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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 Central

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

    2015-01-01

    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. PMID:25737448

  2. 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

    2015-05-08

    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.

  3. 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.

  4. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

    PubMed

    Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

    2015-11-13

    Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.

  5. 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

  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. Antibody responses in New World camelids with tuberculosis caused by Mycobacterium microti.

    PubMed

    Lyashchenko, K P; Greenwald, R; Esfandiari, J; Meylan, M; Burri, I Hengrave; Zanolari, P

    2007-12-15

    Antibody responses in New World camelids (NWC) infected with Mycobacterium microti were studied by two serological methods, multiantigen print immunoassay (MAPIA) and lateral-flow-based rapid test (RT). Serum samples were collected during 2004-2006 from 87 animals including 1 alpaca and 7 llamas with confirmed or suspected M. microti infection, 33 potentially exposed but clinically healthy animals from known infected herds, and 46 control NWC from herds where infection had not been previously diagnosed. The serological assays correctly identified infection status in 97% (MAPIA) or 87% (RT) cases. In three llamas with confirmed M. microti infection and one llama with gross pathology suggestive of disease, for which multiple serum samples collected over time were available, the antibody-based tests showed positive results 1-2 years prior to the onset of clinical signs or being found dead. In MAPIA, MPB83 protein was identified to be an immunodominant serological target antigen recognized in NWC infected with M. microti. With the limited number of animals tested in this study, the serological assays demonstrated the potential for convenient, rapid, and accurate diagnosis of M. microti infection in live llamas and alpacas.

  8. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    PubMed

    Maggi, Maristella; Scotti, Claudia

    2017-02-15

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes.

  9. Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography.

    PubMed

    Franco, Elliott J; Sonneson, Gregory J; DeLegge, Thomas J; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2010-01-15

    This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic separation of small molecules. An anti-caffeine VHH antibody was produced by grafting the complementarity determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold, followed by expression in E. coli. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline, theobromine, and paraxanthine, albeit with lower affinity. Further investigation of the effect of environmental conditions, i.e., temperature, pH, and ionic strength, on the antibody using these methods provided useful information about potential elution conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted in a stationary phase capable of separating caffeine and its metabolites.

  10. Application of Monoclonal Antibodies in Functional and Comparative Investigations of Heavy-Chain Immunoglobulins in New World Camelids

    PubMed Central

    Daley, L. P.; Gagliardo, L. F.; Duffy, M. S.; Smith, M. C.; Appleton, J. A.

    2005-01-01

    Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the γ chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids. PMID:15753251

  11. Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids.

    PubMed

    Daley, L P; Gagliardo, L F; Duffy, M S; Smith, M C; Appleton, J A

    2005-03-01

    Of the three immunoglobulin G (IgG) isotypes described to occur in camelids, IgG2 and IgG3 are distinct in that they do not incorporate light chains. These heavy-chain antibodies (HCAbs) constitute approximately 50% of the IgG in llama serum and as much as 75% of the IgG in camel serum. We have produced isotype-specific mouse monoclonal antibodies (MAbs) in order to investigate the roles of HCAbs in camelid immunity. Seventeen stable hybridomas were cloned, and three MAbs that were specific for epitopes on the gamma chains of llama IgG1, IgG2, or IgG3 were characterized in detail. Affinity chromatography revealed that each MAb bound its isotype in solution in llama serum. The antibodies bound to the corresponding alpaca IgGs, to guanaco IgG1 and IgG2, and to camel IgG1. Interestingly, anti-IgG2 MAbs bound three heavy-chain species in llama serum, confirming the presence of three IgG2 subisotypes. Two IgG2 subisotypes were detected in alpaca and guanaco sera. The MAbs detected llama serum IgGs when they were bound to antigen in enzyme-linked immunosorbent assays and were used to discern among isotypes induced during infection with a parasitic nematode. Diseased animals, infected with Parelaphostrongylus tenuis, did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Our data document the utility of these MAbs in functional and physiologic investigations of the immune systems of New World camelids.

  12. 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

  13. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    PubMed

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  14. Analysis of heavy and light chain sequences of conventional camelid antibodies from Camelus dromedarius and Camelus bactrianus species.

    PubMed

    Griffin, Laura M; Snowden, James R; Lawson, Alastair D G; Wernery, Ulrich; Kinne, Jorg; Baker, Terry S

    2014-03-01

    Camel antibodies have been widely investigated, but work has focused upon the unique heavy chain antibodies found across camelid species. These are homodimers, devoid of light chains and the first constant heavy chain domain. Camelid species also display conventional hetero-tetrameric antibodies with identical pairs of heavy and light chains; in Camelus dromedarius these constitute 25% of circulating antibodies. Few investigations have been made on this subset of antibodies and complete conventional camel IgG sequences have not been reported. Here we study the sequence diversity of functional variable and constant regions observed in 57 conventional heavy, 18 kappa and 35 lambda light chains of C. dromedarius and Camelus bactrianus. We detail sequences of the full kappa and lambda light chain, variable and CH1 region for IgG1a and IgG1b and the CH2 and CH3 region for IgG1a. The majority (60%) of IgG1 variable region sequences aligned with the human IgHV3 family (clan III) and had leader sequences beginning with MELG whereas the remaining sequences aligned with the IgHV4 (clan II) and had leader sequences beginning with MRLL. Distinct differences in CDR length were observed between the two; where CDR1 was typically 5 and 7 residues and CDR2 at 17 and 16 residues, respectively. CDR3 length of IgHV4 (range 11 to 20) was closer to that typical of VHH antibodies than that of IgHV3 (range 3 to 18 residues). Designed oligonucleotide primers have enabled identification of paired heavy and light chains of conventional camel antibodies from individual B cell clones.

  15. Evaluation of a panel of antibodies for the immunohistochemical identification of immune cells in paraffin-embedded lymphoid tissues of new- and old-world camelids.

    PubMed

    Uhde, Ann-Kathrin; Lehmbecker, Annika; Baumgärtner, Wolfgang; Spitzbarth, Ingo

    2017-02-01

    Different species of camelids play an important role in the epidemiology of various emerging infectious diseases such as Middle East respiratory syndrome. For precise investigations of the immunopathogenesis in these host species, appropriate immunohistochemical markers are highly needed in order to phenotype distinct immune cells populations in camelids. So far, specific immunohistochemical markers for camelid immune cells are rarely commercially available, and cross-reactivity studies are restricted to the use of frozen dromedary tissues. To bridge this gap, 14 commercially available primary antibodies were tested for their suitability to demonstrate immune cell populations on formalin fixed paraffin-embedded (FFPE) tissue sections of dromedaries, Bactrian camels, llamas, and alpacas in the present study. Out of these, 9 antibodies directed against CD3, CD20, CD79α, HLA-DR, Iba-1, myeloid/histiocyte antigen, CD204, CD208, and CD68 antigen exhibited distinct immunoreaction patterns to certain camelid immune cell subsets. The distribution of these antigens was comparatively evaluated in different anatomical compartments of thymus, spleen, mesenteric, and tracheobronchial lymph nodes. The presented results will provide a basis for further investigations in camelids, especially with respect to the role of the immune response in certain infectious diseases, which harbor a considerable risk to spill over to other species.

  16. 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

  17. 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.

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

    PubMed Central

    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. PMID:26289739

  19. 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.

  20. Camelid cardiology.

    PubMed

    Margiocco, Marco L; Scansen, Brian A; Bonagura, John D

    2009-07-01

    Cardiovascular disorders, although not thoroughly described in the literature, are frequently diagnosed in South American camelids, causing morbidity, mortality, and loss of production. Definitive confirmation concerning the heritability of cardiac defects in these species is lacking; however, this potential exists and should be taken into account when counseling breeders and owners. This article describes the diagnosis and treatment of cardiovascular diseases in llamas and alpacas and reviews the most recent literature. Unique aspects of the cardiovascular physiology in these species are also reviewed.

  1. 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.

  2. Site-specific labeling of cysteine-tagged camelid single-domain antibody-fragments for use in molecular imaging.

    PubMed

    Massa, Sam; Xavier, Catarina; De Vos, Jens; Caveliers, Vicky; Lahoutte, Tony; Muyldermans, Serge; Devoogdt, Nick

    2014-05-21

    Site-specific labeling of molecular imaging probes allows the development of a homogeneous tracer population. The resulting batch-to-batch reproducible pharmacokinetic and pharmacodynamic properties are of great importance for clinical translation. Camelid single-domain antibody-fragments (sdAbs)-the recombinantly produced antigen-binding domains of heavy-chain antibodies, also called Nanobodies-are proficient probes for molecular imaging. To safeguard their intrinsically high binding specificity and affinity and to ensure the tracer's homogeneity, we developed a generic strategy for the site-specific labeling of sdAbs via a thio-ether bond. The unpaired cysteine was introduced at the carboxyl-terminal end of the sdAb to eliminate the risk of antigen binding interference. The spontaneous dimerization and capping of the unpaired cysteine required a reduction step prior to conjugation. This was optimized with the mild reducing agent 2-mercaptoethylamine in order to preserve the domain's stability. As a proof-of-concept the reduced probe was subsequently conjugated to maleimide-DTPA, for labeling with indium-111. A single conjugated tracer was obtained and confirmed via mass spectrometry. The specificity and affinity of the new sdAb-based imaging probe was validated in a mouse xenograft tumor model using a modified clinical lead compound targeting the human epidermal growth factor receptor 2 (HER2) cancer biomarker. These data provide a versatile and standardized strategy for the site-specific labeling of sdAbs. The conjugation to the unpaired cysteine results in the production of a homogeneous group of tracers and is a multimodal alternative to the technetium-99m labeling of sdAbs.

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

    PubMed Central

    Fanning, Sean W; Horn, James R

    2011-01-01

    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. PMID:21557375

  4. 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.

  5. 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

  6. Stepwise engineering of heterodimeric single domain camelid VHH antibodies that passively protect mice from ricin toxin.

    PubMed

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

    2013-12-20

    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.

  7. A semi-empirical glycosylation model of a camelid monoclonal antibody under hypothermia cell culture conditions.

    PubMed

    Aghamohseni, Hengameh; Spearman, Maureen; Ohadi, Kaveh; Braasch, Katrin; Moo-Young, Murray; Butler, Michael; Budman, Hector M

    2017-03-11

    The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars' levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.

  8. 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

  9. 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.

  10. Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids.

    PubMed

    Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, H Martin; Zanolari, Patrik

    2011-12-01

    Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

  11. Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

    PubMed Central

    Lucas, A H

    1988-01-01

    Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded. PMID:3257499

  12. 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

  13. 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.

  14. 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

  15. Bovine viral diarrhea virus in New World camelids.

    PubMed

    Belknap, E B; Collins, J K; Larsen, R S; Conrad, K P

    2000-11-01

    A virus known to cause multiple problems in cattle, bovine viral diarrhea virus, was isolated from 3 different cases in New World camelids. Virus isolation, immunoperoxidase staining, and fluorescent antibody staining were used to detect the virus. The herds involved were screened for antibody titers to bovine viral diarrhea and virus isolation from the buffy coat. Bovine viral diarrhea virus should be considered as a cause of death in young and old New World camelids.

  16. Methane Emission by Camelids

    PubMed Central

    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. PMID:24718604

  17. 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.

  18. Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

    PubMed

    Tian, Baomin; Wong, Wah Yau; Hegmann, Elda; Gaspar, Kim; Kumar, Praveen; Chao, Heman

    2015-06-17

    A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (<2%), and high purity (>95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

  19. Differential neutralizing activities of a single domain camelid antibody (VHH) specific for ricin toxin's binding subunit (RTB).

    PubMed

    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.

  20. 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

    2015-03-06

    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.

  1. 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.

  2. 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.

  3. Husbandry and diseases of camelids.

    PubMed

    Fowler, M E

    1996-03-01

    Camels of the Old World and the New World have provided the indigenous human population with meat, milk, fibre and fuel, also serving as beasts of burden to carry loads, for millennia. With the advent of motorized vehicles, the use of camelids became obsolete except in isolated situations. The numbers of camelids diminished dramatically. A reversal of that trend is now occurring, with a recognition that these animals still function in their respective environments better than any other species of livestock. Camelids have always been popular animals in zoos. Camels and two of the South American camelids are domestic animals which adapt well to contained management. They have a unique ability to obtain nourishment from harsh forages. Their reproductive physiology is different from that of any other livestock species.

  4. Viral diseases of new world camelids.

    PubMed

    Kapil, Sanjay; Yeary, Teresa; Evermann, James F

    2009-07-01

    The increased popularity and population of New World camelids in the United States requires the development of a broader base of knowledge of the health and disease parameters for these animals by the veterinary livestock practitioner. Although our knowledge regarding infectious diseases of camelids has increased greatly over the past decade, the practice of camelid medicine is a relatively new field in North America, so it is important to seek out seasoned colleagues and diagnostic laboratories that are involved in camelid health treatment and diagnosis.

  5. 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

  6. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  7. Pattern of functional antibody activity against Haemophilus influenzae type B (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine.

    PubMed

    Matos, D C S; Silva, A M V; Neves, P C C; Martins, R M; Homma, A; Marcovistz, R

    2009-12-01

    We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (>or=1.0 microg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (>or=0.15 microg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (>or=1.0 microg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

  8. Embryo transfer in domestic South American camelids.

    PubMed

    Sumar, Julio B

    2013-01-10

    Intraspecific and interspecific embryo transfer in domestic South American camelids is developing into a well-established technique. Reports reveal many benefits of using reproductive biotechnologies to allow rapid propagation of alpacas and llamas of high genetic merit (e.g., high fiber quality, preserve color variation). The objective of this review is to provide up-to-date information about embryo transfer in domestic South American camelids. Specific information is provided on criteria for male selection, donor and recipient synchronization, the practice of single- vs. super-ovulation protocols, embryo recovery and transfer techniques, advances in cryopreservation of embryos, results of intra- and inter-specific transfer, and the future of the embryo transfer in domestic South American camelids.

  9. Seroprevalence and characterization of pestivirus infections in small ruminants and new world camelids in Switzerland.

    PubMed

    Danuser, R; Vogt, H-R; Kaufmann, Th; Peterhans, E; Zanoni, R

    2009-03-01

    The seroprevalence of pestivirus infections in small ruminants and new world camelids in Switzerland was determined. In 5'059 sera of sheep from 382 herds, 503 sera of goats from 54 herds and 109 sera of alpacas and lamas from 53 herds, population prevalences of 16.1% (sheep), 25.4% (goats) and 4.6% (new world camelids), respectively, were found. In order to determine the source of infection, the serological reactions were further characterized by cross-neutralization against two pestiviruses representing the genotypes BVDV (Bovine Virus Diarrhea Virus)-1 and BDV (Border Disease Virus)-1. Based on the ratio of respective antibody titres, 56.1% of the infections in sheep were induced by a BDV-1, 12.9% by a BVDV-1 and 31.0% by an unresolved pestivirus. In goats, the corresponding proportions were 23.4%, 10.2% and 66.4%, respectively. In Alpacas and Lamas, the source of infection of 1 animal was BDV-1 and that of 4 seropositive animals remained unresolved. In view of the phylogenetic relationship between pestiviruses, the unresolved source of infection is most probably attributable to other pestivirus genotypes circulating in small ruminants and new world camelids. Due to the predominance of pestiviral genotypes other than BVDV-1, the risk of transmission of BVDV from persistently infected small ruminants and new world camelids to cattle appears to be moderate, apart from close direct contact in mixed animal husbandry, communal pasturing and grazing in the Alps.

  10. 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.

  11. [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.

  12. [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.

  13. Ecto- and endoparasites of new world camelids.

    PubMed

    Ballweber, Lora Rickard

    2009-07-01

    Parasitism in New World camelids (NWC), which is associated with both ecto- and endoparasites, is a major health concern throughout the world. Clinical disease has been noted as causing severe economic losses; subclinical issues have yet to be addressed. Despite the advances made in the knowledge and understanding of parasites of NWC, old parasites continue to plague producers, and new issues have arisen. This article updates information on the major ecto- and endoparasites of NWC, including diagnostic techniques and issues relative to anthelmintic resistance in nematodes.

  14. In vitro production of embryos in South American camelids.

    PubMed

    Trasorras, V; Giuliano, S; Miragaya, M

    2013-01-10

    Studies in reproductive biotechnology techniques have been minimal in South American camelids (SAC). Complex reproductive characteristics of these species contribute to slow progress. Nevertheless, some techniques, such as in vitro fertilization, intracytoplasmic sperm injection and nuclear transfer have been applied and have produced advances in knowledge on embryo environment and in vitro conditions necessary for development. Embryo production may have a high impact in both domestic and wild camelids population. Studies addressed to improve in vitro embryo production and oocyte collection could be a potential key to develop IVF and embryo production as a routine procedure in camelids.

  15. Adenoviral targeting using genetically incorporated camelid single variable domains

    PubMed Central

    Kaliberov, Sergey A.; Kaliberova, Lyudmila N.; Buggio, Maurizio; Tremblay, Jacqueline M.; Shoemaker, Charles B.; Curiel, David T.

    2014-01-01

    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by employing fiber modifications via genetic incorporation of targeting motifs. In this study we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen. We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH employed in this study retains antigen recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs. PMID:24933423

  16. Adenoviral targeting using genetically incorporated camelid single variable domains.

    PubMed

    Kaliberov, Sergey A; Kaliberova, Lyudmila N; Buggio, Maurizio; Tremblay, Jacqueline M; Shoemaker, Charles B; Curiel, David T

    2014-08-01

    The unique ability of human adenovirus serotype 5 (Ad5) to accomplish efficient transduction has allowed the use of Ad5-based vectors for a range of gene therapy applications. Several strategies have been developed to alter tropism of Ad vectors to achieve a cell-specific gene delivery by using fiber modifications via genetic incorporation of targeting motifs. In this study, we have explored the utility of novel anti-human carcinoembryonic antigen (hCEA) single variable domains derived from heavy chain (VHH) camelid family of antibodies to achieve targeted gene transfer. To obtain anti-CEA VHHs, we produced a VHH-display library from peripheral blood lymphocytes RNA of alpacas at the peak of immune response to the hCEA antigen (Ag). We genetically incorporated an anti-hCEA VHH into a de-knobbed Ad5 fiber-fibritin chimera and demonstrated selective targeting to the cognate epitope expressed on the membrane surface of target cells. We report that the anti-hCEA VHH used in this study retains Ag recognition functionality and provides specificity for gene transfer of capsid-modified Ad5 vectors. These studies clearly demonstrated the feasibility of retargeting of Ad5-based gene transfer using VHHs.

  17. Camelid heat stress: 15 cases (2003–2011)

    PubMed Central

    Norton, Piper L.; Gold, Jenifer R.; Russell, Karen E.; Schulz, Kara L.; Porter, Brian F.

    2014-01-01

    This case series describes novel findings associated with heat stress in 15 cases in South American camelids that had no pre-existing illnesses and which had clinical signs of illness after exposure to a warm environment. Novel findings include decreased packed cell volume and albumin concentration and mild spinal axonal degeneration. Heat stress should be considered in weak camelids with a history of hyperthermia. PMID:25320390

  18. Induction of superovulation in South American camelids.

    PubMed

    Ratto, Marcelo H; Silva, Mauricio E; Huanca, Wilfredo; Huanca, Teodosio; Adams, Gregg P

    2013-01-10

    The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular

  19. 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.

  20. Current status and future direction of cryopreservation of camelid embryos.

    PubMed

    Herrid, M; Vajta, G; Skidmore, J A

    2017-02-01

    Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed.

  1. 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

  2. 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.

  3. 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.

  4. Imaging diagnosis--pulmonary metastases in New World camelids.

    PubMed

    Gall, David A; Zekas, Lisa J; Van Metre, David; Holt, Timothy

    2006-01-01

    The radiographic appearance of pulmonary metastatic disease from carcinoma is described in a llama and an alpaca. In one, a diffuse miliary pattern was seen. In the other, a more atypical unstructured interstitial pattern was recognized. Metastatic pulmonary neoplasia in camelids may assume a generalized miliary or unstructured pattern.

  5. Toxoplasma gondii and Neospora caninum seroprevalences in domestic South American camelids of the Peruvian Andes.

    PubMed

    Chávez-Velásquez, Amanda; Aguado-Martínez, Adriana; Ortega-Mora, Luis M; Casas-Astos, Eva; Serrano-Martínez, Enrique; Casas-Velásquez, Gina; Ruiz-Santa-Quiteria, Jose A; Alvarez-García, Gema

    2014-10-01

    The objective of this study was to investigate the presence of Toxoplasma gondii- and Neospora caninum-specific antibodies in domestic South American camelids (SAC) (llamas and alpacas) from the Peruvian Andes through a cross-sectional study. A wide panel of serum samples collected from 1,845 llamas and 2,874 alpacas from the two main SAC production areas of Peru was selected. Immunofluorescence antibody technique was employed to detect and titrate specific anti-T. gondii and anti-N. caninum immunoglobulins G in serum samples. The association between T. gondii and N. caninum seroprevalence and the geographical origin (Central and South Peruvian Andes) was evaluated. Anti-T. gondii antibodies were found in 460 (24.9 %) llamas and 706 (24.6 %) alpacas, whereas anti-N. caninum antibodies were detected in 153 (8.3 %) llamas and 425 (14.8 %) alpacas. Toxoplasma gondii infection was strongly associated with the South Peruvian Andes where moderate climate conditions, larger human population, compared to the Central region, and the presence of wildlife definitive hosts could favor horizontal transmission to SAC. In contrast, N. caninum infection was not associated with the geographical region. These results indicate that T. gondii and N. caninum infections are highly and moderately widespread, respectively, in both species of domestic SAC studied in the sampled areas and appropriate control measures should be undertaken to reduce the prevalence of both parasitic infections.

  6. Ovarian function in South American camelids (alpacas, llamas, vicunas, guanacos).

    PubMed

    Vaughan, Jane

    2011-04-01

    Ultrasound technology and hormone assays have provided a better understanding of folliculogenesis and ovulation in South American camelids in the last two decades. Females exhibit waves of ovarian follicular growth and are induced ovulators and therefore do not exhibit oestrous cycles in the manner of spontaneously ovulating species such as sheep and cattle. There is much variation in inter-wave interval among camelid species (alpaca/llama 10-22 days, vicuna 4-11 days), within species and within individual animals as the range of each phase of follicular growth is wide. Ovulation occurs 24-30h after mating and luteolysis occurs approximately 10 days later if conception fails to occur.

  7. 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.

  8. 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

  9. 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 ...

  10. Update on reproductive biotechnologies in small ruminants and camelids.

    PubMed

    Tibary, A; Anouassi, A; Khatir, H

    2005-08-01

    Recent advances in reproductive biotechnologies in small ruminants include improvement of methods for in vitro production of embryos and attempts at spermatogonial stem cell transplantation. In vitro production of embryos by IVM/IVF, intra-cytoplasmic sperm injection (ICSI), or nuclear transfer (NT) has been made possible by improvements in oocyte collection and maturation techniques, and early embryo culture systems. However, in vitro embryo production still is not very efficient due to several limiting factors affecting the outcome of each step of the process. This paper discusses factors affecting in vitro embryo production in small ruminants and camelids, as well as preliminary results with the technique of spermatogonial stem cell transplantation.

  11. Development and Application of Camelid Molecular Cytogenetic Tools

    PubMed Central

    Avila, Felipe; Das, Pranab J.; Kutzler, Michelle; Owens, Elaine; Perelman, Polina; Rubes, Jiri; Hornak, Miroslav; Johnson, Warren E.

    2014-01-01

    Cytogenetic chromosome maps offer molecular tools for genome analysis and clinical cytogenetics and are of particular importance for species with difficult karyotypes, such as camelids (2n = 74). Building on the available human–camel zoo-fluorescence in situ hybridization (FISH) data, we developed the first cytogenetic map for the alpaca (Lama pacos, LPA) genome by isolating and identifying 151 alpaca bacterial artificial chromosome (BAC) clones corresponding to 44 specific genes. The genes were mapped by FISH to 31 alpaca autosomes and the sex chromosomes; 11 chromosomes had 2 markers, which were ordered by dual-color FISH. The STS gene mapped to Xpter/Ypter, demarcating the pseudoautosomal region, whereas no markers were assigned to chromosomes 14, 21, 22, 28, and 36. The chromosome-specific markers were applied in clinical cytogenetics to identify LPA20, the major histocompatibility complex (MHC)-carrying chromosome, as a part of an autosomal translocation in a sterile male llama (Lama glama, LGL; 2n = 73,XY). FISH with LPAX BACs and LPA36 paints, as well as comparative genomic hybridization, were also used to investigate the origin of the minute chromosome, an abnormally small LPA36 in infertile female alpacas. This collection of cytogenetically mapped markers represents a new tool for camelid clinical cytogenetics and has applications for the improvement of the alpaca genome map and sequence assembly. PMID:23109720

  12. 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.

  13. Antibody discovery: sourcing of monoclonal antibody variable domains.

    PubMed

    Strohl, William R

    2014-03-01

    Historically, antibody variable domains for therapeutic antibodies have been sourced primarily from the mouse IgG repertoire, and typically either chimerized or humanized. More recently, human antibodies from transgenic mice producing human IgG, phage display libraries, and directly from human B lymphocytes have been used more broadly as sources of antibody variable domains for therapeutic antibodies. Of the total 36 antibodies approved by major maket regulatory agencies, the variable domain sequences of 26 originate from the mouse. Of these, four are marketed as murine antibodies (of which one is a mouse-rat hybrid IgG antibody), six are mouse-human chimeric antibodies, and 16 are humanized. Ten marketed antibodies have originated from human antibody genes, three isolated from phage libraries of human antibody genes and seven from transgenic mice producing human antibodies. Five antibodies currently in clinical trials have been sourced from camelids, as well as two from non-human primates, one from rat, and one from rabbit. Additional sources of antibody variable domains that may soon find their way into the clinic are potential antibodies from sharks and chickens. Finally, the various methods for retrieval of antibodies from humans, mouse and other sources, including various display technologies and amplification directly from B cells, are described.

  14. Single-domain antibodies for biomedical applications.

    PubMed

    Krah, Simon; Schröter, Christian; Zielonka, Stefan; Empting, Martin; Valldorf, Bernhard; Kolmar, Harald

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

  15. [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.

  16. First complete mitochondrial genome data from ancient South American camelids - The mystery of the chilihueques from Isla Mocha (Chile)

    PubMed Central

    Westbury, Michael; Prost, Stefan; Seelenfreund, Andrea; Ramírez, José-Miguel; Matisoo-Smith, Elizabeth A.; Knapp, Michael

    2016-01-01

    In South American societies, domesticated camelids were of great cultural importance and subject to trade and translocation. South American camelids were even found on remote and hard to reach islands, emphasizing their importance to historic and pre-historic South American populations. Isla Mocha, a volcanic island 35 km offshore of Central-South Chile, is an example of such an island. When Dutch and Spanish explorers reached the island in the early 17th century, they found that domesticated camelids called “chilihueque” played a major role in the island’s society. The origin and taxonomy of these enigmatic camelids is unclear and controversial. This study aims to resolve this controversy through genetic analyses of Isla Mocha camelid remains dating from pre-Columbian to early historic times. A recent archaeological excavation of site P21-3 on Isla Mocha yielded a number of camelid remains. Three complete mitochondrial genomes were successfully recovered and analysed. Phylogenetic analyses suggest that “chilihueque” was a local term for a domesticated guanaco. Results from phylogeographic analyses are consistent with Isla Mocha camelids being sourced from Southern Chilean guanaco populations. Our data highlights the capability of ancient DNA to answer questions about extinct populations which includes species identity, potential translocation events and origins of founding individuals. PMID:27929050

  17. First complete mitochondrial genome data from ancient South American camelids - The mystery of the chilihueques from Isla Mocha (Chile).

    PubMed

    Westbury, Michael; Prost, Stefan; Seelenfreund, Andrea; Ramírez, José-Miguel; Matisoo-Smith, Elizabeth A; Knapp, Michael

    2016-12-08

    In South American societies, domesticated camelids were of great cultural importance and subject to trade and translocation. South American camelids were even found on remote and hard to reach islands, emphasizing their importance to historic and pre-historic South American populations. Isla Mocha, a volcanic island 35 km offshore of Central-South Chile, is an example of such an island. When Dutch and Spanish explorers reached the island in the early 17th century, they found that domesticated camelids called "chilihueque" played a major role in the island's society. The origin and taxonomy of these enigmatic camelids is unclear and controversial. This study aims to resolve this controversy through genetic analyses of Isla Mocha camelid remains dating from pre-Columbian to early historic times. A recent archaeological excavation of site P21-3 on Isla Mocha yielded a number of camelid remains. Three complete mitochondrial genomes were successfully recovered and analysed. Phylogenetic analyses suggest that "chilihueque" was a local term for a domesticated guanaco. Results from phylogeographic analyses are consistent with Isla Mocha camelids being sourced from Southern Chilean guanaco populations. Our data highlights the capability of ancient DNA to answer questions about extinct populations which includes species identity, potential translocation events and origins of founding individuals.

  18. 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.

  19. [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.

  20. Cataracts in New World camelids (llamas, alpacas, vicuñas, and guanacos).

    PubMed

    Gionfriddo, Juliet R

    2002-05-01

    Cataracts are the most frequently seen lens diseases in New World camelids. The causes of cataracts are unknown in many animals, but cataracts secondary to intraocular inflammation seem to be common. Congenital or juvenile-onset cataracts, if another cause is not apparent, should be considered as possibly caused by heredity, and the affected animals should not be bred. Persistent hyaloid vascular anomalies may also have an important role in cataract formation and could be heritable or caused by an in utero disturbance. Pedigree analyses, test breedings, and possibly DNA studies of llamas with cataracts will be required to determine their potential heritability in these species. Cataract surgery can be done successfully in camelids. It is important to evaluate the posterior segment with B scan ultrasonography before surgery in animals in which the posterior segment previously has not been seen. This evaluation allows the surgeon to better prepare for the presence of hyaloid vascular anomalies. Use of phacoemulsification, gentle tissue handling, liberal use of anti-inflammatory medications and endothelial protectants (BSS + and viscoelastics) during surgery has increased the success rate of this surgery in camelids. Unlike cataract surgery in dogs and horses undergoing, cataract surgery, in camelids seems to be important to remove much of the posterior lens capsule. This removal prevents severe capsular fibrosis and subsequent vision loss. There is evidence that a posterior capsulectomy and anterior viterectomy can help prevent postoperative glaucoma. Research needs to be done to see whether these species have an increased risk for ciliary-block glaucoma.

  1. [Advances in the study of natural small molecular antibody].

    PubMed

    Zhu, Lei; Zhang, Da-peng

    2012-10-01

    Small molecule antibodies are naturally existed and well functioned but not structurally related to the conventional antibodies. They are only composed of heavy protein chains or light chains, much smaller than common antibody. The first small molecule antibody, called Nanobody was engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, "immunoglobulin new antigen receptor"), from which single-domain antibodies called Vnar fragments can be obtained. In addition, free light chain (FLC) antibodies in human bodies are being developed as therapeutic and diagnostic agents. Comparing to intact antibodies, common advantages of small molecule antibodies are with better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs. This article reviews the structural characteristics and mechanism of action of the Nanobody, IgNAR and FLC.

  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. Diagnosis of tuberculosis in camelids: old problems, current solutions and future challenges.

    PubMed

    Alvarez, J; Bezos, J; Juan, L de; Vordermeier, M; Rodriguez, S; Fernandez-de-Mera, I G; Mateos, A; Domínguez, L

    2012-02-01

    In spite of great efforts for its control and eradication, tuberculosis remains one of the most important zoonosis worldwide. Its causative agents, the members of the Mycobacterium tuberculosis complex, have a wide host range that complicates the epidemiology of this disease. Among susceptible species to these pathogens, camelids from the New World (llama, alpaca and vicuña) and Old World (Bactrian camel and dromedary) are acquiring an increasing importance in several European countries because of its growing number and could act as reservoirs of the disease for livestock and humans in their natural habitat. In addition, tuberculosis caused by a number of M. tuberculosis complex members is a life-threatening disease in these animal species. Although tuberculosis has been known to affect camelids for a long time, ante-mortem diagnosis is still challenging because of the lack of standardized diagnostic techniques and the limited sensitivity and specificity of the most widely applied tests. However, in recent years, several techniques that can at least partially overcome these limitations have been developed. This paper reviews the results and advances achieved in tuberculosis diagnosis in camelids in the last decade as well as the progresses on ongoing investigations, with special attention to the remaining challenges that still have to be faced to assure the availability of reliable tools for the detection of tuberculosis-infected animals and herds.

  4. 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.

  5. Antithyroglobulin antibody

    MedlinePlus

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

  6. Identification and isolation of stimulator of interferon genes (STING): an innate immune sensory and adaptor gene from camelids.

    PubMed

    Premraj, A; Aleyas, A G; Nautiyal, B; Rasool, T J

    2013-10-01

    The mechanism by which type I interferon-mediated antiviral response is mounted by hosts against invading pathogen is an intriguing one. Of late, an endoplasmic reticulum transmembrane protein encoded by a gene called stimulator of interferon genes (STING) is implicated in the innate signalling pathways and has been identified and cloned in few mammalian species including human, mouse and pig. In this article, we report the identification of STING from three different species of a highly conserved family of mammals - the camelids. cDNAs encoding the STING of Old World camels - dromedary camel (Camelus dromedarius) and bactrian camel (Camelus bactrianus) and a New World camel - llama (Llama glama) were amplified using conserved primers and RACE. The complete STING cDNA of dromedary camel is 2171 bp long with a 706-bp 5' untranslated regions (UTR), an 1137-bp open reading frame (ORF) and a 328-bp 3' UTR. Sequence and phylogenetic analysis of the ORF of STING from these three camelids indicate high level of similarity among camelids and conservation of critical amino acid residues across different species. Quantitative real-time PCR analysis revealed high levels of STING mRNA expression in blood, spleen, lymph node and lung. The identification of camelid STING will help in better understanding of the role of this molecule in the innate immunity of the camelids and other mammals.

  7. Prevalence of Eimeria macusaniensis and Eimeria ivitaensis in South American camelids of Northwest Argentina.

    PubMed

    Cafrune, M M; Marín, R E; Rigalt, F A; Romero, S R; Aguirre, D H

    2009-06-10

    Faecal samples from mostly adult llamas (n=626), vicuñas (n=161) and guanacos (n=4) were obtained between December 2004 and July 2008 in three Provinces of Northwest Argentina in order to study the prevalence of Eimeria macusaniensis and Eimeria ivitaensis. Faeces were examined by a flotation technique using a Cl(2)Zn+ClNa solution (specific gravity=1.59). Oocysts of E. macusaniensis occurred in 88.3% of 77 llama herds and in 50.3% of 626 llamas sampled whereas oocysts of E. ivitaensis occurred in only four llamas (herd and llama prevalence of 5.2% and 0.6%, respectively). The individual prevalence of E. macusaniensis in vicuñas and guanacos were of 14.3% and 25.0%, respectively. E. ivitaensis was not detected in these latter species. The results showed a prevalence of E. macusaniensis higher than previously reported in adult domestic camelids (llamas and alpacas). In contrast, the very low prevalence of E. ivitaensis in llamas and its absence in wild camelids (vicuñas and guanacos) was remarkable. Differences between prevalence of both coccidian species are discussed.

  8. 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

  9. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

    PubMed

    Wesolowski, Janusz; Alzogaray, Vanina; Reyelt, Jan; Unger, Mandy; Juarez, Karla; Urrutia, Mariela; Cauerhff, Ana; Danquah, Welbeck; Rissiek, Björn; Scheuplein, Felix; Schwarz, Nicole; Adriouch, Sahil; Boyer, Olivier; Seman, Michel; Licea, Alexei; Serreze, David V; Goldbaum, Fernando A; Haag, Friedrich; Koch-Nolte, Friedrich

    2009-08-01

    Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

  10. Prevalence and significance of gastrointestinal helminths and protozoa in South American Camelids in Switzerland.

    PubMed

    Hertzberg, Hubertus; Kohler, Lucia

    2006-01-01

    A cross sectional study was conducted to determine the prevalence and significance of endoparasitic infections in South American Camelids (SAC) in Switzerland. Qualitative and quantitative coproscopic examinations were performed in 38 farms during the grazing period. Management practices with possible interference with parasitic infections were analyzed. On the farm level prevalences of endoparasitic infections were: trichostrongyles 87%; Trichuris sp. 74%; Capillaria sp. 68%; Nematodirus battus 63%; Nematodirus sp. 53%; Dicrocoelium dendriticum 34%; Moniezia sp. 8%; Fasciola hepatica 5%; protostrongylids 5%; Eimeria macusaniensis 68%. The level of helminth egg excretion was generally low. The highest values were recorded for trichostrongyles with an average of all investigated farms of 53 eggs per gram of faeces. The mean trichostrongyle egg output was approximately three-fold in SAC on farms that also kept sheep and/or goats, although this difference was not significant (P = 0.11). Clinical trichostrongylidosis was not reported from any of the farms. The low infection level with gastrointestinal nematodes is attributed to the defaecation behaviour of the SAC depositing their faeces focally on small spots on pasture. As a consequence, pasture infectivity is largely restricted to the area adjacent to the dung piles. Dicrocoeliosis is regarded as the most relevant parasitic infection of llamas and alpacas in Switzerland causing severe clinical symptoms and death in untreated animals. Sixteen per cent of the owners regularily treated their herds against dicrocoeliosis using praziquantel at a dose of 50 mg/kg body weight orally.

  11. Examination techniques and therapeutic regimens for the ruminant and camelid eye.

    PubMed

    Townsend, Wendy M

    2010-11-01

    A step-wise procedure and necessary equipment for examination of the ruminant and camelid eye are detailed. Restraint techniques and usage of local anesthetics to facilitate examination are described. Common examination findings and their significance are discussed. Finally, therapeutic options for keratoconjunctivitis and uveitis are explored. A complete ocular examination of ruminants is often not performed in the field because of lack of time, lack of appropriate facilities, and/or lack of equipment. Although individual ophthalmic examinations are not frequently performed as part of a herd health program, they can be of value in select cases. Ocular manifestations of systemic diseases may assist the clinician in establishing a diagnosis on the farm and for little additional cost. For patients with a specific ocular complaint, a complete ophthalmic examination is critical. After completion of the examination and arrival at a diagnosis, one must also be cognizant of the therapeutic regimens that are appropriate for use in ruminants, particularly animals that may be used for meat or milk.

  12. 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.

  13. 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%).

  14. 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.

  15. Surgical repair of femoral fractures in New World camelids: five cases (1996-2003).

    PubMed

    Shoemaker, R W; Wilson, D G

    2007-04-01

    Five New World camelids were admitted to the Western College of Veterinary Medicine between 1996 and 2003 for evaluation of femoral fractures. There were three alpacas and two llamas. Four of the animals were female and three were less than 3 months of age. Fracture configurations consisted of distal physeal fractures (three), a comminuted diaphyseal/metaphyseal fracture, and a transverse diaphyseal fracture. Fractures were diagnosed with a combination of physical examination and radiographs in all cases. All five fractures were repaired with internal fixation and three animals were discharged from the hospital with fractures that healed. One cria underwent successful internal fixation but died from pulmonary oedema during recovery from anaesthesia. Postoperative complications were rare and limited to inadequate fracture stability in one alpaca and prolonged recovery to weight bearing in another. One llama with a comminuted metaphyseal fracture, repaired with a 4.5 mm dynamic compression plate, subsequently had catastrophic failure of the bone 17 days after surgery. Overall the clients were pleased with the outcome of discharged animals. Although femoral fractures are considered rare, they pose a unique opportunity for the large animal veterinarian to successfully achieve fracture union with the aid of internal fixation.

  16. Cerebrospinal fluid eosinophilia is a sensitive and specific test for the diagnosis of Parelaphostrongylus tenuis in camelids in the northeastern United States.

    PubMed

    Pinn, Toby L; Bender, Hannah S; Stokol, Tracy; Erb, Hollis N; Schlafer, Donald H; Perkins, Gillian A

    2013-01-01

    Aberrant migration of Parelaphostrongylus tenuis in camelids results in neurologic deficits, recumbency, and sometimes death. An antemortem diagnosis of P. tenuis in camelids is typically based upon the presence of characteristic asymmetric neurologic deficits, known exposure to white-tailed deer, cerebrospinal fluid (CSF) eosinophilia, and response to treatment. The diagnostic accuracy of CSF eosinophil percentage for the diagnosis of P. tenuis in camelids has not been critically examined. The objective of the current study was to determine the sensitivity (Se) and specificity (Sp) of CSF eosinophil percentage, CSF eosinophil concentration, total nucleated cell concentration, and protein concentration for the antemortem diagnosis of P. tenuis. Medical records of camelids admitted to Cornell University with clinical signs of neurologic disease, CSF analysis, and necropsy were examined from January 2000 through December 2009. Se and Sp were determined by receiver operating characteristic curves in camelids diagnosed with P. tenuis (n = 13) or other conditions (n = 24) based on postmortem examination. More than 17% of eosinophils in CSF had a Se of 85% and Sp of 92% for P. tenuis diagnosis (area under the curve [AUC]: 0.87; SE AUC: 0.07; P < 0.0001; 95% confidence interval [CI] AUC: 0.72-0.96), and >1.4 eosinophils/µl of CSF had a Se of 85% and Sp of 96% (AUC: 0.9; SE AUC: 0.06; P < 0.0001; 95% CI AUC: 0.76-0.97). Cerebrospinal fluid eosinophil percentage and concentration are sensitive and specific methods for diagnosing P. tenuis antemortem in camelids residing in regions endemic to white-tailed deer.

  17. 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.

  18. Optimizing Selection of Large Animals for Antibody Production by Screening Immune Response to Standard Vaccines

    PubMed Central

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

    2016-01-01

    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

  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. [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.

  1. 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.

  2. 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

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

    PubMed Central

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

    1999-01-01

    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

  4. 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.

  5. 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

    1999-04-07

    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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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).

  15. 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

  16. Chronic Infection With Camelid Hepatitis E Virus in a Liver Transplant Recipient Who Regularly Consumes Camel Meat and Milk.

    PubMed

    Lee, Guan-Huei; Tan, Boon-Huan; Teo, Esmeralda Chi-Yuan; Lim, Seng-Gee; Dan, Yock-Young; Wee, Aileen; Aw, Pauline Poh Kim; Zhu, Yuan; Hibberd, Martin Lloyd; Tan, Chee-Kiat; Purdy, Michael A; Teo, Chong-Gee

    2016-02-01

    There have been increasing reports of food-borne zoonotic transmission of hepatitis E virus (HEV) genotype 3, which causes chronic infections in immunosuppressed patients. We performed phylogenetic analyses of the HEV sequence (partial and full-length) from 1 patient from the Middle East who underwent liver transplantation, and compared it with other orthohepevirus A sequences. We found the patient to be infected by camelid HEV. This patient regularly consumed camel meat and milk, therefore camelid HEV, which is genotype 7, might infect human beings. Our finding links consumption of camel-derived food products to post-transplantation hepatitis E, which, if detected at early stages, can be cured with antiviral therapy and reduced administration of immunosuppressive agents.

  17. Antibody persistence after primary and booster doses of a pentavalent vaccine against diphtheria, tetanus, acellular pertussis, inactivated poliovirus, haemophilus influenzae type B vaccine among Thai children at 18-19 months of age.

    PubMed

    Chotpitayasunondh, Tawee; Thisyakorn, Usa; Pancharoen, Chitsanu; Chuenkitmongkol, Sunate; Ortiz, Esteban

    2012-03-01

    The World Health Organization recommends a booster dose of a pertussis-containing vaccine for children aged 1-6 years, preferably during the second year of life. This study assessed the immunogenicity and safety of a pentavalent combination vaccine containing diphtheria, tetanus, acellular pertussis, inactivated poliovirus, and conjugated-Hib polysaccharide antigens, [(DTaP-IPV//PRP-T (Pentaxim)], as a booster at 18-19 months of age. Participants had received primary doses of the same vaccine at 2, 4 and 6 months of age. Antibody concentrations were measured immediately before and one month after the booster dose. Adverse events were evaluated from parental reports. Geometric mean concentrations (GMCs) or titers (GMTs) decreased from post-primary to pre-booster vaccination; however, at least 94.4% of children had protective levels of anti-tetanus (> or = 0.01 IU/ml), antipoliovirus (> or = 81/dil) and anti-PRP (Hib, > or = 0.15 microg/ml) antibodies prior to the booster. Anti-diphtheria antibody titers > or = 0.01 IU/ml were also observed in the majority of children pre-booster. One month after the booster, seroprotection rates were 99.4% for PRP (> or = 1.0 microg/ml), 95.0% for diphtheria (> or = 0.10 IU/ml) and 100% for tetanus (> or = 0.1 IU/ml) and poliovirus types 1, 2, 3 (> or = 81/dil). At least 93.1% of subjects had 4 fold post-booster increases in anti-pertussis antibody titers. GMCs increased from 14.0 to 307.3 EU/ml and from 13.9 to 271.9 EU/ml for anti-PT and anti-FHA, respectively. Anti-PRP GMC increased from 1.2 to 62.2 microg/ml. The booster was well tolerated. A booster dose during the second year of life was safe and induced a strong immune response, indicative of long-term protection.

  18. 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

  19. Antimitochondrial antibody

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003529.htm Antimitochondrial antibody To use the sharing features on this page, please enable JavaScript. Antimitochondrial antibodies (AMA) are substances ( antibodies ) that form against mitochondria. ...

  20. 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

  1. Phenotypic lentivirus screens to identify functional single domain antibodies

    PubMed Central

    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. While 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 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

  2. [Serological study of leptospirosis in equids, camelids and bovids from Djibouti].

    PubMed

    Roqueplo, C; Davoust, B; Mulot, B; Lafrance, B; Kodjo, A

    2011-10-01

    Sera obtained from 31 domestic and feral animals in Djibouti were assayed for leptospiral antibodies using the microscopic agglutination test. Antibodies were detected in 26 samples (84%), corresponding to 116 positive reactions. The most common antigen serogroups were Icterohaemorrhagiae and Australis. The highest titre was recorded for serovar Munchen (1:1280) in sera from Somalian wild asses and goats. This study shows a broad dispersion and high prevalence of the different Leptospira serogroups tested. High biodiversity has been previously reported in tropical countries and is thought to be linked to the wide range of reservoir mammals. Additional study will be needed to identify the reservoirs of the different serogroups in this part of Africa.

  3. 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.

  4. Llama-Derived Single Domain Antibodies Specific for Abrus Agglutinin

    PubMed Central

    Goldman, Ellen R.; Anderson, George P.; Zabetakis, Dan; Walper, Scott; Liu, Jinny L.; Bernstein, Rachael; Calm, Alena; Carney, James P.; O’Brien, Thomas W.; Walker, Jennifer L.; Garber, Eric A. E.

    2011-01-01

    Llama derived single domain antibodies (sdAb), the recombinantly expressed variable heavy domains from the unique heavy-chain only antibodies of camelids, were isolated from a library derived from llamas immunized with a commercial abrin toxoid preparation. Abrin is a potent toxin similar to ricin in structure, sequence and mechanism of action. The selected sdAb were evaluated for their ability to bind to commercial abrin as well as abrax (a recombinant abrin A-chain), purified abrin fractions, Abrus agglutinin (a protein related to abrin but with lower toxicity), ricin, and unrelated proteins. Isolated sdAb were also evaluated for their ability to refold after heat denaturation and ability to be used in sandwich assays as both capture and reporter elements. The best binders were specific for the Abrus agglutinin, showing minimal binding to purified abrin fractions or unrelated proteins. These binders had sub nM affinities and regained most of their secondary structure after heating to 95 °C. They functioned well in sandwich assays. Through gel analysis and the behavior of anti-abrin monoclonal antibodies, we determined that the commercial toxoid preparation used for the original immunizations contained a high percentage of Abrus agglutinin, explaining the selection of Abrus agglutinin binders. Used in conjunction with anti-abrin monoclonal and polyclonal antibodies, these reagents can fill a role to discriminate between the highly toxic abrin and the related, but much less toxic, Abrus agglutinin and distinguish between different crude preparations. PMID:22174977

  5. 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)

  6. 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.

  7. 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 ...

  8. Serologic survey for antibodies against three genotypes of bovine parainfluenza 3 virus in unvaccinated ungulates in Alabama.

    PubMed

    Newcomer, Benjamin W; Neill, John D; Galik, Patricia K; Riddell, Kay P; Zhang, Yijing; Passler, Thomas; Velayudhan, Binu T; Walz, Paul H

    2017-02-01

    OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer ≤ 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.

  9. 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).

  10. Single domain camel antibodies: current status.

    PubMed

    Muyldermans, S

    2001-06-01

    The antigen-binding capacity of the paired variable domains of an antibody is well established. The observation that the isolated heavy chains of anti-hapten antibodies retain some antigen-binding capacity in the absence of light chains led to attempts to obtain an even smaller antigen-binding unit in a VH format. Unfortunately, the poor solubility, the reduced affinity for the antigen and the irreproducible outcome showed that additional protein engineering would be required to successfully generate single-domain antibody fragments. By serendipity, it was discovered that this engineering is already performed continuously in nature. Part of the humoral immune response of camels and llamas is based largely on heavy-chain antibodies where the light chain is totally absent. These unique antibody isotypes interact with the antigen by virtue of only one single variable domain, referred to as VHH. Despite the absence of the VH-VL combinatorial diversity, these heavy-chain antibodies exhibit a broad antigen-binding repertoire by enlarging their hypervariable regions. Methods are described to tap the VHH repertoire of an immunised dromedary or llama. These VHH libraries contain a high titre of intact antigen-specific binders that were matured in vivo. Synthetic libraries of a 'camelised' human VH, a mouse VH or a camelid VHH scaffold with a randomised CDR3 could constitute a valid alternative to immune libraries to retrieve useful single-domain antigen binders. The recombinant VHH that are selected from such libraries are well expressed, highly soluble in aqueous environments and very robust. Some in vivo matured VHH were also shown to be potent enzyme inhibitors, and the low complexity of the antigen-binding site is an asset in the design of peptide mimetics. Because of their smaller size and the above properties, the VHH clearly offer added-value over conventional antibody fragments. They are expected to open perspectives as enzyme inhibitors and intrabodies, as modular

  11. Interspecies embryo transfer in camelids: the birth of the first Bactrian camel calves (Camelus bactrianus) from dromedary camels (Camelus dromedarius).

    PubMed

    Niasari-Naslaji, A; Nikjou, D; Skidmore, J A; Moghiseh, A; Mostafaey, M; Razavi, K; Moosavi-Movahedi, A A

    2009-01-01

    Interspecies embryo transfer is a possible approach that can be used to conserve endangered species. It could provide a useful technique to preserve the Iranian and wild Bactrian camels, both of which are threatened with extinction. In the present study, one Bactrian camel was superovulated using decreasing doses of FSH (60, 40, 30, 30, 20, 20 mg, b.i.d.; Folltropin-V; Bioniche, London, ON, Canada) for 6 days, followed by a single injection of FSH (20 mg, i.m.) on Day 7. Daily ovarian ultrasonography was performed until most of the growing follicles had reached a mature size of 13-17 mm, at which time the camel was mated twice, 24 h apart, with a fertile male Bactrian camel. At the time of first mating, female camels were given 20 microg, i.v., buserelin (Receptal; Intervet, Boxmeer, The Netherlands). One day after the donor camel had been mated, the dromedary recipients (n = 8) were injected with 25 mg, i.v., porcine LH (Lutropin-V; Bioniche) to induce ovulation. Embryos were recovered on Day 8.5 after the first mating and transferred non-surgically into recipients on Day 7.5 after LH injection. Pregnancy was diagnosed 25 days after embryo transfer. Healthy Bactrian camel calves (n = 4) were born without any particular complications at the time of parturition (e.g. dystocia and neonatal diseases). The present study is the first report of the birth of Bactrian camel calves from dromedary camels, as well as the first report of interspecies embryo transfer in old world camelids.

  12. Localization of orexin B and receptor 2 for orexins in testicular cytotypes of the camelid alpaca (Vicugna pacos).

    PubMed

    Liguori, G; Squillacioti, C; Assisi, L; Mirabella, N; Langella, E; Costagliola, A; Vittoria, A

    2017-02-08

    The orexins A (OxA) and B (OxB) are two hypothalamic peptides involved in many physiological functions of the mammalian body. They act through the binding of two G-coupled receptors named receptor 1 (OX1 ) and receptor 2 (OX2 ) for orexins. The first receptor is specific for OxA, while the second binds both the substances with equal affinity. The orexins and the relative receptors have been traced by means of different techniques also at the periphery of the body and particularly in the adrenals, and in gastrointestinal and genital organs. Aim of this work was to investigate the presence of OxB and OX2 by means of immunohistochemistry and Western blotting analysis in the testis of the South American camelid alpaca, a species primarily breed in Chile and Ecuador and recently diffused in Europe where the quality of its wool is particularly appreciated. OxB immunoreactivity (IR) was found in the tubular compartment of the testis where spermatogonia (resting), zygotene and pachytene spermatocytes, and spermatids clearly showed differently sized and shaped cytoplasmic positive structures. OX2 -IR was found both in the interstitial and tubular compartments of the testis and particularly in Leydig cells and round and elongated spermatids. Western blotting analysis of testis lysates showed the presence of a protein band whose molecular weight corresponded to that currently assigned to OX2 . Such findings easily translate the hypothesis that OxB and its receptor 2 play a functional role both in the interstitial and tubular compartments of the alpaca testis.

  13. Ricin Detection Using Phage Displayed Single Domain Antibodies

    PubMed Central

    Goldman, Ellen R.; Liu, Jinny L.; Bernstein, Rachael D.; Swain, Marla D.; Mitchell, Stanley Q.; Anderson, George P.

    2009-01-01

    Phage-displayed single domain antibodies (sdAb) were compared to monomeric solubly expressed sdAb and llama polyclonal antibodies for the detection of ricin. SdAb are comprised of the variable domain derived from camelid heavy chain only antibodies (HcAb). Although HcAb lack variable light chains, they as well as their derivative sdAb are able to bind antigens with high affinity. The small size of sdAb (∼16 kDa), while advantageous in many respects, limits the number of labels that can be incorporated. The ability to incorporate multiple labels is a beneficial attribute for reporter elements. Opportunely, sdAb are often selected using phage display methodology. Using sdAb displayed on bacteriophage M13 as the reporter element gives the potential for incorporating a very high number of labels. We have demonstrated the use of both sdAb and phage- displayed sdAb for the detection of ricin using both enzyme linked immunosorbent assays (ELISAs) and Luminex fluid array assays. The phage-displayed sdAb led to five to ten fold better detection of ricin in both the ELISA and Luminex assays, resulting in limits of detection of 1 ng/mL and 64 pg/mL respectively. The phage-displayed sdAb were also dramatically more effective for the visualization of binding to target in nitrocellulose dot blot assays, a method frequently used for epitope mapping. PMID:22389616

  14. 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

  15. 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

  16. Antibody persistence at 18-20 months of age and safety and immunogenicity of a booster dose of a combined DTaP-IPV//PRP∼T vaccine compared to separate vaccines (DTaP, PRP∼T and IPV) following primary vaccination of healthy infants in the People's Republic of China.

    PubMed

    Li, Rong Cheng; Li, Feng Xiang; Li, Yan Ping; Hou, Qi Ming; Li, Chang Gui; Li, Ya Nan; Chen, Fu Sheng; Hu, Xue Zhong; Su, Wen Bin; Zhang, Shu Min; Fang, Han Hua; Ye, Qiang; Zeng, Tian De; Liu, Tao Xuan; Li, Xiu Bi; Huang, Yun Neng; Deng, Man Ling; Zhang, Yan Ping; Ortiz, Esteban

    2011-11-21

    This study assessed the antibody persistence, and the immunogenicity and safety of a booster dose of a DTaP-IPV//PRP∼T (Pentaxim®, Sanofi Pasteur's AcXim family) combined vaccine and of standalone vaccines one year after primary vaccination in the People's Republic of China. Participants (N=719) previously primed with DTaP-IPV//PRP∼T at 2, 3, 4 months (Group A, N=255), 3, 4, 5 months (Group B, N=233), or DTaP (Wuhan Institute of Biological Products), PRP-T (Act-Hib®) and IPV (Imovax® Polio) at 3, 4, 5 months (Group C, N=231) received boosters of the same vaccines at 18-20 months of age. Seroprotection (SP) and seroconversion (SC) were determined before and 1 month after the booster. Safety was monitored from parental reports. In all groups 87.6-100% of participants had pre-booster protective anti-PRP, -diphtheria, -tetanus and -poliovirus antibody titers; post-booster, all SP rates were 100% and SC was ≥ 80.4% for anti-pertussis titers ≥ 4-fold increase. Reactogenicity was low for each group. These data support the use of the DTaP-IPV//PRP∼T vaccine in the People's Republic of China compared to separate DTaP, IPV, and PRP∼T administration in terms of both safety and immunogenicity.

  17. Monoclonal Antibodies.

    PubMed

    Geskin, Larisa J

    2015-10-01

    Use of monoclonal antibodies (mAbs) has revolutionized cancer therapy. Approaches targeting specific cellular targets on the malignant cells and in tumor microenvironment have been proved to be successful in hematologic malignancies, including cutaneous lymphomas. mAb-based therapy for cutaneous T-cell lymphoma has demonstrated high response rates and a favorable toxicity profile in clinical trials. Several antibodies and antibody-based conjugates are approved for use in clinical practice, and many more are in ongoing and planned clinical trials. In addition, these safe and effective drugs can be used as pillars for sequential therapies in a rational stepwise manner.

  18. Crystal structure of a shark single-domain antibody V region in complex with lysozyme.

    PubMed

    Stanfield, Robyn L; Dooley, Helen; Flajnik, Martin F; Wilson, Ian A

    2004-09-17

    Cartilaginous fish are the phylogenetically oldest living organisms known to possess components of the vertebrate adaptive immune system. Key to their immune response are heavy-chain, homodimeric immunoglobulins called new antigen receptors (IgNARs), in which the variable (V) domains recognize antigens with only a single immunoglobulin domain, akin to camelid heavy-chain V domains. The 1.45 angstrom resolution crystal structure of the type I IgNAR V domain in complex with hen egg-white lysozyme (HEL) reveals a minimal antigen-binding domain that contains only two of the three conventional complementarity-determining regions but still binds HEL with nanomolar affinity by means of a binding interface comparable in size to conventional antibodies.

  19. 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.

  20. 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

  1. A general strategy for antibody library screening via conversion of transient target binding into permanent reporter deposition.

    PubMed

    Maaß, Alexander; Heiseler, Tim; Maaß, Franziska; Fritz, Janine; Hofmeyer, Thomas; Glotzbach, Bernhard; Becker, Stefan; Kolmar, Harald

    2014-02-01

    We report here a generally applicable method for the selective covalent attachment of a reporter molecule to a replicating entity that allows one to obtain specific binders from a single round of library screening. We show that selective biotinylation of phage particles displaying a binder to any given target can be achieved by application of a coupled enzyme reaction on the surface of the target-binding phage particles that includes a peroxidase, an oxidase and a catalase. Due to the covalent linkage of biotin together with the tight and stable interaction of biotin with streptavidin, very stringent wash conditions for removal of nonspecific binders can be applied. The method termed (3)CARD (triple catalytic reporter deposition) was successfully applied to single-round screening of a phage display library of camelid single-domain antibodies against three different target proteins.

  2. Expression Cloning and Production of Human Heavy-Chain-Only Antibodies from Murine Transgenic Plasma Cells

    PubMed Central

    Drabek, Dubravka; Janssens, Rick; de Boer, Ernie; Rademaker, Rik; Kloess, Johannes; Skehel, John; Grosveld, Frank

    2016-01-01

    Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH–VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs. PMID:28066429

  3. 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

  4. Serum herpes simplex antibodies

    MedlinePlus

    ... gov/ency/article/003352.htm Serum herpes simplex antibodies To use the sharing features on this page, please enable JavaScript. Serum herpes simplex antibodies is a blood test that looks for antibodies ...

  5. 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.

  6. [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.

  7. Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system.

    PubMed

    Mizukami, Makoto; Tokunaga, Hiroko; Onishi, Hiromasa; Ueno, Yohei; Hanagata, Hiroshi; Miyazaki, Nobuo; Kiyose, Norihiko; Ito, Yuji; Ishibashi, Matsujiro; Hagihara, Yoshihisa; Arakawa, Tsutomu; Miyauchi, Akira; Tokunaga, Masao

    2015-01-01

    Anti-IZUMO1PFF VHH (variable domain of camelid heavy chain antibody) clones, N6 and N15, from immunized alpaca (Lama pacos) phage library were efficiently expressed and their VHH products were secreted into the culture medium of Brevibacillus choshinensis HPD31-SP3, e.g., at a level of 26-95mg in 100ml conventional flask culture. With a 3-L scale fed-batch culture for 65h, the N15 VHH protein with C-terminal His-tag was produced at ∼3g/l culture medium. The N6 and N15 proteins were easily purified to apparent homogeneity by cation exchange and Ni-affinity chromatographies. Both proteins showed specific antigen-binding activity by ELISA and high antigen binding affinity, KD=6.0-8.6nM, by surface plasmon resonance analysis. Size exclusion chromatography-multi-angle laser light scattering analysis revealed that N6 and N15 proteins purified were exclusively monomeric form in phosphate buffered saline. CD spectrum showed beta-sheet rich structure, consistent with a typical antibody structure and also suggested aromatic-aromatic interactions, as indicated by a positive peak at 232nm. Thermal melting analysis of the N15 protein with C-terminal His-tag demonstrated a clear thermal transition with a Tm at 67°C. The heat-denatured sample recovered antigen binding activity upon cooling, indicating a reversible denaturation.

  8. Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments*

    PubMed Central

    Govaert, Jochen; Pellis, Mireille; Deschacht, Nick; Vincke, Cécile; Conrath, Katja; Muyldermans, Serge; Saerens, Dirk

    2012-01-01

    The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond. PMID:22128183

  9. 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.

  10. 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

  11. 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

  12. Platelet associated antibodies

    MedlinePlus

    ... medlineplus.gov/ency/article/003552.htm Platelet-associated antibodies blood test To use the sharing features on ... JavaScript. This blood test shows if you have antibodies against platelets in your blood. Platelets are a ...

  13. Lyme disease antibody

    MedlinePlus

    ... JavaScript. The Lyme disease blood test looks for antibodies in the blood to the bacteria that causes ... needed. A laboratory specialist looks for Lyme disease antibodies in the blood sample using the ELISA test . ...

  14. Antiparietal cell antibody test

    MedlinePlus

    ... Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ... may use this test to help diagnose pernicious anemia. Pernicious anemia is a decrease in red blood ...

  15. 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)

  16. RBC Antibody Screen

    MedlinePlus

    ... Cell Antibody Screen Related tests: Direct Antiglobulin Test ; Blood Typing ; RBC Antibody Identification ; Type and Screen; Crossmatch All content on Lab Tests Online has been reviewed and approved by our Editorial Review Board . At a ... screen is used to screen an individual's blood for antibodies directed against red blood cell (RBC) ...

  17. 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 ...

  18. Analysis of the binding loops configuration and surface adaptation of different crystallized single-domain antibodies in response to various antigens.

    PubMed

    Al Qaraghuli, Mohammed M; Ferro, Valerie A

    2017-04-01

    Monoclonal antibodies have revolutionized the biomedical field through their ubiquitous utilization in different diagnostics and therapeutic applications. Despite this widespread use, their large size and structural complexity have limited their versatility in specific applications. The antibody variable region that is responsible for binding antigen is embodied within domains that can be rescued individually as single-domain antibody (sdAb) fragments. Because of the unique characteristics of sdAbs, such as low molecular weight, high physicochemical stability, and the ability to bind antigens inaccessible to conventional antibodies, they represent a viable alternative to full-length antibodies. Consequently, 149 crystal structures of sdAbs, originating from human (VH), camelids (VHH), or sharks (VNAR), were retrieved from the Protein Data Bank, and their structures were compared. The 3 types of sdAbs displayed complementarity determining regions (CDRs) with different lengths and configurations. CDR3 of the VHH and VNAR domains were dominated by pleated and extended orientations, respectively. Although VNAR showed the smallest average molecular weight and molecular surface area compared with VHH and VH antibodies. However, the solvent accessible surface area measurements of the 3 tested sdAbs types were very similar. All the antihapten VHH antibodies showed pleated CDR3, which were sufficient to create a binding pocket to accommodate haptens (methotrexate and azo dyes) in terms of shape and electrostatic potential. The sdAbs that recognized lysozyme showed more diversity in their CDR3 orientation to enable them to recognize various topographies of lysozyme. Subsequently, the three sdAb classes were different in size and surface area and have shown distinguishable ability to optimize their CDR length and orientation to recognize different antigen classes.

  19. Isolation and epitope mapping of staphylococcal enterotoxin B single-domain antibodies.

    PubMed

    Turner, Kendrick B; Zabetakis, Dan; Legler, Patricia; Goldman, Ellen R; Anderson, George P

    2014-06-19

    Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better.  Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling

    PubMed Central

    Adams, Ralph; Burnley, Rebecca J.; Valenzano, Chiara R.; Qureshi, Omar; Doyle, Carl; Lumb, Simon; del Carmen Lopez, Maria; Griffin, Robert; McMillan, David; Taylor, Richard D.; Meier, Chris; Mori, Prashant; Griffin, Laura M.; Wernery, Ulrich; Kinne, Jörg; Rapecki, Stephen; Baker, Terry S.; Lawson, Alastair D. G.; Wright, Michael; Ettorre, Anna

    2017-01-01

    Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions. PMID:28134246

  5. 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

  6. 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

  7. Antibodies Targeting EMT

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0381 TITLE: Antibodies Targeting EMT PRINCIPAL INVESTIGATOR: Vaughn Smider CONTRACTING ORGANIZATION: The Scripps...REPORT DATE October 2015 2. REPORT TYPE Annual 3. DATES COVERED 30 Sep 2014 - 29 Sep 2015 4. TITLE AND SUBTITLE Antibodies Targeting EMT 5a. CONTRACT ...ABSTRACT Monoclonal antibodies are drugs that can specifically bind targets present on tumor cells. The highly aggressive triple-negative subtype of

  8. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  9. 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.

  10. 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…

  11. 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...

  12. 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.

  13. 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

  14. Antibodies for biodefense

    PubMed Central

    Froude, Jeffrey W; Stiles, Bradley; Pelat, Thibaut

    2011-01-01

    Potential bioweapons are biological agents (bacteria, viruses and toxins) at risk of intentional dissemination. Biodefense, defined as development of therapeutics and vaccines against these agents, has seen an increase, particularly in the US, following the 2001 anthrax attack. This review focuses on recombinant antibodies and polyclonal antibodies for biodefense that have been accepted for clinical use. These antibodies aim to protect against primary potential bioweapons or category A agents as defined by the Centers for Disease Control and Prevention (Bacillus anthracis, Yersinia pestis, Francisella tularensis, botulinum neurotoxins, smallpox virus and certain others causing viral hemorrhagic fevers) and certain category B agents. Potential for prophylactic use is presented, as well as frequent use of oligoclonal antibodies or synergistic effect with other molecules. Capacities and limitations of antibodies for use in biodefense are discussed, and are generally applicable to the field of infectious diseases. PMID:22123065

  15. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

  16. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  17. 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

  18. Therapeutic antibody technology 97.

    PubMed

    Larrick, J W; Gavilondo, J

    1998-01-01

    Almost 200 antibody aficionados attended the Therapeutic Antibody Technology 97 meeting, held September 21-24, 1997 at the Holiday Inn, Union Square in the heart of San Francisco, CA. The meeting was sponsored by the Palo Alto Institute of Molecular Medicine and organized by James W. Larrick (PAIMM) and Dennis R. Burton (Scripps Research Institute). The meeting featured excellent discussions on many interesting talks and a number of poster presentations. It is likely that another meeting will be organized in 2 years, however in the meantime, an effort is underway to organize a 'Virtual Antibody Society' to be set up on the web server at Scripps Research Institute in La Jolla, CA (Questions and comments on this project can be sent to: Jwlarrick@aol.com or Burton@scripps.edu). Richard Lerner (Scripps) gave the keynote address on 'Catalytic Antibodies', describing recent work with Carlos Barbas on so-called reactive immunization to generate a high activity aldolase catalytic antibody. This antibody, soon to be described in an article in Science, is the first commercially available catalytic antibody.

  19. Monoclonal antibody "gold rush".

    PubMed

    Maggon, Krishan

    2007-01-01

    The market, sales and regulatory approval of new human medicines, during the past few years, indicates increasing number and share of new biologics and emergence of new multibillion dollar molecules. The global sale of monoclonal antibodies in 2006 were $20.6 billion. Remicade had annual sales gain of $1 billion during the past 3 years and five brands had similar increase in 2006. Rituxan with 2006 sales of $4.7 billion was the best selling monoclonal antibody and biological product and the 6th among the top selling medicinal brand. It may be the first biologic and monoclonal antibody to reach $10 billion annual sales in the near future. The strong demand from cancer and arthritis patients has surpassed almost all commercial market research reports and sales forecast. Seven monoclonal antibody brands in 2006 had sales exceeding $1 billion. Humanized or fully human monoclonal antibodies with low immunogenicity, enhanced antigen binding and reduced cellular toxicity provide better clinical efficacy. The higher technical and clinical success rate, overcoming of technical hurdles in large scale manufacturing, low cost of market entry and IND filing, use of fully human and humanized monoclonal antibodies has attracted funds and resources towards R&D. Review of industry research pipeline and sales data during the past 3 years indicate a real paradigm shift in industrial R&D from pharmaceutical to biologics and monoclonal antibodies. The antibody bandwagon has been joined by 200 companies with hundreds of new projects and targets and has attracted billions of dollars in R&D investment, acquisitions and licensing deals leading to the current Monoclonal Antibody Gold Rush.

  20. Antiphospholipid antibody syndrome.

    PubMed

    Kutteh, William H; Hinote, Candace D

    2014-03-01

    Antiphospholipid antibodies (aPLs) are acquired antibodies directed against negatively charged phospholipids. Obstetric antiphospholipid antibody syndrome (APS) is diagnosed in the presence of certain clinical features in conjunction with positive laboratory findings. Obstetric APS is one of the most commonly identified causes of recurrent pregnancy loss. Thus, obstetric APS is distinguished from APS in other organ systems where the most common manifestation is thrombosis. Several pathophysiologic mechanisms of action of aPLs have been described. This article discusses the diagnostic and obstetric challenges of obstetric APS, proposed pathophysiologic mechanisms of APS during pregnancy, and the management of women during and after pregnancy.

  1. 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

  2. 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.

  3. 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

  4. Antibodies Targeting EMT

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0382 TITLE: Antibodies Targeting EMT PRINCIPAL INVESTIGATOR: Brunhilde H Felding CONTRACTING ORGANIZATION: The... CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0382 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Vaughn Smider, Brunhilde Felding 5d. PROJECT NUMBER 5e...14. ABSTRACT Monoclonal antibodies are drugs that can specifically bind targets present on tumor cells. The highly aggressive triple-negative subtype

  5. 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.

  6. [New antibodies in cancer treatment].

    PubMed

    Pestalozzi, B C; Knuth, A

    2004-09-22

    Since the development of hybridoma technology in 1975 monoclonal antibodies with pre-defined specificity can be produced. Only twenty years later did it become possible to make therapeutic use of monoclonal antibodies in oncology. To this end it was necessary to attach the antigen-binding site of a mouse antibody onto the scaffold of a human antibody molecule. Such chimeric or "humanized" antibodies may be used in passive immunotherapy without eliciting an immune response. Rituximab and trastuzumab are such humanized antibodies. They are used today routinely in the treatment of malignant lymphoma and breast cancer, respectively. These antibodies are usually used in combination with conventional cytostatic anticancer drugs.

  7. 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.

  8. 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.

  9. Antibody decoration of neurofilaments

    PubMed Central

    1981-01-01

    We have decorated neurofilaments with antibodies against three polypeptides (designated here as H [mol wt = 195,000], 45[mol wt = 145,000], and 46[mol wt = 73,000]) in an effort to understand the arrangement of these polypeptides within neurofilaments. The three polypeptides were purified and antibodies were raised against each. The cross-reactivity of the antibodies suggested that each polypeptide contains both shared and unique antigenic determinants. The differential reactivities of each antibody preparation were enhanced by adsorption with the two heterologous polypeptides, and the resulting preparations were used to decorate purified neurofilaments, which were then negatively stained and examined in an electron microscope. The appearance of the antibody-decorated structures led to the following conclusions: All three polypeptides are physically associated with the same neurofilament. However, the disposition of H and 46 within a filament is different; 46 antigens appear to be associated with a "central core" of the filament, whereas H antigens compose a structure more loosely and peripherally attached to the central core and periodically arranged along its axis. The antibody-decorated H- containing structure assumes variable configurations; in some cases it appears asa bridge connecting two filaments; in other cases it appears as a helix wrapping the central core with a period of approximately 1,000 A and an apparent unit length of approximately 1.5 periods. These configurations suggest several functional implications, including the possibility that H is a component of the cross-bridges observed between filaments in situ. We also note that the central core-helix relationship could be used in the design of an intracellular transport motor. PMID:6788775

  10. Diagnostic nanoparticle targeting of the EGF-receptor in complex biological conditions using single-domain antibodies

    NASA Astrophysics Data System (ADS)

    Zarschler, K.; Prapainop, K.; Mahon, E.; Rocks, L.; Bramini, M.; Kelly, P. M.; Stephan, H.; Dawson, K. A.

    2014-05-01

    For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including antibodies and fragments thereof, through aptamers and glycans to short peptides and small molecules. Here, we analyse the specific nanoparticle targeting capabilities of two previously suggested peptides (D4 and GE11) and a small camelid single-domain antibody (sdAb), representing potential recognition agents for the epidermal growth factor receptor (EGFR). We investigate specificity by way of receptor RNA silencing techniques and look at increasing complexity in vitro by introducing increasing concentrations of human or bovine serum. Peptides D4 and GE11 proved problematic to employ and conjugation resulted in non-receptor specific uptake into cells. Our results show that sdAb-functionalized particles can effectively target the EGFR, even in more complex bovine and human serum conditions where targeting specificity is largely conserved for increasing serum concentration. In human serum however, an inhibition of overall nanoparticle uptake is observed with increasing protein concentration. For highly affine targeting ligands such as sdAbs, targeting a receptor such as EGFR with low serum competitor abundance, receptor recognition function can still be partially realised in complex conditions. Here, we stress the value of evaluating the targeting efficiency of nanoparticle constructs in realistic biological milieu, prior to more extensive in vivo studies.For effective localization of functionalized nanoparticles at diseased tissues such as solid tumours or metastases through biorecognition, appropriate targeting vectors directed against selected tumour biomarkers are a key prerequisite. The diversity of such vector molecules ranges from proteins, including

  11. Analysis of Heavy-Chain Antibody Responses and Resistance to Parelaphostrongylus tenuis in Experimentally Infected Alpacas

    PubMed Central

    Purdy, S. R.; Gagliardo, L. F.; Lefman, S.; Hamel, P. J. S.; Ku, S.; Mainini, T.; Hoyt, G.; Justus, K.; Daley-Bauer, L. P.; Duffy, M. S.

    2012-01-01

    The parasitic nematode Parelaphostrongylus tenuis is an important cause of neurologic disease of camelids in central and eastern North America. The aim of this study was to determine whether alpacas develop resistance to disease caused by P. tenuis in response to a previous infection or a combination of controlled infection and immunization. Alpacas were immunized with a homogenate of third-stage larvae (L3) and simultaneously implanted subcutaneously with diffusion chambers containing 20 live L3. Sham-treated animals received adjuvant alone and empty chambers. The protocol was not effective in inducing resistance to oral challenge with 10 L3, and disease developed between 60 and 71 days following infection. Immediately following the onset of neurologic disease, affected animals were treated with a regimen of anthelmintic and anti-inflammatory drugs, and all recovered. One year later, a subset of alpacas from this experiment was challenged with 20 L3 and the results showed that prior infection induced resistance to disease. Primary and secondary infections induced production of conventional and heavy-chain IgGs that reacted with soluble antigens in L3 homogenates but did not consistently recognize a recombinant form of a parasite-derived aspartyl protease inhibitor. Thus, the latter antigen may not be a good candidate for serology-based diagnostic tests. Antibody responses to parasite antigens occurred in the absence of overt disease, demonstrating that P. tenuis infection can be subclinical in a host that has been considered to be highly susceptible to disease. The potential for immunoprophylaxis to be effective in preventing disease caused by P. tenuis was supported by evidence of resistance to reinfection. PMID:22593238

  12. Compositions, antibodies, asthma diagnosis methods, and methods for preparing antibodies

    DOEpatents

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

    Methods for preparing an antibody are provided with the method including incorporating 3-bromo-4-hydroxy-benzoic acid into a protein to form an antigen, immunizing a mammalian host with the antigen, and recovering an antibody having an affinity for the antigen from the host. Antibodies having a binding affinity for a monohalotyrosine are provided as well as composition comprising an antibody bound with monohalotyrosine. Compositions comprising a protein having a 3-bromo-4-hydroxy-benzoic acid moiety are also provided. Methods for evaluating the severity of asthma are provide with the methods including analyzing sputum of a patient using an antibody having a binding affinity for monohalotyrosine, and measuring the amount of antibody bound to protein. Methods for determining eosinophil activity in bodily fluid are also provided with the methods including exposing bodily fluid to an antibody having a binding affinity for monohalotyrosine, and measuring the amount of bound antibody to determine the eosinophil activity.

  13. Construction of a biotinylated cameloid-like antibody for lable-free detection of apolipoprotein B-100.

    PubMed

    Li, Henan; Yan, Junrong; Ou, Weijun; Liu, Hong; Liu, Songqin; Wan, Yakun

    2015-02-15

    Nanobodies (Nbs), also known as the variable domain of the heavy-chain-only antibody (VHH), are single-domain antigen-binding fragments derived from heavy-chain antibodies that occur naturally in sera of camelids. Due to their unique properties of small size (15 kD), intrinsic stability, high affinity and specificity, Nbs are suitable for detecting clinical relevant antigens. Apolipoprotein B-100 (ApoB-100) is a highly predictive marker for coronary artery disease (CAD), which is frequently detected in clinical diagnosis. Herein, we successfully obtained anti-ApoB-100 Nbs for the first time and further fabricated a label-free and sensitive immunosensor for ApoB-100 based on isolated anti-ApoB-100 nanobody (Nb) using the electrochemical impedance spectroscopy (EIS) technique. We have generated an immunized phage display library against ApoB-100 and isolated four anti-ApoB-100 Nbs with high affinity and stability. The Nb with the highest affinity was biotinylated based on in vivo BirA system. Further, we developed a label-free electrochemical impedance immunosensor for ApoB-100 using this anti-ApoB-100 Nb. The attachment of ApoB-100 onto the anti-ApoB-100 Nb-immobilized sensing layer led to the increased electron-transfer resistance, which was proportional to ApoB-100 concentration in the range from 0.05 to 5 ng mL(-1) with a detection limit of 0.03 ng mL(-1). This proposed immunosensor revealed high specificity to detect ApoB-100, acceptable intra-assay precision and good stability, functioning as a feasible technique for CAD diagnosis.

  14. 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

  15. 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.

  16. Monoclonal Antibodies against Pectin

    PubMed Central

    Liners, Françoise; Letesson, Jean-Jacques; Didembourg, Christian; Van Cutsem, Pierre

    1989-01-01

    Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model. Images Figure 2 PMID:16667195

  17. Monoclonal antibodies against bacteria.

    PubMed

    Macario, A J; Conway de Macario, E

    1988-01-01

    Highlights are presented of most recent work in which monoclonal antibodies have been instrumental in the study of bacteria and their products. Topics summarized pertain to human and veterinary medicines, dentistry, phytopathology, ichthyology, and bacterial ecophysiology, differentiation, evolution and methanogenic biotechnology.

  18. Reshaping Antibody Diversity

    PubMed Central

    Wang, Feng; Ekiert, Damian C.; Ahmad, Insha; Yu, Wenli; Zhang, Yong; Bazirgan, Omar; Torkamani, Ali; Raudsepp, Terje; Mwangi, Waithaka; Criscitiello, Michael F.; Wilson, Ian A.; Schultz, Peter G.; Smider, Vaughn V.

    2014-01-01

    Summary Unlike humans or mice, some species have limited genome encoded combinatorial diversity potential, yet mount a robust antibody response. Cows are unusual in having exceptionally long CDR H3 loops and few V-regions, but the mechanism for creating diversity is not understood. Deep sequencing revealed that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded mini-domains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a β-strand “stalk” that supports a structurally diverse, disulfide-bonded, “knob” domain. Sequence analysis suggests that diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias towards mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of CDR H3s of unprecedented length that fold into a diversity of mini-domains generated through combinations of somatically generated disulfides. PMID:23746848

  19. Therapeutic antibody engineering

    PubMed Central

    Parren, Paul W.H.I.; Lugovskoy, Alexey A.

    2013-01-01

    It is an important event in any knowledge area when an authority in the field decides that it is time to share all accumulated knowledge and learnings by writing a text book. This does not occur often in the biopharmaceutical industry, likely due to both the highly dynamic environment with tight timelines and policies and procedures at many pharmaceutical companies that hamper knowledge sharing. To take on a task like this successfully, a strong drive combined with a desire and talent to teach, but also an accommodating and stimulating environment is required. Luckily for those interested in therapeutic monoclonal antibodies, Dr. William R. Strohl decided about two years ago that the time was right to write a book about the past, present and future of these fascinating molecules. Dr. Strohl’s great expertise and passion for biotechnology is evident from his life story and his strong academic and industry track record. Dr. Strohl pioneered natural product biotechnology, first in academia as a full professor of microbiology and biochemistry at Ohio State University in Columbus, Ohio and later in industry while at Merck. Despite his notable advances in recombinant natural products, industry interest in this area waned and in 2001 Dr. Strohl sought new opportunities by entering the field of antibody therapeutics. He initiated antibody discovery through phage display at Merck, and then moved to Centocor Research and Development Inc. (now Janssen Biotech, Inc.) in 2008 to head Biologics Research, where he now directs the discovery of innovative therapeutic antibody candidates.

  20. 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)

  1. Anti-smooth muscle antibody

    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 presence ...

  2. Lupus anticoagulants and antiphospholipid antibodies

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/000547.htm Lupus anticoagulants and antiphospholipid antibodies To use the sharing features on this page, please enable JavaScript. Lupus anticoagulants are antibodies against substances in the lining ...

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

    2016-08-05

    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.

  8. 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.

  9. 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

  10. [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.

  11. Antibody therapy for Ebola

    PubMed Central

    Qiu, Xiangguo; Kobinger, Gary P

    2014-01-01

    Ebola viruses can cause severe hemorrhagic fever in humans and nonhuman primates with fatality rates up to 90%, and are identified as biosafety level 4 pathogens and CDC Category A Agents of Bioterrorism. To date, there are no approved therapies and vaccines available to treat these infections. Antibody therapy was estimated to be an effective and powerful treatment strategy against infectious pathogens in the late 19th, early 20th centuries but has fallen short to meet expectations to widely combat infectious diseases. Passive immunization for Ebola virus was successful in 2012, after over 15 years of failed attempts leading to skepticism that the approach would ever be of potential benefit. Currently, monoclonal antibody (mAbs)-based therapies are the most efficient at reversing the progression of a lethal Ebola virus infection in nonhuman primates, which recapitulate the human disease with the highest similarity. Novel combinations of mAbs can even fully cure lethally infected animals after clinical symptoms and circulating virus have been detected, days into the infection. These new developments have reopened the door for using antibody-based therapies for filovirus infections. Furthermore, they are reigniting hope that these strategies will contribute to better control the spread of other infectious agents and provide new tools against infectious diseases. PMID:24503566

  12. Antibodies for immunoassays.

    PubMed

    Newman, D J

    2000-01-01

    What is an immunoassay without an antibody? Clearly the name provides the answer to this question; without antibodies there would be no immunoassays. An immunoassay is an analytical technique, quantitative or qualitative, that relies absolutely on the specificity and affinity of the interaction between epitope and paratope for generation of a detectable response. The actual detection of this binding interaction can be via one of literally hundreds of different signal transduction mechanisms, e.g., fluorimetry, chemiluminescence, agglutination (turbidimetry or nephelometry) enzyme reactions, and so forth (1 -4), but these are simply transducing systems for the primary binding interaction. Antibodies thus provide us with an exquisitely sensitive and specific analytical technology for detecting and quantifying epitopic structures. These structures include amino-acid derivatives, e.g., thyroid hormones, peptides, e.g., vasopressin, proteins, e.g., cytokines, as well as carbohydrate structures, e.g., CA-125. Immunoassay technology has developed to such an extent that it is probably the most versatile analytical tool available able to identify and quantify epitopic structures across the milli- to zeptomolar concentration ranges (2).

  13. Antineutrophil cytoplasmic antibodies.

    PubMed

    Bosch, Xavier; Guilabert, Antonio; Font, Josep

    2006-07-29

    Much like other autoantibodies (eg, anti-double stranded DNA in systemic lupus erythematosus or antiglomerular basement membrane antibodies in Goodpasture's syndrome), antineutrophil cytoplasmic antibodies (ANCA) have provided doctors with a useful serological test to assist in diagnosis of small-vessel vasculitides, including Wegener's granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and their localised forms (eg, pauci-immune necrotising and crescentic glomerulonephritis). 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. Present treatments for ANCA-associated vasculitis are not free from side-effects and as many as 50% of patients relapse within 5 years. Accurate understanding of the key pathogenic points of ANCA-associated vasculitis can undoubtedly provide a more rational therapeutic approach.

  14. Microbials for the production of monoclonal antibodies and antibody fragments

    PubMed Central

    Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

    2014-01-01

    Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. PMID:24183828

  15. [Antiglycolipid antibody in inflammatory neuropathy].

    PubMed

    Kusunoki, S

    1995-12-01

    Antiglycolipid antibody is frequently detected in the acute phase sera from patients with acute inflammatory demyelinating polyneuropathy (Guillain-Barré syndrome, GBS). The titer is highest in the serum sample taken first after the neurological onset, and decreases with clinical improvement. Antiglycolipid antibody may play a role in the pathogenetic mechanism of GBS. GM1 and GD1b are the antigens most commonly recognized. Monoclonal anti-GD1b antibody specifically bound to the paranodal myelin of the peripheral nervous system. Serum anti-GD1b antibody may cause demyelinative neuropathy by binding to the paranodal myelin of the peripheral nervous system. Anti-GQ1b IgG antibody is specifically raised in almost all the sera from Fisher syndrome and GBS with ophthalmoplegia. Anti-GQ1b monoclonal antibody immunostained specifically the paranodal myelin of the extramedullary portion of oculomotor, trochlear and abducens nerves, but no such staining was observed in the other peripheral nerves. Anti-GQ1b antibody may cause conduction block in the cranial nerves innervating the muscles for extraocular movement by binding to the paranodal myelin of those nerves. Anti-GalNAc-GD1a antibody is detected in the patients with GBS with very low or inexcitable compound muscle action potentials. The sera from patients with GBS subsequent to mycoplasma infection had antigalactocerebroside antibody. Further study on antiglycolipid antibody is needed for understanding the pathogenetic mechanism of GBS.

  16. From rabbit antibody repertoires to rabbit monoclonal antibodies

    PubMed Central

    Weber, Justus; Peng, Haiyong; Rader, Christoph

    2017-01-01

    In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies. PMID:28336958

  17. 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.

  18. Understanding antinuclear antibodies.

    PubMed

    Pertusi, R M; Rubin, B R

    1991-06-01

    The American College of Rheumatology (formerly the American Rheumatism Association) diagnostic criteria for connective tissue disorders frequently include positive antinuclear antibody (ANA) assays. Proper interpretation of these tests requires an understanding of the principles governing ANA assays. Assay results are reported in two ways: as titers and as descriptions of fluorescent patterns. A titer is a quantitative measure of ANAs in serum. Different patterns of immunofluorescence are associated with different subsets of collagen vascular disease. Positive results can occur in the absence of connective tissue disease. Accurate diagnosis of connective tissue disorders requires judicious use of ANA assays as well as skillful interpretation of the results.

  19. 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

  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. Monoclonal antibodies for treating cancer

    SciTech Connect

    Dillman, R.O. )

    1989-10-01

    The purpose of this study is to assess the current status of in-vivo use of monoclonal antibodies for treating cancer. Publications appearing between 1980 and 1988 were identified by computer searches using MEDLINE and CANCERLIT, by reviewing the table of contents of recently published journals, and by searching bibliographies of identified books and articles. More than 700 articles, including peer-reviewed articles and book chapters, were identified and selected for analysis. The literature was reviewed and 235 articles were selected as relevant and representative of the current issues and future applications for in-vivo monoclonal antibodies for cancer therapy and of the toxicity and efficacy which has been associated with clinical trials. Approaches include using antibody alone (interacting with complement or effector cells or binding directly with certain cell receptors) and immunoconjugates (antibody coupled to radioisotopes, drugs, toxins, or other biologicals). Most experience has been with murine antibodies. Trials of antibody alone and radiolabeled antibodies have confirmed the feasibility of this approach and the in-vivo trafficking of antibodies to tumor cells. However, tumor cell heterogeneity, lack of cytotoxicity, and the development of human antimouse antibodies have limited clinical efficacy. Although the immunoconjugates are very promising, heterogeneity and the antimouse immune response have hampered this approach as has the additional challenge of chemically or genetically coupling antibody to cytotoxic agents. As a therapeutic modality, monoclonal antibodies are still promising but their general use will be delayed for several years. New approaches using human antibodies and reducing the human antiglobulin response should facilitate treatment. 235 references.

  2. 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.

  3. Serosurvey for polio antibodies.

    PubMed

    Biberi-Moroeanu, S; Munţiu, A; Stoiculescu, S

    1988-01-01

    From 1982 to 1988 a serological survey was performed on 573 subjects aged 3-80 years in order to evaluate the polio immunity level of the population. The presence of neutralizing antibodies was tested using the types 1, 2 and 3 Sabin vaccine strains as well as a wild strain of poliovirus type 1 isolated in France in 1978. According to their birth date, the subjects were assigned to 4 groups. The differences between the groups consisted in the different vaccination history of the subjects as well as in their different opportunities of having been in contact with the past epidemics of poliomyelitis. The results obtained indicate a satisfactory polio immunity level in all the 4 groups: seropositives, 96.7%-98.9% for type 2, 91.8%-98.2% for type 1 (Sabin vaccine strain), 89.3%-96.6% for type 3 and 84.2%-96.4% for type 1 wild strain. The highest immunity levels were found in group D (children with recorded history of complete polio vaccination) and in group A (unvaccinated people but contemporary with the past polio epidemics). A special comment is made with respect to 14 subjects showing satisfactory antibody titres for all the three types of Sabin-vaccine strains but who have proved to be seronegative (less than 4) for the wild type 1 poliovirus strain.

  4. [Antineutrophil cytoplasmic antibodies].

    PubMed

    Sebastiani, G D

    2009-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are predominantly IgG autoantibodies directed against constituents of primary granules of neutrophils and monocytes lysosomes. Although several antigenic targets have been identified, those ANCA directed to proteinase 3 or myeloperoxidase are clinically relevant, whereas the importance of other ANCA remains unknown. Both are strongly associated with small vessel vasculitides, the ANCA-associated vasculitides, which include Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and the localised forms of these diseases (eg, pauci-immune necrotising and crescentic glomerulonephritis). ANCA is a useful serological test to assist in diagnosis of small-vessel vasculitides. 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. There is increasing evidence that myeloperoxidase-ANCA are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-ANCA, markers for Wegener's granulomatosis. With respect to proteinase 3-ANCA, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies.

  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. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Radiolabeled antibodies in cancer. Oncology Overview

    SciTech Connect

    Not Available

    1984-11-01

    Oncology Overviews are a service of the International Cancer Research Data Bank (ICRDB) Program of the National Cancer Institute, intended to facilitate and promote the exchange of information between cancer scientists by keeping them aware of literature related to their research being published by other laboratories through the world. Each Oncology Overview represents a survey of the literature associated with a selected area of cancer research. It contains abstracts of articles which have been selected and organized by researchers associated with the field. Contents: Radiolabeled antibodies--labeling and imaging techniques; Radiolabeled antibodies--carcinoembryonic antigen; Radiolabeled antibodies--alpha-fetoprotein; Radiolabeled antibodies--human chorionic gonadotropin; Radiolabeled antibodies--ferritin; Radiolabeled antibodies--imaging of colorectal tumors; Radiolabeled antibodies--imaging of malignant melanoma; Radiolabeled antibodies--imaging of urogenital tumors; Radiolabeled antibodies--imaging of thyroid tumors; Radiolabeled antibodies--other clinical studies; Radiolabeled antibodies--selected preclinical studies; Radiolabeled antibodies--reviews.

  14. Antibodies to Phospholipids and Liposomes: Binding of Antibodies to Cells

    DTIC Science & Technology

    1987-01-01

    LIPOSOMES: BINDING OF ANTIBODIES TO CELLS 12. PERSONAL AUTHOR(S) W.E. FOGLER , G. M. SWARTZ, AND C.R. ALVING 13a TYPE OF REPORT 13b. TIME COVERED 14. DATE...Elsevier BBA 73693 Antibodies to phospholipids and liposomes: binding of antibodies to cells William E. Fogler *, Glenn M. Swartz, Jr. and Carl R. Alving...Immunol. 21. Research Associateship from the U.S. National 12863-86812Hall. T. and Esser, K. (1984) 3. Immunol. 132. 2059-2063 Research Council. 13 Fogler

  15. Encephalitis and GABAB receptor antibodies

    PubMed Central

    Höftberger, Romana; Titulaer, Maarten J.; Sabater, Lidia; Dome, Balazs; Rózsás, Anita; Hegedus, Balazs; Hoda, Mir Alireza; Laszlo, Viktoria; Ankersmit, Hendrik Jan; Harms, Lutz; Boyero, Sabas; de Felipe, Alicia; Saiz, Albert; Dalmau, Josep

    2013-01-01

    Objective: To report the clinical features of 20 newly diagnosed patients with GABAB receptor (GABABR) antibodies and determine the frequency of associated tumors and concurrent neuronal autoantibodies. Methods: Clinical data were retrospectively obtained and evaluated. Serum and CSF samples were examined for additional antibodies using methods previously reported. Results: Seventeen patients presented with seizures, memory loss, and confusion, compatible with limbic encephalitis (LE), one patient presented with ataxia, one patient presented with status epilepticus, and one patient presented with opsoclonus-myoclonus syndrome (OMS). Nineteen (95%) patients eventually developed LE during the course of the disease. Small-cell lung cancer (SCLC) was identified in 10 (50%) patients, all with LE. Treatment and outcome was available from 19 patients: 15 showed complete (n = 7) or partial (n = 8) neurologic improvement after steroids, IV immunoglobulins, or plasma exchange and oncologic treatment when indicated; 1 patient died of tumor progression shortly after the first cycle of immunotherapy, and 3 were not treated. Five patients with SCLC had additional onconeuronal antibodies (Ri, amphiphysin, or SOX1), and 2 without tumor had GAD65 and NMDAR antibodies, respectively. GABABR antibodies were not detected in serum of 116 patients with SCLC without neurologic symptoms. Conclusion: Our study confirms GABABR as an autoantigen of paraneoplastic and nonparaneoplastic LE and expands the phenotype of GABABR antibodies to ataxia, OMS, and status epilepticus. The long-term prognosis is dictated by the presence of a tumor. Recognition of syndromes associated with GABABR antibodies is important because they usually respond to treatment. PMID:24068784

  16. The making of bispecific antibodies

    PubMed Central

    2017-01-01

    ABSTRACT During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The ‘zoo’ of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigen-binding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term ‘developability’. In addition, different ‘target product profiles’, i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not ‘one best format’ for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided. PMID:28071970

  17. [Soluble elastin and antibody formation].

    PubMed

    Stoklasová, A; Randová, Z; Wimmerová, J; Ledvina, M

    1991-01-01

    In the present study, we have obtained antibodies in rabbits to peptides prepared by digestion of elastin with either oxalic acid or phosphoric acid. By Elisa assay, we have shown the lowest production of antibodies against elastin-derived peptides prepared by digestion of N-elastin with phosphoric acid. We have found a strong cross-reactivity between antibodies obtained to peptides prepared by different digestion of insoluble elastin from bovine ligamentum nuchae or aorta and these antigens. A less pronounced cross-reactivity was observed in case of elastin-derived peptides from porcine aorta.

  18. Pneumococcal Antibody Titers

    PubMed Central

    Abghari, Pamella F.; Poowuttikul, Pavadee; Secord, Elizabeth

    2017-01-01

    Purpose: Immunoglobulin replacement is the mainstay treatment in patients with humoral immunodeficiencies, yet a handful of patients continue to develop sinopulmonary infections while on therapy. The objective of our study was to compare immunoglobulin G (IgG) pneumococcal antibody levels in patients with humoral immune deficiencies who have been on intravenous immunoglobulin (IVIG) replacement for at least 1 year to those on subcutaneous immunoglobulin (SCIG) therapy for at least 1 year. Methods: A retrospective chart review was completed on 28 patients. These patients’ ages ranged between 1 and 61 years. Pneumococcal serotype titers obtained at least 1 year after initiating therapy were compared between patients on IVIG (19 patients) and SCIG (9 patients). Results: A comparison between the groups demonstrated that SCIG achieved a higher percentage of serotype titers protective for noninvasive disease (≥1.3) and 100% protection for invasive disease (≥0.2). Our data also demonstrated a similar lack of protection (less than 50% ≥1.3) in 9N, 12F, and 23F on IVIG and 4, 9N, 12F, and 23F on SCIG. Conclusions: Our data demonstrated that serotypes 1, 3, 4, 9N, 12F, and 23F exhibited the lowest random IgG means while on IVIG, which was comparable to other published studies that looked at the mean IgG levels. In addition, our retrospective chart review demonstrated a greater number of therapeutic pneumococcal titers with SCIG in comparison to IVIG. PMID:28321436

  19. STUDIES ON ANTIBODY PRODUCTION

    PubMed Central

    Sainte-Marie, Guy; Coons, Albert H.

    1964-01-01

    Cells from lymph nodes of rabbits injected repeatedly with bovine serum albumin were transferred subcutaneously to previously irradiated rabbits, and the recipients were immediately injected with bovine serum albumin. A good antibody response resulted. In a series of such animals killed on successive days, skin samples at sites of cell deposition were removed and examined by immunofluorescence and by light microscopy. In these tissues abundant plasmocytes were found to have multiplied and differentiated in a regular progression from immature, to medium, to mature plasmocytes. During the 6 days of the experiment the small plasmocytes accumulated until they reached 85 per cent of the total plasmocytic population. The mitotic index of the large and medium plasmocytes averaged 11 per cent, implying a generation time of 6.3 hours on the basis of a 1 hour mitotic time. This rate of growth is sufficiently rapid to account for all the plasmocytes on the 6th day as deriving from less than 1 per cent of the population initially transferred. This rate and the orderly progression in the evolution of the plasmocytic population, make it highly improbable that plasmocytes arise from transformation of lymphocytes, but rather indicate that they spring from specific precursors already present among the transferred cells. PMID:14157028

  20. 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.

  1. 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.

  2. Reproductive physiology of the male camelid.

    PubMed

    Bravo, P W; Johnson, L W

    1994-07-01

    The physiology of reproduction with emphasis on endocrinology of llamas and alpacas is addressed. Information regarding male anatomy, puberty, testicular function, semen description, and sexual behavior is also included.

  3. Fragmentation of monoclonal antibodies

    PubMed Central

    Vlasak, Josef

    2011-01-01

    Fragmentation is a degradation pathway ubiquitously observed in proteins despite the remarkable stability of peptide bond; proteins differ only by how much and where cleavage occurs. The goal of this review is to summarize reports regarding the non-enzymatic fragmentation of the peptide backbone of monoclonal antibodies (mAbs). The sites in the polypeptide chain susceptible to fragmentation are determined by a multitude of factors. Insights are provided on the intimate chemical mechanisms that can make some bonds prone to cleavage due to the presence of specific side-chains. In addition to primary structure, the secondary, tertiary and quaternary structures have a significant impact in modulating the distribution of cleavage sites by altering local flexibility, accessibility to solvent or bringing in close proximity side chains that are remote in sequence. This review focuses on cleavage sites observed in the constant regions of mAbs, with special emphasis on hinge fragmentation. The mechanisms responsible for backbone cleavage are strongly dependent on pH and can be catalyzed by metals or radicals. The distribution of cleavage sites are different under acidic compared to basic conditions, with fragmentation rates exhibiting a minimum in the pH range 5–6; therefore, the overall fragmentation pattern observed for a mAb is a complex result of structural and solvent conditions. A critical review of the techniques used to monitor fragmentation is also presented; usually a compromise has to be made between a highly sensitive method with good fragment separation and the capability to identify the cleavage site. The effect of fragmentation on the function of a mAb must be evaluated on a case-by-case basis depending on whether cleavage sites are observed in the variable or constant regions, and on the mechanism of action of the molecule. PMID:21487244

  4. Antineutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Radice, A; Sinico, R A

    2005-02-01

    Antineutrophil cytoplasmic antibodies (ANCA) are a sensitive and specific marker for ANCA-associated systemic vasculitis. Using indirect immunofluorescence on ethanol-fixed neutrophils, two major fluoroscopic patterns can be recognised: a diffuse cytoplasmic staining (C-ANCA), and a perinuclear/nuclear staining (P-ANCA). In patients with vasculitis, more of 90% of C-ANCA are directed against proteinase 3 (PR3-ANCA) whereas approximately 80-90% of P-ANCA recognise myelperoxidase (MPO-ANCA). Although C-ANCA (PR3-ANCA) is preferentially associated with Wegener's granulomatosis (WG), and P-ANCA (MPO-ANCA) with microscopic polyangiitis (MPA), idiopathic necrotising crescentic glomerulonephritis (iNCGN) and Churg-Strauss syndrome (CSS), there is not absolute specificity. Between 10-20% of patients with classical WG show P-ANCA (MPO-ANCA), and even a larger percentage of patients with MPA or CSS have C-ANCA (PR3-ANCA). Furthermore, it should be stressed that approximately 10-20% of patients with WG or MPA (and 40-50% of cases of CSS) have negative assay for ANCA. The best diagnostic performance is obtained when indirect immunofluorescence is combined with PR3 and MPO-specific ELISAs. ANCA with different and unknown antigen specificity are found in a variety of conditions other than AASV, including inflammatory bowel diseases, other autoimmune diseases, and infections where their clinical significance is unclear. ANCA levels are useful to monitor disease activity but should not be used by themselves to guide treatment. A significant increase in ANCA titres, or the reappearance of ANCA, should alert the clinicians and lead to a stricter patient control.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. Neutralising Antibodies against Ricin Toxin

    PubMed Central

    Prigent, Julie; Panigai, Laetitia; Lamourette, Patricia; Sauvaire, Didier; Devilliers, Karine; Plaisance, Marc; Volland, Hervé; Créminon, Christophe; Simon, Stéphanie

    2011-01-01

    The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC50 of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 µg) protected mice from intranasal challenges with ricin (5 LD50). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention. PMID:21633505

  10. [The pharmacokinetics of monoclonal antibodies].

    PubMed

    Keizer, R J; Huitema, A D R; Damen, C W N; Schellens, J H M; Beijnen, J H

    2007-03-24

    Monoclonal antibodies (MOABs) are, due to their specificity, increasingly being deployed for therapeutic purposes. MOABs are derived from immunoglobulins and are fully or partially of murine or human origin. They are administered parenterally: mostly intravenously, but subcutaneous or intramuscular administration is also possible, in which case absorption probably occurs through the lymphatic system. The distribution of MOABs from the bloodstream into the tissues is slow and is hampered by the high molecular size of the MOABs, which is a lesser problem for fragments of antibodies (Fab fragments). MOABs are metabolised to peptides and amino acids. This process takes place in many tissues of the body, but probably predominantly in epithelial cells. As a consequence of the saturable binding of the MOAB to its target, a dose-dependent (non-linear) elimination is often observed. Immune reactions can accelerate the elimination of antibodies, partially depending on the degree ofhumanisation of the antibody. Antibodies and endogenous immunoglobulins are protected from elimination by binding to protective receptors (neonatal Fc-receptor; FcRn), which explains their long half-lives (up to 4 weeks). Metabolic pharmacokinetic interactions with other drugs have not been reported and are not expected. It is expected that in the years to come, new MOABs directed towards new targets will appear on the market, as well as existing antibodies with improved pharmacokinetic properties.

  11. 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

  12. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    PubMed

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  13. DTPw-HB and Hib primary and booster vaccination: combined versus separate administration to Latin American children.

    PubMed

    Santos, José Ignacio; Martin, Amando; De Leon, Tirza; Rivera, Luis; Gaitán, Maria Elisa García; Del Rio, Carlos; Oselka, Gabriel; Cervantes, Yolanda; Rubio, Pilar; Clemens, Sue Ann Costa; de Mendonça, João Silva

    2002-03-15

    This multicentre study was designed to establish the reactogenicity and immunogenicity profiles of primary and booster vaccination with diphtheria, tetanus, and pertussis whole-cell-hepatitis B/Haemophilus influenzae type-b (DTPw-HB/Hib) administered as either a syringe mix or as separate injections in 400 Latin American children. Both vaccine regimens were equally well tolerated and elicited post-primary excellent seropositivity rates at or close to 100% for all five component antigens. With regard to HB, 100% of subjects in the combined vaccination group, and 98.8% subjects in the separate injection vaccination group reached seroprotective antibody concentrations (>or=10 mIU/ml) 1 month after the primary vaccination course. Equally high anti-PRP antibody concentrations were reached 1 month after vaccination, with 100% of seroprotected subjects in the combined vaccination group (antibody concentrations >or=0.15 microg/ml), against 99.4% in the separate injection vaccination group. Seroprotective anti-HBs and anti-PRP antibody concentration levels persisted approximately 1 year after the primary vaccination course, just prior to booster vaccination. Finally, a significant increase of all antibody concentrations could be observed after the booster vaccination, since all but one subject in the separate injection vaccination group had protective levels of anti-HBs and anti-PRP antibodies 1 month after the booster dose. These results suggest that the combination of DTPw-HB and Hib vaccines provides an effective means for increasing vaccine coverage in childhood vaccination programmes.

  14. Antibodies to watch in 2016

    PubMed Central

    Reichert, Janice M.

    2016-01-01

    ABSTRACT 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Autologous antibodies that bind neuroblastoma cells.

    PubMed

    Sun, Yujing; Sholler, Giselle S; Shukla, Girja S; Pero, Stephanie C; Carman, Chelsea L; Zhao, Ping; Krag, David N

    2015-11-01

    Antibody therapy of neuroblastoma is promising and our goal is to derive antibodies from patients with neuroblastoma for developing new therapeutic antibodies. The feasibility of using residual bone marrow obtained for clinical indications as a source of tumor cells and a source of antibodies was assessed. From marrow samples, neuroblastoma cells were recovered, grown in cell culture and also implanted into mice to create xenografts. Mononuclear cells from the marrow were used as a source to generate phage display antibody libraries and also hybridomas. Growth of neuroblastoma patient cells was possible both in vitro and as xenografts. Antibodies from the phage libraries and from the monoclonal hybridomas bound autologous neuroblastoma cells with some selectivity. It appears feasible to recover neuroblastoma cells from residual marrow specimens and to generate human antibodies that bind autologous neuroblastoma cells. Expansion of this approach is underway to collect more specimens, optimize methods to generate antibodies, and to evaluate the bioactivity of neuroblastoma-binding antibodies.

  20. 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.

  1. Communication: Antibody stability and behavior on surfaces

    NASA Astrophysics Data System (ADS)

    Bush, Derek B.; Knotts, Thomas A.

    2015-08-01

    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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Antibody profiling sensitivity through increased reporter antibody layering

    DOEpatents

    Apel, William A.; Thompson, Vicki S.

    2017-03-28

    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.

  7. Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

    PubMed Central

    Martínez-Arteaga, Rocio; Ruano-Gallego, David; Fraile, Sofía; Margolles, Yago; Teira, Xema; Gutierrez, Carlos; Bodelón, Gustavo; Fernández, Luis Ángel

    2013-01-01

    Screening of antibody (Ab) libraries by direct display on the surface of E. coli cells is hampered by the presence of the outer membrane (OM). In this work we demonstrate that the native β-domains of EhaA autotransporter and intimin, two proteins from enterohemorrhagic E. coli O157:H7 (EHEC) with opposite topologies in the OM, are effective systems for the display of immune libraries of single domain Abs (sdAbs) from camelids (nanobodies or VHH) on the surface of E. coli K-12 cells and for the selection of high affinity sdAbs using magnetic cell sorting (MACS). We analyzed the capacity of EhaA and intimin β-domains to display individual sdAbs and sdAb libraries obtained after immunization with the extracellular domain of the translocated intimin receptor from EHEC (TirMEHEC). We demonstrated that both systems displayed functional sdAbs on the surface of E. coli cells with little proteolysis and cellular toxicity, although E. coli cells displaying sdAbs with the β-domain of intimin showed higher antigen-binding capacity. Both E. coli display libraries were screened for TirMEHEC binding clones by MACS. High affinity binders were selected by both display systems, although more efficiently with the intimin β-domain. The specificity of the selected clones against TirMEHEC was demonstrated by flow cytometry of E. coli cells, along with ELISA and surface plasmon resonance with purified sdAbs. Finally, we employed the E. coli cell display systems to provide an estimation of the affinity of the selected sdAb by flow cytometry analysis under equilibrium conditions. PMID:24086454

  8. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    PubMed

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

  9. 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.

  10. Polyclonal and monoclonal antibodies in clinic.

    PubMed

    Wootla, Bharath; Denic, Aleksandar; Rodriguez, Moses

    2014-01-01

    Immunoglobulins (Ig) or antibodies are heavy plasma proteins, with sugar chains added to amino-acid residues by N-linked glycosylation and occasionally by O-linked glycosylation. The versatility of antibodies is demonstrated by the various functions that they mediate such as neutralization, agglutination, fixation with activation of complement and activation of effector cells. Naturally occurring antibodies protect the organism against harmful pathogens, viruses and infections. In addition, almost any organic chemical induces antibody production of antibodies that would bind specifically to the chemical. These antibodies are often produced from multiple B cell clones and referred to as polyclonal antibodies. In recent years, scientists have exploited the highly evolved machinery of the immune system to produce structurally and functionally complex molecules such as antibodies from a single B clone, heralding the era of monoclonal antibodies. Most of the antibodies currently in the clinic, target components of the immune system, are not curative and seek to alleviate symptoms rather than cure disease. Our group used a novel strategy to identify reparative human monoclonal antibodies distinct from conventional antibodies. In this chapter, we discuss the therapeutic relevance of both polyclonal and monoclonal antibodies in clinic.

  11. Antibody Engineering for Pursuing a Healthier Future

    PubMed Central

    Saeed, Abdullah F. U. H.; Wang, Rongzhi; Ling, Sumei; Wang, Shihua

    2017-01-01

    Since the development of antibody-production techniques, a number of immunoglobulins have been developed on a large scale using conventional methods. Hybridoma technology opened a new horizon in the production of antibodies against target antigens of infectious pathogens, malignant diseases including autoimmune disorders, and numerous potent toxins. However, these clinical humanized or chimeric murine antibodies have several limitations and complexities. Therefore, to overcome these difficulties, recent advances in genetic engineering techniques and phage display technique have allowed the production of highly specific recombinant antibodies. These engineered antibodies have been constructed in the hunt for novel therapeutic drugs equipped with enhanced immunoprotective abilities, such as engaging immune effector functions, effective development of fusion proteins, efficient tumor and tissue penetration, and high-affinity antibodies directed against conserved targets. Advanced antibody engineering techniques have extensive applications in the fields of immunology, biotechnology, diagnostics, and therapeutic medicines. However, there is limited knowledge regarding dynamic antibody development approaches. Therefore, this review extends beyond our understanding of conventional polyclonal and monoclonal antibodies. Furthermore, recent advances in antibody engineering techniques together with antibody fragments, display technologies, immunomodulation, and broad applications of antibodies are discussed to enhance innovative antibody production in pursuit of a healthier future for humans.

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

    PubMed Central

    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. PMID:26418356

  13. 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.

  14. Rickettsial Antibodies in Multiple Sclerosis

    PubMed Central

    Field, E. J.; Chambers, Mavis

    1970-01-01

    The serum of subjects with multiple sclerosis or other degenerative neurological diseases and that of normal controls does not differ significantly in its content of antibodies to several rickettsial antigens. There is no evidence for a rickettsial aetiology of multiple sclerosis, and no grounds for an extended clinical trial of Le Gac's full method of treatment with antibiotics. PMID:4983591

  15. Antibody Isotype Switching in Vertebrates.

    PubMed

    Senger, Kate; Hackney, Jason; Payandeh, Jian; Zarrin, Ali A

    2015-01-01

    The humoral or antibody-mediated immune response in vertebrates has evolved to respond to diverse antigenic challenges in various anatomical locations. Diversification of the immunoglobulin heavy chain (IgH) constant region via isotype switching allows for remarkable plasticity in the immune response, including versatile tissue distribution, Fc receptor binding, and complement fixation. This enables antibody molecules to exert various biological functions while maintaining antigen-binding specificity. Different immunoglobulin (Ig) classes include IgM, IgD, IgG, IgE, and IgA, which exist as surface-bound and secreted forms. High-affinity autoantibodies are associated with various autoimmune diseases such as lupus and arthritis, while defects in components of isotype switching are associated with infections. A major route of infection used by a large number of pathogens is invasion of mucosal surfaces within the respiratory, digestive, or urinary tract. Most infections of this nature are initially limited by effector mechanisms such as secretory IgA antibodies. Mucosal surfaces have been proposed as a major site for the genesis of adaptive immune responses, not just in fighting infections but also in tolerating commensals and constant dietary antigens. We will discuss the evolution of isotype switching in various species and provide an overview of the function of various isotypes with a focus on IgA, which is universally important in gut homeostasis as well as pathogen clearance. Finally, we will discuss the utility of antibodies as therapeutic modalities.

  16. Serum Antibody Biomarkers for ASD

    DTIC Science & Technology

    2014-10-01

    Systemic immunologic alterations in autistic individuals often have been associated with autoimmunity; in particular, the generation of antibodies...Molloy et al., 2006; Braunschweig et al., 2013). Systemic immunologic alterations in autistic individuals often have been associated with autoimmunity...Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. (2005) Dysregulated innate immune responses in young children with autism spectrum disorders: their

  17. Dengue antibodies in blood donors

    PubMed Central

    Ribas-Silva, Rejane Cristina; Eid, Andressa Ahmad

    2012-01-01

    Background Dengue is an urban arbovirus whose etiologic agent is a virus of the genus Flavorius with four distinct antigen serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that is transmitted to humans through the bite of the mosquito Aedes aegypti. The Campo Mourão region in Brazil is endemic for dengue fever. Obtective The aim of this study was to evaluate the presence of IgG and IgM antibodies specific to the four serotypes of dengue in donors of the blood donor service in the city of Campo Mourão. Methods Epidemiological records were evaluated and 4 mL of peripheral blood from 213 blood donors were collected in tubes without anticoagulant. Serum was then obtained and immunochromatographic tests were undertaken (Imuno-Rápido Dengue IgM/IgGTM). Individuals involved in the study answered a social and epidemiological questionnaire on data which included age, gender and diagnosis of dengue. Results Only three (1.4%) of the 213 blood tests were positive for IgG anti-dengue antibodies. No donors with IgM antibody, which identifies acute infection, were identified. Conclusions The results of the current analysis show that the introduction of quantitative or molecular serological methods to determine the presence of anti-dengue antibodies or the detection of the dengue virus in blood donors in endemic regions should be established so that the quality of blood transfusions is guaranteed. PMID:23049418

  18. 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

  19. Encephalitis and AMPA receptor antibodies

    PubMed Central

    Höftberger, Romana; van Sonderen, Agnes; Leypoldt, Frank; Houghton, David; Geschwind, Michael; Gelfand, Jeffrey; Paredes, Mercedes; Sabater, Lidia; Saiz, Albert; Titulaer, Maarten J.; Graus, Francesc

    2015-01-01

    Objective: We report the clinical features, comorbidities, and outcome of 22 newly identified patients with antibodies to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Methods: This was a retrospective review of patients diagnosed between May 2009 and March 2014. Immunologic techniques have been reported previously. Results: Patients' median age was 62 years (range 23–81; 14 female). Four syndromes were identified: 12 (55%) patients presented with distinctive limbic encephalitis (LE), 8 (36%) with limbic dysfunction along with multifocal/diffuse encephalopathy, one with LE preceded by motor deficits, and one with psychosis with bipolar features. Fourteen patients (64%) had a tumor demonstrated pathologically (5 lung, 4 thymoma, 2 breast, 2 ovarian teratoma) or radiologically (1 lung). Additional antibodies occurred in 7 patients (3 onconeuronal, 1 tumor-related, 2 cell surface, and 1 tumor-related and cell surface), all with neurologic symptoms or tumor reflecting the concurrent autoimmunity. Treatment and outcome were available from 21 patients (median follow-up 72 weeks, range 5–266): 5 had good response to immunotherapy and tumor therapy, 10 partial response, and 6 did not improve. Eventually 5 patients died; all had a tumor or additional paraneoplastic symptoms related to onconeuronal antibodies. Coexistence of onconeuronal antibodies predicted a poor outcome (p = 0.009). Conclusion: Anti-AMPAR encephalitis usually manifests as LE, can present with other symptoms or psychosis, and is paraneoplastic in 64% of cases. Complete and impressive neurologic improvement can occur, but most patients have partial recovery. Screening for a tumor and onconeuronal antibodies is important because their detection influences outcome. PMID:25979696

  20. How Is Antiphospholipid Antibody Syndrome Treated?

    MedlinePlus

    ... Is Antiphospholipid Antibody Syndrome Treated? Antiphospholipid antibody syndrome (APS) has no cure. However, medicines can help prevent ... existing clots from getting larger. You may have APS and another autoimmune disorder, such as lupus . If ...

  1. 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.

  2. Antibody reactivity with Skinner HSV vaccine.

    PubMed

    Muniu, E M; Durham, J; Shariff, D; Hartley, C E; Fuller, A; Melling, J; Wiblin, C; Wilkins, G; Buchan, A; Skinner, G R

    1987-01-01

    Antibody reactivity against herpes simplex virus (HSV) was investigated in 15 subjects who received three subcutaneous immunisations with Skinner HSV vaccine. Humoral antibody responses were detected against type 1 HSV in every subject and against type 2 HSV in all but one subject; immuno-precipitating antibody responses were infrequently detected. There was no antibody reactivity against host-cell (MRC-5), foetal calf serum or rubella virus antigen. None of the vaccinated subjects developed clinical evidence of herpes genitalis.

  3. Antidisialosyl antibodies in chronic idiopathic ataxic neuropathy.

    PubMed

    Serrano-Munuera, C; Rojas-García, R; Gallardo, E; De Luna, N; Buenaventura, I; Ferrero, M; García, T; García-Merino, J A; González-Rodríguez, C; Guerriero, A; Marco, M; Márquez, C; Grau, J M; Graus, F; Illa, I

    2002-11-01

    Antidisialosyl antibodies were found in two out of 13 patients with chronic idiopathic ataxic neuropathy (CIAN) and not in 32 patients with different sensory neuropathies of known cause. This finding confirms the association of antidisialosyl antibodies and CIAN regardless of the absence of the M band. These antibodies may have pathogenic relevance; however, larger series are needed to establish their clinical significance.

  4. 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.

  5. 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.

  6. 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

  7. Recombinant antibodies and their use in biosensors.

    PubMed

    Zeng, Xiangqun; Shen, Zhihong; Mernaugh, Ray

    2012-04-01

    Inexpensive, noninvasive immunoassays can be used to quickly detect disease in humans. Immunoassay sensitivity and specificity are decidedly dependent upon high-affinity, antigen-specific antibodies. Antibodies are produced biologically. As such, antibody quality and suitability for use in immunoassays cannot be readily determined or controlled by human intervention. However, the process through which high-quality antibodies can be obtained has been shortened and streamlined by use of genetic engineering and recombinant antibody techniques. Antibodies that traditionally take several months or more to produce when animals are used can now be developed in a few weeks as recombinant antibodies produced in bacteria, yeast, or other cell types. Typically most immunoassays use two or more antibodies or antibody fragments to detect antigens that are indicators of disease. However, a label-free biosensor, for example, a quartz-crystal microbalance (QCM) needs one antibody only. As such, the cost and time needed to design and develop an immunoassay can be substantially reduced if recombinant antibodies and biosensors are used rather than traditional antibody and assay (e.g. enzyme-linked immunosorbant assay, ELISA) methods. Unlike traditional antibodies, recombinant antibodies can be genetically engineered to self-assemble on biosensor surfaces, at high density, and correctly oriented to enhance antigen-binding activity and to increase assay sensitivity, specificity, and stability. Additionally, biosensor surface chemistry and physical and electronic properties can be modified to further increase immunoassay performance above and beyond that obtained by use of traditional methods. This review describes some of the techniques investigators have used to develop highly specific and sensitive, recombinant antibody-based biosensors for detection of antigens in simple or complex biological samples.

  8. Expression studies of catalytic antibodies

    SciTech Connect

    Ulrich, H.D.; Patten, P.A.; Yang, P.L.

    1995-12-05

    We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived form a number of catalytic antibodies. Expression yields of eight hybridoma-and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high-density fermentation. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies. 41 refs., 4 figs., 1 tab.

  9. 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

  10. Phenotypic screening: the future of antibody discovery.

    PubMed

    Gonzalez-Munoz, Andrea L; Minter, Ralph R; Rust, Steven J

    2016-01-01

    Most antibody therapeutics have been isolated from high throughput target-based screening. However, as the number of validated targets diminishes and the target space becomes increasingly competitive, alternative strategies, such as phenotypic screening, are gaining momentum. Here, we review successful phenotypic screens, including those used to isolate antibodies against cancer and infectious agents. We also consider exciting advances in the expression and phenotypic screening of antibody repertoires in single cell autocrine systems. As technologies continue to develop, we believe that antibody phenotypic screening will increase further in popularity and has the potential to provide the next generation of therapeutic antibodies.

  11. Serum Antibody Biomarkers for ASD

    DTIC Science & Technology

    2015-10-01

    system dysfunction has been reported in ASD in many studies. Systemic immunologic alterations in autistic individuals often have been associated with... immunologic alterations in autistic individuals often have been associated with autoimmunity; in particular, the generation of antibodies reactive...2013), Fetuin-A in the developing brain. Devel Neurobio, 73: 354–369. Jyonouchi H, Geng L, Ruby A, Zimmerman-Bier B. (2005) Dysregulated innate

  12. 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.

  13. 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

  14. 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.

  15. Broadly Neutralizing Antibodies for HIV Eradication.

    PubMed

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  16. Solid-phase antibody capture hemadsorption assay for detection of hepatitis A virus immunoglobulin M antibodies.

    PubMed Central

    Summers, P L; Dubois, D R; Cohen, W H; Macarthy, P O; Binn, L N; Sjogren, M H; Snitbhan, R; Innis, B L; Eckels, K H

    1993-01-01

    A solid-phase antibody capture hemadsorption (SPACH) assay was developed to detect hepatitis A virus (HAV)-specific immunoglobulin M (IgM) antibodies in sera from humans recently infected with hepatitis. The assay is performed with microtiter plates coated with anti-human IgM antibodies to capture IgM antibodies from the test sera. HAV-specific IgM antibody is detected by the addition of HAV hemagglutinating antigen and goose erythrocytes. Hemadsorption of erythrocytes to antigen-antibody complexes attached to the solid phase indicate the presence of IgM antibodies. The SPACH assay was compared to a commercial radioimmunoassay and was found to be equally or more sensitive and specific for the detection of HAV IgM antibodies. The SPACH assay is an alternative, rapid assay that doesn't require hazardous substrates or radioactivity for the detection of HAV-specific antibodies. PMID:8388890

  17. 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

  18. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library

    PubMed Central

    Huie, Michael A.; Cheung, Mei-Chi; Muench, Marcus O.; Becerril, Baltazar; Kan, Yuet W.; Marks, James D.

    2001-01-01

    The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia. PMID:11226299

  19. 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.

  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. A simpler sort of antibody.

    PubMed Central

    Masat, L; Wabl, M; Johnson, J P

    1994-01-01

    The monoclonal antibody NEMO is directed against a molecule expressed by human cells of the melanocytic lineage. Although obtained by conventional immunization and fusion procedures, NEMO consists solely of kappa light chain. SDS/PAGE analysis indicates that the kappa chains are present as both monomers and dimers. When these two forms were separated by gel filtration, only the monomeric form bound antigen. As kappa light chains from the myeloma MOPC-41 and the hybridoma MORK do not bind to the melanocytic cells, we conclude that the binding specificity of NEMO resides in the variable region. Images PMID:8302862

  2. Selection, design, and engineering of therapeutic antibodies.

    PubMed

    Presta, Leonard G

    2005-10-01

    mAbs account for an increasing portion of marketed human biological therapeutics. As a consequence, the importance of optimal selection, design, and engineering of these not only has expanded in the past 2 decades but also is now coming into play as a competitive factor. This review delineates the 4 basic areas for optimal therapeutic antibody selection and provides examples of the increasing number of considerations necessary for, and options available for, antibody design. Though some of the advances in antibody technology (eg, antibodies derived from phage-display libraries) have already made it to market, other more recent advances, such as engineering antibodies for enhanced effector functions, may not be far behind, especially given the increasing competition for therapeutic antibodies to the same target (eg, anti-CD20 and anti-TNF-alpha).

  3. 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

  4. 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.

  5. 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.

  6. [Evolution of monoclonal antibodies in cancer treatment].

    PubMed

    Kubczak, Małgorzata; Rogalińska, Małgorzata

    2016-01-01

    Since late 90s of last century the new age of directed therapy began using mainly biological constructs produced in rodents called monoclonal antibodies. The side effects of monoclonal antibodies were a challenge for pharmaceutical companies to improve the biological properties of these biological drugs. The humanization of monoclonal constructs was an idea to improve monoclonal antibodies next generation activity cancer cell reduction in humans. Moreover for some other patients sensitive for monoclonal antibodies therapy could also potentially induce immunological differences that might imply on human health. The new idea related to monoclonal antibodies was to design a small molecule constructs of nanoantibodies with ability to enter into cells. Such small molecules could find their targets inside human cells, even in nuclei leading to differences in cancer cells expression. The existing knowledge on monoclonal antibodies as well as directed activity of nanoantibodies could improve anticancer treatment efficancy of diseases.

  7. Factors determining antibody distribution in tumors.

    PubMed

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

    2008-02-01

    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.

  8. Antibody to dihydropyridine calcium entry blockers

    SciTech Connect

    Thayer, S.; Minaskanian, G.; Fairhurst, A.

    1986-03-05

    Antibodies that recognize dihydropyridine calcium entry blockers were elicited from rabbits. A sensitive and specific radioimmunoassay for dihydropyridines was developed and its specificity compared to the DHP binding site in skeletal muscle membranes. The antibody bound (/sup 3/H)nitrendipine with a higher affinity (K/sub D/ = 0.155 nM) than did the DHP receptor of skeletal muscle (K/sub D/ = 1-3 nM). However, in contrast to the DHP receptor, the antibody recognized only those DHP drugs with meta-nitrophenyl substituents at the 4-position on the DHP ring, and thus reflected the meta position of the nitro group on the DHP hapten used as an antigen. Both the antibody and the receptor exhibited stereospecificity, with each site recognizing the (+) isomer of nicardipine as the more potent. This antibody should prove useful in the studies of some potentially irreversible DHP molecules and for use in the production of anti-idotype antibodies.

  9. [Improved IgG Antibody Diagnostics of Feather Duvet Lung by an Antibody Screening Test].

    PubMed

    Sennekamp, J; Lehmann, E

    2015-11-01

    The underdiagnosed feather duvet lung, an extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by duck and goose feathers, can be more frequently diagnosed, if duck and goose feather antibodies are included in the panel of the routinely applied IgG antibody screening test. This does not necessarily require extending the screening test to include duck and goose feather antigens. By analysing 100 sera with duck and goose antibodies we found that the commonly used pigeon and budgerigar antibodies can also screen for feather duvet antibodies. All examined sera lacking pigeon and budgerigar antibodies also lacked clear-cut duck and goose feather antibodies. The examined sera with strong pigeon or budgerigar antibodies always also contained feather duvet antibodies. However, sera with medium or low concentrated pigeon or budgerigar antibodies are not always associated with feather duvet antibodies. In the light of these observations, we find that 71% of the duck and goose antibody analyses would be dispensable without essential loss of quality, if the results of screening for pigeon and budgerigar antibodies were incorporated into the procedure of a step-by- step diagnostics.

  10. 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.

  11. Monoclonal Antibodies for Lipid Management.

    PubMed

    Feinstein, Matthew J; Lloyd-Jones, Donald M

    2016-07-01

    In recent years, biochemical and genetic studies have identified proprotein convertase subtilisin/kexin type 9 (PCSK9) as a major mediator of low-density lipoprotein cholesterol (LDL-c) levels and thereby a potential novel target for reducing risk of coronary heart disease (CHD). These observations led to the development of PCSK9 inhibitors, which lower LDL-c levels more than any other non-invasive lipid-lowering therapy presently available. The PCSK9 inhibitors furthest along in clinical trials are subcutaneously injected monoclonal antibodies. These PCSK9 inhibitors have demonstrated LDL-c-lowering efficacy with acceptable safety in phase III clinical trials and may offer a useful therapy in addition to maximally tolerated HMG-CoA reductase inhibitors (statins) in certain patient groups. Longer-term data are required to ensure sustained efficacy and safety of this new class of medications. This review provides an overview of the biology, genetics, development, and clinical trials of monoclonal antibodies designed to inhibit PCSK9.

  12. 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.

  13. STUDIES ON THE CONTROL OF ANTIBODY SYNTHESIS

    PubMed Central

    Brody, Neil I.; Walker, J. Geoffrey; Siskind, Gregory W.

    1967-01-01

    In the system studied, antigenic competition between two haptenic determinants was found to be of the same extent whether the haptens were on the same or on separate carrier molecules. Suppression of antibody formation to one determinant by administration of passive antibody partially eliminated the depressive effects of antigenic competition when the two haptens were located on separate carrier molecules but had no effect on antibody production to the second hapten when the two determinants were present on the same molecule. The results are discussed in terms of the mechanisms of suppression, antigenic competition, and control of antibody formation. PMID:4165502

  14. Monoclonal Antibodies against the Drosophila Nervous System

    NASA Astrophysics Data System (ADS)

    Fujita, Shinobu C.; Zipursky, Stephen L.; Benzer, Seymour; Ferrus, Alberto; Shotwell, Sandra L.

    1982-12-01

    A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates of Drosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.

  15. Bioconjugation of Antibodies to Horseradish Peroxidase (HRP).

    PubMed

    Hnasko, Robert M

    2015-01-01

    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 convenient means to maintain antibody affinity while imparting a functional reporter used for antigen detection. In this chapter, we describe a process by which Sulfo-SMCC is used to generate a stable maleimide-activated HRP that is reactive with sulfhydryl groups generated in antibodies by SATA-mediated thiolation.

  16. 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.

  17. 6th Annual European Antibody Congress 2010

    PubMed Central

    2011-01-01

    The 6th European Antibody Congress (EAC), organized by Terrapinn Ltd., was held in Geneva, Switzerland, which was also the location of the 4th and 5th EAC.1,2 As was the case in 2008 and 2009, the EAC was again the largest antibody congress held in Europe, drawing nearly 250 delegates in 2010. Numerous pharmaceutical and biopharmaceutical companies active in the field of therapeutic antibody development were represented, as were start-up and academic organizations and representatives from the US Food and Drug Administration (FDA). The global trends in antibody research and development were discussed, including success stories of recent marketing authorizations of golimumab (Simponi®) and canakinumab (Ilaris®) by Johnson & Johnson and Novartis, respectively, updates on antibodies in late clinical development (obinutuzumab/GA101, farletuzumab/MORAb-003 and itolizumab/T1 h, by Glycart/Roche, Morphotek and Biocon, respectively) and success rates for this fast-expanding class of therapeutics (Tufts Center for the Study of Drug Development). Case studies covering clinical progress of girentuximab (Wilex), evaluation of panobacumab (Kenta Biotech), characterization of therapeutic antibody candidates by protein microarrays (Protagen), antibody-drug conjugates (sanofi-aventis, ImmunoGen, Seattle Genetics, Wyeth/Pfizer), radio-immunoconjugates (Bayer Schering Pharma, Université de Nantes) and new scaffolds (Ablynx, AdAlta, Domantis/GlaxoSmithKline, Fresenius, Molecular Partners, Pieris, Scil Proteins, Pfizer, University of Zurich) were presented. Major antibody structural improvements were showcased, including the latest selection engineering of the best isotypes (Abbott, Pfizer, Pierre Fabre), hinge domain (Pierre Fabre), dual antibodies (Abbott), IgG-like bispecific antibodies (Biogen Idec), antibody epitope mapping case studies (Eli Lilly), insights in FcγRII receptor (University of Cambridge), as well as novel tools for antibody fragmentation (Genovis). Improvements

  18. 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.

  19. 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.

  20. 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.

  1. Reconciling the structural attributes of avian antibodies.

    PubMed

    Conroy, Paul J; Law, Ruby H P; Gilgunn, Sarah; Hearty, Stephen; Caradoc-Davies, Tom T; Lloyd, Gordon; O'Kennedy, Richard J; Whisstock, James C

    2014-05-30

    Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.

  2. 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....

  3. Not All Antibodies Are Created Equal: Factors That Influence Antibody Mediated Rejection

    PubMed Central

    Butler, Carrie L.; Valenzuela, Nicole M.; Thomas, Kimberly A.

    2017-01-01

    Consistent with Dr. Paul Terasaki's “humoral theory of rejection” numerous studies have shown that HLA antibodies can cause acute and chronic antibody mediated rejection (AMR) and decreased graft survival. New evidence also supports a role for antibodies to non-HLA antigens in AMR and allograft injury. Despite the remarkable efforts by leaders in the field who pioneered single antigen bead technology for detection of donor specific antibodies, a considerable amount of work is still needed to better define the antibody attributes that are associated with AMR pathology. This review highlights what is currently known about the clinical context of pre and posttransplant antibodies, antibody characteristics that influence AMR, and the paths after donor specific antibody production (no rejection, subclinical rejection, and clinical dysfunction with AMR). PMID:28373996

  4. Lack of in Vivo Antibody Dependent Cellular Cytotoxicity with Antibody Containing Gold Nanoparticles

    PubMed Central

    2016-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is a cytolytic mechanism that can elicit in vivo antitumor effects and can play a significant role in the efficacy of antibody treatments for cancer. Here, we prepared cetuximab, panitumumab, and rituximab containing gold nanoparticles and investigated their ability to produce an ADCC effect in vivo. Cetuximab treatment of EGFR-expressing H1975 tumor xenografts showed significant tumor regression due to the ADCC activity of the antibody in vivo, while the control antibody, panitumumab, did not. However, all three antibody containing nanoparticles are not able to suppress tumor growth in the same in vivo mouse model. The antibody containing nanoparticles localized in the tumors and did not suppress the immune function of the animals, so the lack of tumor growth suppression of the cetuximab containing nanoparticle suggests that immobilizing antibodies onto a nanoparticle significantly decreases the ability of the antibody to promote an ADCC response. PMID:25879583

  5. Antibody i-Patch prediction of the antibody binding site improves rigid local antibody-antigen docking.

    PubMed

    Krawczyk, Konrad; Baker, Terry; Shi, Jiye; Deane, Charlotte M

    2013-10-01

    Antibodies are a class of proteins indispensable for the vertebrate immune system. The general architecture of all antibodies is very similar, but they contain a hypervariable region which allows millions of antibody variants to exist, each of which can bind to different molecules. This binding malleability means that antibodies are an increasingly important category of biopharmaceuticals and biomarkers. We present Antibody i-Patch, a method that annotates the most likely antibody residues to be in contact with the antigen. We show that our predictions correlate with energetic importance and thus we argue that they may be useful in guiding mutations in the artificial affinity maturation process. Using our predictions as constraints for a rigid-body docking algorithm, we are able to obtain high-quality results in minutes. Our annotation method and re-scoring system for docking achieve their predictive power by using antibody-specific statistics. Antibody i-Patch is available from http://www.stats.ox.ac.uk/research/proteins/resources.

  6. Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies.

    PubMed Central

    Collins, A M; Roberton, D M; Hosking, C S; Flannery, G R

    1991-01-01

    The humoral and mucosal immune responses to oral immunization with xenogeneic antibodies were studied using an animal model in which female rabbits were fed daily doses of the MOPC-315 murine IgA antibody, and were mated during the course of the feeding programme. Serum and colostrum samples were assayed for the presence of anti-idiotypic antibodies by ELISA assay, before and after depletion of anti-IgA antibodies, by affinity chromatography using another murine IgA idiotype. It was shown that all animals responded to exposure to the MOPC-315 idiotype with the production of serum anti-murine immunoglobulin antibodies and that four of six animals produced serum anti-idiotypic antibodies. That the immune response included antibodies directed against the antigen-binding site was confirmed by competition ELISA assay. Mucosal IgG and IgA anti-immunoglobulin antibodies were present in milk from all antibody-fed rabbits tested, and IgA anti-idiotypic antibodies were detectable in the colostrum of one rabbit. The results provide some support for the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine milk immunoglobulins, may provoke the production of anti-idiotypic antibodies, and that such exposure may lead to disturbances of immune regulation. PMID:1916890

  7. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. Maternal antibodies provide early protection from disease, but may interfere with the vaccination efficacy in the chick. MAb are thought to interfere wit...

  8. Passive antibody transfer in chickens to model maternal antibody after avian influenza vaccination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Birds transfer maternal antibodies (MAb) to their offspring through the egg yolk where the antibody is absorbed and enters the circulatory system. These maternal antibodies, depending on the pathogen, can provide early protection from some diseases, but it may also interfere with the vaccination re...

  9. Antibodies to watch in 2017

    PubMed Central

    Reichert, Janice M.

    2017-01-01

    ABSTRACT Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6–9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other ‘antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these

  10. Antibodies to watch in 2017.

    PubMed

    Reichert, Janice M

    Over 50 investigational monoclonal antibody (mAb) therapeutics are currently undergoing evaluation in late-stage clinical studies, which is expected to drive a trend toward first marketing approvals of at least 6-9 mAbs per year in the near-term. In the United States (US), a total of 6 and 9 mAbs were granted first approvals during 2014 and 2015, respectively; all these products are also approved in the European Union (EU). As of December 1, 2016, 6 mAbs (atezolizumab, olaratumab, reslizumab, ixekizumab, bezlotoxumab, oblitoxaximab) had been granted first approvals during 2016 in either the EU or US. Brodalumab, was granted a first approval in Japan in July 2016. Regulatory actions on marketing applications for brodalumab in the EU and US are not expected until 2017. In 2017, first EU or US approvals may also be granted for at least nine mAbs (ocrelizumab, avelumab, Xilonix, inotuzumab ozogamicin, dupilumab, sirukumab, sarilumab, guselkumab, romosozumab) that are not yet approved in any country. Based on announcements of company plans for regulatory submissions and the estimated completion dates for late-stage clinical studies, and assuming the study results are positive, marketing applications for at least 6 antibody therapeutics (benralizumab, tildrakizumab, emicizumab, galcanezumab, ibalizumab, PRO-140) that are now being evaluated in late-stage clinical studies may be submitted during December 2016* or 2017. Other 'antibodies to watch' in 2017 include 20 mAbs are undergoing evaluation in pivotal studies that have estimated primary completion dates in late 2016 or during 2017. Of these, 5 mAbs are for cancer (durvalumab, JNJ-56022473, ublituximab, anetumab ravtansine, glembatumumab vedotin) and 15 mAbs are for non-cancer indications (caplacizumab, lanadelumab, roledumab, tralokinumab, risankizumab, SA237, emapalumab, suptavumab, erenumab, eptinezumab, fremanezumab, fasinumab, tanezumab, lampalizumab, brolucizumab). Positive results from these studies may

  11. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  12. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  13. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  14. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  15. 21 CFR 866.3290 - Gonococcal antibody test (GAT).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Gonococcal antibody test (GAT). 866.3290 Section... antibody test (GAT). (a) Identification. A gonococcal antibody test (GAT) is an in vitro device that..., indirect fluorescent antibody, or radioimmunoassay, antibodies to Neisseria gonorrhoeae in sera...

  16. Antibody humanization methods for development of therapeutic applications.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Feizi, Mohammad Ali Hosseinpour; Nejad, Ramezan Ali Khavari

    2014-04-01

    Recombinant antibody technologies are rapidly becoming available and showing considerable clinical success. However, the immunogenicity of murine-derived monoclonal antibodies is restrictive in cancer immunotherapy. Humanized antibodies can overcome these problems and are considered to be a promising alternative therapeutic agent. There are several approaches for antibody humanization. In this article we review various methods used in the antibody humanization process.

  17. 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

  18. Testing for antineutrophil cytoplasmic antibodies.

    PubMed

    Savige, J

    2001-09-01

    The most common reason to request a test for antineutrophil cytoplasmic antibodies (ANCA) is to diagnose Wegener's granulomatosis and microscopic polyangiitis and to monitor inflammatory activity in these diseases. Several retrospective and prospective studies have suggested that the demonstration of ANCA lacks sensitivity and specificity, but these series have detected ANCA with neutrophil-indirect immunofluorescence alone, have used a disease classification that did not describe microscopic polyangiitis and have included patients with inactive disease. The 'International Consensus Statement on Testing and Reporting ANCA' has been developed to optimize the clinical relevance of ANCA testing by the adoption of standardized testing and reporting procedures. International collaborative efforts continue to focus on improving the tests for ANCA.

  19. Clinical considerations for biosimilar antibodies

    PubMed Central

    Mellstedt, Håkan

    2013-01-01

    Biosimilar agents are approximate copies of branded biologic therapies. Since the first biosimilar was authorized in the European Union in 2006, fifteen additional agents have been approved by the European Medicines Agency, including two biosimilar monoclonal antibodies (mAbs). Biosimilar mAbs represent a distinct class given their large molecular size, complex protein structure, and post-translational modifications. While guidelines have been established for the development, approval, and use of biosimilars, further scrutiny and discussion is necessary to fully understand their potential impact on clinical outcomes. This review takes a critical look at the structural complexity of biosimilar mABs, the feasibility of indication extrapolation, the impact of product variability on immunogenicity, the importance of comprehensive pharmacovigilance, and the potential for ongoing pharmacoeconomic impact. PMID:26217160

  20. The nature of antiphospholipid antibodies.

    PubMed

    Rauch, J; Janoff, A S

    1992-11-01

    Despite the striking clinical manifestations associated with antiphospholipid antibodies (aPL) the role of these autoantibodies in disease and the nature of their true "inducing" and "target" antigens remain elusive. To address these issues, we investigated the immunogenic potential of phospholipid structures. To date, phospholipid immunogens have included hexagonal (II) forms of phosphatidylethanolamine and mixtures of apolipoprotein H (beta 2-glycoprotein I) with cardiolipin. Both hexagonal (II) phosphatidylethanolamine and the cardiolipin/apolipoprotein H mixture were capable of inducing aPL with lupus anticoagulant activity. Bilayer phosphatidylethanolamine and cardiolipin in the absence of apolipoprotein H were nonimmunogenic. Our data support our views that specific phospholipid structures are recognized by the immune system and that such structures serve as inducing and/or target antigens in the pathogenesis of aPL in vivo.

  1. Antinuclear Antibody Negative Systemic Sclerosis

    PubMed Central

    Salazar, GA; Assassi, S; Wigley, F; Hummers, L; Varga, J; Hinchcliff, M; Khanna, D; Schiopu, E; Phillips, K; Furst, DE; Steen, V; Baron, M; Hudson, M; Taillefer, SS; Pope, J; Jones, N; Docherty, P; Khalidi, NA; Robinson, D; Simms, R; Silver, R; Frech, TM; Fessler, B; Molitor, J; Fritzler, M; Segal, B; Al-Kassab, F; Perry, M; Yang, J; Zamanian, S; Reveille, JD; Arnett, FC; Pedroza, C; Mayes, MD

    2014-01-01

    Objective To examine the demographic and clinical characteristics of systemic sclerosis (SSc) patients without antinuclear antibodies (ANA) compared to ANA positive patients. Methods SSc patients enrolled in the Scleroderma Family Registry and DNA Repository were included. Relevant demographic and clinical data were entered by participating sites or obtained by chart review. ANA and SSc related antibodies were determined in all investigated patients using commercially available kits at our laboratories. Results This study included 3249 patients, of whom 208 (6.4%) were ANA negative. The proportion of male patients was higher in the ANA negative group (OR 1.65; p=0.008). ANA negative patients experienced less vasculopathic manifestations of SSc. The percent predicted diffusing capacity of carbon monoxide (DLco) was higher in ANA negative patients (p=0.03). Pulmonary arterial hypertension (PAH) per right heart catheterization was less common in the ANA negative group (OR= 0.28; p=0.03). Furthermore, patients with negative ANA had a lower prevalence of telangiectasias and digital ulcers/pits (OR= 0.59; p=0.03 and OR=0.38; p=0.01, respectively). Although diffuse cutaneous involvement was more common, the modified Rodnan Skin Score (mRSS) was lower in the ANA negative group (2.4 points lower, p=0.05). Furthermore, they experienced more malabsorption (p=0.05). There was no difference in the frequency of pulmonary fibrosis or scleroderma renal crisis. All-cause mortality was not different between the two groups (p=0.28). Conclusions In conclusion, the results of this study suggest that SSc patients who are ANA negative constitute a distinct subset of SSc with less vasculopathy (less PAH, digital ulcers and fewer telangiectasias), a greater proportion of males and possibly, more frequent lower gastrointestinal involvement. PMID:25578738

  2. [Radiolabeled antibodies for cancer treatment].

    PubMed

    Barbet, Jacques; Chatal, Jean-François; Kraeber-Bodéré, Françoise

    2009-12-01

    The first treatment ever by radio-immunotherapy (RIT) was performed by William H. Beierwaltes in 1951 and was a success. Fifty years later, the main question is to find ways of extending the success of radiolabelled anti-CD20 antibodies in indolent non-Hodgkin's lymphoma to other forms of cancer. Solid tumours are much more radioresistant than lymphomas, but they respond to RIT if the lesions are small. Clinical situations of residual or minimal disease are thus the most likely to benefit from RIT in the adjuvant or consolidation settings. For disseminated disease, like leukemias or myelomas, the problem is different: beta- particles emitted by the radioactive atoms classically used for cancer treatment (iodine-131 or yttrium-90) disperse their energy in large volumes (ranges 1 mm to 1 cm) and are not very effective against isolated cells. Advances in RIT progress in two directions. One is the development of pretargeting strategies in which the antibody is not labelled but used to provide binding sites to small molecular weight radioactivity vectors (biotin, haptens). These techniques have been shown to increase tumour to non-target uptake ratios and anti-tumour efficacy has been demonstrated in the clinic. The other approach is the use of radionuclides adapted to the various clinical situations. Lutetium-177 or copper-67, because of the lower energy of their emission, their relatively long half-life and good gamma emission, may significantly improve RIT efficacy and acceptability. Beyond that, radionuclides emitting particles such as alpha particles or Auger electrons, much more efficient to kill isolated tumour cells, are being tested for RIT in the clinic. Finally, RIT should be integrated with other cancer treatment approaches in multimodality protocols. Thus RIT, now a mature technology, should enter a phase of well designed and focused clinical developments that may be expected to afford significant therapeutic advances.

  3. Anti-influenza M2e antibody

    SciTech Connect

    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.

  4. Anti-influenza M2e antibody

    SciTech Connect

    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.

  5. 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.

  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.

  7. 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

  8. Antibody-based immunotherapy for malignant glioma

    PubMed Central

    Gedeon, Patrick C.; Riccione, Katherine A.; Fecci, Peter E.; Sampson, John H.

    2014-01-01

    Conventional therapy for malignant glioma (MG) fails to specifically eliminate tumor cells, resulting in toxicity that limits therapeutic efficacy. In contrast, antibody-based immunotherapy utilizes the immune system to eliminate tumor cells with exquisite specificity. Increased understanding of the pathobiology of MG and the profound immunosuppression present among patients with MG has revealed several biologic targets amenable to antibody-based immunotherapy. Novel antibody engineering techniques allow for the production of fully human antibodies or antibody fragments with vastly reduced antigen-binding dissociation constants, increasing safety when used clinically as therapeutics. In this report, we summarize the use of antibody-based immunotherapy for MG. Approaches currently under investigation include the use of antibodies or antibody fragments to: 1) redirect immune effector cells to target tumor mutations, 2) inhibit immunosuppressive signals and thereby stimulate an immunological response against tumor cells, and 3) provide co-stimulatory signals to evoke immunologic targeting of tumor cells. These approaches demonstrate highly compelling safety and efficacy for the treatment of MG, providing a viable adjunct to current standard-of-care therapy for MG. PMID:25173142

  9. 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...

  10. Epstein-Barr Virus Antibodies Test

    MedlinePlus

    ... and services. Advertising & Sponsorship: Policy | Opportunities Epstein-Barr Virus (EBV) Antibody Tests Share this page: Was this ... EA-D IgG Ab Formal name: Epstein-Barr Virus Antibody to Viral Capsid Antigen, IgM, IgG; Epstein- ...

  11. Antibody Gene Transfer for HIV Immunoprophylaxis

    PubMed Central

    Balazs, Alejandro B.; West, Anthony P.

    2015-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing anti-HIV antibodies, is a promising new strategy to prevent HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field. PMID:23238748

  12. Immunoglobulin classes of Aleutian disease virus antibody.

    PubMed Central

    Porter, D D; Porter, H G; Suffin, S C; Larsen, A E

    1984-01-01

    Aleutian disease virus (ADV) persistently infects mink and causes marked hypergammaglobulinemia. Immunoglobulin class-specific antisera were used to define the total immunoglobulin of each class by radial immunodiffusion and the immunoglobulin class of ADV-specific antibody by immunofluorescence in experimentally and naturally infected mink. Electrophoretic gamma globulin closely reflects the immunoglobulin G (IgG) level in mink, and the majority of the increased immunoglobulin and ADV antibody in infected mink is IgG. IgM becomes elevated within 6 days after infection, reaches peak levels by 15 to 18 days, and returns to normal by 60 days after infection. The first ADV antibody demonstrable is IgM, and most mink have virus-specific IgM antibody for at least 85 days postinfection. Serum IgA levels in normal mink are not normally distributed, and ADV infection causes a marked elevation of IgA. Low levels of ADV-specific IgA antibody can be shown throughout the course of infection. Failure of large amounts of virus-specific IgG antibody to inhibit the reaction of virus-specific IgM and IgA antibodies suggests that the various classes of antibodies are directed against spatially different antigenic determinants. The IgM and IgA were shown not to be rheumatoid factors. PMID:6319283

  13. Methods for Selecting Phage Display Antibody Libraries.

    PubMed

    Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

    2016-01-01

    The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described.

  14. Production of monoclonal antibodies to plant pathogens.

    PubMed

    Thornton, Christopher R

    2009-01-01

    The use of monoclonal antibodies in plant pathology has improved the quality and specificity of detection methods for diseases. Hybridoma technology allows the limitless production of highly specific antibodies which can be used to identify pathogens to the species or even sub-species level.

  15. Antibody humanization methods - a review and update.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh

    2013-01-01

    This article reviews recent advances achieved during recent years on various aspects of antibody humanization theories and techniques. Common methods for producing humanized antibodies including framework-homology-based humanization, germline humanization, complementary determining regions (CDR)-homology-based humanization and specificity determining residues (SDR) grafting, as well as advantages and disadvantages of each of these methods and their applications are discussed.

  16. 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.

  17. 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

  18. 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

  19. Antibody gene transfer for HIV immunoprophylaxis.

    PubMed

    Balazs, Alejandro B; West, Anthony P

    2013-01-01

    Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing antibodies to human immunodeficiency virus (HIV), is a promising new strategy for preventing HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field.

  20. 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

  1. 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.

  2. Radiolabeled antibodies for therapy of infectious diseases

    PubMed Central

    Dadachova, Ekaterina; Casadevall, Arturo

    2014-01-01

    Novel approaches to treatment of infectious diseases are urgently needed. This need has resulted in renewing the interest in antibodies for therapy of infectious diseases. Radioimmunotherapy (RIT) is a cancer treatment modality, which utilizes radiolabeled monoclonal antibodies (mAbs). During the last decade we have translated RIT into the field of experimental fungal, bacterial and HIV infections. In addition, successful proof of principle experiments with radiolabeled pan-antibodies that bind to antigens shared by major pathogenic fungi were performed in vitro. The armamentarium of pan-antibodies would result in reducing the dependence on microorganism-specific antibodies and thus would speed up the development of RIT of infections. We believe that the time is ripe for deploying RIT into the clinic to combat infectious diseases. PMID:25599011

  3. Antibodies to laminin in American cutaneous leishmaniasis.

    PubMed Central

    Avila, J L; Rojas, M; Rieber, M

    1984-01-01

    We found that serum samples from patients with different clinical forms of American cutaneous leishmaniasis (ACL) contained immunoglobulin G and immunoglobulin M antibodies which reacted with laminin but not with various other purified connective tissue components, such as collagen types I, III, IV, and V and fibronectin. Eighty-one percent of ACL patients had high antilaminin antibody levels, with a relationship existing between ACL ulcers and antibody levels. This was not, however, the case with patients having treated and healed ACL ulcers; only 34% of these patients had elevated antilaminin antibodies. Eighty-four percent of chronic Chagas' disease patients were also found to contain antilaminin antibodies that were limited to the immunoglobulin G class, but these were not detected in patients suffering from any of 11 other infectious diseases. PMID:6418660

  4. 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.

  5. 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.

  6. Monoclonal antibodies in chronic lymphocytic leukemia.

    PubMed

    Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J

    2006-09-01

    Multiple options are now available for the treatment of chronic lymphocytic leukemia. Over the last 10 years, monoclonal antibodies have become an integral part of the management of this disease. Alemtuzumab has received approval for use in patients with fludarabine-refractory chronic lymphocytic leukemia. Rituximab has been investigated extensively in chronic lymphocytic leukemia both as a single agent and in combination with chemotherapy and other monoclonal antibodies. Epratuzumab and lumiliximab are newer monoclonal antibodies in the early phase of clinical development. This article will review the monoclonal antibodies more commonly used to treat chronic lymphocytic leukemia, the results obtained with monoclonal antibodies as single agents and in combination with chemotherapy, and other biological agents and newer compounds undergoing clinical trials.

  7. 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

  8. CDR2 antigen and Yo antibodies.

    PubMed

    Totland, Cecilie; Aarskog, Nina K; Eichler, Tilo W; Haugen, Mette; Nøstbakken, Jane K; Monstad, Sissel E; Salvesen, Helga B; Mørk, Sverre; Haukanes, Bjørn I; Vedeler, Christian A

    2011-02-01

    Paraneoplastic cerebellar degeneration (PCD) is often associated with Yo antibodies that are directed against human cerebellar degeneration-related protein 2 (CDR2). Such antibodies may also be found in ovarian cancer patients without PCD. We studied if there was an association between Yo antibody production and differences in CDR2 cDNA sequence, mRNA or CDR2 expression in ovarian cancers. We found similar CDR2 cDNA sequence, mRNA and protein levels in primary ovarian cancers, with or without associated Yo antibodies. CDR2 was also present in other cancers, as well as in normal ovary tissue. The results suggest that Yo antibodies are not only related to the expression of CDR2 alone, but also to immune dysregulation.

  9. Antibodies in the treatment of aplastic anemia.

    PubMed

    Gómez-Almaguer, David; Jaime-Pérez, Jose Carlos; Ruiz-Arguelles, Guillermo J

    2012-04-01

    Antibodies have been the cornerstone of treatment of acquired aplastic anemia for more than 25 years. Treatment with antithymocyte globulin (ATG) is considered pivotal and the addition of cyclosporine improves the overall response rate. This antibody is heterogeneous and horse ATG is apparently more effective than rabbit ATG. Several issues remain unsolved in relation to the combination of ATG and cyclosporine: cost, toxicity and late clonal disorders. In recent years, alternative immunosuppressive therapy has been proposed and new antibodies have emerged: porcine ATG, alemtuzumab, daclizumab, and rituximab. Experience with these antibodies is limited to a few studies with alemtuzumab being the most promising, but the results are interesting and provocative. More studies are needed to find the perfect antibody.

  10. Antibody-Mediated Lung Transplant Rejection

    PubMed Central

    Hachem, Ramsey

    2012-01-01

    Antibody-mediated rejection after lung transplantation remains enigmatic. However, emerging evidence over the past several years suggests that humoral immunity plays an important role in allograft rejection. Indeed, the development of donor-specific antibodies after transplantation has been identified as an independent risk factor for acute cellular rejection and bronchiolitis obliterans syndrome. Furthermore, cases of acute antibody-mediated rejection resulting in severe allograft dysfunction have been reported, and these demonstrate that antibodies can directly injure the allograft. However, the incidence and toll of antibody-mediated rejection are unknown because there is no widely accepted definition and some cases may be unrecognized. Clearly, humoral immunity has become an important area for research and clinical investigation. PMID:23002428

  11. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  12. Trends in Malignant Glioma Monoclonal Antibody Therapy

    PubMed Central

    Chekhonin, Ivan; Gurina, Olga

    2015-01-01

    Although new passive and active immunotherapy methods are emerging, unconjugated monoclonal antibodies remain the only kind of biological preparations approved for high-grade glioma therapy in clinical practice. In this review, we combine clinical and experimental data discussion. As antiangiogenic therapy is the standard of care for recurrent glioblastoma multiforme (GBM), we analyze major clinical trials and possible therapeutic combinations of bevacizumab, the most common monoclonal antibody to vascular endothelial growth factor (VEGF). Another humanized antibody to gain recognition in GBM is epidermal growth factor (EGFR) antagonist nimotuzumab. Other antigens (VEGF receptor, platelet-derived growth factor receptor, hepatocyte growth factor and c-Met system) showed significance in gliomas and were used to create monoclonal antibodies applied in different malignant tumors. We assess the role of genetic markers (isocitrate dehydrogenase, O6-methylguanine-DNA methyltransnsferase) in GBM treatment outcome prediction. Besides antibodies studied in clinical trials, we focus on perspective targets and briefly list other means of passive immunotherapy.

  13. [Auto-antibodies in liver disease].

    PubMed

    Montaño Loza, Aldo J; Angulo, Paul

    2007-01-01

    Autoantibodies are a nonpathogenic manifestation of immune reactivity that may occur in acute and chronic liver diseases. Autoantibodies are the consequence rather than the cause of liver injury, and they can be used as diagnostic tools rather than etiologic markers. Conventional autoantibodies used in the categorization of liver disease are antinuclear antibodies, smooth muscle antibodies, antibodies to liver/kidney microsome type 1, antimitochondrial antibodies, and perinuclear antineutrophil cytoplasmic antibodies. However, the final diagnosis and the treatment strategies do not depend solely on the serological markers. Autoantibodies titles vary overtime and their behavior does not correlate with disease activity. Over-interpretation is the major pitfall in the clinical application of the serological results. Recognition and characterization of new autoantibodies is expected to improve the diagnostic precision, provide diagnostic parameters, and elucidate target autoantigens for the management of liver diseases.

  14. Antibody-based resistance to plant pathogens.

    PubMed

    Schillberg, S; Zimmermann, S; Zhang, M Y; Fischer, R

    2001-01-01

    Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

  15. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis.

  16. Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry.

    PubMed

    Robins, R A; Laxton, R R; Garnett, M; Price, M R; Baldwin, R W

    1986-06-24

    Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.

  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. 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.

  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.

  1. Antiendomysial antibodies in Berger's disease.

    PubMed

    Pierucci, Alessandro; Fofi, Claudia; Bartoli, Benedetta; Simonetti, Bianca Maria; Pecci, Gabriella; Sabbatella, Luigi; Di Tola, Marco; Greco, Rosita; Anania, Maria Cristina; Picarelli, Antonio

    2002-06-01

    The finding of increased levels of immunoglobulin A (IgA) against food antigens in patients with IgA nephropathy prompted the hypothesis of an association between IgA nephropathy and celiac disease (CD). Attention was initially directed to antigliadin antibodies, then to IgA antiendomysial antibodies (IgA-EMA). IgG1-EMA have been found in patients with CD with IgA-EMA-negative results. The presence of IgA- and IgG1-EMA was investigated in 36 patients with IgA nephropathy, 15 patients with other primary glomerulonephritis, and 15 patients with lupus nephritis. IgA-EMA and IgG1-EMA were detected by indirect immunofluorescence analysis. At the time of renal biopsy, the following factors were evaluated: history of macroscopic hematuria, serum creatinine level, urinalysis, 24-hour proteinuria, blood pressure, and histological classification of IgA nephropathy. Sixteen of 36 patients with IgA nephropathy (44.4%) showed EMA positivity. Among patients with positive EMA, 12 patients (75%) were IgG1-EMA positive, 2 patients (12.5%) were IgA-EMA positive, and 2 patients (12.5%) were positive for both isotypes. No significant differences were observed between the two groups (EMA positive versus EMA negative) concerning age, serum creatinine level, macroscopic hematuria, blood pressure, 24-hour proteinuria, or degree of renal histological involvement. IgA- and IgG1-EMA were not detected in patients with other primary nephropathies or lupus nephritis. These results, based on the finding of IgG1-EMA, suggest a common pathogenetic pathway for CD and IgA nephropathy. On this basis, the presence of IgG1-EMA and/or IgA-EMA should be investigated in patients with IgA nephropathy. Furthermore, the role of a gluten-free diet in the natural history of IgA nephropathy, at least in EMA-positive patients, needs to be ascertained.

  2. 21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's...

  3. Glycosylation profiles of therapeutic antibody pharmaceuticals.

    PubMed

    Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

    2011-11-01

    Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth.

  4. Developmental regulation of the human antibody repertoire.

    PubMed

    Schroeder, H W; Mortari, F; Shiokawa, S; Kirkham, P M; Elgavish, R A; Bertrand, F E

    1995-09-29

    The ability to respond to antigen develops in a programmed fashion during ontogeny. In human, "fetal" immunoglobulin gene segment utilization appears biased towards a small set of evolutionarily conserved V gene segments. Many of these gene segments are also used in antibodies with antigen specificities that do not arise until after infancy. The human fetus primarily regulates the diversity of the antibody repertoire through control of the H (heavy) chain CDR 3, which is generated by VDJ joining and forms the center of the antigen-binding site. Molecular modeling suggests that limitations in the length and composition of fetal CDR 3 intervals result in antibodies that contain a relatively "flat" antigen-binding surface that could serve to maximize the number of different interactions possible between the antibody and potential antigens. We propose that these limitations in the sequence and structure of H chain CDR 3 contribute to the low affinity and multireactivity of fetal antibody repertoires. The specific mechanisms used to generate a restricted fetal repertoire appear to differ between human and mouse. Nevertheless, included in the final products of both human and mouse fetal B cells will be antibodies that are quite homologous in composition and structure. The precise role that these antibodies play in the development of immunocompetence remains to be elucidated.

  5. 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.

  6. 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.

  7. Progress towards recombinant anti-infective antibodies

    PubMed Central

    Pai, Jennifer C.; Sutherland, Jamie N.; Maynard, Jennifer A.

    2009-01-01

    The global market for monoclonal antibody therapeutics reached a total of $11.2 billion in 2004, with an impressive 42% growth rate over the previous five years and is expected to reach ~$34 billion by 2010. Coupled with this growth are stream-lined product development, production scale-up and regulatory approval processes for the highly conserved antibody structure. While only one of the 21 current FDA-approved antibodies, and one of the 38 products in advanced clinical trials target infectious diseases, there is increasing academic, government and commercial interest in this area. Synagis, an antibody neutralizing respiratory syncitial virus (RSV), garnered impressive sales of $1.1 billion in 2006 in spite of its high cost and undocumented effects on viral titres in human patients. The success of anti-RSV passive immunization has motivated the continued development of anti-infectives to treat a number of other infectious diseases, including those mediated by viruses, toxins and bacterial/fungal cells. Concurrently, advances in antibody technology suggest that cocktails of several monoclonal antibodies with unique epitope specificity or single monoclonal antibodies with broad serotype specificity may be the most successful format. Recent patents and patent applications in these areas will be discussed as predictors of future anti-infective therapeutics. PMID:19149692

  8. 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.

  9. Antibodies as predictors of complex autoimmune diseases.

    PubMed

    Vojdani, A

    2008-01-01

    Emerging evidence has suggested environmental factors such as infections and xenobiotics and some dietary proteins and peptides in the pathogenesis of many autoimmune diseases. Considering the fact that autoantibodies can often be detected prior to the onset of a disease, in this study an enzyme immunoassay was used for measurement of antibodies against different highly purified antigens or synthetic peptides originating not only from human tissue, but also from cross-reactive epitopes of infectious agents, dietary proteins and xenobiotics. The measurement of antibodies against a panel of antigens allows for identification of patterns or antibody signatures, rather than just one or two markers of autoimmunity, thus establishing the premise for increased sensitivity and specificity of prediction, as well as positive predictive values. This panel of different autoantibodies was applied to 420 patients with different autoimmune diseases, including pernicious anemia, celiac disease, thyroiditis, lupus, rheumatoid arthritis, osteoarthritis, Addison's disease, type 1 diabetes, cardiovascular disease and autoimmunity, which are presented in this article. In all cases, the levels of these antibodies were significantly elevated in patients versus controls. Antibody patterns related to neuroautoimmune disorders, cancer, and patients with somatic hypermutation will be shown in a subsequent article. We believe that this novel 96 antigen-specific autoantibody or predictive antibody screen should be studied for its incorporation into routine medical examinations. Clinicians should be aware that the detection of antibodies should not automatically mean that a patient will definitely become ill, but would rather give a percentage of risk for autoimmune disease over subsequent months or years.

  10. 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

  11. Coeliac Disease-Associated Antibodies in Psoriasis

    PubMed Central

    Akbulut, Sabiye; Gür, Günes; Topal, Firdevs; Topal, Fatih Esad; Alli, Nuran; Saritas, Ülkü

    2013-01-01

    Background The possible relationship between psoriasis and coeliac disease (CD) has been attributed to the common pathogenic mechanisms of the two diseases and the presence of antigliadin antibodies in patients has been reported to increase the incidence of CD. Objective The aim of this report was to study CD-associated antibodies serum antigliadin antibody immunoglobulin (Ig)A, IgG, anti-endomysial antibody IgA and anti-transglutaminase antibody IgA and to demonstrate whether there is an increase in the frequency of those markers of CD in patients with psoriasis. Methods Serum antigliadin antibody IgG and IgA, antiendomysial antibody IgA and anti-transglutaminase antibody IgA were studied in 37 (19 males) patients with psoriasis and 50 (23 males) healthy controls. Upper gastrointestinal endoscopy and duodenal biopsies were performed in patients with at least one positive marker. Results Antigliadin IgA was statistically higher in the psoriasis group than in the controls (p<0.05). Serological markers were found positive in 6 patients with psoriasis and 1 person from the control group. Upper gastrointestinal endoscopy was performed in all these persons, with biopsies collected from the duodenum. The diagnosis of CD was reported in only one patient with psoriasis following the pathological examination of the biopsies. Whereas one person of the control group was found to be positive for antigliadin antibody IgA, pathological examination of the duodenal biopsies obtain from this patient were found to be normal. Conclusion Antigliadin IgA prominently increases in patients diagnosed with psoriasis. Patients with psoriasis should be investigated for latent CD and should be followed up. PMID:24003271

  12. 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.

  13. 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.

  14. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    PubMed

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible.

  15. Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity.

    PubMed

    Tajima, Nobuyuki; Takai, Madoka; Ishihara, Kazuhiko

    2011-03-15

    To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.

  16. Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody.

    PubMed

    Weidner, K M; Denzin, L K; Voss, E W

    1992-05-25

    Anti-metatype antibodies have been described as antibodies which recognize ligand-induced conformational changes in the antibody variable region. Additionally, anti-metatype antibodies, produced by multiple immunizations with liganded high affinity monoclonal anti-fluorescein antibody 4-4-20, enhanced the lifetime of monoclonal antibody 4-4-20-fluorescein complex. To better understand the mechanism of the delayed dissociation rate, deuterium oxide was used to probe the liganded active site. The rate and extent of deuterium oxide-mediated fluorescence enhancement of bound ligand served to monitor the conformational dynamics of the active site in the presence and absence of anti-metatype antibodies. Results showed that anti-metatype antibodies reduced the rate and extent of deuterium oxide-mediated fluorescence enhancement of 4-4-20, a single-chain derivative of 4-4-20 (consisting of the variable domains and a polylinker), and idiotypically related monoclonal anti-fluorescein antibodies suggesting that anti-metatype stabilized the liganded active site. Size exclusion liquid chromatography was utilized to isolate the liganded antibody-anti-metatype complex. Liganded single chain antibody 4-4-20 was mixed with 10-fold molar excess anti-metatype Fab fragments, and a major complex eluted with an apparent M(r) 249,000. The apparent molecular weight of this complex inferred that one liganded single chain antibody was bound by five antimetatype Fab fragments. Spectral analysis confirmed these results and the characteristic delayed rate of ligand dissociation was also observed for the isolated complex. The results suggest that anti-metatype antibodies stabilize the liganded conformation by forming a large, stable, macromolecular complex.

  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. Intellectual property protection: strategies for antibody inventions.

    PubMed

    Storz, Ulrich

    2011-01-01

    In the last decade, therapeutic antibodies have become one of the commercially most successful classes of biopharmaceutical drugs. Major drug manufacturers who have successfully managed to occupy this new market, as well as biotechnology firms, some of which have experienced a quick growth and are now on par with the former, owe part of their success to suitable intellectual property strategies. This article provides an overview of the current thinking on antibody-related patents, and discusses strategies for protecting the antibody products of the future.

  19. Reshaping Human Antibodies: Grafting an Antilysozyme Activity

    NASA Astrophysics Data System (ADS)

    Verhoeyen, Martine; Milstein, Cesar; Winter, Greg

    1988-03-01

    The production of therapeutic human monoclonal antibodies by hybridoma technology has proved difficult, and this has prompted the ``humanizing'' of mouse monoclonal antibodies by recombinant DNA techniques. It was shown previously that the binding site for a small hapten could be grafted from the heavy-chain variable domain of a mouse antibody to that of a human myeloma protein by transplanting the hypervariable loops. It is now shown that a large binding site for a protein antigen (lysozyme) can also be transplanted from mouse to human heavy chain. The success of such constructions may be facilitated by an induced-fit mechanism.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Antibodies to watch in 2010

    PubMed Central

    2010-01-01

    Monoclonal antibodies (mAbs) are a burgeoning class of therapeutics, with more than 25 approved in countries worldwide. Novel molecules are entering clinical study at a rate of nearly 40 per year, and the commercial pipeline includes approximately 240 mAb therapeutics in clinical studies that have not yet progressed to regulatory approval or been approved. Of particular interest are the 26 mAbs that are currently at Phase 3, when safety and efficacy data critical to approval is established. Phase 3 study lengths are typically two to four years, so results for some studies might be announced in 2010, but data from others might not be presented until 2014. This overview of the 26 candidates provides a brief description of the background and the on-going Phase 3 studies of each mAb. Additional mAbs that have progressed to regulatory review or been approved may also be in Phase 3 studies, but these, as well as Fc fusion proteins, have been excluded. Due to the large body of primary literature about the 26 candidates, only selected references are given, with a focus on recent publications and articles that were relevant to Phase 3 studies. Current as of October 2009, the results presented here will serve as a baseline against which future progress can be measured. PMID:20065640

  6. 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.

  7. Polynucleotides encoding anti-sulfotyrosine antibodies

    DOEpatents

    Bertozzi, Carolyn R [Berkeley, CA; Kehoe, John [Saint Davids, PA; Bradbury, Andrew M [Santa Fe, NM

    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.

  8. 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

  9. 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.

  10. Recurrent adverse pregnancy outcome and antiphospholipid antibodies.

    PubMed

    Reece, E A; Gabrielli, S; Cullen, M T; Zheng, X Z; Hobbins, J C; Harris, E N

    1990-07-01

    Antiphospholipid antibodies, which include lupus-like anticoagulant and anticardiolipin antibody, have been linked to a number of adverse pregnancy outcomes, although their exact pathogenic mechanisms remain poorly defined. The relative risk of complications such as intrauterine growth retardation, spontaneous abortions, and stillbirth in patients with antiphospholipid antibodies also remains undetermined. Heightened attention has been focused on the association, leading to investigations into the pathogenesis. Uncontrolled studies have also explored therapeutic regimens such as aspirin, steroids, and heparin, and clinical trials have used various treatment protocols. Although knowledge into the association of antiphospholipid antibodies and recurrent adverse pregnancy outcome is limited and continues to evolve, this association provides new insights into the disease and offers promise for pharmacologic prophylaxis. In this article, current concepts on pathogenesis, diagnosis, and therapy are reviewed and recommendations are made for clinical care of these patients.

  11. 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.

  12. Monoclonal antibodies against metallothioneins and metalloproteins.

    PubMed

    Talbot, B G; Bilodeau, G; Thirion, J P

    1986-10-01

    Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

  13. Antiphospholipid antibodies in critically ill patients

    PubMed Central

    Vassalo, Juliana; Spector, Nelson; de Meis, Ernesto; Soares, Márcio; Salluh, Jorge Ibrain Figueira

    2014-01-01

    Antiphospholipid antibodies are responsible for a wide spectrum of clinical manifestations. Venous, arterial and microvascular thrombosis and severe catastrophic cases account for a large morbidly/mortality. Through the connection between the immune, inflammatory and hemostatic systems, it is possible that these antibodies may contribute to the development of organ dysfunction and are associated with poor short and long-term prognoses in critically ill patients. We performed a search of the PubMed/MedLine database for articles written during the period from January 2000 to February 2013 to evaluate the frequency of antiphospholipid antibodies in critically ill patients and their impact on the outcomes of these patients. Only eight original studies involving critically ill patients were found. However, the development of antiphospholipid antibodies in critically ill patients seems to be frequent, but more studies are necessary to clarify their pathogenic role and implications for clinical practice. PMID:25028953

  14. Creation of catalytic antibodies metabolizing organophosphate compounds.

    PubMed

    Kurkova, I N; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2012-10-01

    Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.

  15. Solid phase synthesis of bifunctional antibodies.

    PubMed

    DeSilva, B S; Wilson, G S

    1995-12-15

    Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.

  16. Synthesis of bifunctional antibodies for immunoassays.

    PubMed

    DeSilva, B S; Wilson, G S

    2000-09-01

    The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.

  17. Advancements in nanosensors using plastic antibodies.

    PubMed

    Volkert, Anna A; Haes, Amanda J

    2014-01-07

    Biosensors possess recognition elements that bind to target molecules which lead to detectable signals. Incorporation of noble metal nanomaterials into biosensors allows for rapid and simple biomolecule detection. Herein, recent developments in affinity nanosensors will be discussed. These sensors often include naturally occurring recognition elements such as antibodies and DNA. As samples become more complex, new recognition elements are sought. For instance, plastic antibodies provide alternative and more environmentally stable recognition elements than traditional antibodies. Molecular imprinted polymers, a class of plastic antibodies, promote biomolecule recognition and detection. The incorporation of noble metal nanomaterials into molecular imprinted polymer biosensors for real world applications will be explored. Further improvements in the design of artificial recognition agents are envisioned to facilitate new methods for complex biological and chemical analyses.

  18. [Antibody formation in type A viral hepatitis].

    PubMed

    Teokharova, M P; Losev, B B; Andonov, A P; Draganov, P I; Mineva, E G

    1978-01-01

    Immune response to hepatitis type A antigen (HAAg) was measured by the passive hemagglutination test (PHA) and by the immune adherence test (IAHA). Specific antibodies found by PHA are of the IgM class which indicates a recent exposure to hepatitis A virus. The antibodies found by IAHA reflect the level of postinfectious immune status. The antibody curve is highest in the age group of 30--49 years (95%). The above-mentioned serological tests were carried out with purified by gel filtration in Sepharose 6B Botevgrad faecal morphologically consisting of 27 nm particles with the buoyant density in CcCl of 1.40 g/ml. The same particles were aggregated with sera positive for antibody to hepatitis type A antigen in immune electron microscopy (IEM).

  19. Deep sequencing and human antibody repertoire analysis

    PubMed Central

    Boyd, Scott D; Crowe, James E

    2016-01-01

    In the past decade, high-throughput DNA sequencing (HTS) methods and improved approaches for isolating antigen-specific B cells and their antibody genes have been applied in many areas of human immunology. This work has greatly increased our understanding of human antibody repertoires and the specific clones responsible for protective immunity or immune-mediated pathogenesis. Although the principles underlying selection of individual B cell clones in the intact immune system are still under investigation, the combination of more powerful genetic tracking of antibody lineage development and functional testing of the encoded proteins promises to transform therapeutic antibody discovery and optimization. Here, we highlight recent advances in this fast-moving field. PMID:27065089

  20. Enzyme mimicry by the antiidiotypic antibody approach

    PubMed Central

    Kolesnikov, Alexander V.; Kozyr, Arina V.; Alexandrova, Elena S.; Koralewski, Frédéric; Demin, Alexander V.; Titov, Mikhail I.; Avalle, Bérangère; Tramontano, Alfonso; Paul, Sudhir; Thomas, Daniel; Gabibov, Alexander G.; Friboulet, Alain

    2000-01-01

    The concept of “internal image” of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches. PMID:11095704

  1. Monoclonal Antibodies for Multiple Sclerosis Treatment.

    PubMed

    Palavra, Filipe

    2015-01-01

    Since their introduction in medical therapy, in the last quarter of the 20th century, monoclonal antibodies have gained an increasing importance in the treatment of various diseases. Neurology has been one of the medical specialties benefiting of the therapeutic potential of these monoclonal antibodies and certain neurological conditions may now contain such drugs in their therapeutic algorithms. Multiple sclerosis is one of these diseases and, in addition to the monoclonal antibodies already licensed for clinical use, several others are in development for future utilization in this specific area. The future will certainly pass through this kind of drugs and, in this article, a review of the most relevant data related to monoclonal antibodies already in use and also in clinical development for multiple sclerosis treatment will be performed.

  2. Detection of Antibodies to Squalene III. Naturally Occurring Antibodies to Squalene in Humans and Mice

    DTIC Science & Technology

    2007-11-02

    14. ABSTRACT An ELISA -based assay is described for the measurement of antibodies to squalene (SQE) in human serum and plasma. The assay was adapted...States Army Medical Department Activities, Fort Knox, KY. (No additional volunteer information is available.) Using this new ELISA method, antibodies to...correlated with vaccination with AVA; and appear to increase in prevalence with age. 15. SUBJECT TERMS squalene, antibody detection, ELISA , Bacillus

  3. 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.

  4. Antibodies to Actin in Autoimmune Neutropenia

    DTIC Science & Technology

    1990-02-01

    protein as actin. Purified Acanthamoeba actin by anti-neutrophil antibodies in autoimmune neutropenia, comigrated with the protein and was specifically...anti-rabbit IgG were obtained from ICN Immunobiolog- formed using purified Acanthamoeba actin (gift of Dr Blair Bowers. icals, Naperville, IL. Cells...preparations𔃼 1 - was the protein recognized by these anti-neutrophil antibody 6 .2- positive sera, lgG, and F(ab’) 2. Purified Acanthamoeba actin

  5. Primary Antiphospholipid Antibody Syndrome: A Case Report.

    PubMed

    Kadeli, Deepak K; Hanjagi, Siddaraya Y

    2015-10-01

    Primary Antiphospholipid antibody syndrome is a rare disease associated with thromboembolic events which may affect either the arterial or the venous vasculature. It presents with an increased risk of thrombosis in pregnant woman leading to repeated fetal losses. We present here a case of primary antiphospholipid antibody syndrome in young women who had previous event of gangrene of toes leading to their amputation and repeated fetal losses.

  6. The current status of neutrophil cytoplasmic antibodies.

    PubMed Central

    van der Woude, F J; Daha, M R; van Es, L A

    1989-01-01

    Several studies in the past 10 years have demonstrated the occurrence of autoantibodies against cytoplasmic constituents in patients with vasculitis and glomerulonephritis. In this review the nomenclature of these antibodies is discussed and assays and clinical associations are summarized. Although the antigens involved are not completely identified, antibodies and T cells reactive with myeloid lysozomal enzymes may both play a significant role in pathogenesis. PMID:12412739

  7. Dengue Antibody and Zika: Friend or Foe?

    PubMed

    Durbin, Anna P

    2016-10-01

    Zika virus is a mosquito-borne Flavivirus related to dengue that is rapidly spreading through the Americas. This outbreak is occurring in dengue-endemic areas where the population has acquired antibodies to dengue. Recent studies reveal that preexisting dengue antibodies may have opposite effects on Zika infection, transmission, and clinical outcome. Discerning these effects is critical to a better understanding of Zika pathogenesis and the prevention of future outbreaks.

  8. Antibody transport and internalization into tumours.

    PubMed Central

    Matzku, S.; Moldenhauer, G.; Kalthoff, H.; Canevari, S.; Colnaghi, M.; Schuhmacher, J.; Bihl, H.

    1990-01-01

    Internalization of monoclonal antibody (MAb) conjugates is an important feature of tumour targeting, both with respect to the therapeutic action of substances coupled to the antibody and to retention of radionuclides. Problems of analysing internalization in vitro and in vivo, of manipulating internalization, and of evaluating the involvement of normal tissues are illustrated by recent experimental data and are discussed in the light of published evidence. Images Figure 4 PMID:2383472

  9. 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

  10. Construction of human antibody gene libraries and selection of antibodies by phage display.

    PubMed

    Frenzel, André; Kügler, Jonas; Wilke, Sonja; Schirrmann, Thomas; Hust, Michael

    2014-01-01

    Antibody phage display is the most commonly used in vitro selection technology and has yielded thousands of useful antibodies for research, diagnostics, and therapy.The prerequisite for successful generation and development of human recombinant antibodies using phage display is the construction of a high-quality antibody gene library. Here, we describe the methods for the construction of human immune and naive scFv gene libraries.The success also depends on the panning strategy for the selection of binders from these libraries. In this article, we describe a panning strategy that is high-throughput compatible and allows parallel selection in microtiter plates.

  11. 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.

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

    DOE PAGES

    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

  13. 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.

  14. 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.

  15. Working towards a consensus for antibody validation.

    PubMed

    Reiss, Peter D; Min, Danxi; Leung, Mei Y

    2014-01-01

    Commercial research antibodies are the most commonly used product in the life science tools market, and their applications represent a significant investment of time and resources for researchers. Frequently however, the quality of antibodies does not meet the expectations of consumers, causing loss of valuable time and money. This can delay research efforts and scientific discovery, or even lead to false, irreproducible results to be published in the scientific literature. This raises the question of whether there should be universal standards for validating antibodies.   During the 1 (st) International Antibody Validation Forum, hosted by St John's Laboratory Ltd on October 15 (th) 2014 at Queen Mary University of London, scientists from academia and industry presented data highlighting quality issues arising from lack of antibody validation. While the forum identified significant current problems in the antibody market, it also discussed future opportunities for improved quality and transparency by encouraging data disclosure and data sharing. This article highlights the key issues and conclusions reached at the forum.

  16. Monoclonal antibodies to leukotoxin of Actinobacillus actinomycetemcomitans.

    PubMed Central

    DiRienzo, J M; Tsai, C C; Shenker, B J; Taichman, N S; Lally, E T

    1985-01-01

    Hybridoma cell lines which produce monoclonal antibodies to a leukotoxin from Actinobacillus actinomycetemcomitans were prepared. The monoclonal antibodies were selected for their ability to neutralize the cytotoxic activity of the leukotoxin and recognize the toxin on nitrocellulose blots. The antibodies belonged to either the immunoglobulin G1 (IgG1) or IgG2 subclass and differed in their ability to bind to the leukotoxin on nitrocellulose blots. However, only slight differences in neutralization titers were observed. Use of the monoclonal antibodies revealed that polymyxin B-extracted or osmotic shock-released leukotoxin could be separated into several high-molecular-weight polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis with the monoclonal antibodies also demonstrated that the leukotoxin was present in eight oral strains of A. actinomycetemcomitans that had been previously classified by a biological assay as leukotoxic. The availability of these monoclonal antibodies should facilitate and expand studies concerning the role of the leukotoxin in the pathogenicity of A. actinomycetemcomitans. Images PMID:3965404

  17. Working towards a consensus for antibody validation

    PubMed Central

    Reiss, Peter D.; Min, Danxi; Leung, Mei Y.

    2014-01-01

    Commercial research antibodies are the most commonly used product in the life science tools market, and their applications represent a significant investment of time and resources for researchers. Frequently however, the quality of antibodies does not meet the expectations of consumers, causing loss of valuable time and money. This can delay research efforts and scientific discovery, or even lead to false, irreproducible results to be published in the scientific literature. This raises the question of whether there should be universal standards for validating antibodies.   During the 1 st International Antibody Validation Forum, hosted by St John’s Laboratory Ltd on October 15 th 2014 at Queen Mary University of London, scientists from academia and industry presented data highlighting quality issues arising from lack of antibody validation. While the forum identified significant current problems in the antibody market, it also discussed future opportunities for improved quality and transparency by encouraging data disclosure and data sharing. This article highlights the key issues and conclusions reached at the forum. PMID:25580232

  18. Antibody-mediated resistance against plant pathogens.

    PubMed

    Safarnejad, Mohammad Reza; Jouzani, Gholamreza Salehi; Tabatabaei, Meisam; Tabatabaie, Meisam; Twyman, Richard M; Schillberg, Stefan

    2011-01-01

    Plant diseases have a significant impact on the yield and quality of crops. Many strategies have been developed to combat plant diseases, including the transfer of resistance genes to crops by conventional breeding. However, resistance genes can only be introgressed from sexually-compatible species, so breeders need alternative measures to introduce resistance traits from more distant sources. In this context, genetic engineering provides an opportunity to exploit diverse and novel forms of resistance, e.g. the use of recombinant antibodies targeting plant pathogens. Native antibodies, as a part of the vertebrate adaptive immune system, can bind to foreign antigens and eliminate them from the body. The ectopic expression of antibodies in plants can also interfere with pathogen activity to confer disease resistance. With sufficient knowledge of the pathogen life cycle, it is possible to counter any disease by designing expression constructs so that pathogen-specific antibodies accumulate at high levels in appropriate sub-cellular compartments. Although first developed to tackle plant viruses and still used predominantly for this purpose, antibodies have been targeted against a diverse range of pathogens as well as proteins involved in plant-pathogen interactions. Here we comprehensively review the development and implementation of antibody-mediated disease resistance in plants.

  19. 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

  20. Secretory antibody following oral influenza immunization.

    PubMed

    Waldman, R H; Stone, J; Bergmann, K C; Khakoo, R; Lazzell, V; Jacknowitz, A; Waldman, E R; Howard, S

    1986-12-01

    Secretory IgA antibody may be important in protection against respiratory viral infections, and the concept of a common mucosal immune system offers the theoretical basis for the convenient stimulation of this antibody. Therefore, the oral route was compared with intramuscular injection in a double-blind, placebo-controlled study in young healthy volunteers. A killed influenza vaccine, given in enteric-coated capsules (total of 98 ug hemagglutinin of A/Bangkok) led to significant salivary and nasal IgA antibody rises in a 4-week period. The preimmunization titers in secretions were inversely correlated with the antibody rise after immunization. The orally administered vaccine was associated with no more side effects than placebo, in contradistinction to reactions following the intramuscular route. The latter route also was without significant effect in regard to a stimulation of secretory antibodies. The observed simultaneous induction of antibodies in saliva and nasal secretions following oral administration of killed vaccine gives further evidence of a common mucosal immune system and its possible clinical use.

  1. A mutagenesis study of a catalytic antibody

    SciTech Connect

    Jackson, D.Y.; Prudent, J.R.; Baldwin, E.P.; Schultz, P.G. )

    1991-01-01

    The authors have generated seven site-specific mutations in the genes encoding the variable region of the heavy chain domain (V{sub H}) of the phosphocholine-binding antibody S107.S107 is a member of a family of well-characterized highly homologous antibodies that bind phosphorylcholine mono- and diesters. Two of these antibodies, MOPC-167 and T15, have previously been shown to catalyze the hydrolysis of 4-nitrophenyl N-trimethylammonioethyl carbonate. Two conserved heavy-chain residues, Tyr-33 and Arg-52, were postulated to be involved in binding and hydrolysis of 4-nitrophenylcholine carbonate esters. To more precisely define the catalytic roles of these residues, three Arg-52 mutants (R52K, R52Q, R52C) and four Tyr-33 mutants (Y33H, Y33F, Y33E, Y33D) of antibody S107 were generated. The genes encoding the V{sub H} binding domain of S107 were inserted into plasmid pUC-fl, and in vitro mutagenesis was performed. These results not only demonstrate the importance of electrostatic interactions in catalysis by antibody S107 but also show that catalytic side chains can be introduced into antibodies to enhance their catalytic efficiency.

  2. 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.

  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. 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...

  5. 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...

  6. Antiphospholipid Antibody Syndrome Presenting with Unilateral Adrenal Hemmorhage.

    PubMed

    Ullah, Kifayat; Butt, Ghias; Neopane, Sippy; Arshi, Shahana

    2016-06-01

    The antiphospholipid antibody syndrome presents with vascular thrombosis which involve both arterial and venous systems. The clinical presentation of antiphospholipid antibody syndrome includes obstetric complications leading to recurrent abortions, presence of circulating antibodies against phospholipids, and multi-organ thromboembolisms. We report a case of a patient who presented with unilateral adrenal hemorrhage and subsequently found to have antiphospholipid antibody syndrome and lupus nephritis.

  7. 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.

  8. [Progress in monoclonal antibody-based immunotherapy for cancer treatment].

    PubMed

    Guo, Yajun

    2015-06-01

    More than 100 years ago, Paul Ehrlich first proposed the "magic bullets" concept in which antibody targeting disease related antigen can fight against human disease. Since then, with the development of hybridoma technology for monoclonal antibody production and cancer serum therapy, immunotherapy based monoclonal antibody bas been used in chinical practice to treat hematological and solid tumor. Up to now, more than 20 recombinant antibody drugs were approved for cancer treatment worldwide. In recent years, the next-generation antibody drug, including immune checkpoint antagonists, bi-specific antibody, and antibody drug conjugates have successfully cured various malignant tumor. This review recalled the history of monoclonal antibody as potent immunotherapy of cancer firstly, and focused on the next-generation antibody drug's mechanism of action, construction strategies, and the side effects in clinic. Lastly, the future trend of anti-tumor antibody drug was also discussed.

  9. Coming-of-Age of Antibodies in Cancer Therapeutics.

    PubMed

    Ayyar, B Vijayalakshmi; Arora, Sushrut; O'Kennedy, Richard

    2016-12-01

    Antibody-based therapies have garnered considerable success in recent years. This is due to the availability of strategies to successfully engineer antibodies into humanized forms, better understanding of the biological processes involved in cancer development, the availability of novel recombinant antibody formats, better antibody selection platforms, and improved antibody conjugation methodologies. Such achievements have led to an explosion in the generation of antibodies and antibody-associated constructs for the treatment of cancer and other diseases. In this review, we critically assess recent trends in the development and applications of bispecific antibodies (bsAbs), antibody-drug conjugates (ADCs), and immune checkpoint inhibitors (ICIs) as cancer therapeutics. We also highlight recent US FDA approvals and clinical trials of antibody-based cancer therapies.

  10. Cross-reactive broadly neutralizing antibodies: timing is everything.

    PubMed

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  11. Immunoglobulin VH determinants defined by monoclonal antibodies

    PubMed Central

    1982-01-01

    Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts. PMID:6185604

  12. The significance of anti-streptokinase antibodies.

    PubMed Central

    Lynch, M; Pentecost, B L; Littler, W A; Stockley, R A

    1994-01-01

    Antibodies to streptokinase (SK) are widespread in the population, but reports of their effect on the action of SK are conflicting. Specific anti-SK IgG was purified from the sera of 10 patients, five with low titres of anti-SK IgG and five with high titres. The effect of increasing specific anti-SK IgG antibodies on the action of SK was evaluated in vitro using a fluorimetric assay for plasmin and by a fibrin plate lysis assay. The inhibition of SK by whole plasma from a further group of patients was also assessed by the fibrin plate assay. There was a positive correlation between the serum antibody concentration and the quantity of specific anti-SK eluted (r = 0.797; P < 0.005). The addition of specific anti-SK IgG caused a dose-related decrease in SK activity (fluorimetric assay r = -0.93; P = 0.02; fibrin plate assay r = -0.98; P < 0.001). The addition of patient plasma to the fibrin plate assay also resulted in decreased lysis, which was dependent upon antibody titre (r = -0.95; P < 0.0001). Significant in vitro reduction of the activity of SK by specific antibody was demonstrated, and this was similar with plasma containing comparable amounts of antibody. The findings suggest that treatment with SK would be unlikely to induce an effective thrombolytic state when antibody titres are high (such as those seen within 2 years of an initial dose of SK). PMID:8004811

  13. Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose.

    PubMed

    Liu, B; Marks, J D

    2000-11-01

    Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.

  14. Vimentin antibodies: a non-HLA antibody as a potential risk factor in renal transplantation.

    PubMed

    Carter, V; Shenton, B K; Jaques, B; Turner, D; Talbot, D; Gupta, A; Chapman, C E; Matthews, C J; Cavanagh, G

    2005-03-01

    Chronic allograft rejection is the major problem encountered in solid organ transplantation and is the end point of several complex processes. A number of recent studies show both alloimmune and autoimmune responses may have roles to play. The importance of HLA antibodies in transplantation is well documented, but despite the introduction of very sensitive HLA screening assays, antibody-mediated allograft rejection still occurs without detectable HLA antibodies. The target for antibody-mediated allograft rejection in these circumstances remains elusive, perhaps due to the variety of potential targets presented on endothelial cells. Recent studies identifying C4d and immunoglobulin deposits in patients undergoing late allograft loss provide evidence that chronic rejection involves humoral as well as cellular components. Several endothelial cell antigens that might be important in chronic rejection have been suggested, including MHC class I chain-related genes; Lewis; and the intermediate filament protein, vimentin. Vimentin is an ideal candidate antigen for antibody-mediated rejection as it is found in endothelial cells and is exposed to the immune system following surgery or by chronic allograft rejection due to endothelial cell breakdown, where the development of antibodies may cause further damage. We have developed a flow cytometric assay for the detection of antibodies to vimentin and have investigated whether HLA or vimentin antibodies are present in renal transplant recipients undergoing chronic rejection.

  15. 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.

  16. 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.

  17. Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

    PubMed

    Schwimmer, Lauren J; Huang, Betty; Giang, Hoa; Cotter, Robyn L; Chemla-Vogel, David S; Dy, Francis V; Tam, Eric M; Zhang, Fangjiu; Toy, Pamela; Bohmann, David J; Watson, Susan R; Beaber, John W; Reddy, Nithin; Kuan, Hua-Feng; Bedinger, Daniel H; Rondon, Isaac J

    2013-05-31

    Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

  18. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells

    PubMed Central

    2013-01-01

    Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline. PMID:23802841

  19. 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

  20. 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

  1. Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates

    PubMed Central

    Catcott, Kalli C.; McShea, Molly A.; Bialucha, Carl Uli; Miller, Kathy L.; Hicks, Stuart W.; Saxena, Parmita; Gesner, Thomas G.; Woldegiorgis, Mikias; Lewis, Megan E.; Bai, Chen; Fleming, Michael S.; Ettenberg, Seth A.; Erickson, Hans K.; Yoder, Nicholas C.

    2016-01-01

    ABSTRACT Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates. PMID:26752675

  2. 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.

  3. Antibody Fragments as Probe in Biosensor Development

    PubMed Central

    Saerens, Dirk; Huang, Lieven; Bonroy, Kristien; Muyldermans, Serge

    2008-01-01

    Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed. PMID:27873779

  4. Platelet antibodies in idiopathic thrombocytopenic purpura.

    PubMed Central

    Veenhoven, W A; Van der Schans, G S; Nieweg, H O

    1980-01-01

    An immunofluorescence (IF) technique for the detection of antibodies was applied to idiopathic thrombocytopenic purpura (ITP). Serum platelet antibodies were found in thirteen out of twenty-two patients (59 percent) with active disease, but in only four out of fifteen patients (27 percent) who had attained remission. Direct tests for platelet-associated IgG were positive in 36 and 44 percent of these patients respectively. In two cases IgM was observed on the patients' platelet membranes. C3 was not detedted on patients' platelets. Platelet-associated IgG was also found in several other disorders and its occurrence is not therefore diagnostic of ITP. In addition, serum platelet antibodies do not indicate specifically ITP as they may also be due to previous isoimmunization. Antibodies in the sera of patients with ITP generally did not fix Clq and in most cases bound to platelets only in the presence of EDTA. In contrast, isoantibodies often fixed Clq and they had equal affinity for platelets suspended in ACD or EDTA plasma. This was confirmed by quantitative data on IgG binding by platelets obtained by measuring 125-I-labelled protein A uptake. The simplicity of the IF technique permits its routine application and the technique may give useful information with respect to the nature of the antibodies. It must, however, be considered of limited value in the diagnosis of ITP. PMID:6991171

  5. Use of antibody fragments (Fv) in immunocytochemistry.

    PubMed

    Kleymann, G; Ostermeier, C; Heitmann, K; Haase, W; Michel, H

    1995-06-01

    We developed a novel antibody fragment (Fv) technique for localization and determination of the surface topology of membrane protein complexes by immunogold electron microscopy. Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) raised against bacterial membrane proteins were established. The cDNAs coding for the variable domains of the MAbs were cloned and expressed in Escherichia coli. The engineered Fv fragments served as trifunctional adapter molecules. The Fv fragment binds to the epitope of the membrane protein. The Strep tag fused to the VH chain was used for one-step affinity purification of the Fv fragments. Immunological detection of the membrane protein-bound Fv fragments in electron microscopy was accomplished either via the Strep tag with colloidal gold-labeled streptavidin or via the c-myc tag, which was fused to the VL chain, in combination with the c-myc tag-specific antibody 9E10 and a colloidal gold-labeled secondary antibody. We examined four Fv fragments directed against the cytochrome c oxidase or the ubiquinol-cytochrome c oxidoreductase of Paracoccus denitrificans and bacteriorhodopsin of Halobacterium halobium to show that this method is generally applicable. In all cases the Fv fragments showed the same results as their corresponding parent antibodies in electron microscopic immunostaining and other applications.

  6. Neuronal Surface Antibody-Mediated Autoimmune Encephalitis

    PubMed Central

    Linnoila, Jenny J.; Rosenfeld, Myrna R.; Dalmau, Josep

    2016-01-01

    In the past few years, many autoimmune encephalitides have been identified, with specific clinical syndromes and associated antibodies against neuronal surface antigens. There is compelling evidence that many of these antibodies are pathogenic and most of these encephalitides are highly responsive to immunotherapies. The clinical spectra of some of these antibody-mediated syndromes, especially those reported in only a few patients, are evolving. Others, such as anti-N-methyl-D-aspartate (NMDA) receptor encephalitis, are well characterized. Diagnosis involves recognizing the specific syndromes and identifying the antibody in a patient’s cerebrospinal fluid (CSF) and/or serum. These syndromes are associated with variable abnormalities in CSF, magnetic resonance imaging, and electroencephalography. Treatment is often multidisciplinary and should be focused upon neutralizing the effects of antibodies and eliminating their source. Overlapping disorders have been noted, with some patients having more than one neurologic autoimmune disease. In other patients, viral infections such as herpes simplex virus encephalitis trigger robust antineuronal autoimmune responses. PMID:25369441

  7. Adaptive responses to antibody based therapy.

    PubMed

    Rodems, Tamara S; Iida, Mari; Brand, Toni M; Pearson, Hannah E; Orbuch, Rachel A; Flanigan, Bailey G; Wheeler, Deric L

    2016-02-01

    Receptor tyrosine kinases (RTKs) represent a large class of protein kinases that span the cellular membrane. There are 58 human RTKs identified which are grouped into 20 distinct families based upon their ligand binding, sequence homology and structure. They are controlled by ligand binding which activates intrinsic tyrosine-kinase activity. This activity leads to the phosphorylation of distinct tyrosines on the cytoplasmic tail, leading to the activation of cell signaling cascades. These signaling cascades ultimately regulate cellular proliferation, apoptosis, migration, survival and homeostasis of the cell. The vast majority of RTKs have been directly tied to the etiology and progression of cancer. Thus, using antibodies to target RTKs as a cancer therapeutic strategy has been intensely pursued. Although antibodies against the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) have shown promise in the clinical arena, the development of both intrinsic and acquired resistance to antibody-based therapies is now well appreciated. In this review we provide an overview of the RTK family, the biology of EGFR and HER2, as well as an in-depth review of the adaptive responses undertaken by cells in response to antibody based therapies directed against these receptors. A greater understanding of these mechanisms and their relevance in human models will lead to molecular insights in overcoming and circumventing resistance to antibody based therapy.

  8. Going adaptive: the saga of antibodies.

    PubMed

    Danilova, Nadia; Amemiya, Chris T

    2009-06-01

    Because of their extreme importance to human health, we probably know more about the structure and function of antibodies than practically any other molecule. Despite all the knowledge that has been accrued in the understanding of antibodies, modern approaches, especially comparative genomics, continue to yield novel findings regarding their underlying biology and evolution. In this review, we describe recent research that led to these revelations, and discuss the broad evolutionary implications of these findings. We have restricted our discussion to three vignettes. Considerable attention has been paid to the recent discovery that the teleost IgH locus is highly similar in organization to the Tcra-Tcrd locus, implicating an evolutionary common ancestor and parallels between the functions of B and T cells during development. Second, we discuss how a new type of antibody, recently discovered in jawless vertebrates, composed not of immunoglobulins but leucine-rich repeats, sheds new light on the overall forces driving evolution of all adaptive antigen receptors. Lastly, we discuss how accumulation of genomic sequences of various human subpopulations leads to better understanding of the directionality of antibody evolution. There is always more to learn from the unfolding saga of antibodies.

  9. Detection of antibodies against paramyxoviruses in tortoises.

    PubMed

    Rösler, Reinhild; Abbas, Maha Diekan; Papp, Tibor; Marschang, Rachel E

    2013-06-01

    Sera from a total of 202 tortoises from six countries and nine species were tested for antibodies against four different reptilian paramyxoviruses (ferlaviruses, ferlaVs) by hemagglutination inhibition (HI) test. The viruses used were a tortoise PMV (tPMV) and three squamatid PMV isolates, each belonging to a different subgroup of ferlaV within the genus Ferlavirus. HI tests revealed that antibodies against ferlaVs occurred regularly in the tested samples (5.5%). One and a half percent of the tested samples have measurable antibody titers against the group A isolate, 3% had antibodies against the group B isolate, and 1% had antibodies against the group C isolate. The significantly highest number of positive reactions was detected against the tortoise isolate (5%). Most of the animals that tested positive for one of the snake isolates also tested positive in HI assays with the tortoise isolate. Of the samples from different origins, the sera from Great Britain showed the highest percentage of positive tested animals (10.3%, n = 39), followed by those from Spain (10%, n = 10), while none of the samples from Madagascar or Italy scored positive. Since in most cases animals from one country came from the same collection, this does not represent the real prevalence of ferlaV in tortoises in these countries but rather indicates that ferlaVs occur in a number of different countries and tortoise species.

  10. 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.

  11. Antibodies to selected minor target antigens in patients with anti-neutrophil cytoplasmic antibodies (ANCA).

    PubMed

    Talor, M V; Stone, J H; Stebbing, J; Barin, J; Rose, N R; Burek, C L

    2007-10-01

    In patients with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, indirect immunofluorescence (IF) distinguishes between cytoplasmic (C-ANCA) and perinuclear (P-ANCA) neutrophil staining patterns. In patients with primary systemic vasculitis such as Wegener's granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, these IF staining patterns correspond broadly with antibodies to the two major antigens: the C-ANCA pattern is associated generally with antibodies to serine protease 3 (PR3) and the P-ANCA pattern with antibodies to myeloperoxidase (MPO). However, some sera positive for ANCA by IF are negative for anti-PR3 and anti-MPO antibodies, suggesting the presence of antibodies to minor antigens of PMN granules. We tested sera from a previously well-defined clinical cohort of patients for antibodies to four possible minor antigens: bactericidal permeability increasing protein, elastase, cathepsin G and lactoferrin. IF-positive (+) sera had significantly higher antibody frequencies to the minor antigens than did the IF-negative (-) sera (P < 0.01). Patients with IF(+) PR3(-)MPO(-) sera showed the most varied reactivity to the minor antigens. Among the IF(+) groups, the IF(+) PR3(+)/MPO(-) sera showed the lowest reactivity to the minor antigens. Patients with well-defined ANCA specificities, e.g. the PR3-ANCA response associated with Wegener's granulomatosis, are less likely than are other patient subsets to have antibodies to minor antigen targets. Autoantibodies to these minor antigens contribute to the overall pattern of ANCA identified by IF and help to explain why the correlation between IF and enzyme immunoassays show discrepancies. While the pathophysiological significance of antibodies to minor target antigens needs further evaluation, they may be markers of inflammation associated with disease processes.

  12. Method for preparation of single chain antibodies

    SciTech Connect

    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.

  13. Current status of antibody therapy in ALL.

    PubMed

    Ai, Jing; Advani, Anjali

    2015-02-01

    Despite the significant advances in modern chemotherapy, it remains challenging to treat adult patients with acute lymphoblastic leukaemia (ALL). The relapse rate remains high, and the outcome at the time of relapse is dismal. Antibody-based therapies have demonstrated promising results in this patient group. Variable mechanisms have been applied to target surface antigens (CD20 [also termed MS4A1], CD22, CD52 and CD19) that are commonly expressed on malignant leukaemia cells. In this review, we will focus on the clinical application of such therapies in adult ALL, including the naked antibodies: Rituximab, Ofatumumab, Epratuzumab and Alemtuzumab; the immunotoxins: BL22 and Combotox; the immunoconjugates: inotuzumab and SAR 3419; as well as the Bi-specific T cell engaging (BiTE)-specific antibody, Blinatumomab.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Adsorption of monoclonal antibodies to glass microparticles.

    PubMed

    Hoehne, Matthew; Samuel, Fauna; Dong, Aichun; Wurth, Christine; Mahler, Hanns-Christian; Carpenter, John F; Randolph, Theodore W

    2011-01-01

    Microparticulate glass represents a potential contamination to protein formulations that may occur as a result of processing conditions or glass types. The effect of added microparticulate glass to formulations of three humanized antibodies was tested. Under the three formulation conditions tested, all three antibodies adsorbed irreversibly at near monolayer surface coverages to the glass microparticles. Analysis of the secondary structure of the adsorbed antibodies by infrared spectroscopy reveal only minor perturbations as a result of adsorption. Likewise, front-face fluorescence quenching measurements reflected minimal tertiary structural changes upon adsorption. In contrast to the minimal effects on protein structure, adsorption of protein to suspensions of glass microparticles induced significant colloidal destabilization and flocculation of the suspension.

  19. Are natural antibodies involved in tumour defence?

    PubMed

    Bohn, J

    1999-09-01

    Natural antibodies (NAb) are found in the serum of healthy individuals. These antibodies are produced without any apparent specific antigenic stimulation. They are one part of the circulating immunoglobulins and are found in virtually all vertebrate species. NAb react to various self- and non-self antigens. A protective function in different infection models could be demonstrated. Several groups have reported the ability of NAb to bind to tumour cells. Their possible role in tumour defence is documented in mice. The present status of attempts to characterise the role of NAb in tumour defence is discussed, particularly as regards the human immune system. This paper focuses on antibody cell interactions and discusses the genetic background of the Nab-producing B-cells.

  20. Sensitive neutralization test for rubella antibody.

    PubMed Central

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  1. 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.

  2. Antibody-based bacterial toxin detection

    NASA Astrophysics Data System (ADS)

    Menking, Darrell E.; Heitz, Jonathon M.; Anis, Nabil A.; Thompson, Roy G.

    1994-03-01

    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 one step assay, antibodies against Cholera toxin or Staphylococcus Enterotoxin B were noncovalently immobilized on quartz fibers and probed with fluorescein-isothiocyanate (FITC)-labeled toxins. In the two-step assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified antitoxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or antitoxin IgG in a dose-dependent manner and the detection of the toxins was in the nanomolar range.

  3. 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

  4. 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

  5. Quantitative cumulative biodistribution of antibodies in mice

    PubMed Central

    Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

    2014-01-01

    The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level. PMID:24572100

  6. Process economics of industrial monoclonal antibody manufacture.

    PubMed

    Farid, Suzanne S

    2007-03-15

    Pressures for cost-effective manufacture of antibodies are growing given their high doses and increasing market potential that have resulted in significant increases in total site capacities of up to 200,000 L. This paper focuses on the process economic issues associated with manufacturing antibodies and reviews the cost studies published in the literature; many of the issues highlighted are not only specific to antibodies but also apply to recombinant proteins. Data collated at UCL suggest current benchmark investment costs of $660-$1580/ft2 ($7130-$17,000/m2) and $1765-$4220/L for antibody manufacturing facilities with total site capacities in the range of 20,000-200,000 L; the limitations of the data are highlighted. The complications with deriving benchmark cost of goods per gram (COG/g) values are discussed, stressing the importance of stating the annual production rate and either titre or fermentation capacity with the cost so as to allow comparisons. The uses and limitations of the methods for cost analysis and the available software tools for process economics are presented. Specific examples found in the literature of process economic studies related to antibody manufacture for different expression systems are reviewed. The key economic drivers are identified; factors such as fermentation titre and overall yield are critical determinants of economic success. Future trends in antibody manufacture that are driven by economic pressures are discussed, such as the use of alternative expression systems (e.g. transgenics, E. coli and yeast), disposables, and improvements to downstream technology. The hidden costs and the challenges in each case are highlighted.

  7. 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.

  8. Utility of feline coronavirus antibody tests.

    PubMed

    Addie, Diane D; le Poder, Sophie; Burr, Paul; Decaro, Nicola; Graham, Elizabeth; Hofmann-Lehmann, Regina; Jarrett, Oswald; McDonald, Michael; Meli, Marina L

    2015-02-01

    Eight different tests for antibodies to feline coronavirus (FCoV) were evaluated for attributes that are important in situations in veterinary practice. We compared four indirect immunofluorescent antibody tests (IFAT), one enzyme-linked immunosorbent assay (ELISA) (FCoV Immunocomb; Biogal) and three rapid immunochromatographic (RIM) tests against a panel of samples designated by consensus as positive or negative. Specificity was 100% for all but the two IFATs based on transmissible gastroenteritis virus (TGEV), at 83.3% and 97.5%. The IFAT and ELISA tests were best for obtaining an antibody titre and for working in the presence of virus. The RIM tests were the best for obtaining a result quickly (10-15 mins); of these, the Speed F-Corona was the most sensitive, at 92.4%, followed by FASTest feline infectious peritonitis (FIP; 84.6%) and Anigen Rapid FCoV antibody test (64.1%). Sensitivity was 100% for the ELISA, one FCoV IFAT and one TGEV IFAT; and 98.2% for a second TGEV IFA and 96.1% for a second FCoV IFAT. All tests worked with effusions, even when only blood products were stipulated in the instruction manual. The ELISA and Anigen RIM tests were best for small quantities of sample. The most appropriate FCoV antibody test to use depends on the reason for testing: in excluding a diagnosis of FIP, sensitivity, specificity, small sample quantity, rapidity and ability to work in the presence of virus all matter. For FCoV screening, speed and sensitivity are important, and for FCoV elimination antibody titre is essential.

  9. 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

  10. Basic immunology of antibody targeted radiotherapy

    SciTech Connect

    Wong, Jeffrey Y.C. . E-mail: jwong@coh.org

    2006-10-01

    Antibody targeted radiotherapy brings an important new treatment modality to Radiation oncology clinic. Radiation dose to tumor and normal tissues are determined by a complex interplay of antibody, antigen, tumor, radionuclide, and host-related factors. A basic understanding of these immunologic and physiologic factors is important to optimally utilize this therapy in the clinic. Preclinical and clinical studies need to be continued to broaden our understanding and to develop new strategies to further improve the efficacy of this promising form of targeted therapy.

  11. 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.

  12. (Auto)antibodies in inflammatory bowel diseases.

    PubMed

    Vermeire, Severine; Vermeulen, Nathalie; Van Assche, Gert; Bossuyt, Xavier; Rutgeerts, Paul

    2008-06-01

    Patients who have inflammatory bowel diseases (IBD) express strong antibody responses to a variety of epitopes. A number of (auto)antibodies have been described in patients who have Crohn's disease or ulcerative colitis. These markers reflect a loss of tolerance toward bacterial and fungal flora and have been studied for their clinical value in IBD patients. However, currently, they have no place in the diagnostic work up. Their real promise may lie in their use as surrogate markers of complicated aggressive disease as shown in various retrospective studies, but prospective data are lacking.

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

    PubMed Central

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

    2012-01-01

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

  14. 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.

  15. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  16. Therapeutic assessment of SEED: a new engineered antibody platform designed to generate mono- and bispecific antibodies.

    PubMed

    Muda, Marco; Gross, Alec W; Dawson, Jessica P; He, Chaomei; Kurosawa, Emmi; Schweickhardt, Rene; Dugas, Melanie; Soloviev, Maria; Bernhardt, Anna; Fischer, David; Wesolowski, John S; Kelton, Christie; Neuteboom, Berend; Hock, Bjoern

    2011-05-01

    The strand-exchange engineered domain (SEED) platform was designed to generate asymmetric and bispecific antibody-like molecules, a capability that expands therapeutic applications of natural antibodies. This new protein engineered platform is based on exchanging structurally related sequences of immunoglobulin within the conserved CH3 domains. Alternating sequences from human IgA and IgG in the SEED CH3 domains generate two asymmetric but complementary domains, designated AG and GA. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of AG and GA SEED CH3 domains. Using a clinically validated antibody (C225), we tested whether Fab derivatives constructed on the SEED platform retain desirable therapeutic antibody features such as in vitro and in vivo stability, favorable pharmacokinetics, ligand binding and effector functions including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. In addition, we tested SEED with combinations of binder domains (scFv, VHH, Fab). Mono- and bivalent Fab-SEED fusions retain full binding affinity, have excellent biochemical and biophysical stability, and retain desirable antibody-like characteristics conferred by Fc domains. Furthermore, SEED is compatible with different combinations of Fab, scFv and VHH domains. Our assessment shows that the new SEED platform expands therapeutic applications of natural antibodies by generating heterodimeric Fc-analog proteins.

  17. Heterophile antibodies in the serum of children with nephrotic syndrome.

    PubMed

    Satoh, P S; Elberg, A J; Fleming, W E; Baluarte, H J; Gruskin, A B

    1980-09-01

    Serum of children with the nephrotic syndrome contained high titers of a (19S) IgM antibody against sheep, horse, guinea pig, rat, and rabbit red blood cells but not against cow red blood cells. There was high correlation between high titers of antisheep antibodies and active nephrotic syndrome in the children with minimal change nephrotic syndrome. The antibody differed from the Paul-Bunnell antibody found in patients with infectious mononucleosis and from the anti-Forssman, Hangautziu-Deicher antibody found in patients with horse serum sickness. Rabbit red blood cells completely absorbed the antibody, but horse or guinea pig red blood cells removed only the anti-Forssman activity.

  18. Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies to Toxoplasma gondii

    PubMed Central

    Hackett, John; Hoff-Velk, Jane; Golden, Alan; Brashear, Jeff; Robinson, John; Rapp, Margaret; Klass, Michael; Ostrow, David H.; Mandecki, Wlodek

    1998-01-01

    In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays. PMID:9574691

  19. 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.

  20. Mouse x pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies.

    PubMed

    Jar, Ana M; Osorio, Fernando A; López, Osvaldo J

    2009-01-01

    The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse x pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs.