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Sample records for affinity human antibodies

  1. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  2. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  3. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-03-03

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  4. The identification of affinity peptide ligands specific to the variable region of human antibodies.

    PubMed

    Akiyama, Yasuto; Miyata, Haruo; Komiyama, Masaru; Nogami, Masahiro; Ozawa, Kazumichi; Oshita, Chie; Kume, Akiko; Ashizawa, Tadashi; Sakura, Naoki; Mochizuki, Tohru; Yamaguchi, Ken

    2014-01-01

    Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becoming the dominant focus of clinical research. In particular, smaller recombinant antibody fragments such as single-chain variable fragments (scFv) have become the subject of intense focus. However, an efficient affinity ligand for antibody fragment purification has not been developed. In the present study, we designed a consensus sequence for the human antibody heavy or light chain-variable regions (Fv) based on the antibody sequences available in the ImMunoGeneTics information system (IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screened peptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody using a 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VH template and 8 peptides for the VK template were selected as the candidate ligands after 4 rounds of panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 and code-13 peptides showed recovery rates of the VH and VK templates that were 20-30% and 40-50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately 40%). If it were possible to identify the best combination of VH and VK-binding peptides among the ligand peptides suitable for the human mAb Fv sequence, the result could be a promising purification tool that might greatly improve the cost efficiencies of the purification process.

  5. Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis.

    PubMed

    Lippok, Svenja; Seidel, Susanne A I; Duhr, Stefan; Uhland, Kerstin; Holthoff, Hans-Peter; Jenne, Dieter; Braun, Dieter

    2012-04-17

    The direct quantification of both the binding affinity and absolute concentration of disease-related biomarkers in biological fluids is particularly beneficial for differential diagnosis and therapy monitoring. Here, we extend microscale thermophoresis to target immunological questions. Optically generated thermal gradients were used to deplete fluorescently marked antigens in 2- and 10-fold-diluted human serum. We devised and validated an autocompetitive strategy to independently fit the concentration and dissociation constant of autoimmune antibodies against the cardiac β1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen, the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibody concentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding in its native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix and probe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine.

  6. Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.

    PubMed

    Ding, Ling; Azam, Mark; Lin, Yu-Huei; Sheridan, James; Wei, Shuanghong; Gupta, Gigi; Singh, Rakesh K; Pauling, Michelle H; Chu, Waihei; Tran, Antares; Yu, Nai-Xuan; Hu, Jiefeng; Wang, Wei; Long, Hao; Xiang, Dong; Zhu, Li; Hua, Shao-Bing

    2010-10-01

    We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.

  7. 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...

  8. Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.

    PubMed

    Kim, Jin Hong; Gripon, Philippe; Bouezzedine, Fidaa; Jeong, Mun Sik; Chi, Seung-Wook; Ryu, Seong-Eon; Hong, Hyo Jeong

    2015-01-16

    To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)-specific preS1 humanized antibody (HzKR127), we further humanized it through specificity-determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy-chain complementarity-determining regions (CDR), on the basis of the crystal structure of the antigen-antibody complex. HzKR127-3.2 exhibited 2.5-fold higher affinity and enhanced virus-neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus-neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127-3.2 may be a good candidate for HBV immunoprophylaxis.

  9. Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

    PubMed

    Keck, Zhen-Yong; Wang, Yong; Lau, Patrick; Lund, Garry; Rangarajan, Sneha; Fauvelle, Catherine; Liao, Grant C; Holtsberg, Frederick W; Warfield, Kelly L; Aman, M Javad; Pierce, Brian G; Fuerst, Thomas R; Bailey, Justin R; Baumert, Thomas F; Mariuzza, Roy A; Kneteman, Norman M; Foung, Steven K H

    2016-12-01

    Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation.

  10. Limits for antibody affinity maturation and repertoire diversification in hypervaccinated humans.

    PubMed

    Poulsen, Tine Rugh; Jensen, Allan; Haurum, John S; Andersen, Peter S

    2011-10-15

    The immune system is known to generate a diverse panel of high-affinity Abs by adaptively improving the recognition of pathogens during ongoing immune responses. In this study, we report the biological limits for Ag-driven affinity maturation and repertoire diversification by analyzing Ab repertoires in two adult volunteers after each of three consecutive booster vaccinations with tetanus toxoid. Maturation of on-rates and off-rates occurred independently, indicating a kinetically controlled affinity maturation process. The third vaccination induced no significant changes in the distribution of somatic mutations and binding rate constants implying that the limits for affinity maturation and repertoire diversification had been reached. These fully matured Ab repertoires remained similar in size, genetically diverse, and dynamic. Somatic mutations and kinetic rate constants showed normal and log-normal distribution profiles, respectively. Mean values can therefore be considered as biological constants defining the observed boundaries. At physiological temperature, affinity maturation peaked at k(on) = 1.6 × 10(4) M(-1) s(-1) and k(off) = 1.7 × 10(-4) s(-1) leading to a maximum mean affinity of K(D) = 1.0 × 10(-9) M. At ambient temperature, the average affinity increased to K(D) = 3.4 × 10(-10) M mainly due to slower off-rates. This experimentally determined set of constants can be used as a benchmark for analysis of the maturation level of human Abs and Ab responses.

  11. Monoclonal antibodies to human interferon-gamma: production, affinity purification and radioimmunoassay.

    PubMed Central

    Novick, D; Eshhar, Z; Fischer, D G; Friedlander, J; Rubinstein, M

    1983-01-01

    Human interferon-gamma (IFN-gamma) purified to electrophoretic homogeneity by a cation exchange h.p.l.c., was used for the development of monoclonal antibodies. Following immunization, spleen lymphocytes of two mice showing the highest binding and neutralizing titers were isolated, fused with NSO mouse myeloma cells and cloned. The screening of hybridomas was based on precipitation of the immune complexes with a second antibody and recovery of the biological activity of IFN-gamma from the precipitate. Twenty nine independent hybridomas secreting antibodies specific to IFN-gamma were obtained. Twelve out of these 29 hybridomas produced antibodies that neutralized the antiviral activity of pure as well as crude IFN-gamma. Moreover, IFN-gamma obtained by various induction procedures was neutralized as well, indicating that these various IFN-gamma subtypes are immunologically cross-reactive. Immune precipitation of partially purified 125I-labelled IFN-gamma by several monoclonal antibodies revealed two protein bands of 26,000 and 21,000 daltons. Immunoaffinity chromatography of IFN-gamma gave a 50-fold purification to a specific activity > or = 4 x 10(7) units/mg. Two of the monoclonal antibodies were found suitable for a sensitive and rapid double antibody solid-phase radioimmunoassay, allowing the detection of IFN-gamma at concentrations of at least 4 ng/ml (150 units/ml) within 8 h. Images Fig. 1. Fig. 2. PMID:11892806

  12. An illustration of the clinical relevance of detecting human antimouse antibody interference by affinity chromatography.

    PubMed

    Koper, N P; Massuger, L F; Thomas, C M; Beyer, C; Crooy, M J

    1999-10-01

    Elevated Cancer antigen 125 (CA 125) serum concentrations (up to 221 kU/1) were measured in a 39 year old woman with a positive family history of breast cancer. The serum determinations were performed with the automated Immulite OM-MA chemiluminescent enzyme immunoassay system (Diagnostic Products). Laparoscopic evaluation of the ovaries did not reveal any abnormalities. CA 125 measurements in the same patient using the automated IMx immunoassay system (Abbott) demonstrated normal serum levels. Using a previously reported chromatography procedure IgG type human antimouse antibody activity was found to be present in the serum samples explaining the falsely elevated levels. To prevent this interference the manufacturer modified the assay system by replacing the monoclonal M11 detection antibody with a rabbit polyclonal antibody. Using the modified OM-MA CA 125 assay results were comparable with the IMx values.

  13. Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1.

    PubMed

    Douthwaite, Julie A; Sridharan, Sudharsan; Huntington, Catherine; Hammersley, Jayne; Marwood, Rose; Hakulinen, Jonna K; Ek, Margareta; Sjögren, Tove; Rider, David; Privezentzev, Cyril; Seaman, Jonathan C; Cariuk, Peter; Knights, Vikki; Young, Joyce; Wilkinson, Trevor; Sleeman, Matthew; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2015-01-01

    Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding VH CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long VH CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long VH CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.

  14. Human Monoclonal Antibody Fragments Binding to Insulin-like Growth Factors 1 and 2 with Picomolar Affinity

    PubMed Central

    Zhao, Qi; Feng, Yang; Zhu, Zhongyu; Dimitrov, Dimiter S.

    2011-01-01

    The type 1 insulin-like growth factor receptor (IGF1R) and its ligands (IGF1 and IGF2) have been implicated in a variety of physiological processes and in diseases such as cancer. In addition to IGF1R, IGF2 also activates the insulin receptor (IR) isoform A and therefore antibodies against IGF2 can inhibit cell proliferation mediated by the signaling through both IGF1R and IR triggered by IGF2. We identified a new human monoclonal antibody (mAb), m708.2, which bound to IGF1 and IGF2 but not to insulin. m708.2 potently inhibited signal transduction mediated by the interaction of IGF1 or IGF2 with the IGF1R and IGF2 with the IR. It also inhibited the growth of the breast cancer cell line MCF-7. An affinity-matured derivative of m708.2, m708.5, bound to IGF1 with equilibrium dissociation constant, KD = 200 pM and to IGF2 with KD = 60 pM. m708.5 inhibited signal transduction mediated by IGF1 and IGF2 and cancer cell growth more potently than m708.2. These results suggest that m708.5 could have potential as a candidate therapeutic for cancers driven by the IGF1,2 interactions with IGF1R and IR. PMID:21750218

  15. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  16. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  17. 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.

  18. 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.

  19. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  20. Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

    PubMed

    Merten, O W; Reiter, S; Scheirer, W; Katinger, H

    1983-01-01

    The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

  1. Selective targeting of the IL23 pathway: Generation and characterization of a novel high-affinity humanized anti-IL23A antibody

    PubMed Central

    Singh, Sanjaya; Kroe-Barrett, Rachel R; Canada, Keith A; Zhu, Xiang; Sepulveda, Eliud; Wu, Helen; He, Yaqin; Raymond, Ernest L; Ahlberg, Jennifer; Frego, Lee E; Amodeo, Laura M; Catron, Katrina M; Presky, David H; Hanke, Jeffrey H

    2015-01-01

    Herein, we describe the generation and characterization of BI 655066, a novel, highly potent neutralizing anti-interleukin-23 (IL23) monoclonal antibody in clinical development for autoimmune conditions, including psoriasis and Crohn's disease. IL23 is a key driver of the differentiation, maintenance, and activity of a number of immune cell subsets, including T helper 17 (Th17) cells, which are believed to mediate the pathogenesis of several immune-mediated disorders. Thus, IL23 neutralization is an attractive therapeutic approach. Designing an antibody for clinical activity and convenience for the patient requires certain properties, such as high affinity, specificity, and solubility. These properties were achieved by directed design of the immunization, lead identification, and humanization procedures. Favorable substance and pharmacokinetic properties were established by biophysical assessments and studies in cynomolgus monkeys. PMID:25905918

  2. Naturally occurring human phosphorylcholine antibodies are predominantly products of affinity-matured B cells in the adult.

    PubMed

    Fiskesund, Roland; Steen, Johanna; Amara, Khaled; Murray, Fiona; Szwajda, Agnieszka; Liu, Anquan; Douagi, Iyadh; Malmström, Vivianne; Frostegård, Johan

    2014-05-15

    Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

  3. Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

    PubMed

    Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

    2007-02-01

    Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

  4. Isoelectric focusing-affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Roebber, Marianne; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    Isoelectric focusing (IEF)/affinity immunoblotting and enzyme-linked immunosorbent assay (ELISA) were used for parallel analysis of murine monoclonal antihuman IgG-subclass antisera (MoAbs). Coomassie Blue-stained protein bands in the pH region 5.5-8.0 were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated antimouse IgG. Use of IgG myeloma antigen-coated nitrocellulose in the IEF-affinity immunoblot allowed detection of the charge microheterogeneity of MoAbs. The MoAb group contained one to five major dense bands flanked by up to four minor fainter bands, all with pIs ranging from 6.1 to 7.8. Semiquantitative estimates of binding specificity in the IEF-affinity blot compared well with cross-reactivity data obtained from a quantitative ELISA.

  5. A High-Affinity Native Human Antibody Disrupts Biofilm from Staphylococcus aureus Bacteria and Potentiates Antibiotic Efficacy in a Mouse Implant Infection Model

    PubMed Central

    Estellés, Angeles; Woischnig, Anne-Kathrin; Liu, Keyi; Stephenson, Robert; Lomongsod, Evelene; Nguyen, Da; Zhang, Jianzhong; Heidecker, Manfred; Yang, Yifan; Simon, Reyna J.; Tenorio, Edgar; Ellsworth, Stote; Leighton, Anton; Ryser, Stefan; Gremmelmaier, Nina Khanna

    2016-01-01

    Many serious bacterial infections are difficult to treat due to biofilm formation, which provides physical protection and induces a sessile phenotype refractory to antibiotic treatment compared to the planktonic state. A key structural component of biofilm is extracellular DNA, which is held in place by secreted bacterial proteins from the DNABII family: integration host factor (IHF) and histone-like (HU) proteins. A native human monoclonal antibody, TRL1068, has been discovered using single B-lymphocyte screening technology. It has low-picomolar affinity against DNABII homologs from important Gram-positive and Gram-negative bacterial pathogens. The disruption of established biofilm was observed in vitro at an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068 in vivo was evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistant Staphylococcus aureus (MRSA; ATCC 43300). Treatment of the established biofilm by combination therapy of TRL1068 (15 mg/kg of body weight, intraperitoneal [i.p.] administration) with daptomycin (50 mg/kg, i.p.) significantly reduced adherent bacterial count compared to that after daptomycin treatment alone, accompanied by significant reduction in planktonic bacterial numbers. The quantification of TRL1068 in sample matrices showed substantial penetration of TRL1068 from serum into the cage interior. TRL1068 is a clinical candidate for combination treatment with standard-of-care antibiotics to overcome the drug-refractory state associated with biofilm formation, with potential utility for a broad spectrum of difficult-to-treat bacterial infections. PMID:26833157

  6. Generation of recombinant antibodies and means for increasing their affinity.

    PubMed

    Altshuler, E P; Serebryanaya, D V; Katrukha, A G

    2010-12-01

    Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.

  7. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  8. Affinity maturation of antibodies requires integrity of the adult thymus.

    PubMed

    AbuAttieh, Mouhammed; Bender, Diane; Liu, Esther; Wettstein, Peter; Platt, Jeffrey L; Cascalho, Marilia

    2012-02-01

    The generation of B-cell responses to proteins requires a functional thymus to produce CD4(+) T cells which helps in the activation and differentiation of B cells. Because the mature T-cell repertoire has abundant cells with the helper phenotype, one might predict that in mature individuals, the generation of B-cell memory would proceed independently of the thymus. Contrary to that prediction, we show here that the removal of the thymus after the establishment of the T-cell compartment or sham surgery without removal of the thymus impairs the affinity maturation of antibodies. Because removal or manipulation of the thymus did not decrease the frequency of mutation of the Ig variable heavy chain exons encoding antigen-specific antibodies, we conclude that the thymus controls affinity maturation of antibodies in the mature individual by facilitating the selection of B cells with high-affinity antibodies.

  9. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  10. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  11. Heteroantibody-mediated cytotoxicity: antibody to the high affinity Fc receptor for IgG mediates cytotoxicity by human monocytes that is enhanced by interferon-gamma and is not blocked by human IgG.

    PubMed

    Shen, L; Guyre, P M; Anderson, C L; Fanger, M W

    1986-12-01

    An IgG1 monoclonal antibody, 32.2, raised against the 72,000 dalton monocyte high affinity Fc receptor, was used to examine the role of this receptor in ADCC. This antibody did not inhibit the binding of human IgG1 to monocytes or to the U937 cell line, nor did it block or stimulate their killing of IgG-coated chicken erythrocytes (CE). Whole 32.2 or its Fab fragments were cross-linked to Fab fragments of rabbit anti-CE by using the agent SPDP. The resulting heteroantibodies (32.2 X Fab anti-CE) mediated monocyte and U937 cytotoxicity against CE, whereas an anti-HLA X anti-CE reagent did not. Both FcR expression and heteroantibody-mediated cytotoxicity were increased by culturing monocytes or U937 with IFN-gamma. Although IgG-mediated ADCC was significantly inhibited by 40 micrograms/ml human IgG1, cytotoxicity mediated by 32.2 X Fab anti-CE was not blocked by 2 mg/ml human IgG1, suggesting that such cytotoxicity might not be blocked by IgG in vivo. These data indicate the potential of 32.2 heteroantibodies in analysis of FcR function and in therapy.

  12. The use of ammonium sulphate precipitation method for the determination of antigen-binding capacity and affinity of anti-tetanus antibodies in human serum.

    PubMed

    Alausa, O K

    1975-01-01

    Radioimmunoassay of anti-tetanus antibodies produced in human serum in response to tetanus toxoid immunization was carried out by employing the non-specific precipitation effect of ammonium sulphate described by Farr (1958). The results showed that the method was sensitive and was able to differentiate between immune and non-immune persons. The effects of booster dose injection on the quantity and quality of antibodies produced during immunization were discussed, and the possible use of the method to predict the amount of immunogen, the timing and number of injections required for optimal host response in immunization schedules was suggested.

  13. 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.

  14. 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

  15. Solution Equilibrium Titration for High-Throughput Affinity Estimation of Unpurified Antibodies and Antibody Fragments.

    PubMed

    Della Ducata, Daniela; Jaehrling, Jan; Hänel, Cornelia; Satzger, Marion; Wolber, Meike; Ostendorp, Ralf; Pabst, Stefan; Brocks, Bodo

    2015-12-01

    The generation of therapeutic antibodies with extremely high affinities down to the low picomolar range is today feasible with state-of-the art recombinant technologies. However, reliable and efficient identification of lead candidates with the desired affinity from a pool of thousands of antibody clones remains a challenge. Here, we describe a high-throughput procedure that allows reliable affinity screening of unpurified immunoglobulin G or antibody fragments. The method is based on the principle of solution equilibrium titration (SET) using highly sensitive electrochemiluminescence as a readout system. Because the binding partners are not labeled, the resulting KD represents a sound approximation of the real affinity. For screening, diluted bacterial lysates or cell culture supernatants are equilibrated with four different concentrations of a soluble target molecule, and unbound antibodies are subsequently quantified on 384-well Meso Scale Discovery (MSD) plates coated with the respective antigen. For determination of KD values from the resulting titration curves, fit models deduced from the law of mass action for 1:1 and 2:1 binding modes are applied to assess hundreds of interactions simultaneously. The accuracy of the method is demonstrated by comparing results from different screening campaigns from affinity optimization projects with results from detailed affinity characterization.

  16. Mitogenicity and down-regulation of high-affinity interleukin 2 receptor by YTA-1 and YTA-2, monoclonal antibodies that recognize 75-kDa molecules on human large granular lymphocytes.

    PubMed Central

    Nakamura, Y; Inamoto, T; Sugie, K; Masutani, H; Shindo, T; Tagaya, Y; Yamauchi, A; Ozawa, K; Yodoi, J

    1989-01-01

    A large number of interleukin 2 receptors lacking the Tac epitope (IL-2R/p75) were found to be constitutively expressed on the human large granular lymphocyte/natural killer cell line YT, which bears inducible IL-2R/p55 associated with Tac antigen. Two anti-YT IgG1 monoclonal antibodies, YTA-1 and YTA-2, recognizing different epitopes of the same 75- to 80-kDa molecule, were established. The 75-kDa antigen recognized by these monoclonal antibodies was strongly expressed on the large granular lymphocytes of normal peripheral blood mononuclear cells and on various lymphoid cell lines bearing IL-2R/p75. The YTA-1 and YTA-2 antibodies were mitogenic and were different from other mitogenic monoclonal antibodies such as anti-T3 (CD3), anti-T11 (CD2), and KOLT-2 (CD28). Further, they down-regulated the high-affinity IL-2R of peripheral blood mononuclear cells within 24 hr in culture. The relationship between the YTA-1/2 antigen and the IL-2R system is discussed. Images PMID:2465549

  17. The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas.

    PubMed Central

    Velders, M. P.; van Rhijn, C. M.; Oskam, E.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

    1998-01-01

    Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease. PMID:9716030

  18. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200.

    PubMed

    Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom; Machado, Camila Maria L; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.

  19. Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

    PubMed Central

    Lindegren, Sture; Andrade, Luciana N. S.; Bäck, Tom; Machado, Camila Maria L.; Horta, Bruno Brasil; Buchpiguel, Carlos; Moro, Ana Maria; Okamoto, Oswaldo Keith; Jacobsson, Lars; Cederkrantz, Elin; Washiyama, Kohshin; Aneheim, Emma; Palm, Stig; Jensen, Holger; Tuma, Maria Carolina B.; Chammas, Roger; Hultborn, Ragnar; Albertsson, Per

    2015-01-01

    The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with 211At-Rebmab200. Here, the biodistribution of 211At-Rebmab200 was evaluated, as was the utility of 99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that 99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics. PMID:25970341

  20. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  1. Efficient affinity maturation of antibody variable domains requires co-selection of compensatory mutations to maintain thermodynamic stability

    PubMed Central

    Julian, Mark C.; Li, Lijuan; Garde, Shekhar; Wilen, Rebecca; Tessier, Peter M.

    2017-01-01

    The ability of antibodies to accumulate affinity-enhancing mutations in their complementarity-determining regions (CDRs) without compromising thermodynamic stability is critical to their natural function. However, it is unclear if affinity mutations in the hypervariable CDRs generally impact antibody stability and to what extent additional compensatory mutations are required to maintain stability during affinity maturation. Here we have experimentally and computationally evaluated the functional contributions of mutations acquired by a human variable (VH) domain that was evolved using strong selections for enhanced stability and affinity for the Alzheimer’s Aβ42 peptide. Interestingly, half of the key affinity mutations in the CDRs were destabilizing. Moreover, the destabilizing effects of these mutations were compensated for by a subset of the affinity mutations that were also stabilizing. Our findings demonstrate that the accumulation of both affinity and stability mutations is necessary to maintain thermodynamic stability during extensive mutagenesis and affinity maturation in vitro, which is similar to findings for natural antibodies that are subjected to somatic hypermutation in vivo. These findings for diverse antibodies and antibody fragments specific for unrelated antigens suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability. PMID:28349921

  2. Comparison of monoclonal antibodies 17-1A and 323/A3: the influence of the affinity on tumour uptake and efficacy of radioimmunotherapy in human ovarian cancer xenografts.

    PubMed Central

    Kievit, E.; Pinedo, H. M.; Schlüper, H. M.; Haisma, H. J.; Boven, E.

    1996-01-01

    The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. Both MAbs are directed against the same pancarcinoma glycoprotein. In vitro, the number of binding sites on tumour cells at 4 degrees C was similar for both MAbs, but m-323/A3 had an approximately 5-fold higher affinity (1.3-3.0x10(9) M-1) than c-17-1A (3.0-5.4x10(8) M-1). This difference in affinity was more extreme at 37 degrees C, when no binding of c-17-1A could be observed. MAb m-323/A3 completely blocked binding of c-17-1A to tumour cells, whereas the reverse was not observed. Immunohistochemistry showed a similar but more intense staining pattern of m-323/A3 in human ovarian cancer xenografts than of c-17-1A. In vivo, the blood clearance in non-tumour-bearing nude mice was similar for both MAbs with terminal half-lives of 71.4 h for m-323/A3 and 62.7 h for c-17-1A. MAb m-323/A3 targeted better to tumour tissue, but was more heterogeneously distributed than c-17-1A. The cumulative absorbed radiation dose delivered by m-323/A3 to tumour tissue was 2.5- to 4.7-fold higher than that delivered by c-17-1A. When mice were treated with equivalent radiation doses of 131(I)m-323/A3 and 131(I)c-17-1A, based on a correction for the immunoreactivity of the radiolabelled MAbs, m-323/A3 induced a better growth inhibition in two of the three xenografts. When the radiation doses were adjusted to obtain a similar amount of radiation in the tumour c-17-1A was more effective in tumour growth inhibition in all three xenografts. Images Figure 3 Figure 4 PMID:8595159

  3. Generation of metastatic melanoma specific antibodies by affinity purification

    PubMed Central

    Schütz, Birgit; Koppensteiner, Anita; Schörghofer, David; Kinslechner, Katharina; Timelthaler, Gerald; Eferl, Robert; Hengstschläger, Markus; Missbichler, Albert; Hundsberger, Harald; Mikula, Mario

    2016-01-01

    Melanoma is the most aggressive type of skin cancer and one of the most frequent tumours in young adults. Identification of primary tumours prone to develop metastasis is of paramount importance for further patient stratification. However, till today, no markers exist that are routinely used to predict melanoma progression. To ameliorate this problem, we generated antiserum directed against metastatic melanoma tissue lysate and applied a novel approach to purify the obtained serum via consecutive affinity chromatography steps. The established antibody, termed MHA-3, showed high reactivity against metastatic melanoma cell lines both in vitro and in vivo. We also tested MHA-3 on 227 melanoma patient samples and compared staining with the melanoma marker S100b. Importantly, MHA-3 was able to differentiate between metastatic and non-metastatic melanoma samples. By proteome analysis we identified 18 distinct antigens bound by MHA-3. Combined expression profiling of all identified proteins revealed a significant survival difference in melanoma patients. In conclusion, we developed a polyclonal antibody, which is able to detect metastatic melanoma on paraffin embedded sections. Hence, we propose that this antibody will represent a valuable additional tool for precise melanoma diagnosis. PMID:27853253

  4. Humanized Antibodies for Antiviral Therapy

    NASA Astrophysics Data System (ADS)

    Co, Man Sung; Deschamps, Marguerite; Whitley, Richard J.; Queen, Cary

    1991-04-01

    Antibody therapy holds great promise for the treatment of cancer, autoimmune disorders, and viral infections. Murine monoclonal antibodies are relatively easy to produce but are severely restricted for therapeutic use by their immunogenicity in humans. Production of human monoclonal antibodies has been problematic. Humanized antibodies can be generated by introducing the six hypervariable regions from the heavy and light chains of a murine antibody into a human framework sequence and combining it with human constant regions. We humanized, with the aid of computer modeling, two murine monoclonal antibodies against herpes simplex virus gB and gD glycoproteins. The binding, virus neutralization, and cell protection results all indicate that both humanized antibodies have retained the binding activities and the biological properties of the murine monoclonal antibodies.

  5. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity

    PubMed Central

    Kamali, Ali N.; Marín-García, Patricia; Azcárate, Isabel G.; Puyet, Antonio; Diez, Amalia; Bautista, José M.

    2015-01-01

    Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites. PMID:26539558

  6. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    PubMed

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  7. 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.

  8. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity.

    PubMed

    Maynard, Jennifer A; Maassen, Catharina B M; Leppla, Stephen H; Brasky, Kathleen; Patterson, Jean L; Iverson, Brent L; Georgiou, George

    2002-06-01

    The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

  9. Protein A affinity precipitation of human immunoglobulin G.

    PubMed

    Janoschek, Lars; Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-08-15

    The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.

  10. Production of human or humanized antibodies in mice.

    PubMed

    Laffleur, Brice; Pascal, Virginie; Sirac, Christophe; Cogné, Michel

    2012-01-01

    Mice are widely available laboratory animals that can easily be used for the production of antibodies against a broad range of antigens, using well-defined immunization protocols. Such an approach allows optimal in vivo affinity maturation of the humoral response. In addition, high-affinity antibodies arising in this context can readily be further characterized and produced as monoclonals after immortalizing and selecting specific antibody-producing cells through hybridoma derivation. Using such conventional strategies combined with mice that are either genetically engineered to carry humanized immunoglobulin (Ig) genes or engrafted with a human immune system, it is thus easy to obtain and immortalize clones that produce either fully human Ig or antibodies associating variable (V) domains with selected antigen specificities to customized human-like constant regions, with defined effector functions. In some instances, where there is a need for in vivo functional assays of a single antibody with a known specificity, it might be of interest to transiently express that gene in mice by in vivo gene transfer. This approach allows a rapid functional assay. More commonly, mice are used to obtain a diversified repertoire of antibody specificities after immunization by producing antibody molecules in the mouse B cell lineage from mouse strains with transgene Ig genes which are of human, humanized, or chimeric origin. After in vivo maturation of the immune response, this will lead to the secretion of antibodies with optimized antigen binding sites, associated to the desired human constant domains. This chapter focuses on two simple methods: (1) to obtain such humanized Ig mice and (2) to transiently express a human Ig gene in mice using hydrodynamics-based transfection.

  11. Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering.

    PubMed

    Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C; Varughese, Kottayil I

    2014-01-14

    Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP.

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

    PubMed Central

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

    1990-01-01

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

  13. 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.

  14. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    PubMed Central

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  15. Nonchromatographic affinity precipitation method for the purification of bivalently active pharmaceutical antibodies from biological fluids.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Alves, Nathan J; Bilgicer, Basar

    2013-05-21

    This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.

  16. Recombinant genetic libraries and human monoclonal antibodies.

    PubMed

    Adams, Jarrett J; Nelson, Bryce; Sidhu, Sachdev S

    2014-01-01

    In order to comprehensively manipulate the human proteome we require a vast repertoire of pharmacological reagents. To address these needs we have developed repertoires of synthetic antibodies by phage display, where diversified oligonucleotides are used to modify the complementarity-determining regions (CDRs) of a human antigen-binding fragment (Fab) scaffold. As diversity is produced outside the confines of the mammalian immune system, synthetic antibody libraries allow us to bypass several limitations of hybridoma technology while improving the experimental parameters under which pharmacological reagents are produced. Here we describe the methodologies used to produce synthetic antibody libraries from a single human framework with diversity restricted to four CDRs. These synthetic repertoires can be extremely functional as they produce highly selective, high affinity Fabs to the majority of soluble human antigens. Finally we describe selection methodologies that allow us to overcome immuno-dominance in our selections to target a variety of epitopes per antigen. Together these methodologies allow us to produce human monoclonal antibodies to manipulate the human proteome.

  17. New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

    PubMed

    Zhang, Yi-Fan; Phung, Yen; Gao, Wei; Kawa, Seiji; Hassan, Raffit; Pastan, Ira; Ho, Mitchell

    2015-05-21

    Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

  18. Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide.

    PubMed

    Zeng, Zhiyang; Hoshino, Yu; Rodriguez, Andy; Yoo, Hoseong; Shea, Kenneth J

    2010-01-26

    Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide have been prepared by inverse microemulsion polymerization. Peptide affinity was achieved in part by incorporating the target (imprint) peptide in the polymerization reaction mixture. Incorporation of the imprint peptide assists in the creation of complementary binding sites in the resulting polymer nanoparticle (NP). To orient the imprint peptide at the interface of the water and oil domains during polymerization, the peptide target was coupled with fatty acid chains of varying length. The peptide--NP binding affinities (ca. 90-900 nM) were quantitatively evaluated by a quartz crystal microbalance (QCM). The optimal chain length was established that created high affinity peptide binding sites on the surface of the nanoparticles. This method can be used for the preparation of nanosized synthetic polymers with antibody-like affinity for hydrophilic peptides and proteins ("plastic antibodies").

  19. A new method for the evaluation of antibody affinity based on serial dilutions of studied antibody-antigen mixture.

    PubMed

    Bobrovnik, S A

    2002-01-01

    A new method for the evaluation of the affinity of bivalent antibodies were suggested. This method is based on the previously published by the author the idea of using so-called coordinates of dilutions. It was shown that the suggested method allows to evaluate the affinity of antibodies with high accuracy using this simple approach. It is supposed that at some conditions the suggested method could have substantial advantages in comparison to the traditional methods. This method allows to analyze situations when antibodies are already in a mixture with antigen, for example in the bloodstream in the case of infections or autoimmune diseases. The method provides useful approach for the evaluation not only antibody affinity, but also the concentration of circulated antigen.

  20. High-affinity IgG antibodies develop naturally in Ig-knockout rats carrying germline human IgH/Igκ/Igλ loci bearing the rat CH region.

    PubMed

    Osborn, Michael J; Ma, Biao; Avis, Suzanne; Binnie, Ashleigh; Dilley, Jeanette; Yang, Xi; Lindquist, Kevin; Ménoret, Séverine; Iscache, Anne-Laure; Ouisse, Laure-Hélène; Rajpal, Arvind; Anegon, Ignacio; Neuberger, Michael S; Buelow, Roland; Brüggemann, Marianne

    2013-02-15

    Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human V(H)s, all human D and J(H) segments in natural configuration linked to the rat C(H) locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

  1. Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design.

    PubMed

    Clark, Louis A; Boriack-Sjodin, P Ann; Eldredge, John; Fitch, Christopher; Friedman, Bethany; Hanf, Karl J M; Jarpe, Matthew; Liparoto, Stefano F; Li, You; Lugovskoy, Alexey; Miller, Stephan; Rushe, Mia; Sherman, Woody; Simon, Kenneth; Van Vlijmen, Herman

    2006-05-01

    Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.

  2. Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn.

    PubMed

    Maltezos, Anastasios; Platis, Dimitris; Vlachakis, Dimitrios; Kossida, Sophia; Marinou, Marigianna; Labrou, Nikolaos E

    2014-01-01

    The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

  3. Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence

    PubMed Central

    Mazor, Yariv; Sachsenmeier, Kris F.; Yang, Chunning; Hansen, Anna; Filderman, Jessica; Mulgrew, Kathy; Wu, Herren; Dall’Acqua, William F.

