colorectal cancer-related metastases resection. Among the most promising approaches to activating therapeutic antitumor immunity is the blockade of immune checkpoints, exemplified by the recently FDA-approved agent, Ipilimumab, an antibody that blocks the coinhibitory receptor CTLA-4. Capitalizing on the success of Ipilimumab, agents that target a second coinhibitory receptor, PD-1, or its ligand, PD-L1, are in clinical development. This section attempts to

  13. Therapeutic antibodies in breast cancer.

    PubMed

    Pérez-Garcia, José; Muñoz-Couselo, Eva; Cortés, Javier; Scaltriti, Maurizio

    2014-10-01

    The discovery of HER2 and development of trastuzumab pioneered the field of targeted therapy in breast cancer. Hoping to emulate the same clinical success, pharmaceutical companies have developed several antibodies against newly identified membrane-bound targets. Unfortunately, none of these agents has yet matched the thousands of lives saved by trastuzumab. In this article we review the most advanced therapeutic antibodies in breast cancer. While acknowledging their unquestionable benefit, we emphasize the need to better understand their biology and mechanisms of action in order to optimize their use in defined patient populations.

  14. Antibody profiling sensitivity through increased reporter antibody layering

    DOEpatents

    Apel, William A.; Thompson, Vicki S.

    2013-02-26

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  15. Antibody profiling sensitivity through increased reporter antibody layering

    SciTech Connect

    Apel, William A.; Thompson, Vicki S

    2010-04-13

    A method for analyzing a biological sample by antibody profiling for identifying forensic samples or for detecting the presence of an analyte. In an embodiment of the invention, the analyte is a drug, such as marijuana, Cocaine (crystalline tropane alkaloid), methamphetamine, methyltestosterone, or mesterolone. The method comprises attaching antigens to a surface of a solid support in a preselected pattern to form an array wherein locations of the antigens are known; contacting the array with the biological sample such that a portion of antibodies in the sample reacts with and binds to the antigens in the array to form immune complexes; washing away antibodies that do form immune complexes; and detecting the immune complexes, to form an antibody profile. Forensic samples are identified by comparing a sample from an unknown source with a sample from a known source. Further, an assay, such as a test for illegal drug use, can be coupled to a test for identity such that the results of the assay can be positively correlated to the subject's identity.

  16. Monoclonal Antibodies in Diagnosis and Therapy

    NASA Astrophysics Data System (ADS)

    Waldmann, Thomas A.

    1991-06-01

    Monoclonal antibodies have been applied clinically to the diagnosis and therapy of an array of human disorders, including cancer and infectious diseases, and have been used for the modulation of immune responses. Effective therapy using unmodified monoclonal antibodies has, however, been elusive. Recently, monoclonal antibody-mediated therapy has been revolutionized by advances such as the definition of cell-surface structures on abnormal cells as targets for effective monoclonal antibody action, genetic engineering to create less immunogenic and more effective monoclonal antibodies, and the arming of such antibodies with toxins or radionuclides to enhance their effector function.

  17. The European antibody network's practical guide to finding and validating suitable antibodies for research.

    PubMed

    Roncador, Giovanna; Engel, Pablo; Maestre, Lorena; Anderson, Amanda P; Cordell, Jacqueline L; Cragg, Mark S; Šerbec, Vladka Č; Jones, Margaret; Lisnic, Vanda J; Kremer, Leonor; Li, Demin; Koch-Nolte, Friedrich; Pascual, Núria; Rodríguez-Barbosa, Jose-Ignacio; Torensma, Ruurd; Turley, Helen; Pulford, Karen; Banham, Alison H

    2016-01-01

    Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.

  18. Pharmacokinetics interactions of monoclonal antibodies.

    PubMed

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction. PMID:27438459

  19. Anti-acetylcholine receptor antibodies.

    PubMed Central

    Vincent, A; Newsom Davis, J

    1980-01-01

    Early suggestions that a humoral factor might be implicated in the disorder of neuromuscular transmission in myasthenia gravis have been confirmed by the detection of anti-AChR antibody in 85-90% of the patients with generalised disease and in 75% of cases with restricted ocular myasthenia. Plasma exchange reveals that serum anti-AChR usually has an inverse relationship to muscle strength and present evidence indicates that patients responding to thymectomy and immunosuppressive durg treatment usually show a consistent decline in serum anti-AChR titres. The antibody is heterogeneous and can lead to a loss of muscle AChR by several mechanisms. Anti-AChR is produced in the thymus in relatively small amounts. Anti-AChR antibody synthesis by thymic lymphocytes and pokeweed stimulated peripheral lymphocytes in culture provides a means of studying the effect of different lymphocyte populations in vitro. Analysis of clinical, immunological and HLA antigen characteristics in MG suggest that more than one mechanism may underlie the breakdown in tolerance to AChR, leading to the production of anti-AChR antibodies. PMID:7400823

  20. Pharmacokinetics interactions of monoclonal antibodies.

    PubMed

    Ferri, Nicola; Bellosta, Stefano; Baldessin, Ludovico; Boccia, Donatella; Racagni, Giorgi; Corsini, Alberto

    2016-09-01

    The clearance of therapeutic monoclonal antibodies (mAbs) typically does not involve cytochrome P450 (CYP450)-mediated metabolism or interaction with cell membrane transporters, therefore the pharmacokinetics interactions of mAbs and small molecule drugs are limited. However, a drug may affect the clearance of mAbs through the modulation of immune response (e.g., methotrexate reduces the clearance of infliximab, adalimumab, and golimumab, possibly due to methotrexate's inhibitory effect on the formation of antibodies against the mAbs). In addition, mAbs that are cytokine modulators may modify the metabolism of drugs through their effects on P450 enzymes expression. For example, cytokine modulators such as tocilizumab (anti-IL-6 receptor antibody) may reverse the "inhibitory" effect of IL-6 on CYP substrates, resulting in a "normalization" of CYP activities. Finally, a drug may alter the clearance of mAbs by either increasing or reducing the levels of expression of targets of mAbs on the cell surface. For instance, statins and fibrates induce PCSK9 expression and therefore increase cellular uptake and clearance of alirocumab and evolocumab, anti-PCSK9 antibodies. In the present review, we will provide an overview on the pharmacokinetics properties of mAbs as related to the most relevant examples of mAbs-small molecule drug interaction.

  1. Anti-Sulfoglucuronosyl Paragloboside Antibody

    PubMed Central

    Li, Dongpei; Usuki, Seigo; Quarles, Brandy; Rivner, Michael H.; Ariga, Toshio

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. Although the etiology of ALS is obscure, genetic studies of familiar ALS suggest a multifactorial etiology for this condition. Similarly, there probably are multiple causes for sporadic ALS. Autoimmune-mediated motor neuron dysfunction is one proposed etiology for sporadic ALS. In the present study, anti-glycolipid antibodies including GM1, GD1b, GD3, and sulfoglucuronosyl paragloboside (SGPG) were investigated in the sera of a large number of patient samples, including 113 ALS patients and 50 healthy controls, by means of enzyme-linked immunosorbent assay with affinity parametric complex criterion evaluation and thin-layer chromatography immunooverlay (immuno-TLC). Anti-SGPG antibodies were found in the sera of 13.3% ALS patients (15 out of 113). The highest titer reached 1:1600. The presence of anti-SGPG antibodies in the serum samples was also confirmed by immuno-TLC. Importantly, a multiple logistic regression analysis showed that the presence of anti-SGPG antibody was positively correlated with age (p < .01) and negatively correlated with ALS Functional Rating Scale score (p < .05). Moreover, the localization of SGPG-immunoreactivity on the motor neurons of rat spinal cord and a mouse motor neuronal cell line, NSC-34 was observed by an immunofluorescence method. These data suggest that SGPG could represent a specific pathogenic antigen in those ALS patients. The presence of anti-SGPG antibodies in the serum of ALS patients should represent a diagnostic biomarker of ALS, and it could reflect the severity of the disease. PMID:27683876

  2. Detection of Campylobacter species using monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Young, Colin R.; Lee, Alice; Stanker, Larry H.

    1999-01-01

    A panel of species specific monoclonal antibodies were raised to Campylobacter coli, Campylobacter jejuni and Campylobacter lari. The isotypes, and cross-reactivity profiles of each monoclonal antibody against an extensive panel of micro- organisms, were determined.

  3. [Neuroimmunological diseases associated with VGKC complex antibodies].

    PubMed

    Watanabe, Osamu

    2013-05-01

    Antibodies to voltage-gated potassium channels(VGKC) were first identified by radioimmunoassay of radioisotope labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were found only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in Morvan's syndrome and in a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins(for example LGI-1, Caspr-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now usually known as VGKC-complex antibodies. In general, LGI-1 antibodies are most common in limbic encephalitis with SIADH. Caspr-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability.

  4. Production and Screening of Monoclonal Peptide Antibodies.

    PubMed

    Trier, Nicole Hartwig; Mortensen, Anne; Schiolborg, Annette; Friis, Tina

    2015-01-01

    Hybridoma technology is a remarkable and indispensable tool for generating high-quality monoclonal antibodies. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents, but have also emerged as the most rapidly expanding class of therapeutic biologicals. In this chapter, an overview of hybridoma technology and the laboratory procedures used routinely for hybridoma production and antibody screening are presented, including characterization of peptide antibodies.

  5. The antibody response in seabather's eruption.

    PubMed

    Burnett, J W; Kumar, S; Malecki, J M; Szmant, A M

    1995-01-01

    Thirty-six of 44 patients with seabather's eruption had specific IgG antibodies against Linuche unguilata (thimble jelly) medusae antigen. ELISA detected antibodies in serum stored for 10 years. The extent of the cutaneous eruption or sting severity was correlated with antibody titer. Antibodies were detected in patients acquiring the eruption in Florida, the Bahamas and Aruba, reflecting the habitat of these jellyfish. This serological assay can be useful to confirm the clinical diagnosis. PMID:7778134

  6. Anti-DNA antibodies in SLE

    SciTech Connect

    Voss, E.W.

    1988-01-01

    This book contains 8 chapters. Some of the titles are: Anti-DNA Antibodies in SLE: Historical Perspective; Specificity of Anti-DNA Antibodies in Systemic Lupus Erythematosus; Monoclonial Autoimmune Anti-DNA Antibodies; and Structure--Function Analyses of Anti-DNA Autoantibodies.

  7. Effects of medium concentration on antibody production

    NASA Technical Reports Server (NTRS)

    Williams, J.

    1984-01-01

    Antibody production by two different cell lines was measured as the media were supplemented with varied amounts of glucose and fetal bovine serum. Both cell lines elaborated antidinitrophenyl hapten antibodies. Two basic media were used: RPMI 1640 and Dulbecco's modified Eagle's medium. The production of antibodies was followed from 0 to 180 h and was assayed by radioimmunoassay.

  8. Immunology update: symposium on antiphospholipid antibodies.

    PubMed

    Silver, R M

    1997-02-01

    The seventh international symposium on antiphospholipid antibodies was held in New Orleans, LA, USA, on October 9-13, 1996. The meeting was attended by over 230 people and was hosted by Azzudin E. Gharavi and Wendell A. Wilson. Workshops were also conducted on assays for lupus anticoagulant, antiphospholipid antibodies, and anti-endothelial cell antibodies. PMID:9080388

  9. Antibody biosimilars: Fears or opportunities?

    PubMed Central

    Guillon-Munos, Audrey; Daguet, Arnaud; Watier, Hervé

    2014-01-01

    The annual “LabEx MAbImprove industrial workshops” are primarily intended to provide scientists involved in therapeutic antibodies, a comprehensive view about topics of interest for the pharmaceutical industry. They are organized by the “LabEx MAbImprove industrial committee”, for this first edition especially in partnership with ARITT, the regional agency for innovation and technology transfer which operates in the French Région Centre, the 1st French region for pharmaceutical production. The 2013 edition, held May 28 at the Vinci Center of Tours, was dedicated to antibody biosimilars. Depending on opinions, the impending expiry of antibody patents and the imminent marketing approval of competitors to blockbusters can be perceived as good or bad things. Fears or opportunities? Risks for patients? Breath of fresh air for the health systems? Opportunity for re-industrializing France? In this context, it is necessary for people to form a fair and informed opinion on the current landscape of antibody biosimilars. In particular, this is especially important for scientists from the academic world, from the industry or from the regulation agencies, for pharmacists, for pharmacovigilance specialists, for health authorities, and staff from health insurance and decision makers. The first session was devoted to market and regulatory issues, and included both an overview of the evolution of the patent landscape and a description of biosimilars regulation in the European Union (EU). This session was closed by a talk on manufacturing processes for biosimilars. In the next session, quality control attributes of biosimilars were discussed and compared with the consistent quality of biotechnology products to raise the question: “How close is close enough?” In vitro assays for evaluating the Fc function of therapeutic antibodies were also discussed. The third session focused on development of biosimilars and primarily on the stepwise process for introducing an antibody

  10. Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

    NASA Astrophysics Data System (ADS)

    Massey, Richard J.; Schochetman, Gerald

    1981-07-01

    The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

  11. Therapeutic antibodies: Discovery, design and deployment.

    PubMed

    Ramsland, Paul A; Hutchinson, Andrew T; Carter, Paul J

    2015-10-01

    Therapeutic antibodies have come of age with major progress being made in cancer, autoimmunity and chronic inflammation, as well as a wide range of other human diseases. Antibody engineering is further driving development of novel antibody formats and genetically modified cell-based therapies that harness the power of the immune system to progress cures in otherwise intractable human diseases. Nevertheless, there are still significant challenges ahead for basic and applied research relating to therapeutic antibodies. This special issue of the journal provides reviews and opinions that relate to the discovery, design and deployment of antibodies as therapeutics.

  12. Antibody engineering and therapeutics conference

    PubMed Central

    Larrick, James W; Parren, Paul WHI; Huston, James S; Plückthun, Andreas; Bradbury, Andrew; Tomlinson, Ian M; Chester, Kerry A; Burton, Dennis R; Adams, Gregory P; Weiner, Louis M; Scott, Jamie K; Alfenito, Mark R; Veldman, Trudi; Reichert, Janice M

    2014-01-01

    The 25th anniversary of the Antibody Engineering & Therapeutics Conference, the Annual Meeting of The Antibody Society, will be held in Huntington Beach, CA, December 7–11, 2014. Organized by IBC Life Sciences, the event will celebrate past successes, educate participants on current activities and offer a vision of future progress in the field. Keynote addresses will be given by academic and industry experts Douglas Lauffenburger (Massachusetts Institute of Technology), Ira Pastan (National Cancer Institute), James Wells (University of California, San Francisco), Ian Tomlinson (GlaxoSmithKline) and Anthony Rees (Rees Consulting AB and Emeritus Professor, University of Bath). These speakers will provide updates of their work, placed in the context of the substantial growth of the industry over the past 25 years. PMID:25517297

  13. [Industrial production of monoclonal antibodies].

    PubMed

    Baron, D

    1990-10-01

    Murine monoclonal antibodies (mabs) are produced in either mouse ascites or bioreactors (spinner culture, stirred-tank reactor, airlift reactor, hollow-fiber reactor). Human mabs are produced solely in bioreactors. Encapsulation represents a special technology. Hybridoma cells have to be adapted prior to growth in bioreactors. Of crucial importance is the construction of over-producing cell lines by cell- and gene-technological methods. Manipulated cell lines often produce modified mabs.

  14. Improved monoclonal antibodies to halodeoxyuridine

    DOEpatents

    Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

    1983-10-18

    The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

  15. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

    Researchers at Pacific Northwest National Laboratory (PNNL) have constructed a nonimmune library consisting of 109 human antibody scFv fragments, which have been cloned and expressed on the surface of yeast. Nanomolar-affinity scFvs are routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010 fold without measurable loss of clonal diversity. This allows for indefinite expansion of the library. All scFv clones can be assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps. The ability to use multiplex library screening demonstrates the utility of this approach for high-throughput antibody isolation for proteomic applications. The yeast library may be used for research projects or teaching performed for U.S. Government purposes only. If you would like to request an aliquot of the single-chain antibody library for your research, please print and fill out the Materials Transfer Agreement (MTA) [PDF, 20K]. The website provides the contact information for mailing the MTA. [copied from http://www.sysbio.org/dataresources/singlechain.stm

  16. Methods of identification employing antibody profiles

    DOEpatents

    Francoeur, Ann-Michele

    1993-12-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of the set of individual-specific antibodies that are part of the unique antibody repertoire present in animals, by reacting an effective amount of such antibodies with a particular panel, of n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly be used to distinguish genetically, or otherwise similar individuals, or their body parts containing individual-specific antibodies.

  17. Natural human antibodies to amyloid beta peptide.

    PubMed

    Szabo, Paul; Relkin, Norman; Weksler, Marc E

    2008-06-01

    Properties of human, natural anti-Abeta antibodies and commercially available intravenous immunoglobulin (IVIg) have been examined in light of the beneficial effects of passive immunotherapy with IVIg for patients with mild to moderate Alzheimer's disease (AD). Anti-Abeta antibodies in IVIg recognize conformation-specific epitopes as well as linear epitopes from different regions of the Abeta peptide. Anti-Abeta antibodies in circulation, especially those with high avidity, are often masked by ligands and the avidity of these antibodies increases upon dissociation of the bound ligands from the antibodies. Such natural anti-Abeta antibodies have the capacity to prevent Abeta oligomer-induced neurotoxicity in N2A neuroblastoma cells. This neuro-protective effect may reflect the therapeutic potential of the natural anti-Abeta antibodies found in IVIg for the treatment of patients with AD.

  18. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

    Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

  19. A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

    PubMed

    Dong, Jianbo; Thompson, Aaron A; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Stevens, Raymond C; Marks, James D

    2010-04-01

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

  20. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    SciTech Connect

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D.

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  1. Measurement of affinity of viral monoclonal antibodies by ELISA titration of free antibody in equilibrium mixtures.

    PubMed

    Azimzadeh, A; Van Regenmortel, M H

    1991-08-01

    The binding affinity of a monoclonal antibody (Mab) to tobacco mosaic virus (TMV) was determined by measuring, in an enzyme-linked immunosorbent assay, the amount of free antibody present after ultracentrifugation of virus-antibody complexes at equilibrium. In antibody excess, univalent binding of Mabs was observed and the affinity constant was K = 3.2 +/- 0.4 10(8) l/mol; in antigen excess, bivalent antibody binding was observed and the antibody avidity was about 15 times higher. In antigen excess, it was imperative to correct experimental data for the presence of 0.55% inactive molecules in the immunopurified antibody preparation. Modelling studies suggest that in the case of antibodies of increasing affinity, it becomes increasingly important to correct for the presence of inactive antibody in the binding assay.

  2. Distinction between MOG antibody-positive and AQP4 antibody-positive NMO spectrum disorders

    PubMed Central

    Sato, Douglas Kazutoshi; Callegaro, Dagoberto; Lana-Peixoto, Marco Aurelio; Waters, Patrick J.; Jorge, Frederico M. de Haidar; Takahashi, Toshiyuki; Nakashima, Ichiro; Apostolos-Pereira, Samira Luisa; Talim, Natalia; Simm, Renata Faria; Lino, Angelina Maria Martins; Misu, Tatsuro; Leite, Maria Isabel; Aoki, Masashi

    2014-01-01

    Objective: To evaluate clinical features among patients with neuromyelitis optica spectrum disorders (NMOSD) who have myelin oligodendrocyte glycoprotein (MOG) antibodies, aquaporin-4 (AQP4) antibodies, or seronegativity for both antibodies. Methods: Sera from patients diagnosed with NMOSD in 1 of 3 centers (2 sites in Brazil and 1 site in Japan) were tested for MOG and AQP4 antibodies using cell-based assays with live transfected cells. Results: Among the 215 patients with NMOSD, 7.4% (16/215) were positive for MOG antibodies and 64.7% (139/215) were positive for AQP4 antibodies. No patients were positive for both antibodies. Patients with MOG antibodies represented 21.1% (16/76) of the patients negative for AQP4 antibodies. Compared with patients with AQP4 antibodies or patients who were seronegative, patients with MOG antibodies were more frequently male, had a more restricted phenotype (optic nerve more than spinal cord), more frequently had bilateral simultaneous optic neuritis, more often had a single attack, had spinal cord lesions distributed in the lower portion of the spinal cord, and usually demonstrated better functional recovery after an attack. Conclusions: Patients with NMOSD with MOG antibodies have distinct clinical features, fewer attacks, and better recovery than patients with AQP4 antibodies or patients seronegative for both antibodies. PMID:24415568

  3. Individual-specific antibody identification methods

    DOEpatents

    Francoeur, Ann -Michele

    1989-11-14

    An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of a hithertofore unknown set of individual-specific, or IS antibodies, that are part of the unique antibody repertoire present in animals, by reacting an effective amount of IS antibodies with a particular panel, or n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly by used to distinguish genetically, or otherwise similar individuals, or their body parts containing IS antibodies. Identification of inanimate objects, particularly security documents, is similarly affected by associating with the documents, an effective amount of a particular individual's IS antibodies, or conversely, a particular panel of antigens, and forming antibody-antigen complexes with a particular panel of antigens, or a particular individual's IS antibodies, respectively. One embodiment of the instant identification method, termed the blocked fingerprint assay, has applications in the area of allergy testing, autoimmune diagnostics and therapeutics, and the detection of environmental antigens such as pathogens, chemicals, and toxins.

  4. Stabilization of antibody fragments in adverse environments.

    PubMed

    Dooley, H; Grant, S D; Harris, W J; Porter, A J

    1998-08-01

    Antibody fragments have the potential to be used as sensitive and specific binding agents in a broad range of industrial applications. Genetic manipulation has been used to design a series of antibody fragment configurations with a flexible linker and/or a disulphide bond between the heavy chain and light chain of an antibody fragment against the herbicide atrazine. The thermostability and stability to a range of denaturants, polar and non-polar solvents, surfactants and proteases have been compared. It has been found that a novel antibody fragment construct (STAB: stabilized antibody) containing both a flexible linker and a disulphide bond can be effectively produced and shows greatly improved stability in these diverse environments. These STABs should be useful in environmental diagnostics and remediation, and may provide a generic approach for stabilizing antibody fragments in formulations containing detergents and penetrants for topical application in the pharmaceutical and cosmetic industries.

  5. Factors determining antibody distribution in tumors

    PubMed Central

    Thurber, Greg M.; Schmidt, Michael M.; Wittrup, K. Dane

    2009-01-01

    The development of antibody therapies for cancer is increasing rapidly, primarily owing to their specificity. Antibody distribution in tumors is often extremely uneven, however, leading to some malignant cells being exposed to saturating concentrations of antibody, whereas others are completely untargeted. This is detrimental because large regions of cells escape therapy, whereas other regions might be exposed to suboptimal concentrations that promote a selection of resistant mutants. The distribution of antibody depends on a variety of factors, including dose, affinity, antigens per cell and molecular size. Because these parameters are often known or easily estimated, a quick calculation based on simple modeling considerations can predict the uniformity of targeting within a tumor. Such analyses should enable experimental researchers to identify in a straightforward way the limitations in achieving evenly distributed antibody, and design and test improved antibody therapeutics more rationally. PMID:18179828

  6. The antibody crossmatch in liver transplantation

    PubMed Central

    Gordon, Robert D.; Fung, John J.; Markus, Bernd; Fox, Ira; Iwatsuki, Shunzaburo; Esquivel, Carlos O.; Tzakis, Andreas; Todo, Satoru; Starzl, Thomas E.

    2011-01-01

    Six hundred sixty-seven first, second, and third orthotopic liver allografts in 520 patients were reviewed to determine the effect of recipient panel-reactive antibody (PRA) and donor-recipient antibody crossmatch on 2-year patient and liver allograft survival rates. Neither a high panel-reactive antibody nor a positive crossmatch for donor-specific preformed antibody was associated with decreased patient or liver allograft survival for primary grafts or retransplants. Two patients have been given kidney transplants immediately after a liver allograft from a donor with whom each patient had an initial strongly positive donor-specific antibody crossmatch. The liver apparently removed or neutralized circulating anti-donor antibody, since the renal allografts functioned promptly and did not experience hyperacute rejection. PMID:3532391

  7. Phase Separation in Solutions of Monoclonal Antibodies

    NASA Astrophysics Data System (ADS)

    Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

    2012-02-01

    We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

  8. Peptide Antibodies: Past, Present, and Future.

    PubMed

    Houen, Gunnar

    2015-01-01

    Peptide antibodies recognize epitopes with amino acid residues adjacent in sequence ("linear" epitopes). Such antibodies can be made to virtually any sequence and have been immensely important in all areas of molecular biology and diagnostics due to their versatility and to the rapid growth in protein sequence information. Today, peptide antibodies can be routinely and rapidly made to large numbers of peptides, including peptides with posttranslationally modified residues, and are used for immunoblotting, immunocytochemistry, immunohistochemistry, and immunoassays. In the future, peptide antibodies will continue to be immensely important for molecular biology, TCR- and MHC-like peptide antibodies may be produced routinely, peptide antibodies with predetermined conformational specificities may be designed, and peptide-based vaccines may become part of vaccination programs.

  9. Radioimmunotherapy of malignancy using antibody targeted radionuclides.

    PubMed Central

    Cobb, L. M.; Humm, J. L.

    1986-01-01

    Antibodies directed against tumour associated antigens provide a means for delivering preferentially cytotoxic radionuclides to the cells of primary and secondary tumours. The factors that influence the effectiveness of the radiation in the tumour compared with its effect on the radiosensitive normal tissues include the specificity of the antibody, the distribution of targeted energy within the tumour and the host's response to the injected foreign antibody. Recently some encouraging results from clinical trials of radioimmunotherapy have been reported in the literature. There is a continual search for more avid and specific antibodies, and the techniques of genetic engineering are being applied to the problem of reducing the antigenicity and mass of the carrier antibody. The improved efficiency of the labelled antibody needs to be supplemented by an identification of those tumours most likely to respond to this form of therapy. PMID:3542006

  10. Antibody Based Imaging Strategies of Cancer

    PubMed Central

    Warram, Jason M; de Boer, Esther; Sorace, Anna G; Chung, Thomas K; Kim, Hyunki; Pleijhuis, Rick G; van Dam, Gooitzen M; Rosenthal, Eben L

    2014-01-01

    Although mainly developed for preclinical research and therapeutic use, antibodies have high antigen specificity, which can be used as a courier to selectively deliver a diagnostic probe or therapeutic agent to cancer. It is generally accepted that the optimal antigen for imaging will depend on both the expression in the tumor relative to normal tissue and the homogeneity of expression throughout the tumor mass and between patients. For the purpose of diagnostic imaging, novel antibodies can be developed to target antigens for disease detection, or current FDA-approved antibodies can be repurposed with the covalent addition of an imaging probe. Reuse of therapeutic antibodies for diagnostic purposes reduces translational costs since the safety profile of the antibody is well defined and the agent is already available under conditions suitable for human use. In this review, we will explore a wide range of antibodies and imaging modalities that are being translated to the clinic for cancer identification and surgical treatment. PMID:24913898

  11. Controlled delivery of antibodies from injectable hydrogels.

    PubMed

    Fletcher, Nathan A; Babcock, Lyndsey R; Murray, Ellen A; Krebs, Melissa D

    2016-02-01

    Therapeutic antibodies are currently used for the treatment of various diseases, but large doses delivered systemically are typically required. Localized controlled delivery techniques would afford major benefits such as decreasing side effects and required doses. Injectable biopolymer systems are an attractive solution due to their minimally invasive potential for controlled release in a localized area. Here, alginate-chitosan hydrogels are demonstrated to provide controlled delivery of IgG model antibodies and also of Fab antibody fragments. Also, an alternate delivery system comprised of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with antibodies and encapsulated in alginate was shown to successfully provide another level of control over release. These biopolymer systems that offer controlled delivery for antibodies and antibody fragments will be promising for many applications in drug delivery and regenerative medicine.

  12. Nanoliposome-mediated targeting of antibodies to tumors: IVIG antibodies as a model.

    PubMed

    Nikpoor, Amin Reza; Tavakkol-Afshari, Jalil; Gholizadeh, Zahra; Sadri, Kayvan; Babaei, Mohammad Hossein; Chamani, Jamshidkhan; Badiee, Ali; Jalali, Seyed Amir; Jaafari, Mahmoud Reza

    2015-11-10

    Monoclonal antibodies are routinely used as tools in immunotherapies against solid tumors. However, administration of monoclonal antibodies may cause undesired side effects due to their accumulation in non-targeted organs. Nanoliposomes of less than 200 nm can target antibodies to tumors by enhanced permeation and retention (EPR) mechanisms. To direct monoclonal antibodies to tumors, nanoliposomes encapsulating intravenous immunoglobulin (IVIG) as a model antibody were prepared. The liposomes had average diameters of 100 nm and encapsulation efficiencies of 31 to 46%. They showed less than 10% release in plasma at 37°C up to seven days. The secondary and tertiary structures of liposome-encapsulated antibodies were analyzed by circular dichroism (CD) spectroscopy. The near and far-UV spectra analyses revealed no obvious conformational changes in the structures of the encapsulated antibodies. The biodistribution of free and liposome-encapsulated iodinated antibodies was investigated in mice bearing C-26 colon carcinoma tumors. The accumulation of liposome-encapsulated antibodies in tumors was significantly greater than that of free antibodies due to the EPR effect. The PEGylated liposomes were more efficient in the delivery of antibodies to the tumor site than non-PEGylated liposomes. We conclude that administration of monoclonal antibodies in PEGylated liposomes is more efficient than administration of non-encapsulated monoclonal antibodies for solid tumor immunotherapy. PMID:26302860

  13. Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

    PubMed

    Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix; Scott, Jamie K; Sok, Devin; Pauthner, Matthias; Reichert, Janice M; Helguera, Gustavo; Andrabi, Raiees; Mabry, Robert; Bléry, Mathieu; Voss, James E; Laurén, Juha; Abuqayyas, Lubna; Barghorn, Stefan; Ben-Jacob, Eshel; Crowe, James E; Huston, James S; Johnston, Stephen Albert; Krauland, Eric; Lund-Johansen, Fridtjof; Marasco, Wayne A; Parren, Paul W H I; Xu, Kai Y

    2014-01-01

    The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

  14. Anti-enterobacteria antibodies in psoriatic arthritis.

    PubMed

    Lapadula, G; Iannone, F; Covelli, M; Numo, R; Pipitone, V

    1992-01-01

    The occurrence of certain antibacterial antibodies was studied in the sera of 22 healthy donors (HD) and 66 patients with different diseases. The cases investigated included 22 rheumatoid arthritis (RA), 22 non-arthritic-psoriasis (NAP), and 22 psoriatic arthritis (PA) patients. A complement fixation test was used with Yersinia enterocolitica 0:3 type (YEC), Yersinia pseudotuberculosis (YPT), Campylobacter jejuni (CJ), and Campylobacter fetus (CF) antigens; the detection of anti-Chlamydia trachomatis (CT) antibodies was carried out using an immunoperoxidase colorimetric slide test that allowed the detection of isotypes of specific antibodies. It was found that the synthesis of anti-CF, CJ, YEC, and YPT antibodies in NAP patients does not differ significantly from that of the HD group; on the contrary, the antibody levels were statistically higher in PA than in the other disease groups or in the healthy controls, although only anti-CF antibodies seemed to significantly differentiate (p = 0.000003) the PA group from the others. Anti-CT IgA antibody titers were found to be significantly higher in the PA as well as in the RA groups when compared with the controls, while the antibody levels in NAP patients showed no clear-cut difference with respect to those of either the arthritic patients or the healthy controls. By showing that anti-enterobacterial antibodies are increased in PA but not in NAP patients, our data furnish additional support to the thesis of a pathogenic role of bacterial infections in psoriatic arthritis.

  15. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2010-06-15

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  16. Uses of monoclonal antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V

    2013-08-06

    This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

  17. 5th Annual Monoclonal Antibodies Conference

    PubMed Central

    2009-01-01

    The conference, which was organized by Visiongain and held at the BSG Conference Center in London, provided an excellent opportunity for participants to exchange views on the development, production and marketing of therapeutic antibodies, and discuss the current business environment. The conference included numerous interactive panel and group discussions on topics such as isotyping for therapeutic antibodies (panel chair: Nick Pullen, Pfizer), prospects for fully human monoclonal antibodies (chair: Christian Rohlff, Oxford BioTherapeutics), perspectives on antibody manufacturing and development (chair: Bo Kara, Avecia), market impact and post-marketing issues (chair: Keith Rodgers, Bodiam Consulting) and angiogenesis inhibitors (chair: David Blakey, AstraZeneca). PMID:20073132

  18. Exceptional Antibodies Produced by Successive Immunizations.

    PubMed

    Gearhart, Patricia J; Castiblanco, Diana P; Russell Knode, Lisa M

    2015-12-01

    Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations. PMID:26641938

  19. Preparation of astatine-labeled monoclonal antibodies

    SciTech Connect

    Milesz, S.; Norseev, Yu.V.; Szucs, Z. |

    1995-07-01

    In the cationic state astatine forms a stable complex with diethylenetriaminepentaacetic acid. Thanks to this complex, astatine can be bound to monoclonal antibodies of the RYa{sub 1} type. The most favorable conditions for preparing astatine-labeled antibodies are established. The chromatographic analysis and electromigration experiments showed that astatine is firmly linked to a biomolecule in vitro and it did not escape from labeled monoclonal antibodies even under treatment with such highly effective astatine-complexing agent as thiourea. The immune activity of astatine-labeled antibodies did not change even after 20 h.

  20. Reducing the cost of HIV antibody testing.

    PubMed

    Tamashiro, H; Maskill, W; Emmanuel, J; Fauquex, A; Sato, P; Heymann, D

    1993-07-10

    Available tests to detect antibody to human immunodeficiency virus (HIV) have a range of applications, and injudicious selection and inappropriate use can add a significant financial burden to budgets for AIDS programmes in developing countries. There are several ways by which the cost of HIV antibody testing can be reduced; they include use of tests appropriate for existing laboratory capabilities; adoption of cost-effective testing strategies; pooling of serum samples before testing; and ensuring best possible purchase prices. Each approach can significantly reduce the cost of HIV antibody testing alone or in combination, which increases the potential sustainability of antibody testing programmes, even in settings of limited resources. PMID:8100916

  1. Studies on the control of antibody synthesis

    PubMed Central

    Werblin, T. P.; Kim, Young Tai; Quagliata, F.; Siskind, G. W.

    1973-01-01

    The distribution and heterogeneity of antibody affinity has been followed with time after immunization. Initially, a symmetrical distribution of heterogeneous low affinity antibody molecules is present. With time the population becomes skewed towards the high affinity end of the distribution, the average affinity increases, and the bulk of the antibody generally comes to be present in a subpopulation of high affinity and of relatively restricted heterogeneity. Still later after immunization, the proportion of high affinity antibody decreases and a highly heterogeneous population of antibody molecules is present with a somewhat decreased average affinity. Low affinity subpopulations were found to persist throughout the course of the immune response. In addition it was noted that by day 42 after immunization a significant amount of the highest affinity antibody that a given rabbit would synthesize at any time during its immune response was already present. Thus, changes in average affinity can be accounted for by shifts in the relative amounts of those antibody subpopulations already present by day 42 and do not require the appearance of any new antibody species. The results can be interpreted as consistent with a selection theory of antibody synthesis in which cells of high affinity are preferentially selected in what amounts to a micro-evolutionary system. PMID:4574576

  2. Exceptional Antibodies Produced by Successive Immunizations

    PubMed Central

    Gearhart, Patricia J.; Castiblanco, Diana P.; Russell Knode, Lisa M.

    2015-01-01

    Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations. PMID:26641938

  3. Antibodies in myasthenia gravis and related disorders.

    PubMed

    Vincent, Angela; McConville, John; Farrugia, Maria Elena; Bowen, John; Plested, Paul; Tang, Teresa; Evoli, Amelia; Matthews, Ian; Sims, Gary; Dalton, Paolo; Jacobson, Leslie; Polizzi, Agata; Blaes, Frans; Lang, Bethan; Beeson, David; Willcox, Nick; Newsom-Davis, John; Hoch, Werner

    2003-09-01

    Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

  4. Monoclonal Antibody That Defines Human Myoepithelium

    NASA Astrophysics Data System (ADS)

    Dairkee, Shahnaz Hashmi; Blayney, Carlene; Smith, Helene S.; Hackett, Adeline J.

    1985-11-01

    We have isolated a mouse monoclonal antibody that, upon immunohistochemical localization in frozen sections, displays specificity for human myoepithelial cells in the resting mammary gland, sweat glands, and salivary glands. Furthermore, this antibody was strongly and homogeneously reactive with frozen sections of 3 of 60 breast carcinoma specimens. Using immunolocalization techniques in conjunction with polyacrylamide gel electrophoresis, we have determined that the reactivity of this monoclonal antibody is directed toward a 51,000-dalton keratin polypeptide. The potential uses of this antibody in the prognosis of human mammary carcinoma and in understanding the role of the myoepithelium in development and differentiation are discussed.

  5. Snake venom antibodies in Ecuadorian Indians.

    PubMed

    Theakston, R D; Reid, H A; Larrick, J W; Kaplan, J; Yost, J A

    1981-10-01

    Serum samples from 223 Waorani Indians, a tribe in eastern Ecuador, were investigated by enzyme-linked immunosorbent assay for antibodies to snake venom. Seventy-eight per cent were positive, confirming the highest incidence and mortality from snake bite poisoning yet recorded in the world. Most samples were positive for more than one venom antibody. Antibodies were found to venoms of Bothrops viper in 60% of positive cases, of Micrurus coral snake in 21%, and of the bushmaster, Lachesis muta, in 18%. Further studies are needed to determine whether high venom-antibody levels afford protection against further snake envenoming. PMID:7299877

  6. Somatic diversification of antibody responses

    SciTech Connect

    Zheng, B.; Kelsoe, G.; Han, S.

    1996-01-01

    The humoral immune response is the culmination of a complex series of cellular interactions and migrations that define specific pathways of antigen-driven B cell differentiation. There are about 5 x 10{sup 8} and 10{sup 12} B lymphocyte lineage cells in the mouse and human, respectively, after the immune system has been established. B lymphocytes perceive antigen in their environment by virtue of their surface receptors (B cell receptor; BCR), in which membrane-associated immunoglobulin (mIg) is the antigen recognition substructure and the associated Ig{alpha} and Ig{beta} molecules transduce the activation signal. mIgs have extensive structural homology to immunoglobulins which are secreted by differentiated daughter cells following antigen stimulation. The specificity of a given BCR or antibody is created by a programmed successive process of gene rearrangements, first of D to J segments, then of V to DJ segments of the Ig heavy (H)-chain gene loci, and finally, of V to J segments of the Ig light (L)-chain gene loci. V,D, and J elements are assembled in a enormous number of combinations; variability is further achieved at the break-points of joining segments, including the insertion of non-germline-encoded nucleotide (N)sequences at the borders of V(D)J points, hence the almost unlimited specificities of the B cells or antibody repertoire. 129 refs.

  7. The second life of antibodies.

    PubMed

    Navolotskaya, E V

    2014-01-01

    Antibodies (immunoglobulins, Ig) are used by the immune system to identify and neutralize foreign objects and are responsible for antigen-binding and effector functions. Immunoglobulin G (IgG) is the major serum immunoglobulin of a healthy human (~75% of the total Ig fraction). The discovery in 1970 of the endogenous tetrapeptide tuftsin (Thr-Lys-Pro-Arg, fragment 289-292 of the C(H2)-domain of the heavy (H) chain of IgG), possessing both immunostimulatory and neurotrophic activities, was an impetus for the search for new biologically active peptides of immunoglobulin origin. As a result, fragments of the H-chain of IgG produced as a result of enzymatic cleavage of IgG within the antigen-antibody complex were discovered, synthesized, and studied. These fragments include rigin (341-344), immunorphin (364-373), immunocortin (11-20), and peptide p24 (335-358) and its fragments. In this review the properties of these peptides and their role in regulating the immune response are analyzed. PMID:24512657

  8. QUANTITATIVE INVESTIGATIONS OF IDIOTYPIC ANTIBODIES

    PubMed Central

    Spring, Susan B.; Schroeder, Kenneth W.; Nisonoff, Alfred

    1971-01-01

    The effect of challenge by antigen on persistence of clones of antibody-producing cells and on the induction of new clones was investigated through quantitative measurements of idiotypic specificities. In each of nine rabbits idiotypic specificities present in the earliest bleedings were completely replaced after a few months; subsequent changes occurred much more slowly. On a quantitative basis the population of molecules used as immunogen always reacted most effectively with the homologous anti-idiotypic antiserum. Little effect of increased antigen dose on the rate of change of idiotype was observed. Even large amounts of antigen administered every 2 wk caused only gradual changes in idiotypic specificities. This was attributed either to more effective capture of antigen by memory cells, as compared to precursor cells, or to the induction of tolerance in those clones that were not expressed. In two of three rabbits on a monthly injection schedule, the idiotypic specificities identified underwent very slow changes over a period as long as 17 months. Changes occurred more rapidly when antigen was administered every 2 wk. In each of four rabbits investigated, all idiotypic specificities identified before a 5 month rest period were still present afterwards, indicating the survival of essentially all clones of antibody-producing cells during that interval. Quantitative inhibition data indicated that some new clones of cells were initiated. PMID:15776574

  9. Antibody Staining in Drosophila Germaria.

    PubMed

    Lie-Jensen, Anette; Haglund, Kaisa

    2016-01-01

    Drosophila oogenesis is a powerful model for studying a wide spectrum of cellular and developmental processes in vivo. Oogenesis starts in a specialized structure called the germarium, which harbors the stem cells for both germ and somatic cells. The germarium produces egg chambers, each of which will develop into an egg. Active areas of research in Drosophila germaria include stem cell self-renewal, division, and maintenance, cell cycle control and differentiation, oocyte specification, intercellular communication, and signaling, among others. The solid knowledge base, the genetic tractability of the Drosophila model, as well as the availability and fast development of tools and imaging techniques for oogenesis research ensure that studies in this model will keep being instrumental for novel discoveries within cell and developmental biology also in the future. This chapter focuses on antibody staining in Drosophila germaria and provides a protocol for immunostaining as well as an overview of commonly used antibodies for visualization of different cell types and cellular structures. The protocol is well-suited for subsequent confocal microscopy analyses, and in addition we present key adaptations of the protocol that are useful when performing structured illumination microscopy (SIM) super-resolution imaging. PMID:27557571

  10. Monoclonal antibodies with group specificity toward sulfonamides: Selection of hapten and antibody selectivity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although many antibodies to sulfonamides have been generated, immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have properties dependent on the immunizing hapten structure and have always suffered from high selectivity for individual sulfonamides....

  11. Competitive binding of antibodies and antigen—antibody complexes to receptors on spleen lymphocytes

    PubMed Central

    Niederer, W.

    1974-01-01

    We have confirmed that adherence of antibodies to spleen lymphocytes is stabilized when the antibodies form complexes with antigen. Furthermore, there is evidence that antibodies having both binding sites occupied are less easily supplanted from the lymphocyte receptor by unspecific IgG than antibodies carrying one antigen only. Based on these results a hypothetical model for a feed-back control of the humoral immune response is put forward. PMID:4846174

  12. Induction and detection of antibodies to squalene.

    PubMed

    Matyas, G R; Wassef, N M; Rao, M; Alving, C R

    2000-11-01

    An enzyme-linked immunosorbent assay (ELISA) utilizing antigen coated on hydrophobic polyvinyldiene fluoride (PVDF) membranes is described for detecting antibodies that bind to squalene (SQE). Because of the prior lack of availability of validated antibodies to SQE, positive controls for the assay were made by immunization with formulations containing SQE to create monoclonal antibodies (mAbs) that reacted with SQE. Among eight immunogens tested, only two induced detectable murine antibodies to SQE: liposomes containing dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, 71% SQE, and lipid A [L(71% SQE+LA)], and, to a much lesser extent, an oil-in-water emulsion containing SQE, Tween 80, Span 85, and lipid A. In each case, lipid A served as an adjuvant, but neither SQE alone, SQE mixed with lipid A, liposomes containing 43% SQE and lipid A, nor several other emulsions containing both SQE and lipid A, induced antibodies that reacted with SQE. Monoclonal antibodies produced after immunizing mice with [L(71% SQE+LA)] served as positive controls for developing the ELISA. Monoclonal antibodies were produced that either recognized SQE alone but did not recognize squalane (SQA, the hydrogenated form of SQE), or that recognized both SQE and SQA. As found previously with other liposomal lipid antigens, liposomes containing lipid A also induced antibodies that reacted with the liposomal phospholipids. However, mAbs were also identified that reacted with SQE on PVDF membranes, but did not recognize either SQA or liposomal phospholipid. The polyclonal antiserum produced by immunizing mice with [L(71% SQE+LA)] therefore contained a mixed population of antibody specificities and, as expected, the ELISA of polyclonal antiserum with PVDF membranes detected antibodies both to SQE and SQA. We conclude that SQE is a weak antigen, but that antibodies that specifically bind to SQE can be readily induced by immunization with [L(71% SQE+LA)] and detected by ELISA with PVDF

  13. Quality Issues of Research Antibodies

    PubMed Central

    Weller, Michael G.

    2016-01-01

    According to several recent studies, an unexpectedly high number of landmark papers seem to be not reproducible by independent laboratories. Nontherapeutic antibodies used for research, diagnostic, food analytical, environmental, and other purposes play a significant role in this matter. Although some papers have been published offering suggestions to improve the situation, they do not seem to be comprehensive enough to cover the full complexity of this issue. In addition, no obvious improvements could be noticed in the field as yet. This article tries to consolidate the remarkable variety of conclusions and suggested activities into a more coherent conception. It is concluded that funding agencies and journal publishers need to take first and immediate measures to resolve these problems and lead the way to a more sustainable way of bioanalytical research, on which all can rely with confidence. PMID:27013861

  14. B Cells, Antibodies, and More

    PubMed Central

    Hoffman, William; Lakkis, Fadi G.

    2016-01-01

    B cells play a central role in the immunopathogenesis of glomerulonephritides and transplant rejection. B cells secrete antibodies that contribute to tissue injury via multiple mechanisms. In addition, B cells contribute to disease pathogenesis in autoimmunity and alloimmunity by presenting antigens as well as providing costimulation and cytokines to T cells. B cells also play an immunomodulatory role in regulating the immune response by secreting cytokines that inhibit disease onset and/or progression. B cell–targeted approaches for treating immune diseases of the kidney and other organs have gained significant momentum. However, much remains to be understood about B-cell biology in order to determine the timing, duration, and context of optimal therapeutic response to B cell–targeted approaches. In this review, we discuss the multifaceted roles of B cells as enhancers and regulators of immunity with relevance to kidney disease and transplantation. PMID:26700440

  15. IBC’s 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics International Conferences and the 2012 Annual Meeting of The Antibody Society

    PubMed Central

    Klöhn, Peter-Christian; Wuellner, Ulrich; Zizlsperger, Nora; Zhou, Yu; Tavares, Daniel; Berger, Sven; Zettlitz, Kirstin A.; Proetzel, Gabriele; Yong, May; Begent, Richard H.J.; Reichert, Janice M

    2013-01-01

    The 23rd Annual Antibody Engineering, 10th Annual Antibody Therapeutics international conferences, and the 2012 Annual Meeting of The Antibody Society, organized by IBC Life Sciences with contributions from The Antibody Society and two Scientific Advisory Boards, were held December 3–6, 2012 in San Diego, CA. The meeting drew over 800 participants who attended sessions on a wide variety of topics relevant to antibody research and development. As a prelude to the main events, a pre-conference workshop held on December 2, 2012 focused on intellectual property issues that impact antibody engineering. The Antibody Engineering Conference was composed of six sessions held December 3–5, 2012: (1) From Receptor Biology to Therapy; (2) Antibodies in a Complex Environment; (3) Antibody Targeted CNS Therapy: Beyond the Blood Brain Barrier; (4) Deep Sequencing in B Cell Biology and Antibody Libraries; (5) Systems Medicine in the Development of Antibody Therapies/Systematic Validation of Novel Antibody Targets; and (6) Antibody Activity and Animal Models. The Antibody Therapeutics conference comprised four sessions held December 4–5, 2012: (1) Clinical and Preclinical Updates of Antibody-Drug Conjugates; (2) Multifunctional Antibodies and Antibody Combinations: Clinical Focus; (3) Development Status of Immunomodulatory Therapeutic Antibodies; and (4) Modulating the Half-Life of Antibody Therapeutics. The Antibody Society’s special session on applications for recording and sharing data based on GIATE was held on December 5, 2012, and the conferences concluded with two combined sessions on December 5–6, 2012: (1) Development Status of Early Stage Therapeutic Antibodies; and (2) Immunomodulatory Antibodies for Cancer Therapy. PMID:23575266

  16. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M.

    2011-12-20

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  17. Anti-influenza M2e antibody

    DOEpatents

    Bradbury, Andrew M.

    2013-04-16

    Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

  18. Antibody-drug conjugates: Intellectual property considerations

    PubMed Central

    Storz, Ulrich

    2015-01-01

    Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs. PMID:26292154

  19. How to successfully patent therapeutic antibodies.

    PubMed

    Lahrtz, Fritz

    2015-04-01

    Therapeutic antibodies have become an established class of drugs for the treatment of a variety of diseases, especially cancer and autoimmune/inflammatory disorders, and a sufficient patent protection is a prerequisite for their successful commercialization. As monoclonal antibodies and their therapeutic potential have been well known for decades, the mere production of yet another therapeutic antibody is in many jurisdictions not considered a patentable invention. In contrast, antibodies with novel structural features and/or improved properties may be patentable. When drafting the claims, care should be taken to obtain a broad patent scope that protects both the antibody of interest and related antibodies having the same functional features, thereby preventing competitors from marketing a functionally equivalent antibody. Furthermore, the application should contain experimental evidence showing the improved properties of the claimed antibody. After the filing of a priority patent application, patent protection should be initiated at least in countries that are of particular commercial importance. Subsequent inventions relating to novel uses, formulations, dosage regimens, and combinations with other treatment modalities should be protected by further patent applications to extend patent term. PMID:25614506

  20. Bioconjugation of antibodies to horseradish peroxidase (hrp)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The bioconjugation of an antibody to an enzymatic reporter such as horseradish peroxidase (HRP) affords an effective mechanism by which immunoassay detection of a target antigen can be achieved. The use of heterobifunctional cross—linkers to covalently link antibodies to HRP provides a simple and c...

  1. Monoclonal Antibody Therapy for Advanced Neuroblastoma

    Cancer.gov

    NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

  2. 8th Annual European Antibody Congress 2012

    PubMed Central

    Beck, Alain; Carter, Paul J.; Gerber, Hans-Peter; Lugovskoy, Alexey A.; Wurch, Thierry; Junutula, Jagath R.; Kontermann, Roland E; Mabry, Robert

    2013-01-01

    The 8th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The new agenda format for 2012 included three parallel tracks on: (1) naked antibodies; (2) antibody drug conjugates (ADCs); and (3) bispecific antibodies and alternative scaffolds. The meeting started and closed with three plenary lectures to give common background and to share the final panel discussion and conclusions. The two day event included case studies and networking for nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The monoclonal antibody track was focused on understanding the structure-function relationships, optimization of antibody design and developability, and processes that allow better therapeutic candidates to move through the clinic. Discussions on novel target identification and validation were also included. The ADC track was dedicated to evaluation of the ongoing success of the established ADC formats alongside the rise of the next generation drug-conjugates. The bispecific and alternative scaffold track was focused on taking stock of the multitude of bispecific formats being investigated and gaining insight into recent innovations and advancements. Mechanistic understanding, progression into the clinic and the exploration of multispecifics, redirected T cell killing and alternative scaffolds were extensively discussed. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:23493119

  3. How to successfully patent therapeutic antibodies.

    PubMed

    Lahrtz, Fritz

    2015-04-01

    Therapeutic antibodies have become an established class of drugs for the treatment of a variety of diseases, especially cancer and autoimmune/inflammatory disorders, and a sufficient patent protection is a prerequisite for their successful commercialization. As monoclonal antibodies and their therapeutic potential have been well known for decades, the mere production of yet another therapeutic antibody is in many jurisdictions not considered a patentable invention. In contrast, antibodies with novel structural features and/or improved properties may be patentable. When drafting the claims, care should be taken to obtain a broad patent scope that protects both the antibody of interest and related antibodies having the same functional features, thereby preventing competitors from marketing a functionally equivalent antibody. Furthermore, the application should contain experimental evidence showing the improved properties of the claimed antibody. After the filing of a priority patent application, patent protection should be initiated at least in countries that are of particular commercial importance. Subsequent inventions relating to novel uses, formulations, dosage regimens, and combinations with other treatment modalities should be protected by further patent applications to extend patent term.

  4. 7th Annual European Antibody Congress 2011

    PubMed Central

    2012-01-01

    The 7th European Antibody Congress (EAC), organized by Terrapin Ltd., was again held in Geneva, Switzerland, following on the tradition established with the 4th EAC. The 2011 version of the EAC was attended by nearly 250 delegates who learned of the latest advances and trends in the global development of antibody-based therapeutics. The first day focused on advances in understanding structure-function relationships, choosing the best format, glycoengineering biobetter antibodies, improving the efficacy and drugability of mAbs and epitope mapping. On the second day, the discovery of novel targets for mAb therapy, clinical pipeline updates, use of antibody combinations to address resistance, generation and identification of mAbs against new targets and biosimilar mAb development were discussed. Antibody-drug conjugates, domain antibodies and new scaffolds and bispecific antibodies were the topics of the third day. In total, nearly 50 speakers provided updates of programs related to antibody research and development on-going in the academic, government and commercial sectors. PMID:22453093

  5. Microscale purification of antigen-specific antibodies

    PubMed Central

    Brown, Eric P.; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E.; Chan, Ying N.; Lai, Jennifer I.; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E.

    2015-01-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain. PMID:26078040

  6. Antibody-drug conjugates: Intellectual property considerations.

    PubMed

    Storz, Ulrich

    2015-01-01

    Antibody-drug conjugates are highly complex entities that combine an antibody, a linker and a toxin. This complexity makes them demanding both technically and from a regulatory point of view, and difficult to deal with in their patent aspects. This article discusses different issues of patent protection and freedom to operate with regard to this promising new class of drugs. PMID:26292154

  7. Microscale purification of antigen-specific antibodies.

    PubMed

    Brown, Eric P; Normandin, Erica; Osei-Owusu, Nana Yaw; Mahan, Alison E; Chan, Ying N; Lai, Jennifer I; Vaccari, Monica; Rao, Mangala; Franchini, Genoveffa; Alter, Galit; Ackerman, Margaret E

    2015-10-01

    Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain.

  8. Anti-miroestrol polyclonal antibodies: a comparison of immunogen preparations used to obtain desired antibody properties.

    PubMed

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Yusakul, Gorawit; Tanaka, Hiroyuki; Putalun, Waraporn

    2016-04-01

    Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays. PMID:26563142

  9. Anti-miroestrol polyclonal antibodies: a comparison of immunogen preparations used to obtain desired antibody properties.

    PubMed

    Kitisripanya, Tharita; Jutathis, Kamonthip; Inyai, Chadathorn; Komaikul, Jukrapun; Udomsin, Orapin; Yusakul, Gorawit; Tanaka, Hiroyuki; Putalun, Waraporn

    2016-04-01

    Immunogen quality is one important factor that contributes to desirable antibody characteristics. Highly specific antibodies against miroestrol can be used to develop a quality control immunoassay for Pueraria candollei products. In this study, we investigated how various immunogen preparations affect antibody properties. The results show that immunogen prepared using the Mannich reaction provides antibodies with higher specificity and sensitivity against miroestrol than immunogen prepared with the periodate reaction. The results suggest the Mannich reaction maintains the original structure of miroestrol and generates useful antibodies for developing immunoassays.

  10. Edelman's view on the discovery of antibodies.

    PubMed

    Ribatti, Domenico

    2015-04-01

    Gerald M. Edelman began working to the structure of antibodies when joined as graduate student the laboratory of Henry Kunkel in 1958 at the "Rockefeller University" in New York, obtaining his doctorate in 1960. Edelman's focus on the structure of antibodies led to the 1972 Nobel Prize in Physiology or Medicine along with Rodney R. Porter. Edelman and Porter decided to approach the problem of antibodies structure by splitting. In 1959, Porter published a report in which he used the enzyme papain to cleave the antibody molecule into three pieces of about 50,000 Da, corresponding to the two Fab (antigen-binding) and constant Fc (crystallizable) fragments. In the same year, Edelman showed that reduction of the disulfide bonds of antibodies in the presence of denaturizing agents led to dissociation of the molecule into smaller pieces, now known to be the light (L) and heavy (H) chains.

  11. Clearance of pathological antibodies using biomimetic nanoparticles

    PubMed Central

    Copp, Jonathan A.; Fang, Ronnie H.; Luk, Brian T.; Hu, Che-Ming J.; Gao, Weiwei; Zhang, Kang; Zhang, Liangfang

    2014-01-01

    Pathological antibodies have been demonstrated to play a key role in type II immune hypersensitivity reactions, resulting in the destruction of healthy tissues and leading to considerable morbidity for the patient. Unfortunately, current treatments present significant iatrogenic risk while still falling short for many patients in achieving clinical remission. In the present work, we explored the capability of target cell membrane-coated nanoparticles to abrogate the effect of pathological antibodies in an effort to minimize disease burden, without the need for drug-based immune suppression. Inspired by antibody-driven pathology, we used intact RBC membranes stabilized by biodegradable polymeric nanoparticle cores to serve as an alternative target for pathological antibodies in an antibody-induced anemia disease model. Through both in vitro and in vivo studies, we demonstrated efficacy of RBC membrane-cloaked nanoparticles to bind and neutralize anti-RBC polyclonal IgG effectively, and thus preserve circulating RBCs. PMID:25197051

  12. Development and Detection of Kidd Antibodies.

    PubMed

    Sanford, Kimberly Williams; Bourikian, Seda; McClain, Aryn; Curtis, Kyle

    2015-01-01

    Kidd antibodies have a reputation for causing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We present a case of an untransfused male patient who developed anti-Kidd(a) (Jk(a)) antibodies after receiving an allogenic renal transplant. The formation of this antibody was associated with exposure to the Kidd antigen expressed on the tubular epithelium of the transplanted kidney. The 59-year-old white male patient had received a cadaveric renal transplant at our clinic and returned 5 years later with proteinuria and elevated serum creatinine levels, consistent with nephrotic syndrome. We review the expression of Kidd antigens and the development and detection of Kidd antibodies, and discuss the case reports from the literature of Kidd antibodies associated with kidney-graft rejection that suggest Kidd antigens play a role as a minor histocompatibility antigen. PMID:26199265

  13. Genetically engineered antibody molecules and their application.

    PubMed

    Morrison, S L; Wims, L; Wallick, S; Tan, L; Oi, V T

    1987-01-01

    Immunoglobulin genes can be efficiently expressed following transfection into myeloma cells. Using protoplast fusion, transfection frequencies greater than 10(-3) can be achieved. Compatible plasmids containing two different selectible markers are used to simultaneously deliver heavy and light chain genes to the same cell. To produce molecules with differing specificities the rearranged and expressed variable regions can be cloned from the appropriate hybridoma. In some cases, variable regions from cDNAs can be inserted into the expression vectors. It is possible to manipulate the immunoglobulin genes and produce novel antibody molecules. Antibodies have been produced in which the variable regions from mouse antibodies have been joined to human constant regions. In addition, antibodies with altered constant regions have been produced. These genetically engineered antibodies provide a unique set of reagents to study structure-function relationships within the molecule. They also can potentially be used in the diagnosis and therapy of human disease.

  14. Development and Detection of Kidd Antibodies.

    PubMed

    Sanford, Kimberly Williams; Bourikian, Seda; McClain, Aryn; Curtis, Kyle

    2015-01-01

    Kidd antibodies have a reputation for causing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. We present a case of an untransfused male patient who developed anti-Kidd(a) (Jk(a)) antibodies after receiving an allogenic renal transplant. The formation of this antibody was associated with exposure to the Kidd antigen expressed on the tubular epithelium of the transplanted kidney. The 59-year-old white male patient had received a cadaveric renal transplant at our clinic and returned 5 years later with proteinuria and elevated serum creatinine levels, consistent with nephrotic syndrome. We review the expression of Kidd antigens and the development and detection of Kidd antibodies, and discuss the case reports from the literature of Kidd antibodies associated with kidney-graft rejection that suggest Kidd antigens play a role as a minor histocompatibility antigen.

  15. Dual targeting strategies with bispecific antibodies

    PubMed Central

    2012-01-01

    Monoclonal antibodies are widely used for the treatment of cancer, inflammatory and infectious diseases and other disorders. Most of the marketed antibodies are monospecific and therefore capable of interacting and interfering with a single target. However, complex diseases are often multifactorial in nature, and involve redundant or synergistic action of disease mediators or upregulation of different receptors, including crosstalk between their signaling networks. Consequently, blockade of multiple, different pathological factors and pathways may result in improved therapeutic efficacy. This result can be achieved by combining different drugs, or use of the dual targeting strategies applying bispecific antibodies that have emerged as an alternative to combination therapy. This review discusses the various dual targeting strategies for which bispecific antibodies have been developed and provides an overview of the established bispecific antibody formats. PMID:22453100

  16. Principles and application of antibody libraries for infectious diseases.

    PubMed

    Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

    2014-12-01

    Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

  17. Radiohalogenated half-antibodies and maleimide intermediate therefor

    DOEpatents

    Kassis, Amin I.; Khawli, Leslie A.

    1991-01-01

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabelled half-antibody having immunological specific binding characteristics of whole antibody.

  18. Radiohalogenated half-antibodies and maleimide intermediate therefor

    DOEpatents

    Kassis, A.I.; Khawli, L.A.

    1991-02-19

    N-(m-radiohalophenyl) maleimide can be conjugated with a reduced antibody having a mercapto group to provide a radiolabeled half-antibody having immunological specific binding characteristics of whole antibody. No Drawings

  19. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    PubMed

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries.

  20. Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society December 7-10, 2015, San Diego, CA, USA.

    PubMed

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M; Lund-Johansen, Fridtjof; Bradbury, Andrew R M; Carter, Paul J; Melis, Joost P M

    2016-01-01

    The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6-10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on "Antibodies to watch" in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  1. Combinatorial antibody libraries: new advances, new immunological insights.

    PubMed

    Lerner, Richard A

    2016-08-01

    Immunochemists have become quite proficient in engineering existing antibody molecules to control their pharmacological properties. However, in terms of generating new antibodies, the combinatorial antibody library has become a central feature of modern immunochemistry. These libraries are essentially an immune system in a test tube and enable the selection of antibodies without the constraints of whole animal or cell-based systems. This Review provides an overview of how antibody libraries are constructed and discusses what can be learnt from these synthetic systems. In particular, the Review focuses on new biological insights from antibody libraries - such as the concept of 'SOS antibodies' - and the growing use of intracellular antibodies to perturb cellular functions.

  2. Antiphospholipid Antibodies in Lupus Nephritis

    PubMed Central

    Arnaud, Laurent; Gerhardsson, Jakob; Zickert, Agneta; Sundelin, Birgitta; Malmström, Vivianne; Svenungsson, Elisabet; Gunnarsson, Iva

    2016-01-01

    Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE). It remains unclear whether antiphospholipid antibodies (aPL) alter the course of LN. We thus investigated the impact of aPL on short-term and long-term renal outcomes in patients with LN. We assessed levels of aPL cross-sectionally in SLE patients diagnosed with (n = 204) or without (n = 294) LN, and prospectively in 64 patients with active biopsy-proven LN (52 proliferative, 12 membranous), before and after induction treatment (short-term outcomes). Long-term renal outcome in the prospective LN cohort was determined by the estimated glomerular filtration rate (eGFR) and the Chronic Kidney Disease (CKD) stage, after a median follow-up of 11.3 years (range: 3.3–18.8). Cross-sectional analysis revealed no association between LN and IgG/IgM anticardiolipin or anti-β2-glycoprotein I antibodies, or lupus anticoagulant. Both aPL positivity and levels were similar in patients with active LN and non-renal SLE. Following induction treatment for LN, serum IgG/IgM aPL levels decreased in responders (p<0.005 for all), but not in non-responders. Both at active LN and post-treatment, patients with IgG, but not IgM, aPL had higher creatinine levels compared with patients without IgG aPL. Neither aPL positivity nor levels were associated with changes in eGFR from either baseline or post-treatment through long-term follow-up. Moreover, aPL positivity and levels both at baseline and post-treatment were similar in patients with a CKD stage ≥3 versus 1–2 at the last follow-up. In conclusion, neither aPL positivity nor levels were found to be associated with the occurrence of LN in SLE patients. However, IgG aPL positivity in LN patients was associated with a short-term impairment of the renal function while no effect on long-term renal outcome was observed. Furthermore, IgG and IgM aPL levels decreased following induction treatment only in responders, indicating that aPL levels are affected by

  3. Antibody production in the bullfrog (Rana catesbeiana)

    PubMed Central

    Coe, J. E.; Peel, L. F.

    1970-01-01

    Serum antibody was found by radioimmunoelectrophoresis (RIEP) in thirty-one of thirty-five bullfrogs (BF) immunized with one of four protein antigens. Rabbit γ-globulin (RGG) and hen egg albumin were the best antigens, whereas human serum albumin and bovine serum albumin induced a less consistent immune response. Although a IgM to IgG sequence of antibody production usually was detected with RGG as antigen, a similar sequence was infrequent with the other antigens and the initial response was usually a IgG antibody. IgM antibody was detected in the serum for a prolonged interval (>100 days) and precipitating quantities of IgG antibody were found more than 1 year after antigen inoculation. As measured by haemagglutination, the IgM antibody was routinely inactivated by mercaptoethanol (ME) treatment and IgG antibody was frequently inactivated by ME, although it still had antigen binding capacity by RIEP. IgG hemagglutinins, which were resistant to ME treatment, were found in some sera obtained from BF after booster injections of antigen. Immunoelectrophoretic examination of normal or immune BF sera revealed a prominent precipitin line of slow γ-mobility which did not bind 131I-labelled antigen but did bind 59Fe. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5 PMID:4097586

  4. Next generation of antibody therapy for cancer

    PubMed Central

    Zhu, Zhenping; Yan, Li

    2011-01-01

    Monoclonal antibodies (mAbs) have become a major class of therapeutic agents providing effective alternatives to treating various human diseases. To date, 15 mAbs have been approved by regulatory agencies in the world for clinical use in oncology indications. The selectivity and specificity, the unique pharmacokinetics, and the ability to engage and activate the host immune system differentiate these biologics from traditional small molecule anticancer drugs. mAb-based regimens have brought clinical benefits, including improvements in overall survival, to patients with a variety of cancers. Many challenges still remain, however, to fully realize the potential of these new medicines. With our further understanding of cancer biology, mechanism of antibody action, and advancement of antibody engineering technologies, many novel antibody formats or antibody-derived molecules are emerging as promising new generation therapeutics. Carefully designed and engineered, they retain the advantage of specificity and selectivity of original antibodies, but in the meantime acquire additional special features such as improved pharmacokinetics, increased selectivity, and enhanced anticancer efficacy. Promising clinical results are being generated with these newly improved antibody-based therapeutics. PMID:21527062

  5. Baculovirus display of functional antibody Fab fragments.

    PubMed

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

    2015-08-01

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

  6. 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. PMID:27472861

  7. Monoclonal antibodies in the treatment of cancer

    SciTech Connect

    Dillman, R.O.

    1984-01-01

    Potential uses of monoclonal antibodies in anti-cancer treatment include passive serotherapy, radioisotope conjugates, toxin-linked conjugates, and chemotherapy-monoclonal antibody conjugates. The bases for these applications have been founded in research with heterologous antisera, and in some cases with monoclonal antibodies in animal tumor models. Human trials with passive serotherapy have already begun in both hematopoietic and solid tumor malignancies. Promising results have been reported in cutaneous T cell lymphoma with anti-T cell monoclonal antibody, and in nodular lymphoma with anti-idiotype monoclonal antibody. Radioisotope conjugate work appears promising for imaging in both animals and humans, and this work will lay the foundation for possible therapeutic application of radio-immunotherapy. Toxin-linked conjugates are promising in vitro and may have application in autologous bone marrow transplantation. Research with chemotherapy conjugates is also underway. Preliminary results suggest that murine monoclonal antibodies will be well tolerated clinically except in the setting of circulating cells which bear the target antigen, where rapid infusions may be associated with intolerable side effects. In certain diseases, production of endogenous anti-mouse antibodies may also limit application. Advances in the technology for human-human hybridoma production may help solve some of these problems. 132 references.

  8. Distinct Therapeutic Mechanisms of Tau Antibodies

    PubMed Central

    Funk, Kristen E.; Mirbaha, Hilda; Jiang, Hong; Holtzman, David M.; Diamond, Marc I.

    2015-01-01

    Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo. PMID:26126828

  9. Antibodies Against Three Forms of Urokinase

    NASA Technical Reports Server (NTRS)

    Morrison, Dennis R.; Atassi, M. Zouhair

    2007-01-01

    Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

  10. Labeling of monoclonal antibodies with radionuclides

    SciTech Connect

    Bhargava, K.K.; Acharya, S.A. )

    1989-07-01

    Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as EDTA or DTPA are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides. 78 references.

  11. Uses of monoclonial antibody 8H9

    DOEpatents

    Cheung, Nai-Kong V.

    2015-06-23

    This invention provides an antibody that binds the same antigen as that of monoclonal antibody 8H9, wherein the heavy chain CDR (Complementary Determining Region)1 comprises NYDIN, heavy chain CDR2 comprises WIFPGDGSTQY, heavy chain CDR3 comprises QTTATWFAY, and the light chain CDR1 comprises RASQSISDYLH, light chain CDR2 comprises YASQSIS, and light chain CDR3 comprises QNGHSFPLT. In another embodiment, there is provided a polypeptide that binds the same antigen as that of monoclonal antibody 8H9, wherein the polypeptide comprises NYDIN, WIFPGDGSTQY, QTTATWFAY, RASQSISDYLH, YASQSIS, and QNGHSFPLT.

  12. AIDS antibody tests on inpatient psychiatric units.

    PubMed

    Binder, R L

    1987-02-01

    An antibody test for the causative virus of the acquired immune deficiency syndrome (AIDS) became commercially available in 1985. The author discusses the use of the AIDS antibody test on inpatient psychiatric units. She reviews the controversial legal and ethical questions related to its use, addressing such questions as Who should be tested for the AIDS antibody? When and to whom should the results of the test be disclosed? and How should the doctrine of "right to privacy" be balanced with the "duty to warn"?

  13. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  14. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1991-01-01

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

  15. The antibody approach of labeling blood cells

    SciTech Connect

    Srivastava, S.C.

    1992-12-31

    Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

  16. Emerging antibody products and Nicotiana manufacturing.

    PubMed

    Whaley, Kevin J; Hiatt, Andrew; Zeitlin, Larry

    2011-03-01

    Antibody based products are not widely available to address multiple global health challenges due to high costs, limited manufacturing capacity, and long manufacturing lead times. Nicotiana-based manufacturing of antibody products may now begin to address these challenges as a result of revolutionary advances in transient expression and altered glycosylation pathways. This review provides examples of emerging antibody-based products (mucosal and systemic) that could be competitive and commercially viable when the attributes of Nicotiana-based manufacturing (large scale, versatile, rapid, low cost) are utilized.

  17. Immunocytochemical and Immunohistochemical Staining with Peptide Antibodies.

    PubMed

    Friis, Tina; Pedersen, Klaus Boberg; Hougaard, David; Houen, Gunnar

    2015-01-01

    Peptide antibodies are particularly useful for immunocytochemistry (ICC) and immunohistochemistry (IHC), where antigens may denature due to fixation of tissues and cells. Peptide antibodies can be made to any defined sequence, including unknown putative proteins and posttranslationally modified sequences. Moreover, the availability of large amounts of the antigen (peptide) allows inhibition/adsorption controls, which are important in ICC/IHC, due to the many possibilities for false-positive reactions caused by immunoglobulin Fc receptors, nonspecific reactions, and cross-reactivity of primary and secondary antibodies with other antigens and endogenous immunoglobulins, respectively. Here, simple protocols for ICC and IHC are described together with recommendations for appropriate controls.

  18. Antiphospholipid antibody syndrome complicated by Grave's disease.

    PubMed

    Takahashi, Ayumi; Tamura, Atsushi; Ishikawa, Osamu

    2002-12-01

    The report describes a woman with primary antiphospholipid antibody syndrome complicated with Grave's disease. Developing symptoms included a small cutaneous nodule on her finger and subsequently ecchymotic purpura on the cheeks, ears, buttocks and lower legs. Histological examinations showed thrombosed vessels in the dermis without or with hemorrhage, respectively. Laboratory investigation revealed positive lupus anticoagulant and immunogenic hyperthyroidism due to Grave's disease. There is a close relationship between the cutaneous manifestation of antiphospholipid antibody syndrome and the activities of Grave's disease and a possible link of antiphospholipid antibody syndrome with Grave's disease was suggested both by the etiology of the disease as well as the disease activity. PMID:12532043

  19. Advances in recombinant antibody manufacturing.

    PubMed

    Kunert, Renate; Reinhart, David

    2016-04-01

    Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today.

  20. [Anti-basal ganglia antibody].

    PubMed

    Hayashi, Masaharu

    2013-04-01

    Sydenham's chorea (SC) is a major manifestation of rheumatic fever, and the production of anti-basal ganglia antibodies (ABGA) has been proposed in SC. The pathogenesis is hypothesized as autoimmune targeting of the basal ganglia via molecular mimicry, triggered by streptococcal infection. The spectrum of diseases in which ABGA may be involved has been broadened to include other extrapyramidal movement disorders, such as tics, dystonia, and Parkinsonism, as well as other psychiatric disorders. The autoimmune hypothesis in the presence and absence of ABGA has been suggested in Tourette's syndrome (TS), early onset obsessive-compulsive disorders (OCD), and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS). Recently, the relationship between ABGA and dopamine neurons in the basal ganglia has been examined, and autoantibodies against dopamine receptors were detected in the sera from patients with basal ganglia encephalitis. In Japan, the occurrence of subacute encephalitis, where patients suffer from episodes of altered behavior and involuntary movements, has increased. Immune-modulating treatments are effective, indicating the involvement of an autoimmune mechanism. We aimed to detect the anti-neuronal autoantibodies in such encephalitis, using immunohistochemical assessment of patient sera. The sera from patients showing involuntary movements had immunoreactivity for basal ganglia neurons. Further epitopes for ABGA will be investigated in basal ganglia disorders other than SC, TS, OCD, and PANDAS. PMID:23568985

  1. Optimising testing for phospholipid antibodies

    PubMed Central

    Helbert, M; Bodger, S; Cavenagh, J; D'Cruz, D; Thomas, J; MacCallum, P

    2001-01-01

    Aim—To compare anticardiolipin (ACL) and anti-ß2 glycoprotein 1 (ß2gp1) enzyme linked immunosorbent assays (ELISAs) in the diagnosis of antiphospholipid syndrome (APS) and to incorporate these results into a meta-analysis of published data. Method—Three representative commercial ACL ELISAs and an in house ß2gp1 assay were optimised and then assessed on 124 sera from normal donors, patients with infection, or patients with APS. A Medline search was screened for papers meeting defined criteria to conduct a meta-analysis. The performance of the assays used in this study was included. Results—A non-quantitative ACL assay performed at least as well as the anti-ß2gp1 assay in the diagnosis of APS. Meta-analysis confirmed that neither assay is perfect, although the anti-ß2gp1 assay had a higher specificity and lower sensitivity than the ACL assay. Conclusions—The pooled data suggest that the ACL assay is used to investigate thrombosis without overt underlying pathology and that the improved specificity of the anti-ß2gp1 assay is exploited where infection, connective tissue disease, or atheroma are present. Key Words: antiphospholipid syndrome • anticardiolipin antibodies • anti-ß2 glycoprotein 1 • sensitivity PMID:11533076

  2. Antiphospholipid antibodies: Paradigm in transition

    PubMed Central

    Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Ahn, Yeon S; Kelley, Roger E; Zivadinov, Robert; Maghzi, Amir H; Etemadifar, Masoud; Mousavi, Seyed Ali; Minagar, Alireza

    2009-01-01

    Objectives This is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP. Organization After a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed. Conclusion The current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology. PMID:19154576

  3. Antibody Response to Hypervariable Region 1 Interferes with Broadly Neutralizing Antibodies to Hepatitis C Virus

    PubMed Central

    Keck, Zhen-yong; Girard-Blanc, Christine; Wang, Wenyan; Lau, Patrick; Zuiani, Adam; Rey, Felix A.; Krey, Thomas; Diamond, Michael S.

    2015-01-01

    ABSTRACT Hypervariable region 1 (HVR1) (amino acids [aa] 384 to 410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412 to 423 revealed two subsets, designated HC33 HMAbs. While both subsets have contact residues within aa 412 to 423, alanine-scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412 to 423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412 to 423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside aa 412 to 423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection. IMPORTANCE HVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from

  4. Antisperm antibodies and in vitro fertilization failure.

    PubMed

    Pexieder, T; Boillat, E; Janecek, P

    1985-12-01

    A new enzyme-linked immunosorbent assay (ELISA)-based test (Zer, Jerusalem) has allowed us to show the presence of antisperm antibodies (1:32 to 1:64) in the blood of 14 (33%) of 32 patients undergoing in vitro fertilization and embryo transfer (IVF-ET) under gonadotropin stimulation. Observation of the morphology of fertilization in eight patients with and seven patients without antisperm antibodies has shown a significant association (P less than or equal to 0.025, Fisher exact probability test) among the cumulus/corona coagulation, the absence of fertilization, and the presence of these antibodies. Cumulus/oocyte complex washing and/or enzymatic cumulus removal are considered as elective interventions in the case of antisperm immunity. Each patient entering an IVF-ET program should have the antisperm antibody assay performed as a preliminary screening.

  5. Broadly neutralizing antibodies against influenza viruses

    PubMed Central

    Laursen, Nick S.; Wilson, Ian A.

    2014-01-01

    Despite available antivirals and vaccines, influenza infections continue to be a major cause of mortality worldwide. Vaccination generally induces an effective, but strain-specific antibody response. As the virus continually evolves, new vaccines have to be administered almost annually when a novel strain becomes dominant. Furthermore, the sporadic emerging resistance to neuraminidase inhibitors among circulating strains suggests an urgent need for new therapeutic agents. Recently, several cross-reactive antibodies have been described, which neutralize an unprecedented spectrum of influenza viruses. These broadly neutralizing antibodies generally target conserved functional regions on the major influenza surface glycoprotein hemagglutinin (HA). The characterization of their neutralization breadth and epitopes on HA could stimulate the development of new antibody-based antivirals and broader influenza vaccines. PMID:23583287

  6. Antibodies as Mediators of Brain Pathology.

    PubMed

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T; Diamond, Betty

    2015-11-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease.

  7. Gut Microbial Metabolites Fuel Host Antibody Responses.

    PubMed

    Kim, Myunghoo; Qie, Yaqing; Park, Jeongho; Kim, Chang H

    2016-08-10

    Antibody production is a metabolically demanding process that is regulated by gut microbiota, but the microbial products supporting B cell responses remain incompletely identified. We report that short-chain fatty acids (SCFAs), produced by gut microbiota as fermentation products of dietary fiber, support host antibody responses. In B cells, SCFAs increase acetyl-CoA and regulate metabolic sensors to increase oxidative phosphorylation, glycolysis, and fatty acid synthesis, which produce energy and building blocks supporting antibody production. In parallel, SCFAs control gene expression to express molecules necessary for plasma B cell differentiation. Mice with low SCFA production due to reduced dietary fiber consumption or microbial insufficiency are defective in homeostatic and pathogen-specific antibody responses, resulting in greater pathogen susceptibility. However, SCFA or dietary fiber intake restores this immune deficiency. This B cell-helping function of SCFAs is detected from the intestines to systemic tissues and conserved among mouse and human B cells, highlighting its importance. PMID:27476413

  8. Polynucleotides encoding anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R.; Kehoe, John; Bradbury, Andrew M.

    2011-01-11

    The invention provides anti-sulfotyrosine specific antibodies capable of detecting and isolating polypeptides that are tyrosine-sulfated. The sulfotyrosine antibodies and antibody fragments of the invention may be used to discriminate between the non-sulfated and sulfated forms of such proteins, using any number of immunological assays, such ELISAs, immunoblots, Western Blots, immunoprecipitations, and the like. Using a phage-display system, single chain antibodies (scFvs) were generated and screened against tyrosine-sulfated synthetic peptide antigens, resulting in the isolation of scFvs that specifically recognize sulfotyrosine-containing peptides and/or demonstrate sulfotyrosine-specific binding in tyrosine sulfated proteins. The VH and VL genes from one such sulfotyrosine-specific scFv were employed to generate a full length, sulfotyrosine-specific immunoglobulin.

  9. Brain-Reactive Antibodies and Disease

    PubMed Central

    Diamond, B.; Honig, G.; Mader, S.; Brimberg, L.; Volpe, B.T.

    2015-01-01

    Autoimmune diseases currently affect 5–7% of the world's population; in most diseases there are circulating autoantibodies. Brain-reactive antibodies are present in approximately 2–3% of the general population but do not usually contribute to brain pathology. These antibodies penetrate brain tissue only early in development or under pathologic conditions. This restriction on their pathogenicity and the lack of correlation between serum titers and brain pathology have, no doubt, contributed to a delayed appreciation of the contribution of autoantibodies in diseases of the central nervous system. Nonetheless, it is increasingly clear that antibodies can cause damage in the brain and likely initiate or aggravate multiple neurologic conditions; brain-reactive antibodies contribute to symptomatology in autoimmune disease, infectious disease, and malignancy. PMID:23516983

  10. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  11. Antibodies as Mediators of Brain Pathology.

    PubMed

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T; Diamond, Betty

    2015-11-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  12. Host versus flu: antibodies win a round?

    PubMed Central

    Brooke, Christopher B; Yewdell, Jonathan W

    2013-01-01

    Structural and functional analyses of three neutralizing antibodies against influenza virus H2 HA may explain why this HA subtype has disappeared from circulation in the human population and point to a potential new avenue for antiflu therapeutics. PMID:23463305

  13. Antibodies against the calcium-binding protein

    SciTech Connect

    Chou, Mei; Jensen, K.G.; Sjolund, R.D. ); Krause, K.H.; Campbell, K.P. )

    1989-12-01

    Plant microsomes contain a protein clearly related to a calcium-binding protein, calsequestrin, originally found in the sarcoplasmic reticulum of muscle cells, responsible for the rapid release and uptake of Ca{sup 2+} within the cells. The location and role of calsequestrin in plant cells is unknown. To generate monoclonal antibodies specific to plant calsequestrin, mice were immunized with a microsomal fraction from cultured cells of Streptanthus tortuosus (Brassicaceae). Two clones cross-reacted with one protein band with a molecular weight equal to that of calsequestrin (57 kilodaltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. This band is able to bind {sup 45}Ca{sup 2+} and can be recognized by a polyclonal antibody against the canine cardiac muscle calsequestrin. Rabbit skeletal muscle calsequestrin cross-reacted with the plant monoclonal antibodies. The plant monoclonal antibodies generated here are specific to calsequestrin protein.

  14. Analyzing antibody-Fc-receptor interactions.

    PubMed

    Nimmerjahn, Falk; Ravetch, Jeffrey V

    2008-01-01

    Cellular receptors for immunoglobulins (Fc-receptors; FcR) are central mediators of antibody-triggered effector functions. Immune complex (IC) binding to FcRs results in a variety of reactions such as the release of inflammatory mediators, antibody dependent cellular cytotoxicity (ADCC) and phagocytosis of ICs. Analyzing antibody-FcR (Ab-FcR) interactions in vitro is essential to determine the effector mechanisms, binding characteristics and affinity parameters that will impact and predict antibody activity in vivo. The methods described in this chapter include the generation of ICs and soluble FcR variants, as well as ELISA and FACS-based assays to study Ab-FcR interactions.

  15. Antibody Fc: Linking Adaptive and Innate Immunity

    PubMed Central

    Reichert, Janice M.

    2014-01-01

    Antibody Fc: Linking Adaptive and Innate Immunity, edited by Margaret E. Ackerman and Falk Nimmerjahn and published by Academic Press, provides a highly detailed examination of the involvement of the antibody Fc in mechanisms critical to both innate and adaptive immune responses. Despite a recent increase in format diversity, most marketed antibodies are full-length IgG molecules and the majority of the commercial clinical pipeline of antibody therapeutics is composed of Fc-containing IgG molecules, which underscores the importance of understanding how the Fc domain affects biological responses. The book is divided into six sections that include a total of 20 chapters. In order of their appearance, the sections provide extensive coverage of effector mechanisms, effector cells, Fc receptors, variability of the Fc domain, genetic associations, and evolving areas.

  16. Chemical biology: How to minimalize antibodies

    NASA Astrophysics Data System (ADS)

    Rader, Christoph

    2015-02-01

    The success of antibodies as pharmaceuticals has triggered interest in crafting much smaller mimics. A crucial step forward has been taken with the chemical synthesis of small molecules that recruit immune cells to attack cancer cells.

  17. Monoclonal antibodies: their importance to surgeons.

    PubMed

    Estabrook, A; Mesa-Tejada, R

    1989-01-01

    A tremendous technological advance occurred in 1975 when a method was developed to fuse two cells producing a "hybridoma" which secretes a single clone of antibody, having one immunoglobulin (Ig) class, one structure, one affinity, and one specificity for an antigenic determinant. Because monoclonal antibodies are more precise reagents than conventional antisera they open new doors to diagnosis and therapy of disease, and they are useful tools in research. The pathologist uses monoclonals in immunocytochemistry to determine tumor type; the surgeon uses monoclonals for immunosuppression in renal transplantation; the immunologist uses monoclonals to decipher cellular and humoral interactions that could not be appreciated with polyclonal reagents. This review outlines the background of monoclonal antibodies and some of their clinically important uses, both in vitro and in vivo. We also project into the future and describe chimeric antibodies and their possible uses.

  18. Antibodies as Mediators of Brain Pathology

    PubMed Central

    Brimberg, Lior; Mader, Simone; Fujieda, Yuichiro; Arinuma, Yoshiyuki; Kowal, Czeslawa; Volpe, Bruce T.; Diamond, Betty

    2016-01-01

    The brain is normally sequestered from antibody exposure by the blood brain barrier. However, antibodies can access the brain during fetal development before the barrier achieves full integrity, and in disease states when barrier integrity is compromised. Recent studies suggest that antibodies contribute to brain pathology associated with autoimmune diseases such as systemic lupus erythematosus and neuromyelitis optica, and can lead to transient or permanent behavioral or cognitive abnormalities. We review these findings here and examine the circumstances associated with antibody entry into the brain, the routes of access and the mechanisms that then effect pathology. Understanding these processes and the nature and specificity of neuronal autoantibodies may reveal therapeutic strategies toward alleviating or preventing the neurological pathologies and behavioral abnormalities associated with autoimmune disease. PMID:26494046

  19. Autoimmune encephalitis: Clinical diagnosis versus antibody confirmation

    PubMed Central

    Cyril, Asha Caroline; Nair, Sruthi S.; Mathai, Annamma; Kannoth, Sudheeran; Thomas, Sanjeev V.

    2015-01-01

    Context: Autoimmune encephalitis is a heterogeneous disorder which is being diagnosed with increasing frequency. The diagnosis of these disorders is based on the detection of autoantibodies and characteristic clinical profiles. Aims: We aimed to study the antibody profile in encephalitis patients with suspected autoimmune etiology presenting to a tertiary care center. Settings and Design: The subjects were selected by screening all patients with clinical profile suggesting autoimmune encephalitis admitted in the neuromedical intensive care unit (ICU) of a tertiary care center in South India. Materials and Methods: Patients who fulfilled modified Zuliani et al.'s, criteria for autoimmune encephalitis were identified during the period December 2009–June 2013. Blood samples from these subjects were screened for six neuronal antibodies. Statistical analysis used: Chi-square test was applied to compare the antibody positive and negative patients. Results: Out of 1,227 patients screened, 39 subjects (14 males: 25 females) were identified with a mean age of 15.95 years and 19 cases were assessed in the acute and 20 in the convalescent phase of the illness. Seizure (87.8 %) was the most common presenting symptom; status epilepticus occurred in 23 (60.5%) patients during the course of the illness. Fourteen (35.9%) patients were N-methyl-D-aspartate receptor (NMDAR) antibody-positive and all were negative for the other antibodies tested. Conclusions: One-third of patients presenting with acute noninfective encephalitis would be positive for NMDAR antibodies with the remaining two-thirds with clinically suspected autoimmune encephalitis being antibody-negative. There are few markers in the clinical and investigative profiles to distinguish antibody-positive and -negative patients. PMID:26713011

  20. Indirect hemagglutination test for chlamydial antibodies.

    PubMed

    Lewis, V J; Thacker, W L; Engelman, H M

    1972-07-01

    An indirect hemagglutination (IHA) test is described for chlamydial antibodies in psittacosis diagnostic sera; for this test tanned sheep erythrocytes sensitized with a deoxycholate extract of Chlamydia psittaci grown in Vero cell monolayers were used. Adaptation of the IHA test to the Microtiter system decreased sensitivity; nevertheless, the Microtiter-IHA test was more sensitive than the complement fixation test. Lymphogranuloma venereum antibodies also were detected by using antigen extracted from C. psittaci. PMID:4626906

  1. Broadening Horizons: New Antibodies Against Influenza.

    PubMed

    Jackson, Katherine J L; Boyd, Scott D

    2016-07-28

    Seasonal influenza vaccine formulation efforts struggle to keep up with viral antigenic variation. Two studies now report engineered or naturally occurring human antibodies targeting the influenza hemagglutinin (HA) stem, with exceptional neutralizing breadth (Joyce et al., 2016; Kallewaard et al., 2016). Antibodies with similar structural features are elicited in multiple subjects, suggesting that modified vaccine regimens could provide broad protection. PMID:27471961

  2. History and Practice: Antibodies in Infectious Diseases.

    PubMed

    Hey, Adam

    2015-04-01

    Antibodies and passive antibody therapy in the treatment of infectious diseases is the story of a treatment concept which dates back more than 120 years, to the 1890s, when the use of serum from immunized animals provided the first effective treatment options against infections with Clostridium tetani and Corynebacterium diphtheriae. However, after the discovery of penicillin by Fleming in 1928, and the subsequent introduction of the much cheaper and safer antibiotics in the 1930s, serum therapy was largely abandoned. However, the broad and general use of antibiotics in human and veterinary medicine has resulted in the development of multi-resistant strains of bacteria with limited to no response to existing treatments and the need for alternative treatment options. The combined specificity and flexibility of antibody-based treatments makes them very valuable tools for designing specific antibody treatments to infectious agents. These attributes have already caused a revolution in new antibody-based treatments in oncology and inflammatory diseases, with many approved products. However, only one monoclonal antibody, palivizumab, for the prevention and treatment of respiratory syncytial virus, is approved for infectious diseases. The high cost of monoclonal antibody therapies, the need for parallel development of diagnostics, and the relatively small markets are major barriers for their development in the presence of cheap antibiotics. It is time to take a new and revised look into the future to find appropriate niches in infectious diseases where new antibody-based treatments or combinations with existing antibiotics, could prove their value and serve as stepping stones for broader acceptance of the potential for and value of these treatments.

  3. Immunology. RNA editing AIDs antibody diversification?

    PubMed

    Neuberger, M S; Scott, J

    2000-09-01

    How do B cells generate the enormous diversity of antibodies that are able to recognize and bind to whichever antigen a B cell might happen to encounter in the body? Several genetic mechanisms that manipulate different combinations of immunoglobulin genes are known. In their Perspective, Neuberger and Scott, highlight another genetic mechanism called RNA editing now shown to be involved in the production of antibody diversity.

  4. Cold denaturation of monoclonal antibodies

    PubMed Central

    Lazar, Kristi L; Patapoff, Thomas W

    2010-01-01

    The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔGu, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔGu versus temperature data from fluorescence gave a ΔCp of 8.0 kcal mol−1 K−1, a maximum apparent stability of 23.7 kcal mol−1 at 18°C, and an apparent cold denaturation temperature (TCD) of −23°C. ΔGu values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative TCD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs. PMID:20093856

  5. Therapeutic monoclonal antibody for sporotrichosis

    PubMed Central

    Almeida, Sandro R.

    2012-01-01

    Sporotrichosis is a chronic subcutaneous mycosis that affects both humans and animals worldwide. This subcutaneous mycosis had been attributed to a single etiological agent, Sporothrix schenckii. S. schenckii exhibits considerable genetic variability, and recently, it was suggested that this taxon consists of a complex of species. Sporotrichosis is caused by traumatic inoculation of the fungus, which is a ubiquitous environmental saprophyte that can be isolated from soil and plant debris. The infection is limited to cutaneous forms, but recently, more severe clinical forms of this mycosis have been described, especially among immunocompromised individuals. The immunological mechanisms involved in the prevention and control of sporotrichosis are not well understood. Some studies suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. In contrast, the role of the humoral immune response in protection against this fungus has not been studied in detail. In a previous study, we showed that antigens secreted by S. schenckii induced a specific humoral response in infected animals, primarily against a 70-kDa molecule, indicating a possible role of specific antibodies against this molecule in infection control. In another study by our group, we produced a mAb against a 70-kDa glycoprotein of S. schenckii to better understand the effect of the passive immunization of mice infected with S. schenckii. The results showed a significant reduction in the number of CFUs in various mice organs when the mAb was injected before or during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. The drugs of choice in the treatment of sporotrichosis require long periods, and relapses are frequently observed, primarily in immunocompromised patients. The strong protection induced by the mAb against a 70-kDa glycoprotein makes it a strong candidate as a therapeutic vaccine against sporotrichosis. PMID

  6. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

    SciTech Connect

    Luo, Shishi; Perelson, Alan S.

    2015-08-31

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinct strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. As a result, if less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.

  7. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

    DOE PAGESBeta

    Luo, Shishi; Perelson, Alan S.

    2015-08-31

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinctmore » strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. As a result, if less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains.« less

  8. Competitive exclusion by autologous antibodies can prevent broad HIV-1 antibodies from arising

    PubMed Central

    Luo, Shishi; Perelson, Alan S.

    2015-01-01

    The past decade has seen the discovery of numerous broad and potent monoclonal antibodies against HIV type 1 (HIV-1). Eliciting these antibodies via vaccination appears to be remarkably difficult, not least because they arise late in infection and are highly mutated relative to germline antibody sequences. Here, using a computational model, we show that broad antibodies could in fact emerge earlier and be less mutated, but that they may be prevented from doing so as a result of competitive exclusion by the autologous antibody response. We further find that this competitive exclusion is weaker in infections founded by multiple distinct strains, with broadly neutralizing antibodies emerging earlier than in infections founded by a single strain. Our computational model simulates coevolving multitype virus and antibody populations. Broadly neutralizing antibodies may therefore be easier for the adaptive immune system to generate than previously thought. If less mutated broad antibodies exist, it may be possible to elicit them with a vaccine containing a mixture of diverse virus strains. PMID:26324897

  9. A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities.

    PubMed Central

    Babcook, J S; Leslie, K B; Olsen, O A; Salmon, R A; Schrader, J W

    1996-01-01

    We report a novel approach to the generation of monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from single rabbit or murine lymphocytes that were selected for the production of specific antibodies. Single cells secreting antibodies for a specific peptide either from gp116 of the human cytomegalovirus or from gp120 of HIV-1 or for sheep red blood cells were selected using antigen-specific hemolytic plaque assays. Sheep red blood cells were coated with specific peptides in a procedure applicable to any antigen that can be biotinylated. Heavy- and light-chain variable region cDNAs were rescued from single cells by reverse transcription-PCR and expressed in the context of human immunoglobulin constant regions. These chimeric murine and rabbit monoclonal antibodies replicated the target specificities of the original antibody-forming cells. The selected lymphocyte antibody method exploits the in vivo mechanisms that generate high-affinity antibodies. This method can use lymphocytes from peripheral blood, can exploit a variety of procedures that identify individual lymphocytes producing a particular antibody, and is applicable to the generation of monoclonal antibodies from many species, including humans. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755564

  10. PASSIVE ANTIBODY AND THE IMMUNE RESPONSE

    PubMed Central

    McBride, Raymond A.; Schierman, Louis W.

    1971-01-01

    The isoimmune response of fowl inoculated with RBC coated with antibody was investigated. Anti-B antiserum from a single animal was used to coat different donor type RBC. With each donor type RBC the immune response to the coated determinants is suppressed. Enhancement of the immune response to noncoated determinants occurs when they are products of an allelic gene or belong to a different blood group system. Coating some B antigen determinants suppresses the response to noncoated determinants of the same antigen, i.e., determinants which are products of the same B gene. Varying the quantity of passive antibody revealed that the degree of suppression and the degree of enhancement are negatively correlated. These findings support the concept that antibody-coated determinants function as carrier for noncoated determinants, provided a certain physical association exists between them. A further interpretation of these studies is that in certain situations an antibody to one antigen may interfere with events which lead to an immune response to a different antigen. The possibility, that the protection afforded by ABO incompatibility against Rh isoimmunization is because of a similar phenomenon, is discussed. A hypothesis is presented which states that where the immune response to certain antigens behaves as a dominantly inherited trait, and is associated with histocompatibility type, the nonresponder animals possess an antibody (perhaps cell bound) which interferes with the response to determinants for which it does not have specificity. Responders are assumed to lack this antibody because it has specificity for their major histocompatibility antigens. PMID:4106486

  11. Production, characterization, and use of serpin antibodies.

    PubMed

    Kummer, J Alain; Strik, Merel C M; Bladergroen, Bellinda A; Hack, C Erik

    2004-02-01

    Serine protease inhibitors (serpins) constitute a still expanding superfamily of structural similar proteins, which are localized extracellularly and intracellularly. Serpins play a central role in the regulation of a wide variety of (patho) physiological processes including coagulation, fibrinolysis, inflammation, development, tumor invasion, and apoptosis. Serpins have a unique mechanism of inhibition that involves a profound change in conformational state upon interaction with their protease. This conformational change enables the production of monoclonal antibodies specific for native, complexed, and inactivated serpins. Antibodies, and assays based on these antibodies, have been helpful in elucidating the (patho) physiological function of serpins in the last decade. Serpin-specific antibodies can be used for: (1) structure-function studies such as detection of conformational changes; (2) identification of target-proteases; (3) the detection and quantification of serpin and serpin-protease complexes in bodily fluids by immunoassays such as ELISA, RIA or FACS; (4) detection of serpins in tissues by immunohistochemistry; and (5) possible therapeutical interventions. This review summarizes the techniques we have used to obtain and screen antibodies against extra- and intracellular serpins, as well as the use of these antibodies for some of the above-mentioned purposes.

  12. Boosting antibody developability through rational sequence optimization

    PubMed Central

    Seeliger, Daniel; Schulz, Patrick; Litzenburger, Tobias; Spitz, Julia; Hoerer, Stefan; Blech, Michaela; Enenkel, Barbara; Studts, Joey M; Garidel, Patrick; Karow, Anne R

    2015-01-01

    The application of monoclonal antibodies as commercial therapeutics poses substantial demands on stability and properties of an antibody. Therapeutic molecules that exhibit favorable properties increase the success rate in development. However, it is not yet fully understood how the protein sequences of an antibody translates into favorable in vitro molecule properties. In this work, computational design strategies based on heuristic sequence analysis were used to systematically modify an antibody that exhibited a tendency to precipitation in vitro. The resulting series of closely related antibodies showed improved stability as assessed by biophysical methods and long-term stability experiments. As a notable observation, expression levels also improved in comparison with the wild-type candidate. The methods employed to optimize the protein sequences, as well as the biophysical data used to determine the effect on stability under conditions commonly used in the formulation of therapeutic proteins, are described. Together, the experimental and computational data led to consistent conclusions regarding the effect of the introduced mutations. Our approach exemplifies how computational methods can be used to guide antibody optimization for increased stability. PMID:25759214

  13. Standardization of anti-DNA antibody assays.

    PubMed

    Pisetsky, David S

    2013-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and represent important biomarkers for clinical and research purposes. These antibodies are part of a family of antibodies to nucleosomes and bind to conserved sites widely present on DNA. While the value of anti-DNA as a biomarker is well established, the assay for these antibodies has involved a variety of DNA sources and systems to detect DNA-anti-DNA interactions. The influence of these variations on antibody detection has complicated assay standardization. As an antigen, DNA has unique features since it is a highly charged polymer that has structural heterogeneity. This heterogeneity can affect antigenicity which can vary on the basis of DNA origin, size, conformation and mobility. In addition, as a polymer, DNA can promote patterns of antibody binding based on monogamous or bivalent interaction which require an extended polynucleotide structure. Understanding the nature of DNA as an antigen can facilitate interpretation of serological tests and underpin efforts at better standardization.

  14. Anti Transglutaminase Antibodies Cause Ataxia in Mice

    PubMed Central

    Boscolo, Sabrina; Lorenzon, Andrea; Sblattero, Daniele; Florian, Fiorella; Stebel, Marco; Marzari, Roberto; Not, Tarcisio; Aeschlimann, Daniel; Ventura, Alessandro; Hadjivassiliou, Marios; Tongiorgi, Enrico

    2010-01-01

    Background Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. Methods We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. Conclusion The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia. PMID:20300628

  15. Discovery of functional antibodies targeting ion channels.

    PubMed

    Wilkinson, Trevor C I; Gardener, Matthew J; Williams, Wendy A

    2015-04-01

    Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

  16. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  17. Antinuclear antibodies in patients on anticonvulsant therapy

    PubMed Central

    Alarcón-Segovia, D.; Fishbein, Eugenia; Reyes, P. A.; Díes, H.; Shwadsky, S.

    1972-01-01

    Antinuclear antibodies to calf thymus nuclei, NP, DNA, sDNA, sNP and Sm antigen were investigated in sera from 170 patients on various programmes of prolonged anticonvulsant treatment. Findings were compared to those on 214 tuberculous patients on isoniazid, 109 SLE patients and 66 healthy subjects. Patients on anticonvulsants had a significantly higher incidence of ANA to DNA, sDNA, sNP and Sm antigen than the controls but had a lower incidence of ANA to all antigens, except sNP, than the SLE patients. Patients on isoniazid did not have DNA antibodies, but had antibodies to whole nuclei and to NP which were practically absent in the anticonvulsant group. Of all patients on anticonvulsants only those receiving hydantoins had ANA to Sm antigen, while those receiving only primidone had antibodies to sNP but no antibodies to DNA. Alteration of sNP with isoniazid did not result in an increased incidence of ANA in the anticonvulsant group as it does in isoniazid treated subjects. It is concluded that the SLE-activating properties of diverse anticonvulsants probably resides in their potential to induce ANA. Although all anticonvulsants elicit ANA directed primarily to sNP, each may do so by different mechanisms or by altering different sites in the sNP molecule. The mechanisms by which anticonvulsant and isoniazid intake results in ANA probably differ. Presence of DNA antibodies in some patients on anticonvulsants may indicate that their convulsions were due to SLE. PMID:4117275

  18. Fetal arthrogryposis and maternal serum antibodies.

    PubMed

    Dalton, Paola; Clover, Linda; Wallerstein, Robert; Stewart, Helen; Genzel-Boroviczeny, Orsolya; Dean, Andrew; Vincent, Angela

    2006-08-01

    Arthrogryposis multiplex congenital (AMC) describes multiple joint contractures resulting from lack of movement in utero. Antibodies directed at the fetal isoform of the muscle acetylcholine receptor (AChR) have been reported in a small number of asymptomatic mothers of AMC babies. We examined sera from 179 mothers of AMC babies and 20 parous and non-parous controls to look for antibodies to AChR or undefined muscle or neuronal proteins. We found positive AChR antibodies in only three sera (1.5%) from asymptomatic AMC mothers. However, there was reactivity with muscle or with neuronal antigens in 33% of the sera, and reactivity to undefined neuronal antigens was more common in sera from mothers of AMC babies with CNS involvement (p=0.001) than those without. The offspring of mothers with AChR antibodies may benefit from treatment during pregnancy. Other maternal antibodies require further study, but these observations add to the emerging literature on maternal antibodies associated with developmental intrauterine disorders.

  19. Introduction to Antigen and Antibody Assays.

    PubMed

    Day, Michael J

    2015-12-01

    Serological tests are used widely in veterinary practice; most often in the diagnosis of infectious disease. Such tests may be used to detect antigen from an infectious agent within a biological sample or to detect the presence of serum antibody specific for the pathogen as evidence of immunological exposure. These tests are all based on the fundamental principles of interaction between antigenic epitopes and antibodies of either the immunoglobulin (Ig) G, IgM, IgA, or IgE classes. The relative concentration of specific antibody within a sample is traditionally determined by calculation of the titer of antibody. With few exceptions, the primary interaction between an antigen and antibody in vitro cannot be visualized and so serological tests generally employ a secondary indicator system based on the use of a polyclonal antiserum or monoclonal antibody. A range of such tests has been developed, but many in veterinary medicine are based on the principle of the enzyme-linked immunosorbent assay, which is described in detail in this article. The interpretation of serological tests must be made carefully, taking into consideration the sensitivity and specificity of the test and the possible reasons for false-positive and false-negative outcomes. PMID:27154595

  20. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular...

  1. Avian Diagnostic and Therapeutic Antibodies to Viral Emerging Pathogens

    SciTech Connect

    David Bradley

    2011-03-31

    During the current period the following key objectives were achieved: demonstration of high titer antibody production by geese following immunization with inactived H1N1 virus; completion of the epitope mapping of West Nile Virus-specific goose antibodies and initiation of epitope mapping of H1N1 flu-specific goose antibodies; advancement in scalable purification of goose antibodies.

  2. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular...

  3. 21 CFR 866.5100 - Antinuclear antibody immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Antinuclear antibody immunological test system....5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular...

  4. Persistence of Antibodies against Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Iblan, Ibrahim; Rha, Brian; Alqasrawi, Sultan; Haddadin, Aktham; Al Nsour, Mohannad; Alsanouri, Tarek; Ali, Sami Sheikh; Harcourt, Jennifer; Miao, Congrong; Tamin, Azaibi; Gerber, Susan I.; Haynes, Lia M.; Al Abdallat, Mohammad Mousa

    2016-01-01

    To determine how long antibodies against Middle East respiratory syndrome coronavirus persist, we measured long-term antibody responses among persons serologically positive or indeterminate after a 2012 outbreak in Jordan. Antibodies, including neutralizing antibodies, were detectable in 6 (86%) of 7 persons for at least 34 months after the outbreak. PMID:27332149

  5. Engineering the variable region of therapeutic IgG antibodies

    PubMed Central

    Tsunoda, Hiroyuki; Kuramochi, Taichi; Sampei, Zenjiro; Ishii, Shinya; Hattori, Kunihiro

    2011-01-01

    Since the first generation of humanized IgG1 antibodies reached the market in the late 1990s, IgG antibody molecules have been extensively engineered. The success of antibody therapeutics has introduced severe competition in developing novel therapeutic monoclonal antibodies, especially for promising or clinically validated targets. Such competition has led researchers to generate so-called second or third generation antibodies with clinical differentiation utilizing various engineering and optimization technologies. Parent IgG antibodies can be engineered to have improved antigen binding properties, effector functions, pharmacokinetics, pharmaceutical properties and safety issues. Although the primary role of the antibody variable region is to bind to the antigen, it is also the main source of antibody diversity and its sequence affects various properties important for developing antibody therapeutics. Here we review recent research activity in variable region engineering to generate superior antibody therapeutics. PMID:21406966

  6. Heterologous flavivirus infection-enhancing antibodies in sera of Nigerians.

    PubMed

    Fagbami, A; Halstead, S B; Marchette, N; Larsen, K

    1988-01-01

    Human sera collected from Nigerians were examined for plaque reduction neutralizing and infection-enhancing antibodies against dengue 2, yellow fever, and West Nile viruses. Neutralization tests showed that 17 of 19 sera contained flavivirus neutralizing antibody; 11 were positive to all 3 viruses, 5 to dengue and yellow fever, and 1 to dengue virus only. Two sera had no detectable neutralizing antibody to any of the flaviviruses. Enhancement assays showed that 17 flavivirus neutralizing antibody-positive sera contained infection-enhancing antibodies to dengue 2, and 16 had antibody to yellow fever. Although 11 sera were positive for West Nile neutralizing antibody, 17 enhanced this virus. Heterologous infection-enhancing antibody titers were lower than the homologous ones. Broadly reacting sera and those with high neutralizing antibody titers produced the highest infection-enhancing antibody titers.

  7. [Current situations and the future prospect of monoclonal antibody products].

    PubMed

    Yamaguchi, Teruhide

    2014-01-01

    Monoclonal antibody products and monoclonal antibody-based biopharmaceuticals have shown considerable effectiveness in the treatment for variety of diseases; cancer, auto-immune/auto-inflammation diseases and so on. Significant advance in monoclonal antibody products for cancer treatments was made with antibody-drug conjugates (ADC), and antibodies for blockade of immune checkpoints. Already 3 ADCs and 2 anti-immune-checkpoint antibodies products have been approved, and these monoclonal antibody-related product pipelines reach over 30. On the other hand, EU approved first monoclonal-antibody biosimilar, RemsimaTM (infliximab), suggesting that other monoclonal-antibody biosmilars will follow to the market. In this paper, several new issues about monoclonal antibody products will be discussed. PMID:25707201

  8. Antibodies to Trichomonas vaginalis surface glycolipid

    PubMed Central

    Bastida-Corcuera, F D; Singh, B N; Gray, G C; Stamper, P D; Davuluri, M; Schlangen, K; Corbeil, R R; Corbeil, L B

    2015-01-01

    Background Human trichomoniasis is the most common non-viral sexually transmitted disease, yet immune responses are not well studied. Methods Since the Trichomonas vaginalis lipophosphoglycan (TvLPG) is an important virulence factor, a bank of eight monoclonal antibodies was generated to define the antigen in clinical isolates. The TvLPG-specific antibody response of women who were culture positive (n=33) or negative (n=33) for T vaginalis infection was determined by isotype-specific ELISA. Results The bank of monoclonal antibodies reacted with conserved surface TvLPG epitopes in 27 isolates from pregnant women at their first prenatal visit. Conserved TvLPG epitopes were shown to be surface exposed by immunofluorescence. Sera collected from the same patients at the same time were assayed for specific antibodies. Serum and vaginal secretions from 33 T vaginalis-positive women had statistically higher IgG anti-TvLPG levels than age-matched and race-matched negative controls in the same clinical study (p<0.01). Vaginal IgA anti-TvLPG levels of the women with trichomoniasis were almost significantly higher than controls (p=0.055). Infected women with normal pregnancies had significantly higher vaginal IgG anti-TvLPG values than infected women with adverse outcomes of pregnancy. Conclusions These antibody responses show that infected women can respond to the conserved TvLPG antigen. Since antibodies to trichomonad surface LPG protect in a bovine model of trichomoniasis, the role of these antibodies in the human disease should be investigated. PMID:23785040

  9. Neutralization of HIV by Milk Expressed Antibody

    PubMed Central

    Yu, Xiaocong; Pollock, Daniel; Duval, Mark; Lewis, Christopher; Joseph, Kristin; Meade, Harry; Cavacini, Lisa

    2012-01-01

    Background In some areas of the world mother-to-child transmission of HIV remains a significant problem in part due to widespread breastfeeding which is essential due to scarce supply of a safe replacement, protection conferred by breast milk against many enteric illnesses, and cultural norms. We propose that sustained, adequate levels of protective antibodies in breast milk will prevent transmission of HIV. Methods The HIV neutralizing human monoclonal antibody b12 (IgG1) has been expressed as an IgA2 in CHO cells and shown to retain full immunoreactivity and neutralizing activity as the parental IgG1. The expression plasmids containing the b12 heavy and light chains were also used to construct milk specific expression vectors using the GTC goat β-casein expression vector to direct expression of linked genes to the mammary gland with subsequent secretion into the milk. Female transgenic mice were generated and following parturition, their milk was tested for antibody immunoreactivity with gp120 and neutralization of HIV. Results When compared to CHO derived b12 IgA2 (or IgG1), immunoreactivity was retained. When tested for neutralization, milk derived b12 IgA2 was at least comparable to CHO derived antibody and in some cases superior to CHO derived antibody. Furthermore, milk that expressed b12 IgA2 was significantly more effective at mediating antibody dependent cell killing. Conclusions These results suggest it is possible to achieve functional HIV-specific mAb in the milk of transgenic mice and further investigations are warranted to explore ways for inducing this type of antibody response in the breast milk of HIV infected women. PMID:23269241

  10. Patient With Homer-3 Antibodies and Cerebellitis

    PubMed Central

    Höftberger, Romana; Sabater, Lidia; Ortega, Angel; Dalmau, Josep; Graus, Francesc

    2013-01-01

    Importance Homer proteins are a family of scaffolding proteins of the postsynaptic density. Homer-3 colocalizes and modulates the activity of group I metabotropic glutamate receptors (mGluR1 and mGluR5). Cerebellitis has been reported in association with antibodies to mGluR1. We describe the second patient with cerebellitis and Homer-3 antibodies and report a novel, highly specific immunoblot assay. Observations A 38-year-old man had acute onset of headache, nausea, vomiting, and confusion. He developed a pancerebellar syndrome during the ensuing week. Extensive studies did not reveal any tumor. Cerebrospinal fluid analysis showed a white blood cell count of 60/µL (to convert to ×109 per liter, multiply by 0.001). Brain magnetic resonance imaging findings were normal. For 2 years, the patient was treated with intravenous immunoglobulins and steroids, with partial improvement of the cerebellar ataxia. The patient was negative for onconeural (Hu, Yo, Ri, CV2, Tr, amphiphysin, and Ma2), glutamic acid decarboxylase, and mGluR1 antibodies. Immunohistochemistry on rat brain revealed immunostaining of the cerebellar molecular layer. Homer-3 antibodies were demonstrated by immunoblot of recombinant Homer-3. The clinical features of this patient and a previously described patient with Homer-3 antibodies are similar to those of patients with mGluR1 antibodies. Conclusions and Relevance We report the second case of autoimmune cerebellar ataxia associated with Homer-3 antibodies. The presence of Homer-3 autoantibodies should be considered in the differential diagnosis of patients with subacute cerebellar ataxia of unknown cause. PMID:23400636

  11. A multi-Fc-species system for recombinant antibody production

    PubMed Central

    Moutel, Sandrine; El Marjou, Ahmed; Vielemeyer, Ole; Nizak, Clément; Benaroch, Philippe; Dübel, Stefan; Perez, Franck

    2009-01-01

    Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies. Results We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody. Conclusion Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use. PMID:19245715

  12. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    SciTech Connect

    Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

    2009-04-24

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  13. Thermodynamics of antibody-antigen interaction revealed by mutation analysis of antibody variable regions.

    PubMed

    Akiba, Hiroki; Tsumoto, Kouhei

    2015-07-01

    Antibodies (immunoglobulins) bind specific molecules (i.e. antigens) with high affinity and specificity. In order to understand their mechanisms of recognition, interaction analysis based on thermodynamic and kinetic parameters, as well as structure determination is crucial. In this review, we focus on mutational analysis which gives information about the role of each amino acid residue in antibody-antigen interaction. Taking anti-hen egg lysozyme antibodies and several anti-small molecule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed in terms of thermodynamics. Here, thermodynamics of the contribution from aromatic, charged and hydrogen bond-forming amino acids are discussed, and their different characteristics have been elucidated. The information gives fundamental understanding of the antibody-antigen interaction. Furthermore, the consequences of antibody engineering are analysed from thermodynamic viewpoints: humanization to reduce immunogenicity and rational design to improve affinity. Amino acid residues outside hot-spots in the interface play important roles in these cases, and thus thermodynamic and kinetic parameters give much information about the antigen recognition. Thermodynamic analysis of mutant antibodies thus should lead to advanced strategies to design and select antibodies with high affinity.

  14. The antibody mining toolbox: an open source tool for the rapid analysis of antibody repertoires.

    PubMed

    D'Angelo, Sara; Glanville, Jacob; Ferrara, Fortunato; Naranjo, Leslie; Gleasner, Cheryl D; Shen, Xiaohong; Bradbury, Andrew R M; Kiss, Csaba

    2014-01-01

    In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1-2 million reads can be accomplished in 10-15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries. PMID:24423623

  15. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

    PubMed Central

    McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.

    2016-01-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  16. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    PubMed

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  17. Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

    PubMed

    Batonick, Melissa; Holland, Erika G; Busygina, Valeria; Alderman, Dawn; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

    2016-09-25

    Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

  18. Metabolic engineering of monoclonal antibody carbohydrates for antibody-drug conjugation.

    PubMed

    Okeley, Nicole M; Toki, Brian E; Zhang, Xinqun; Jeffrey, Scott C; Burke, Patrick J; Alley, Stephen C; Senter, Peter D

    2013-10-16

    The role that carbohydrates play in antibody function and pharmacokinetics has made them important targets for modification. The terminal fucose of the N-linked glycan structure, which has been shown to be involved in modulation of antibody-directed cellular cytotoxicity, is a particularly interesting location for potential modification through incorporation of alternative sugar structures. A library of fucose analogues was evaluated for their ability to incorporate into antibody carbohydrates in place of the native fucose. A number of efficiently incorporated molecules were identified, demonstrating the ability of fucosyltransferase VIII to utilize a variety of non-natural sugars as substrates. Among these structures was a thiolated analogue, 6-thiofucose, which was incorporated into the antibody carbohydrate with good efficiency. This unnatural thio-sugar could then be used for conjugation using maleimide chemistry to produce antibody-drug conjugates with pronounced cytotoxic activities and improved homogeneity compared to drug attachment through hinge disulfides.

  19. Antibody mapping of functional domains in vinculin.

    PubMed Central

    Westmeyer, A; Ruhnau, K; Wegner, A; Jockusch, B M

    1990-01-01

    We have analyzed the functional domain structure of vinculin, a protein involved in linking microfilaments to the cytoplasmic face of cell membranes in animal cells. For this purpose, we used several monoclonal antibodies raised against chicken gizzard vinculin whose epitopes could be assigned to discrete regions in the vinculin sequence by immunoblotting of proteolytic fragments combined with N-terminal amino acid sequencing. Two of these antibodies induced the disruption of stress fibers and changed the number of morphology of focal contacts after microinjection in chicken embryo fibroblasts. Based on the location of its epitope in comparison with vinculin domains previously identified by other groups, we propose that one of these antibodies (15B7) interferes with the binding of vinculin to talin, the most peripheral of the microfilament proteins. The second antibody (14C10) binds within a region comprising three internal repeats and might therefore distort the inner architecture of vinculin. A third antibody (As3) inhibited the binding of F-actin to vinculin in an in vitro assay but had no effect on the microfilament system in cells. These data emphasize the role of vinculin as a key protein in microfilament-membrane linkage and support previous work on a direct interaction between vinculin and actin. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1694125

  20. Homogeneity of Antibody Responses in Tuberculosis Patients

    PubMed Central

    Samanich, K.; Belisle, J. T.; Laal, S.

    2001-01-01

    The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936–3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients. PMID:11402004

  1. Antibody Phage Display Libraries: Contributions to Oncology

    PubMed Central

    Dantas-Barbosa, Carmela; de Macedo Brigido, Marcelo; Maranhao, Andrea Queiroz

    2012-01-01

    Since the advent of phage display technology, dating back to 1985, antibody libraries displayed on filamentous phage surfaces have been used to identify specific binders for many different purposes, including the recognition of tumors. Phage display represents a high-throughput technique for screening billions of random fusion antibodies against virtually any target on the surface or inside cancer cells, or even soluble markers found in patient serum. Many phage display derived binders targeting important tumor markers have been identified. Selection directed to tumoral cells’ surfaces lead to the identification of unknown tumoral markers. Also the improvement of methods that require smaller amounts of cells has opened the possibility to use this approach on patient samples. Robust techniques combining an antibody library displayed on the phage surface and protein microarray allowed the identification of auto antibodies recognized by patient sera. Many Ab molecules directly or indirectly targeting angiogenesis have been identified, and one of them, ramucirumab, has been tested in 27 phase I–III clinical trials in a broad array of cancers. Examples of such antibodies will be discussed here with emphasis on those used as probes for molecular imaging and other clinical trials. PMID:22754305

  2. Modeling antibody hypervariable loops: a combined algorithm.

    PubMed Central

    Martin, A C; Cheetham, J C; Rees, A R

    1989-01-01

    To be of any value, a predicted model of an antibody combining site should have an accuracy approaching that of antibody structures determined by x-ray crystallography (1.6-2.7 A). A number of modeling protocols have been proposed, which fall into two main categories--those that adopt a knowledge-based approach and those that attempt to construct the hypervariable loop regions of the antibody ab initio. Here we present a combined algorithm requiring no arbitrary decisions on the part of the user, which has been successfully applied to the modeling of the individual loops in two systems: the anti-lysozyme antibody HyHel-5, the crystal structure of which is as a complex with lysozyme [Sheriff, S., Silverton, E. W., Padlan, E. A., Cohen, G. H., Smith-Gill, S. J., Finzel, B. C. & Davies, D. R. (1987) Proc. Natl. Acad. Sci. USA 84, 8075-8079], and the free antigen binding fragment (Fab) of the anti-lysozyme peptide antibody, Gloop2. This protocol may be used with a high degree of confidence to model single-loop replacements, insertions, deletions, and side-chain replacements. In addition, it may be used in conjunction with other modeling protocols as a method by which to model particular loops whose conformations are predicted poorly by these methods. PMID:2594766

  3. 4th European Antibody Congress 2008

    PubMed Central

    2009-01-01

    The Fourth European Antibody meeting, organized by Terrapin Ltd., was held in Geneva, a center of the European biopharmaceutical industry. Merck-Serono, NovImmune, Pierre Fabre and Therapeomic are located nearby, as are R&D centers of Boehringer-Ingelheim, Novartis, Roche and Sanofi-Aventis. Over 40 speakers and more than 200 delegates attended the event. Companies represented included Abbott, Ablynx, Adnexus/ BMS, Astra-Zeneca/ CAT/ Medimmune, BiogenIdec, BioRad, Centocor (Johnson & Johnson), Crucell/DSM, Domantis, Dyax, Genmab, Genzyme, Glycart/ Roche, Haptogen, Immunogen, Kyowa-Kirin, LFB, Medarex, Merck-Serono, Micromet, Novartis, Pierre Fabre Laboratories, Roche, Sanofi-Aventis, Seattle-Genetics, Transgene, UCB Celltech and Wyeth. Other attendees included those based in academe or government (University of Amsterdam, University of Zurich, Univeristy Hospital-Lyon, Ecole Polytechnique Federale de Lausanne, INSERM, Tufts University, US National Institutes of Health), consultants, and patent attorneys (Edwards, Angell, Palmer & Dodge). The meeting was very interactive and included exchanges during the many scheduled networking times (exhibitions, speed-networking, lunches and evening receptions). The first day of the three day conference was dedicated to advances in understanding antibody structure-function relationships. Challenges and opportunities in antibody development were the focus of the second day and the third day featured discussion of innovative antibodies and antibody alternatives. PMID:20061813

  4. Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function

    PubMed Central

    Chung, Amy W.; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K.; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E.; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

    2015-01-01

    Objective To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Design Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein–Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4+ binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Methods Each mAb was assayed for antibody-binding affinity to gp140SF162, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcgRIIa, FcgRIIb and FcgRIIIa receptors. Antibody glycan profiles were determined by HPLC. Results Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcgRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcgRIIIa and ADCC activity, independent of the specificity of the mAb. Conclusions Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection. PMID:25160934

  5. Antigen-Antibody Interaction Database (AgAbDb): a compendium of antigen-antibody interactions.

    PubMed

    Kulkarni-Kale, Urmila; Raskar-Renuse, Snehal; Natekar-Kalantre, Girija; Saxena, Smita A

    2014-01-01

    Antigen-Antibody Interaction Database (AgAbDb) is an immunoinformatics resource developed at the Bioinformatics Centre, University of Pune, and is available online at http://bioinfo.net.in/AgAbDb.htm. Antigen-antibody interactions are a special class of protein-protein interactions that are characterized by high affinity and strict specificity of antibodies towards their antigens. Several co-crystal structures of antigen-antibody complexes have been solved and are available in the Protein Data Bank (PDB). AgAbDb is a derived knowledgebase developed with an objective to compile, curate, and analyze determinants of interactions between the respective antigen-antibody molecules. AgAbDb lists not only the residues of binding sites of antigens and antibodies, but also interacting residue pairs. It also helps in the identification of interacting residues and buried residues that constitute antibody-binding sites of protein and peptide antigens. The Antigen-Antibody Interaction Finder (AAIF), a program developed in-house, is used to compile the molecular interactions, viz. van der Waals interactions, salt bridges, and hydrogen bonds. A module for curating water-mediated interactions has also been developed. In addition, various residue-level features, viz. accessible surface area, data on epitope segment, and secondary structural state of binding site residues, are also compiled. Apart from the PDB numbering, Wu-Kabat numbering and explicit definitions of complementarity-determining regions are provided for residues of antibodies. The molecular interactions can be visualized using the program Jmol. AgAbDb can be used as a benchmark dataset to validate algorithms for prediction of B-cell epitopes. It can as well be used to improve accuracy of existing algorithms and to design new algorithms. AgAbDb can also be used to design mimotopes representing antigens as well as aid in designing processes leading to humanization of antibodies. PMID:25048123

  6. Method for altering antibody light chain interactions

    DOEpatents

    Stevens, Fred J.; Stevens, Priscilla Wilkins; Raffen, Rosemarie; Schiffer, Marianne

    2002-01-01

    A method for recombinant antibody subunit dimerization including modifying at least one codon of a nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in the interface segment of the light polypeptide variable region, the charged amino acid having a first polarity; and modifying at least one codon of the nucleic acid sequence to replace an amino acid occurring naturally in the antibody with a charged amino acid at a position in an interface segment of the heavy polypeptide variable region corresponding to a position in the light polypeptide variable region, the charged amino acid having a second polarity opposite the first polarity. Nucleic acid sequences which code for novel light chain proteins, the latter of which are used in conjunction with the inventive method, are also provided.

  7. Antibody-based biological toxin detection

    SciTech Connect

    Menking, D.E.; Goode, M.T.

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  8. Methods for conjugating antibodies to nanocarriers.

    PubMed

    Wagh, Anil; Law, Benedict

    2013-01-01

    Antibodies are one of the most commonly used targeting ligands for nanocarriers, mainly because they are specific, have a strong binding affinity, and are available for a number of disease biomarkers. The bioconjugation chemistry can be a crucial factor in determining the targeting efficiency of drug delivery and should be chosen on a case-by-case basis. An antibody consists of a number of functional groups which offer many flexible options for bioconjugation. This chapter focuses on discussing some of the approaches including periodate oxidation, carbodiimide, maleimide, and heterofunctional linkers, for conjugating antibodies to different nanocarriers. The advantages and limitations are described herein. Specific examples are selected to demonstrate the experimental procedures and to illustrate the potential for applying to other nanocarrier system. PMID:23913152

  9. Methods for conjugating antibodies to nanocarriers.

    PubMed

    Wagh, Anil; Law, Benedict

    2013-01-01

    Antibodies are one of the most commonly used targeting ligands for nanocarriers, mainly because they are specific, have a strong binding affinity, and are available for a number of disease biomarkers. The bioconjugation chemistry can be a crucial factor in determining the targeting efficiency of drug delivery and should be chosen on a case-by-case basis. An antibody consists of a number of functional groups which offer many flexible options for bioconjugation. This chapter focuses on discussing some of the approaches including periodate oxidation, carbodiimide, maleimide, and heterofunctional linkers, for conjugating antibodies to different nanocarriers. The advantages and limitations are described herein. Specific examples are selected to demonstrate the experimental procedures and to illustrate the potential for applying to other nanocarrier system.

  10. Antibody-Mediated Pathogen Resistance in Plants.

    PubMed

    Peschen, Dieter; Schillberg, Stefan; Fischer, Rainer

    2016-01-01

    The methods described in this chapter were developed in order to produce transgenic plants expressing pathogen-specific single-chain variable fragment (scFv) antibodies fused to antifungal peptides (AFPs), conferring resistance against fungal pathogens. We describe the selection from a phage display library of avian scFv antibodies that recognize cell surface proteins on fungi from the genus Fusarium, and the construction of scFv-AFP fusion protein constructs followed by their transient expression in tobacco (Nicotiana spp.) plants and stable expression in Arabidopsis thaliana plants. Using these techniques, the antibody fusion with the most promising in vitro activity can be used to generate transgenic plants that are resistant to pathogens such as Fusarium oxysporum f. sp. matthiolae.

  11. An immunosuppressive antibody-drug conjugate.

    PubMed

    Wang, Rongsheng E; Liu, Tao; Wang, Ying; Cao, Yu; Du, Jintang; Luo, Xiaozhou; Deshmukh, Vishal; Kim, Chan Hyuk; Lawson, Brian R; Tremblay, Matthew S; Young, Travis S; Kazane, Stephanie A; Wang, Feng; Schultz, Peter G

    2015-03-11

    We have developed a novel antibody-drug conjugate (ADC) that can selectively deliver the Lck inhibitor dasatinib to human T lymphocytes. This ADC is based on a humanized antibody that selectively binds with high affinity to CXCR4, an antigen that is selectively expressed on hematopoietic cells. The resulting dasatinib-antibody conjugate suppresses T-cell-receptor (TCR)-mediated T-cell activation and cytokine expression with low nM EC50 and has minimal effects on cell viability. This ADC may lead to a new class of selective immunosuppressive drugs with improved safety and extend the ADC strategy to the targeted delivery of kinase inhibitors for indications beyond oncology.

  12. Antidrug Antibodies: B Cell Immunity Against Therapy.

    PubMed

    Fogdell-Hahn, A

    2015-09-01

    Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review.

  13. The expanding role of therapeutic antibodies.

    PubMed

    Salemi, Simonetta; Markovic, Milica; Martini, Gabriella; D'Amelio, Raffaele

    2015-05-01

    Therapeutic antibodies have been used since the end of nineteenth century, but their use is progressively increased and recently, with the availability of monoclonal antibodies, they are successfully employed in a large disease spectrum, which transversally covers different fields of medicine. Hyperimmune polyclonal immune globulin has been used against infectious diseases, in a period in which anti-microbial drugs were not yet available, and it still maintains a relevant place in prophylaxis/therapy. Although immune globulin should be considered life-saving as replacement therapy in humoral immunodeficiencies, its place in the immune-modulating treatment is not usually first-choice, but it should be considered as support to standard approved treatments. Despite therapeutic monoclonal antibodies have been lastly introduced in therapy, their extreme potentiality is reflected by the large number of approved molecules, addressed toward different immunological targets and able to heavily influence the prognosis and quality of life of a wide range of different diseases.

  14. Method for preparation of single chain antibodies

    DOEpatents

    Cheung, Nai-Kong V.; Guo, Hong-fen

    2012-04-03

    This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

  15. Monoclonal antibodies specific for sickle cell hemoglobin

    SciTech Connect

    Jensen, R.H.; Vanderlaan, M.; Grabske, R.J.; Branscomb, E.W.; Bigbee, W.L.; Stanker, L.H.

    1985-01-01

    Two mouse hybridoma cell lines were isolated which produce monoclonal antibodies that bind hemoglobin S. The mice were immunized with peptide-protein conjugates to stimulate a response to the amino terminal peptide of the beta chain of hemoglobin S, where the single amino acid difference between A and S occurs. Immunocharacterization of the antibodies shows that they bind specifically to the immunogen peptide and to hemoglobin S. The specificity for S is high enough that one AS cell in a mixture with a million AA cells is labeled by antibody, and such cells can be analyzed by flow cytometry. Immunoblotting of electrophoretic gels allows definitive identification of hemoglobin S as compared with other hemoglobins with similar electrophoretic mobility. 12 references, 4 figures.

  16. Antibody microarrays as tools for biomarker discovery.

    PubMed

    Sanchez-Carbayo, Marta

    2011-01-01

    The cancer biomarkers field is being enriched by molecular profiling obtained by high-throughput approaches. Targeted antibody arrays are strongly contributing to the identification of protein cancer -biomarker candidates and functional proteomic analyses. Due to their versatility, novel technological and experimental design implementations are broadening the applications of antibody arrays. However, the cancer biomarker candidates delivered to date using this technology are still in their early developmental phase, requiring validation with high number of specimens focusing on specific clinical endpoints. Innovative strategies multiplexing protein measurements of protein extracts of cultured cells, tissue and body fluids using antibody arrays combined with appropriate validation approaches are enabling the -discovery of cancer-associated biomarkers. This review describes these strategies and cancer biomarker candidates reported to date that may assist in the diagnosis, surveillance, prognosis, and potentially for predictive and therapeutic purposes for patients affected with solid and hematological neoplasias.

  17. Non-antibody protein-based biosensors

    PubMed Central

    2016-01-01

    Biosensors that depend on a physical or chemical measurement can be adversely affected by non-specific interactions. For example, a biosensor designed to measure specifically the levels of a rare analyte can give false positive results if there is even a small amount of interaction with a highly abundant but irrelevant molecule. To overcome this limitation, the biosensor community has frequently turned to antibody molecules as recognition elements because they are renowned for their exquisite specificity. Unfortunately antibodies can often fail when immobilised on inorganic surfaces, and alternative biological recognition elements are needed. This article reviews the available non-antibody-binding proteins that have been successfully used in electrical and micro-mechanical biosensor platforms. PMID:27365032

  18. Back to the future: recombinant polyclonal antibody therapeutics

    PubMed Central

    Wang, Xian-zhe; Coljee, Vincent W.; Maynard, Jennifer A.

    2013-01-01

    Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed. PMID:24443710

  19. Back to the future: recombinant polyclonal antibody therapeutics.

    PubMed

    Wang, Xian-Zhe; Coljee, Vincent W; Maynard, Jennifer A

    2013-11-01

    Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed. PMID:24443710

  20. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  1. The maturation of antibody technology for the HIV epidemic.

    PubMed

    Winnall, Wendy R; Beasley, Matthew D; Center, Rob J; Parsons, Matthew S; Kiefel, Ben R; Kent, Stephen J

    2014-08-01

    Antibodies are one of our most useful biological tools. Indeed, improvements in antibody-based technologies have ushered in a new era of antibody-based therapeutics, research and diagnostic tools. Although improved technologies have led to the development of therapeutic antibodies for treatment of malignancies and inflammatory conditions, the use of advanced antibody technology in the therapy of viral infections is in its infancy. Non-human primate studies have demonstrated that antibodies against the HIV envelope can both prevent viral infection and control viremia. Despite the obvious potential of antibody therapies against HIV, there remain limitations in production and purification capacity that require further research. Recent advances in recombinant antibody technology have led to the development of a range of novel antibody fragments, such as single-domain nanobodies and bispecific antibodies, that are capable of targeting cancer cells to cytotoxic T cells. Novel antibody production techniques have also been designed, allowing antibodies to be obtained from non-mammalian cells, bovine colostrum and the periplasm and cytoplasm of bacteria. These advances may allow large-scale production of HIV antibodies that are capable of protecting against HIV infection or serving as therapeutics that reduce the need for life-long antiretroviral treatment. This review summarises recent advances in antibody-based technologies and discusses the possibilities and challenges of using these advances to design prophylactics and therapeutics against HIV. PMID:24797582

  2. Anti-DNA antibodies--quintessential biomarkers of SLE.

    PubMed

    Pisetsky, David S

    2016-02-01

    Antibodies that recognize and bind to DNA (anti-DNA antibodies) are serological hallmarks of systemic lupus erythematosus (SLE) and key markers for diagnosis and disease activity. In addition to common use in the clinic, anti-DNA antibody testing now also determines eligibility for clinical trials, raising important questions about the nature of the antibody-antigen interaction. At present, no 'gold standard' for serological assessment exists, and anti-DNA antibody binding can be measured with a variety of assay formats, which differ in the nature of the DNA substrates and in the conditions for binding and detection of antibodies. A mechanism called monogamous bivalency--in which high avidity results from simultaneous interaction of IgG Fab sites with a single polynucleotide chain--determines anti-DNA antibody binding; this mechanism might affect antibody detection in different assay formats. Although anti-DNA antibodies can promote pathogenesis by depositing in the kidney or driving cytokine production, they are not all alike, pathologically, and anti-DNA antibody expression does not necessarily correlate with active disease. Levels of anti-DNA antibodies in patients with SLE can vary over time, distinguishing anti-DNA antibodies from other pathogenic antinuclear antibodies. Elucidation of the binding specificities and the pathogenic roles of anti-DNA antibodies in SLE should enable improvements in the design of informative assays for both clinical and research purposes.

  3. Antibody engineering & therapeutics, the annual meeting of the antibody society December 7–10, 2015, San Diego, CA, USA

    PubMed Central

    Pauthner, Matthias; Yeung, Jenny; Ullman, Chris; Bakker, Joost; Wurch, Thierry; Reichert, Janice M.; Lund-Johansen, Fridtjof; Bradbury, Andrew R.M.; Carter, Paul J.; Melis, Joost P.M.

    2016-01-01

    ABSTRACT The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries. PMID:26909869

  4. Human antibody production in transgenic animals.

    PubMed

    Brüggemann, Marianne; Osborn, Michael J; Ma, Biao; Hayre, Jasvinder; Avis, Suzanne; Lundstrom, Brian; Buelow, Roland

    2015-04-01

    Fully human antibodies from transgenic animals account for an increasing number of new therapeutics. After immunization, diverse human monoclonal antibodies of high affinity can be obtained from transgenic rodents, while large animals, such as transchromosomic cattle, have produced respectable amounts of specific human immunoglobulin (Ig) in serum. Several strategies to derive animals expressing human antibody repertoires have been successful. In rodents, gene loci on bacterial artificial chromosomes or yeast artificial chromosomes were integrated by oocyte microinjection or transfection of embryonic stem (ES) cells, while ruminants were derived from manipulated fibroblasts with integrated human chromosome fragments or human artificial chromosomes. In all strains, the endogenous Ig loci have been silenced by gene targeting, either in ES or fibroblast cells, or by zinc finger technology via DNA microinjection; this was essential for optimal production. However, comparisons showed that fully human antibodies were not as efficiently produced as wild-type Ig. This suboptimal performance, with respect to immune response and antibody yield, was attributed to imperfect interaction of the human constant region with endogenous signaling components such as the Igα/β in mouse, rat or cattle. Significant improvements were obtained when the human V-region genes were linked to the endogenous CH-region, either on large constructs or, separately, by site-specific integration, which could also silence the endogenous Ig locus by gene replacement or inversion. In animals with knocked-out endogenous Ig loci and integrated large IgH loci, containing many human Vs, all D and all J segments linked to endogenous C genes, highly diverse human antibody production similar to normal animals was obtained.

  5. Detection of antibodies against botulinum toxins.

    PubMed

    Sesardic, Dorothea; Jones, Russell G A; Leung, Tong; Alsop, Toni; Tierney, Robert

    2004-03-01

    After immunisation with botulinum vaccine, antibodies to multiple epitopes are produced. Only some of these will have the capacity to neutralise the toxin activity. In fact, the ability of toxoid vaccine to induce toxin neutralising antibodies has provided the basis for the use of therapeutic antitoxins and immunoglobulins for the prophylaxis and treatment of diseases caused by bacterial toxins. Increasing indications for the chronic use of botulinum toxin for therapy have inevitably resulted in concern for patients becoming unresponsive because of the presence of circulating toxin-specific antibodies. Highly sensitive and relevant assays to detect only clinically relevant toxin neutralising antibodies are essential. Although immunoassays often provide the sensitivity, their relevance and specificity is often questioned. The mouse protection LD(50) bioassay is considered most relevant but can often only detect 10 mIU/ml of antitoxin. This sensitivity, although sufficient for confirming protective immunity, is inadequate for patients undergoing toxin therapy. An intramuscular paralysis assay improves the sensitivity to ca. 1 mIU/ml, and a mouse ex vivo diaphragm assay, with sensitivity of < 0.5 mIU/ml, is the most sensitive functional assay to date for this purpose. Alternative approaches for the detection of antibodies to botulinum toxin have included in vitro endopeptidase activity neutralisation. Unlike any other functional assay, this approach is not reliant on serotype-specific antibodies for specificity. Most recent promising developments are focused on cellular assays utilising primary rat embryonic cord cells or more conveniently in vitro differentiated established cell lines such as human neuroblastoma cells.

  6. Molecular Imaging of Pancreatic Cancer with Antibodies

    PubMed Central

    2015-01-01

    Development of novel imaging probes for cancer diagnostics remains critical for early detection of disease, yet most imaging agents are hindered by suboptimal tumor accumulation. To overcome these limitations, researchers have adapted antibodies for imaging purposes. As cancerous malignancies express atypical patterns of cell surface proteins in comparison to noncancerous tissues, novel antibody-based imaging agents can be constructed to target individual cancer cells or surrounding vasculature. Using molecular imaging techniques, these agents may be utilized for detection of malignancies and monitoring of therapeutic response. Currently, there are several imaging modalities commonly employed for molecular imaging. These imaging modalities include positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance (MR) imaging, optical imaging (fluorescence and bioluminescence), and photoacoustic (PA) imaging. While antibody-based imaging agents may be employed for a broad range of diseases, this review focuses on the molecular imaging of pancreatic cancer, as there are limited resources for imaging and treatment of pancreatic malignancies. Additionally, pancreatic cancer remains the most lethal cancer with an overall 5-year survival rate of approximately 7%, despite significant advances in the imaging and treatment of many other cancers. In this review, we discuss recent advances in molecular imaging of pancreatic cancer using antibody-based imaging agents. This task is accomplished by summarizing the current progress in each type of molecular imaging modality described above. Also, several considerations for designing and synthesizing novel antibody-based imaging agents are discussed. Lastly, the future directions of antibody-based imaging agents are discussed, emphasizing the potential applications for personalized medicine. PMID:26620581

  7. Antibodies for Treatment of Clostridium difficile Infection

    PubMed Central

    Wilcox, Mark H.

    2014-01-01

    Antibodies for the treatment of Clostridium difficile infection (CDI) have been demonstrated to be effective in the research and clinical environments. Early uncertainties about molecular and treatment modalities now appear to have converged upon the systemic dosing of mixtures of human IgG1. Although multiple examples of high-potency monoclonal antibodies (MAbs) exist, significant difficulties were initially encountered in their discovery. This minireview describes historical and contemporary MAbs and highlights differences between the most potent MAbs, which may offer insight into the pathogenesis and treatment of CDI. PMID:24789799

  8. Effects of interferon on antibody formation

    NASA Technical Reports Server (NTRS)

    Sonnenfeld, G.

    1984-01-01

    Studies of the effects of interferon on primary and secondary antibody responses and of the relationship of interferon to other cytokines, or cell products, are presented. Dosage- and timing-dependent immunoenhancing and immunosuppressive activities of interferon are documented for mouse spleen cell cultures and for mice infected with murine hepatitis virus (MHV-3). A possibility that altered interferon production might lead to immunopathological disorders, such as lupus erythematosus, AIDS, arthritis, etc., is discussed. Latest technological developments are presented that indicate that interferon does apparently play a major role in the regulation of antibody responses.

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

    PubMed

    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; Murray, Christopher J

    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 (IgG 1) 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.

  10. Sequencing Antibody Repertoires Provides Evidence for Original Antigenic Sin Shaping the Antibody Response to Influenza Vaccination

    PubMed Central

    Tan, Yann-Chong; Scalfone, Lisa K.; Kongpachith, Sarah; Ju, Chia-Hsin; Cai, Xiaoyong; Lindstrom, Tamsin M.; Sokolove, Jeremy; Robinson, William H.

    2014-01-01

    We used a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of expressed antibodies. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy chain VJ and light chain VJ sequence usage, do so most effectively. Vaccine-induced heavy chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years’ seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years’ seasonal influenza, suggesting that ‘original antigenic sin’ shapes the antibody response to influenza vaccination. PMID:24525048

  11. Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies

    PubMed Central

    Seidel, Ursula J. E.; Schlegel, Patrick; Lang, Peter

    2013-01-01

    In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function. PMID:23543707

  12. Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier.

    PubMed Central

    Friden, P M; Walus, L R; Musso, G F; Taylor, M A; Malfroy, B; Starzyk, R M

    1991-01-01

    Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate approximately 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26. Images PMID:2052557

  13. What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins.

    PubMed

    Vanderven, Hillary A; Ana-Sosa-Batiz, Fernanda; Jegaskanda, Sinthujan; Rockman, Steven; Laurie, Karen; Barr, Ian; Chen, Weisan; Wines, Bruce; Hogarth, P Mark; Lambe, Teresa; Gilbert, Sarah C; Parsons, Matthew S; Kent, Stephen J

    2016-06-01

    The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential. PMID:27428437

  14. Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

    SciTech Connect

    Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M. )

    1991-06-01

    Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate {approximately} 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.

  15. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates. PMID:27040182

  16. Modeling single cell antibody excretion on a biosensor.

    PubMed

    Stojanović, Ivan; Baumgartner, Wolfgang; van der Velden, Thomas J G; Terstappen, Leon W M M; Schasfoort, Richard B M

    2016-07-01

    We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

  17. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    PubMed

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

  18. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

    PubMed

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  19. Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

    PubMed Central

    Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

    2015-01-01

    Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs. PMID:25996084

  20. Cross-reactive and pre-existing antibodies to therapeutic antibodies--Effects on treatment and immunogenicity.

    PubMed

    van Schie, Karin A; Wolbink, Gerrit-Jan; Rispens, Theo

    2015-01-01

    The potential for immunogenicity is an ever-present concern during the development of biopharmaceuticals. Therapeutic antibodies occasionally elicit an antibody response in patients, which can result in loss of response or adverse effects. However, antibodies that bind a drug are sometimes found in pre-treatment serum samples, with the amount depending on drug, assay, and patient population. This review summarizes published data on pre-existing antibodies to therapeutic antibodies, including rheumatoid factors, anti-allotype antibodies, anti-hinge antibodies, and anti-glycan antibodies. Unlike anti-idiotype antibodies elicited by the drug, pre-formed antibodies in general appear to have little consequences during treatment. In the few cases where (potential) clinical consequences were encountered, antibodies were characterized and found to bind a distinct, unusual epitope of the therapeutic. Immunogenicity testing strategies should therefore always include a proper level of antibody characterization, especially when pre-formed antibodies are present. This minimizes false-positives, particularly due to rheumatoid factors, and helps to judge the potential threat in case a genuine pre-dose antibody reactivity is identified.

  1. Nutritional requirements and assessing nutritional status in camelids.

    PubMed

    Van Saun, Robert J

    2009-07-01

    It has been nearly 30 years since the first imported llamas and alpacas have been commercially raised in the United States. Nutritional requirements for these animals have not been well understood and most feeding practices were based on extrapolated and experiential information. Only recently has a National Research Council committee reviewed the available published information relative to nutrient requirements of llamas and alpacas. This article summarizes current nutrient requirement recommendations and provides some practical feeding recommendations and methods to assess nutritional status.

  2. Infectious diseases of New-World camelids (NWC).

    PubMed

    Thedford, T R; Johnson, L W

    1989-03-01

    Although there are notable infectious conditions that are capable of producing clinical disease in the NWC, overall, these species are quite healthy. Of the bacterial diseases, enterotoxemia caused by Clostridium perfringens types C and D would be deemed the most significant in North America, while type A also would be regarded as important in South America. Other important bacterial infections of potential concern are tuberculosis, Johne's disease, anthrax, malignant edema, actinomycosis, tetanus, and the South American condition referred to as alpaca fever, which, to date, has not been observed in North America. Fungal infections include classical ringworm, principally caused by Trichophyton spp., and the cases of coccidioidomycosis that are associated with the arid desert lands of the southwestern United States. Most notable of naturally occurring viral infections in the NWC would be rabies, ecthyma, and a recently described blindness neuropathy that has been associated with the equine herpesvirus I. NWC can be infected experimentally with agents causing hoof-and-mouth disease and vesicular stomatitis, but naturally occurring cases do not seem to occur. Serological evidence of exposure to many viral agents, including blue tongue, parainfluenza 3, bovine respiratory syncytial virus, bovine herpesvirus I, bovine viral diarrhea, influenza A, and rotavirus, has been demonstrated; however, no clinical disease associated with these agents, as yet, is apparent. PMID:2647231

  3. Treatment of leukemia with radiolabeled monoclonal antibodies.

    PubMed

    Sgouros, G; Scheinberg, D A

    1993-01-01

    In contrast to radioimmunotherapy of solid disease, wherein the primary obstacle to success is access of radiolabeled antibody to antigen-positive cells, in the treatment of leukemia delivering a lethal absorbed dose to the isolated cell appears to be the primary obstacle. The isolated cell is defined as one that is exposed only to self-irradiation (from internalized or surface-bound radiolabeled antibody) and to irradiation from free antibody in the blood. It is isolated in the sense that the particulate (beta, electron, alpha) emissions from its nearest neighboring antigen-positive cell do not contribute to its absorbed dose. Disease in the bone marrow and other tissues, since it is confined to a smaller volume, is more easily eradicated because the absorbed dose to a given cell nucleus is enhanced by emissions from adjacent cells (a smaller fraction of the emission energy is 'wasted'). The optimization simulations presented above for the M195 antibody suggest that the optimum dose of antibody that should be administered is that required to yield a concentration within the distribution volume of the antibody that is approximately equal to the concentration of antigen sites as determined by the tumor burden. Although not specifically considered in the modeling example presented above, antibody internalization and catabolism may be expected to play an important role in radioimmunotherapy treatment planning of leukemia. Depending upon the kinetics of internalization and catabolism, the absorbed dose to the red marrow and to antigen-positive cells may be reduced considerably, since catabolism, assuming that it is followed by rapid extrusion of the radioactive label, would decrease the cells' exposure time considerably. The recently demonstrated effectiveness of radioimmunotherapy in certain cases of B-cell lymphoma and in reducing tumor burden in acute myelogenous leukemia suggests that radioimmunotherapy is beginning to fulfill the promise held when it was initially

  4. Phase Transitions in Antibody Solutions: from Pharmaceuticals to Human Disease

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Lomakin, Aleksey; Benedek, George; Dana Farber Cancer Institute Collaboration; Amgen Inc. Collaboration

    2014-03-01

    Antibodies are very important proteins. Natural antibodies play essential role in the immune system of human body. Pharmaceutical antibodies are used as drugs. Antibodies are also indispensable tools in biomedical research and diagnostics. Recently, a number of observations of phase transitions of pharmaceutical antibodies have been reported. These phase transitions are undesirable from the perspective of colloid stability of drug solutions in processing and storage, but can be used for protein purification, X-ray crystallography, and improving pharmokinetics of drugs. Phase transitions of antibodies can also take place in human body, particularly in multiple myeloma patients who overproduce monoclonal antibodies. These antibodies, in some cases, crystallize at body temperature and cause severe complications called cryoglobulinemia. I will present the results of our current studies on phase transitions of both pharmaceutical antibodies and cryoglobulinemia-associated antibodies. These studies have shown that different antibodies have different propensity to undergo phase transitions, but their phase behavior has universal features which are remarkably different from those of spherical proteins. I will discuss how studies of phase behavior can be useful in assessing colloid stability of pharmaceutical antibodies and in early diagnostics of cryoglobulinemia, as well as general implications of the fact that some antibodies can precipitate at physiological conditions.

  5. Antitubulin antibody in healthy adults and patients with infectious mononucleosis and its relationship to smooth muscle antibody (SMA).

    PubMed Central

    Mead, G M; Cowin, P; Whitehouse, J M

    1980-01-01

    Antibody to tubulin in man has been studied using a specific radioimmunoassay, affinity chromatography radioimmunoassay but markedly increased levels were noted in patients with infectious mononucleosis where the antibody was predominantly IgM in type. This finding was confirmed on fluorescence microscopy. Affinity chromatography purified antibody produced characteristic microtubular staining of fixed 3T3 cells, but in addition, produced weak staining of cryostat sections of rat tissue, similar in distribution to that of smooth muscle antibody. Our studies indicate that the IgM smooth muscle antibody found in infectious mononucleosis by IF techniques is at least in part due to an antitubulin antibody. Images FIG. 1 PMID:6993069

  6. Monoclonal antibody technologies and rapid detection assays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

  7. Monoclonal antibody-based therapy for neuroblastoma.

    PubMed

    Cheung, N K

    2000-11-01

    Dose-intensive combination chemotherapy can improve the clinical response of many pediatric solid tumors. However, cure remains elusive. Stage 4 neuroblastoma stands out as an exception. Part of this success is a result of antibody-based strategies, which include immunomagnetic purging of autologous marrow prior to autologous marrow transplantation and immunotherapy directed at minimal residual disease. It is striking that treatment with monoclonal antibodies, even when targeted at a single antigen, namely, ganglioside G(D2), can affect long-term progression-free survival among these patients. The potential role of the idiotype network in tumor control can be exploited clinically. The genetic engineering of these antibodies into novel forms holds great promise for more specific and effective targeting possibilities, including the delivery of cytokines and cells. Preclinical results are also promising. It is expected that the availability of novel antibodies directed at a broader spectrum of pediatric solid tumors will facilitate the successful application of this approach to more patients. Experience with metastatic neuroblastoma has provided proof of this principle. It is likely that other tumors will fall.

  8. Standardization of poliovirus neutralizing antibody tests.

    PubMed

    Albrecht, P; van Steenis, G; van Wezel, A L; Salk, J

    1984-01-01

    Recently the Forum for Advancement of Immunization Research sponsored a Collaborative Study on Poliovirus Antibody Titration. Twenty laboratories from 12 countries participated. There were considerable differences in detail of test performance and test results among laboratories. The sensitivity of the tests varied over a 10-fold range (geometric mean titer from 21 to 288). The average coefficient of variation ranged from 4.5% to 13.5%. Tests performed at the Food and Drug Administration indicated that Hep-2 cells were more suitable than Vero cells for poliovirus titration by cytopathic effect end point or plaque assay. However, the cell lines were equally suitable for polio antibody titration by neutralization of cytopathic effect. A high degree of sensitivity and optimal reproducibility of antibody assay were observed in tests utilizing a medium to low dose of virus and serum-virus incubation overnight at 36 C. On the basis of current experience, a protocol is proposed for a test that provides high sensitivity and reproducibility and is practical for large-scale determinations of poliovirus antibody.

  9. The emergence of antibody therapies for Ebola.

    PubMed

    Hiatt, Andrew; Pauly, Michael; Whaley, Kevin; Qiu, Xiangguo; Kobinger, Gary; Zeitlin, Larry

    2015-12-23

    This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing challenges in the long effort to develop therapeutic antibody strategies. Since the first report of a neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs, including the first reported, failed to protect non-human primates (NHPs) in spite of protection in rodents. An appreciation of the role of effector functions to antibody efficacy has contributed significantly to understanding mechanisms of in vivo protection. However a crucial contribution, as measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails. This effort was aided by the use of plant production technology where various combinations of mAbs could be rapidly produced and tested. Introduction of appropriate modifications, such as specific glycan profiles, also improved therapeutic efficacy. The resulting cocktail, ZMapp™, consists of three mAbs that were identified from numerous mAb candidates. ZMapp™ \\ is now being evaluated in human clinical trials but has already played a role in bringing awareness to the potential of antibody therapy for Ebola. PMID:27472862

  10. Orthobunyavirus Antibodies in Humans, Yucatan Peninsula, Mexico

    PubMed Central

    Saiyasombat, Rungrat; Talavera-Aguilar, Lourdes G.; Garcia-Rejon, Julian E.; Farfan-Ale, Jose A.; Machain-Williams, Carlos; Loroño-Pino, Maria A.

    2012-01-01

    We performed a serologic investigation to determine whether orthobunyaviruses commonly infect humans in the Yucatan Peninsula of Mexico. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 146 (18%) of 823 persons tested. Further studies are needed to determine health risks for humans from this potentially deadly group of viruses. PMID:23017592

  11. Human antibody Fc deamidation in vivo.

    PubMed

    Liu, Y Diana; van Enk, Jian Zhang; Flynn, Gregory C

    2009-10-01

    Protein and peptide deamidation occurs spontaneously in vitro under relatively mild conditions. For antibodies and other therapeutic proteins, great effort is placed in manufacturing and storage to minimize this form of degradation. Concern has been especially great in cases where deamidation has been shown to impact protein activity. Here we monitored asparagine deamidation from a recombinant human antibody in humans and found that among the conserved sites, only Asn 384 deamidated at an appreciable rate. Under physiological temperature and pH conditions, in vitro antibody deamidation followed similar kinetics, indicating that simple incubation reactions may be used to model in vivo behavior. Endogenous IgG isolated from human serum possessed 23% deamidation at this site, further demonstrating that this modification is naturally occurring. Thus, deamidation generated in manufacturing and storage does not fully determine the patient exposure to the attribute. Instead, pharmacokinetic data along with the deamidation kinetics are combined to predict patient exposure. The deamidation rate can also be used to estimate the serum lifetime of antibodies. This approach could potentially be used to estimate turnover for other cellular or extracellular proteins.

  12. Electron Microscopic Observations of Rabbit Antibodies.

    PubMed

    Hall, C E; Nisonoff, A; Slayter, H S

    1959-12-01

    Electron micrographs were obtained showing the individual, shadow-cast macromolecules from solutions of purified anti-p-azobenzoate rabbit antibody and of normal gamma-globulin. The two materials look alike and consist mainly of asymmetrical rod-like particles about 30 to 40 A in diameter. Lengths are not constant but the weight average is about 250 A for the antibodies and about 200 A for the gamma-globulin. The average observed dimensions are reasonably consistent with values deduced from physical-chemical methods, although the shape is more nearly that of a cylindrical rod rather than the ellipsoid employed in hydrodynamical theory. Mixtures of antibody and specific dihaptenic dye were examined in attempts to establish the mode of the specific aggregation. At the high dilutions necessary for electron microscopy (0.1 mg./ml.), the effect of the dye was small and tended to be masked by non-specific aggregation on drying. The evidence suggests that under these conditions the specific reaction involves an end-to-end aggregation of the elementary particles to produce a weight average length about twice that of the pure antibody.

  13. Nonpathogenicity of antiintestinal antibody in the rabbit.

    PubMed Central

    Rabin, B. S.; Rogers, S. J.

    1976-01-01

    Rabbits were immunized with intestinal extract prepared from rabbits, guinea pigs, and germ-free rats. The resultant serum antibody response to intestinal antigen was determined by gel precipitation and direct tissue immunofluorescence. Forty-eight hours prior to sacrifice of each immunized animal, a portion of the duodenum, ileum, and colon were traumatized to bring circulating antibody into contact with the tissue. Sections for histology and direct immunofluorescence were taken from the area of trauma, just adjacent to the area, and 10 cm from it. The humoral immune response, the presence of tissue bound immunoglobulin, and tissue histology were compared. The area of trauma in normal and immunized animals showed the same histologic changes. In each animal, tissue just adjacent to the area of trauma and 10 cm from it were histologically identical. The pathologic alterations in the immunized animals were similar to those associated with malabsorption in man. Direct immunofluorescence of the intestinal tissue revealed bound immunoglobulin in histologically normal and abnormal tissue. Precipitating antibody to intestine was present in the serum of rabbits with normal and abnormal histology. Thus, antiintestinal antibody as dected by precipitation in gel and direct tissue immunofluorescence does not appear to be a factor in the pathogenesis of this model of immunologically induced histologic changes in the intestine. Images Figure 5 Figure 6 Figure 7 Figure 8 Figure 1 Figure 2 Figure 3 Figure 4 PMID:1266943

  14. IgA Antibodies in Rett Syndrome

    ERIC Educational Resources Information Center

    Reichelt, K. L.; Skjeldal, O.

    2006-01-01

    The level of IgA antibodies to gluten and gliadin proteins found in grains and to casein found in milk, as well as the level of IgG to gluten and gliadin, have been examined in 23 girls with Rett syndrome and 53 controls. Highly statistically significant increases were found for the Rett population compared to the controls. The reason for this…

  15. The emergence of antibody therapies for Ebola.

    PubMed

    Hiatt, Andrew; Pauly, Michael; Whaley, Kevin; Qiu, Xiangguo; Kobinger, Gary; Zeitlin, Larry

    2015-12-23

    This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing challenges in the long effort to develop therapeutic antibody strategies. Since the first report of a neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs, including the first reported, failed to protect non-human primates (NHPs) in spite of protection in rodents. An appreciation of the role of effector functions to antibody efficacy has contributed significantly to understanding mechanisms of in vivo protection. However a crucial contribution, as measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails. This effort was aided by the use of plant production technology where various combinations of mAbs could be rapidly produced and tested. Introduction of appropriate modifications, such as specific glycan profiles, also improved therapeutic efficacy. The resulting cocktail, ZMapp™, consists of three mAbs that were identified from numerous mAb candidates. ZMapp™ \\ is now being evaluated in human clinical trials but has already played a role in bringing awareness to the potential of antibody therapy for Ebola.

  16. Enterovirus-Specific Anti-peptide Antibodies.

    PubMed

    Poh, Chit Laa; Kirk, Katherine; Chua, Hui Na; Grollo, Lara

    2015-01-01

    Enterovirus 71 (EV-71) is the main causative agent of hand, foot, and mouth disease (HFMD) which is generally regarded as a mild childhood disease. In recent years, EV71 has emerged as a significant pathogen capable of causing high mortalities and severe neurological complications in large outbreaks in Asia. A formalin-inactivated EV71 whole virus vaccine has completed phase III trial in China but is currently unavailable clinically. The high cost of manufacturing and supply problems may limit practical implementations in developing countries. Synthetic peptides representing the native primary structure of the viral immunogen which is able to elicit neutralizing antibodies can be made readily and is cost effective. However, it is necessary to conjugate short synthetic peptides to carrier proteins to enhance their immunogenicity. This review describes the production of cross-neutralizing anti-peptide antibodies in response to immunization with synthetic peptides selected from in silico analysis, generation of B-cell epitopes of EV71 conjugated to a promiscuous T-cell epitope from Poliovirus, and evaluation of the neutralizing activities of the anti-peptide antibodies. Besides neutralizing EV71 in vitro, the neutralizing antibodies were cross-reactive against several Enteroviruses including CVA16, CVB4, CVB6, and ECHO13.

  17. 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.

  18. Platelet antibody: review of detection methods

    SciTech Connect

    Schwartz, K.A.

    1988-10-01

    The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with /sup 125/I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated. 61 references.

  19. Viral antibody dynamics in a chiropteran host

    PubMed Central

    Baker, Kate S; Suu-Ire, Richard; Barr, Jennifer; Hayman, David T S; Broder, Christopher C; Horton, Daniel L; Durrant, Christopher; Murcia, Pablo R; Cunningham, Andrew A; Wood, James L N

    2014-01-01

    Bats host many viruses that are significant for human and domestic animal health, but the dynamics of these infections in their natural reservoir hosts remain poorly elucidated. In these, and other, systems, there is evidence that seasonal life-cycle events drive infection dynamics, directly impacting the risk of exposure to spillover hosts. Understanding these dynamics improves our ability to predict zoonotic spillover from the reservoir hosts. To this end, we followed henipavirus antibody levels of >100 individual E. helvum in a closed, captive, breeding population over a 30-month period, using a powerful novel antibody quantitation method. We demonstrate the presence of maternal antibodies in this system and accurately determine their longevity. We also present evidence of population-level persistence of viral infection and demonstrate periods of increased horizontal virus transmission associated with the pregnancy/lactation period. The novel findings of infection persistence and the effect of pregnancy on viral transmission, as well as an accurate quantitation of chiropteran maternal antiviral antibody half-life, provide fundamental baseline data for the continued study of viral infections in these important reservoir hosts. PMID:24111634

  20. Dengue antibody prevalence in German travelers.

    PubMed

    Wichmann, Ole; Lauschke, Annekathrin; Frank, Christina; Shu, Pei-Yun; Niedrig, Matthias; Huang, Jyh-Hsiung; Stark, Klaus; Jelinek, Tomas

    2005-05-01

    We studied 2,259 German citizens after they returned from dengue-endemic countries from 1996 to 2004. Serotype-specific dengue antibodies indicated acute infections in 51 (4.7%) travelers with recent fever and 13 (1.1%) travelers with no recent fever, depending largely on destination and epidemic activity in the countries visited.

  1. SPECT assay of radiolabeled monoclonal antibodies

    SciTech Connect

    Jaszczak, R.J.

    1992-02-01

    The accurate determination of the biodistribution of radiolabeled monoclonal antibodies (MoAbs) is important for calculation of dosimetry and evaluation of pharmacokinetic variables such as antibody dose and route of administration. The hypothesis of this application is that the biodistribution of radiolabeled monoclonal antibodies (MoAbs) can be quantitatively determined using single photon emission computed tomography (SPECT). The major thrusts during the third year include the continued development and evaluation of improved 3D SPECT acquisition and reconstruction approaches to improve quantitative imaging of radiolabeled monoclonal antibodies (MoAbs), and the implementation and evaluation of algorithms to register serial SPECT image data sets, or to register 3D SPECT images with 3D image data sets acquired from positron emission tomography (PEI) and magnetic resonance images (MRI). The research has involved the investigation of statistical models and iterative reconstruction algorithms that accurately account for the physical characteristics of the SPECT acquisition system. It is our belief that SPECT quantification can be improved by accurately modeling the physical processes such as attenuation, scatter, geometric collimator response, and other factors that affect the measured projection data.

  2. Chemoenzymatic glyco-engineering of monoclonal antibodies

    PubMed Central

    Giddens, John P.; Wang, Lai-Xi

    2016-01-01

    Summary Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications. PMID:26082235

  3. Pyridoxal-5'-phosphate-dependent catalytic antibodies.

    PubMed

    Gramatikova, Svetlana; Mouratou, Barbara; Stetefeld, Jörg; Mehta, Perdeep K; Christen, Philipp

    2002-11-01

    Strategies for expanding the catalytic scope of antibodies include the incorporation of inorganic or organic cofactors into their binding sites. An obvious choice is pyridoxal-5'-phosphate (PLP), which is probably the most versatile organic cofactor of enzymes. Monoclonal antibodies against the hapten N(alpha)-(5'-phosphopyridoxyl)-L-lysine, a stable analog of the covalent coenzyme-substrate adducts were screened by a competition ELISA for binding of the PLP-amino acid Schiff base adduct. The Schiff base with its C4'-N alpha double bond is, in contrast to the hapten, a planar compound and is an obligatory intermediate in all PLP-dependent reactions of amino acids. This highly discriminating screening step eliminated all but 5 of 24 hapten-binding antibodies. The five remaining antibodies were tested for catalysis of the PLP-dependent alpha,beta-elimination reaction of beta-chloroalanine. Antibody 15A9 complied with this selection criterion and catalyzed in addition the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k(cat)'=0.42 min(-1) with D-alanine at 25 degrees C). Homology modeling together with alanine scanning yielded a 3D model of Fab 15A9. The striking analogy between antibody 15A9 and PLP-dependent enzymes includes the following features: (1) The binding sites accommodate the planar coenzyme-amino acid adduct. (2) The bond at C alpha to be broken lies together with the C alpha-N bond in a plane orthogonal to the plane of coenzyme and imine bond. (3) The alpha-carboxylate group of the substrate is bound by an arginine residue. (4) The coenzyme-substrate adduct assumes a cisoid conformation. (5) PLP markedly contributes to catalytic efficiency, being a 10(4) times more efficient amino group acceptor than pyruvate. The protein moiety, however, ensures reaction as well as substrate specificity, and further accelerates the reaction (in 15A9 k(cat (Ab x PLP))'/k(cat (PLP))'=5 x 10(3)). The analogies of antibody 15A9 with

  4. A new tool for monoclonal antibody analysis

    PubMed Central

    An, Yan; Zhang, Ying; Mueller, Hans-Martin; Shameem, Mohammed; Chen, Xiaoyu

    2014-01-01

    Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')2 and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications. PMID:24927271

  5. Antibody diffusion in human cervical mucus.

    PubMed Central

    Saltzman, W M; Radomsky, M L; Whaley, K J; Cone, R A

    1994-01-01

    The mucosal immune system actively transports large quantities of antibodies into all mucus secretions, and these secreted antibodies help prevent infectious entry of many pathogens. Mucus is generally thought to protect epithelial cells by forming a diffusional barrier through which only small molecules can pass. However, electron microscopy indicates that the pore size in mucus is approximately 100 nm, which suggests that antibodies as well as other large molecules might also diffuse through mucus. We measured the diffusion coefficients for antibodies and other proteins within human midcycle cervical mucus using two techniques: fluorescence imaging of concentration profiles and fluorescence photobleaching recovery. The two techniques are complementary, since the rates of diffusion are observed over millimeter distances with fluorescence imaging of concentration profiles and micron distances with fluorescence photobleaching recovery. Both methods yielded essentially the same diffusion coefficients. In contrast to previous reports indicating mucus significantly impedes diffusion of small molecules, antibody diffusion in mucus was relatively unimpeded. In our observations IgG, IgG fragments, IgA, and IgM diffused almost as rapidly in cervical mucus as in water (1.0 > Dmucus/Dwater > 0.7). Simple models for diffusion through water-filled pores suggest that the hydrodynamic pore size for cervical mucus is approximately 100 nm, smaller than the approximately 1000 nm pore size of a collagen gel (at 1 mg/ml) and larger than the approximately 10 nm pore size of gelatin (at 100 mg/ml). This estimated pore size is consistent both with electron micrographs and geometric models of interfiber spacing. Based on these results, we predict that particles as large as viruses can diffuse rapidly through human midcycle cervical mucus, provided the particle forms no adhesive interactions with mucus glycoproteins. Images FIGURE 4 PMID:8161703

  6. An indirect fluorescent antibody test for antibodies to transmissible gastroenteritis of swine.

    PubMed

    Benfield, D A; Haelterman, E O; Burnstein, T

    1978-10-01

    The indirect fluorescent antibody test was modified to provide a rapid technique for the detection, screening and titration of antibodies to transmissible gastroenteritis of pigs. Large numbers of slides containing transmissible gastroenteritis antigen were prepared by planting mixtures of infected and uninfected swine testicular cells onto multiwelled teflon-coated slides. After overnight incubation, about one-half of the cells in each well were infected which provided contrast to aid in detecting specific fluorescence in the presence of varying degrees of background staining. Following fixation, antigen slides were stored at -20 degrees C until used. The indirect fluorescent antibody test was compared to the virus neutralization test in both the screening and titration of swine sera containing transmissible gastroenteritis antibodies. The test was found to be sensitive and reliable and to offer certain advantages over the virus neutralization test.

  7. Antibody avidity in swine lymphocyte antigen-defined miniature pigs.

    PubMed Central

    Appleyard, G D; Mallard, B A; Kennedy, B W; Wilkie, B N

    1992-01-01

    Antibody avidity to hen egg white lysozyme (HEWL) was measured by thiocyanate ion elution enzyme-linked immunosorbent assay (ELISA) in swine lymphocyte antigen (SLA) defined miniature pigs. Serum antibody avidity was evaluated on day 14 and 30 after primary (day 0) and secondary (day 14) immunizations in eight to ten week old miniature pigs previously typed for swine lymphocyte antigen genotype. The effect of SLA genotype, litter, and gender on anti-HEWL antibody avidity was determined by least squares. Antibody avidity varied amongst individuals. Antibody avidity maturation was observed as a mean rise in antibody avidity from primary response (0.89 +/- 0.64) to secondary response (1.23 +/- 0.54) (p < 0.0005). Overall, SLA genotype did not significantly influence antibody avidity or avidity maturation, but pigs of dd genotype had greater avidity maturation between primary and secondary responses than other genotypes. Litter effects significantly affected antibody avidity and maturation. PMID:1477799

  8. Development trends for generation of single-chain antibody fragments.

    PubMed

    Farajnia, Safar; Ahmadzadeh, Vahideh; Tanomand, Asghar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2014-10-01

    Recombinant antibodies are increasingly being employed as therapeutic agents especially in combination with anti-cancer drugs. The single-chain antibody fragments are small antigen-binding proteins which provide the most commonly used antibody formats for diagnostic and therapeutic purposes. These antibody fragments have more rapid tumor penetration and clearance from the serum relative to full-length monoclonal antibodies. There are in vitro antibody-display technologies such as phage display, cell surface display, ribosome display and mRNA display that can be used to isolate high specificity and affinity single-chain antibodies against a wide variety of targets. We review these strategies for generation of stable and active antibody fragments in the present article.

  9. Antibody-based therapies for emerging infectious diseases.

    PubMed Central

    Casadevall, A.

    1996-01-01

    In the 19th century, it was discovered that immune sera were useful in treating infectious diseases. Serum therapy was largely abandoned in the 1940s because of the toxicity associated with the administration of heterologous sera and the introduction of effective antimicrobial chemotherapy. Recent advances in the technology of monoclonal antibody production provide the means to generate human antibody reagents and reintroduce antibody therapies, while avoiding the toxicities associated with serum therapy. Because of the versatility of antibodies, antibody-based therapies could, in theory, be developed against any existing pathogen. The advantages of antibody-based therapies include versatility, low toxicity, pathogen specificity, enhancement of immune function, and favorable pharmacokinetics; the disadvantages include high cost, limited usefulness against mixed infections, and the need for early and precise microbiologic diagnosis. The potential of antibodies as antiinfective agents has not been fully tapped. Antibody-based therapies constitute a potentially useful option against newly emergent pathogens. PMID:8903230

  10. Antibody Request - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  11. Monoclonal antibodies: new agents for cancer detection and targeted therapy

    SciTech Connect

    Baldwin, R.W.; Byers, V.S. )

    1991-01-01

    Antibodies directed against markers on cancer cells are gaining in importance for the purpose of targeting diagnostic and therapeutic agents. In the past, this approach has had very limited success principally because the classical methods for producing antibodies from blood serum of animals immunized with cancer cells or extracts were unsatisfactory. The situation has changed dramatically since 1975 following the design of procedures for 'immortalizing' antibody-producing cells (lymphocytes) by fusing them with cultured myeloma cells to form hybridomas which continuously secrete antibodies. Since these hybridomas produce antibodies coded for by a single antibody-producing cell, the antibodies are called monoclonal. Building on these advances in biomedical research, it is now possible to reproducibly manufacture monoclonal antibodies on a scale suitable for use in cancer detection and therapy.

  12. Induction and detection of antibodies to squalene. II. Optimization of the assay for murine antibodies.

    PubMed

    Matyas, Gary R; Rao, Mangala; Alving, Carl R

    2002-09-15

    An improved high throughput assay for measuring murine antibodies to squalene (SQE) is described. The assay is highly reproducible and sensitive and can detect 80 ng/ml of antibody to SQE. The assay, an ELISA, is similar to our previously described assay in which plates containing PVDF membranes were used [J. Immunol. Methods 245 (2000) 1]. The PVDF plates worked well for detection of murine monoclonal antibodies (mAbs) to SQE, but substantial PVDF plate variation was observed, resulting in significant loss of signal and reproducibility between different lots of plates. In the new assay, the PVDF plates were replaced with Costar round bottom 96-well sterile tissue culture plates. These latter plates, which are not normally used for ELISA assay, gave high absorbances for monoclonal antibodies and anti-SQE serum binding to SQE and low absorbances for solvent-treated wells. Other commercially available polystyrene ELISA plates were unsuitable, in that either the background was high or the absorbance for antibodies binding to SQE was low, or both. This change in plate from PVDF to polystyrene allowed the use of an ELISA plate washer, which dramatically increased the throughput rate over the hand-washed PVDF plates. The improved assay also replaced fetal bovine serum (FBS), which contained SQE in lipoproteins, with fatty acid-free bovine serum albumin (BSA) as the blocker/diluent. Fifteen nanomoles of SQE were selected as the optimal amount of SQE to add to the wells. The binding of monoclonal antibodies and anti-SQE serum was dependent upon both the amount of antibody added to the wells and the amount of SQE added to the wells. Antibody concentration curves were hyperbolic in shape, as seen with most other antibodies. Antibody binding first increased with SQE amount and then reached a plateau around 10 nmol of SQE/well. At high SQE amounts (>75 nmol/well), antibody binding decreased with the amount of SQE added. Using 3H-SQE, the amount of SQE bound to the wells

  13. CiteAb: a searchable antibody database that ranks antibodies by the number of times they have been cited

    PubMed Central

    2014-01-01

    Background Research antibodies are used by thousands of scientists working in diverse disciplines, but it is common to hear concerns about antibody quality. This means that researchers need to carefully choose the antibodies they use to avoid wasting time and money. A well accepted way of selecting a research antibody is to identify one which has been used previously, where the associated data has been peer-reviewed and the results published. Description CiteAb is a searchable database which ranks antibodies by the number of times they have been cited. This allows researchers to easily find antibodies that have been used in peer-reviewed publications and the accompanying citations are listed, so users can check the data contained within the publications. This makes CiteAb a useful resource for identifying antibodies for experiments and also for finding information to demonstrate antibody validation. The database currently contains 1,400,000 antibodies which are from 90 suppliers, including 87 commercial companies and 3 academic resources. Associated with these antibodies are 140,000 publications which provide 306,000 antibody citations. In addition to searching, users can also browse through the antibodies and add their own publications to the CiteAb database. Conclusions CiteAb provides a new way for researchers to find research antibodies that have been used successfully in peer-reviewed publications. It aims to assist these researchers and will hopefully help promote progress in many areas of life science research. PMID:24528853

  14. Antibody Engineering & Therapeutics 2015: The Antibody Society's annual meeting December 7-10, 2015, San Diego, CA.

    PubMed

    Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Weiner, Louis M; Adams, Gregory P; Reichert, Janice M

    2015-01-01

    Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on "Antibodies to watch" in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meeting will commence with keynote presentations by Erica Ollmann Saphire (The Scripps Research Institute), Wayne A. Marasco (Dana-Farber Cancer Institute/Harvard Medical School), Joe W. Gray (Oregon Health & Science University), and Anna M. Wu (University of California Los Angeles), and it will conclude with workshops on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries and on computational antibody design. PMID:26421752

  15. Antibody Engineering & Therapeutics 2015: The Antibody Society's annual meeting December 7–10, 2015, San Diego, CA

    PubMed Central

    Parren, Paul W H I; Burton, Dennis R; Bradbury, Andrew; Huston, James S; Carter, Paul J; Veldman, Trudi; Chester, Kerry A; Larrick, James W; Alfenito, Mark R; Scott, Jamie K; Weiner, Louis M; Adams, Gregory P; Reichert, Janice M

    2015-01-01

    Antibody Engineering & Therapeutics, the annual meeting of The Antibody Society, will be held in San Diego, CA in early December 2015. In this meeting preview, the chairs provide their thoughts on the importance of their session topics, which include antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, overcoming resistance to clinical immunotherapy, and building comprehensive IGVH-gene repertoires through discovering, confirming and cataloging new germline IGVH genes. The Antibody Society's special session will focus on “Antibodies to watch” in 2016, which are a subset of the nearly 50 antibodies currently in Phase 3 clinical studies. Featuring over 100 speakers in total, the meeting will commence with keynote presentations by Erica Ollmann Saphire (The Scripps Research Institute), Wayne A. Marasco (Dana-Farber Cancer Institute/Harvard Medical School), Joe W. Gray (Oregon Health & Science University), and Anna M. Wu (University of California Los Angeles), and it will conclude with workshops on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries and on computational antibody design. PMID:26421752

  16. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  17. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  18. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    PubMed Central

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  19. [Indirect immunofluorescence microtest in the determination of antirabies antibodies].

    PubMed

    Buonavoglia, C; Pestalozza, S; Ciuchini, F

    1982-01-01

    The A.A. describe an indirect fluorescent antibody microtest for the rapid detection and titration of antirabies antibodies. Slides containing rabies antigen were prepared by planting 50% infected cells onto multiwelled teflon-coated slides. Following fixation, slides were stored at -20 degrees C until used. The indirect fluorescent antibody microtest was compared to the RFFIT in titration of sera containing rabies antibodies. The test was found to be rapid, easy and reliable.

  20. Maternal antibodies and developing blood-brain barrier.

    PubMed

    Kowal, Czeslawa; Athanassiou, Andrew; Chen, Huiyi; Diamond, Betty

    2015-12-01

    We briefly review the protective role of maternal antibodies during fetal development and at early postnatal stages. We describe antibody delivery to fetuses, particularly in the context of the developing blood-brain barrier (BBB), and present the essential concepts regarding the adult BBB, together with existing information on the prenatal developing BBB. We focus on maternal antibody transfer to the developing brain and the consequences of the presence of pathogenic antibodies at early stages of brain development on subsequent brain dysfunction.