    2017-01-01

    Bispecific antibodies are considered attractive bio-therapeutic agents owing to their ability to target two distinct disease mediators. Cross-arm avidity targeting of antigen double-positive cancer cells over single-positive normal tissue is believed to enhance the therapeutic efficacy, restrict major escape mechanisms and increase tumor-targeting selectivity, leading to reduced systemic toxicity and improved therapeutic index. However, the interplay of factors regulating target selectivity is not well understood and often overlooked when developing clinically relevant bispecific therapeutics. We show in vivo that dual targeting alone is not sufficient to endow selective tumor-targeting, and report the pivotal roles played by the affinity of the individual arms, overall avidity and format valence. Specifically, a series of monovalent and bivalent bispecific IgGs composed of the anti-HER2 trastuzumab moiety paired with affinity-modulated VH and VL regions of the anti-EGFR GA201 mAb were tested for selective targeting and eradication of double-positive human NCI-H358 non-small cell lung cancer target tumors over single-positive, non-target NCI-H358-HER2 CRISPR knock out tumors in nude mice bearing dual-flank tumor xenografts. Affinity-reduced monovalent bispecific variants, but not their bivalent bispecific counterparts, mediated a greater degree of tumor targeting selectivity, while the overall efficacy against the targeted tumor was not substantially affected. PMID:28067257

  4. Protection against measles virus-induced encephalitis by anti-mimotope antibodies: the role of antibody affinity.

    PubMed

    Olszewska, W; Obeid, O E; Steward, M W

    2000-06-20

    Synthetic peptides mimicking a conformational B-cell epitope (M2) of the measles virus fusion protein (MVF) were used for the immunization of BALB/c mice and the anti-peptide and anti-virus antibody titers induced were compared. Of the panel of tested peptides, a chimeric peptide consisting of two copies of a T-helper epitope (residues 288-302 of MVF) and one copy of the mimotope M2 (TTM2) and a multiple antigen peptide with eight copies of M2 (MAP-M2) induced the highest titers of anti-M2 and anti-MV antibodies. Furthermore, peptides TTM2 and MAP-M2 induced antibodies with highest affinity for the mimotope and highest avidity for measles virus. Immunization with the MAP-M2 construct induced high titers of high-affinity anti-M2 antibody despite the absence of a T-helper epitope, and lymphocyte proliferation data suggest that the addition of M2 to the MAP resulted in the generation of a structure capable of stimulating T-cell help. Sera with anti-M2 reactivity were pooled according to affinity values for binding to M2, and high- and low-affinity pools were tested for their ability to prevent MV-induced encephalitis in a mouse model. The high-affinity serum pool conferred protection in 100% of mice, whereas the lower affinity pool conferred protection to only 50% of animals. These results indicate the potential of mimotopes for use as synthetic peptide immunogens and highlight the importance of designing vaccines to induce antibodies of high affinity.

  5. Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

    PubMed

    Ngo, That T; Narinesingh, Dyer

    2008-01-01

    The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

  6. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    PubMed

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  7. Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

    PubMed

    Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

    2014-09-07

    In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

  8. Quality control of coated antibodies: new, rapid determination of binding affinity.

    PubMed

    Ricoux, R; Chazaud, B; Tresca, J P; Pontet, M

    2000-03-01

    A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.

  9. Co-evolution of affinity and stability of grafted amyloid-motif domain antibodies

    PubMed Central

    Julian, Mark C.; Lee, Christine C.; Tiller, Kathryn E.; Rabia, Lilia A.; Day, Evan K.; Schick, Arthur J.; Tessier, Peter M.

    2015-01-01

    An attractive approach for designing lead antibody candidates is to mimic natural protein interactions by grafting peptide recognition motifs into the complementarity-determining regions (CDRs). We are using this approach to generate single-domain (VH) antibodies specific for amyloid-forming proteins such as the Alzheimer's Aβ peptide. Here, we use random mutagenesis and yeast surface display to improve the binding affinity of a lead VH domain grafted with Aβ residues 33–42 in CDR3. Interestingly, co-selection for improved Aβ binding and VH display on the surface of yeast yields antibody domains with improved affinity and reduced stability. The highest affinity VH domains were strongly destabilized on the surface of yeast as well as unfolded when isolated as autonomous domains. In contrast, stable VH domains with improved affinity were reliably identified using yeast surface display by replacing the display antibody that recognizes a linear epitope tag at the terminus of both folded and unfolded VH domains with a conformational ligand (Protein A) that recognizes a discontinuous epitope on the framework of folded VH domains. Importantly, we find that selection for improved stability using Protein A without simultaneous co-selection for improved Aβ binding leads to strong enrichment for stabilizing mutations that reduce antigen binding. Our findings highlight the importance of simultaneously optimizing affinity and stability to improve the rapid isolation of well-folded and specific antibody fragments. PMID:26386257

  10. Modification of the Fc Region of a Human Anti-oncostatin M Monoclonal Antibody for Higher Affinity to FcRn Receptor and Extension of Half-life in Cynomolgus Monkeys.

    PubMed

    Nnane, Ivo P; Han, Chao; Jiao, Qun; Tam, Susan H; Davis, Hugh M; Xu, Zhenhua

    2017-01-28

    The purpose of this study was to evaluate the pharmacokinetics (PK) of anti-oncostatin M (OSM) IgG1 monoclonal antibodies, CNTO 1119 and its Fc variant (CNTO 8212), which incorporates the LS(Xtend) mutation to extend terminal half-life (T1/2 ), after a single intravenous (IV) or subcutaneous (SC) administration in cynomolgus monkeys, and to predict human PK. In study 1, single doses of CNTO 1119 and CNTO 8212 were administered IV or SC at 3 mg/kg to cynomolgus monkeys (n = 3 per group). In study 2, single doses of CNTO 8212 were administered IV at 1, 5 or 20 mg/kg, or SC at 5 mg/kg to cynomolgus monkeys (n = 5 per group). Serial blood samples were collected for assessment of serum concentrations of CNTO 1119 and/or CNTO 8212. A two-compartment population PK model with first-order elimination was utilized to simultaneously describe the serum concentrations of CNTO 1119 and CNTO 8212 over time after IV and SC administration in cynomolgus monkeys. The typical population PK parameter estimates for CNTO 1119 in cynomolgus monkeys were clearance (CL) = 2.81 mL/day/kg, volume of distribution of central compartment (V1 ) = 31.3 mL/kg, volume of distribution of peripheral compartment (V2 ) = 23.3 mL/kg, absolute bioavailability (F) = 0.84 and T1/2 = 13.4 days. In comparison, the typical population PK parameter estimates for CNTO 8212 in cynomolgus monkeys were CL = 1.41 mL/day/kg, V1 = 39.8 mL/kg, V2 = 32.6 mL/kg, F = 0.75 and T1/2 = 35.7 days. The mean CL of CNTO 8212 was ~50% lower compared with that for CNTO 1119 in cynomolgus monkeys. The overall volume of distribution (V1 +V2 ) for CNTO 8212 was about 32% larger compared with that for CNTO 1119, but generally similar to the vascular volume in cynomolgus monkeys. The T1/2 of CNTO 8212 was significantly (p < 0.05) longer by about 2.7-fold than that for CNTO 1119 in cynomolgus monkeys. Thus, the modification of the Fc portion of an anti-OSM IgG1 mAb for higher FcRn binding affinity resulted in lower systemic clearance and

  11. Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

    NASA Technical Reports Server (NTRS)

    Knisley, Keith A.; Rodkey, L. Scott

    1986-01-01

    A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

  12. Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

    NASA Technical Reports Server (NTRS)

    Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

    1987-01-01

    The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

  13. Generation of an affinity column for antibody purification by intein-mediated protein ligation.

    PubMed

    Sun, Luo; Ghosh, Inca; Xu, Ming-Qun

    2003-11-01

    Coupling an antigenic peptide to a solid support is a crucial step in the affinity purification of a peptide-specific antibody. Conventional methods for generating reactive agarose, cellulose or other matrices for peptide conjugation are laborious and can result in a significant amount of chemical waste. In this report, we present a novel method for the facile production of a peptide affinity column by employing intein-mediated protein ligation (IPL) in conjunction with chitin affinity chromatography. A reactive thioester was generated at the C-terminal of the chitin binding domain (CBD) from the chitinase A1 of Bacillus circulans WL-2 by thiol-induced cleavage of the peptide bond between the CBD and a modified intein. Peptide epitopes possessing an N-terminal cysteine were ligated to the chitin bound CBD tag. We demonstrate that the resulting peptide columns permit the highly specific and efficient affinity purification of antibodies from animal sera.

  14. 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.

  15. Measuring the sequence-affinity landscape of antibodies with massively parallel titration curves

    PubMed Central

    Adams, Rhys M; Mora, Thierry; Walczak, Aleksandra M; Kinney, Justin B

    2016-01-01

    Despite the central role that antibodies play in the adaptive immune system and in biotechnology, much remains unknown about the quantitative relationship between an antibody’s amino acid sequence and its antigen binding affinity. Here we describe a new experimental approach, called Tite-Seq, that is capable of measuring binding titration curves and corresponding affinities for thousands of variant antibodies in parallel. The measurement of titration curves eliminates the confounding effects of antibody expression and stability that arise in standard deep mutational scanning assays. We demonstrate Tite-Seq on the CDR1H and CDR3H regions of a well-studied scFv antibody. Our data shed light on the structural basis for antigen binding affinity and suggests a role for secondary CDR loops in establishing antibody stability. Tite-Seq fills a large gap in the ability to measure critical aspects of the adaptive immune system, and can be readily used for studying sequence-affinity landscapes in other protein systems. DOI: http://dx.doi.org/10.7554/eLife.23156.001 PMID:28035901

  16. Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

    SciTech Connect

    Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

    2008-03-07

    Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.

  17. 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.

  18. Maximum-Entropy Models of Sequenced Immune Repertoires Predict Antigen-Antibody Affinity

    PubMed Central

    Marcatili, Paolo; Pagnani, Andrea

    2016-01-01

    The immune system has developed a number of distinct complex mechanisms to shape and control the antibody repertoire. One of these mechanisms, the affinity maturation process, works in an evolutionary-like fashion: after binding to a foreign molecule, the antibody-producing B-cells exhibit a high-frequency mutation rate in the genome region that codes for the antibody active site. Eventually, cells that produce antibodies with higher affinity for their cognate antigen are selected and clonally expanded. Here, we propose a new statistical approach based on maximum entropy modeling in which a scoring function related to the binding affinity of antibodies against a specific antigen is inferred from a sample of sequences of the immune repertoire of an individual. We use our inference strategy to infer a statistical model on a data set obtained by sequencing a fairly large portion of the immune repertoire of an HIV-1 infected patient. The Pearson correlation coefficient between our scoring function and the IC50 neutralization titer measured on 30 different antibodies of known sequence is as high as 0.77 (p-value 10−6), outperforming other sequence- and structure-based models. PMID:27074145

  19. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  20. Affinity-purified respiratory syncytial virus antibodies from intravenous immunoglobulin exert potent antibody-dependent cellular cytotoxicity.

    PubMed

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F; Kazatchkine, Michel D; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

  1. Affinity-Purified Respiratory Syncytial Virus Antibodies from Intravenous Immunoglobulin Exert Potent Antibody-Dependent Cellular Cytotoxicity

    PubMed Central

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F.; Kazatchkine, Michel D.; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V.

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections. PMID:23894466

  2. Manipulating the selection forces during affinity maturation to generate cross-reactive HIV antibodies

    PubMed Central

    Wang, Shenshen; Mata-Fink, Jordi; Kriegsman, Barry; Hanson, Melissa; Irvine, Darrell J.; Eisen, Herman N.; Burton, Dennis R.; Wittrup, K. Dane; Kardar, Mehran; Chakraborty, Arup K.

    2015-01-01

    Summary Generation of potent antibodies by a mutation-selection process called affinity maturation is a key component of effective immune responses. Antibodies that protect against highly mutable pathogens must neutralize diverse strains. Developing effective immunization strategies to drive their evolution requires understanding how affinity maturation happens in an enviroment where variants of the same antigen are present. We present an in silico model of affinity maturation driven by antigen variants which reveals that induction of cross-reactive antibodies often occurs with low probability because conflicting selection forces, imposed by different antigen variants, can frustrate affinity maturation. We describe how variables such as temporal pattern of antigen administration influence the outcome of this frustrated evolutionary process. Our calculations predict, and experiments in mice with variant gp120 constructs of the HIV envelope protein confirm, that sequential immunization with antigen variants is preferred over a cocktail for induction of cross-reactive antibodies focused on the shared CD4 binding site epitope. PMID:25662010

  3. A human monoclonal antibody that binds serotype A botulinum neurotoxin.

    PubMed

    Adekar, Sharad P; Jones, R Mark; Elias, M D; Al-Saleem, Fetweh H; Root, Michael J; Simpson, Lance L; Dessain, Scott K

    2008-02-01

    Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

  4. Disturbance of hapten-antibody equilibria by ammonium sulphate solutions. A source of error in antibody affinity determinations.

    PubMed

    Seppälä, I J

    1975-12-01

    The affinity of anti-hapten antibody can be conveniently measured by precipitating immune complexes with ammonium sulphate. The method has, however, not proved very reproducible. Here is described one variable difficult to control in the assay: the ammonium sulphate was found to cause dissociation of ligands from hapten (NIP)--antibody complexes. The reason was the volume increase caused by addition of ammonium sulphate. The study suggested that in the calculation of the free hapten concentration the final volume during precipitation should be used. The precipitate should not be washed when hapten binding capacities are measured.

  5. Deconvolution of antibody affinities and concentrations by non-linear regression analysis of competitive ELISA data.

    SciTech Connect

    Stevens, F. J.; Bobrovnik, S. A.; Biosciences Division; Palladin Inst. Biochemistry

    2007-12-01

    Physiological responses of the adaptive immune system are polyclonal in nature whether induced by a naturally occurring infection, by vaccination to prevent infection or, in the case of animals, by challenge with antigen to generate reagents of research or commercial significance. The composition of the polyclonal responses is distinct to each individual or animal and changes over time. Differences exist in the affinities of the constituents and their relative proportion of the responsive population. In addition, some of the antibodies bind to different sites on the antigen, whereas other pairs of antibodies are sterically restricted from concurrent interaction with the antigen. Even if generation of a monoclonal antibody is the ultimate goal of a project, the quality of the resulting reagent is ultimately related to the characteristics of the initial immune response. It is probably impossible to quantitatively parse the composition of a polyclonal response to antigen. However, molecular regression allows further parameterization of a polyclonal antiserum in the context of certain simplifying assumptions. The antiserum is described as consisting of two competing populations of high- and low-affinity and unknown relative proportions. This simple model allows the quantitative determination of representative affinities and proportions. These parameters may be of use in evaluating responses to vaccines, to evaluating continuity of antibody production whether in vaccine recipients or animals used for the production of antisera, or in optimizing selection of donors for the production of monoclonal antibodies.

  6. Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

    PubMed

    Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

    2014-01-01

    Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

  7. Affinity Chromatography of Native and Recombinant Proteins from Receptors for Insulin and IGF-I to Recombinant Single Chain Antibodies

    PubMed Central

    Fujita-Yamaguchi, Yoko

    2015-01-01

    Affinity chromatography is an efficient method to isolate proteins by taking advantage of their affinities for specific molecules such as substrates, inhibitors, antigens, ligands, antibodies, and other interacting molecules, including subunits. Nowadays, we take the effectiveness and excellence of this technology for granted. This essay will mainly cover the use of affinity chromatography based on my experience. PMID:26579073

  8. 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.

  9. [Obtaining of the affinity purified antibodies against survivin for the structure functional study of the protein].

    PubMed

    Akhidova, E V; Volkova, T D; Koroev, D O; Yakupov, I Iu; Kalintseva, M V; Zavalishina, L E; Kaplun, A P; Zharskaia, O O; Zatsepina, O V; Vol'pina, O M

    2013-01-01

    Tumor-associated protein survivin is the bifunctional protein which can participate either in cell division regulation or in apoptosis inhibition depending on its localization and structure state. The aim of this work was to obtain monospecific antibodies useful for investigation of protein structure and functional features. Six affinity purified antibodies directed to different protein regions were obtained. The ability of antibodies obtained to detect survivin in tumor cells and breast cancer tissues was studied. It was shown that antibodies to (1-22) and (95-105) survivin fragments have the highest specific activity. In western-blot antibodies to (1-22) region predominantly binds with survivin-containing complex, which may be the survivin dimer as we suppose, while antibodies to (95-105) region detects only monomeric form of the protein. Breast cancer tissues study demonstrated that survivin monomer presents only in the tumor core tissues, while survivin-containing complex is expressed both in tumor core and tumor periphery tissues. It was shown that antibodies to (1-22) fragment detect predominantly nuclear survivin, which participates in mitosis regulation, while antibodies to (95-105) fragment gave nucleoplasm and cytoplasm staining at all stages of cell cycle. Thereby antibodies obtained are the useful tool for structure-functional study of survivin.

  10. Monoclonal antibodies to human glycophorin A and cell lines for the production thereof

    DOEpatents

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

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A.sup.N and differentiate between the M and N forms of human glycophorin A.

  11. 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.

  12. Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

    PubMed

    Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

    2016-12-23

    High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

  13. Polystyrene as an affinity chromatography matrix for the purification of antibodies.

    PubMed

    Staak, C; Salchow, F; Clausen, P H; Luge, E

    1996-08-14

    Affinity chromatography is used for the purification of diagnostic polyclonal antibodies in order to ensure specificity. Most commonly, activated bead-formed agarose or its derivatives are used as gel matrices. Alternative matrix materials have been described, but as yet they do not appear to offer important advantages. In this study, pulverized polystyrene (PS 158K, BASF, Mannheim, Germany) was used as a solid phase for the immobilisation of bovine immunoglobulins (Ig). Affinity chromatography was performed using these coated polystyrene beads as the column matrix material in the purification of anti-bovine Ig. The polystyrene binding capacity for the different bovine Ig classes was compared using the Mancini single radial immunodiffusion technique, and ELISA procedures were used to monitor the antibody reactivity of purified and unpurified antibodies. The degree of purification was comparable to the most commonly used procedure using gel matrices from activated bead-formed agarose (e.g. CNBr-activated Sepharose 4B, Pharmacia/LKB Biotechnology, Uppsala, Sweden), but the antibody yield per ml column volume was distinctly lower. In order to raise the yield, such polystyrene bead columns with immobilized antigen can be re-used without loss of activity or larger column volumes can be used to raise the binding capacity. The polystyrene material is quite durable, chemically and immunologically inert and has a long shelf life. We conclude that polystyrene based affinity chromatography is efficient, simple and cheap.

  14. Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

    PubMed Central

    Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

    2016-01-01

    High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

  15. Antibody humanization by structure-based computational protein design.

    PubMed

    Choi, Yoonjoo; Hua, Casey; Sentman, Charles L; Ackerman, Margaret E; Bailey-Kellogg, Chris

    2015-01-01

    Antibodies derived from non-human sources must be modified for therapeutic use so as to mitigate undesirable immune responses. While complementarity-determining region (CDR) grafting-based humanization techniques have been successfully applied in many cases, it remains challenging to maintain the desired stability and antigen binding affinity upon grafting. We developed an alternative humanization approach called CoDAH ("Computationally-Driven Antibody Humanization") in which computational protein design methods directly select sets of amino acids to incorporate from human germline sequences to increase humanness while maintaining structural stability. Retrospective studies show that CoDAH is able to identify variants deemed beneficial according to both humanness and structural stability criteria, even for targets lacking crystal structures. Prospective application to TZ47, a murine anti-human B7H6 antibody, demonstrates the approach. Four diverse humanized variants were designed, and all possible unique VH/VL combinations were produced as full-length IgG1 antibodies. Soluble and cell surface expressed antigen binding assays showed that 75% (6 of 8) of the computationally designed VH/VL variants were successfully expressed and competed with the murine TZ47 for binding to B7H6 antigen. Furthermore, 4 of the 6 bound with an estimated KD within an order of magnitude of the original TZ47 antibody. In contrast, a traditional CDR-grafted variant could not be expressed. These results suggest that the computational protein design approach described here can be used to efficiently generate functional humanized antibodies and provide humanized templates for further affinity maturation.

  16. High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization.

    PubMed

    Lunn, M P; Johnson, L A; Fromholt, S E; Itonori, S; Huang, J; Vyas, A A; Hildreth, J E; Griffin, J W; Schnaar, R L; Sheikh, K A

    2000-07-01

    Gangliosides, sialic acid-bearing glycosphingolipids, are highly enriched in the vertebrate nervous system. Anti-ganglioside antibodies are associated with various human neuropathies, although the pathogenicity of these antibodies remains unproven. Testing the pathogenic role of anti-ganglioside antibodies will be facilitated by developing high-affinity IgG-class complement-fixing monoclonal anti-bodies against major brain gangliosides, a goal that has been difficult to achieve. In this study, mice lacking complex gangliosides were used as immune-naive hosts to raise anti-ganglioside antibodies. Wild-type mice and knockout mice with a disrupted gene for GM2/GD2 synthase (UDP-N-acetyl-D-galactosamine : GM3/GD3 N-acetyl-D-glactosaminyltransferase) were immunized with GD1a conjugated to keyhole limpet hemocyanin. The knockout mice produced a vigorous anti-GD1a IgG response, whereas wildtype littermates failed to do so. Fusion of spleen cells from an immunized knockout mouse with myeloma cells yielded numerous IgG anti-GD1a antibody-producing colonies. Ganglioside binding studies revealed two specificity classes; one colony representing each class was cloned and characterized. High-affinity monoclonal antibody was produced by each hybridoma : an IgG1 that bound nearly exclusively to GD1a and an IgG2b that bound GD1a, GT1b, and GT1aalpha. Both antibodies readily readily detected gangliosides via ELISA, TLC immune overlay, immunohistochemistry, and immunocytochemistry. In contrast to prior reports using anti-GD1a and anti-GT1b IgM class monoclonal antibodies, the new antibodies bound avidly to granule neurons in brain tissue sections and cell cultures. Mice lacking complex gangliosides are improved hosts for raising high-affinity, high-titer anti-ganglioside IgG antibodies for probing for the distribution and physiology of gangliosides and the pathophysiology of anti-ganglioside antibodies.

  17. SNAP-Tag Technology: A Useful Tool To Determine Affinity Constants and Other Functional Parameters of Novel Antibody Fragments.

    PubMed

    Niesen, Judith; Sack, Markus; Seidel, Melanie; Fendel, Rolf; Barth, Stefan; Fischer, Rainer; Stein, Christoph

    2016-08-17

    Antibody derivatives, such as the single chain fragment variable (scFv), can be developed as diagnostic and therapeutic tools in cancer research, especially in the form of fusion proteins. Such derivatives are easier to produce and modify than monoclonal antibodies (mAbs) and achieve better tissue/tumor penetration. The genetic modification of scFvs is also much more straightforward than the challenging chemical modification of mAbs. Therefore, we constructed two scFvs derived from the approved monoclonal antibodies cetuximab (scFv2112) and panitumumab (scFv1711), both of which are specific for the epidermal growth factor receptor (EGFR), a well-characterized solid tumor antigen. Both scFvs were genetically fused to the SNAP-tag, an engineered version of the human DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase that allows the covalent coupling of benzylguanine (BG)-modified substrates such as fluorescent dyes. The SNAP-tag achieves controllable and irreversible protein modification and is an important tool for experimental studies in vitro and in vivo. The affinity constant of a scFv is a key functional parameter, especially in the context of a fusion protein. Therefore, we developed a method to define the affinity constants of scFv-SNAP fusion proteins by surface plasmon resonance (SPR) spectroscopy. We could confirm that both scFvs retained their functionality after fusion to the SNAP-tag in a variety of procedures and assays, including ELISA, flow cytometry, and confocal microscopy. The experimental procedures described herein, and the new protocol for affinity determination by SPR spectroscopy, are suitable for the preclinical evaluation of diverse antibody formats and derivatives.

  18. Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

    PubMed

    Ahlqvist, Josefin; Kumar, Ashok; Sundström, Heléne; Ledung, Erika; Hörnsten, E Gunnar; Enfors, Sven-Olof; Mattiasson, Bo

    2006-03-23

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  19. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    PubMed

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction.

  20. 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.

  1. A novel antibody humanization method based on epitopes scanning and molecular dynamics simulation.

    PubMed

    Zhang, Ding; Chen, Cai-Feng; Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

    2013-01-01

    1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization.

  2. Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain.

    PubMed

    Mahmood, Rubab

    2016-01-01

    Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)-bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.

  3. Shortened Engineered Human Antibody CH2 Domains

    PubMed Central

    Gong, Rui; Wang, Yanping; Feng, Yang; Zhao, Qi; Dimitrov, Dimiter S.

    2011-01-01

    The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. D., Prabakaran, P., Veenstra, T. D., Tarasov, S. G., Ishima, R., and Dimitrov, D. S. (2009) J. Biol. Chem. 284, 14203–14210). We have hypothesized that the stability of this engineered antibody domain could be further increased by removing unstructured residues. To test our hypothesis, we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric, and remarkably stable, with a melting temperature (Tm) of 82.6 °C, which is about 10 and 30 °C higher than those of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody that recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human neonatal Fc receptor at pH 6.0 more strongly than CH2. These data suggest that shortening the m01 N terminus significantly increases stability without disrupting its conformation and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins. PMID:21669873

  4. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  5. Broad epitope coverage of a human in vitro antibody library.

    PubMed

    Sivasubramanian, Arvind; Estep, Patricia; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Niles, Rebecca Hurley; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

  6. Subcutaneous bioavailability of therapeutic antibodies as a function of FcRn binding affinity in mice

    PubMed Central

    Meng, Y Gloria; Hoyte, Kwame; Lutman, Jeff; Lu, Yanmei; Iyer, Suhasini; DeForge, Laura E; Theil, Frank-Peter; Fielder, Paul J; Prabhu, Saileta

    2012-01-01

    The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies. PMID:22327433

  7. Performance of protein-A-based affinity membranes for antibody purification.

    PubMed

    Uzun, Lokman; Türkmen, Deniz; Karakoç, Veyis; Yavuz, Handan; Denizli, Adil

    2011-01-01

    The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5-10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL-protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL-protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL-protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption-elution cycles.

  8. Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

    PubMed Central

    Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

    2014-01-01

    Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

  9. Design, expression and characterization of a single chain anti-CD20 antibody; a germline humanized antibody derived from Rituximab.

    PubMed

    Ahmadzadeh, Vahideh; Farajnia, Safar; Hosseinpour Feizi, Mohammad Ali; Khavarinejad, Ramazan Ali

    2014-10-01

    CD20 is a B cell lineage specific surface antigen involved in various B cell malignancies. So far, several murine and chimeric antibodies have been produced against this antigen among which Rituximab is a commercially approved antibody widely used in treatment of cancers associated with CD20 overexpression. The current study reports the production and characterization of a humanized single chain version of Rituximab through CDR grafting method. For either heavy or light chain variable domains, a human antibody with the highest sequence homology to Rituximab was selected from human germline sequences and used as framework donors. Vernier zone residues in framework regions were replaced with those of Rituximab to retain the antigen binding affinity of parental antibody. The reactivity of humanized single chain antibody with CD20 was examined by ELISA and dot blot assays. The ability of antibody to suppress the growth of CD20 overexpressing Raji cells was tested by MTT assay. Analysis of reactivity with CD20 antigen revealed that the humanized single chain antibody reacted to the target antigen with high affinity. Proliferation inhibition assay showed that humanized scFv could suppress the proliferation of Raji cells efficiently in a dose-dependent manner. This successful production of a humanized scFv with the ability to inhibit growth of CD20-expressing cancer cell may provide a promising alternative strategy for CD20 targeted therapy.

  10. Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis.

    PubMed Central

    Casipit, C. L.; Tal, R.; Wittman, V.; Chavaillaz, P. A.; Arbuthnott, K.; Weidanz, J. A.; Jiao, J. A.; Wong, H. C.

    1998-01-01

    Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity. PMID:10082364

  11. Antibody to FcεRIα Suppresses Immunoglobulin E Binding to High-Affinity Receptor I in Allergic Inflammation

    PubMed Central

    Hong, Jung Yeon; Bae, Jong-Hwan; Lee, Kyung Eun; Kim, Mina; Kim, Min Hee; Kang, Hyun Jung; Park, Eun Hye; Yoo, Kyung Sook; Jeong, Se Kyoo; Kim, Kyung Won; Kim, Kyu-Earn

    2016-01-01

    Purpose High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. Materials and Methods Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1atm1Knt Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. Results NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. Conclusion Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases. PMID:27593869

  12. Affinity purification of egg yolk immunoglobulins (IgY) using a human mycoplasma protein.

    PubMed

    Jiang, Xuemei; Diraviyam, Thirumalai; Zhang, Xiaoying

    2016-02-15

    Egg yolk immunoglobulin (IgY) is a superior functional equivalent to mammalian IgG. However, the preparation of refined and highly purified IgY is still attributed as difficult task. Protein M (a transmembrane protein from human mycoplasma) has been newly demonstrated as an ideal affinity regent for mammalian antibody purification. This study aimed to evaluate the interaction between protein M and IgY. The results showed protein M could be a superior affinity reagent for IgY, scFv as well as IgYΔFc, based on pull down and western blot investigations; in addition, it was found that ∼125 times increase of effective IgY in the elutent was obtained using protein M affinity chromatography column compared with traditional IgY extraction methods. This indicates, the purification strategy of protein M is entirely different to traditional IBPs and the salient purification feature of protein M would be a breakthrough for purifying not only non-mammalian antibodies, but also monoclonal antibodies and engineered antibodies based on variable region.

  13. Antibody affinities and relative titers in polyclonal populations: surface plasmon resonance analysis of anti-DNA antibodies.

    PubMed

    Sem, D S; McNeeley, P A; Linnik, M D

    1999-12-01

    This paper presents the equations and methodology for the measurement and interpretation of apparent dissociation constants for polyclonal populations of antibodies, where antigen is kept trace relative to antibody concentration. Surface plasmon resonance is used to determine K(d)s for the binding of anti-DNA antibodies to trace amounts of DNA antigen on a chip. Since the approach taken relies on equilibrium measurements, kinetic mass transport artifacts are avoided. The apparent K(d) is a weighted average of all the K(d)s for the clonally related subpopulations within the polyclonal pool, where each weighting factor is the relative titer (fractional presence) of the subpopulation. Titration curves appear as if there is one monoclonal population with that titer-weighted-average K(d). Implications of changes in the antibody affinity distribution within the population are discussed. The equations described herein provide a better physical understanding of the apparent K(d) that is obtained when a heterogeneous population of receptors is titrated against a trace ligand.

  14. Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

    PubMed

    Kiyoshi, Masato; Caaveiro, Jose M M; Miura, Eri; Nagatoishi, Satoru; Nakakido, Makoto; Soga, Shinji; Shirai, Hiroki; Kawabata, Shigeki; Tsumoto, Kouhei

    2014-01-01

    The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody.

  15. Strategy for affinity maturation of an antibody with high evolvability to (4-hydroxy-3-nitrophenyl) acetyl hapten.

    PubMed

    Furukawa, Koji; Shimizu, Takeyuki; Murakami, Akikazu; Kono, Ryo; Nakagawa, Masatoshi; Sagawa, Takuma; Yamato, Ichiro; Azuma, Takachika

    2007-03-01

    In order to quantitate the contribution of amino acid replacements to an increase in affinity during affinity maturation, we measured thermodynamic parameters of the antigen-antibody interaction for a group of anti-(4-hydroxy-3-nitrophenyl) acetyl monoclonal antibodies whose differences in amino acid sequences had arisen only from somatic hypermutation. We prepared a common ancestor and hypothetical intermediate clones that might occur on the affinity maturation pathway, by employing site-directed mutagenesis. Isothermal calorimetric titration of the antigen-antibody reaction revealed that antibody evolution proceeds in two steps. The first step is driven by a decrease in enthalpy, in which two amino acid replacements in the VL region play an essential role. Further accumulation of amino acid replacements in VH and VL regions during the second step induce a progressive increase in affinity, which is driven by an increase in entropy, which has a cooperative mutational effect.

  16. Have we overestimated the benefit of human(ized) antibodies?

    PubMed Central

    Getts, Meghann T; McCarthy, Derrick P; Chastain, Emily ML; Miller, Stephen D

    2010-01-01

    The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account. PMID:20935511

  17. Monoclonal Antibodies Against Human Cardiac Troponin I for Immunoassays II.

    PubMed

    Lee, Gregory; Liu, Suefay

    2015-06-01

    Human cardiac troponin I (cTnI) is one of the most specific biomarkers for detection of acute myocardial infarction (AMI). To formulate immunoassay kits for rapid immunodiagnosis of AMI, monoclonal antibodies with high affinity and specificity were generated against cTnI and subsequently tested through a series of experiments. C57BL/6 mice were immunized with cTnI as the immunogen and cell fusions with myeloma cells of BALB/c origin were performed to generate hybridomas. The supernatants of the hybridoma cell culture were routinely screened for antibody secretions against intact cTnI and synthetic peptides from the N-terminal half of cTnI (amino acid residues N1-30, N24-40, N59-79, and N80-95). Monoclonal antibodies specific to different epitope regions were then determined and selected, according to their respective affinity and specificity, for formulation of enzyme immunoassay kits. The results of this study found that most of the selected antibodies revealed comparable binding affinity to cTnI and to the corresponding synthetic peptides. Optimal sandwich enzyme immunoassays with high sensitivity could be achieved through proper combinations of the epitope-distinct monoclonal antibodies in different capture-detection pairs; signal enhancements were frequently observed when a mixture of epitope-distinct anti-cTnI monoclonal antibodies was used for coating. This indicates that a combination of epitope-distinct anti-cTnI monoclonal antibodies recognizing the N-terminal half of cTnI yield reliable detection and greater sensitivity for cTnI in AMI patients.

  18. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  19. Characterization of Humanized Antibodies Secreted by Aspergillus niger

    PubMed Central

    Ward, Michael; Lin, Cherry; Victoria, Doreen C.; Fox, Bryan P.; Fox, Judith A.; Wong, David L.; Meerman, Hendrik J.; Pucci, Jeff P.; Fong, Robin B.; Heng, Meng H.; Tsurushita, Naoya; Gieswein, Christine; Park, Minha; Wang, Huaming

    2004-01-01

    Two different humanized immunoglobulin G1(κ) antibodies and an Fab′ fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex6GlcNAc2 to Hex15GlcNAc2. An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function. PMID:15128505

  20. Microdevices integrating affinity columns and capillary electrophoresis for multi-biomarker analysis in human serum

    PubMed Central

    Yang, Weichun; Yu, Ming; Sun, Xiuhua; Woolley, Adam T.

    2010-01-01

    Summary Biomarkers in human body fluids have great potential for use in screening for diseases such as cancer and diabetes, diagnosis, determining the effectiveness of treatments, and detecting recurrence. Present 96-well immunoassay technology effectively analyzes large numbers of samples; however, this approach is more expensive and less time effective on single or a few samples. In contrast, microfluidic systems are well suited for assaying small numbers of specimens in a point-of-care setting, provided suitable procedures are developed to work within peak capacity constraints when analyzing complex mixtures like human blood serum. Here, we developed integrated microdevices with an affinity column and capillary electrophoresis channels to isolate and quantitate a panel of proteins in complex matrices. To form an affinity column, a thin film of a reactive polymer was photopolymerized in a microchannel, and four antibodies were covalently immobilized to it. The retained protein amounts were consistent from chip to chip, demonstrating reproducibility. Furthermore, the signals from four fluorescently labeled proteins captured on-column were in the same range after rinsing, indicating the column has little bias toward any of the four antibodies or their antigens. These affinity columns have been integrated with capillary electrophoresis separation, enabling us to simultaneously quantify four protein biomarkers in human blood serum in the low ng/mL range using either a calibration curve or standard addition. Our systems provide a fast, integrated and automated platform for multiple biomarker quantitation in complex media such as human blood serum. PMID:20664867

  1. Affinity separation using an Fv antibody fragment-"smart" polymer conjugate.

    PubMed

    Fong, Robin B; Ding, Zhongli; Hoffman, Allan S; Stayton, Patrick S

    2002-08-05

    Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.

  2. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-08

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals.

  3. Low antigen dose favours selection of somatic mutants with hallmarks of antibody affinity maturation.

    PubMed Central

    González-Fernández, A; Milstein, C

    1998-01-01

    The immunization schedule is critical for the derivation of high-affinity antibodies, low antigen dose being particularly favourable for the development of a more efficient memory response. To analyse the molecular events underpinning this preference, we analysed the early maturation of the response to the hapten 2-phenyloxazolone (phOx) using low and high doses of immunogen. The phOx response is initially dominated by antibodies expressing the VkOx1-Jk5 light chain and the hallmark of the early stages of maturation is the substitution of His 34 by Asn or Gln increasing affinity 10- or eightfold, respectively, and of Tyr 36 by Phe. High-affinity antibodies express mutations at both sites. We cloned and sequenced VkOx1-Jk5 light chains from antigen-specific B cells taken 14 and 21 days after immunization with high and low antigen doses. We found that overall, the derived sequences were more mutated both at longer times and at higher dose. At day 14, His 34 was more frequently mutated at the higher than at the lower dose, while at day 21 the reverse was true. On the other hand, the His 34/Tyr 36 mutation pair was more frequent at low than high doses at both 14 and 21 days. Furthermore, at both times, the low immunization protocol yielded double mutants in cells with a lower mutation background. It appears therefore that while the higher dose may favour the acquisition of individual critical mutations, low-dose immunization favours the selection of a more focused mutational pattern, whereby advantageous mutations are associated with a low mutational background. Images Figure 1 PMID:9616362

  4. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  5. A complex water network contributes to high-affinity binding in an antibody-antigen interface.

    PubMed

    Marino, S F; Olal, D; Daumke, O

    2016-03-01

    This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment), J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA). The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: "Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma", Mol. Oncol. 9 (7) (2015) pp. 1348-58.

  6. Deficient for endoplasmic reticulum calcium sensors Stim1 and Stim2 affects aberrant antibody affinity maturation in B cells

    PubMed Central

    Mao, Xuhua; Zhang, Jianfeng; Han, Yue; Luan, Chao; Hu, Yu; Hao, Zhimin; Chen, Min

    2016-01-01

    Antigen specific B cells undergo a process termed affinity maturation in the germinal centers of secondary lymphoid organs where B cells with high affinity receptors are selected to mature into antibody-producing cells or to the memory B cell pool. It is known that B cell antigen receptor (BCR) signaling plays pivotal role in this selection process. Calcium influx is an essential component of BCR signaling. The current report is to determine the effect of calcium influx on antibody affinity maturation. In our studies, mice deficient for both endoplasmic reticulum calciumsensor Stim1 and Stim2 was immunized with T-cell dependent and independent antigens. Antibody affinity was measured by ELISA. We demonstrated that Stim1 &Stim2 deficient B cells exhibit accelerated pace of affinity maturation compared to wild type controls while the overall antibody production was not dramatically impaired to T-independent antigen immunization. In conclusion, calcium influx plays an important role in antibody affinity maturation in humoral immune responses. The knowledge can be used in manipulate humoral immune response for the design of effective vaccines. PMID:27572320

  7. Mass Spectrometric Detection of Neuropeptides Using Affinity-Enhanced Microdialysis with Antibody-Coated Magnetic Nanoparticles

    PubMed Central

    Schmerberg, Claire M.; Li, Lingjun

    2012-01-01

    Microdialysis (MD) is a useful sampling tool for many applications due to its ability to permit sampling from an animal concurrent with normal activity. MD is of particular importance in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the animal is behaving in order to correlate dynamic changes in NTs with behavior. One important class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinity-enhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional mass transport driving force for analyte to pass through the dialysis membrane, and thus increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in vitro and in vivo, including particles with C18 surface functionality and antibody-coated particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement in vitro, with statistically significant (p<0.05) enhancements for 4 out of 6 NP standards tested, and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab, Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were detected in a 30 min collection sample, compared to 17 when no AA was used. The use of AbMnP also increased the temporal resolution from 4–18 hrs in previous studies to just 30 min in this study. The levels of NPs detected were also sufficient for reliable quantitation with the MS system in use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30 min time course during feeding. PMID:23249250

  8. Immunological and structural characterization of a high affinity anti-fluorescein single-chain antibody.

    PubMed

    Bedzyk, W D; Weidner, K M; Denzin, L K; Johnson, L S; Hardman, K D; Pantoliano, M W; Asel, E D; Voss, E W

    1990-10-25

    Single-chain antibody of the (NH2) VL-linker-VH (COOH) design, was constructed based on prototype high affinity anti-fluorescein monoclonal antibody (mAb) 4-4-20. Purified single-chain antibody (SCA) 4-4-20/212 was studied relative to Ig mAb 4-4-20 in terms of ligand binding, kinetics, idiotypy, metatypy, and stability in denaturing agents. Ligand-binding data correlated with metatypic relatedness of the liganded site. Anti-metatypic reagents reacted preferentially with the liganded conformer of the 4-4-20 antibody active site and were unreactive with free ligand and the non-liganded (idiotypic) state. All results were consistent with the conclusion that SCA 4-4-20/212, with a 14-amino acid linker folded into a native conformational state that closely simulated the prototypical mAb. Furthermore, GndHCl unfolding and refolding studies demonstrated H and L chain variable domain intrinsic stability between SCA 4-4-20/212 and a 50 kDa antigen-binding fragment were nearly identical. This suggested CH1 and CL domain interactions may be more prevalent in V region molecular dynamics than structure.

  9. Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease

    PubMed Central

    Aliesky, Holly A.; Chen, Chun-Rong; McLachlan, Sandra M.

    2015-01-01

    Context: The TSH receptor (TSHR) A-subunit shed from the cell surface contributes to the induction and/or affinity maturation of pathogenic TSHR autoantibodies in Graves' disease. Objective: This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. Design: The isolated TSHR A-subunit generated by transfected mammalian cells exists in two forms; one (active) is recognized only by Graves' TSHR autoantibodies, the second (inactive) is recognized only by mouse monoclonal antibody (mAb) 3BD10. Recent evidence suggests that both Graves' TSHR autoantibodies and mAb 3BD10 recognize the A-subunit monomer. Therefore, if the A-subunit monomer is an immunogen, Graves' sera should have antibodies to both active and inactive A-subunits. Conversely, restriction of TSHR autoantibodies to active A-subunits would be evidence of a role for shed A-subunit multimers, not monomers, in the pathogenesis of Graves' disease. Therefore, we tested a panel of Graves' sera for their relative recognition of active and inactive A-subunits. Results: Of 34 sera from unselected Graves' patients, 28 were unequivocally positive in a clinical TSH binding inhibition assay. None of the latter sera, as well as 8/9 sera from control individuals, recognized inactive A-subunits on ELISA. In contrast to Graves' sera, antibodies induced in mice, not by shedding from the TSHR holoreceptor, but by immunization with adenovirus expressing the free human A-subunit, were directed to both the active and inactive A-subunit forms. Conclusions: The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers. PMID:25856215

  10. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  11. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  12. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  13. High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

    PubMed Central

    Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

    2016-01-01

    Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

  14. 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.

  15. Antibody Fragments for On-Site Testing of Cannabinoids Generated via in Vitro Affinity Maturation.

    PubMed

    Morita, Izumi; Oyama, Hiroyuki; Yasuo, Mayumi; Matsuda, Kazuhisa; Katagi, Kengo; Ito, Aya; Tatsuda, Hiroka; Tanaka, Hiroyuki; Morimoto, Satoshi; Kobayashi, Norihiro

    2017-01-01

    Law enforcement against illicit use of cannabis and related substances requires rapid, feasible, and reliable tools for on-site testing of cannabinoids. Notably, methods based on cannabinoid-specific antibodies enable efficient screening of multiple specimens. Antibody engineering may accelerate development of modern and robust testing systems. Here, we used in vitro affinity maturation to generate a single-chain Fv fragment (scFv) that recognizes with high affinity the psychoactive cannabinoid, Δ(9)-tetrahydrocannabinol (THC). A mouse monoclonal antibody against THC, Ab-THC#33, with Ka 6.2×10(7) M(-1) (as Fab fragment) was established by the hybridoma technique. Then, a "wild-type" scFv (wt-scFv) with Ka, 1.1×10(7) M(-1) was prepared by bacterial expression of a fusion gene combining the VH and VL genes for Ab-THC#33. Subsequently, random point mutations in VH and VL were generated separately, and the resulting products were assembled into mutant scFv genes, which were then phage-displayed. Repeated panning identified a mutant scFv (scFv#m1-36) with 10-fold enhanced affinity (Ka 1.1×10(8) M(-1)) for THC, in which only a single conservative substitution (Ser50Thr) was present at the N-terminus of the VH-complementarity-determining region 2 (CDR2) sequence. In competitive enzyme-linked immunosorbent assay (ELISA), the mutant scFv generated dose-response curves with midpoint 0.27 ng/assay THC, which was 3-fold lower than that of wt-scFv. Even higher reactivity with a major THC metabolite, 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol, indicated that the mutant scFv will be useful for testing not only THC in confiscated materials, but also the metabolite in urine. Indeed, the antibody fragment is potentially suitable for use in advanced on-site testing platforms for cannabinoids.

  16. 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.

  17. Single-chain antibody-based immunotoxins targeting Her2/neu: design optimization and impact of affinity on antitumor efficacy and off-target toxicity.

    PubMed

    Cao, Yu; Marks, James D; Huang, Qian; Rudnick, Stephen I; Xiong, Chiyi; Hittelman, Walter N; Wen, Xiaoxia; Marks, John W; Cheung, Lawrence H; Boland, Kim; Li, Chun; Adams, Gregory P; Rosenblum, Michael G

    2012-01-01

    Recombinant immunotoxins, consisting of single-chain variable fragments (scFv) genetically fused to polypeptide toxins, represent potentially effective candidates for cancer therapeutics. We evaluated the affinity of various anti-Her2/neu scFv fused to recombinant gelonin (rGel) and its effect on antitumor efficacy and off-target toxicity. A series of rGel-based immunotoxins were created from the human anti-Her2/neu scFv C6.5 and various affinity mutants (designated ML3-9, MH3-B1, and B1D3) with affinities ranging from 10(-8) to 10(-11) mol/L. Against Her2/neu-overexpressing tumor cells, immunotoxins with increasing affinity displayed improved internalization and enhanced autophagic cytotoxicity. Targeting indices were highest for the highest affinity B1D3/rGel construct. However, the addition of free Her2/neu extracellular domain (ECD) significantly reduced the cytotoxicity of B1D3/rGel because of immune complex formation. In contrast, ECD addition had little impact on the lower affinity constructs in vitro. In vivo studies against established BT474 M1 xenografts showed growth suppression by all immunotoxins. Surprisingly, therapy with the B1D3-rGel induced significant liver toxicity because of immune complex formation with shed Her2/neu antigen in circulation. The MH3-B1/rGel construct with intermediate affinity showed effective tumor growth inhibition without inducing hepatotoxicity or complex formation. These findings show that while high-affinity constructs can be potent antitumor agents, they may also be associated with mistargeting through the facile formation of complexes with soluble antigen leading to significant off-target toxicity. Constructs composed of intermediate-affinity antibodies are also potent agents that are more resistant to immune complex formation. Therefore, affinity is an exceptionally important consideration when evaluating the design and efficacy of targeted therapeutics.

  18. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  19. Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity

    PubMed Central

    Derer, Stefanie; Glorius, Pia; Schlaeth, Martin; Lohse, Stefan; Klausz, Katja; Muchhal, Umesh; Desjarlais, John R; Humpe, Andreas; Valerius, Thomas; Peipp, Matthias

    2014-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC. Here, non-fucosylated or protein-engineered anti-EGFR antibodies with optimized FcγRIIIa affinities demonstrated the expected benefit in NK cell-mediated ADCC, but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Furthermore, eosinophils and PMN from paroxysmal nocturnal hemoglobinuria patients that expressed no or low levels of FcγRIIIb mediated effective ADCC with FcγRIII-optimized anti-EGFR antibody. Additional experiments with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared with single FcγRIII-optimized antibody. In conclusion, our data demonstrate that FcγRIIIb engagement impairs PMN-mediated ADCC activity of FcγRIII-optimized anti-EGFR antibodies, while further optimization of FcγRIIa binding significantly restores PMN recruitment. PMID:24492248

  20. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    PubMed

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  1. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    PubMed

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  2. Broad epitope coverage of a human in vitro antibody library

    PubMed Central

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  3. Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

    PubMed Central

    Diebolder, Philipp; Keller, Armin; Haase, Stephanie; Schlegelmilch, Anne; Kiefer, Jonathan D; Karimi, Tamana; Weber, Tobias; Moldenhauer, Gerhard; Kehm, Roland; Eis-Hübinger, Anna M; Jäger, Dirk; Federspil, Philippe A; Herold-Mende, Christel; Dyckhoff, Gerhard; Kontermann, Roland E; Arndt, Michaela AE; Krauss, Jürgen

    2014-01-01

    The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)2. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential. PMID:24256717

  4. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  5. Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform.

    PubMed

    Landry, J P; Ke, Yaohuang; Yu, Guo-Liang; Zhu, X D

    2015-02-01

    Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare the results obtained from the microarray-based platform with those from a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

  6. 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.

  7. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    PubMed

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  8. Optimal Affinity of a Monoclonal Antibody: Guiding Principles Using Mechanistic Modeling.

    PubMed

    Tiwari, Abhinav; Abraham, Anson K; Harrold, John M; Zutshi, Anup; Singh, Pratap

    2017-03-01

    Affinity optimization of monoclonal antibodies (mAbs) is essential for developing drug candidates with the highest likelihood of clinical success; however, a quantitative approach for setting affinity requirements is often lacking. In this study, we computationally analyzed the in vivo mAb-target binding kinetics to delineate general principles for defining optimal equilibrium dissociation constant ([Formula: see text]) of mAbs against soluble and membrane-bound targets. Our analysis shows that in general [Formula: see text] to achieve 90% coverage for a soluble target is one tenth of its baseline concentration ([Formula: see text]), and is independent of the dosing interval, target turnover rate or the presence of competing ligands. For membrane-bound internalizing targets, it is equal to the ratio of internalization rate of mAb-target complex and association rate constant ([Formula: see text]). In cases where soluble and membrane-bound forms of the target co-exist, [Formula: see text] lies within a range determined by the internalization rate ([Formula: see text]) of the mAb-membrane target complex and the ratio of baseline concentrations of soluble and membrane-bound forms ([Formula: see text]). Finally, to demonstrate practical application of these general rules, we collected target expression and turnover data to project [Formula: see text] for a number of marketed mAbs against soluble (TNFα, RANKL, and VEGF) and membrane-bound targets (CD20, EGFR, and HER2).

  9. Efficient generation of human IgA monoclonal antibodies.

    PubMed

    Lorin, Valérie; Mouquet, Hugo

    2015-07-01

    Immunoglobulin A (IgA) is the most abundant antibody isotype produced in humans. IgA antibodies primarily ensure immune protection of mucosal surfaces against invading pathogens, but also circulate and are present in large quantities in blood. IgAs are heterogeneous at a molecular level, with two IgA subtypes and the capacity to form multimers by interacting with the joining (J) chain. Here, we have developed an efficient strategy to rapidly generate human IgA1 and IgA2 monoclonal antibodies in their monomeric and dimeric forms. Recombinant monomeric and dimeric IgA1/IgA2 counterparts of a prototypical IgG1 monoclonal antibody, 10-1074, targeting the HIV-1 envelope protein, were produced in large amounts after expression cloning and transient transfection of 293-F cells. 10-1074 IgAs were FPLC-purified using a novel affinity-based resin engrafted with anti-IgA chimeric Fabs, followed by a monomers/multimers separation using size exclusion-based FPLC. ELISA binding experiments confirmed that the artificial IgA class switching of 10-1074 did not alter its antigen recognition. In summary, our technical approach allows the very efficient production of various forms of purified recombinant human IgA molecules, which are precious tools in dissecting IgA B-cell responses in physiological and pathophysiological conditions, and studying the biology, function and therapeutic potential of IgAs.

  10. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  11. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  12. Poly(hydroxyethyl methacrylate) based affinity membranes for in vitro removal of anti-dsDNA antibodies from SLE plasma.

    PubMed

    Uzun, Lokman; Yavuz, Handan; Osman, Bilgen; Celik, Hamdi; Denizli, Adil

    2010-07-01

    The preparation of polymeric membrane using affinity technology for application in blood filtration devices is described here. DNA attached poly(hydroxyethyl methacrylate) (PHEMA) based microporous affinity membrane was prepared for selective removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in in vitro. In order to further increase blood-compatibility of affinity membrane, aminoacid based comonomer N-methacryloyl-L-alanine (MAAL) was included in the polymerization recipe. PHEMAAL membrane was produced by a photopolymerization technique and then characterized by swelling tests and scanning electron microscope (SEM) studies. Blood-compatibility tests were also performed. The water swelling ratio of PHEMAAL membrane increased significantly (133.2%) compared with PHEMA (58%). PHEMAAL membrane has large pores around in the range of 5-10 microm. All the clotting times increased when compared with PHEMA membrane. Loss of platelets and leukocytes was very low. DNA loading was 7.8 mg/g. There was a very low anti-dsDNA-antibody adsorption onto the plain PHEMAAL membrane, about 78 IU/g. The PHEMAAL-DNA membrane adsorbed anti-dsDNA-antibody in the range of 10-68 x 10(3)IU/g from SLE plasma. Anti-dsDNA-antibody concentration decreased significantly from 875 to 144 IU/ml with the time. Anti-dsDNA-antibodies could be repeatedly adsorbed and eluted without noticeable loss in the anti-dsDNA-antibody adsorption amount.

  13. An improved affinity tag based on the FLAG peptide for the detection and purification of recombinant antibody fragments.

    PubMed

    Knappik, A; Plückthun, A

    1994-10-01

    The commercially available monoclonal antibodies M1 and M2 were raised against and bind the FLAG sequence DYKDDDDK with high specificity. Using the calcium-dependent M1 antibody and the FLAG tag attached to the N terminus of various fragments of the antibody McPC603 expressed in Escherichia coli, we found that the M1 antibody binds with almost the same affinity to a much shorter version of this sequence (DYKD). Since most antibody light chains start with an aspartate, the addition of only three additional amino acids to the N terminus is sufficient to detect and quantify the expressed antibody fragments using standard immunological methods. Similarly, the heavy chain can be detected specifically with the sequence DYKD, which requires four additional amino acids since most heavy chains do not start with Asp. The signal sequence of both chains that is necessary for the transport of the chains to the periplasm of E. coli is processed correctly. Furthermore, we investigated the influence of the amino acid at the fifth position of the FLAG sequence on the binding affinity of the M1 antibody and found that a glutamate at this position increased the sensitivity in Western blots sixfold over the original long FLAG sequence containing an aspartate residue at this position. Together, the improved FLAG is a versatile tool for both sensitive detection and one-step purification of recombinant proteins.

  14. 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

  15. 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.

  16. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

    PubMed Central

    Schanzer, Juergen M.; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J.; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H.; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    ABSTRACT The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  17. Statistical geometric affinity in human brain electric activity

    NASA Astrophysics Data System (ADS)

    Chornet-Lurbe, A.; Oteo, J. A.; Ros, J.

    2007-05-01

    The representation of the human electroencephalogram (EEG) records by neurophysiologists demands standardized time-amplitude scales for their correct conventional interpretation. In a suite of graphical experiments involving scaling affine transformations we have been able to convert electroencephalogram samples corresponding to any particular sleep phase and relaxed wakefulness into each other. We propound a statistical explanation for that finding in terms of data collapse. As a sequel, we determine characteristic time and amplitude scales and outline a possible physical interpretation. An analysis for characteristic times based on lacunarity is also carried out as well as a study of the synchrony between left and right EEG channels.

  18. Optimization of pore structure and particle morphology of mesoporous silica for antibody adsorption for use in affinity chromatography

    NASA Astrophysics Data System (ADS)

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-10-01

    Antibodies have received significant attention for use as antibody drugs, because they bind the objective protein (antigen) via antigen-antibody reactions. Recently, many reports have appeared on various monoclonal antibodies that recognize a single antigen. In this study, monoclonal antibodies are used as adsorbates on mesoporous silica (MPS) for affinity chromatography. MPS has high surface area and large pore volume; moreover, pore diameter, pore structure, and particle morphology are relatively easy to tune by adjusting the conditions of synthesis. The pore structure (two-dimensional (2D) hexagonal and three-dimensional cubic) and particle morphology (spherical and polyhedral) of MPS are optimized for use in a monoclonal antibody/MPS composite. When anti-IgG (one of the monoclonal antibodies) adsorbs on the MPS material and IgG (antigen) binds to anti-IgG/MPS composites, MCM-41p with a 2D-hexagonal pore structure and polyhedral particle morphology has the highest IgG binding efficiency. In addition, the antibody/MPS composites remain stable in chaotropic and low-pH solutions and can be cycled at least five times without decreasing IgG elution. In purification and removal tests, the use of the antibody/MPS composites allows only the objective protein from protein mixtures to be bound and eluted.

  19. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  20. Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner.

    PubMed

    Ohta, Akane; Fujita, Ayano; Murayama, Tsugiya; Iba, Yoshitaka; Kurosawa, Yoshikazu; Yoshikawa, Tetsushi; Asano, Yoshizo

    2009-11-01

    Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

  1. Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcgammaRIII and antibodies lacking core fucose.

    PubMed

    Ferrara, Claudia; Grau, Sandra; Jäger, Christiane; Sondermann, Peter; Brünker, Peter; Waldhauer, Inja; Hennig, Michael; Ruf, Armin; Rufer, Arne Christian; Stihle, Martine; Umaña, Pablo; Benz, Jörg

    2011-08-02

    Antibody-mediated cellular cytotoxicity (ADCC), a key immune effector mechanism, relies on the binding of antigen-antibody complexes to Fcγ receptors expressed on immune cells. Antibodies lacking core fucosylation show a large increase in affinity for FcγRIIIa leading to an improved receptor-mediated effector function. Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies is currently being developed to exploit this finding. In this study, the crystal structures of a glycosylated Fcγ receptor complexed with either afucosylated or fucosylated Fc were determined allowing a detailed, molecular understanding of the regulatory role of Fc-oligosaccharide core fucosylation in improving ADCC. The structures reveal a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent, explaining the decreased affinity for the receptor. These findings allow us to understand the higher efficacy of therapeutic antibodies lacking the core fucose and also suggest a unique mechanism by which the immune system can regulate antibody-mediated effector functions.

  2. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.

    PubMed Central

    Liu, A Y; Robinson, R R; Hellström, K E; Murray, E D; Chang, C P; Hellström, I

    1987-01-01

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man. PMID:3106970

  3. Characterization of the native and denatured herceptin by enzyme linked immunosorbent assay and quartz crystal microbalance using a high-affinity single chain fragment variable recombinant antibody.

    PubMed

    Shang, Yuqin; Mernaugh, Ray; Zeng, Xiangqun

    2012-10-02

    Herceptin/Trastuzumab is a humanized IgG1κ light chain antibody used to treat some forms of breast cancer. A phage-displayed recombinant antibody library was used to obtain a single chain fragment variable (scFv, designated 2B4) to a linear synthetic peptide representing Herceptin's heavy chain CDR3. Enzyme linked immunosorbent assays (ELISAs) and piezoimmunosensor/quartz crystal microbalance (QCM) assays were used to characterize 2B4-binding activity to both native and heat denatured Herceptin. The 2B4 scFv specifically bound to heat denatured Herceptin in a concentration dependent manner over a wide (35-220.5 nM) dynamic range. Herceptin denatures and forms significant amounts of aggregates when heated. UV-vis characterization confirms that Herceptin forms aggregates as the temperature used to heat Herceptin increases. QCM affinity assay shows that binding stoichiometry between 2B4 scFv and Herceptin follows a 1:2 relationship proving that 2B4 scFv binds strongly to the dimers of heat denatured Herceptin aggregates and exhibits an affinity constant of 7.17 × 10(13) M(-2). The 2B4-based QCM assay was more sensitive than the corresponding ELISA. Combining QCM with ELISA can be used to more fully characterize nonspecific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of the use of QCM to characterize human therapeutic antibodies in samples are also discussed.

  4. Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus.

    PubMed

    Kusano-Arai, Osamu; Fukuda, Rie; Kamiya, Wakana; Iwanari, Hiroko; Hamakubo, Takao

    2016-07-01

    The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (Kd, ∼10(-11) M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R(2) = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R(2) = 0.834). These results suggest that the BV-KinExA method described here provides a suitably accurate Kd evaluation of mAbs against proteins on the cell surface.

  5. Prospective Design of Anti‐Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain

    PubMed Central

    Kanodia, JS; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, RK; Luk, W; Hoyte, K; Lu, Y; Wildsmith, KR; Couch, JA; Watts, RJ; Dennis, MS; Ernst, JA; Scearce‐Levie, K; Atwal, JK; Joseph, S

    2016-01-01

    Anti‐transferrin receptor (TfR)‐based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR‐based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti‐TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic‐pharmacodynamic (PK‐PD) model for bispecific anti‐TfR/BACE1 antibodies that accounts for antibody‐TfR interactions at the blood‐brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti‐BACE1 arm. The calibrated model correctly predicted the optimal anti‐TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti‐TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti‐TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

  6. The Use of Monoclonal Antibodies in Human Prion Disease

    NASA Astrophysics Data System (ADS)

    Bodemer, Walter

    Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

  7. Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity.

    PubMed

    Groves, Maria A T; Amanuel, Lily; Campbell, Jamie I; Rees, D Gareth; Sridharan, Sudharsan; Finch, Donna K; Lowe, David C; Vaughan, Tristan J

    2014-01-01

    In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn

  8. Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

    PubMed Central

    Géraudie, Solène; Scheffler, Ulrike; Griep, Remko A.; Reiersen, Herald; Duncan, Alexander R.; Kiprijanov, Sergej M.

    2014-01-01

    Background CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer. PMID:25080123

  9. Autoreactivity of primary human immunoglobulins ancestral to hypermutated human antibodies that neutralize HCMV.

    PubMed

    McLean, Gary R; Cho, Chin-wen; Schrader, John W

    2006-05-01

    The human antibody response to the AD-2S1 epitope of glycoprotein B (gB) of human cytomegalovirus (HCMV) is dominated by a family of closely related somatically mutated antibodies. These antibodies neutralize viral infectivity and the genes encoding them are derived from two commonly used germ-line variable (V) region genes, IGHV3-30 and IGKV3-11. Recombination of these V genes with the appropriate junctional diversity generates genes that encode primary immunoglobulins that bind to AD-2S1. To further understand the initial primary immunoglobulin response to AD-2S1 we synthesized the germ-line-based ancestor of one such family of antibodies and showed that it bound gB at the AD-2S1 epitope. Here we show that the germ-line ancestor of a second family of antibodies likewise binds to gB. We further show that one of the ancestral primary immunoglobulins, but not the other, also recognized autoantigens. In contrast, the hypermutated derivatives did not demonstrate autoreactivity and minor structural changes in the primary immunoglobulin were sufficient to generate or abolish autoreactivity or to change specificity. Thus, our demonstration that the ancestor of a highly mutated, non-autoreactive antiviral IgG antibody binds nuclear and cell-surface autoantigens indicates for the first time that self-reactivity is not necessarily a barrier to development into a follicular B lymphocyte that undergoes antigen-initiated affinity maturation.

  10. Titre and affinity of propylthiouracil-induced anti-myeloperoxidase antibodies are closely associated with the development of clinical vasculitis.

    PubMed

    Ye, Hua; Gao, Ying; Guo, Xiao-Hui; Zhao, Ming-Hui

    2005-10-01

    Substantial evidences suggested that propylthiouracil (PTU) could induced anti-myeloperoxidase (MPO) antibodies in sera from patients with hyperthyroidism, however, only a subgroup of the PTU-induced anti-MPO antibody positive patients developed clinical evident vasculitis. The aim of this study is to compare the titres and affinities of PTU induced anti-MPO antibodies in sera from patients with hyperthyroidism with and without clinical vasculitis. Anti-MPO antibody positive sera from patients diagnosed hyperthyroidism with (n = 13) and without (n = 14) clinical evident vasculitis were collected. The titre was determined by MPO-ELISA and expressed as logarithm value (lgT). The affinity constant (aK) of anti-MPO IgG was measured by antigen inhibition assay. The titre and aK values were compared between patients with and without vasculitis. In patients with vasculitis, the mean lgT of anti-MPO antibodies was 3.62 +/- 0.66; the median aK was 4.47 x 10(7)M(-1). In patients without vasculitis, the mean lgT was 2.54 +/- 0.29; the median aK was 0.14 x 10(7)M(-1), and both were significant lower than those in patients with vasculitis (t = 5.464; P = 0.000 & z = -4.373; P = 0.000, respectively). We concluded that the titre and affinity of anti-MPO antibodies might be associated with the development of clinical vasculitis in patients with PTU-induced ANCA.

  11. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

    PubMed Central

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-01-01

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research. PMID:28266535

  12. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique

    PubMed Central

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L.; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J.; Casquel, Rafael

    2015-01-01

    The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. PMID:26287192

  13. Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique.

    PubMed

    Laguna, Maríafe; Holgado, Miguel; Hernandez, Ana L; Santamaría, Beatriz; Lavín, Alvaro; Soria, Javier; Suarez, Tatiana; Bardina, Carlota; Jara, Mónica; Sanza, Francisco J; Casquel, Rafael

    2015-08-13

    The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique.

  14. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    PubMed Central

    Avila-Alonso, Ana; González-Rivera, Milagros; Tamayo, Eduardo; Eiros, Jose María; Almansa, Raquel

    2016-01-01

    Diphtheria antitoxin for therapeutic use is in limited supply. A potential source might be affinity-purified antibodies originally derived from plasma of adults who received a booster dose of a vaccine containing diphtheria toxoid. These antibodies might be useful for treating even severe cases of diphtheria. PMID:27314309

  15. An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

    PubMed

    Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

    2015-03-31

    Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples.

  16. Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

    PubMed Central

    Sherwood, Laura J.; Hayhurst, Andrew

    2013-01-01

    Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent

  17. Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies.

    PubMed

    Mao, S J; Patton, J G; Badimon, J J; Kottke, B A; Alley, M C; Cardin, A D

    1983-11-01

    Four monoclonal antibodies (IgG2b) to human plasma low-density lipoproteins (LDL) have been characterized. The binding affinities of each monoclonal antibody to 125I-labeled LDL were moderately high, ranging from 10(8) to 10(10) L/mol at 4 degrees C, but were reduced by at least 50-70% at 37 degrees C. The maximum binding of each monoclonal antibody was unique, ranging from 20 to 95% of total 125I-labeled LDL, suggesting that LDL particles were immunochemically heterogeneous. One antibody, LP-34, had both high and low binding affinities to LDL. Another, LP-47, exhibited high affinity for isolated LDL, yet reacted poorly with native LDL in plasma, indicating that the conformation of isolated LDL differs from that of native LDL in plasma. Unlike polyclonal serum antibodies, a mixture of four monoclonal antibodies failed to precipitate LDL, but did show a drastic increase in binding to LDL. We found that only two of our monoclonal antibodies were necessary for such synergistic enhancement. We propose that one of the monoclonal antibodies may serve as a catalytic reagent, and discuss the clinical significance of this finding.

  18. Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

    PubMed

    Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

    2014-09-01

    Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

  19. Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation.

    PubMed

    Van Deventer, James A; Wittrup, Karl Dane

    2014-01-01

    Antibodies play key roles as reagents, diagnostics, and therapeutics in numerous biological and biomedical research settings. Although many antibodies are commercially available, oftentimes, specific applications require the development of antibodies with customized properties. Yeast surface display is a robust, versatile, and quantitative method for generating these antibodies and is accessible to single-investigator laboratories. This protocol details the key aspects of yeast surface display library construction and screening.

  20. Validation of radioimmunoassay for human lactalbumin in the serum by testing the endogenous antibodies interference.

    PubMed

    Zangerle, P F; Franchimont, P

    1985-10-01

    For re-establishing the value of human lactalbumin as a functional marker of normal and pathological activity of the breast a sensitive and specific radioimmunoassay was established with the prior important control of the interference of endogenous antibodies. The specificity of the assay was assessed by the absence of interference from other proteins in milk or in breast cyst fluid, various hormones and tumor markers. Bovine lactalbumin showed incomplete and weak cross-reactivity. By an enzymoimmunoassay method it was shown that all the 222 human sera studied contain IgG immunoglobulins which bind bovine and human lactalbumin with greater reactivity of children's serum and without relationship to the blood groups. The maximum affinity constant of these endogenous immunoglobulins determined by the radioimmunoassay method is 4.5 times greater for bovine (Kd = 18 X 4(-11) M) than for human (Kd = 4 X 10(-11) M) lactalbumin. These endogenous anti-lactalbumin immunoglobulins caused no interference in the radioimmunoassay as shown by the complete correlation between the concentrations of human lactalbumin previously incubated and added to sera containing high-affinity antibodies and those measured directly in the radioimmunoassay. This lack of interference was explained by the higher (22-fold) affinity constant of the rabbit antiserum against human lactalbumin (Kd = 9 X 10(-12) M). The study of endogenous antibodies by the two enzymes and radioimmunoassay methods is needed before assessing and using a radioimmunoassay of human lactalbumin in serum.

  1. Purification of antibodies against N-homocysteinylated proteins by affinity chromatography on Nomega-homocysteinyl-aminohexyl-Agarose.

    PubMed

    Perła, Joanna; Undas, Anetta; Twardowski, Tomasz; Jakubowski, Hieronim

    2004-08-05

    Modification with homocysteine (Hcy)-thiolactone leads to the formation of N-Hcy-Lys-protein. Although N-Hcy-Lys-proteins are immunogenic, pure antibodies have not yet been obtained. Here we describe synthesis and application of Nomega-homocysteinyl-aminohexyl-Agarose for affinity purification of anti-N-Hcy-Lys-protein antibodies. Nomega-homocysteinyl-aminohexyl-Agarose was prepared by N-homocysteinylation of omega-aminohexyl-Agarose with Hcy-thiolactone. Immune serum was obtained from rabbits inoculated with N-Hcy-Lys-keyhole limpet hemocyanine and IgG fraction prepared by chromatography on protein A-Agarose. Anti-N-Hcy-Lys-protein IgG was adsorbed on Nomega-homocysteinyl-aminohexyl-Agarose column at pH 8.6 and eluted with a pH 2.3 buffer. Enzyme-linked immunosorbent assays demonstrate that the antibody recognizes specifically N-homocysteinylated variants of hemoglobin, albumin, transferrin, and antitrypsin.

  2. Preferential germline usage and VH/VL pairing observed in human antibodies selected by mRNA display.

    PubMed

    Chen, Lei; Kutskova, Yuliya A; Hong, Feng; Memmott, John E; Zhong, Suju; Jenkinson, Megan D; Hsieh, Chung-Ming

    2015-10-01

    Since the invention of phage display, in vitro antibody display technologies have revolutionized the field of antibody discovery. In combination with antibody libraries constructed with sequences of human origin, such technologies enable accelerated therapeutic antibody discovery while bypassing the laborious animal immunization and hybridoma generation processes. Many in vitro display technologies developed since aim to differentiate from phage display by displaying full-length IgG proteins, utilizing eukaryotic translation system and codons, increasing library size or real-time kinetic selection by fluorescent activated cell sorting. We report here the development of an mRNA display technology and an accompanying HCDR3 size spectratyping monitor for human antibody discovery. Importantly, the mRNA display technology maintains a monovalent linkage between the mRNA (genotype) and display binding protein (phenotype), which minimizes avidity effect common in other display systems and allows for a stringent affinity and off-rate selection. The mRNA display technology successfully identified 100 human antibodies in 15 different selections against various targets from naïve human antibody libraries. These antibodies in general have high affinity and diversity. By analyzing the germline usage and combination of antibodies selected by the mRNA display technology, we identified trends and determined the productivity of each germline subgroup in the libraries that could serve as the knowledge base for constructing fully synthetic, next generation antibody libraries.

  3. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  4. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

    PubMed

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C; Van Bergen En Henegouwen, Paul M P; Fernández, Luis Ángel

    2016-10-01

    Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

  5. Influenza virus resistance to human neutralizing antibodies.

    PubMed

    Crowe, James E

    2012-01-01

    The human antibody repertoire has an exceptionally large capacity to recognize new or changing antigens through combinatorial and junctional diversity established at the time of V(D)J recombination and through somatic hypermutation. Influenza viruses exhibit a relentless capacity to escape the human antibody response by altering the amino acids of their surface proteins in hypervariable domains that exhibit a high level of structural plasticity. Both parties in this high-stakes game of shape shifting drive structural evolution of their functional proteins (the B cell receptor/antibody on one side and the viral hemagglutinin and neuraminidase proteins on the other) using error-prone polymerase systems. It is likely that most of the genetic mutations that occur in these systems are deleterious, resulting in the failure of the B cell or virus with mutations to propagate in the immune repertoire or viral quasispecies. A subset of mutations is tolerated in functional surface proteins that enter the B cell or virus progeny pool. In both cases, selection occurs in the population of mutated and unmutated species. In cases where the functional avidity of the B cell receptor is increased significantly, that clone may be selected for preferential expansion. In contrast, an influenza virus that "escapes" the inhibitory effect of secreted antibodies may represent a high proportion of the progeny virus in that host. The recent paper by O'Donnell et al. [C. D. O'Donnell et al., mBio 3(3):e00120-12, 2012] identifies a mechanism for antibody resistance that does not require escape from binding but rather achieves a greater efficiency in replication.

  6. 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

  7. 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.

  8. Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.

    PubMed

    Conway de Macario, E; Macario, A J; Tosi, R M; Celada, F; Landucci-Tosi, S

    1978-08-01

    A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were

  9. Long-term antibody synthesis in vitro. VI. Anti-allotype sera as probes of clonal products in affinity maturation.

    PubMed Central

    Conway de Macario, E; Macario, A J; Tosi, R M; Celada, F; Landucci-Tosi, S

    1978-01-01

    A new experimental system is described for measuring the allotypic product of rabbit B cells during long-lasting in vitro antibody responses. The immunoenzymatic assays described allow determination of several parameters mapping in different regions of the same molecule, which can be measured and combined to yield a multidimensional picture of the time-course dynamics of antibody synthesis. The rabbit immune system responding to Escherichia coli beta-D-galactosidase was sample and disassembled by (a) culturing lymph node microfragments and (b) sorting out from among all anti-enzyme antibodies only those activating a mutant enzyme, AMEF, which bore the b4 or b9 allotype. A considerable simplification of the response was achieved in the microcultures as documented by cultures of heterozygous cells which produced only one allotype and by the fact that each culture showed a distinctive pattern when antibody titre, association constant, heterogeneity index, L-chain type, and k-chain allotype were considered together. This array of patterns was not an artifact but the result of disassembling a representative sample of the rabbit immune system into small components, since the b4/b9 ratio obtained by averaging the results of all cultures from a heterozygous rabbit lymph node was the same as the serum ratio. Despite the Poisson distribution of the responder microcultures, none of them was monoclonal; i.e. no antibodies homogeneous by all parameters tested were observed, This finidng supports the notion that in normal lymphoid tissue in its native tridimensional arrangement, one T cell can trigger several B cells clustered in one antibody-forming unit. This natural arrangement would ensure the monospecificity of the cluster (dictated by the T cell) while allowing for variation in affinity (depending upon the array of B cells in the unit). Accordingly our findings would results from the fact that as the size of the microfragments was reduced, the cells diluted out first were

  10. Development of monoclonal antibodies against parathyroid hormone: genetic control of the immune response to human PTH

    SciTech Connect

    Nussbaum, S.R.; Lin, C.S.; Potts, J.T. Jr.; Rosenthal, A.S.; Rosenblatt, M.

    1985-01-01

    Seventeen monocloanl antibodies against the aminoterminal portion of parathyroid hormone (PTH) were generated by using BALB/c mouse for immunization fully biologically active synthetic human PTH-(1-34) and bovine PTH-(1-84) as immunogens, monoclonal antibody methods, and a solid-phase screening assay. Isotypic analysis of these monoclonal antibodies was performed using affinity purified goat antimouse immunoglobulins specific for IgG heavy chains and ..mu..(IgM). All antibodies were IgM as evidenced by 40 times greater than background activity when 25,000 cpm of /sup 125/I-labelled goat anti-mouse IgM was used as second antibody in a radioimmunoassay.

  11. A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

    PubMed

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

    2014-01-01

    A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

  12. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  13. Human whole-blood oxygen affinity: effect of temperature.

    PubMed

    Zwart, A; Kwant, G; Oeseburg, B; Zijlstra, W G

    1984-08-01

    phe effect of temperature changes on human whole-blood O2 affinity was measured in the blood of six healthy donors over almost the entire O2 saturation (SO2) range (1-99%). The results showed that temperature has no influence on the shape of the O2 dissociation curve, implying that the temperature coefficient (delta log PO2/delta T) is independent of SO2. Simultaneous measurements of the total (proton) Haldane factor (delta[HbH]/[delta HbO2]) at the five temperatures under study (22, 27, 32, 37, and 42 degrees C) revealed that this factor depends on temperature. The liberation of protons from hemoglobin appeared to be linear with respect to changes in SO2. We therefore conclude that the (proton) Bohr factor (H+ factor) is dependent on temperature over the entire SO2 range in the same way as previously described for SO2 = 50%. The exothermic oxygenation reaction in whole blood was accompanied by a heat evolution (delta HO2) of 42.7 kJ/mol (monomeric) hemoglobin.

  14. Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin.

    PubMed

    Naik, Amith D; Menegatti, Stefano; Reese, Hannah R; Gurgel, Patrick V; Carbonell, Ruben G

    2012-10-19

    The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.

  15. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    SciTech Connect

    Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

    1988-01-01

    The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

  16. Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

    DOEpatents

    Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

    1984-11-29

    The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

  17. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  18. Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies.

    PubMed

    Batalha, Iris L; Hussain, Abid; Roque, A C A

    2010-01-01

    A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non-functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q(máx) of 344 mg IgG/g support and K(a) of 1.5 x 10(5) M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine-NaOH (pH 11) buffer.

  19. VH-VL orientation prediction for antibody humanization candidate selection: A case study

    PubMed Central

    Bujotzek, Alexander; Lipsmeier, Florian; Harris, Seth F; Benz, Jörg; Kuglstatter, Andreas; Georges, Guy

    2016-01-01

    abstract Antibody humanization describes the procedure of grafting a non-human antibody's complementarity-determining regions, i.e., the variable loop regions that mediate specific interactions with the antigen, onto a β-sheet framework that is representative of the human variable region germline repertoire, thus reducing the number of potentially antigenic epitopes that might trigger an anti-antibody response. The selection criterion for the so-called acceptor frameworks (one for the heavy and one for the light chain variable region) is traditionally based on sequence similarity. Here, we propose a novel approach that selects acceptor frameworks such that the relative orientation of the 2 variable domains in 3D space, and thereby the geometry of the antigen-binding site, is conserved throughout the process of humanization. The methodology relies on a machine learning-based predictor of antibody variable domain orientation that has recently been shown to improve the quality of antibody homology models. Using data from 3 humanization campaigns, we demonstrate that preselecting humanization variants based on the predicted difference in variable domain orientation with regard to the original antibody leads to subsets of variants with a significant improvement in binding affinity. PMID:26637054

  20. Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein.

    PubMed

    Sun, Lina; Chen, Zhe; Yu, Li; Wei, Jingshuang; Li, Chuan; Jin, Jing; Shen, Xinxin; Lv, Xinjun; Tang, Qing; Li, Dexin; Liang, Mifang

    2012-10-01

    The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

  1. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    PubMed

    Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

    2007-06-01

    Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P < 0.0001). Blocking Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  2. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    PubMed

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  3. Framework selection can influence pharmacokinetics of a humanized therapeutic antibody through differences in molecule charge

    PubMed Central

    Li, Bing; Tesar, Devin; Boswell, C Andrew; Cahaya, Hendry S; Wong, Anne; Zhang, Jianhuan; Meng, Y Gloria; Eigenbrot, Charles; Pantua, Homer; Diao, Jinyu; Kapadia, Sharookh B; Deng, Rong; Kelley, Robert F

    2014-01-01

    Pharmacokinetic (PK) testing of a humanized (κI, VH3 framework) and affinity matured anti-hepatitis C virus E2-glycoprotein (HCV-E2) antibody (hu5B3.κ1VH3.v3) in rats revealed unexpected fast clearance (34.9 mL/day/kg). This antibody binds to the rat recycling receptor FcRn as expected for a human IgG1 antibody and does not display non-specific binding to baculovirus particles in an assay that is correlated with fast clearance in cynomolgus monkey. The antigen is not expressed in rat so target-dependent clearance does not contribute to PK. Removal of the affinity maturation changes (hu5B3.κ1VH3.v1) did not restore normal clearance. The antibody was re-humanized on a κ4, VH1 framework and the non-affinity matured version (hu5B3.κ4VH1.v1) was shown to have normal clearance (8.5 mL/day/kg). Since the change in framework results in a lower pI, primarily due to more negative charge on the κ4 template, the effect of additional charge variation on antibody PK was tested by incorporating substitutions obtained through phage display affinity maturation of hu5B3.κ1VH3.v1. A variant having a pI of 8.61 gave very fast clearance (140 mL/day/kg) whereas a molecule with pI of 6.10 gave slow clearance (5.8 mL/kg/day). Both antibodies exhibited comparable binding to rat FcRn, but biodistribution experiments showed that the high pI variant was catabolized in liver and spleen. These results suggest antibody charge can have an effect on PK through alterations in antibody catabolism independent of FcRn-mediated recycling. Furthermore, introduction of affinity maturation changes into the lower pI framework yielded a candidate with PK and virus neutralization properties suitable for clinical development. PMID:25517310

  4. SDR grafting--a new approach to antibody humanization.

    PubMed

    Kashmiri, Syed V S; De Pascalis, Roberto; Gonzales, Noreen R; Schlom, Jeffrey

    2005-05-01

    A major impediment to the clinical utility of the murine monoclonal antibodies is their potential to elicit human anti-murine antibody (HAMA) response in patients. To circumvent this problem, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts. To that end, murine antibodies have been humanized by grafting their complementarity determining regions (CDRs) onto the variable light (V(L)) and variable heavy (V(H)) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients. To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies has been described that is based on grafting, onto the human frameworks, only the specificity determining residues (SDRs), the CDR residues that are most crucial in the antibody-ligand interaction. The SDRs are identified through the help of the database of the three-dimensional structures of the antigen-antibody complexes of known structures or by mutational analysis of the antibody-combining site. An alternative approach to humanization, which involves retention of more CDR residues, is based on grafting of the 'abbreviated' CDRs, the stretches of CDR residues that include all the SDRs. A procedure to assess the reactivity of the humanized antibody to sera from patients who had been administered the murine antibody has also been described.

  5. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

    PubMed Central

    Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

    2015-01-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

  6. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins

    PubMed Central

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-01-01

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins. PMID:28224978

  7. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins.

    PubMed

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-02-22

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using (14)N/(15)N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.

  8. Magnetic poly(2-hydroxyethyl methacrylate) microspheres for affinity purification of antibodies for early diagnosis of multiple sclerosis patients.

    PubMed

    Horak, Daniel; Hlidkova, Helena; Kit, Yurii; Stoika, Rostyslav; Antonyuk, Volodymyr; Myronovsky, Severyn

    2017-03-28

    The aim of this work is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ca. 4 µm in diameter and containing ~ 1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and [(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier-transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS-PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.

  9. humMR1, a highly specific humanized single chain antibody for targeting EGFRvIII.

    PubMed

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

    2014-02-01

    Production of an efficient humanized single chain antibody is reported here to specifically target EGFRvIII, a truncated receptor expressed in a wide variety of human cancers. CDR loops of MR1, a phage display-derived murine single chain antibody developed against this mutant receptor, were grafted on human frameworks that had been selected based on similarity to MR1 in terms of two distinct parameters, variable domain protein sequence and CDR canonical structures. Moreover, two point mutations were introduced in CDR-H2 and CDR-H3 loops of the humanized antibody to destroy its cross-reactivity to wild-type EGFR. The resultant antibody, referred to as humMR1, was found by MTT assay, ELISA and western blot techniques to be highly specific for EGFRvIII. The affinity of this antibody for EGFRvIII-specific 14-amino acid synthetic peptide and HC2 cells were measured to be 1.87 × 10(10) and 2.17 × 10(10)/M respectively. This humanized antibody leads to 78.5% inhibition in proliferation of EGFRvIII-overexpressing cells.

  10. Generation and characterization of human B lymphocyte stimulator blocking monoclonal antibody.

    PubMed

    Zhuang, Weiliang; Zhang, Jianjun; Pei, Lili; Fang, Shuping; Liu, Honghao; Wang, Ruixue; Su, Yunpeng

    2016-09-01

    The cytokine, B lymphocyte stimulator (Blys) is essential for activation and proliferation of B cells and is involved in the pathogenesis of B-cell mediated autoimmune diseases. Based on its essential activity, Blys may be a potential therapeutic target for human autoimmune diseases. In this article, we have described the development of a novel humanized anti-Blys antibody, NMB04, that binds with high affinity and specificity to both soluble and membrane bound Blys. This monoclonal antibody has the potential to block Blys binding to all its three receptors, TACI, BCMA and BR-3. Further in vivo studies revealed that NMB04 possessed more potent inhibitory activity against human Blys as compared to an existing antibody, Belimumab. Therefore, NMB04 may have potential as a therapeutic candidate targeting autoimmune diseases.

  11. Localisation of malignant glioma by a radiolabelled human monoclonal antibody.

    PubMed Central

    Phillips, J; Alderson, T; Sikora, K; Watson, J

    1983-01-01

    Human monoclonal antibodies were produced by fusing intratumoral lymphocytes from patients with malignant gliomas with a human myeloma line. One antibody was selected for further study after screening for binding activity to glioma cell lines. The patient from whom it was derived developed recurrent glioma. 1 mg of antibody was purified, radiolabelled with 131I, and administered intravenously. The distribution of antibody was determined in the blood, CSF and tumour cyst fluid and compared with that of a control human monoclonal immunoglobulin. Antibody localisation in the tumour was observed and confirmed by external scintiscanning. Images PMID:6101173

  12. Emerging studies of human HIV-specific antibody repertoires

    PubMed Central

    Hicar, Mark D.; Kalams, Spyros A.; Spearman, Paul W.; Crowe, James E.

    2010-01-01

    There has been an explosion of interest in the human B cell response to HIV infection of late. Recent advances in techniques for isolation of human antibodies and antibody secreting cell lines have facilitated a rapid expansion in the number of antibodies available for study. Early analysis of these repertoires reveals interesting features of the HIV-specific antibody response. HIV-specific repertoires exhibit a high level of clonality in circulating cells, and high levels of somatic mutations within the antibody variable gene segments. It appears that many if not most antibodies in circulation bind to virus envelope conformations that are found only in complex oligomeric structures on virion particles or virus-like particles. The rapid isolation of large panels of novel human neutralizing antibodies promises to reveal new insights into the fundamental principles underlying antibody-mediated neutralization of HIV. PMID:20510738

  13. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env.

    PubMed

    Heydarchi, Behnaz; Center, Rob J; Bebbington, Jonathan; Cuthbertson, Jack; Gonelli, Christopher; Khoury, Georges; Mackenzie, Charlene; Lichtfuss, Marit; Rawlin, Grant; Muller, Brian; Purcell, Damian

    2017-04-01

    We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection.

  14. Trimeric gp120-specific bovine monoclonal antibodies require cysteine and aromatic residues in CDRH3 for high affinity binding to HIV Env

    PubMed Central

    Center, Rob J.; Bebbington, Jonathan; Cuthbertson, Jack; Khoury, Georges; Lichtfuss, Marit; Rawlin, Grant; Purcell, Damian

    2017-01-01

    ABSTRACT We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titer polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env. Characterization of both the fully bovine and human chimeric isoforms of these two mAbs revealed them as highly type-specific and capable of binding only to soluble AD8 uncleaved gp140 trimers and covalently stabilized AD8 SOSIP gp140 cleaved trimers, but not monomeric gp120. Genomic sequence analysis of the V genes showed the third heavy complementarity-determining region (CDRH3) of 6A mAb was 21 amino acids in length while 8C CDRH3 was 14 amino acids long. The entire V heavy (VH) region was 27% and 25% diverged for 6A and 8C, respectively, from the best matched germline V genes available, and the CDRH3 regions of 6A and 8C were 47.62% and 78.57% somatically mutated, respectively, suggesting a high level of somatic hypermutation compared with CDRH3 of other species. Alanine mutagenesis of the VH genes of 6A and 8C, showed that CDRH3 cysteine and tryptophan amino acids were crucial for antigen binding. Therefore, these bovine vaccine-induced anti-HIV antibodies shared some of the notable structural features of elite human broadly neutralizing antibodies, such as CDRH3 size and somatic mutation during affinity-maturation. However, while the 6A and 8C mAbs inhibited soluble CD4 binding to gp140 Env, they did not recapitulate the neutralizing activity of the polyclonal antibodies against HIV infection. PMID:27996375

  15. A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

    PubMed

    Scalley-Kim, Michelle L; Hess, Bruce W; Kelly, Ryan L; Krostag, Anne-Rachel F; Lustig, Kurt H; Marken, John S; Ovendale, Pamela J; Posey, Aaron R; Smolak, Pamela J; Taylor, Janelle D L; Wood, C L; Bienvenue, David L; Probst, Peter; Salmon, Ruth A; Allison, Daniel S; Foy, Teresa M; Raport, Carol J

    2012-01-01

    Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

  16. A novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: a case study using monoclonal antibodies against the West Nile virus E protein.

    PubMed

    Denisova, Galina F; Denisov, Dimitri A; Yeung, Jeffrey; Loeb, Mark B; Diamond, Michael S; Bramson, Jonathan L

    2008-11-01

    Understanding antibody function is often enhanced by knowledge of the specific binding epitope. Here, we describe a computer algorithm that permits epitope prediction based on a collection of random peptide epitopes (mimotopes) isolated by antibody affinity purification. We applied this methodology to the prediction of epitopes for five monoclonal antibodies against the West Nile virus (WNV) E protein, two of which exhibit therapeutic activity in vivo. This strategy was validated by comparison of our results with existing F(ab)-E protein crystal structures and mutational analysis by yeast surface display. We demonstrate that by combining the results of the mimotope method with our data from mutational analysis, epitopes could be predicted with greater certainty. The two methods displayed great complementarity as the mutational analysis facilitated epitope prediction when the results with the mimotope method were equivocal and the mimotope method revealed a broader number of residues within the epitope than the mutational analysis. Our results demonstrate that the combination of these two prediction strategies provides a robust platform for epitope characterization.

  17. Effect of VK framework-1 glycosylation on the binding affinity of lymphoma-specific murine and chimeric LL2 antibodies and its potential use as a novel conjugation site.

    PubMed

    Leung, S O; Dion, A S; Pellegrini, M C; Losman, M J; Grebenau, R C; Goldenberg, D M; Hansen, H J

    1995-02-08

    A potential asparagine (Asn)-linked glycosylation site was identified in the VK FRI sequence of an anti-B lymphoma monoclonal antibody (MAb), LL2.SDS-PAGE analysis and endo-F treatment of both murine and chimeric LL2 antibodies indicated that this site was glycosylated; however, no differences in the binding affinity to Raji cells were observed between the native murine LL2 and the endo-F-deglycosylated murine LL2 antibodies. Elimination of the glycosylation site from the chimeric LL2 antibody was accomplished by an Asn to Gln mutation in the tri-acceptor site found in the light chain. The resultant aglycosylated chimeric LL2 exhibited a similar Raji cell binding affinity to that of the glycosylated form. The results are in agreement with computer modeling studies which suggested the lack of interactions between the oligosaccharide moiety and the CDRs. The finding is interesting because it enables a wider choice of human framework sequences, which in most cases do not have a corresponding glycosylation site, for the humanization of the LL2 VK domain, as well as a greater latitude of host expression systems. Most importantly, the LL2 VK carbohydrate moiety might be used as a novel conjugation site for drugs and radionuclides without compromising the immunoreactivity of the antibody.

  18. Physiologically relevant binding affinity quantification of monoclonal antibody PF‐00547659 to mucosal addressin cell adhesion molecule for in vitro in vivo correlation

    PubMed Central

    Wang, Mengmeng; Kussrow, Amanda K; Ocana, Mireia Fernandez; Chabot, Jeffrey R; Lepsy, Christopher S

    2016-01-01

    Background and Purpose A monoclonal antibody (PF‐00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans‐membrane (mMAdCAM) target forms, showed over 30‐fold difference in antibody‐target KD between in vitro (Biacore) and clinically derived (KD,in‐vivo) values. Back‐scattering interferometry (BSI) was applied to acquire physiologically relevant KD values which were used to establish in vitro and in vivo correlation (IVIVC). Experimental Approach BSI was applied to obtain KD values between PF‐00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue. CHO cells and tissue were minimally processed to yield homogenate containing membrane vesicles and soluble proteins. A series of binding affinities in serum with various dilution factors was used to estimate both KD,in‐vivo and target concentrations; MAdCAM concentrations were also measured using LC–MS/MS. Key Results BSI measurements revealed low KD values (higher affinity) for sMAdCAM in buffer and serum, yet a 20‐fold higher KD value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted KD,in‐vivo in serum was similar to clinically derived KD,in‐vivo, and the BSI‐estimated serum sMAdCAM concentration also matched the measured concentration by LC–MS/MS. Conclusions and Implications Our results successfully demonstrated that BSI measurements of physiologically relevant KD values can be used to establish IVIVC, for PF‐00547659 to MAdCAM despite the lack of correlation when using Biacore measured KD and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development. PMID:27760281

  19. Phase Transitions in Antibody Solutions: from Pharmaceuticals to Human Disease

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Lomakin, Aleksey; Benedek, George; Dana Farber Cancer Institute Collaboration; Amgen Inc. Collaboration

    2014-03-01

    Antibodies are very important proteins. Natural antibodies play essential role in the immune system of human body. Pharmaceutical antibodies are used as drugs. Antibodies are also indispensable tools in biomedical research and diagnostics. Recently, a number of observations of phase transitions of pharmaceutical antibodies have been reported. These phase transitions are undesirable from the perspective of colloid stability of drug solutions in processing and storage, but can be used for protein purification, X-ray crystallography, and improving pharmokinetics of drugs. Phase transitions of antibodies can also take place in human body, particularly in multiple myeloma patients who overproduce monoclonal antibodies. These antibodies, in some cases, crystallize at body temperature and cause severe complications called cryoglobulinemia. I will present the results of our current studies on phase transitions of both pharmaceutical antibodies and cryoglobulinemia-associated antibodies. These studies have shown that different antibodies have different propensity to undergo phase transitions, but their phase behavior has universal features which are remarkably different from those of spherical proteins. I will discuss how studies of phase behavior can be useful in assessing colloid stability of pharmaceutical antibodies and in early diagnostics of cryoglobulinemia, as well as general implications of the fact that some antibodies can precipitate at physiological conditions.

  20. A fully human anti-CD47 blocking antibody with therapeutic potential for cancer

    PubMed Central

    Chen, Ang; Fan, Jiangfeng; Yang, Xiaopeng; Xu, Lei; Du, Peng; Qiu, Weiyi; Zhang, Weicai; Wang, Shuang; Sun, Zhiwei

    2016-01-01

    CD47/SIRPα interaction serves as an immune checkpoint for macrophage-mediated phagocytosis. Mouse anti-CD47 blocking antibodies had demonstrated potent efficacy in the treatment of both leukemic and solid tumors in preclinical experimentations, and therefore had moved forward rapidly into clinical trials. However, a fully human blocking antibody, which meets clinical purpose better, has not been reported for CD47 up to date. In this study, we reported the isolation of a fully human anti-CD47 blocking antibody, ZF1, from a phage display library. ZF1 displayed high specificity and affinity for CD47 protein, which were comparable to those for humanized anti-CD47 blocking antibody B6H12. Importantly, ZF1 treatment could induce robust, or even stronger than B6H12, phagocytosis of leukemic cancer cells by macrophage in vitro, and protect BALB/c nude mice from cancer killing by engrafted leukemic cells (CCRF and U937) to a similar extent as B6H12 did. Thus, these data provide primary early pre-clinical support for the development of ZF1 as a fully human blocking antibody to treat human leukemia by targeting CD47 molecule. PMID:27863402

  1. Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc).

    PubMed

    Niccheri, Francesca; Real-Fernàndez, Feliciana; Ramazzotti, Matteo; Lolli, Francesco; Rossi, Giada; Rovero, Paolo; Degl'Innocenti, Donatella

    2014-10-01

    Multiple sclerosis (MS) is a chronic auto-immune disease characterized by a damage to the myelin component of the central nervous system. Self-antigens created by aberrant glycosylation have been described to be a key component in the formation of auto-antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS-specific auto-antibodies, and it is actively used in diagnostics and research to monitor and quantify MS-associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His-tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide-clone interaction were calculated obtaining a value of KD in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies.

  2. 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.

  3. Affinity purification of antibodies using immobilized FB domain of protein A.

    PubMed

    Solomon, B; Raviv, O; Leibman, E; Fleminger, G

    1992-04-24

    A continuous method for the efficient digestion of protein A into active fragments (FB, Mr = 7000) using immobilized trypsin was developed. These fragments originate from almost identical five-repeated monovalent Fc-binding units of 58 residues each. The fragments obtained were found to be similar to the recently described genetically engineered fragment B. Antibody-binding characteristics of the FB domain and also of intact protein A, immobilized on to adipic dihydrazide-modified Eupergit CB6200 beads, were investigated. Based on the experimental data obtained, a high-performance liquid chromatographic column containing C30N Eupergit C-immobilized FB domain was prepared and its performance in antibody purification was compared with that of Eupergit C-immobilized intact protein A.

  4. High affinity antibodies against Lex and sialyl Lex from a phage display library.

    PubMed

    Dinh, Q; Weng, N P; Kiso, M; Ishida, H; Hasegawa, A; Marcus, D M

    1996-07-15

    Our previous studies of seven murine mAbs against the carbohydrate Lex Ag demonstrated that they were all encoded by VH441 and V kappa 24B. To obtain higher affinity Abs, and to ascertain whether their L chains could be encoded by other genes, we constructed a phage display library in a modified pComb 8 vector. The library contained random L chains, and Fd segments enriched in VH domains encoded by the VHX24 gene family. We selected phage with an Lex-BSA Ag, and obtained two Fab mAbs, clones 23 and 24, whose affinities were more than 100-fold higher than hybridoma mAb PM81. Both new mAbs were encoded by VH441, and their L chains were encoded by genes of the V kappa Ox1 and V kappa 9 families. In contrast to hybridoma mAb PM81, which binds only Lex, clones 23 and 24 bound sialyl Lex (SLex) as well as Lex, and clone 23 also binds the backbone carbohydrate structure nLacCer. Analysis of the binding of these three mAbs to synthetic glycolipids that contained structural modifications indicated that they recognize different aspects of the Lex structure, and suggested that they bind to limited regions of the oligosaccharide.

  5. Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo.

    PubMed

    Cao, Jingyuan; Meng, Shufang; Li, Chuan; Ji, Yan; Meng, Qingling; Zhang, Quanfu; Liu, Feng; Li, Jiandong; Bi, Shengli; Li, Dexin; Liang, Mifang

    2008-07-01

    Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.

  6. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    PubMed

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  7. Purification of human immunoglobulin G autoantibodies to tumor necrosis factor using affinity chromatography and magnetic separation.

    PubMed

    Sennikov, S V; Golikova, E A; Kireev, F D; Lopatnikova, J A

    2013-04-30

    Autoantibodies to cytokines are important biological effector molecules that can regulate cytokine activities. The aim of the study was to develop a protocol to purify autoantibodies to tumor necrosis factor from human serum, for use as a calibration material to determine the absolute content of autoantibodies to tumor necrosis factor by enzyme-linked immunosorbent assay. The proposed protocol includes a set of affinity chromatography methods, namely, Bio-Gel P6DG sorbent to remove albumin from serum, Protein G Sepharose 4 Fast Flow to obtain a total immunoglobulin G fraction of serum immunoglobulins, and Affi-Gel 15 to obtain specifically antibodies to tumor necrosis factor. The addition of a magnetic separation procedure to the protocol eliminated contaminant tumor necrosis factor from the fraction of autoantibodies to tumor necrosis factor. The protocol generated a pure fraction of autoantibodies to tumor necrosis factor, and enabled us to determine the absolute concentrations of different subclasses of immunoglobulin G autoantibodies to tumor necrosis factor in apparently healthy donors.

  8. Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response.

    PubMed

    Siegrist, Claire-Anne; Pihlgren, Maria; Tougne, Chantal; Efler, Sue M; Morris, Mary Lou; AlAdhami, Mohammed J; Cameron, D William; Cooper, Curtis L; Heathcote, Jenny; Davis, Heather L; Lambert, Paul-Henri

    2004-12-16

    We assessed the avidity maturation process elicited by human immunization with alum-adsorbed HBsAg alone or with a novel adjuvant containing CpG motifs (CpG 7909). Mean avidity indexes and distribution of low- and high-avidity anti-HBs indicated that avidity maturation essentially takes place late after priming. CpG 7909 markedly enhanced this affinity maturation process, increasing the pool of high-avidity antibodies. The influence of CpG 7909 was antigen-specific, isotype-specific and distinct from the influence on anti-HBs production, as avidity did not correlate with anti-HBs IgG titers. This is the first demonstration that a novel human adjuvant may induce antibodies with higher antigen-binding affinity.

  9. Identification of a high-affinity monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay.

    PubMed

    Zhang, Xian; Sun, Mengjiao; Kang, Yue; Xie, Hui; Wang, Xin; Song, Houhui; Li, Xiaoliang; Fang, Weihuan

    2015-11-01

    Ochratoxin A (OTA) is one of the most commonly occurring mycotoxins produced by some species of Aspergillus and can contaminate cereal and cereal products. A high-affinity anti-OTA monoclonal antibody (mAb) was generated from a hybridoma cell line 2D8 using splenocytes from a BALB/c mouse immunized with synthesized OTA-bovine serum albumin conjugate. The mAb 2D8 is specific with high affinity (3.75 × 10(9) L/M). An indirect competitive ELISA (ic-ELISA) was then developed using this mAb for quantitative determination of OTA in corn and feed samples. Using the optimized conditions, there was good linearity between OTA concentration and competitive inhibition (y = -0.6076x + 0.2441, R(2) = 0.9923) with the working range from 2.4 to 23.6 μg/kg, IC50 at 7.6 μg/kg and lower limit of detection at 1.4 μg/kg. The recovery rates in spiked samples were 91.2-110.3%. Of the 56 corn and feed samples, this ic-ELISA and a commercial kit both found the same 13 samples positive for OTA with good linear correlation between the two methods in OTA quantification (R(2) = 0.9706). We conclude that this ic-ELISA can be used for rapid and quantitative screening of corn and feed samples for the presence of OTA.

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

    PubMed

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

    2016-01-01

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

  11. Single-step Antibody-based Affinity Cryo-Electron Microscopy for Imaging and Structural Analysis of Macromolecular Assemblies

    PubMed Central

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E.; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J.; Serwer, Philip; Thompson, David H.; Jiang, Wen

    2014-01-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (nonpurified His-tagged bacteriophage T7, His-tagged E. coli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures. PMID:24780590

  12. Affinity precipitation of a monoclonal antibody from an industrial harvest feedstock using an ELP-Z stimuli responsive biopolymer.

    PubMed

    Sheth, Rahul D; Jin, Mi; Bhut, Bharat V; Li, Zhengjian; Chen, Wilfred; Cramer, Steven M

    2014-08-01

    In this work, a proof of concept elastin-like polypeptide-Z domain fusion (ELP-Z) based affinity precipitation process is developed for monoclonal antibody (mAb) purification from industrial harvest feeds. Greater than 99% mAb recoveries are obtained during the initial binding step of the process for both pure mAb and the mAb harvest feeds. Great than 90% overall mAb yields are also obtained for the subsequent elution step of the process with no measurable mAb aggregation. The process is shown to result in more than 2 logs of host cell protein (HCP) and more than 4 logs of DNA clearance from the harvest feed. While the overall mAb yield and HCP clearance for the affinity precipitation process was comparable to Protein A chromatography the DNA clearance was clearly superior. Performance is maintained for mAb final elution concentrations up to 20 g/L, demonstrating the ability of the process to both concentrate and purify the mAb. Effective ELP-Z regeneration is also demonstrated using 0.1 M NaOH with no adverse effect on subsequent capture efficiency. Finally, the reusability of the ELP-Z construct and robustness of the process is demonstrated for up to three purification-regeneration cycles with minimal product and impurity carryover and high yields and purity. This work demonstrates that the ELP-Z based precipitation approach can be successfully employed as an affinity capture step for industrial mAbs.

  13. Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

    PubMed

    Yu, Guimei; Vago, Frank; Zhang, Dongsheng; Snyder, Jonathan E; Yan, Rui; Zhang, Ci; Benjamin, Christopher; Jiang, Xi; Kuhn, Richard J; Serwer, Philip; Thompson, David H; Jiang, Wen

    2014-07-01

    Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

  14. 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

  15. Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

    PubMed Central

    Grimberg, Brian T; Udomsangpetch, Rachanee; Xainli, Jia; McHenry, Amy; Panichakul, Tasanee; Sattabongkot, Jetsumon; Cui, Liwang; Bockarie, Moses; Chitnis, Chetan; Adams, John; Zimmerman, Peter A; King, Christopher L

    2007-01-01

    Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection. PMID:18092885

  16. A new kind of carbohydrate array, its use for profiling antiglycan antibodies, and the discovery of a novel human cellulose-binding antibody.

    PubMed

    Schwarz, Mikael; Spector, Larissa; Gargir, Ari; Shtevi, Avraham; Gortler, Monica; Altstock, Rom T; Dukler, Avinoam A; Dotan, Nir

    2003-11-01

    In this study, we use a novel glycan array to analyze the glycan-binding antibody repertoire in a pool of affinity-purified IgG collected from a healthy human population. The glycan array used is based on mono- and oligosaccharides covalently linked to the surface via a long linker at their reducing ends. They are thus presented to the medium with a well-defined orientation and are accessible for specific binding by glycan-binding proteins, such as antibodies and lectins. A novel anticellulose antibody was detected that binds specifically to beta4-linked saccharides with a preference for glucopyranose over galactopyranose residues. We also found previously known antiglycan antibodies against mono- and oligosaccharides that are constituents of commonly occurring bacterial polysaccharides. We propose that this array can facilitate high-throughput screening of glycan-binding proteins and the search for biomarkers for personalized medicine.

  17. Evaluation of affinity-based serum clean-up in mass spectrometric analysis: Plastic vs monoclonal antibodies.

    PubMed

    Rossetti, Cecilia; Levernæs, Maren C S; Reubsaet, Léon; Halvorsen, Trine G

    2016-11-04

    Mass spectrometric assays are now of great relevance for trace compound analysis in complex matrices such as serum and plasma samples. Especially in the quantification of low abundant protein-biomarkers, the choice of the sample preparation is crucial. In the present paper immunocapture and Molecular Imprinted Polymers (MIPs) have been applied in the determination of pro-gastrin-releasing peptide, a Small Cell Lung Cancer marker. These affinity-based techniques were compared in terms of matrix effect, limits of detection, repeatability and extraction specificity. In addition, protein precipitation was included for comparison as it is a typical sample preparation method of biological matrices. The results highlighted differences in the methods' performance and specificity, strongly affecting the outcome of the mass spectrometric determination. Plastic and monoclonal antibodies confirmed to be sensitive and specific sample preparations able to determine ProGRP at clinical relevant concentration, although only the use of monoclonal antibodies allowed the reliable quantification of ProGRP at reference levels (8pM). In addition better insight in the specificity of the three sample preparation techniques was gained. This might also be of interest for other biological applications.

  18. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    PubMed

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

  19. Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

    PubMed

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

    2012-07-20

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

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

    PubMed Central

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

    2012-01-01

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

  1. Human immunoglobulin repertoires against tetanus toxoid contain a large and diverse fraction of high-affinity promiscuous V(H) genes.

    PubMed

    de Kruif, John; Kramer, Arjen; Visser, Therèse; Clements, Carina; Nijhuis, Roy; Cox, Freek; van der Zande, Vanessa; Smit, Renate; Pinto, Daniel; Throsby, Mark; Logtenberg, Ton

    2009-04-03

    To study the contribution of antibody light (L) chains to the diversity and binding properties of immune repertoires, a phage display repertoire was constructed from a single human antibody L chain and a large collection of antibody heavy (H) chains harvested from the blood of two human donors immunized with tetanus toxoid (TT) vaccine. After selection for binding to TT, 129 unique antibodies representing 53 variable immunoglobulin H chain (V(H)) gene rearrangements were isolated. This panel of anti-TT antibodies restricted to a single variable immunoglobulin L chain (V(L)) could be organized into 17 groups binding non-competing epitopes on the TT molecule. Comparison of the V(H) regions in this V(L)-restricted panel with a previously published repertoire of anti-TT V(H) regions with cognate V(H)-V(L) pairing showed a very similar distribution of V(H), D(H) and J(H) gene segment utilization and length of the complementarity-determining region 3 of the H chain. Surface plasmon resonance analysis of the single-V(L) anti-TT repertoire unveiled a range of affinities, with a median monovalent affinity of 2 nM. When the single-V(L) anti-TT V(H) repertoire was combined with a collection of naïve V(L) regions and again selected for binding to TT, many of the V(H) genes were recovered in combination with a diversity of V(L) regions. The affinities of a panel of antibodies consisting of a single promiscuous anti-TT V(H) combined with 15 diverse V(L) chains were determined and found to be identical to each other and to the original isolate restricted to a single-V(L) chain. Based on previous estimates of the clonal size of the human anti-TT repertoire, we conclude that up to 25% of human anti-TT-encoding V(H) regions from an immunized repertoire have promiscuous features. These V(H) regions readily combine with a single antibody L chain to result in a large panel of anti-TT antibodies that conserve the expected epitope diversity, V(H) region diversity and affinity of a

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

    PubMed

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

    2012-12-01

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

  3. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  4. RNA recognition by a human antibody against brain cytoplasmic 200 RNA.

    PubMed

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

    2014-06-01

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

  5. 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

  6. The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library.

    PubMed

    Tikunova, Nina; Dubrovskaya, Viktoriya; Morozova, Vera; Yun, Tatiana; Khlusevich, Yana; Bormotov, Nikolai; Laman, Aleksandr; Brovko, Fedor; Shvalov, Aleksandr; Belanov, Eugeni

    2012-01-01

    A panel of recombinant human antibodies to orthopoxviruses was isolated from a combinatorial phage display library of human scFv antibodies constructed from the Vh and Vl genes cloned from the peripheral blood lymphocytes of Vaccinia virus (VACV) immune donors. Plaque-reduction neutralization tests showed that seven selected phage-displaying scFv antibodies (pdAbs) neutralized both CPXV and VACV, and five of them neutralized Monkeypox virus (MPXV). Western blot analysis of VACV and CPXV proteins demonstrated that seven neutralizing antibodies recognized a 35 kDa protein. To identify this target protein, we produced a recombinant J3L protein of CPXV and showed that all the selected neutralizing antibodies recognized this protein. Neutralizing pdAb b9 was converted into fully human mAb b9 (fh b9), and scFv b9 displayed high binding affinities (K(d) of 0.7 and 3.2 nM). The fh b9 reduced VACV plaque formation in a dose-dependent manner.

  7. Mechanism of human antibody-mediated neutralization of Marburg virus.

    PubMed

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  8. Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus

    PubMed Central

    Flyak, Andrew I.; Ilinykh, Philipp A.; Murin, Charles D.; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L.; Hashiguchi, Takao; Bornholdt, Zachary A.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Ward, Andrew B.; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of Antibody-GP complexes reveals that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP but not to MARV GP. The data suggest that MARV neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition. PMID:25723164

  9. A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth

    PubMed Central

    Jia, Xuelian; Wang, Wenyi; Xu, Zhuobin; Wang, Shijing; Wang, Tong; Wang, Min; Wu, Min

    2016-01-01

    Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications. PMID:27301650

  10. Purification of human copper, zinc superoxide dismutase by copper chelate affinity chromatography

    SciTech Connect

    Weslake, R.J.; Chesney, S.L.; Petkau, A.; Friesen, A.D.

    1986-05-15

    Copper, zinc superoxide dismutase was isolated from human red blood cell hemolysate by DEAE-Sepharose and copper chelate affinity chromatography. Enzyme preparations had specific activities ranging from 3400 to 3800 U/mg and recoveries were approximately 60% of the enzyme activity in the lysate. Copper chelate affinity chromatography resulted in a purification factor of about 60-fold. The homogeneity of the superoxide dismutase preparation was analyzed by sodium dodecyl sulfate-gel electrophoresis, analytical gel filtration chromatography, and isoelectric focusing.

  11. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

    PubMed

    Nguyen, Annalee W; Wagner, Ellen K; Laber, Joshua R; Goodfield, Laura L; Smallridge, William E; Harvill, Eric T; Papin, James F; Wolf, Roman F; Padlan, Eduardo A; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A

    2015-12-02

    Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

  12. Immunoglobulin gene insertions and deletions in the affinity maturation of HIV-1 broadly reactive neutralizing antibodies.

    PubMed

    Kepler, Thomas B; Liao, Hua-Xin; Alam, S Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Yandava, Chandri; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S Abdool; Cohen, Myron S; Walter, Emmanuel; Moody, M Anthony; Wu, Xueling; Altae-Tran, Han R; Georgiev, Ivelin S; Kwong, Peter D; Boyd, Scott D; Fire, Andrew Z; Mascola, John R; Haynes, Barton F

    2014-09-10

    Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events.

  13. A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

    PubMed Central

    Thie, Holger

    2017-01-01

    ABSTRACT Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs. PMID:28055297

  14. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  15. Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

    PubMed

    Matsumoto, Yutaka; Takizawa, Hisao; Gong, Xiaoqi; Le, Sang; Lockyer, Simon; Okuyama, Keiji; Tanaka, Michinori; Yoshitake, Masuhiro; Tandon, Narendra N; Kambayashi, Junichi

    2007-01-01

    Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

  16. Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses

    PubMed Central

    Pascale, Juan M.; Moreno, Brechla; Chackerian, Bryce; Peabody, David S.

    2017-01-01

    Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets. PMID:28152075

  17. Characterization of a novel inhibitory human monoclonal antibody directed against Plasmodium falciparum Apical Membrane Antigen 1

    PubMed Central

    Maskus, Dominika J.; Królik, Michał; Bethke, Susanne; Spiegel, Holger; Kapelski, Stephanie; Seidel, Melanie; Addai-Mensah, Otchere; Reimann, Andreas; Klockenbring, Torsten; Barth, Stefan; Fischer, Rainer; Fendel, Rolf

    2016-01-01

    Malaria remains a major challenge to global health causing extensive morbidity and mortality. Yet, there is no efficient vaccine and the immune response remains incompletely understood. Apical Membrane Antigen 1 (AMA1), a leading vaccine candidate, plays a key role during merozoite invasion into erythrocytes by interacting with Rhoptry Neck Protein 2 (RON2). We generated a human anti-AMA1-antibody (humAbAMA1) by EBV-transformation of sorted B-lymphocytes from a Ghanaian donor and subsequent rescue of antibody variable regions. The antibody was expressed in Nicotiana benthamiana and in HEK239-6E, characterized for binding specificity and epitope, and analyzed for its inhibitory effect on Plasmodium falciparum. The generated humAbAMA1 shows an affinity of 106–135 pM. It inhibits the parasite strain 3D7A growth in vitro with an expression system-independent IC50-value of 35 μg/ml (95% confidence interval: 33 μg/ml–37 μg/ml), which is three to eight times lower than the IC50-values of inhibitory antibodies 4G2 and 1F9. The epitope was mapped to the close proximity of the RON2-peptide binding groove. Competition for binding between the RON2-peptide and humAbAMA1 was confirmed by surface plasmon resonance spectroscopy measurements. The particularly advantageous inhibitory activity of this fully human antibody might provide a basis for future therapeutic applications. PMID:28000709

  18. Pathogen-specific deep sequence-coupled biopanning: A method for surveying human antibody responses.

    PubMed

    Frietze, Kathryn M; Pascale, Juan M; Moreno, Brechla; Chackerian, Bryce; Peabody, David S

    2017-01-01

    Identifying the targets of antibody responses during infection is important for designing vaccines, developing diagnostic and prognostic tools, and understanding pathogenesis. We developed a novel deep sequence-coupled biopanning approach capable of identifying the protein epitopes of antibodies present in human polyclonal serum. Here, we report the adaptation of this approach for the identification of pathogen-specific epitopes recognized by antibodies elicited during acute infection. As a proof-of-principle, we applied this approach to assessing antibodies to Dengue virus (DENV). Using a panel of sera from patients with acute secondary DENV infection, we panned a DENV antigen fragment library displayed on the surface of bacteriophage MS2 virus-like particles and characterized the population of affinity-selected peptide epitopes by deep sequence analysis. Although there was considerable variation in the responses of individuals, we found several epitopes within the Envelope glycoprotein and Non-Structural Protein 1 that were commonly enriched. This report establishes a novel approach for characterizing pathogen-specific antibody responses in human sera, and has future utility in identifying novel diagnostic and vaccine targets.

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

    PubMed

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

    2010-11-01

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

  20. Characterization of a novel inhibitory human monoclonal antibody directed against Plasmodium falciparum Apical Membrane Antigen 1.

    PubMed

    Maskus, Dominika J; Królik, Michał; Bethke, Susanne; Spiegel, Holger; Kapelski, Stephanie; Seidel, Melanie; Addai-Mensah, Otchere; Reimann, Andreas; Klockenbring, Torsten; Barth, Stefan; Fischer, Rainer; Fendel, Rolf

    2016-12-21

    Malaria remains a major challenge to global health causing extensive morbidity and mortality. Yet, there is no efficient vaccine and the immune response remains incompletely understood. Apical Membrane Antigen 1 (AMA1), a leading vaccine candidate, plays a key role during merozoite invasion into erythrocytes by interacting with Rhoptry Neck Protein 2 (RON2). We generated a human anti-AMA1-antibody (humAbAMA1) by EBV-transformation of sorted B-lymphocytes from a Ghanaian donor and subsequent rescue of antibody variable regions. The antibody was expressed in Nicotiana benthamiana and in HEK239-6E, characterized for binding specificity and epitope, and analyzed for its inhibitory effect on Plasmodium falciparum. The generated humAbAMA1 shows an affinity of 106-135 pM. It inhibits the parasite strain 3D7A growth in vitro with an expression system-independent IC50-value of 35 μg/ml (95% confidence interval: 33 μg/ml-37 μg/ml), which is three to eight times lower than the IC50-values of inhibitory antibodies 4G2 and 1F9. The epitope was mapped to the close proximity of the RON2-peptide binding groove. Competition for binding between the RON2-peptide and humAbAMA1 was confirmed by surface plasmon resonance spectroscopy measurements. The particularly advantageous inhibitory activity of this fully human antibody might provide a basis for future therapeutic applications.

  1. [Anti Shiga-like toxin II(SLT-II) humanized monoclonal antibody].

    PubMed

    Matsumoto, Yoh-ichi

    2002-03-01

    Anti-Shiga-Like Toxin II(SLT-II) Humanized Monoclonal Antibody(TMA-15) was constructed from Mouse Monoclonal Antibody(MuVTm1.1) recognizing the same antigen using recombinant and CDR grafting technology. TMA-15 had almost the same affinity to SLT-II as MuVTm1.1 and showed the good protective activity of mice challenged either with SLT-II or with SLT-II secreting Shiga-like Toxin producing E. coli(STEC). TMA-15 showed no acute toxicity to monkeys and no cross-reactivity to human tissues in pre-clinical safety studies. From the preliminary results of Phase 1 clinical trial using healthy adult volunteers, doses up to planned maximum dose were well tolerated and TMA-15 showed long half life in blood almost comparable to gamma globulin preparations. Therefore, TMA-15 is expected to show clinical efficacy in coming clinical trial using pediatric STEC patients.

  2. Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

    PubMed

    Bidlingmaier, Scott; Su, Yang; Liu, Bin

    2015-01-01

    Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

  3. Development and characterization of a human antibody reference panel against erythropoietin suitable for the standardization of ESA immunogenicity testing.

    PubMed

    Mytych, Daniel T; Barger, Troy E; King, Chadwick; Grauer, Stephanie; Haldankar, Raj; Hsu, Eric; Wu, Michelle Min; Shiwalkar, Mukta; Sanchez, Sergio; Kuck, Andrew; Civoli, Francesca; Sun, Jilin; Swanson, Steven J

    2012-08-31

    Recombinant human erythropoietin (EPO) has been used therapeutically for more than two decades in the treatment of anemia. Although EPO is generally well tolerated, in rare cases, patients have developed anti-EPO antibodies that can negatively impact safety and efficacy. Therefore, the detection of antibodies against EPO is a regulatory requirement during clinical development and post-approval. Although it is a rare phenomenon, antibody-mediated pure red cell aplasia (PRCA) is a serious complication than can result from antibodies that develop and neutralize EPO as well as endogenous erythropoietin. Currently, there are no universally accepted analytical methods to detect the full repertoire of binding and neutralizing anti-EPO antibodies. A number of different methods that differ in terms of antibodies detected and assay sensitivities are used by different manufacturers. There is also a lack of antibody reference reagents, and therefore no consistent basis for detecting and measuring anti-EPO antibodies. Reference reagents, with established ranges, are essential to monitor the safety and efficacy of all erythropoiesis-stimulating agents (ESAs) structurally related to human erythropoietin. This is the first report of the development and characterization of a panel of fully human antibodies against EPO suitable as reference reagents. The characteristics of antibodies within the panel were selected based on the prevalence of non-neutralizing IgG and IgM antibodies in non-PRCA patients and neutralizing IgG antibodies, including IgG1 and IgG4, in antibody-mediated PRCA subjects. The reference panel includes antibodies of high- and low-affinity with binding specificity to neutralizing and non-neutralizing erythropoietin epitopes. The subclass of human antibodies in this reference panel includes an IgG1, IgG2, and IgG4, as well as an IgM isotype. This antibody panel could help select appropriate immunogenicity assays, guide validation, and monitor assay performance

  4. Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR).

    PubMed

    Akbari, Bahman; Farajnia, Safar; Zarghami, Nosratollah; Mahdieh, Nejat; Rahmati, Mohammad; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

    2016-11-01

    Various strategies have been attempted for targeting of epidermal growth factor receptor (EGFR), as an essential biomarker in a variety of cancers. Several anti-EGFR antibodies including cetuximab are used in clinics for treatment of EGFR-overexpressing colorectal and head and neck cancers but the efficiency of these antibodies is threatened by their large size and chimeric nature. Humanized single chains antibodies (huscFv) are smaller generation of antibodies with lower immunogenicity may overcome these limitations. This article reports production and evaluation of a novel humanized anti-EGFR scFv. The CDRs of cetuximab heavy and light chains were grafted onto human antibody frameworks as framework donors. To maintain the antigen binding affinity of murine antibody, the murine vernier zone residues were retained in framework regions of huscFv. Additionally, two point mutations in CDR-L1 and CDR-L3 and three point mutations in CDR-H2 and CDR-H3 loops of the humanized scFv (huscFv) were introduced to increase affinity of the huscFv to EGFR. Analysis of results demonstrated that the humanness degree of resultant huscFv was increased as 19%. HuscFv was expressed in BL21 (DE3) and affinity purified via Ni-NTA column. The reactivity of huscFv with EGFR was evaluated by ELISA and dot blot techniques. Analysis by ELISA and dot blot showed that the huscFv was able to recognize and react with EGFR. Toxicity analysis by MTT assay indicated an inhibitory effect on growth of EGFR-overexpressing A431 cells. In conclusion, the huscFv produced in this study revealed decreased immunogenicity while retained growth inhibitory effect on EGFR-overexpressing tumor cells.

  5. Autoimmune anti-androgen-receptor antibodies in human serum.

    PubMed Central

    Liao, S; Witte, D

    1985-01-01

    Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9- to 12S units. Other serum samples, however, formed 14- to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate alpha-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance of finding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization. PMID:3866227

  6. Human-like antibodies neutralizing Western equine encephalitis virus

    PubMed Central

    Hülseweh, Birgit; Rülker, Torsten; Pelat, Thibaut; Langermann, Claudia; Frenzel, Andrè; Schirrmann, Thomas; Dübel, Stefan; Thullier, Philippe; Hust, Michael

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development. PMID:24518197

  7. Expression and antigenicity of recombinant human respiratory syncytial virus glycoproteins having different affinity tags.

    PubMed

    Lee, Han Saem; Kim, A-Reum; Kim, Kisoon; Lee, Wan-Ji; Kim, Sung Soon; Kim, You-Jin

    2016-12-29

    Human respiratory syncytial virus (HRSV) is a main cause of lower respiratory tract infections in infants and the elderly. Glycoprotein (G) is major antigen on the viral surface, and plays a key role for virus entry. Therefore, purification of the glycoprotein of HRSV is critical for the development of HRSV vaccine and serological diagnosis. In this study, we report the design and characterization of glycoprotein engineered rationally to enhance the protein solubility and to facilitate efficient purification. We permuted HRSV glycoproteins with two tags: (i) an immunoglobulin (Ig) M signal peptide and a protein A B domain tag to render HRSV glycoprotein secret into the culture media and (ii) a foldon and 6 × histidine tag with or without transmembrane domain. Three recombinant baculoviruses were constructed: (i) transmembrane-truncated HRSV glycoprotein (amino acid positions 66-298) inserted with the N-terminal IgM signal peptide and protein A B domain (MG-GΔTM), (ii) truncated HRSV glycoprotein (amino acid positions 66-298) fused with a C-terminal foldon and 6 × histidine tag (GΔTM-FH), and (iii) full-length HRSV glycoprotein (amino acid positions 1-298) fused with a C-terminal foldon and 6 × histidine tag (G-FH). Highly soluble recombinant MG-GΔTM protein was clearly purified using one-step affinity chromatography with IgG-sepharose resin, whereas the recombinant G-FH protein and truncated GΔTM-FH were purified partially using nickel-resin. Although, the antigenicity of GΔTM-FH was stronger than highly mannose-rich MG-GΔTM protein, MG-GΔTM induced neutralizing antibodies efficiently in the mice to protect from infectious HRSV.

  8. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  9. Induction of in vitro heart block is not restricted to affinity purified anti-52 kDa Ro/SSA antibody from mothers of children with neonatal lupus.

    PubMed

    Viana, V S; Garcia, S; Nascimento, J H; Elkon, K B; Brot, N; Campos de Carvalho, A C; Bonfá, E

    1998-01-01

    The ability of affinity purified anti-52 kDa Ro/SSA antibody from patients without obstetric history of neonatal lupus to cause heart block using an experimental model was investigated. IgG-enriched fractions from sera of 20 systemic lupus erythematosus (SLE) and one Sjögren's syndrome (SS) all positives for anti-Ro/SSA antibodies as detected by CIE, were perfused on isolated whole rabbit hearts. Only six (29%) samples induced A-V block, five of them presenting low anti-Ro/SSA titre. All of them recognized the 52 kDa isoform on ELISA whereas only one had a concomitant binding to the 60 kDa protein. Moreover, affinity purified antibodies from two sera previously known to induce A-V block were obtained by affinity chromatography using a column containing the full-length 52 kDa Ro/SSA fusion protein. Paired eluate and effluent devoid of anti-52 kDa activity from the same patient were individually perfused in whole hearts. The ability to cause cardiac blockade was restricted to the affinity anti-52 kDa eluates. In addition, anti-52 kDa eluates from three IgG fractions that primarily failed to induce blockade remained ineffective. The present study has added to our knowledge that affinity anti-52 kDa Ro/SSA antibodies from mothers with healthy infants are capable of causing in vitro cardiac conduction disturbances. A prospective follow up of these patients will better delineate the clinical usefulness of this experimental model.

  10. Initial Characterization of Monoclonal Antibodies against Human Monocytes

    NASA Astrophysics Data System (ADS)

    Ugolini, Valentina; Nunez, Gabriel; Smith, R. Graham; Stastny, Peter; Capra, J. Donald

    1980-11-01

    Three monoclonal antibodies against human monocytes have been produced by somatic cell fusion. Extensive specificity analysis suggests that these antibodies react with most if not all human peripheral blood monocytes and not with highly purified T or B cells. Initial chemical characterization of the monocyte antigen recognized by two of these antibodies is presented. The molecule is a single polypeptide chain with an apparent molecular weight of 200,000. These reagents should prove useful in the clinical definition of disorders of monocyte differentiation, in studies of monocyte function, and in the elucidation of the genetics and structure of monocyte cell surface antigens.

  11. Monoclonal Antibody Cross-Reactions between Drosophila and Human Brain

    NASA Astrophysics Data System (ADS)

    Miller, Carol A.; Benzer, Seymour

    1983-12-01

    A panel of 146 monoclonal antibodies (MAbs), obtained with Drosophila melanogaster tissue as primary immunogen, was tested for cross-reactivity with the human central nervous system. Sites examined included spinal cord, cerebellum, hippocampus, and optic nerve. Nonnervous tissues tested were liver and lymph node. Approximately half of the antibodies reacted with one or more sites in the human central nervous system, identifying regional, cell class, and subcellular antigens. Some recognized neuronal, glial, or axonal subsets. Immunoblot analysis revealed that some antibodies reacted with similar antigen patterns in both species.

  12. Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

    PubMed Central

    Shigeta, M; Watanabe, T; Maruyama, S; Koyama, K; Isojima, S

    1980-01-01

    Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6783353

  13. Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies.

    PubMed

    O'Nuallain, Brian; Acero, Luis; Williams, Angela D; Koeppen, Helen P McWilliams; Weber, Alfred; Schwarz, Hans P; Wall, Jonathan S; Weiss, Deborah T; Solomon, Alan

    2008-11-25

    Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.

  14. Purification of human monoclonal antibodies and their fragments.

    PubMed

    Müller-Späth, Thomas; Morbidelli, Massimo

    2014-01-01

    This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have been proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

  15. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    PubMed Central

    Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

    2016-01-01

    Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

  16. A simple nonradioactive method for the determination of the binding affinities of antibodies induced by hapten bioconjugates for drugs of abuse.

    PubMed

    Torres, Oscar B; Antoline, Joshua F G; Li, Fuying; Jalah, Rashmi; Jacobson, Arthur E; Rice, Kenner C; Alving, Carl R; Matyas, Gary R

    2016-02-01

    The accurate analytical measurement of binding affinities of polyclonal antibody in sera to heroin, 6-acetylmorphine (6-AM), and morphine has been a challenging task. A simple nonradioactive method that uses deuterium-labeled drug tracers and equilibrium dialysis (ED) combined with ultra performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to measure the apparent dissociation constant (K d) of antibodies to 6-AM and morphine is described. The method can readily detect antibodies with K d in the low nanomolar range. Since heroin is rapidly degraded in sera, esterase inhibitors were included in the assay, greatly reducing heroin hydrolysis. MS/MS detection directly measured the heroin in the assay after overnight ED, thereby allowing the quantitation of % bound heroin in lieu of K d as an alternative measurement to assess heroin binding to polyclonal antibody sera. This is the first report that utilizes a solution-based assay to quantify heroin-antibody binding without being confounded by the presence of 6-AM and morphine and to measure K d of polyclonal antibody to 6-AM. Hapten surrogates 6-AcMorHap, 6-PrOxyHap, MorHap, DiAmHap, and DiPrOxyHap coupled to tetanus toxoid (TT) were used to generate high affinity antibodies to heroin, 6-AM, and morphine. In comparison to competition ED-UPLC/MS/MS which gave K d values in the nanomolar range, the commonly used competition enzyme-linked immunosorbent assay (ELISA) measured the 50% inhibition concentration (IC50) values in the micromolar range. Despite the differences in K d and IC50 values, similar trends in affinities of hapten antibodies to heroin, 6-AM, and morphine were observed by both methods. Competition ED-UPLC/MS/MS revealed that among the five TT-hapten bioconjugates, TT-6-AcMorHap and TT-6-PrOxyHap induced antibodies that bound heroin, 6-AM, and morphine. In contrast, TT-MorHap induced antibodies that poorly bound heroin, while TT-DiAmHap and TT-DiPrOxyHap induced antibodies either did not

  17. The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

    SciTech Connect

    Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji; Jin, Aishun; Kishi, Hiroyuki; Muraguchi, Atsushi

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

  18. 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

  19. A Fully Human Inhibitory Monoclonal Antibody to the Wnt Receptor RYK

    PubMed Central

    Parish, Clare L.; Takano, Elena A.; Fox, Stephen; Layton, Daniel; Nice, Edouard; Stacker, Steven A.

    2013-01-01

    RYK is an unusual member of the receptor tyrosine kinase (RTK) family that is classified as a putative pseudokinase. RYK regulates fundamental biological processes including cell differentiation, migration and target selection, axon outgrowth and pathfinding by transducing signals across the plasma membrane in response to the high affinity binding of Wnt family ligands to its extracellular Wnt inhibitory factor (WIF) domain. Here we report the generation and initial characterization of a fully human inhibitory monoclonal antibody to the human RYK WIF domain. From a naïve human single chain fragment variable (scFv) phage display library, we identified anti-RYK WIF domain–specific scFvs then screened for those that could compete with Wnt3a for binding. Production of a fully human IgG1κ from an inhibitory scFv yielded a monoclonal antibody that inhibits Wnt5a-responsive RYK function in a neurite outgrowth assay. This antibody will have immediate applications for modulating RYK function in a range of settings including development and adult homeostasis, with significant potential for therapeutic use in human pathologies. PMID:24058687

  20. CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display.

    PubMed

    Miescher, S; Zahn-Zabal, M; De Jesus, M; Moudry, R; Fisch, I; Vogel, M; Kobr, M; Imboden, M A; Kragten, E; Bichler, J; Mermod, N; Stadler, B M; Amstutz, H; Wurm, F

    2000-10-01

    Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.

  1. Humanization of an anti-human TNF-alpha antibody by variable region resurfacing with the aid of molecular modeling.

    PubMed

    Zhang, Wei; Feng, Jiannan; Li, Yan; Guo, Ning; Shen, Beifen

    2005-08-01

    The murine monoclonal antibody Z12 is of therapeutic interest for its neutralizing biological activity against human tumor necrosis factor-alpha (hTNF-alpha). We attempted to humanize Z12 with variable domain resurfacing guided by computer modeling. First, the genes of heavy and light chain variable region (VH, VL) of Z12 were cloned and the whole three-dimensional structure of Fv fragment was constructed by using homology-based modeling and molecular docking methods. Then the complex model of Fv interacting with hTNF-alpha whose crystal structure derived from PDB database was gained with computer-guided docking program. Based on this model, a humanized version was designed. The humanized Fab antibody was constructed, expressed and purified in the pComb3H vector system and it showed unaltered binding affinity to the antigen as determined by ELISA and atomic force microscopy (AFM). The method described here can be used to humanize other anti-hTNF-alpha antibodies.

  2. Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans: Potential implications for disease

    PubMed Central

    Padler-Karavani, Vered; Yu, Hai; Cao, Hongzhi; Chokhawala, Harshal; Karp, Felix; Varki, Nissi; Chen, Xi; Varki, Ajit

    2008-01-01

    Human heterophile antibodies that agglutinate animal erythrocytes are known to detect the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc). This monosaccharide cannot by itself fill the binding site (paratope) of an antibody and can also be modified and presented in various linkages, on diverse underlying glycans. Thus, we hypothesized that the human anti-Neu5Gc antibody response is diverse and polyclonal. Here, we use a novel set of natural and chemoenzymatically synthesized glycans to show that normal humans have an abundant and diverse spectrum of such anti-Neu5Gc antibodies, directed against a variety of Neu5Gc-containing epitopes. High sensitivity and specificity assays were achieved by using N-acetylneuraminic acid (Neu5Ac)-containing probes (differing from Neu5Gc by one less oxygen atom) as optimal background controls. The commonest anti-Neu5Gc antibodies are of the IgG class. Moreover, the range of reactivity and Ig classes of antibodies vary greatly amongst normal humans, with some individuals having remarkably large amounts, even surpassing levels of some well-known natural blood group and xenoreactive antibodies. We purified these anti-Neu5Gc antibodies from individual human sera using a newly developed affinity method and showed that they bind to wild-type but not Neu5Gc-deficient mouse tissues. Moreover, they bind back to human carcinomas that have accumulated Neu5Gc in vivo. As dietary Neu5Gc is primarily found in red meat and milk products, we suggest that this ongoing antigen-antibody reaction may generate chronic inflammation, possibly contributing to the high frequency of diet-related carcinomas and other diseases in humans. PMID:18669916

  3. Evidence for a human-specific mechanism for diet and antibody-mediated inflammation in carcinoma progression

    PubMed Central

    Hedlund, Maria; Padler-Karavani, Vered; Varki, Nissi M.; Varki, Ajit

    2008-01-01

    Patients with cancer have circulating heterophile antibodies that agglutinate animal red cells via recognition of the mammalian cell surface sialic acid N-glycolylneuraminic acid (Neu5Gc), which was long considered an oncofetal antigen in humans. However, humans are genetically deficient in Neu5Gc production and instead metabolically accumulate Neu5Gc from dietary sources, particularly red meats and milk products. Moreover, mice with a human-like defect showed no alternate pathway for Neu5Gc synthesis and even normal humans express anti-Neu5Gc antibodies. We show here that human tumors accumulate Neu5Gc that is covalently attached to multiple classes of glycans. The paradox of human tumor Neu5Gc accumulation in the face of circulating anti-Neu5Gc antibodies was hypothesized to be due to facilitation of tumor progression by the resulting low-grade chronic inflammation. Indeed, murine tumors expressing human-like levels of Neu5Gc show accelerated growth in syngeneic mice with a human-like Neu5Gc deficiency, coincident with the induction of anti-Neu5Gc antibodies and increased infiltration of inflammatory cells. Transfer of polyclonal monospecific syngeneic mouse anti-Neu5Gc serum also enhanced growth of transplanted syngeneic tumors bearing human-like levels of Neu5Gc, with tumors showing evidence for antibody deposition, enhanced angiogenesis and chronic inflammation. These effects were suppressed by a cyclooxygenase-2 inhibitor, a drug type known to reduce human carcinoma risk. Finally, affinity-purified human anti-Neu5Gc antibodies also accelerate growth of Neu5Gc-containing tumors in Neu5Gc-deficient mice. Taken together, the data suggest that the human propensity to develop diet-related carcinomas is contributed to by local chronic inflammation, resulting from interaction of metabolically-accumulated dietary Neu5Gc with circulating anti-Neu5Gc antibodies. PMID:19017806

  4. [Production of human monoclonal antibody reactive with gastrointestinal carcinoma].

    PubMed

    Soyama, N; Ohyanagi, H; Saitoh, Y

    1990-12-01

    Lymphocytes obtained from regional lymph nodes and spleen in the patients with gastrointestinal carcinoma were fused with the human B lymphoblastoid cell line GC01 and human hybridomas producing human monoclonal antibody (MoAb) were derived. Human MoAb No. 235 (IgM) derived from spleen cell of a gastric cancer patient reacted with adenocarcinoma of stomach, colon, and pancreas in the new immunohistochemical assay, modified direct immunoperoxidase method, and reacted with KATO III cells in cultured cell lines. The antigenic determinant of this antibody was suspected to be protein moiety after enzyme treatment. The competitive binding inhibition assay indicated that its epitope was different from anti-CEA monoclonal antibodies (KM10, A10, B9, AH3, JA4) and KM01. These findings suggested the possible use of human MoAb No. 235 for clinical application of targeting cancer chemotherapy in the future.

  5. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    PubMed Central

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  6. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  7. Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: A study using the hydatid vaccine EG95

    PubMed Central

    Read, A.J.; Gauci, C.G.; Lightowlers, M.W.

    2009-01-01

    The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95. PMID:19349218

  8. Adjuvant dependence of APS pathology-related low-affinity antibodies during secondary immune response to tetanus toxoid in BALB/c mice.

    PubMed

    Zivković, Irena; Petrušić, Vladimir; Dimitrijević, Rajna; Stojanović, Marijana; Dimitrijević, Ljiljana

    2013-05-01

    One of the established animal models for autoimmune disease antiphospholipid syndrome (APS) is TTd hyperimmunization of mice. Tetanus toxoid (TTd) and plasma protein β2GPI share structural homology so that immunization with TTd induces appearance of cross-reactive antibodies. In this paper, we have investigated the presence and dynamic of fluctuation of specific (anti-TTd) and auto (anti-β2GPI) antibodies induced in BALB/c mice during secondary immune response after TTd immunization with alhydrogel or glycerol as adjuvants. In addition, we followed the induced reproductive pathology as a sign of autoimmune outcome. We show undoubtedly adjuvant dependance of (1) level of induced anti-TTd IgG antibodies, (2) changes in levels of low-affinity anti-β2GPI IgG antibodies, and (3) change in fecundity and fertility during secondary immune response. These findings once more indicate the importance of chosen adjuvants used for successful immunization and eventual autoantibody outcome, this time associated with the processes involving low affinity, natural antibodies.

  9. Isolation of a Trypanosoma cruzi antigen by affinity chromatography with a monoclonal antibody. Preliminary evaluation of its possible applications in serological tests.

    PubMed Central

    Carbonetto, C H; Malchiodi, E L; Chiaramonte, M; Durante de Isola, E; Fossati, C A; Margni, R A

    1990-01-01

    By affinity chromatography with a monoclonal antibody (163B6), obtained in our laboratory, we have isolated a T. cruzi antigen which could be useful for differential diagnosis of Chagas' disease from leishmaniasis. This antigen, a 52-kD protein, reacted with all sera from Chagas' disease patients tested but not with sera from patients with leishmania, in ELISA. The 52-kD antigen is widely distributed in the Trypanosoma genus since the 163B6 monoclonal antibody reacts with T. rangeli and 8 strains and a clone of T. cruzi epimastigotes. Images Fig. 1 Fig. 2 PMID:2119921

  10. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  11. Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

    SciTech Connect

    Clagett-Dame, M.; Chung, C.; Chao, M.V.; DiStefano, P.S. )

    1990-12-01

    Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface. Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to {sup 125}I-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added {sup 125}I-labeled NGF.

  12. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro.

    PubMed

    Ding, Guipeng; Chen, Ximin; Zhu, Jin; Duesbery, Nicholas S; Cheng, Xunjia; Cao, Brian

    2013-01-01

    Human anthrax infection caused by exposure to Bacillus anthracis cannot always be treated by antibiotics. This is mostly because of the effect of the remaining anthrax toxin in the body. Lethal factor (LF) is a component of lethal toxin (LeTx), which is the major virulence of anthrax toxin. A murine IgG monoclonal antibody (mAb) against LF with blocking activity (coded LF8) was produced in a previous study. In this report, a human/murine chimeric Fab mAb (coded LF8-Fab) was developed from LF8 by inserting murine variable regions into human constant regions using antibody engineering to reduce the incompatibility of the murine antibody for human use. The LF8-Fab expressed in Escherichia coli could specifically identify LF with an affinity of 3.46 × 10(7) L/mol and could neutralize LeTx with an EC50 of 85  μ g/mL. Even after LeTx challenge at various time points, the LF8-Fab demonstrated protection of J774A.1 cells in vitro. The results suggest that the LF8-Fab might be further characterized and potentially be used for clinical applications against anthrax infection.

  13. Human monoclonal antibodies targeting carbonic anhydrase IX for the molecular imaging of hypoxic regions in solid tumours

    PubMed Central

    Ahlskog, J K J; Schliemann, C; Mårlind, J; Qureshi, U; Ammar, A; Pedley, R B; Neri, D

    2009-01-01

    Background: Hypoxia, which is commonly observed in areas of primary tumours and of metastases, influences response to treatment. However, its characterisation has so far mainly been restricted to the ex vivo analysis of tumour sections using monoclonal antibodies specific to carbonic anhydrase IX (CA IX) or by pimonidazole staining, after the intravenous administration of this 2-nitroimidazole compound in experimental animal models. Methods: In this study, we describe the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, using phage technology. Results: These antibodies were able to stain CA IX ex vivo and to target the cognate antigen in vivo. In one of the two animal models of colorectal cancer studied (LS174T), CA IX imaging closely matched pimonidazole staining, with a preferential staining of tumour areas characterised by little vascularity and low perfusion. In contrast, in a second animal model (SW1222), distinct staining patterns were observed for pimonidazole and CA IX targeting. We observed a complementary pattern of tumour regions targeted in vivo by the clinical-stage vascular-targeting antibody L19 and the anti-CA IX antibody A3, indicating that a homogenous pattern of in vivo tumour targeting could be achieved by a combination of the two antibodies. Conclusion: The new human anti-CA IX antibodies are expected to be non-immunogenic in patients with cancer and may serve as broadly applicable reagents for the non-invasive imaging of hypoxia and for pharmacodelivery applications. PMID:19623173

  14. Human papillomavirus vaccination induces neutralising antibodies in oral mucosal fluids

    PubMed Central

    Handisurya, A; Schellenbacher, C; Haitel, A; Senger, T; Kirnbauer, R

    2016-01-01

    Background: Mucosal human papillomaviruses (HPV) are a major cause of cancers and papillomas of the anogenital and oropharyngeal tract. HPV-vaccination elicits neutralising antibodies in sera and cervicovaginal secretions and protects uninfected individuals from persistent anogenital infection and associated diseases caused by the vaccine-targeted HPV types. Whether immunisation can prevent oropharyngeal infection and diseases and whether neutralising antibodies represent the correlate of protection, is still unclear. Methods: We determined IgG and neutralising antibodies against low-risk HPV6 and high-risk HPV16/18 in sera and oral fluids from healthy females (n=20) before and after quadrivalent HPV-vaccination and compared the results with non-vaccinated controls. Results: HPV-vaccination induced type-specific antibodies in sera and oral fluids of the vaccinees. Importantly, the antibodies in oral fluids were capable of neutralising HPV pseudovirions in vitro, indicating protection from infection. The increased neutralising antibody levels against HPV16/18 in sera and oral fluids post-vaccination correlated significantly within an individual. Conclusions: We provide experimental proof that HPV-vaccination elicits neutralising antibodies to the vaccine-targeted types in oral fluids. Hence, immunisation may confer direct protection against type-specific HPV infection and associated diseases of the oropharyngeal tract. Measurement of antibodies in oral fluids represents a suitable tool to assess vaccine-induced protection within the mucosal milieu of the orophayrynx. PMID:26867163

  15. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  16. Targeting of human glioma xenografts in vivo utilizing radiolabeled antibodies

    SciTech Connect

    Williams, J.A.; Wessels, B.W.; Wharam, M.D.; Order, S.E.; Wanek, P.M.; Poggenburg, J.K.; Klein, J.L. )

    1990-06-01

    Radiolabeled antibodies provide a potential basis for selective radiotherapy of human gliomas. We have measured tumor targeting by radiolabeled monoclonal and polyclonal antibodies directed against neuroectodermal and tumor-associated antigens in nude mice bearing human glioma xenografts. Monoclonal P96.5, a mouse IgG2a immunoglobulin, defines an epitope of a human melanoma cell surface protein, and specifically binds the U-251 human glioma as measured by immunoperoxidase histochemistry. 111In-radiolabeled P96.5 specifically targets the U-251 human glioma xenograft and yields 87.0 microCuries (microCi) of tumor activity per gram per 100 microCi injected activity compared to 4.5 microCi following administration of radiolabeled irrelevant monoclonal antibody. Calculations of targeting ratios demonstrate deposited dose to be 11.6 times greater with radiolabeled P96.5 administration compared to irrelevant monoclonal antibody. The proportion of tumor dose found in normal organs is less than 10%, further supporting specific targeting of the human glioma xenograft by this antibody. Monoclonal antibody ZME018, which defines a second melanoma-associated antigen, and polyclonal rabbit antiferritin, which defines a tumor-associated antigen, demonstrate positive immunoperoxidase staining of the tumor, but comparatively decreased targeting. When compared to the 111In-radiolabeled antibody, 90Y-radiolabeled P96.5 demonstrates comparable tumor targeting and percentages of tumor dose found in normal organs. To test the therapeutic potential of 90Y-radiolabeled P96.5, tumors and normal sites were implanted with miniature thermoluminescent dosimeters (TLD). Seven days following administration of 100 microCi 90Y-radiolabeled P96.5, average absorbed doses of 3770, 980, 353, and 274 cGy were observed in tumor, liver, contralateral control site, and total body, respectively.

  17. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  18. Monoclonal antibody to microtubule-associated STOP protein: affinity purification of neuronal STOP activity and comparison of antigen with activity in neuronal and nonneuronal cell extracts.

    PubMed

    Pirollet, F; Rauch, C T; Job, D; Margolis, R L

    1989-01-24

    Microtubules, ordinarily cold-labile structures, are made entirely resistant to cold temperature by the presence of substoichiometric amounts of STOP (stable tubule only polypeptide), a microtubule-associated protein. We have produced a monoclonal antibody which specifically recognizes a 145-kDa protein previously implicated in STOP activity in rat brain extracts. An antibody affinity column removes both the 145-kDa protein and STOP activity from solution. A urea eluate from the affinity column contains the 145-kDa protein and exhibits substantial STOP activity. We conclude the 145-kDa protein accounts for all measurable STOP activity in rat neuronal extracts. For this work, we have developed an assay of microtubule cold stability which is generally applicable to the detection of STOP activity in various tissues. Using this assay, we show STOP activity is most abundant in neuronal tissue but is detectable in all tissues tested, with the exception of heart muscle. In all tissues that we have examined, STOP activity elutes as a single peak from heparin affinity columns, and in common with brain STOP, all activity is Ca2+-calmodulin sensitive. The monoclonal antibody recognizes the 145-kDa STOP in rat neuronal extracts but reacts with no protein in active fractions from other tissue. A similar, but not identical, analogue of brain STOP thus appears to be widespread in mammalian tissues.

  19. Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

    PubMed

    Covaceuszach, Sonia; Marinelli, Sara; Krastanova, Ivet; Ugolini, Gabriele; Pavone, Flaminia; Lamba, Doriano; Cattaneo, Antonino

    2012-01-01

    Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

  20. Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

    PubMed

    Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y

    2007-01-01

    The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

  1. Evolution of promoter affinity for transcription factors in the human lineage.

    PubMed

    Molineris, Ivan; Grassi, Elena; Ala, Ugo; Di Cunto, Ferdinando; Provero, Paolo

    2011-08-01

    Changes in gene regulation are believed to play an important role in the evolution of animals. It has been suggested that changes in cis-regulatory regions are responsible for many or most of the anatomical and behavioral differences between humans and apes. However, the study of the evolution of cis-regulatory regions is made problematic by the degeneracy of transcription factor (TF) binding sites and the shuffling of their positions. In this work, we use the predicted total affinity of a promoter for a large collection of TFs as the basis for studying the evolution of cis-regulatory regions in mammals. We introduce the human specificity of a promoter, measuring the divergence between the affinity profile of a human promoter and its orthologous promoters in other mammals. The promoters of genes involved in functional categories such as neural processes and signal transduction, among others, have higher human specificity compared with the rest of the genome. Clustering of the human-specific affinities (HSAs) of neural genes reveals patterns of promoter evolution associated with functional categories such as synaptic transmission and brain development and to diseases such as bipolar disorder and autism.

  2. Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library.

    PubMed

    Smith, Richard G; Missailidis, Sotiris; Price, Michael R

    2002-01-05

    A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.

  3. Development and characterization of new monoclonal antibodies against human recombinant CA XII.

    PubMed

    Dekaminaviciute, Dovile; Lasickiene, Rita; Parkkila, Seppo; Jogaite, Vaida; Matuliene, Jurgita; Matulis, Daumantas; Zvirbliene, Aurelija

    2014-01-01

    Carbonic anhydrases (CAs) are enzymes that catalyse the reversible hydration of CO2 to bicarbonate. CA XII is considered a potential biomarker of tumor cells and a promising target for specific therapies. The aim of the current study was to develop new monoclonal antibodies (MAbs) against human recombinant CA XII and evaluate their diagnostic potential. An extracellular catalytic domain of human CA XII was expressed in E. coli and used as an immunogen. Seven stable hybridoma cell lines producing high-affinity IgG antibodies against human CA XII were generated. The majority of MAbs were highly specific to CA XII and did not cross-react with human recombinant CA I, CA II, CA VII, and CA XIII. In order to demonstrate the diagnostic value of the MAbs, they were employed for the immunohistochemistry analysis of CA XII expression in tissues. Two MAbs (15A4 and 4A6) demonstrated a strong and specific immunostaining of CA XII in human tissue specimens. Flow cytometry analysis of 5 human tumor cell lines with the MAb 15A4 revealed its immunoreactivity with cellular CA XII. In conclusion, the MAbs raised against recombinant catalytic domain of CA XII recognize cellular CA XII and represent a promising diagnostic tool for the immunodetection of CA XII-expressing cells.

  4. Expression of human antibodies in eukaryotic micro-algae.

    PubMed

    Mayfield, Stephen P; Franklin, Scott E

    2005-03-07

    Protein based therapeutics have enjoyed great success over the past decade. Unfortunately, with this clinical success comes a heavy price tag, owing to the inherently high costs of capitalization and production using mammalian cell fermentation. To address this problem, we have begun developing a system for the expression of recombinant proteins in the unicellular eukaryotic green algae, Chlamydomonas reinhardtii, leading to the production of human IgA single chain antibodies. The expression of human monoclonal antibodies in C. reinhardtii offers an attractive alternative to traditional mammalian based expression systems for several reasons, including an ability to rapidly obtain stable plastid and nuclear transformants, coupled with inherently low costs of capitalization and production.

  5. Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

    PubMed

    Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-04-22

    High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies.

  6. Profiling human serum antibodies with a carbohydrate antigen microarray

    PubMed Central

    Oyelaran, Oyindasola; McShane, Lisa M.; Dodd, Lori; Gildersleeve, Jeffrey C.

    2009-01-01

    Carbohydrate antigen arrays (glycan arrays) have been recently developed for the high-throughput analysis of carbohydrate macromolecule interactions. When profiling serum, information about experimental variability, inter-individual biological variability, and intra-individual temporal variability is critical. In this report, we describe the characterization of a carbohydrate antigen array and assay for profiling human serum. Through optimization of assay conditions and development of a normalization strategy, we obtain highly reproducible results with a within-experiment coefficient of variation (CV) of 10.8% and an overall CV (across multiple batches of slides and days) of 28.5%. We also report antibody profiles for 48 human subjects and evaluate for the first time the effects of age, race, sex, geographic location, and blood type on antibody profiles for a large set of carbohydrate antigens. We found significant dependence on age and blood type of antibody levels for a variety of carbohydrates. Finally, we conducted a longitudinal study with a separate group of 7 serum donors to evaluate the variation in anti-carbohydrate antibody levels within an individual over a period ranging from 3 to 13 weeks and found that, for nearly all antigens on our array, antibody levels are generally stable over this period. The results presented here provide the most comprehensive evaluation of experimental and biological variation reported to date for a glycan array and have significant implications for studies involving human serum profiling. PMID:19624168

  7. The high-affinity receptor for IgG, FcγRI, of humans and non-human primates.

    PubMed

    Chenoweth, Alicia M; Trist, Halina M; Tan, Peck-Szee; Wines, Bruce D; Hogarth, P Mark

    2015-11-01

    Non-human primate (NHP) models, especially involving macaques, are considered important models of human immunity and have been essential in preclinical testing for vaccines and therapeutics. Despite this, much less characterization of macaque Fc receptors has occurred compared to humans or mice. Much of the characterization of macaque Fc receptors so far has focused on the low-affinity Fc receptors, particularly FcγRIIIa. From these studies, it is clear that there are distinct differences between the human and macaque low-affinity receptors and their interaction with human IgG. Relatively little work has been performed on the high-affinity IgG receptor, FcγRI, especially in NHPs. This review will focus on what is currently known of how FcγRI interacts with IgG, from mutation studies and recent crystallographic studies of human FcγRI, and how amino acid sequence differences in the macaque FcγRI may affect this interaction. Additionally, this review will look at the functional consequences of differences in the amino acid sequences between humans and macaques.

  8. Preparation of immuno-affinity membranes for cholesterol removal from human plasma.

    PubMed

    Denizli, Adil

    2002-06-05

    Anti-low density lipoprotein antibody (anti-LDL) immobilized polyhydroxyethylmethacrylate (pHEMA) based membrane was prepared for selective removal of cholesterol from hypercholesterolemic human plasma. In order to further increase blood-compatibility, a newly synthesized comonomer, methacryloylamidophenylalanine (MAPA) was included in the membrane formulation. p(HEMA-MAPA) membranes were produced by a photopolymerization and then characterized by swelling tests, SEM and contact angle studies. Blood-compatibility tests were also investigated. The water swelling ratio of the p(HEMA-MAPA) membrane increases significantly (133.2.9%) compared with pHEMA (58%). p(HEMA-MAPA) membranes have large pores around in the range of 5-10 microm. All the clotting times increased when compared with pHEMA membranes. Loss of platelets and leukocytes was very low. The maximum anti-LDL antibody immobilization was achieved around pH 7.0. Immobilization of anti-LDL antibody was 12.6 mg/ml. There was a very low non-specific cholesterol adsorption onto the plain p(HEMA-MAPA) membranes, about 0.36 mg/ml. Anti-LDL antibody immobilized membranes adsorbed in the range of 4.5-7.2 mg cholesterol/ml from hypercholesterolemic human plasma. Up to 95% of the adsorbed LDL antibody was desorbed. The adsorption-desorption cycle was repeated 10 times using the same membrane. There was no significant loss in the adsorption capacity.

  9. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

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

    PubMed Central

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

    2015-01-01

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

  11. Human Antibodies against a Purified Glucosylceramide from Cryptococcus neoformans Inhibit Cell Budding and Fungal Growth

    PubMed Central

    Rodrigues, Marcio L.; Travassos, Luiz R.; Miranda, Kildare R.; Franzen, Anderson J.; Rozental, Sonia; de Souza, Wanderley; Alviano, Celuta S.; Barreto-Bergter, Eliana

    2000-01-01

    A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a β-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the β-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal β-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro. PMID:11083830

  12. Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

    SciTech Connect

    Mullinax, R.L.; Gross, E.A.; Amberg, J.R.; Hogrefe, H.H.; Kubitz, M.M.; Greener, A.; Alting-Mees, M.; Ardourel, D.; Short, J.M.; Sorge, J.A. ); Hay, B.N.; Shopes, B. )

    1990-10-01

    The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

  13. In vitro improvement of a shark IgNAR antibody by Qbeta replicase mutation and ribosome display mimics in vivo affinity maturation.

    PubMed

    Kopsidas, George; Roberts, Anthony S; Coia, Gregory; Streltsov, Victor A; Nuttall, Stewart D

    2006-11-15

    We have employed a novel mutagenesis system, which utilizes an error-prone RNA dependent RNA polymerase from Qbeta bacteriophage, to create a diverse library of single domain antibody fragments based on the shark IgNAR antibody isotype. Coupling of these randomly mutated mRNA templates directly to the translating ribosome allowed in vitro selection of affinity matured variants showing enhanced binding to target, the apical membrane antigen 1 (AMA1) from Plasmodium falciparum. One mutation mapping to the IgNAR CDR1 loop was not readily additive to other changes, a result explained by structural analysis of aromatic interactions linking the CDR1, CDR3, and Ig framework regions. This combination appeared also to be counter-selected in experiments, suggesting that in vitro affinity maturation is additionally capable of discriminating against incorrectly produced protein variants. Interestingly, a further mutation was directed to a position in the IgNAR heavy loop 4 which is also specifically targeted during the in vivo shark response to antigen, providing a correlation between natural processes and laboratory-based affinity maturation systems.

  14. Human cysticercosis: antigens, antibodies and non-responders.

    PubMed Central

    Flisser, A; Woodhouse, E; Larralde, C

    1980-01-01

    Immunoelectrophoresis of sera from patients with brain cysticercosis against a crude antigenic extract from Cysticercus cellulosae indicates that nearly 50% of the patients do not make sufficient antibodies to ostensively precipitate. The other 50% of the patients who do make precipitating antibodies show a very heterogeneous response in the number of antigens they recognize as well as in the type of antigen--as classified by their electrophoretic mobilities. The most favoured, called antigen B, is recognized by 84% of positive sera and corresponds to one or a limited number of antigens isoelectric at pH 8.6. Indirect immunofluorescence with monospecific anti-human immunoglobulins, performed upon the immunoelectrophoretic preparations, reveal that all cysticercus antigens induced the synthesis of antibodies in the immunoglobulin classes in the order G greater than M greater than E greater than A greater than D. Finally, antigen H (an anodic component) seems to favour IgE relative to its ability to induce IgG. Thus, although in natural infection a good proportion of cysticercotic patients do not seem to mount an energetic antibody response against the parasite, giving rise to some speculations about immunosuppression, the fact that 50% do synthesize antibodies allows for some optimistic expectations from vaccination of humans--in view of the good results of vaccination in experimental animals mediated by IgG antibodies. A likely prospect for a human vaccine would be antigen B because it is the most frequently detected by humans, although its immunizing and toxic properties remain to be properly studied. Images FIG. 1 FIG. 3 FIG. 6 PMID:7389197

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

    PubMed Central

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

    1986-01-01

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

  16. Generation of Human Antigen-Specific Monoclonal IgM Antibodies Using Vaccinated “Human Immune System” Mice

    PubMed Central

    van Geelen, Caroline M. M.; Noerder, Miriam; Huntington, Nicholas D.; Lim, Annick; Yasuda, Etsuko; Diehl, Sean A.; Scheeren, Ferenc A.; Ott, Michael; Weijer, Kees; Wedemeyer, Heiner; Di Santo, James P.; Beaumont, Tim; Guzman, Carlos A.; Spits, Hergen

    2010-01-01

    Background Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the ‘humanization’ of murine monoclonal antibodies (mAbs) is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. Methodology/Principal Findings After transplantation with CD34+CD38− human hematopoietic progenitor cells, BALB/c Rag2−/−IL-2Rγc−/− mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. “Human Immune System” mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL)-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. Conclusion/Significance This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens. PMID:20957227

  17. Facile fabrication and instant application of miniaturized antibody-decorated affinity columns for higher-order structure and functional characterization of TRIM21 epitope peptides.

    PubMed

    Al-Majdoub, M; Opuni, K F M; Koy, C; Glocker, M O

    2013-11-05

    Both epitope excision and epitope extraction methods, combined with mass spectrometry, generate precise informations on binding surfaces of full-length proteins, identifying sequential (linear) or assembled (conformational) epitopes, respectively. Here, we describe the one-step fabrication and application of affinity columns using reversibly immobilized antibodies with highest flexibility with respect to antibody sources and lowest sample amount requirements (fmol range). Depending on the antibody source, we made use of protein G- or protein A-coated resins as support materials. These materials are packed in pipet tips and in combination with a programmable multichannel pipet form a highly efficient epitope mapping system. In addition to epitope identification, the influence of epitope structure modifications on antibody binding specificities could be studied in detail with synthetic peptides. Elution of epitope peptides was optimized such that mass spectrometric analysis was feasible after a single desalting step. Epitope peptides were identified by accurate molecular mass determinations or by partial amino acid sequence analysis. In addition, charge state comparison or ion mobility analysis of eluted epitope peptides enabled investigation of higher-order structures. The epitope peptide of the TRIM21 (TRIM: tripartite motif) autoantigen that is recognized by a polyclonal antibody was determined as assembling an "L-E-Q-L" motif on an α-helix. Secondary structure determination by circular dichroism spectroscopy and structure modeling are in accordance with the mass spectrometric results and the antigenic behavior of the 17-mer epitope peptide variants from the full-length autoantigen.

  18. Immunohistochemical identification of cytotoxic lymphocytes using human perforin monoclonal antibody.

    PubMed Central

    Hameed, A.; Olsen, K. J.; Cheng, L.; Fox, W. M.; Hruban, R. H.; Podack, E. R.

    1992-01-01

    Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforin in cardiac allograft rejection was observed. The detection and quantitative analysis of perforin-associated cytotoxic cells by the human anti-perforin monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. Images Figure 1 PMID:1374586

  19. Constant domains influence binding of mouse–human chimeric antibodies to the capsular polypeptide of Bacillus anthracis

    PubMed Central

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-01-01

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct “rim” quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse–human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared “puffy” in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site. PMID:23863605

  20. Constant domains influence binding of mouse-human chimeric antibodies to the capsular polypeptide of Bacillus anthracis.

    PubMed

    Hubbard, Mark A; Thorkildson, Peter; Kozel, Thomas R; AuCoin, David P

    2013-08-15

    Our laboratory previously described the binding characteristics of the murine IgG3 monoclonal antibody (MuAb) F26G3. This antibody binds the poly-glutamic acid capsule (PGA) of Bacillus anthracis, an essential virulence factor in the progression of anthrax. F26G3 IgG3 MuAb binds PGA with a relatively high functional affinity (10 nM), produces a distinct "rim" quellung reaction, and is protective in a murine model of pulmonary anthrax. This study engineered an IgG subclass family of F26G3 mouse-human chimeric antibodies (ChAb). The F26G3 ChAbs displayed 9- to 20-fold decreases in functional affinity, as compared with the parent IgG3 MuAb. Additionally, the quellung reactions that were produced by the ChAbs all differed from the parent IgG3 MuAb in that they appeared "puffy" in nature. This study demonstrates that human constant domains may influence multiple facets of antibody binding to microbial capsular antigens despite their spatial separation from the traditional antigen-binding site.

  1. Therapeutic monoclonal antibodies in human breast milk: a case study.

    PubMed

    Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

    2014-04-01

    Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

  2. Serum or breast milk immunoglobulins mask the self-reactivity of human natural IgG antibodies.

    PubMed

    Djoumerska-Alexieva, Iglika; Manoylov, Iliyan; Dimitrov, Jordan D; Tchorbanov, Andrey

    2014-04-01

    B cells producing IgG antibodies specific to a variety of self- or foreign antigens are a normal constituent of the immune system of all healthy individuals. These naturally occurring IgG antibodies are found in the serum, external secretions, and pooled human immunoglobulin preparations. They bind with low affinity to antigens, which can also be targets for pathologic autoantibodies. An enhancement of naturally occurring IgG autoantibody activity was observed after treatment of human IgG molecules with protein-destabilizing agents. We have investigated the interactions of human immunoglobulins that were obtained from serum or from breast milk of healthy individuals or IVIg with human liver antigens. Proteins from an individual serum or milk were isolated by two methods, one of which included exposure to low pH and the other did not. Purified serum, mucosal IgM, IgA, and the fraction containing immunoglobulin G F(ab')2 fragments each inhibited the binding of a single donor or pooled IgG to human liver antigens. Our study presents findings regarding the role of the breast milk or serum antibodies in blocking the self-reactivity of IgG antibodies. It supports the suggestion that not IVIg only, but also the pooled human IgM and IgA might possess a potent beneficial immunomodulatory activity in autoimmune patients.

  3. Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.

    PubMed

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

    1991-07-25

    Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.

  4. Humanization of the anti-CEA T84.66 antibody based on crystal structure data.

    PubMed

    Yazaki, Paul J; Sherman, Mark A; Shively, John E; Ikle, David; Williams, Lawrence E; Wong, Jeffrey Y C; Colcher, David; Wu, Anna M; Raubitschek, Andrew A

    2004-05-01

    Chimeric T84.66 (cT84.66) is a monoclonal antibody (mAb) of high specificity and affinity for the tumor-associated carcinoembryonic antigen (CEA). Radiolabeled cT84.66 has demonstrated utility in the clinic as a reagent for the radioimmunoscintigraphy and radioimmunotherapy of CEA-positive colorectal and breast malignancies. To extend the therapeutic efficacy of T84.66, humanization by complementary determining region (CDR) grafting was employed. CDR grafting is a well-established technique, though often a series of framework back-mutations is required to restore high affinity. Recently, the crystal structure of the T84.66 diabody (scFv dimer) derived from the murine T84.66 mAb was determined, facilitating the humanization process by the availability of crystal structure data for both the graft donor and graft acceptor. A search of the Protein Data Bank revealed close structural similarity (r.m.s.d. of 1.07 A) between the Fv of T84.66 and the Fv of 4D5v8, a humanized anti-p185HER2 antibody marketed as Herceptin (Trastuzumab). This resulted in two humanized versions of the T84.66 M5A and M5B mAbs that differed only in the number of murine residues present in the C-terminal half of CDR-H2. Biochemical analysis and animal biodistribution studies were conducted to evaluate the humanized mAbs. The M5A, M5B and cT84.66 mAbs showed sub-nanomolar affinity for CEA and as radiolabeled mAbs exhibited specific tumor localization in tumor bearing mice. The T84.66 M5A mAb was selected for clinical development due to a slightly higher tumor uptake and a larger content of human residues, and was renamed hT84.66. A limited-scale production and animal imaging study have demonstrated hT84.66's ability to support clinical trials. Planned clinical trials will determine the effective utilization of this structure-based approach in the development of a promising new therapeutic.

  5. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  6. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    SciTech Connect

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  7. A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4.

    PubMed

    Sangawa, Takeshi; Nogi, Terukazu; Takagi, Junichi

    2008-10-01

    Abstract A monoclonal antibody that recognizes native G protein coupled receptors (GPCR) is generally difficult to obtain. Protease-activated receptor-4 (PAR4) is a GPCR that plays an important role in platelet activation as a low-affinity thrombin receptor. By immunizing peptide corresponding to the N-terminal segment of human PAR4, we obtained a monoclonal antibody that recognizes cell surface expressed PAR4. Epitope mapping using a series of artificial fusion proteins that carry PAR4-derived peptide revealed that the recognition motif is fully contained within the 6-residue portion adjacent to the thrombin cleavage site. The antibody blocked PAR4 peptide cleavage by thrombin, suggesting its utility in the functional study of PAR4 signaling.

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

    PubMed

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

    2016-10-01

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

  9. An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody

    SciTech Connect

    Krause, Jens C.; Ekiert, Damian C.; Tumpey, Terrence M.; Smith, Patricia B.; Wilson, Ian A.; Crowe, Jr., James E.

    2011-09-02

    The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.

  10. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    PubMed Central

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region. PMID:25692880

  11. Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

    PubMed

    Kirley, Terence L; Norman, Andrew B

    2015-01-01

    Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

  12. Human receptor kinetics and lung tissue retention of the enhanced-affinity glucocorticoid fluticasone furoate

    PubMed Central

    Valotis, Anagnostis; Högger, Petra

    2007-01-01

    Fluticasone furoate (FF) – USAN approved name, a new topically active glucocorticoid has been recently identified. The aim of this study was to characterise the binding affinity of this compound to the human lung glucocorticoid receptor in relation to other glucocorticoids. Additionally, we sought to determine the binding behaviour of fluticasone furoate to human lung tissue. The glucocorticoid receptor binding kinetics of fluticasone furoate revealed a remarkably fast association and a slow dissociation resulting in a relative receptor affinity (RRA) of 2989 ± 135 with reference to dexamethasone (RRA: 100 ± 5). Thus, the RRA of FF exceeds the RRAs of all currently clinically used corticosteroids such as mometasone furoate (MF; RRA 2244), fluticasone propionate (FP; RRA 1775), ciclesonide's active metabolite (RRA 1212 – rat receptor data) or budesonide (RRA 855). FP and FF displayed pronounced retention in human lung tissue in vitro. Lowest tissue binding was found for MF. There was no indication of instability or chemical modification of FF in human lung tissue. These advantageous binding attributes may contribute to a highly efficacious profile for FF as a topical treatment for inflammatory disorders of the respiratory tract. PMID:17650349

  13. Recognition of N-glycoforms in human chorionic gonadotropin by monoclonal antibodies and their interaction motifs.

    PubMed

    Li, Daoyuan; Zhang, Ping; Li, Fei; Chi, Lequan; Zhu, Deyu; Zhang, Qunye; Chi, Lianli

    2015-09-11

    The glycosylation of human chorionic gonadotropin (hCG) plays an important role in reproductive tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related cancers. However, developing an immunoassay directly against the N-linked oligosaccharides is unlikely because of the heterogeneity and low immunogenicity of carbohydrates. Here, we report a hydrogen/deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of antibodies against different hCG glycoforms. Hyperglycosylated hCG was purified from the urine of invasive mole patients, and the structure of its N-linked oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG antibodies MCA329 and MCA1024 against hCG and hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked oligosaccharides. Hydrogen/deuterium exchange-MS reveals that the peptide β65-83 of the hCG β subunit is the epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some antibodies are sensitive to the structural change of N-linked oligosaccharides, whereas others are not affected by N-glycosylation. It is promising to improve glycoprotein biomarker-based cancer diagnostics by developing combined immunoassays that can determine the level of protein and measure the degree of N-glycosylation simultaneously.

  14. The Human Antibody Response to the Surface of Mycobacterium tuberculosis

    PubMed Central

    Perley, Casey C.; Frahm, Marc; Click, Eva M.; Dobos, Karen M.; Ferrari, Guido; Stout, Jason E.; Frothingham, Richard

    2014-01-01

    Background Vaccine-induced human antibodies to surface components of Haemophilus influenzae and Streptococcus pneumonia are correlated with protection. Monoclonal antibodies to surface components of Mycobacterium tuberculosis are also protective in animal models. We have characterized human antibodies that bind to the surface of live M. tuberculosis. Methods Plasma from humans with latent tuberculosis (TB) infection (n = 23), active TB disease (n = 40), and uninfected controls (n = 9) were assayed by ELISA for reactivity to the live M. tuberculosis surface and to inactivated M. tuberculosis fractions (whole cell lysate, lipoarabinomannan, cell wall, and secreted proteins). Results When compared to uninfected controls, patients with active TB disease had higher antibody titers to the surface of live M. tuberculosis (Δ = 0.72 log10), whole cell lysate (Δ = 0.82 log10), and secreted proteins (Δ = 0.62 log10), though there was substantial overlap between the two groups. Individuals with active disease had higher relative IgG avidity (Δ = 1.4 to 2.6) to all inactivated fractions. Surprisingly, the relative IgG avidity to the live M. tuberculosis surface was lower in the active disease group than in uninfected controls (Δ = –1.53, p = 0.004). Patients with active disease had higher IgG than IgM titers for all inactivated fractions (ratios, 2.8 to 10.1), but equal IgG and IgM titers to the live M. tuberculosis surface (ratio, 1.1). Higher antibody titers to the M. tuberculosis surface were observed in active disease patients who were BCG-vaccinated (Δ = 0.55 log10, p = 0.008), foreign-born (Δ = 0.61 log10, p = 0.004), or HIV-seronegative (Δ = 0.60 log10, p = 0.04). Higher relative IgG avidity scores to the M. tuberculosis surface were also observed in active disease patients who were BCG-vaccinated (Δ = 1.12, p<0.001) and foreign-born (Δ = 0.87, p = 0.01). Conclusions/Significance Humans

  15. An Integrated Peptide-Antigen Microarray on Plasmonic Gold Films for Sensitive Human Antibody Profiling

    PubMed Central

    Price, Jordan V.; Tabakman, Scott M.; Li, Yanguang; Gong, Ming; Hong, Guosong; Feng, Ju; Utz, Paul J.; Dai, Hongjie

    2013-01-01

    High-throughput screening for interactions of peptides with a variety of antibody targets could greatly facilitate proteomic analysis for epitope mapping, enzyme profiling, drug discovery and biomarker identification. Peptide microarrays are suited for such undertaking because of their high-throughput capability. However, existing peptide microarrays lack the sensitivity needed for detecting low abundance proteins or low affinity peptide-protein interactions. This work presents a new peptide microarray platform constructed on nanostructured plasmonic gold substrates capable of metal enhanced NIR fluorescence enhancement (NIR-FE) by hundreds of folds for screening peptide-antibody interactions with ultrahigh sensitivity. Further, an integrated histone peptide and whole antigen array is developed on the same plasmonic gold chip for profiling human antibodies in the sera of systemic lupus erythematosus (SLE) patients, revealing that collectively a panel of biomarkers against unmodified and post-translationally modified histone peptides and several whole antigens allow more accurate differentiation of SLE patients from healthy individuals than profiling biomarkers against peptides or whole antigens alone. PMID:23923050

  16. Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire

    PubMed Central

    Yeung, Yik Andy; Foletti, Davide; Deng, Xiaodi; Abdiche, Yasmina; Strop, Pavel; Glanville, Jacob; Pitts, Steven; Lindquist, Kevin; Sundar, Purnima D.; Sirota, Marina; Hasa-Moreno, Adela; Pham, Amber; Melton Witt, Jody; Ni, Irene; Pons, Jaume; Shelton, David; Rajpal, Arvind; Chaparro-Riggers, Javier

    2016-01-01

    Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition. PMID:27857134

  17. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    SciTech Connect

    Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cindy J.

    2009-09-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment was demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.

  18. Production, characterization, and applications of two novel monoclonal antibodies against human interleukin-28A.

    PubMed

    Li, M; Chen, Y; Huang, T; Liu, Y; He, S

    2006-12-01

    Interleukin-28A (IL-28A) is a novel cytokine discovered in recent years and has been shown to have antiviral activity. In this study, IL-28A complementary DNA was inserted into prokaryotic expression vector pET-44 Ek/LIC. The Nus-S-His-tagged IL-28A fusion protein was expressed in Escherichia coli BL21 (DE3) in the soluble fraction. The fusion protein was purified by S-protein agarose affinity chromatography, and the fusion tag was removed from recombinant IL-28A by cleavage with thrombin. To prepare specific monoclonal antibody against human IL-28A, BALB/c mice were immunized with IL-28A, and hybridoma cell lines were obtained by fusing mouse spleen cells with myeloma NS-1 cells. Two strains of hybridoma cells, which produced the anti-human IL-28A antibodies 1B9 and 4B5 were obtained. They are IgM isotype and working in western blot analysis and enzyme-linked immunosorbent assay. In the present study, it was shown for the first time that human umbilical vein endothelial cells treated with interferon-alpha and poly(I:C) express IL-28A protein assessed by flow cytometry and immunofluorescent staining techniques. Immunohistochemistry showed that macrophage-like cells in colon and lung tissue and alveolar epithelial cells in lung tissue contain IL-28A, indicating a novel mechanism for both cell types to carry out their antivirus or antitumor functions.

  19. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    PubMed

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  20. An SF1 affinity model to identify branch point sequences in human introns

    PubMed Central

    Pastuszak, Alexander W.; Joachimiak, Marcin P.; Blanchette, Marco; Rio, Donald C.; Brenner, Steven E.; Frankel, Alan D.

    2011-01-01

    Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing. PMID:21071404

  1. Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure.

    PubMed

    Patton, J G; Alley, M C; Mao, S J

    1982-12-17

    Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.

  2. [One amino acid mutation in an anti-CD20 antibody fragment that affects the yield bacterial secretion and the affinity].

    PubMed

    Liu, Yin-Xing; Xiong, Dong-Sheng; Fan, Dong-Mei; Shao, Xiao-Feng; Xu, Yuan-Fu; Zhu, Zhen-Ping; Yang, Chun-Zheng

    2003-05-01

    Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

  3. High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

    SciTech Connect

    Nielsen, F.C.; Gammeltoft, S.; Westermark, B.; Fahrenkrug, J. )

    1990-11-01

    Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin, PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and (des-His1)VIP bound with 10 and 100 times lower affinity. The fragment VIP(7-28) displaced 25% of the receptor-bound {sup 125}I-VIP whereas VIP(16-28) and VIP(1-22-NH2) were inactive. The binding of {sup 125}I-VIP could be completely inhibited by 10 mumol/l of the antagonists (N-Ac-Tyr1,D-Phe2)GRF(1-29)-NH2, (pCl-D-Phe6,Leu17)VIP and VIP(10-28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated {sup 125}I-VIP was bound to receptors on the cell surface. The internalized {sup 125}I-VIP was completely degraded to {sup 125}I-tyrosine which was released from the cells. Degradation of internalized {sup 125}I-VIP was significantly reduced by chloroquine phenanthroline and pepstatin-A. Surface binding and internalization of {sup 125}I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound {sup 125}I-VIP, but caused retention of internalized {sup 125}I-VIP.

  4. Method for removal of human antibodies to native DNA from serum

    SciTech Connect

    Diamond, B.A.

    1987-09-01

    A method is described for removing human anti-native DNA antibody from a liquid sample comprising coupling monoclonal, antiidiotypic antibodies capable of binding to a shared idiotype on human anti-native DNA antibody to a medium. The idiotype shares between genetically nonidentical individuals, contacting a liquid sample to the medium to permit binding of human anti-native DNA antibody in the sample to the anti-idiotypic antibodies and separating the sample from the medium to remove the human anti-native DNA antibodies therefrom.

  5. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities

    PubMed Central

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG’ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG’ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG’ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells

  6. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    PubMed

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

  7. High-throughput screening for the development of a monoclonal antibody affinity precipitation step using ELP-z stimuli responsive biopolymers.

    PubMed

    Sheth, Rahul D; Madan, Bhawna; Chen, Wilfred; Cramer, Steven M

    2013-10-01

    This study provides a detailed investigation into the performance of a stimuli responsive ELP-Z based process for monoclonal antibody (mAb) affinity precipitation. A multidimensional high-throughput screening (HTS) protocol was developed and employed to investigate the effects of a variety of operating conditions on mAb yield and aggregation during the process. Precipitation efficiency of ELP-Z in the absence of mAb was first determined as a function of temperature and sodium sulfate concentration and conditions producing high yields were identified. HTS was then employed to determine appropriate conditions for the initial capture and co-precipitation of mAbs at high yields using ELP-Z. mAb elution from ELP-Z was then examined using HTS and the mAb yields and aggregate content of the overall process were determined. It was observed that mAb aggregation was sensitive primarily to the elution conditions and that this behavior was antibody specific and a strong function of operating temperature and elution pH. Importantly, for both mAbs examined in this study, the results indicated that room temperature operation and appropriate elution pH could be readily employed to produce both high mAb yields and low aggregate content using this approach. This study demonstrates the ability of ELP-Z based affinity precipitation for mAb purification and shows that HTS can be successfully employed to rapidly develop a robust and high yield process.

  8. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography.

    PubMed

    Machado, Gleyce Alves; Oliveira, Heliana Batista de; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-05-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (J unbound ) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJ unbound ) and aqueous (AJ unbound ) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for J unbound , 92.5% and 93.5% for DJ unbound and 82.5% and 82.6% for AJ unbound . By immunoblot, the DJ unbound fraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJ unbound fraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot.

  9. Serodiagnosis of human neurocysticercosis using antigenic components of Taenia solium metacestodes derived from the unbound fraction from jacalin affinity chromatography

    PubMed Central

    Machado, Gleyce Alves; de Oliveira, Heliana Batista; Gennari-Cardoso, Margareth Leitão; Mineo, José Roberto; Costa-Cruz, Julia Maria

    2013-01-01

    The aim of the present study was to analyse Taenia solium metacestode antigens that were derived from the unbound fraction of jacalin affinity chromatography and subsequent tert-octylphenoxy poly (oxyethylene) ethanol Triton X-114 (TX-114) partitioning in the diagnosis of human neurocysticercosis (NCC). Immunoassays were designed to detect T. solium-specific IgG antibodies by ELISA and immunoblot. Serum samples were collected from 132 individuals who were categorised as follows: 40 had NCC, 62 presented Taenia spp or other parasitic diseases and 30 were healthy individuals. The jacalin-unbound (Junbound) fraction presented higher sensitivity and specificity rates than the jacalin-bound fraction and only this fraction was subjected to subsequent TX-114 partitioning, resulting in detergent (DJunbound) and aqueous (AJunbound) fractions. The ELISA sensitivity and specificity were 85% and 84.8% for Junbound, 92.5% and 93.5% for DJunboundand 82.5% and 82.6% for AJunbound. By immunoblot, the DJunboundfraction showed 100% sensitivity and specificity and only serum samples from patients with NCC recognised the 50-70 kDa T. solium-specific components. We conclude that the DJunboundfraction can serve as a useful tool for the differential immunodiagnosis of NCC by immunoblot. PMID:23778661

  10. Belimumab: anti-BLyS human monoclonal antibody, anti-BLyS monoclonal antibody, BmAb, human monoclonal antibody to B-lymphocyte stimulator.

    PubMed

    2008-01-01

    Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of B-lymphocyte stimulator, or BLyS. Belimumab is in phase III trials for the treatment of systemic lupus erythematosus (SLE) and has completed a phase II trial in rheumatoid arthritis (RA); the product may also have potential in the treatment of other autoimmune disorders. In May 2001, Cambridge Antibody Technology (now MedImmune) completed its discovery programme and Human Genome Sciences identified belimumab as a candidate for clinical development. More than 1000 distinct human antibodies specific to BLyS were characterized by the collaboration.B-lymphocyte stimulator is a naturally occurring protein discovered by Human Genome Sciences that stimulates B-lymphocytes to develop into mature B cells. Laboratory studies have indicated that higher than normal levels of B-lymphocyte stimulator may contribute to the pathogenesis of autoimmune diseases, such as SLE and RA. Human Genome Sciences (HGS) and Cambridge Antibody Technology signed a collaborative agreement in August 1999 to study the B-lymphocyte stimulator as a human protein target. HGS is also developing other BLyS products. In March 2000, HGS and Cambridge Antibody Technology expanded their agreement into a 10-year collaboration and product development alliance, providing Human Genome Sciences with the right to use the antibody technology of Cambridge Antibody Technology to fully develop human antibodies for therapeutic and diagnostic purposes. Cambridge Antibody Technology will receive royalty payments on product sales from HGS, as well as the development and milestone payments it has already received. Belimumab will be manufactured in Human Genome Sciences' manufacturing facility, located in Rockville, MD, USA. HGS holds commercial rights to the drug. In July 2005, GlaxoSmithKline (GSK) exercised its co-development and co-promotion option to belimumab. In an agreement made in June 1996, HGS had

  11. 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 ...

  12. Human Immunodeficiency Virus Antibody Testing and the Right of Privacy.

    DTIC Science & Technology

    1987-09-30

    test, the Western blot is done. Western blot is more specific than ELISA for antibodies but it is more expensive and complicated to run. Unfortunately...the error rates or the reliability of the ELISA test varies from laboratory to laboratory. The S interpretation of the Western blot also can vary...Immunodeficiency Virus, HIV (formerly known as Human T-Lymphotropic Virus Type III, HTLV III and Lymphadenopathy Associated Virus, LAV or a combination of those

  13. Therapeutic Human Hyperimmune Polyclonal Antibodies Against Staphylococcal Enterotoxin B

    DTIC Science & Technology

    2008-05-01

    AD_________________ Award Number: W81XWH-08-C-0004 TITLE: Therapeutic Human Hyperimmune Polyclonal Antibodies against Staphylococcal...01-05-2008 2. REPORT TYPE Final, Phase I 3. DATES COVERED 19 OCT 2007 - 18 APR 2008 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic ...aerosol in a biowarfare scenario. The primary goal of Phase I was to demonstrate the feasibility of therapeutic intervention with immunoglobulin

  14. Genetically Engineered Humanized Mouse Models for Preclinical Antibody Studies

    PubMed Central

    Proetzel, Gabriele; Wiles, Michael V.; Roopenian, Derry C.

    2015-01-01

    The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies. PMID:24150980

  15. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

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

    PubMed Central

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

    2016-01-01

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

  17. Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology

    PubMed Central

    Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Akbari, Bahman; Ahdi khosroshahi, Shiva

    2016-01-01

    Purpose: EGFRvIII as the most common mutant variant of the epidermal growth factor receptor is resulting from deletion of exons 2–7 in the coding sequence and junction of exons 1 and 8 through a novel glycine residue. EGFRvIII is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. The aim of the present study was identification of a novel single chain antibody against EGFRvIII as a promising target for cancer therapy. Methods: In this study, a synthetic peptide corresponding to EGFRvIII protein was used for screening a naive human scFv phage library. A novel five-round selection strategy was used for enrichment of rare specific clones. Results: After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, only three clones had expected size in PCR reaction. The specific interaction of two of the scFv clones with EGFRvIII was confirmed by indirect ELISA. One phage clone with higher affinity in scFv ELISA was purified for further analysis. The purity of the produced scFv antibody was confirmed using SDS-PAGE and Western blotting analyses. Conclusion: In the present study, a human anti- EGFRvIII scFv with high affinity was first identified from a scFv phage library. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. PMID:28101463

  18. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  19. A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

    PubMed Central

    Weber, Tobias; Mavratzas, Athanasios; Kiesgen, Stefan; Haase, Stephanie; Bötticher, Benedikt; Exner, Evelyn; Mier, Walter; Grosse-Hovest, Ludger; Jäger, Dirk; Arndt, Michaela A. E.; Krauss, Jürgen

    2015-01-01

    Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies. PMID:26605343

  20. Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

    PubMed

    Leong, S R; DeForge, L; Presta, L; Gonzalez, T; Fan, A; Reichert, M; Chuntharapai, A; Kim, K J; Tumas, D B; Lee, W P; Gribling, P; Snedecor, B; Chen, H; Hsei, V; Schoenhoff, M; Hale, V; Deveney, J; Koumenis, I; Shahrokh, Z; McKay, P; Galan, W; Wagner, B; Narindray, D; Hébert, C; Zapata, G

    2001-11-07

    A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

  1. Generation of human monoclonal antibodies reactive with cellular antigens.

    PubMed Central

    Cote, R J; Morrissey, D M; Houghton, A N; Beattie, E J; Oettgen, H F; Old, L J

    1983-01-01

    Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer. Images PMID:6572959

  2. [IgE antibodies in human Fasciola hepatica distomiasis].

    PubMed

    Sampaio Silva, M L; Vindimian, M; Wattré, P; Capron, A

    1985-09-01

    In patients infected by Fasciola hepatica, total IgE and specific IgE antibodies have been determined by radioimmunoassays, and IgG, IgA, IgM levels by radial immunodiffusion test (Mancini, 1965). Moreover, total and specific IgE levels have been related to parasite egg burden, age, clinical features and eosinophilia. Elevated total IgE and specific IgE antibodies levels have been found respectively in 76% and 48% of the patients whereas there was no significant variations in other immunoglobulins levels. However, though the amount of total and specific IgE was lower than in other helminthic diseases, it appears to be a significant data of the immune response to parasites as it has been reported and discussed previously. It has been shown a significant relationship between total and specific IgE levels, the number of lines by immunoelectrophoresis, and the results of the indirect haemagglutination and indirect fluorescent antibody techniques; each method appeared to be in equal value to perform the early diagnosis of human Fasciola hepatica. In addition, specific IgE antibodies levels were correlated with eosinophilia specially when it exceeds 15%. This results demonstrate the availability of their measurement in the diagnosis of fascioliasis versus other diseases with marked eosinophilia.

  3. 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

  4. Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modelling.

    PubMed

    Hou, Sheng; Li, Bohua; Wang, Ling; Qian, Weizhu; Zhang, Dapeng; Hong, Xueyu; Wang, Hao; Guo, Yajun

    2008-07-01

    4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modelling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically.

  5. Fully human antibodies from transgenic mouse and phage display platforms.

    PubMed

    Lonberg, Nils

    2008-08-01

    Over the past two decades, technologies have emerged for generating monoclonal antibodies (MAbs) derived from human immunoglobulin gene sequences. These fully human MAbs provide an alternative to re-engineered, or de-immunized, rodent MAbs as a source of low immunogenicity therapeutic antibodies. There are now two marketed fully human therapeutic MAbs, adalimumab and panitumumab, and several dozen more in various stages of human clinical testing. Most of the drugs, including adalimumab and panitumumab, were generated using either phage display or transgenic mouse platforms. The reported clinical experience with fully human MAbs demonstrates that these two platforms are, and should continue to be, a significant source of active and well tolerated experimental therapeutics. While this body of reported clinical data does not yet provide a clear distinction between the platforms, the available descriptions of the drug discovery processes used to identify the clinical candidates highlight one difference. It appears that lead optimization is more commonly applied to phage display derived leads than transgenic mouse derived leads.

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

    PubMed Central

    1990-01-01

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

  7. Affinity separation of human plasma gelsolin on Affi-Gel Blue.

    PubMed

    Yamamoto, H; Terabayashi, M; Egawa, T; Hayashi, E; Nakamura, H; Kishimoto, S

    1989-05-01

    Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

  8. Yawn contagion in humans and bonobos: emotional affinity matters more than species

    PubMed Central

    Norscia, Ivan; Demuru, Elisa

    2014-01-01

    In humans and apes, yawn contagion echoes emotional contagion, the basal layer of empathy. Hence, yawn contagion is a unique tool to compare empathy across species. If humans are the most empathic animal species, they should show the highest empathic response also at the level of emotional contagion. We gathered data on yawn contagion in humans (Homo sapiens) and bonobos (Pan paniscus) by applying the same observational paradigm and identical operational definitions. We selected a naturalistic approach because experimental management practices can produce different psychological and behavioural biases in the two species, and differential attention to artificial stimuli. Within species, yawn contagion was highest between strongly bonded subjects. Between species, sensitivity to others’ yawns was higher in humans than in bonobos when involving kin and friends but was similar when considering weakly-bonded subjects. Thus, emotional contagion is not always highest in humans. The cognitive components concur in empowering emotional affinity between individuals. Yet, when they are not in play, humans climb down from the empathic podium to return to the “understory”, which our species shares with apes. PMID:25165630

  9. The Fingerprint of Anti-Bromodeoxyuridine Antibodies and Its Use for the Assessment of Their Affinity to 5-Bromo-2'-Deoxyuridine in Cellular DNA under Various Conditions

    PubMed Central

    Ligasová, Anna; Liboska, Radek; Rosenberg, Ivan; Koberna, Karel

    2015-01-01

    We have developed a simple system for the analysis of the affinity of anti-bromodeoxyuridine antibodies. The system is based on the anchored oligonucleotides containing 5-bromo-2'-deoxyuridine (BrdU) at three different positions. It allows a reliable estimation of the reactivity of particular clones of monoclonal anti-bromodeoxyuridine antibodies with BrdU in fixed and permeabilized cells. Using oligonucleotide probes and four different protocols for the detection of BrdU incorporated in cellular DNA, we identified two antibody clones that evinced sufficient reactivity to BrdU in all the tested protocols. One of these clones exhibited higher reactivity to 5-iodo-2'-deoxyuridine (IdU) than to BrdU. It allowed us to increase the sensitivity of the used protocols without a negative effect on the cell physiology as the cytotoxicity of IdU was comparable with BrdU and negligible when compared to 5-ethynyl-2'-deoxyuridine. The combination of IdU and the improved protocol for oxidative degradation of DNA provided a sensitive and reliable approach for the situations when the low degradation of DNA and high BrdU signal is a priority. PMID:26161977

  10. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  11. Human immunodeficiency virus antibodies and the vaccine problem.

    PubMed

    Chiodi, F; Weiss, R A

    2014-05-01

    Despite the great advances made in controlling human immunodeficiency virus type 1 (HIV-1) infection with antiretroviral drug treatment, a safe and efficacious HIV vaccine has yet to be developed. Here, we discuss why clinical trials and vaccine development for HIV have so far been disappointing, with an emphasis on the lack of protective antibodies. We review approaches for developing appropriate HIV immunogens and the stimulation of long-lasting B-cell responses with antibody maturation. We conclude that candidate reagents in the pipeline for HIV vaccine development are unlikely to be particularly effective. Although the major funders of HIV vaccine research and development are placing increasing emphasis on clinical product development, a genuine breakthrough in preventing HIV infection through vaccines is more likely to come from novel immunogen research.

  12. Antibody-dependent cellular cytotoxicity by allosensitized human T cells

    PubMed Central

    1978-01-01

    Peripheral human T cells, isolated by sheep erythrocyte-rosette formation and density centrifugation, were highly cytotoxic to both Ab- coated autologous lymphocytes and antibody (Ab)-coated chicken erythrocytes when stimulated in mixed lymphocyte culture, but were not lytic when freshly purified, or when unstimulated in 6-day culture. Allosensitized T cells were shown to effect this activity by a specific effector-target cell interaction dependent on Ab, as indicated by: (a) induction of killing by Ab to target cells not lysed in the absence of Ab. (b) inhibition of Ab-dependent killing by aggregated Ig. The mechanism by which allosensitized T cells effect antibody-dependent cellular cytotoxicity is discussed. PMID:146728

  13. A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough

    PubMed Central

    Nguyen, Annalee W.; Wagner, Ellen K.; Laber, Joshua R.; Goodfield, Laura L.; Smallridge, William E.; Harvill, Eric T.; Papin, James F.; Wolf, Roman F.; Padlan, Eduardo A.; Bristol, Andy; Kaleko, Michael; Maynard, Jennifer A.

    2016-01-01

    In spite of wide-spread vaccination, pertussis rates are rising in industrialized countries and remain high world-wide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. Here, we humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human IgG1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the B. pertussis-induced rise in white blood cell count and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated but not control animals experienced a blunted rise in white blood cell count and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care. PMID:26631634

  14. NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

    PubMed Central

    Moutel, Sandrine; Bery, Nicolas; Bernard, Virginie; Keller, Laura; Lemesre, Emilie; de Marco, Ario; Ligat, Laetitia; Rain, Jean-Christophe; Favre, Gilles; Olichon, Aurélien; Perez, Franck

    2016-01-01

    In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications. DOI: http://dx.doi.org/10.7554/eLife.16228.001 PMID:27434673

  15. Radiotracers for Cardiac Sympathetic Innervation: Transport Kinetics and Binding Affinities for the Human Norepinephrine Transporter

    PubMed Central

    Raffel, David M.; Chen, Wei; Jung, Yong-Woon; Jang, Keun Sam; Gu, Guie; Cozzi, Nicholas V.

    2013-01-01

    Introduction Most radiotracers for imaging of cardiac sympathetic innervation are substrates of the norepinephrine transporter (NET). The goal of this study was to characterize the NET transport kinetics and binding affinities of several sympathetic nerve radiotracers, including [11C]-(−)-meta-hydroxyephedrine, [11C]-(−)-epinephrine, and a series of [11C]-labeled phenethylguanidines under development in our laboratory. For comparison, the NET transport kinetics and binding affinities of some [3H]-labeled biogenic amines were also determined. Methods Transport kinetics studies were performed using rat C6 glioma cells stably transfected with the human norepinephrine transporter (C6-hNET cells). For each radiolabeled NET substrate, saturation transport assays with C6-hNET cells measured the Michaelis-Menten transport constants Km and Vmax for NET transport. Competitive inhibition binding assays with homogenized C6-hNET cells and [3H]mazindol provided estimates of binding affinities (KI) for NET. Results Km, Vmax and KI values were determined for each NET substrate with a high degree of reproducibility. Interestingly, C6-hNET transport rates for ‘tracer concentrations’ of substrate, given by the ratio Vmax/Km, were found to be highly correlated with neuronal transport rates measured previously in isolated rat hearts (r2 = 0.96). This suggests that the transport constants Km and Vmax measured using the C6-hNET cells accurately reflect in vivo transport kinetics. Conclusion The results of these studies show how structural changes in NET substrates influence NET binding and transport constants, providing valuable insights that can be used in the design of new tracers with more optimal kinetics for quantifying regional sympathetic nerve density. PMID:23306137

  16. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  17. Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

    PubMed

    Rossant, Christine J; Carroll, Danielle; Huang, Ling; Elvin, John; Neal, Frances; Walker, Edward; Benschop, Joris J; Kim, Eldar E; Barry, Simon T; Vaughan, Tristan J

    2014-01-01

    Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and β-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.

  18. Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

    PubMed Central

    Xu, Chen; Lo, Agnes; Yammanuru, Anuradha; Tallarico, Aimee St. Clair; Brady, Kristen; Murakami, Akikazu; Barteneva, Natasha; Zhu, Quan; Marasco, Wayne A.

    2010-01-01

    Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule. PMID:20224781

  19. 78 FR 7438 - Prospective Grant of Exclusive License: Development of Human Monoclonal Antibodies Against DR4

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-01

    ... Human Monoclonal Antibodies Against DR4 AGENCY: National Institutes of Health, Public Health Service... Monoclonal Antibodies Against DR4'' (HHS Ref. No. E-158-2010/0) to Customized Biosciences, Inc., which is... relates to the development of two human monoclonal antibodies (mAbs) that bind to death receptor 4...

  20. Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies.

    PubMed

    Venet, Sophie; Kosco-Vilbois, Marie; Fischer, Nicolas

    2013-01-01

    The plasticity of natural immunoglobulin repertoires can be exploited for the generation of phage display libraries. Secondary lymphoid organs, such as the spleen and the lymph nodes, constitute interesting sources of diversity because they are rich in B cells, part of which can be affinity matured. These organs, however, differ in their anatomical structure, reflecting the different fluids they drain, which affects the B cell repertoires. The CDRH3 repertoires from these organs, extracted from naïve or immunized mice, were compared in the context of phage display libraries using human antibody framework families. Deep sequencing analysis revealed that all libraries displayed different CDRH3 repertoires, but the one derived from lymph nodes of naïve mice was the most diverse. Library performance was assessed by in vitro selection. For both organs, immunization increased substantially the frequency of molecules able to bind to the immunogen. The library derived from lymph nodes from naïve mice, however, was the most effective in generating diverse and high affinity candidates. These results illustrate that the use of a biased CDRH3 repertoire increases the performance of libraries, but reduces the clonal diversity, which may be detrimental for certain strategies.

  1. The affinities of human platelet and Acanthamoeba profilin isoforms for polyphosphoinositides account for their relative abilities to inhibit phospholipase C.

    PubMed Central

    Machesky, L M; Goldschmidt-Clermont, P J; Pollard, T D

    1990-01-01

    In light of recent work implicating profilin from human platelets as a possible regulator of both cytoskeletal dynamics and inositol phospholipid-mediated signaling, we have further characterized the interaction of platelet profilin and the two isoforms of Acanthamoeba profilin with inositol phospholipids. Profilin from human platelets binds to phosphatidylinositol-4-monophosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) with relatively high affinity (Kd approximately 1 microM for PIP2 by equilibrium gel filtration), but interacts only weakly (if at all) with phosphatidylinositol (PI) or inositol trisphosphate IP3) in small-zone gel-filtration assays. The two isoforms of Acanthamoeba profilin both have a lower affinity for PIP2 than does human platelet profilin, but the more basic profilin isoform from Acanthamoeba (profilin-II) has a much higher (approximately 10-microM Kd) affinity than the acidic isoform (profilin-I, 100 to 500-microM Kd). None of the profilins bind to phosphatidylserine (PS) or phosphatidylcholine (PC) in small-zone gel-filtration experiments. The differences in affinity for PIP2 parallel the ability of these three profilins to inhibit PIP2 hydrolysis by soluble phospholipase C (PLC). The results show that the interaction of profilins with PIP2 is specific with respect to both the lipid and the proteins. In Acanthamoeba, the two isoforms of profilin may have specialized functions on the basis of their identical (approximately 10 microM) affinities for actin monomers and different affinities for PIP2. PMID:1966040

  2. Detection and quantification of affinity ligand leaching and specific antibody fragment concentration within chromatographic fractions using surface plasmon resonance.

    PubMed

    Thillaivinayagalingam, Pranavan; Newcombe, Anthony R; O'Donovan, Kieran; Francis, Richard; Keshavarz-Moore, Eli

    2007-12-01

    Rapid analyses of chromatographic steps within a biopharmaceutical manufacturing process are often desirable to evaluate column performance, provide mass balance data and to permit accurate calculations of yields and recoveries. Using SPR (surface plasmon resonance) biosensor (Biacore) technology, we have developed a sandwich immunoassay to quantify polyclonal anti-digoxin Fab fragments used for the production of the FDA (Food and Drug Administration)-approved biotherapeutic DigiFab. The results show that specific Fab may be quantified in all affinity process streams and accurate yield and mass balance data calculated. Control experiments using sheep Fab and Fc indicate that the assay is specific to DigiFab. The quantification of potential leached ligand within chromatographic fractions may also be technically challenging, particularly when low-molecular-mass ligands are covalently coupled with an affinity absorbent. Typical methods to assess ligand leakage such as DDMA (digoxin-dicarboxymethoxylamine; digoxin analogue) often involve the use of labelled ligands and relatively complex and labour-intensive analytical techniques. Using the same analytical methodologies, an assay to detect leached or eluted ligand off the column was developed. The results indicate minimal levels of leached ligand in all chromatographic fractions, with total levels of leached DDMA calculated to be 1.52 microg. This is less than 0.01% of the total amount of DDMA coupled with the laboratory-scale affinity column. The SPR methods described in the present study may be applicable for the rapid in-process analysis of specific polyclonal Fab fragments (within a polyclonal mixture) and to rapidly assess leakage of small molecule ligands covalently attached to chromatographic supports.

  3. A safe, effective, and facility compatible cleaning in place procedure for affinity resin in large-scale monoclonal antibody purification.

    PubMed

    Wang, Lu; Dembecki, Jill; Jaffe, Neil E; O'Mara, Brian W; Cai, Hui; Sparks, Colleen N; Zhang, Jian; Laino, Sarah G; Russell, Reb J; Wang, Michelle

    2013-09-20

    Cleaning-in-place (CIP) for column chromatography plays an important role in therapeutic protein production. A robust and efficient CIP procedure ensures product quality, improves column life time and reduces the cost of the purification processes, particularly for those using expensive affinity resins, such as MabSelect protein A resin. Cleaning efficiency, resin compatibility, and facility compatibility are the three major aspects to consider in CIP process design. Cleaning MabSelect resin with 50mM sodium hydroxide (NaOH) along with 1M sodium chloride is one of the most popular cleaning procedures used in biopharmaceutical industries. However, high concentration sodium chloride is a leading cause of corrosion in the stainless steel containers used in large scale manufacture. Corroded containers may potentially introduce metal contaminants into purified drug products. Therefore, it is challenging to apply this cleaning procedure into commercial manufacturing due to facility compatibility and drug safety concerns. This paper reports a safe, effective and environmental and facility-friendly cleaning procedure that is suitable for large scale affinity chromatography. An alternative salt (sodium sulfate) is used to prevent the stainless steel corrosion caused by sodium chloride. Sodium hydroxide and salt concentrations were optimized using a high throughput screening approach to achieve the best combination of facility compatibility, cleaning efficiency and resin stability. Additionally, benzyl alcohol is applied to achieve more effective microbial control. Based on the findings, the recommended optimum cleaning strategy is cleaning MabSelect resin with 25 mM NaOH, 0.25 M Na2SO4 and 1% benzyl alcohol solution every cycle, followed by a more stringent cleaning using 50 mM NaOH with 0.25 M Na2SO4 and 1% benzyl alcohol at the end of each manufacturing campaign. A resin life cycle study using the MabSelect affinity resin demonstrates that the new cleaning strategy

  4. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  5. Determination of interdependent ligand effects on human red cell oxygen affinity.

    PubMed

    Zijlstra, W G; Oeseburg, B; Kwant, G; Zwart, A

    1982-06-01

    Human whole blood oxygen affinity was determined as P50, i.e. PO2 for oxygen saturation (SO2) = 0.5, in a new system which allows the measurement of SO2, PO2 and pH continuously and independently, with control of PCO2 and temperature. The influence of pH on P50 expressed as the H+ factor (delta log P50/delta pH)PCO2 was measured under conditions of varying PCO2, temperature and concentration of 2,3-diphosphoglycerate (2,3-DPG), resulting in a set of data expressing second-order inter-ligand interactions. The H+ factor appeared to be only slightly dependent on PCO2. Similarly, the CO2 factor (delta log P50/delta log PCO2)pH shows only a minor dependence on pH. The H+ factor in linearly related to the temperature: at 17 degrees C and 42 degrees C the H+ factor is about -0.53 and -0.36, respectively. Likewise, the temperature factor (delta log P50/delta T)PCO2,pH is linearly related to pH. A pilot study on the effect of varying intra-erythrocytic 2,3-DPG concentrations on the oxygen affinity showed that a very low 2,3-DPH/Hb4 ratio apparently does not influence the H+ factor. A high ratio, however, seems to lower the H+ factor considerably.

  6. 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.

  7. Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1

    DTIC Science & Technology

    2014-07-01

    subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE...Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6...Induced by the Polyvalent Malaria Vaccine AMA1 Sidhartha Chaudhury, Jaques Reifman, and Anders Wallqvist Polyvalent vaccines use a mixture of Ags

  8. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    PubMed Central

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  9. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells

    PubMed Central

    Jeffries, Thomas L; Sacha, CR; Pollara, Justin; Himes, Jon; Jaeger, Frederick H; Dennison, S Moses; McGuire, Erin; Kunz, Erika; Eudailey, Joshua A; Trama, Ashley M; LaBranche, Celia; Fouda, Genevieve G; Wiehe, Kevin; Montefiori, David C; Haynes, Barton F; Liao, Hua-Xin; Ferrari, Guido; Alam, S Munir; Moody, M Anthony; Permar, Sallie R

    2015-01-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants due to beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 Envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. Interestingly, we also identified divergent patterns of colostrum Env-specific B cell lineage evolution with respect to cross-reactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk IgG repertoire. Maternal vaccine strategies to specifically target this breast milk B cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants. PMID:26242599

  10. The function and affinity maturation of HIV-1 gp120-specific monoclonal antibodies derived from colostral B cells.

    PubMed

    Jeffries, T L; Sacha, C R; Pollara, J; Himes, J; Jaeger, F H; Dennison, S M; McGuire, E; Kunz, E; Eudailey, J A; Trama, A M; LaBranche, C; Fouda, G G; Wiehe, K; Montefiori, D C; Haynes, B F; Liao, H-X; Ferrari, G; Alam, S M; Moody, M A; Permar, S R

    2016-03-01

    Despite the risk of transmitting HIV-1, mothers in resource-poor areas are encouraged to breastfeed their infants because of beneficial immunologic and nutritional factors in milk. Interestingly, in the absence of antiretroviral prophylaxis, the overwhelming majority of HIV-1-exposed, breastfeeding infants are naturally protected from infection. To understand the role of HIV-1 envelope (Env)-specific antibodies in breast milk in natural protection against infant virus transmission, we produced 19 HIV-1 Env-specific monoclonal antibodies (mAbs) isolated from colostrum B cells of HIV-1-infected mothers and investigated their specificity, evolution, and anti-HIV-1 functions. Despite the previously reported genetic compartmentalization and gp120-specific bias of colostrum HIV Env-specific B cells, the colostrum Env-specific mAbs described here demonstrated a broad range of gp120 epitope specificities and functions, including inhibition of epithelial cell binding and dendritic cell-mediated virus transfer, neutralization, and antibody-dependent cellular cytotoxicity. We also identified divergent patterns of colostrum Env-specific B-cell lineage evolution with respect to crossreactivity to gastrointestinal commensal bacteria, indicating that commensal bacterial antigens play a role in shaping the local breast milk immunoglobulin G (IgG) repertoire. Maternal vaccine strategies to specifically target this breast milk B-cell population may be necessary to achieve safe breastfeeding for all HIV-1-exposed infants.

  11. Phage display-derived human antibodies in clinical development and therapy

    PubMed Central

    Frenzel, André; Schirrmann, Thomas; Hust, Michael

    2016-01-01

    ABSTRACT Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of “fully” human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years. PMID:27416017

  12. Phage display-derived human antibodies in clinical development and therapy.

    PubMed

    Frenzel, André; Schirrmann, Thomas; Hust, Michael

    2016-10-01

    Over the last 3 decades, monoclonal antibodies have become the most important class of therapeutic biologicals on the market. Development of therapeutic antibodies was accelerated by recombinant DNA technologies, which allowed the humanization of murine monoclonal antibodies to make them more similar to those of the human body and suitable for a broad range of chronic diseases like cancer and autoimmune diseases. In the early 1990s in vitro antibody selection technologies were developed that enabled the discovery of "fully" human antibodies with potentially superior clinical efficacy and lowest immunogenicity. Antibody phage display is the first and most widely used of the in vitro selection technologies. It has proven to be a robust, versatile platform technology for the discovery of human antibodies and a powerful engineering tool to improve antibody properties. As of the beginning of 2016, 6 human antibodies discovered or further developed by phage display were approved for therapy. In 2002, adalimumab (Humira®) became the first phage display-derived antibody granted a marketing approval. Humira® was also the first approved human antibody, and it is currently the best-selling antibody drug on the market. Numerous phage display-derived antibodies are currently under advanced clinical investigation, and, despite the availability of other technologies such as human antibody-producing transgenic mice, phage display has not lost its importance for the discovery and engineering of therapeutic antibodies. Here, we provide a comprehensive overview about phage display-derived antibodies that are approved for therapy or in clinical development. A selection of these antibodies is described in more detail to demonstrate different aspects of the phage display technology and its development over the last 25 years.

  13. RA8, A human anti-CD25 antibody against human treg cells

    SciTech Connect

    Arias, Robyn; Flanagan, Meg; Miller, Keith D.; Nien, Yu-Chih; Hu, Peisheng; Gray, Dixon; Khawli, Leslie A.; Epstein, Alan L.

    2007-06-01

    Although anti-CD25 antibodies exist for clinical use in patients, there is a need for the development of a human Treg antibody that will abrogate the immunosuppressive function of this small but critical T cell subtype. Based upon mounting evidence that the level of Treg cells in the tumor microenvironment correlates with clinical prognosis and stage in man, it appears that Treg cells play an important role in the tumor's ability to overcome host immune responses. In mice, the rat anti-mouse CD25 antibody PC61 causes depletion of CD25-bearing Treg cells both peripherally in lymphatic tissues and in the tumor microenvironment, without inducing symptoms of autoimmunity. A similar antibody, though with the ability to delete Treg cells specifically, would be an important new tool for reversing tumor escape associated with Treg immunosuppression in man. To begin to generate such a reagent, we now describe the development of a human anti-CD25 antibody using a novel yeast display library. The target antigen CD25-Fc was constructed and used for five rounds of selection using a non-immune yeast display library that contained as many as 109 single chain variable fragments (scFv). Two unique clones with low KD values (RA4 and RA8) were then selected to construct fully human anti-CD25 antibodies (IgG1/kappa) for stable expression. One antibody, RA8, showed excellent binding to human CD25+ cell lines and to human Treg cells and appears to be an excellent candidate for the generation of a human reagent that may be used in man for the immunotherapy of cancer.

  14. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

  15. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  16. High-resolution crystal structure of the therapeutic antibody pembrolizumab bound to the human PD-1

    PubMed Central

    Horita, Shoichiro; Nomura, Yayoi; Sato, Yumi; Shimamura, Tatsuro; Iwata, So; Nomura, Norimichi

    2016-01-01

    Pembrolizumab is an FDA-approved therapeutic antibody that targets the programmed cell death-1 (PD-1) to block the immune checkpoint pathway for the treatment of various types of cancer. It receives remarkable attention due to the high degree of efficacy. Very recently, the crystal structure of the Fab fragment of pembrolizumab (PemFab) in complex with the extracellular domain of human PD-1 (PD-1ECD) was reported at a resolution of 2.9 Å. However, this relatively low-resolution structural data fails to provide sufficient information on interfacial water molecules at the binding interface that substantially contribute to affinity and specificity between the therapeutic antibody and target. Here, we present the independently determined crystal structure of the Fv fragment of pembrolizumab (PemFv) in complex with the PD-1ECD at a resolution of 2.15 Å. This high-resolution structure allows the accurate mapping of the interaction including water-mediated hydrogen bonds and provides, for the first time, a coherent explanation of PD-1 antagonism by pembrolizumab. Our structural data also provides new insights into the rational design of improved anti-PD-1 therapeutics. PMID:27734966

  17. Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

    PubMed

    Nishiya, Casey T; Boxx, Gayle M; Robison, Kerry; Itatani, Carol; Kozel, Thomas R; Zhang, Mason X

    2015-11-16

    Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

  18. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    PubMed

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  19. Application of affinity aqueous two-phase systems for the fractionation of CD133(+) stem cells from human umbilical cord blood.

    PubMed

    González-González, Mirna; Rito-Palomares, Marco

    2015-03-01

    In a further attempt to establish a novel stem cell primary recovery strategy, the use of aqueous two-phase systems (ATPS) complemented with the use of antibodies (known as immunoaffinity ATPS) is explored in this work. This type of liquid-liquid extraction systems exploits antigen-antibody affinity and represents a novel and selective approach for the purification of stem cells. The proposed bioengineering strategies include the implementation of traditional [polyethylene glycol (PEG), dextran (DEX) and ficoll] and novel (Ucon) immunoaffinity ATPS to prove the viability of cluster of differentiation 133 (CD133(+) ) stem cells from human umbilical cord blood. Furthermore, the addition of the antibody is implemented to identify conditions under which contaminants and stem cells of interest concentrate in opposite phases. The objective of this work is to establish the initial basis for the development of a novel and scalable purification bioprocess for the selective recovery of CD133(+) stem cells employing immunoaffinity ATPS. The reported methodology allows a partitioning of 62% CD133(+) stem cells to the top phase of the ficoll 400,000-DEX 70,000 immunoaffinity ATPS. In PEG 8,000-DEX 500,000 and Ucon-DEX 75,000 systems, no difference was observed when compared with the conventional ATPS (without antibody addition), as the CD133 antibody does not have preference for the desired clean top phase. In all experiments, cell viability was at least 98% after ATPS recovery. This research highlights the challenges that must be addressed to allow the potential establishment of a separation process using immunoaffinity ATPS for the recovery and purification of stem cells.

  20. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  1. A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

    PubMed

    Ebert, Tim; Smith, Sharon; Pancari, Greg; Clark, Desmond; Hampton, Richard; Secore, Susan; Towne, Victoria; Fan, Hongxia; Wang, Xin-Min; Wu, Xiaoqing; Ernst, Robin; Harvey, Barrett R; Finnefrock, Adam C; Wang, Fubao; Tan, Charles; Durr, Eberhard; Cope, Leslie; Anderson, Annaliesa; An, Zhiqiang; McNeely, Tessie

    2010-01-01

    A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

  2. Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure.

    PubMed

    Brazzolotto, Xavier; Wandhammer, Marielle; Ronco, Cyril; Trovaslet, Marie; Jean, Ludovic; Lockridge, Oksana; Renard, Pierre-Yves; Nachon, Florian

    2012-08-01

    Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ≈ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.

  3. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  4. 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

  5. Efficient production and purification of recombinant human interleukin-12 (IL-12) overexpressed in mammalian cells without affinity tag

    PubMed Central

    Jayanthi, Srinivas; Koppolu, Bhanu prasanth; Smith, Sean G.; Jalah, Rashmi; Bear, Jenifer; Rosati, Margherita; Pavlakis, George N.; Felber, Barbara K.; Zaharoff, David A.; Kumar, Thallapuranam Krishnaswamy Suresh

    2014-01-01

    Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous.Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be ~98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichrosim, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31 ± 0.05 mg/ mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app) = 69 ± 1 μM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics. PMID:25123642

  6. Humanization of an anti-CCR4 antibody that kills cutaneous T-cell lymphoma cells and abrogates suppression by T-regulatory cells.

    PubMed

    Chang, De-Kuan; Sui, Jianhua; Geng, Shusheng; Muvaffak, Asli; Bai, Mei; Fuhlbrigge, Robert C; Lo, Agnes; Yammanuru, Anuradha; Hubbard, Luke; Sheehan, Jared; Campbell, James J; Zhu, Quan; Kupper, Thomas S; Marasco, Wayne A

    2012-11-01

    Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4(+) CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. mAb1567 also exerts human NK cell-mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4(+)CD25(high) Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity-optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development based on its higher binding affinity and more potent ADCC and CDC activities. Taken together, this high-affinity humanized mAb2-3 with potent antitumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors.

  7. Chimeric Anti-Human Podoplanin Antibody NZ-12 of Lambda Light Chain Exerts Higher Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity Compared with NZ-8 of Kappa Light Chain.

    PubMed

    Kaneko, Mika K; Abe, Shinji; Ogasawara, Satoshi; Fujii, Yuki; Yamada, Shinji; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Nishioka, Yasuhiko; Kato, Yukinari

    2017-02-01

    Podoplanin (PDPN), a type I transmembrane 36-kDa glycoprotein, is expressed not only in normal cells, such as renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, but also in cancer cells, including brain tumors and lung squamous cell carcinomas. Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and the podoplanin/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced neutralizing anti-human podoplanin monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda), which neutralizes the podoplanin/CLEC-2 interaction and inhibits platelet aggregation and cancer metastasis. Human-rat chimeric antibody, NZ-8, was previously developed using variable regions of NZ-1 and human constant regions of heavy chain (IgG1) and light chain (kappa chain). Although NZ-8 showed high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cells, the binding affinity of NZ-8 was lower than that of NZ-1. Herein, we produced a novel human-rat chimeric antibody, NZ-12, the constant regions of which consist of IgG1 heavy chain and lambda light chain. Using flow cytometry, we demonstrated that the binding affinity of NZ-12 was much higher than that of NZ-8. Furthermore, ADCC and CDC activities of NZ-12 were significantly increased against glioblastoma cell lines (LN319 and D397) and lung cancer cell line (PC-10). These results suggested that NZ-12 could become a promising therapeutic antibody against podoplanin-expressing brain tumors and lung cancers.

  8. Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin

    PubMed Central

    Nguyen, Hung Huy; Varadi, Mihaly; Tompa, Peter

    2017-01-01

    Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies. PMID:28319173

  9. Observation of interactions of human serum components with transferrin by affinity capillary electrophoresis.

    PubMed

    Taga, Atsushi; Maruyama, Rie; Yamamoto, Yuka; Honda, Susumu

    2008-01-22

    Interaction of human transferrin (TF) with human serum components was investigated by affinity capillary electrophoresis. It was found that any peaks of human serum protein fractions did not give migration time change on addition of intact TF to running buffer (50mM phosphate buffer, pH 7.5), whereas two peaks belonging to alpha-globulin fraction showed marked acceleration upon addition of desialylated TF. These results provide strong evidence that the sialic acid residue in TF masks its binding ability to serum proteins. The association constants of desialylated TF to these interactive components, estimated based on the double reciprocal plot of migration time change vs. glycoprotein concentration, were at a high level of 10(7)M(-1). TF is well known as a ferric ion transfer protein, and hence formation of this protein might be changed by ferric ion. The presence of iron(II) played no essential role in this interaction, though its influence was not negligible.

  10. Intracellular and circulating neuronal antinuclear antibodies in human epilepsy.

    PubMed

    Iffland, Philip H; Carvalho-Tavares, Juliana; Trigunaite, Abhishek; Man, Shumei; Rasmussen, Peter; Alexopoulos, Andreas; Ghosh, Chaitali; Jørgensen, Trine N; Janigro, Damir

    2013-11-01

    There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, the human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from the brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in the serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n=4/4). In the brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated

  11. Intracellular and circulating neuronal antinuclear antibodies in human epilepsy

    PubMed Central

    Iffland, Philip H.; Carvalho-Tavares, Juliana; Trigunaite, Abhishek; Man, Shumei; Rasmussen, Peter; Alexopoulos, Andreas; Ghosh, Chaitali; Jørgensen, Trine N.; Janigro, Damir

    2013-01-01

    There are overwhelming data supporting the inflammatory origin of some epilepsies (e.g., Rasmussen's encephalitis and limbic encephalitis). Inflammatory epilepsies with an autoimmune component are characterized by autoantibodies against membrane-bound, intracellular or secreted proteins (e.g., voltage gated potassium channels). Comparably, little is known regarding autoantibodies targeting nuclear antigen. We tested the hypothesis that in addition to known epilepsy-related autoantigens, human brain tissue and serum from patients with epilepsy contain autoantibodies recognizing nuclear targets. We also determined the specific nuclear proteins acting as autoantigen in patients with epilepsy. Brain tissue samples were obtained from patients undergoing brain resections to treat refractory seizures, from brain with arteriovenous malformations or from post-mortem multiple sclerosis brain. Patients with epilepsy had no known history of autoimmune disease and were not diagnosed with autoimmune epilepsy. Tissue was processed for immunohistochemical staining. We also obtained subcellular fractions to extract intracellular IgGs. After separating nuclear antibody-antigen complexes, the purified autoantigen was analyzed by mass spectrometry. Western blots using autoantigen or total histones were probed to detect the presence of antinuclear antibodies in the serum of patients with epilepsy. Additionally, HEp-2 assays and antinuclear antibody ELISA were used to detect the staining pattern and specific presence of antinuclear antibodies in serum of patients with epilepsy. Brain regions from patients with epilepsy characterized by blood-brain barrier disruption (visualized by extravasated albumin) contained extravasated IgGs. Intracellular antibodies were found in epilepsy (n=13/13) but not in multiple sclerosis brain (n= 4/4). In brain from patients with epilepsy, neurons displayed higher levels of nuclear IgGs compared to glia. IgG colocalized with extravasated albumin. All

  12. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    PubMed

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  13. Production of human anti-HLA monoclonal antibodies

    SciTech Connect

    Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

    1986-03-01

    Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

  14. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-05-10

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design.

  15. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  16. Gonadotropin-releasing hormone/human chorionic gonadotropin beta based recombinant antibodies and vaccines.

    PubMed

    Talwar, G P; Vyas, Hemant K; Purswani, Shilpi; Gupta, Jagdish C

    2009-12-01

    Gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are unique targets for the control of fertility. Immunological approaches to neutralizing these hormones have additional utility in cancer treatment. Vaccines have been developed against both GnRH and hCG and these have undergone Phase I/II clinical trials documenting their safety, reversibility and efficacy. The heterospecies dimer hCG vaccine prevented pregnancy in women of proven fertility without impairment of ovulation or derangement of menstrual regularity and bleeding profiles. The protective threshold of antibody titers to achieve efficacy was determined in these first-ever trials. Recently, a recombinant vaccine against the beta subunit of hCG linked to the B subunit of heat labile enterotoxin has been made and expressed as a glycosylated conjugate in Pichia pastoris. Experiments indicate its ability to generate antibodies above the protective threshold in all immunized Balb/c mice. Ectopic expression of hCG/hCGbeta is observed in many advanced stage cancers of various origins. A chimeric high affinity and specific recombinant antibody against hCGbeta linked to curcumin kills hCGbeta expressing T lymphoblastic leukemia cells without any deleterious effect. Several synthetic and recombinant vaccines have been developed against GnRH. These reduce serum testosterone to castration levels causing atrophy of the prostate. Three Phase I/II clinical trials conducted in India and Austria have shown that these vaccines elicit non-surgical reduction of testosterone, a fall in prostate specific antigen and clinical improvement of prostate carcinoma patients. A multimer recombinant vaccine against GnRH has high efficacy for sterilization of pigs and other animals.

  17. [Obtaining of ScFv-CBD fusion protein and its application for affinity purification of recombinant human interferon alpha2b].

    PubMed

    Hil'chuk, P V; Okuniev, O V; Pavlova, M V; Irodov, D M; Horbatiuk, O B

    2006-01-01

    The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.

  18. Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching

    PubMed Central

    Horns, Felix; Vollmers, Christopher; Croote, Derek; Mackey, Sally F; Swan, Gary E; Dekker, Cornelia L; Davis, Mark M; Quake, Stephen R

    2016-01-01

    Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state. DOI: http://dx.doi.org/10.7554/eLife.16578.001 PMID:27481325

  19. Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells.

    PubMed

    Nogales-Gadea, Gisela; Saxena, Abhishek; Hoffmann, Carolin; Hounjet, Judith; Coenen, Daniëlle; Molenaar, Peter; Losen, Mario; Martinez-Martinez, Pilar

    2015-06-05

    Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

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

    NASA Astrophysics Data System (ADS)

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

    1993-05-01

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

  1. A chromosome-centric analysis of antibodies directed toward the human proteome using Antibodypedia.

    PubMed

    Alm, Tove; von Feilitzen, Kalle; Lundberg, Emma; Sivertsson, Åsa; Uhlén, Mathias

    2014-03-07

    Antibodies are crucial for the study of human proteins and have been defined as one of the three pillars in the human chromosome-centric Human Proteome Project (C-HPP). In this article the chromosome-centric structure has been used to analyze the availability of antibodies as judged by the presence within the portal Antibodypedia, a database designed to allow comparisons and scoring of publicly available antibodies toward human protein targets. This public database displays antibody data from more than one million antibodies toward human protein targets. A summary of the content in this knowledge resource reveals that there exist more than 10 antibodies to over 70% of all the putative human genes, evenly distributed over the 24 human chromosomes. The analysis also shows that at present, less than 10% of the putative human protein-coding genes (n = 1882) predicted from the genome sequence lack antibodies, suggesting that focused efforts from the antibody-based and mass spectrometry-based proteomic communities should be encouraged to pursue the analysis of these missing proteins. We show that Antibodypedia may be used to track the development of available and validated antibodies to the individual chromosomes, and thus the database is an attractive tool to identify proteins with no or few antibodies yet generated.

  2. Crystal Structures of the Pro-Inflammatory Cytokine Interleukin-23 and Its Complex with a High-Affinity Neutralizing Antibody

    SciTech Connect

    Beyer, Brian M.; Ingram, Richard; Ramanathan, Lata; Reichert, Paul; Le, Hung V.; Madison, Vincent; Orth, Peter

    2009-06-25

    Interleukin (IL)-23 is a pro-inflammatory cytokine playing a key role in the pathogenesis of several autoimmune and inflammatory diseases. We have determined the crystal structures of the heterodimeric p19-p40 IL-23 and its complex with the Fab (antigen-binding fragment) of a neutralizing antibody at 2.9 and 1.9 {angstrom}, respectively. The IL-23 structure closely resembles that of IL-12. They share the common p40 subunit, and IL-23 p19 overlaps well with IL-12 p35. Along the hydrophilic heterodimeric interface, fewer charged residues are involved for IL-23 compared with IL-12. The binding site of the Fab is located exclusively on the p19 subunit, and comparison with published cytokine-receptor structures suggests that it overlaps with the IL-23 receptor binding site.

  3. Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library.

    PubMed

    Li, Feng; Liu, Yan-Hong; Li, Yan-Wen; Ju, Qian; Chen, Lin; Xie, Ping-Li; Li, Yue-Hui; Li, Guan-Cheng

    2012-06-01

    Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

  4. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  5. The role of human cytochrome P450 enzymes in the formation of 2-hydroxymetronidazole: CYP2A6 is the high affinity (low Km) catalyst.

    PubMed

    Pearce, Robin E; Cohen-Wolkowiez, Michael; Sampson, Mario R; Kearns, Gregory L

    2013-09-01

    Despite metronidazole's widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a "therapeutic concentration" of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo.

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

    DOEpatents

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

    1988-01-01

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

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

    DOEpatents

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

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

  8. Strains of Mycobacterium tuberculosis differ in affinity for human osteoblasts and alveolar cells in vitro.

    PubMed

    Sarkar, Shrabanti; Dlamini, Muyalo G; Bhattacharya, Debapriya; Ashiru, Olubisi T; Sturm, A Willem; Moodley, Prashini

    2016-01-01

    Although the lung is the primary site of infection of tuberculosis, Mycobacterium tuberculosis is capable of causing infection at other sites. In 5-10 % such extra-pulmonary tuberculosis is located in bone tissue of the spine. It is unknown whether host or microbial factors are responsible for the site where extra-pulmonary tuberculosis manifests itself. One MDR isolate belonging to strain F28, one susceptible F11 and one isolate each of susceptible, MDR and XDR F15/LAM4/KZN were cultured in Middlebrook 7H9 media. Human osteoblasts (SaOS-2) and human alveolar epithelial cells (A549) were exposed to these different isolates of M. tuberculosis and invasion capacity and intra-cellular multiplication rates were established. Mouse macrophage (MHS) cells exposed to M. tuberculosis H37Rv served as control. The invasion capacity of F15/LAM4/KZN representatives increased with the level of resistance. The F28 MDR strain showed similar invasion capacity as the XDR F15/LAM4/KZN for pulmonary epthelial cells, whilst the fully susceptible F11 strain displayed a propensity for osteoblasts. The differences observed may in part explain why certain strains are able to cause infection at specific extra-pulmonary sites. We postulated that the development of extra-pulmonary tuberculosis depends on the ability of the microbe to pass effectively through the alveolar epithelial lining and its affinity for cells other than those in pulmonary tissue.

  9. Generation, characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

    PubMed Central

    Cho, Seulki; Park, Insoo; Kim, Haejung; Jeong, Mun Sik; Lim, Mooney; Lee, Eung Suk; Kim, Jin Hong; Kim, Semi; Hong, Hyo Jeong

    2016-01-01

    ABSTRACT L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent. PMID:26785809

  10. Neanderthal and Denisova genetic affinities with contemporary humans: introgression versus common ancestral polymorphisms.

    PubMed

    Lowery, Robert K; Uribe, Gabriel; Jimenez, Eric B; Weiss, Mark A; Herrera, Kristian J; Regueiro, Maria; Herrera, Rene J

    2013-11-01

    Analyses of the genetic relationships among modern humans, Neanderthals and Denisovans have suggested that 1-4% of the non-Sub-Saharan African gene pool may be Neanderthal derived, while 6-8% of the Melanesian gene pool may be the product of admixture between the Denisovans and the direct ancestors of Melanesians. In the present study, we analyzed single nucleotide polymorphism (SNP) diversity among a worldwide collection of contemporary human populations with respect to the genetic constitution of these two archaic hominins and Pan troglodytes (chimpanzee). We partitioned SNPs into subsets, including those that are derived in both archaic lineages, those that are ancestral in both archaic lineages and those that are only derived in one archaic lineage. By doing this, we have conducted separate examinations of subsets of mutations with higher probabilities of divergent phylogenetic origins. While previous investigations have excluded SNPs from common ancestors in principal component analyses, we included common ancestral SNPs in our analyses to visualize the relative placement of the Neanderthal and Denisova among human populations. To assess the genetic similarities among the various hominin lineages, we performed genetic structure analyses to provide a comparison of genetic patterns found within contemporary human genomes that may have archaic or common ancestral roots. Our results indicate that 3.6% of the Neanderthal genome is shared with roughly 65.4% of the average European gene pool, which clinally diminishes with distance from Europe. Our results suggest that Neanderthal genetic associations with contemporary non-Sub-Saharan African populations, as well as the genetic affinities observed between Denisovans and Melanesians most likely result from the retention of ancient mutations in these populations.

  11. Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So

    2014-01-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins. PMID:25545206

  12. Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor.

    PubMed

    Suharni; Nomura, Yayoi; Arakawa, Takatoshi; Hino, Tomoya; Abe, Hitomi; Nakada-Nakura, Yoshiko; Sato, Yumi; Iwanari, Hiroko; Shiroishi, Mitsunori; Asada, Hidetsugu; Shimamura, Tatsuro; Murata, Takeshi; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Nomura, Norimichi

    2014-12-01

    The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

  13. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

    PubMed Central

    Kirsch, Martina Inga; Hülseweh, Birgit; Nacke, Christoph; Rülker, Torsten; Schirrmann, Thomas; Marschall, Hans-Jürgen; Hust, Michael; Dübel, Stefan

    2008-01-01

    Background Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent. Conclusion For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak