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Sample records for antibody affinity maturation

  1. 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

  2. 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.

  3. 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

  4. Memory in the B-cell compartment: antibody affinity maturation.

    PubMed Central

    Neuberger, M S; Ehrenstein, M R; Rada, C; Sale, J; Batista, F D; Williams, G; Milstein, C

    2000-01-01

    In the humoral arm of the immune system, the memory response is not only more quickly elicited and of greater magnitude than the primary response, but it is also different in quality. In the recall response to antigen, the antibodies produced are of higher affinity and of different isotype (typically immunoglobulin G rather than immunoglobulin M). This maturation rests on the antigen dependence of B-cell maturation and is effected by programmed genetic modifications of the immunoglobulin gene loci. Here we consider how the B-cell response to antigen depends on the affinity of the antigen receptor interaction. We also compare and draw parallels between the two processes, which underpin the generation of secondary-response antibodies: V gene somatic hypermutation and immunoglobulin heavy-chain class switching. PMID:10794054

  5. Dynamics behind affinity maturation of an anti-HCMV antibody family influencing antigen binding.

    PubMed

    Di Palma, Francesco; Tramontano, Anna

    2017-08-03

    The investigation of antibody affinity maturation and its effects on antigen binding is important with respect to understanding the regulation of the immune response. To shed light on this crucial process, we analyzed two Igs neutralizing the human cytomegalovirus: the primary germline antibody M2J1 and its related mature antibody 8F9. Both antibodies target the AD-2S1 epitope of the gB envelope protein and are considered to establish similar interactions with the cognate antigen. We used molecular dynamics simulations to understand the effect of mutations on the antibody-antigen interactions. The results provide a qualitative explanation for the increased 8F9 peptide affinity compared with that of M2J1. The emerging atomistic-detailed description of these complexes reveals the molecular effects of the somatic hypermutations occurring during affinity maturation. © 2017 Federation of European Biochemical Societies.

  6. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  7. The influence of antibody fragment format on phage display based affinity maturation of IgG

    PubMed Central

    Steinwand, Miriam; Droste, Patrick; Frenzel, Andrè; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

    2014-01-01

    Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.   In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab. PMID:24262918

  8. The influence of antibody fragment format on phage display based affinity maturation of IgG.

    PubMed

    Steinwand, Miriam; Droste, Patrick; Frenzel, André; Hust, Michael; Dübel, Stefan; Schirrmann, Thomas

    2014-01-01

    Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly,single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs.Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.

  9. 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

  10. 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.

  11. 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.

  12. Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

    PubMed Central

    Pavoni, Emiliano; Flego, Michela; Dupuis, Maria Luisa; Barca, Stefano; Petronzelli, Fiorella; Anastasi, Anna Maria; D'Alessio, Valeria; Pelliccia, Angela; Vaccaro, Paola; Monteriù, Giorgia; Ascione, Alessandro; De Santis, Rita; Felici, Franco; Cianfriglia, Maurizio; Minenkova, Olga

    2006-01-01

    Background CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration. Methods The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized. Results The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells. Conclusion The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance. PMID:16504122

  13. 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

  14. 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

  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. Key mutations stabilize antigen-binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage

    PubMed Central

    Xu, Huafeng; Schmidt, Aaron G; O'Donnell, Timothy; Therkelsen, Matthew D; Kepler, Thomas B; Moody, M Anthony; Haynes, Barton F; Liao, Hua-Xin; Harrison, Stephen C; Shaw, David E

    2015-01-01

    Affinity maturation, the process in which somatic hypermutation and positive selection generate antibodies with increasing affinity for an antigen, is pivotal in acquired humoral immunity. We have studied the mechanism of affinity gain in a human B-cell lineage in which two main maturation pathways, diverging from a common ancestor, lead to three mature antibodies that neutralize a broad range of H1 influenza viruses. Previous work showed that increased affinity in the mature antibodies derives primarily from stabilization of the CDR H3 loop in the antigen-binding conformation. We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively. In the two maturation pathways, different contacts between light and heavy chains stabilize the CDR H3 loop. As few as two single-site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own. Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies. Proteins 2014; 83:771–780. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:25524709

  17. 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

  18. 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.

  19. 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.

  20. 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. The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933). © 2016 by the American Association for the Study of Liver Diseases.

  1. Preconfiguration of the antigen-binding site during affinity maturation of a broadly neutralizing influenza virus antibody.

    PubMed

    Schmidt, Aaron G; Xu, Huafeng; Khan, Amir R; O'Donnell, Timothy; Khurana, Surender; King, Lisa R; Manischewitz, Jody; Golding, Hana; Suphaphiphat, Pirada; Carfi, Andrea; Settembre, Ethan C; Dormitzer, Philip R; Kepler, Thomas B; Zhang, Ruijun; Moody, M Anthony; Haynes, Barton F; Liao, Hua-Xin; Shaw, David E; Harrison, Stephen C

    2013-01-02

    Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.

  2. 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.

  3. Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

    PubMed Central

    Eylenstein, Roy; Weinfurtner, Daniel; Härtle, Stefan; Strohner, Ralf; Böttcher, Jark; Augustin, Martin; Ostendorp, Ralf; Steidl, Stefan

    2016-01-01

    X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11–24), a short second sub-epitope (residues 83–89) and a third at the C-terminus (residues 112–123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF. PMID:26406987

  4. 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.

  5. Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

    PubMed

    Rosenfeld, Ronit; Alcalay, Ron; Mechaly, Adva; Lapidoth, Gideon; Epstein, Eyal; Kronman, Chanoch; J Fleishman, Sarel; Mazor, Ohad

    2017-05-03

    While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. 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

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

    PubMed

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

    2015-06-01

    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. This study aimed to determine whether the quaternary structure (multimerization) of shed A-subunits influences pathogenic TSHR autoantibody generation. 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. 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. The present study supports the concept that pathogenic TSHR autoantibody affinity maturation in Graves' disease is driven by A-subunit multimers, not monomers.

  8. 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.

  9. 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.

  10. Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies

    PubMed Central

    Kepler, Thomas B.; Liao, Hua-Xin; Alam, S. Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; 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.

    2014-01-01

    Summary 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. PMID:25211073

  11. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments.

    PubMed

    Blaise, Lydia; Wehnert, Anita; Steukers, Mieke P G; van den Beucken, Twan; Hoogenboom, Hennie R; Hufton, Simon E

    2004-11-24

    Yeast display is a powerful technology for the affinity maturation of human antibody fragments. However, the technology thus far has been limited by the size of antibody libraries that can be generated, as using current transformation protocols libraries of only between 10(6) and 10(7) are typically possible. We have recently shown that Fab antibodies can be displayed on the cell surface of Saccharomyces cerevisiae [van den Beucken, T., Pieters, H., Steukers, M., van der Vaart, M., Ladner, R.C., Hoogenboom, H.R., Hufton, S.E., 2003. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288-294]. This discovery and the knowledge that Fab antibodies are heterodimeric suggest that independent repertoires of heavy chain (HC) and light chain (LC) can be constructed in haploid yeast strains of opposite mating type. These separate repertoires can then be combined by highly efficient yeast mating. Using this approach, we have rapidly generated a naive human Fab yeast display library of over 10(9) clones. In addition, utilizing error-prone polymerase chain reaction, we have diversified Fab sequences and generated combinatorial and hierarchical chain shuffled libraries with complexities of up to 5 x 10(9) clones. These libraries have been selected for higher affinity using a repeating process of mating-driven chain shuffling and flow cytometric sorting.

  12. 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.

  13. 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

  14. 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

  15. Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3.

    PubMed

    Ahmed, Mahiuddin; Cheng, Ming; Zhao, Qi; Goldgur, Yehuda; Cheal, Sarah M; Guo, Hong-fen; Larson, Steven M; Cheung, Nai-kong V

    2015-12-11

    B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nm KD) compared with parental hu8H9 scFv (144 nm KD). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar KD) had 2-9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1-0.3 μg/ml EC50), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  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. 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.

  18. Affinity purification of antibodies

    USDA-ARS?s Scientific Manuscript database

    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...

  19. Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity.

    PubMed

    Kwong, Ka Yin; Baskar, Sivasubramanian; Zhang, Hua; Mackall, Crystal L; Rader, Christoph

    2008-12-31

    In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.

  20. 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.

  1. 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

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

    PubMed Central

    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. PMID:25484051

  4. 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.

  5. 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.

  6. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Genetic and structural analyses of affinity maturation in the humoral response to HIV-1.

    PubMed

    Kepler, Thomas B; Wiehe, Kevin

    2017-01-01

    Most broadly neutralizing antibodies (BNAbs) elicited in response to HIV-1 infection are extraordinarily mutated. One goal of HIV-1 vaccine development is to induce antibodies that are similar to the most potent and broad BNAbs isolated from infected subjects. The most effective BNAbs have very high mutation frequencies, indicative of the long periods of continual activation necessary to acquire the BNAb phenotype through affinity maturation. Understanding the mutational patterns that define the maturation pathways in BNAb development is critical to vaccine design efforts to recapitulate through vaccination the successful routes to neutralization breadth and potency that have occurred in natural infection. Studying the mutational changes that occur during affinity maturation, however, requires accurate partitioning of sequence data into B-cell clones and identification of the starting point of a B-cell clonal lineage, the initial V(D)J rearrangement. Here, we describe the statistical framework we have used to perform these tasks. Through the recent advancement of these and similar computational methods, many HIV-1 ancestral antibodies have been inferred, synthesized and their structures determined. This has allowed, for the first time, the investigation of the structural mechanisms underlying the affinity maturation process in HIV-1 antibody development. Here, we review what has been learned from this atomic-level structural characterization of affinity maturation in HIV-1 antibodies and the implications for vaccine design.

  8. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    PubMed

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

  9. What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination? Is Affinity Maturation a Self-Defeating Process for Eliciting Broad Protection?

    PubMed

    Stamper, Christopher T; Wilson, Patrick C

    2017-06-19

    Vaccinations are one of the greatest success stories of modern medicine, saving millions of lives since their widespread adoption. However, several diseases continue to elude highly effective vaccination strategies. Chief among these are human immunodeficiency virus (HIV) and influenza (flu), both of which will require vaccines that can guide the creation of highly mutated, broadly neutralizing antibodies (bnAbs). The generation of bnAbs is hindered by our inability to effectively drive the high levels of affinity maturation required to achieve them in a large number of cells. Major limitations placed on affinity maturation derives from the inherent mutability of immunoglobulin genes, the evolved activation-induced cytidine deaminase (AID) targeting mechanisms that exist within them, and biases in targeting of particular epitope B cells. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  10. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation

    PubMed Central

    Kepler, Thomas B.; Munshaw, Supriya; Wiehe, Kevin; Zhang, Ruijun; Yu, Jae-Sung; Woods, Christopher W.; Denny, Thomas N.; Tomaras, Georgia D.; Alam, S. Munir; Moody, M. Anthony; Kelsoe, Garnett; Liao, Hua-Xin; Haynes, Barton F.

    2014-01-01

    Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain–light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases. PMID:24795717

  11. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation.

    PubMed

    Kepler, Thomas B; Munshaw, Supriya; Wiehe, Kevin; Zhang, Ruijun; Yu, Jae-Sung; Woods, Christopher W; Denny, Thomas N; Tomaras, Georgia D; Alam, S Munir; Moody, M Anthony; Kelsoe, Garnett; Liao, Hua-Xin; Haynes, Barton F

    2014-01-01

    Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain-light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases.

  12. Oral priming with replicating adenovirus serotype 4 followed by subunit H5N1 vaccine boost promotes antibody affinity maturation and expands H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Manischewitz, Jody; King, Lisa R; Ishioka, Glenn; Alexander, Jeff; Smith, Jon; Gurwith, Marc; Golding, Hana

    2015-01-01

    A Phase I trial conducted in 2009-2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 107-1011 viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.

  13. H5 N-terminal β sheet promotes oligomerization of H7-HA1 that induces better antibody affinity maturation and enhanced protection against H7N7 and H7N9 viruses compared to inactivated influenza vaccine.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Verma, Swati; King, Lisa R; Manischewitz, Jody; Crevar, Corey J; Carter, Donald M; Ross, Ted M; Golding, Hana

    2014-11-12

    Initiation of mass vaccination is critical in response to influenza pandemic. There is an urgent need of a simple, rapid method for production of influenza vaccine that is more effective than current traditional influenza vaccines. Recent H7N9 transmissions to humans in China with high morbidity/mortality initiated extensive vaccine evaluation. We produced the HA1 domains (amino acids 1-320) from H7N9 and H7N7 strains in E. coli. Both were found to contain primarily monomers/trimers with low oligomeric content. However, when residues from the N-terminal β sheet (first 8 amino acid) of H7 HA1 domains were swapped with the corresponding amino acids from H5N1, functional oligomeric H7 HA1 were produced (HA1-DS), demonstrating strong receptor binding and hemagglutination. In rabbits, the HA1-DS from either H7N9 or H7N7 generated high neutralization titers against both homologous and heterologous H7 strains, superior to the unmodified H7 HA1 proteins. In ferrets, HA1-DS from H7N7 elicited higher (and faster) HI titers, better protected ferrets from lethality, weight loss, and reduced viral loads following challenge with wild-type highly pathogenic H7N7 virus compared with inactivated H7N7 subunit vaccine. HA1-DS vaccinated ferrets were also better protected from weight loss after challenge with the heterologous H7N9 virus compared with inactivated H7N7 subunit vaccine. Importantly, the H7N7 HA1-DS vaccine induced antibody affinity maturation far superior to the inactivated H7N7 subunit vaccine, which strongly correlated with control of viral loads in the nasal washes after challenge with either H7N7 or H7N9 strains. We conclude that N-terminus β sheet domain-swap can be used to produce stable functional oligomeric forms of better recombinant HA1 vaccines in simple, inexpensive bacterial system for rapid response to emerging pandemic threat for the global population.

  14. Determinism and stochasticity during maturation of the zebrafish antibody repertoire

    PubMed Central

    Jiang, Ning; Weinstein, Joshua A.; Penland, Lolita; White, Richard A.; Fisher, Daniel S.; Quake, Stephen R.

    2011-01-01

    It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity. PMID:21393572

  15. Heterologous Prime-Boost Vaccination with MF59-Adjuvanted H5 Vaccines Promotes Antibody Affinity Maturation towards the Hemagglutinin HA1 Domain and Broad H5N1 Cross-Clade Neutralization

    PubMed Central

    Khurana, Surender; Coyle, Elizabeth M.; Dimitrova, Milena; Castellino, Flora; Nicholson, Karl; Giudice, Giuseppe Del; Golding, Hana

    2014-01-01

    In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions. PMID:24755693

  16. Heterologous prime-boost vaccination with MF59-adjuvanted H5 vaccines promotes antibody affinity maturation towards the hemagglutinin HA1 domain and broad H5N1 cross-clade neutralization.

    PubMed

    Khurana, Surender; Coyle, Elizabeth M; Dimitrova, Milena; Castellino, Flora; Nicholson, Karl; Del Giudice, Giuseppe; Golding, Hana

    2014-01-01

    In an open label clinical study (2007), MF59-adjuvanted hemagglutinin (HA) vaccine from H5N1-A/Vietnam/1194/2004 (clade 1) was administered to subjects previously vaccinated (primed) with clade 0 H5N3 (A/duck/Singapore/97) vaccine at least 6 years earlier (in 1999 or 2001). The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR). Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity) of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2) and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.

  17. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

  18. Antigen-antibody interactions and structural flexibility of a femtomolar-affinity antibody.

    PubMed

    Fukunishi, Hiroaki; Shimada, Jiro; Shiraishi, Kenji

    2012-03-27

    The femtomolar-affinity mutant antibody (4M5.3) generated by directed evolution is interesting because of the potential of antibody engineering. In this study, the mutant and its wild type (4-4-20) were compared in terms of antigen-antibody interactions and structural flexibility to elucidate the effects of directed evolution. For this purpose, multiple steered molecular dynamics (SMD) simulations were performed. The pulling forces of SMD simulations elucidated the regions that form strong attractive interactions in the binding pocket. Structural analysis in these regions showed two important mutations for improving attractive interactions. First, mutation of Tyr102(H) to Ser (sequence numbering of Protein Data Bank entry 1FLR ) played a role in resolving the steric hindrance on the pathway of the antigen in the binding pocket. Second, mutation of Asp31(H) to His played a role in resolving electrostatic repulsion. Potentials of mean force (PMFs) of both the wild type and the mutant showed landscapes that do not include obvious intermediate states and go directly to the bound state. These landscapes were regarded as funnel-like binding free energy landscapes. Furthermore, the structural flexibility based on the fluctuations of the positions of atoms was analyzed. It was shown that the fluctuations in the positions of the antigen and residues in contact with antigen tend to be smaller in the mutant than in the wild type. This result suggested that structural flexibility decreases as affinity is improved by directed evolution. This suggestion is similar to the relationship between affinity and flexibility for in vivo affinity maturation, which was suggested by Romesberg and co-workers [Jimenez, R., et al. (2003) Proc. Natl. Acad. Sci. U.S.A.100, 92-97]. Consequently, the relationship was found to be applicable up to femotomolar affinity levels.

  19. 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.

  20. The assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach.

    PubMed

    Ferreira, M U; Katzin, A M

    1995-12-01

    We describe a simple dose-response approach to assess the affinity distribution of polyclonal antibodies. The proportion of antigen-specific antibodies dissociated by increasing concentrations of the mild chaotropic agent ammonium thiocyanate (NH4SCN) was measured by enzyme immunoassay, and the distribution of tolerances to this agent was presented in a histogram form. Such 'tolerance distribution', which is analogous to that described in classical dose-response bioassays, is proposed as a representation of the actual antibody affinity distribution. To test this approach, we assessed affinity maturation patterns of anti-Plasmodium falciparum IgG antibodies in paired sera obtained from 22 malaria patients during the acute infection and convalescence. We obtained patterns of antibody affinity distributions consistent with those previously described in immunization experiments with the aid of more complex laboratory and computational approaches. Therefore, we suggest the thiocyanate elution technique as an alternative method for rapid assessment of affinity distributions of polyclonal antibodies elicited against complex antigens, readily applicable to large number of serum samples.

  1. Off-rate screening for selection of high-affinity anti-drug antibodies.

    PubMed

    Ylera, Francisco; Harth, Stefan; Waldherr, Dirk; Frisch, Christian; Knappik, Achim

    2013-10-15

    The rapidly increasing number of therapeutic antibodies in clinical development and on the market requires corresponding detection reagents for monitoring the concentration of these drugs in patient samples and as positive controls for measurement of anti-drug antibodies. Phage display of large recombinant antibody libraries has been shown to enable the rapid development of fully human anti-idiotypic antibodies binding specifically to antibody drugs, since the in vitro panning approach allows for incorporation of suitable blockers to drive selection toward the paratope of the drug. A typical bottleneck in antibody generation projects is ranking of the many candidates obtained after panning on the basis of antibody binding strength. Ideally, such method will work without prior labeling of antigens and with crude bacterial lysates. We developed an off-rate screening method of crude Escherichia coli lysates containing monovalent Fab fragments obtained after phage display of the HuCAL PLATINUM® antibody library. We used the antibody drugs trastuzumab and cetuximab as antigen examples. Using the Octet® RED384 label-free sensor instrument we show that antibody off rates can be reliably determined in crude bacterial lysates with high throughput. We also demonstrate that the method can be applied to screening for high-affinity antibodies typically obtained after affinity maturation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Arginine mutations in antibody complementarity-determining regions display context-dependent affinity/specificity trade-offs.

    PubMed

    Tiller, Kathryn E; Li, Lijuan; Kumar, Sandeep; Julian, Mark C; Garde, Shekhar; Tessier, Peter M

    2017-08-04

    Antibodies commonly accumulate charged mutations in their complementarity-determining regions (CDRs) during affinity maturation to enhance electrostatic interactions. However, charged mutations can mediate non-specific interactions and it is unclear to what extent CDRs can accumulate charged residues to increase antibody affinity without compromising specificity. This is especially concerning for positively charged CDR mutations that are linked to antibody polyspecificity. To better understand antibody affinity/specificity trade-offs, we have selected single-chain antibody fragments specific for the negatively charged and hydrophobic Alzheimer's Aβ peptide using weak and stringent selections for antibody specificity. Antibody variants isolated using weak selections for specificity were enriched in arginine CDR mutations and displayed low specificity. Alanine scanning mutagenesis revealed that the affinities of these antibodies were strongly dependent on their arginine mutations. Antibody variants isolated using stringent selections for specificity were also enriched in arginine CDR mutations, but these antibodies possessed significant improvements in specificity. Importantly, the affinities of the most specific antibodies were much less dependent on their arginine mutations, suggesting that overreliance on arginine for affinity leads to reduced specificity. Structural modeling and molecular simulations reveal unique hydrophobic environments near the arginine CDR mutations. The more specific antibodies contained arginine mutations in the most hydrophobic portions of the CDRs, while the less specific antibodies contained arginine mutations in more hydrophilic regions. These findings demonstrate that arginine mutations in antibody CDRs display context-dependent impacts on specificity and that affinity/specificity trade-offs are governed by the relative contribution of arginine CDR residues to the overall antibody affinity. Copyright © 2017, The American Society for

  3. 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

  4. 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 Central

    Groves, Maria AT; 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. PMID:24256948

  5. The genetic control of antibody affinity in mice.

    PubMed Central

    Katz, F E; Steward, M W

    1975-01-01

    Random-bred TO mice have been selectively bred into two lines on the basis of the relative affinity (KR) of antibody produced to protein antigens, one line producing high and the other low KR antibody. After four generations of selective breeding the difference in KR between the two lines was highly significant (P less than 0-001). The selection on the basis of KR did not result in a corresponding selection for antibody levels (Abt), which were not significantly different in the two lines. These results indicate that antibody affinity is a genetically controlled parameter of the immune response. Furthermore, this control appears to be expressed by a mechanism which is independent of the amount of antibody produced. PMID:1165110

  6. MF59 Adjuvant Enhances Diversity and Affinity of Antibody-Mediated Immune Response to Pandemic Influenza Vaccines

    PubMed Central

    Khurana, Surender; Verma, Nitin; Yewdell, Jonathan W.; Hilbert, Anne Katrin; Castellino, Flora; Lattanzi, Maria; Del Giudice, Giuseppe; Rappuoli, Rino; Golding, Hana

    2012-01-01

    Oil-in-water adjuvants have been shown to improve immune responses against pandemic influenza vaccines as well as reduce the effective vaccine dose, increasing the number of doses available to meet global vaccine demand. Here, we use genome fragment phage display libraries and surface plasmon resonance to elucidate the effects of MF59 on the quantity, diversity, specificity, and affinity maturation of human antibody responses to the swine-origin H1N1 vaccine in different age groups. In adults and children, MF59 selectively enhanced antibody responses to the hemagglutinin 1 (HA1) globular head relative to the more conserved HA2 domain in terms of increased antibody titers as well as a more diverse antibody epitope repertoire. Antibody affinity, as inferred by greatly diminished (≥10-fold) off-rate constants, was significantly increased in toddlers and children who received the MF59-adjuvanted vaccine. Moreover, MF59 also improved antibody affinity maturation after each sequential vaccination against avian H5N1 in adults. For both pandemic influenza vaccines, there was a close correlation between serum antibody affinity and virus-neutralizing capacity. Thus, MF59 quantitatively and qualitatively enhances functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity, demonstrating the added value of such adjuvants for influenza vaccines. PMID:21632986

  7. MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines.

    PubMed

    Khurana, Surender; Verma, Nitin; Yewdell, Jonathan W; Hilbert, Anne Katrin; Castellino, Flora; Lattanzi, Maria; Del Giudice, Giuseppe; Rappuoli, Rino; Golding, Hana

    2011-06-01

    Oil-in-water adjuvants have been shown to improve immune responses against pandemic influenza vaccines as well as reduce the effective vaccine dose, increasing the number of doses available to meet global vaccine demand. Here, we use genome fragment phage display libraries and surface plasmon resonance to elucidate the effects of MF59 on the quantity, diversity, specificity, and affinity maturation of human antibody responses to the swine-origin H1N1 vaccine in different age groups. In adults and children, MF59 selectively enhanced antibody responses to the hemagglutinin 1 (HA1) globular head relative to the more conserved HA2 domain in terms of increased antibody titers as well as a more diverse antibody epitope repertoire. Antibody affinity, as inferred by greatly diminished (≥10-fold) off-rate constants, was significantly increased in toddlers and children who received the MF59-adjuvanted vaccine. Moreover, MF59 also improved antibody affinity maturation after each sequential vaccination against avian H5N1 in adults. For both pandemic influenza vaccines, there was a close correlation between serum antibody affinity and virus-neutralizing capacity. Thus, MF59 quantitatively and qualitatively enhances functional antibody responses to HA-based vaccines by improving both epitope breadth and binding affinity, demonstrating the added value of such adjuvants for influenza vaccines.

  8. First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

    PubMed

    Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F

    2006-02-07

    The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

  9. 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

  10. 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.

  11. The genetic control of antibody affinity. Evidence from breeding studies with mice selectively bred for either high or low affinity antibody production.

    PubMed Central

    Steward, M W; Reinhardt, M C; Staines, N A

    1979-01-01

    The genetic control of antibody affinity has been studied in mice selectively bred on the basis of the affinity of antibody they produce to protein antigens injected in saline. Two lines of mice have been obtained, one producing predominantly high and the other predominantly low affinity antibody. Breeding experiments have been performed with these two lines after ten generations of selection and the level and affinity of antibody to protein measured in parents, F1 hybrids and backcross offspring. The results indicate that antibody affinity is a genetically controlled parameter of the immune response and that this control is exerted independently of that controlling antibody levels. Furthermore, high and low affinity line mice have been typed for major histocompatibility complex antigens and the results show that the two lines are not significantly different. This therefore suggests that genes controlling antibody affinity are not linked to the major histocompatibility locus. PMID:500124

  12. 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

  13. 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.

  14. 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.

  15. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

    PubMed Central

    Goenka, Radhika; Matthews, Andrew H.; Zhang, Bochao; O’Neill, Patrick J.; Scholz, Jean L.; Migone, Thi-Sau; Leonard, Warren J.; Stohl, William; Hershberg, Uri

    2014-01-01

    We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. PMID:24367004

  16. Selection and affinity maturation of IgNAR variable domains targeting Plasmodium falciparum AMA1.

    PubMed

    Nuttall, Stewart D; Humberstone, Karen S; Krishnan, Usha V; Carmichael, Jennifer A; Doughty, Larissa; Hattarki, Meghan; Coley, Andrew M; Casey, Joanne L; Anders, Robin F; Foley, Michael; Irving, Robert A; Hudson, Peter J

    2004-04-01

    The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.

  17. High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

    PubMed Central

    Kaur, Kaval; Zheng, Nai-Ying; Smith, Kenneth; Huang, Min; Li, Lie; Pauli, Noel T.; Henry Dunand, Carole J.; Lee, Jane-Hwei; Morrissey, Michael; Wu, Yixuan; Joachims, Michelle L.; Munroe, Melissa E.; Lau, Denise; Qu, Xinyan; Krammer, Florian; Wrammert, Jens; Palese, Peter; Ahmed, Rafi; James, Judith A.; Wilson, Patrick C.

    2015-01-01

    Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus. PMID:25951191

  18. DNA priming prior to inactivated influenza A(H5N1) vaccination expands the antibody epitope repertoire and increases affinity maturation in a boost-interval-dependent manner in adults.

    PubMed

    Khurana, Surender; Wu, Jian; Dimitrova, Milena; King, Lisa R; Manischewitz, Jody; Graham, Barney S; Ledgerwood, Julie E; Golding, Hana

    2013-08-01

    DNA priming improves the response to inactivated influenza A(H5N1) vaccination. We compared the immunogenicity of an H5 DNA prime (using strain A/Indonesia/5/2005) followed by an H5N1 monovalent inactivated vaccine boost at 4, 8, 12, 16, or 24 weeks to that of 2 doses of H5N1 monovalent inactivated vaccine in adults. Antibody epitope repertoires were elucidated by genome-fragment phage-display library analysis, and antibody avidities for HA1 and HA2 domains were measured by surface plasmon resonance. H5 DNA priming expanded the H5-specific antibody epitope repertoire and enhanced antibody avidity to the HA1 (but not the HA2) domain in an interval-dependent manner. Enhanced HA1 binding and avidity after an interval of ≥12 weeks between prime and boost correlated with improved neutralization of homologous and heterologous H5N1 strains. Clinical trials registration NCT01086657.

  19. Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding.

    PubMed

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

    2007-03-23

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

  20. Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

    SciTech Connect

    Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

    2007-07-13

    Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

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

    PubMed

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

    2012-09-01

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

  2. 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.

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

    PubMed Central

    Steward, M W; Petty, R E

    1976-01-01

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

  4. Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders.

    PubMed

    Hu, Francis Jingxin; Volk, Anna-Luisa; Persson, Helena; Säll, Anna; Borrebaeck, Carl; Uhlen, Mathias; Rockberg, Johan

    2017-08-01

    Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Streamlining the Pipeline for Generation of Recombinant Affinity Reagents by Integrating the Affinity Maturation Step

    PubMed Central

    Huang, Renhua; Gorman, Kevin T.; Vinci, Chris R.; Dobrovetsky, Elena; Gräslund, Susanne; Kay, Brian K.

    2015-01-01

    Often when generating recombinant affinity reagents to a target, one singles out an individual binder, constructs a secondary library of variants, and affinity selects a tighter or more specific binder. To enhance the throughput of this general approach, we have developed a more integrated strategy where the “affinity maturation” step is part of the phage-display pipeline, rather than a follow-on process. In our new schema, we perform two rounds of affinity selection, followed by error-prone PCR on the pools of recovered clones, generation of secondary libraries, and three additional rounds of affinity selection, under conditions of off-rate competition. We demonstrate the utility of this approach by generating low nanomolar fibronectin type III (FN3) monobodies to five human proteins: ubiquitin-conjugating enzyme E2 R1 (CDC34), COP9 signalosome complex subunit 5 (COPS5), mitogen-activated protein kinase kinase 5 (MAP2K5), Splicing factor 3A subunit 1 (SF3A1) and ubiquitin carboxyl-terminal hydrolase 11 (USP11). The affinities of the resulting monobodies are typically in the single-digit nanomolar range. We demonstrate the utility of two binders by pulling down the targets from a spiked lysate of HeLa cells. This integrated approach should be applicable to directed evolution of any phage-displayed affinity reagent scaffold. PMID:26437402

  6. 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

  7. Affinity chromatography matures as bioinformatic and combinatorial tools develop.

    PubMed

    Clonis, Yannis D

    2006-01-06

    Affinity chromatography has the reputation of a more expensive and less robust than other types of liquid chromatography. Furthermore, the technique is considered to stand a modest chance of large-scale purification of proteinaceous pharmaceuticals. This perception is changing because of the pressure for quality protein therapeutics, and the realization that higher returns can be expected when ensuring fewer purification steps and increased product recovery. These developments necessitated a rethinking of the protein purification processes and restored the interest for affinity chromatography. This liquid chromatography technique is designed to offer high specificity, being able to safely guide protein manufactures to successfully cope with the aforementioned challenges. Affinity ligands are distinguished into synthetic and biological. These can be generated by rational design or selected from ligand libraries. Synthetic ligands are generated by three methods. The rational method features the functional approach and the structural template approach. The combinatorial method relies on the selection of ligands from a library of synthetic ligands synthesized randomly. The combined method employs both methods, that is, the ligand is selected from an intentionally biased library based on a rationally designed ligand. Biological ligands are selected by employing high-throughput biological techniques, e.g. phage- and ribosome-display for peptide and microprotein ligands, in addition to SELEX for oligonucleotide ligands. Synthetic mimodyes and chimaeric dye-ligands are usually designed by rational approaches and comprise a chloro-triazinlyl scaffold. The latter substituted with various amino acids, carbocyclic, and heterocyclic groups, generates libraries from which synthetic ligands can be selected. A 'lead' compound may help to generating a 'focused' or 'biased' library. This can be designed by various approaches, e.g.: (i) using a natural ligand-protein complex as a

  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. 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.

  10. 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

  11. Quantifying evolutionary constraints on B-cell affinity maturation

    PubMed Central

    McCoy, Connor O.; Bedford, Trevor; Minin, Vladimir N.; Bradley, Philip; Robins, Harlan; Matsen, Frederick A.

    2015-01-01

    The antibody repertoire of each individual is continuously updated by the evolutionary process of B-cell receptor (BCR) mutation and selection. It has recently become possible to gain detailed information concerning this process through high-throughput sequencing. Here, we develop modern statistical molecular evolution methods for the analysis of B-cell sequence data, and then apply them to a very deep short-read dataset of BCRs. We find that the substitution process is conserved across individuals but varies significantly across gene segments. We investigate selection on BCRs using a novel method that side-steps the difficulties encountered by previous work in differentiating between selection and motif-driven mutation; this is done through stochastic mapping and empirical Bayes estimators that compare the evolution of in-frame and out-of-frame rearrangements. We use this new method to derive a per-residue map of selection, which provides a more nuanced view of the constraints on framework and variable regions. PMID:26194758

  12. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  13. Robotic QM/MM-driven maturation of antibody combining sites.

    PubMed

    Smirnov, Ivan V; Golovin, Andrey V; Chatziefthimiou, Spyros D; Stepanova, Anastasiya V; Peng, Yingjie; Zolotareva, Olga I; Belogurov, Alexey A; Kurkova, Inna N; Ponomarenko, Natalie A; Wilmanns, Matthias; Blackburn, G Michael; Gabibov, Alexander G; Lerner, Richard A

    2016-10-01

    In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24419156

  15. 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.

  16. Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice

    PubMed Central

    De Trez, Carl; Van Acker, Annette; Vansanten, Georgette; Urbain, Jacques; Brait, Maryse

    2002-01-01

    Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed. The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected. PMID:12885152

  17. 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

  18. Guiding the evolution to catch the virus: An in silico study of affinity maturation against rapidly mutating antigen

    NASA Astrophysics Data System (ADS)

    Wang, Shenshen; Burton, Dennis; Kardar, Mehran; Chakraborty, Arup

    2014-03-01

    The immune system comprises an intricate and evolving collection of cells and molecules that enables a defense against pathogenic agents. Its workings present a rich source of physical problems that impact human health. One intriguing example is the process of affinity maturation (AM) through which an antibody (Ab)--a component of the host immune system--evolves to more efficiently bind an antigen (Ag)--a unique part of a foreign pathogen such as a virus. Sufficiently strong binding to the Ag enables recognition and neutralization. A major challenge is to contain a diversifying mixture of Ag variants, that arise in natural infection, from evading Ab neutralization. This entails a thorough understanding of AM against multiple Ag species and mutating Ag. During AM, Ab-encoding cells undergo cycles of mutation and selection, a process reminiscent of Darwinian evolution yet occurring in real time. We first cast affinity-dependent selection into an extreme value problem and show how the binding characteristics scale with Ag diversity. We then develop an agent-based residue-resolved computational model of AM which allows us to track the evolutionary trajectories of individual cells. This dynamic model not only reveals significant stochastic effects associated with the relatively small and highly dynamic population size, it also uncovers the markedly distinct maturation outcomes if designed Ag variants are presented in different temporal procedures. Insights thus obtained would guide rational design of vaccination protocols.

  19. 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.

  20. 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").

  1. 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.

  2. Robotic QM/MM-driven maturation of antibody combining sites

    PubMed Central

    Smirnov, Ivan V.; Golovin, Andrey V.; Chatziefthimiou, Spyros D.; Stepanova, Anastasiya V.; Peng, Yingjie; Zolotareva, Olga I.; Belogurov, Alexey A.; Kurkova, Inna N.; Ponomarenko, Natalie A.; Wilmanns, Matthias; Blackburn, G. Michael; Gabibov, Alexander G.; Lerner, Richard A.

    2016-01-01

    In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis. PMID:27774510

  3. 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

  4. 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.

  5. What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination? Breaking through Immunity's Glass Ceiling.

    PubMed

    Kelsoe, Garnett; Haynes, Barton F

    2017-06-19

    A key goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs) targeted to the vulnerable regions of the HIV envelope. BnAbs develop over time in ∼50% of HIV-1-infected individuals. However, to date, no vaccines have induced bnAbs and few or none of these vaccine-elicited HIV-1 antibodies carry the high frequencies of V(D)J mutations characteristic of bnAbs. Do the high frequencies of mutations characteristic of naturally induced bnAbs represent a fundamental barrier to the induction of bnAbs by vaccines? Recent studies suggest that high frequencies of V(D)J mutations can be achieved by serial vaccination strategies. Rather, it appears that, in the absence of HIV-1 infection, physiologic immune tolerance controls, including a germinal center process termed affinity reversion, may limit vaccine-driven bnAb development by clonal elimination or selecting for mutations incompatible with bnAb activity. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  6. 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.

  7. 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.

  8. 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.

  9. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  10. 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

  11. 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.

  12. 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

  13. Suppression of neuro inflammation in experimental autoimmune encephalomyelitis by glia maturation factor antibody.

    PubMed

    Zaheer, Smita; Wu, Yanghong; Sahu, Shailendra K; Zaheer, Asgar

    2011-02-10

    Glia maturation factor (GMF), a protein primarily localized in the central nervous system (CNS) was isolated, sequenced and cloned in our laboratory. We previously demonstrated that GMF mediates the experimental autoimmune encephalomyelitis (EAE)-induced production of pro-inflammatory cytokines and chemokines in the central nervous system of mice. In the present study we show that immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) caused an early onset (days 7-9 post immunization) and severe EAE with a mean peak score of 3.5 ± 0.5 in mice. Neutralization of GMF with four injections of anti-GMF antibody 5 to 11 days post immunization delayed the time of onset (days 12-14 post immunization) and significantly reduced the severity of EAE (mean peak score of 1.5 ± 0.4). Consistent with these clinical scores, histological examination of the CNS of these mice revealed profound differences between GMF-antibody treated mice and isotype matched control-antibody treated mice. Histological analysis of the spinal cord and brain showed severe inflammation and demyelination in the control antibody-treated mice whereas significantly reduced inflammation and demyelination was detected in GMF-antibody-treated mice at days 8, 16, and 24 post immunization. The decreased incidence and reduced severity of EAE in GMF-antibody-treated mice was consistent with the significantly reduced expressions of pro-inflammatory cytokines and chemokines. Our overall results demonstrate that neutralization of endogenous GMF with an affinity purified GMF antibody significantly decreased the inflammation, severity and progression of immunization induced active, passive and relapsing-remitting EAE. Treatment of mice with isotype-matched control antibody did not have any effect on EAE. Taken together, these results demonstrate the critical role of GMF in EAE, and GMF antibody as a potent anti-inflammatory therapeutic agent for effectively suppressing EAE in mouse models of

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

    PubMed

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

    2016-12-30

    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.

  15. Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies.

    PubMed

    Mudgett-Hunter, M; Anderson, W; Haber, E; Margolies, M N

    1985-04-01

    High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  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. A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus

    PubMed Central

    Frank, Gregory M.; Ince, William L.; Gibbs, James S.; Khurana, Surender; Wheatley, Adam K.; Max, Edward E.; McDermott, Adrian B.; Golding, Hana; Stevens, James; Bennink, Jack R.

    2015-01-01

    ABSTRACT Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. PMID:26242629

  5. 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

  6. Affinity-based methodologies and ligands for antibody purification: advances and perspectives.

    PubMed

    Roque, Ana C A; Silva, Cláudia S O; Taipa, M Angela

    2007-08-10

    Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification "paradigm" still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

  7. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    SciTech Connect

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  8. Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications.

    PubMed

    Gonzalez-Sapienza, Gualberto; Rossotti, Martín A; Tabares-da Rosa, Sofía

    2017-01-01

    With just three CDRs in their variable domains, the antigen-binding site of camelid heavy-chain-only antibodies (HcAbs) has a more limited structural diversity than that of conventional antibodies. Even so, this does not seem to limit their specificity and high affinity as HcAbs against a broad range of structurally diverse antigens have been reported. The recombinant form of their variable domain [nanobody (Nb)] has outstanding properties that make Nbs, not just an alternative option to conventional antibodies, but in many cases, these properties allow them to reach analytical or diagnostic performances that cannot be accomplished with conventional antibodies. These attributes include comprehensive representation of the immune specificity in display libraries, easy adaptation to high-throughput screening, exceptional stability, minimal size, and versatility as affinity building block. Here, we critically reviewed each of these properties and highlight their relevance with regard to recent developments in different fields of immunosensing applications.

  9. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies

    PubMed Central

    Hjelm, Barbara; Forsström, Björn; Igel, Ulrika; Johannesson, Henrik; Stadler, Charlotte; Lundberg, Emma; Ponten, Fredrik; Sjöberg, Anna; Rockberg, Johan; Schwenk, Jochen M; Nilsson, Peter; Johansson, Christine; Uhlén, Mathias

    2011-01-01

    A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies. PMID:21898641

  10. 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.

  11. 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

  12. Preparation and characterization of a monoclonal antibody with specific affinity for pyrimidine dimers in RNA

    SciTech Connect

    Phillips, R.G.

    1986-01-01

    A monoclonal antibody was prepared which shows specific affinity for pyrimidine dimers in RNA. The antibody meets three criteria for pyrimidine dimer-dependent affinity. (1) Antibody binding is dependent upon UV irradiation of nucleic acids. Irradiation both at 313 nm in the presence of a sensitizer and at 270 nm without sensitizer promotes antibody binding to RNA and polyribonucleotides. (2) Antibody binding is dependent upon the presence of adjacent pyrimidine nucleotides. Irradiated polyuridylic acid and polycytidylic acid are bound while irradiated polyadenylic acid and polyguanidylic acid are not bound. An alternating polymer of adenylic and uridylic acids is not bound regardless of irradiation. (3) The antigenic determinants are reduced in number by exposure to UV radiation of short wavelength (240 nm). Antibody binding increases with increasing length of irradiated oligonucleotides. The antibody binds irradiated oligonucleotides which contain a single pair of pyrimidines surrounded by purines. An oligonucleotide with a terminal pyrimidine pair is bound to a much lower degree. I propose that the minimal determinant is a pyrimidine dimer with at least one adjacent nucleotide on either side.

  13. 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.

  14. 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.

  15. Cross-reactivity of Aspergillus, Penicillium, and Stachybotrys antigens using affinity-purified antibodies and immunoassay.

    PubMed

    Vojdani, Aristo

    2004-05-01

    In this study, the author examined the cross-reactivities of Stachybotrys chartarum, Aspergillus niger/fumigatus, and Penicillium notatum with affinity-purified rabbit sera. The molds were grown for expression of maximum numbers of antigens, after which they were extracted and mixed with commercially available extracts. The mixture was used for antibody preparation in rabbits, measurement of antibody levels, and for the demonstration of the degree of cross-reactivity. Control rabbits were injected with saline, yet they produced significant levels of immunoglobulin G antibodies against all mold extracts tested. The author interpreted this result to mean that sera obtained from rabbits immunized with pure mold extracts likely reflected cross-reactivity with other molds. Therefore, only affinity-purified antibodies and the most sensitive immunoassay technique (i.e., enzyme-linked immunosorbent assay [ELISA]) were used for the cross-inhibition studies. The antigenic cross-reactivities were as follows: (a) between Aspergillus and Penicillium, 19.6-21.0%; (b) between Stachybotrys and Aspergillus, 8.2-8.7%; and (c) between Stachybotrys and Penicillium, 7.0-9.6%. The findings of this study demonstrate that cross-reactivity studies between different molds require the use of affinity-purified antibodies and a sensitive and quantitative assay with untreated antigens. With the use of such an assay, it was determined that the cross-reactivity between Stachybotrys, Aspergillus, and Penicillium was at approximately 10%, which is less widespread than previously believed.

  16. Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum.

    PubMed

    Dooley, Helen; Flajnik, Martin F

    2005-03-01

    The cartilaginous fish are the oldest phylogenetic group in which all of the molecular components of the adaptive immune system have been found. Although early studies clearly showed that sharks could produce an IgM-based response following immunization, evidence for memory, affinity maturation and roles for the other isotypes (notably IgNAR) in this group remained inconclusive. The data presented here illustrate that the nurse shark (Ginglymostoma cirratum) is able to produce not only an IgM response, but we also show for the first time a highly antigen-specific IgNAR response. Additionally, under appropriate conditions, a memory response for both isotypes can be elicited. Analysis of the response shows differential expression of pentameric and monomeric IgM. Pentameric IgM provides the 'first line of defense' through high-avidity, low-affinity interaction with antigen. In contrast, monomeric IgM and IgNAR seem responsible for the specific, antigen-driven response. We propose the presence of distinct lineages of B cells in sharks. As there is no conventional isotype switching, each lineage seems pre-determined to express a single isotype (IgM versus IgNAR). However, our data suggest that there may also be specific lineages for the different forms (pentameric versus monomeric) of the IgM isotype.

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

    PubMed

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

    2015-10-01

    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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  18. Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

    PubMed Central

    Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

    2013-01-01

    Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

  19. 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

  20. 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.

  1. [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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

    PubMed

    Rahbarizadeh, F; Rasaee, M J; Madani, R; Rahbarizadeh, M H; Omidfar, K

    2000-10-01

    A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.

  8. Affinity chromatography of a binder of 1-methyladenine, the maturation-inducing hormone for starfish oocytes.

    PubMed

    Toraya, Tetsuo; Kuyama, Atsushi; Tanaka, Seiichi; Yamamoto, Masatoyo; Ohmiya, Tadamasa; Saito, Yuri; Tanabe, Tomoko

    2017-05-13

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary. They resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the maturation-inducing hormone for starfish oocytes. Putative 1-MeAde receptors have been suggested to be present on the oocyte surface, but not yet been characterized biochemically. As reported recently (T. Toraya, T. Kida, A. Kuyama, S. Matsuda, S. Tanaka, Y. Komatsu, T. Tsurukai, Biochem. Biophys. Res. Commun. 485 (2017) 41-46), it became possible to detect unknown 1-MeAde binders of starfish oocytes by immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection. We designed and synthesized water-soluble and insoluble polymer-bound 1-MeAde derivatives. A water-soluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to dextran through an N(6)-substituent, triggered the germinal-vesicle breakdown toward follicle-free oocytes, dejellied oocytes, and denuded oocytes. This is consistent with the idea that putative 1-MeAde receptors are located on the cell surface of starfish oocytes. A water-insoluble polymer-bound 1-MeAde derivative, in which 1-MeAde is bound to Sepharose 4B through an N(6)-substituent, served as an effective affinity adsorbent for the partial purification of a 1-MeAde binder with Mr of 47.5 K that might be a possible candidate of the maturation-inducing hormone receptors of starfish oocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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.

  10. 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.

  11. Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation.

    PubMed

    Wichert, Moreno; Krall, Nikolaus; Decurtins, Willy; Franzini, Raphael M; Pretto, Francesca; Schneider, Petra; Neri, Dario; Scheuermann, Jörg

    2015-03-01

    In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5' and 3' ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

  12. Dual-display of small molecules enables the discovery of ligand pairs and facilitates affinity maturation

    NASA Astrophysics Data System (ADS)

    Wichert, Moreno; Krall, Nikolaus; Decurtins, Willy; Franzini, Raphael M.; Pretto, Francesca; Schneider, Petra; Neri, Dario; Scheuermann, Jörg

    2015-03-01

    In contrast to standard fragment-based drug discovery approaches, dual-display DNA-encoded chemical libraries have the potential to identify fragment pairs that bind simultaneously and benefit from the chelate effect. However, the technology has been limited by the difficulty in unambiguously decoding the ligand pairs from large combinatorial libraries. Here we report a strategy that overcomes this limitation and enables the efficient identification of ligand pairs that bind to a target protein. Small organic molecules were conjugated to the 5‧ and 3‧ ends of complementary DNA strands that contain a unique identifying code. DNA hybridization followed by an inter-strand code-transfer created a stable dual-display DNA-encoded chemical library of 111,100 members. Using this approach we report the discovery of a low micromolar binder to alpha-1-acid glycoprotein and the affinity maturation of a ligand to carbonic anhydrase IX, an established marker of renal cell carcinoma. The newly discovered subnanomolar carbonic anhydrase IX binder dramatically improved tumour targeting performance in vivo.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. BIOINTERACTION ANALYSIS BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY: KINETIC STUDIES OF IMMOBILIZED ANTIBODIES

    PubMed Central

    Nelson, Mary Anne; Moser, Annette; Hage, David S.

    2009-01-01

    A system based on high-performance affinity chromatography was developed for characterizing the binding, elution and regeneration kinetics of immobilized antibodies and immunoaffinity supports. This information was provided by using a combination of frontal analysis, split-peak analysis and peak decay analysis to determine the rate constants for antibody-antigen interactions under typical sample application and elution conditions. This technique was tested using immunoaffinity supports that contained monoclonal antibodies for 2,4-dichlorophenoxyacetic acid (2,4-D). Association equilibrium constants measured by frontal analysis for 2,4-D and related compounds with the immobilized antibodies were 1.7–12 × 106 M−1 at pH 7.0 and 25°C. Split-peak analysis gave association rate constants of 1.4–12 × 105 M−1s−1 and calculated dissociation rate constants of 0.01–0.4 s−1 under the application conditions. Elution at pH 2.5 for the analytes from the antibodies was examined by peak decay analysis and gave dissociation rate constants of 0.056–0.17 s−1. A comparison of frontal analysis results after various periods of column regeneration allowed the rate of antibody regeneration to be examined, with the results giving a first-order regeneration rate constant of 2.4 × 10−4 s−1. This combined approach and the information it provides should be useful in the design and optimization of immunoaffinity chromatography and other analytical methods that employ immobilized antibodies. The methods described are not limited to the particular analytes and antibodies employed in this study but should be useful in characterizing other targets, ligands and supports. PMID:19394281

  2. 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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Site-specific photocoupling of pBpa mutated scFv antibodies for use in affinity proteomics.

    PubMed

    Brofelth, Mattias; Städe, Lars Wagner; Ekstrand, Anna Isinger; Edfeldt, Linn Petersson; Kovačič, Rebeka; Nielsen, Thorbjørn Terndrup; Larsen, Kim Lambertsen; Duroux, Laurent; Wingren, Christer

    2017-08-01

    Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics. Copyright © 2017. Published by Elsevier B.V.

  4. Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling

    PubMed Central

    Riese, Sebastian B; Kuehne, Christian; Tedder, Thomas F; Hallmann, Rupert; Hohenester, Erhard; Buscher, Konrad

    2013-01-01

    Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and EGF-like domains. Conformation-specific antibodies have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. Here we show that the anti-human L-selectin monoclonal antibodies DREG-55 and LAM1.5 but not DREG-56, -200 or LAM1.1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin antibodies and introduce the new concept of heterotropic modulation of receptor function. PMID:24431230

  5. Oligonucleotide Hybridization Combined with Competitive Antibody Binding for the Truncation of a High-Affinity Aptamer.

    PubMed

    Vu, Cong Quang; Rotkrua, Pichayanoot; Tantirungrotechai, Yuthana; Soontornworajit, Boonchoy

    2017-10-09

    Truncation can enhance the affinity of aptamers for their targets by limiting nonessential segments and therefore limiting the molecular degrees of freedom that must be overcome in the binding process. This study demonstrated a truncation protocol relying on competitive antibody binding and the hybridization of complementary oligonucleotides, using platelet derived growth factor BB (PDGF-BB) as the model target. On the basis of the immunoassay results, an initial long aptamer was truncated to a number of sequences with lengths of 36-40 nucleotides (nt). These sequences showed apparent KD values in the picomolar range, with the best case being a 36-nt truncated aptamer with a 150-fold increase in affinity over the full-length aptamer. The observed binding energies correlated well with relative energies calculated by molecular dynamics simulations. The effect of the truncated aptamer on PDGF-BB-stimulated fibroblasts was found to be equivalent to that of the full-length aptamer.

  6. 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.

  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. De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.

    PubMed

    Shi, Lei; Wheeler, John C; Sweet, Raymond W; Lu, Jin; Luo, Jinquan; Tornetta, Mark; Whitaker, Brian; Reddy, Ramachandra; Brittingham, Raymond; Borozdina, Lina; Chen, Qiang; Amegadzie, Bernard; Knight, David M; Almagro, Juan C; Tsui, Ping

    2010-03-26

    Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.

  9. Antibody Maturation in Women Who Acquire HIV Infection While Using Antiretroviral Preexposure Prophylaxis.

    PubMed

    Laeyendecker, Oliver; Redd, Andrew D; Nason, Martha; Longosz, Andrew F; Karim, Quarraisha Abdool; Naranbhai, Vivek; Garrett, Nigel; Eshleman, Susan H; Abdool Karim, Salim S; Quinn, Thomas C

    2015-09-01

    The CAPRISA 004 preexposure prophylaxis (PrEP) randomized trial demonstrated that women who used a vaginal gel containing the antiretroviral drug tenofovir (TFV) had a 39% lower risk of acquiring human immunodeficiency virus (HIV). It is not known whether topical TFV alters the antibody response to breakthrough HIV infection. In this study, antibody maturation was evaluated using 3 serologic assays: the BED capture enzyme immunoassay (CEIA), the Bio-Plex (Luminex) assay, and the Bio-Rad avidity assay. Tests were performed using serum samples collected 3, 6, 9, 12, 24, 36, 48, and >48 months after seroconversion from 95 women in the CAPRISA 004 trial (35 in the TFV gel arm and 60 in the placebo arm). For the BED CEIA and Luminex assay, linear mixed effects models were used to examine test results by study arm. Cox proportional hazard analysis was used to examine time to avidity cutoff. Anti-HIV antibody titers did not differ between study arms. Women assigned to TFV gel demonstrated slower antibody avidity maturation, as determined by the Bio-Rad (P = .04) and gp120 Bio-Plex (P = .028) assays. Women who were assigned to receive topical TFV but became infected had slower antibody avidity maturation, with potential implications for diagnosis and antibody-based incidence assays as access to antiretroviral therapy-based PrEP is increased.

  10. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    PubMed Central

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD <10−8 M), a new challenge arises: to measure these values accurately. Isothermal titration calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  11. 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.

  12. Maturation of human B lymphocytes--studies with a panel of monoclonal antibodies against membrane antigens.

    PubMed Central

    Zola, H; McNamara, P J; Moore, H A; Smart, I J; Brooks, D A; Beckman, I G; Bradley, J

    1983-01-01

    The expression of six different membrane markers by cells of the human B lymphocyte lineage has been studied, using monoclonal antibodies. B cells representing various stages of differentiation/maturation have been examined, using normal cells, leukaemia cells, and continuous cell lines. The expression of the six markers has been compared with maturation stages defined by immunoglobulin expression. The HLA/beta 2-microglobulin complex is present throughout the B cell lineage, whilst the Ia (p28,33) marker is present from the earliest stage that can be attributed to the B lineage, but is lost during plasma cell differentiation. A marker detected by monoclonal antibody FMC 1 is present only on mature B lymphocytes, being absent from pre-B cells or plasma cells. FMC 7 detects an antigen found on a relatively mature subpopulation, whereas FMC 8 detects early as well as mature B cells. FMC 3 expression is found on a proportion of cells at any maturation stage, suggesting that expression of this marker is controlled by factors unrelated to maturation. Images Fig. 1 PMID:6191892

  13. 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.

  14. 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

  15. 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.

  16. 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

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

    PubMed

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

    2017-01-09

    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.

  18. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    PubMed Central

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  19. Parallel kinetic analysis and affinity determination of hundreds of monoclonal antibodies using the ProteOn XPR36.

    PubMed

    Nahshol, Oded; Bronner, Vered; Notcovich, Ariel; Rubrecht, Laetitia; Laune, Daniel; Bravman, Tsafrir

    2008-12-01

    The production of antibodies for diagnostic and therapeutic applications is a major focus for biotechnology and pharmaceutical companies, and it requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. These candidates must have very high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR 36 protein interaction array system for the rapid screening and selection of high-affinity antibodies--"one-shot" kinetics. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies simultaneously in one experiment. We have used this new methodology to screen hundreds of monoclonal supernatants containing antibodies against two proteins of potential clinical interest: human interleukin 12 (IL-12) and a human hemoglobin (Hb) variant. In fact, approximately 250 supernatants raised against each antigen were screened in approximately 17 h, providing several high-affinity candidate monoclonal antibodies for each of these antigens.

  20. 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

  1. Affinity maturation of single-chain variable fragment specific for aflatoxin B(1) using yeast surface display.

    PubMed

    Min, Won-Ki; Kim, Sung-Gun; Seo, Jin-Ho

    2015-12-01

    As aflatoxin B1 is one of the most toxic mycotoxins, it is important to detect and to quantify aflatoxin B1 accurately by immunological methods. To enhance aflatoxin B1-binding affinity of the single-chain variable fragment, yeast surface display technique combined with fluorescence-activated cell sorting was applied. A randomly mutated scFv library was subjected to 4 rounds of fluorescence-activated cell sorting, resulting in isolation of 5 scFv variants showing an affinity improvement compared to the parental wild type scFv. The best scFv with a 9-fold improvement in affinity for aflatoxin B1 exhibited similar specificity to the monoclonal antibody. Most of the mutations in scFv-M37 were located outside of the canonical antigen-contact loops, suggesting that its affinity improvement might be driven by an allosteric effect inducing scFv-M37 to form a more favorable binding pocket for aflatoxin B1 than the wild type scFv.

  2. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    DOE PAGES

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; ...

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level ofmore » somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.« less

  3. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

    PubMed

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A; Wiehe, Kevin; Alam, S Munir; Bradley, Todd; Gladden, Morgan A; Hwang, Kwan-Ki; Iyengar, Sheelah; Kumar, Amit; Lu, Xiaozhi; Luo, Kan; Mangiapani, Michael C; Parks, Robert J; Song, Hongshuo; Acharya, Priyamvada; Bailer, Robert T; Cao, Allen; Druz, Aliaksandr; Georgiev, Ivelin S; Kwon, Young D; Louder, Mark K; Zhang, Baoshan; Zheng, Anqi; Hill, Brenna J; Kong, Rui; Soto, Cinque; Mullikin, James C; Douek, Daniel C; Montefiori, David C; Moody, Michael A; Shaw, George M; Hahn, Beatrice H; Kelsoe, Garnett; Hraber, Peter T; Korber, Bette T; Boyd, Scott D; Fire, Andrew Z; Kepler, Thomas B; Shapiro, Lawrence; Ward, Andrew B; Mascola, John R; Liao, Hua-Xin; Kwong, Peter D; Haynes, Barton F

    2016-04-07

    Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

  4. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    SciTech Connect

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M.  Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A.; Wiehe, Kevin; Alam, S.  Munir; Bradley, Todd; Gladden, Morgan A.; Hwang, Kwan-Ki; Iyengar, Sheelah; Kumar, Amit; Lu, Xiaozhi; Luo, Kan; Mangiapani, Michael C.; Parks, Robert J.; Song, Hongshuo; Acharya, Priyamvada; Bailer, Robert T.; Cao, Allen; Druz, Aliaksandr; Georgiev, Ivelin S.; Kwon, Young D.; Louder, Mark K.; Zhang, Baoshan; Zheng, Anqi; Hill, Brenna J.; Kong, Rui; Soto, Cinque; Mullikin, James C.; Douek, Daniel C.; Montefiori, David C.; Moody, Michael A.; Shaw, George M.; Hahn, Beatrice H.; Kelsoe, Garnett; Hraber, Peter T.; Korber, Bette T.; Boyd, Scott D.; Fire, Andrew Z.; Kepler, Thomas B.; Shapiro, Lawrence; Ward, Andrew B.; Mascola, John R.; Liao, Hua-Xin; Kwong, Peter D.; Haynes, Barton F.

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.

  5. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    DOE PAGES

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; ...

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level ofmore » somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.« less

  6. Polyclonal antibody localizes glia maturation factor beta-like immunoreactivity in neurons and glia.

    PubMed

    Wang, B R; Zaheer, A; Lim, R

    1992-09-18

    A rabbit polyclonal antibody (91-01) was raised against recombinant human glia maturation factor beta (r-hGMF-beta). The antibody did not cross-react with a number of other growth factors on ELISA test. When compared with the monoclonal antibody G2-09 previously obtained, 91-01 immunoblotted the same protein band in rat brain extract. However, unlike G2-09 which immunostained only astrocytes and Bergmann glia, 91-01 stained neurons as well. Many but not all neurons in the central and peripheral nervous system were positive for GMF-beta. The larger cell population stained by the polyclonal antibody was most likely due to its increased sensitivity, although other explanations are possible. The presence of GMF-beta-like immunoreactivity in both neurons and glia raises the possibility of a wider range of cell-cell interaction than was previously considered.

  7. Combining different design strategies for rational affinity maturation of the MICA-NKG2D interface

    PubMed Central

    Henager, Samuel H; Hale, Melissa A; Maurice, Nicholas J; Dunnington, Erin C; Swanson, Carter J; Peterson, Megan J; Ban, Joseph J; Culpepper, David J; Davies, Luke D; Sanders, Lisa K; McFarland, Benjamin J

    2012-01-01

    We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed-backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity-enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross-validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50-fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement. PMID:22761154

  8. Steroid binding by antibodies and artificial receptors: Exploration of theoretical methods to determine the origins of binding affinities and specificities

    NASA Astrophysics Data System (ADS)

    Handschuh, Sandra; Goldfuss, Bernd; Chen, Jiangang; Gasteiger, Johann; Houk, K. N.

    2000-10-01

    Binding mode calculations for complexes between an artificial paracyclophane receptor and digoxins, cholic acids as well as cortisone steroids show encapsulation of different ring combinations. Docking experiments were performed between the 26-10 antibody and digoxins. Coordination affinity arises from hydrophobic desolvation and van der Waals interactions rather than from hydrogen bonds. The specificity and affinity arises mainly from shape complementarity. Computed binding free energies and Kohonen neural network computations both point to physicochemical and structural similarities of natural antibodies and artificial receptors.

  9. A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins.

    PubMed

    Zhang, Siyan; Long, Angel; Link, A James

    2012-03-16

    The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-x(L) and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a pro-death protein to a hydrophobic cleft on the surface of the pro-survival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics. Here we apply a high-throughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-x(L) and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-x(L) and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-x(L) and increased specificity toward Bcl-x(L). Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.

  10. 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).

  11. 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).

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  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. 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.

  20. 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.

  1. Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

    PubMed

    Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Fernandez Rodriguez, Blanca M; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

    2015-11-01

    We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Affinity of HIV-1 antibody 2G12 with monosaccharides: a theoretical study based on explicit and implicit water models.

    PubMed

    Koyama, Yuka; Ueno-Noto, Kaori; Takano, Keiko

    2014-04-01

    In order to develop potential ligands to HIV-1 antibody 2G12 toward HIV-1 vaccine, binding mechanisms of the antibody 2G12 with the glycan ligand of D-mannose and D-fructose were theoretically examined. D-Fructose, whose molecular structure is slightly different from D-mannose, has experimentally shown to have stronger binding affinity to the antibody than that of D-mannose. To clarify the nature of D-fructose's higher binding affinity over D-mannose, we studied interaction between the monosaccharides and the antibody using ab initio fragment molecular orbital (FMO) method considering solvation effect as implicit model (FMO-PCM) as well as explicit water model. The calculated binding free energies of the glycans were qualitatively well consistent with the experimentally reported order of their affinities with the antibody 2G12. In addition, the FMO-PCM calculation elucidated the advantages of D-fructose over D-mannose in the solvation energy as well as the entropic contribution term obtained by MD simulations. The effects of explicit water molecules observed in the X-ray crystal structure were also scrutinized by means of FMO methods. Significant pair interaction energies among D-fructose, amino acids, and water molecules were uncovered, which indicated contributions from the water molecules to the strong binding ability of D-fructose to the antibody 2G12. These FMO calculation results of explicit water model as well as implicit water model indicated that the strong binding of D-fructose over D-mannose was due to the solvation effects on the D-fructose interaction energy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. 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.

  4. Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

    PubMed

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

    2014-01-01

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

  5. Facile Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy.

    PubMed

    Geng, Steven B; Wu, Jiemin; Alam, Magfur E; Schultz, Jason S; Dickinson, Craig D; Seminer, Carly R; Tessier, Peter M

    2016-10-19

    Protein-nanoparticle conjugates are widely used for conventional applications such as immunohistochemistry and biomolecular detection as well as emerging applications such as therapeutics and advanced materials. Nevertheless, it remains challenging to reproducibly prepare stable protein-nanoparticle conjugates with highly similar optical properties. Here we report an improved physisorption method for reproducibly preparing stable antibody-gold conjugates at acidic pH using polyclonal antibodies from a wide range of species (human, goat, rabbit, mouse, and rat). We find that gold particles synthesized using citrate alone or in combination with tannic acid are similar in size but display variable colloidal stability when conjugated to polyclonal antibodies. The variability in conjugate stability is due to differences in the pH and composition of the original gold colloid, which prevents reproducible preparation of stable antibody conjugates without additional purification of the particles prior to conjugation. Sedimentation-based purification of gold particles synthesized using different methods enabled reproducible generation of antibody-gold conjugates with high stability and similar plasmon wavelengths. We also find that antibody conjugates prepared using our improved procedure display excellent performance when applied to a high-throughput immunogold assay (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) for identifying monoclonal antibodies with low self-association, high solubility, and low viscosity. The stable antibody conjugates prepared with various types of gold colloid result in robust and reproducible AC-SINS measurements of antibody self-association using extremely dilute (microgram per mL) and unpurified antibody solutions. We expect that this improved methodology will be useful for reproducibly preparing stable antibody-gold conjugates for diverse applications.

  6. 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

  7. 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.

  8. Peptide-based protein capture agents with high affinity, selectivity, and stability as antibody replacements in biodetection assays

    NASA Astrophysics Data System (ADS)

    Coppock, Matthew B.; Farrow, Blake; Warner, Candice; Finch, Amethist S.; Lai, Bert; Sarkes, Deborah A.; Heath, James R.; Stratis-Cullum, Dimitra

    2014-05-01

    Current biodetection assays that employ monoclonal antibodies as primary capture agents exhibit limited fieldability, shelf life, and performance due to batch-to-batch production variability and restricted thermal stability. In order to improve upon the detection of biological threats in fieldable assays and systems for the Army, we are investigating protein catalyzed capture (PCC) agents as drop-in replacements for the existing antibody technology through iterative in situ click chemistry. The PCC agent oligopeptides are developed against known protein epitopes and can be mass produced using robotic methods. In this work, a PCC agent under development will be discussed. The performance, including affinity, selectivity, and stability of the capture agent technology, is analyzed by immunoprecipitation, western blotting, and ELISA experiments. The oligopeptide demonstrates superb selectivity coupled with high affinity through multi-ligand design, and improved thermal, chemical, and biochemical stability due to non-natural amino acid PCC agent design.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Studies on the affinity chromatography purification of anti-patulin polyclonal antibodies by enzyme linked immunosorbent assay and electrophoresis.

    PubMed

    Mhadhbi, H; Benrejeb, S; Martel, A

    2005-12-01

    Patulin is a mycotoxin produced by fungal species that frequently grow on fruit and vegetables. It presents risks, particularly for children consuming compotes and fruit juices. Thus, it is important to have methods such as immunoassays to screen a large number of samples. In the relevant literature, previous studies on the production of antibodies against patulin derivatives described qualitative tests for a patulin derivative or showed slight responses. The present study reinvestigated the production of polyclonal antibodies against patulin and their purification since crude antiserum could react non-specifically in immunoassays. Patulin-hemiglutarate was synthesized and conjugated to bovine serum albumin as the immunogen for the immunization of five New Zealand white rabbits. The immunoglobulin G (IgG) fraction was isolated twice by affinity chromatography using Sepharose-LS gel and recombinant G-protein. Classic affinity chromatography using Sepharose-LS gel was unable to eliminate serum albumin from the IgG fraction and the use of recombinant G-protein was efficient to isolate the purified IgG. Titres and specificity were determined by indirect competitive enzyme-linked immunosorbent assay. Patulin-hemiglutarate-ovalbumin gave complete displacement, while patulin displaced 30% of bound antibodies. Thus, a fraction of the antibodies are specific for free patulin. The non-specific binding increased with patulin concentrations. The electrophilic properties of patulin might also induce intermolecular cross-links in vitro that hinder the possibility of responses displacement when free patulin is used.

  15. 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.

  16. Development and Preclinical Testing of a High Affinity Single Chain Antibody against (+)-Methamphetamine

    PubMed Central

    Peterson, Eric C.; Laurenzana, Elizabeth M.; Atchley, William T.; Hendrickson, Howard; Owens, S. Michael

    2009-01-01

    Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, κ light chain, KD = 11 nM) and found to have similar ligand affinity (KD = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified and formulated as a naturally occurring mixture of monomer (~75%) and dimer (~25%). To test the in vivo efficacy of the scFv6H4, male Sprague Dawley rats (n=5) were implanted with 3-day sc osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [3H]-scFv6H4 tracer. Serum pharmacokinetic (PCKN) analysis of METH and [3H]-scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0–480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t1/2λz of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t1/2λz (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum. PMID:18192498

  17. 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.

  18. 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.

  19. 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

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

    PubMed

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

    2016-01-01

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

  1. Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection

    PubMed Central

    Gillis, Jacqueline; Wong, Fay E.; Yu, Yi; Camp, Jeremy V.; Li, Qingsheng; Connole, Michelle; Li, Yuan; Lifson, Jeffrey D.; Li, Wenjun; Keele, Brandon F.; Kozlowski, Pamela A.; Desrosiers, Ronald C.; Haase, Ashley T.

    2016-01-01

    Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination. PMID:27959961

  2. Maturation and Diversity of the VRC01-Antibody Lineage over 15 Years of Chronic HIV-1 Infection.

    PubMed

    Wu, Xueling; Zhang, Zhenhai; Schramm, Chaim A; Joyce, M Gordon; Kwon, Young Do; Zhou, Tongqing; Sheng, Zizhang; Zhang, Baoshan; O'Dell, Sijy; McKee, Krisha; Georgiev, Ivelin S; Chuang, Gwo-Yu; Longo, Nancy S; Lynch, Rebecca M; Saunders, Kevin O; Soto, Cinque; Srivatsan, Sanjay; Yang, Yongping; Bailer, Robert T; Louder, Mark K; Mullikin, James C; Connors, Mark; Kwong, Peter D; Mascola, John R; Shapiro, Lawrence

    2015-04-23

    HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.

  3. Preparation and application of an immunoaffinity column based on an antibody with strong affinity and packing material with good stability.

    PubMed

    Zhang, Hongyan; Wang, Mengmeng; Wang, Yao; Li, Qingling; Zhou, Jianhua; Huo, Feng; Tang, Bo

    2013-01-01

    This article describes the production of an anti-citrinin antibody that showed a high affinity constant (Ka) of 6.28 × 10⁹ and good tolerance to organic solvent and low pH, the synthesis of a Cu (II)-embedded polymer that showed strong binding with this antibody and the preparation of packing material for an immunoaffinity column (IAC) that show good stability. Most of the IACs reported either use harsh elution conditions and are used only once or use gentle elution conditions and are reused many times. Here, through the combined use of a strong-affinity antibody and packing material with good stability, high recoveries during clean-up and yet simultaneously good stability of the IAC were successfully achieved. Under optimised conditions of 80% methanol (pH 3), the IACs were used to clean-up the extracts of Monascus colour and red yeast rice samples, followed by HPLC detection. The recoveries of citrinin from spiked samples at levels of 50-200 μg kg⁻¹ were in the range of 84-97%.

  4. 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.

  5. 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

  6. γδTFH cells promote B cell maturation and antibody production in neuroblastoma.

    PubMed

    Mou, Wenjun; Han, Wei; Ma, Xiaoli; Wang, Xiaolin; Qin, Hong; Zhao, Wen; Ren, Xiaoya; Chen, Xi; Yang, Wei; Cheng, Haiyan; Wang, Xisi; Zhang, Hui; Ni, Xin; Wang, Huanmin; Gui, Jingang

    2017-07-07

    Previous studies have shown that γδ TFH cells are capable of modulating antibody production in immunized and infected mouse model. In recent studies, human γδ TFH cells are shown to contribute to the activation of humoral immunity and promote the maturation of B cells. However, little information is available on their involvement in neuroblastoma (NB) pathogenesis. In the present study, the frequency of γδ TFH cells in 74 NB patients was significantly higher compared with that in 60 healthy controls. Moreover, most γδ TFH cells in NB patients had a naive phenotype with up-regulation of CD25, CD69, HLA-DR and CD40L and down-regulation of ICOS. Importantly, γδ TFH cells in NB patients produced more IL-4 and IL-10 than those in healthy controls. Furthermore, serum total IgG level was significantly increased in NB patients compared with healthy controls. The expression of CD23 on B cells was up-regulated while CD80 expression was significantly down-regulated in NB patients. Further analysis of B cell compartment showed that the frequency of CD19(+)CD27(hi) plasma cells was enhanced in NB patients. Spearman's correlation analysis revealed that the frequency of γδ TFH cells was positively correlated to serum total IgG level and CD19(+)CD27(hi) plasma cells in NB patients, but negatively correlated to CD19(+) B cells. We concluded that γδ TFH cells might promote B cell maturation and antibody production in NB patients.

  7. 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.

  8. The significance of intestinal flow in the maturing of B lymphocytes and the chicken antibody response.

    PubMed Central

    Dolfi, A; Bianchi, F; Lupetti, M; Michelucci, S

    1989-01-01

    The aim of the present study was to evaluate the influence of intestinal material on the maturing of B lymphocytes in the bursa of Fabricius and on the antibody response to SRBC. Experiments were carried out on chicks whose burso-cloacal stalk had been ligated at hatching, in order to avoid any contact between the bursal tissues and intestinal material. Chicks subjected to a sham operation, or not operated at all, were used as controls. Results obtained by immunoperoxidase test, using an anti-chicken Ig antiserum, indicate that burso-cloacal stalk ligature leads to an increase in B cells in the bursa of Fabricius 20 days after the operation and a slight decrease after 60 days. The response to SRBC reveals an increase after 20 days, though no variations are found with respect to controls after 60 days. Thus, B lymphocytes mature independently of intestinal stimuli. Furthermore, observations carried out at the age of 20 days indicate that B lymphocytes disappear almost completely from the area of cloacal lymphoid infiltration in chicks with a ligated bursa. It is hypothesised that together with a systemic peripheralisation of B lymphocytes in secondary lymphoid organs and in blood, there exists an intramural local peripheralisation, which is interrupted by ligature of the burso-cloacal stalk. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:2621141

  9. Obstruction of dengue virus maturation by Fab fragments of the 2H2 antibody.

    PubMed

    Wang, Zhiqing; Li, Long; Pennington, Janice G; Sheng, Ju; Yap, Moh Lan; Plevka, Pavel; Meng, Geng; Sun, Lei; Jiang, Wen; Rossmann, Michael G

    2013-08-01

    The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.

  10. HIV-specific antibody-dependent phagocytosis matures during HIV infection.

    PubMed

    Ana-Sosa-Batiz, Fernanda; Johnston, Angus P R; Liu, Haiyin; Center, Robert J; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayaphan, Sorachai; Kaewkungwal, Jaranit; Kim, Jerome H; Michael, Nelson L; Kelleher, Anthony D; Stratov, Ivan; Kent, Stephen J; Kramski, Marit

    2014-09-01

    Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV.

  11. Early Postnatal B Cell Ontogeny and Antibody Repertoire Maturation in the Opossum, Monodelphis domestica

    PubMed Central

    Wang, Xinxin; Sharp, Alana R.; Miller, Robert D.

    2012-01-01

    Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their “fetal” development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species. PMID:23029324

  12. Early postnatal B cell ontogeny and antibody repertoire maturation in the opossum, Monodelphis domestica.

    PubMed

    Wang, Xinxin; Sharp, Alana R; Miller, Robert D

    2012-01-01

    Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their "fetal" development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species.

  13. 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

  14. 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.

  15. 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

  16. Improved Glucose Metabolism In Vitro and In Vivo by an Allosteric Monoclonal Antibody That Increases Insulin Receptor Binding Affinity

    PubMed Central

    Corbin, John A.; Bhaskar, Vinay; Goldfine, Ira D.; Bedinger, Daniel H.; Lau, Angela; Michelson, Kristen; Gross, Lisa M.; Maddux, Betty A.; Kuan, Hua F.; Tran, Catarina; Lao, Llewelyn; Handa, Masahisa; Watson, Susan R.; Narasimha, Ajay J.; Zhu, Shirley; Levy, Raphael; Webster, Lynn; Wijesuriya, Sujeewa D.; Liu, Naichi; Wu, Xiaorong; Chemla-Vogel, David; Lee, Steve R.; Wong, Steve; Wilcock, Diane; White, Mark L.

    2014-01-01

    Previously we reported studies of XMetA, an agonist antibody to the insulin receptor (INSR). We have now utilized phage display to identify XMetS, a novel monoclonal antibody to the INSR. Biophysical studies demonstrated that XMetS bound to the human and mouse INSR with picomolar affinity. Unlike monoclonal antibody XMetA, XMetS alone had little or no agonist effect on the INSR. However, XMetS was a strong positive allosteric modulator of the INSR that increased the binding affinity for insulin nearly 20-fold. XMetS potentiated insulin-stimulated INSR signaling ∼15-fold or greater including; autophosphorylation of the INSR, phosphorylation of Akt, a major enzyme in the metabolic pathway, and phosphorylation of Erk, a major enzyme in the growth pathway. The enhanced signaling effects of XMetS were more pronounced with Akt than with Erk. In cultured cells, XMetS also enhanced insulin-stimulated glucose transport. In contrast to its effects on the INSR, XMetS did not potentiate IGF-1 activation of the IGF-1 receptor. We studied the effect of XMetS treatment in two mouse models of insulin resistance and diabetes. The first was the diet induced obesity mouse, a hyperinsulinemic, insulin resistant animal, and the second was the multi-low dose streptozotocin/high-fat diet mouse, an insulinopenic, insulin resistant animal. In both models, XMetS normalized fasting blood glucose levels and glucose tolerance. In concert with its ability to potentiate insulin action at the INSR, XMetS reduced insulin and C-peptide levels in both mouse models. XMetS improved the response to exogenous insulin without causing hypoglycemia. These data indicate that an allosteric monoclonal antibody can be generated that markedly enhances the binding affinity of insulin to the INSR. These data also suggest that an INSR monoclonal antibody with these characteristics may have the potential to both improve glucose metabolism in insulinopenic type 2 diabetes mellitus and correct compensatory

  17. Measurement of Free Versus Total Therapeutic Monoclonal Antibody in Pharmacokinetic Assessment is Modulated by Affinity, Incubation Time, and Bioanalytical Platform.

    PubMed

    Talbot, Jeffrey J; Calamba, Dominador; Pai, Melody; Ma, Mark; Thway, Theingi M

    2015-11-01

    Decisions about efficacy and safety of therapeutic proteins (TP) designed to target soluble ligands are made in part by their ex vivo quantification. Ligand binding assays (LBAs) are critical tools in measuring serum TP levels in pharmacokinetic, toxicokinetic, and pharmacodynamic studies. This study evaluated the impact of reagent antibody affinities, assay incubation times, and analytical platform on free or total TP quantitation. An ELISA-based LBA that measures monoclonal anti-sclerostin antibody (TPx) was used as the model system. To determine whether the method measures free or total TPx, the effects of K on, K off, and K D were determined. An 8:1 molar ratio of sclerostin (Scl) to TPx compared to a 1:1 molar ratio produced by rabbit polyclonal antibodies to TPx was required to achieve IC50, a measure of TPx interference effectiveness, making it unclear whether the ELISA truly measured free TPx. Kinetic analysis revealed that Scl had a rapid dissociation rate (K off) from TPx and that capture and detection antibodies had significantly higher binding affinities (K D) to TPx. These kinetic limitations along with long ELISA incubation times lead to the higher molar ratios (8:1) required for achieving 50% inhibition of TPx. However, a microfluidic platform with the same reagent pairs required shorter incubations to achieve a lower Scl IC50 molar ratio (1:1). The findings from this study provide the bioanalytical community with a deeper understanding of how reagent and platform selection for LBAs can affect what a particular method measures, either free or total TP concentrations.

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

    PubMed

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

    2015-09-04

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. The effects of hitchhiker antigens co-eluting with affinity-purified research antibodies.

    PubMed

    Mechetner, Lilly; Sood, Radhika; Nguyen, Van; Gagnon, Pete; Parseghian, Missag H

    2011-09-01

    The popularity of Protein G for the purification of antibodies has given rise to an entire industry that supplies scientists with research grade immunoreagents; however, many times the supplied product is contaminated with antigens bound to the antibody's complementarity-determining regions (CDRs). These "hitchhikers" are a category of host cell proteins that are elusive to detect due to their interaction with the antibody in the final product and yet their impact on an experiment or an entire field of study can be far reaching. In an earlier work, the role of hitchhikers on a human anti-histone antibody destined for clinical usage was explored and a stringent purification scheme developed. Here we use a murine monoclonal, which reflects the type of commercial antibody usually purchased for research. We evaluate three purification schemes: a traditional approach using a one-step, low pH elution buffer (pH 2.5); a gentler approach using a pH gradient elution scheme (pH 7 down to pH 2.5); and finally, a more stringent purification patterned on our earlier published method that uses a quaternary amine guard column and a high salt wash during antibody immobilization on the Protein G. We stress that the stringent purification incorporates the pH gradient scheme and is gentler than the low-pH approach. The resulting product from all three purifications is directly compared for binding potency, histone content (using an ELISA based assay) and residual DNA (using quantitative PCR). The results demonstrate that the first two methods are inadequate for hitchhiker removal. The traditional one-step, low pH approach produces a single elution peak containing histone contaminated antibody with picogram quantities of residual DNA, however, the trailing end of the same peak is loaded with antibody complexed to nanogram amounts of DNA, in some cases, over 100 ng. The pH gradient approach provided antibodies accompanied by only picograms of residual DNA and, on average, 1 out of

  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. 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.

  6. Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats. Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities.

    PubMed

    Day, E D; Varitek, V A; Paterson, P Y

    1981-01-01

    MBP-SF, previously described as an endogenous myelin basic protein-serum factor in Lewis rats with a suggested function as a neuroautotolerogen, appears not to be a single factor but a heterogeneous collection of serum factors (MBP-SFs), most probably small fragments of MBP, each cross-reactive with a different region of the multideterminant parent molecule. The heterogeneity of the MBP-SFs in any serum sample is defined and limited by the spectrum of binding affinities of the antibody populations represented in a given reagent anti-MBP antiserum. Some samples of normal Lewis rat serum have been found to contain high affinity MBP-SFs which coexist with low affinity anti-MBP antibodies whereas other sera have shown the reversed pattern, viz. low affinity MBP-SFs and high affinity antibodies. Additional sera have been found to contain MBP-SFs of several different affinities. In time-course studies of rats sensitized to neuroantigen-adjuvant a variety of MBP-SFs and anti-MBP antibodies of different affinities may be observed in sequentially collected sera from a given animal. In no animal has any serum sample been found to contain the full spectrum of MBP-SFs. Although some MBP-SFs have been found to increase temporarily during the 2nd week after neuroantigen/CFA sensitization, all MBP-SFs tend to disappear in the 2nd week and to be replaced by anti-MBP antibodies of differing affinities 3-4 weeks following sensitization.

  7. Affinity purification and characterization of Shiga-like toxin II and production of toxin-specific monoclonal antibodies.

    PubMed Central

    Downes, F P; Barrett, T J; Green, J H; Aloisio, C H; Spika, J S; Strockbine, N A; Wachsmuth, I K

    1988-01-01

    Shiga-like toxin (SLT-II) was purified to apparent homogeneity from Escherichia coli K-12 strain NM522 containing the cloned toxin genes on recombinant plasmid pEB1. Purification was accomplished by a series of column chromatography techniques: anion-exchange, chromatofocusing, cation-exchange, and monoclonal antibody affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the pure toxin showed that SLT-II consisted of A and B subunits with apparent molecular weights of 32,000 and 10,200 +/- 800, respectively. A band of molecular weight 25,000 was also observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as the A1 subunit by Western immunoblot analysis with toxin-specific monoclonal antibodies (MAbs). The pI of the purified toxin was 5.2. Approximately 1 pg of pure SLT-II, but was not neutralized by polyclonal antibodies or MAbs to SLT-I. Five hybridomas against SLT-II were produced (BC5 BB12, DC1 EH5, EA5 BA3, ED5 DF3, and GB6 BA4). Culture supernatant fluids containing MAbs from these hybridomas did not neutralize the cytotoxicity of SLT-I or Shiga toxin. Western blot analysis showed that two MAbs (MAb DC1 EH5 and MAb GB6 BA4) recognized the A and A1 subunits of SLT-II and three MAbs (MAb BC5 BB12, MAb EA5 BA3, and MAb ED5 DF3) recognized the B subunit of SLT-II. MAb BC5 BB12 was used to prepare an affinity column for toxin purification. Images PMID:3294179

  8. Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.

    PubMed

    Sarikhani, Sina; Mirshahi, Manouchehr; Gharaati, Mohammad Reza; Mirshahi, Tooran

    2010-11-01

    As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG(1)kappa.

  9. 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.

  10. Protection by immunoglobulin dual-affinity retargeting antibodies against dengue virus.

    PubMed

    Brien, James D; Sukupolvi-Petty, Soila; Williams, Katherine L; Lam, Chia-Ying Kao; Schmid, Michael A; Johnson, Syd; Harris, Eva; Diamond, Michael S

    2013-07-01

    Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and ∼3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.

  11. 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.

  12. Bivalent binding by intermediate affinity of nimotuzumab: a contribution to explain antibody clinical profile.

    PubMed

    Garrido, Greta; Tikhomirov, Ilia A; Rabasa, Ailem; Yang, Eric; Gracia, Elías; Iznaga, Normando; Fernández, Luis E; Crombet, Tania; Kerbel, Robert S; Pérez, Rolando

    2011-02-15

    Nimotuzumab is an EGFR-targeting antibody that has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. We investigated whether different clinical behavior of nimotuzumab is related to its bivalent/monovalent binding profile. Binding properties of nimotuzumab and cetuximab, the most development of anti-EGFR antibodies, were studied in vitro using chip surfaces and cells with varying EGFR expression levels. Experimental observations demonstrated that in contrast to cetuximab, the intrinsic properties of nimotuzumab required bivalent binding for stable attachment to the cellular surface, leading to nimotuzumab selectively binding to cells that express moderate to high EGFR expression levels. At these conditions, both antibodies bound bivalently, and accumulated to similar degrees. When EGFR density is low, nimotuzumab monovalent interaction was transient, whereas cetuximab continued to interact strongly with the receptors. We compared the in vitro anti-tumor efficacy of nimotuzumab and cetuximab. Cetuximab decreased the cell viability and induced apoptosis for all the tested cell lines, effects which did not depend on EGFR expression level. In contrast, nimotuzumab also provoked significant anti-cellular effects, but its anti-tumor capacity decreased together with EGFR expression level. Cetuximab Fab fragment was able to impact tumor cell survival, whereas nimotuzumab fragment totally lost this effect. Tumor-xenograft experiments using cells with a high EGFR expression revealed similar tumor growth inhibiting effects for both antibodies. This study suggests an explanation for nimotuzumab clinical profile, whereby anti-tumor activity is obtained in absence of severe toxicities due to its properties of bivalent binding to EGFR.

  13. 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.

  14. 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.

  15. 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

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

    PubMed

    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; Meeks, Shannon L

    2016-10-20

    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. © 2016 by The American Society of Hematology.

  17. 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.

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

    PubMed Central

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

    2015-01-01

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

  19. Influence of the antibody purification method on immunoassay performance: hapten-antibody binding in accordance with the structure of the affinity column ligand.

    PubMed

    Choi, J; Kim, C; Choi, M J

    1999-10-01

    The effects of ligands for immunoaffinity chromatography on the immunoassay were investigated with three goat anti-methamphetamine (anti-MA) antibodies (Abs). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with proteins and used as immunogens. All the antisera produced were purified by affinity chromatography with various ligands of 4-ABMA-proteins and of haptens as well as protein G: 4-ABMA-bovine serum albumin (4-ABMA-BSA), 4-ABMA-keyhole limpet hemocyanine (4-ABMA-KLH), 4-ABMA-ovalbumin (4-ABMA-OVA), MA, 4-ABMA, and amphetamine were used as ligands. Enzyme-linked immunosorbent assay (ELISA) was conducted to examine characteristics of the purified Abs with the 4-ABMA-OVA competitor coated. The results obtained revealed that characters of the purified Abs were closely related with chemical structures of ligands used. The Abs from the MA and the amphetamine columns showed better sensitivities than those from the others in each antiserum. Particularly, the Ab from the amphetamine column gave the best results in terms of sensitivity and specificity. The recognition or the affinity of the Ab selected was considered to be affected by the structure of the ligand concerned. These results suggest that the Ab purification method should be considered as an important parameter which has great influence on the performance of immunoassays with polyclonal Abs. Copyright 1999 Academic Press.

  20. Measuring Affinity Constants of 1,450 Monoclonal Antibodies to Peptide Targets with a Microarray-based Label-Free Assay Platform

    PubMed Central

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

    2014-01-01

    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 1,410 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 results of the microarray-based platform with those of a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000). PMID:25536073

  1. Allosteric beta1 integrin antibodies that stabilize the low affinity state by preventing the swing-out of the hybrid domain.

    PubMed

    Luo, Bing-Hao; Strokovich, Konstantin; Walz, Thomas; Springer, Timothy A; Takagi, Junichi

    2004-06-25

    The ligand binding function of integrins can be modulated by various monoclonal antibodies by both direct and indirect mechanisms. We have characterized an anti-beta(1) antibody, SG/19, that had been reported to inhibit the function of the beta(1) integrin on the cell surface. SG/19 recognized the wild type beta(1) subunit that exists in a conformational equilibrium between the high and low affinity states but bound poorly to a mutant beta(1) integrin that had been locked in a high affinity state. Epitope mapping of SG/19 revealed that Thr(82) in the beta(1) subunit, located at the outer face of the boundary between the I-like and hybrid domains, was the key binding determinant for this antibody. Direct visualization of the alpha (5)beta(1) headpiece fragment in complex with SG/19 Fab with electron microscopy confirmed the location of the binding surface and showed that the ligand binding site is not occluded by the bound Fab. Surface plasmon resonance showed that alpha (5)beta(1) integrin bound by SG/19 maintained a low affinity toward its physiological ligand fibronectin (Fn) whereas binding by function-blocking anti-alpha(5) antibodies resulted in a complete loss of fibronectin binding. Thus a class of the anti-beta antibodies represented by SG/19 attenuate the ligand binding function by restricting the conformational shift to the high affinity state involving the swing-out of the hybrid domain without directly interfering with ligand docking.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Maturation and Diversity of the VRC01-Antibody Lineage over 15 Years of Chronic HIV-1 Infection

    SciTech Connect

    Wu, Xueling; Zhang, Zhenhai; Schramm, Chaim A.; Joyce, M.  Gordon; Do Kwon, Young; Zhou, Tongqing; Sheng, Zizhang; Zhang, Baoshan; O’Dell, Sijy; McKee, Krisha; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Longo, Nancy S.; Lynch, Rebecca M.; Saunders, Kevin O.; Soto, Cinque; Srivatsan, Sanjay; Yang, Yongping; Bailer, Robert T.; Louder, Mark K.; Mullikin, James C.; Connors, Mark; Kwong, Peter D.; Mascola, John R.; Shapiro, Lawrence; Benjamin, Betty; Blakesley, Robert; Bouffard, Gerry; Brooks, Shelise; Coleman, Holly; Dekhtyar, Mila; Gregory, Michael; Guan, Xiaobin; Gupta, Jyoti; Han, Joel; Hargrove, April; Ho, Shi-ling; Legaspi, Richelle; Maduro, Quino; Masiello, Cathy; Maskeri, Baishali; McDowell, Jenny; Montemayor, Casandra; Park, Morgan; Riebow, Nancy; Schandler, Karen; Schmidt, Brian; Sison, Christina; Stantripop, Mal; Thomas, James; Thomas, Pam; Vemulapalli, Meg; Young, Alice

    2015-04-09

    HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here in this paper, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ~2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.

  9. Maturation and Diversity of the VRC01-Antibody Lineage over 15 Years of Chronic HIV-1 Infection

    DOE PAGES

    Wu, Xueling; Zhang, Zhenhai; Schramm, Chaim A.; ...

    2015-04-09

    HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here in this paper, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of themore » study period, VRC01-lineage clades showed continuous evolution, with rates of ~2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.« less

  10. 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.

  11. Antigen affinity discrimination is an intrinsic function of the B cell receptor

    PubMed Central

    Liu, Wanli; Meckel, Tobias; Tolar, Pavel; Sohn, Hae Won

    2010-01-01

    Antibody affinity maturation, a hallmark of adaptive immune responses, results from the selection of B cells expressing somatically hypermutated B cell receptors (BCRs) with increased affinity for antigens. Despite the central role of affinity maturation in antibody responses, the molecular mechanisms by which the increased affinity of a B cell for antigen is translated into a selective advantage for that B cell in immune responses is incompletely understood. We use high resolution live-cell imaging to provide evidence that the earliest BCR-intrinsic events that follow within seconds of BCR–antigen binding are highly sensitive to the affinity of the BCR for antigen. High affinity BCRs readily form oligomers and the resulting microclusters grow rapidly, resulting in enhanced recruitment of Syk kinase and calcium fluxes. Thus, B cells are able to read the affinity of antigen by BCR-intrinsic mechanisms during the earliest phases of BCR clustering, leading to the initiation of B cell responses. PMID:20404102

  12. Crystal structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex with testosterone. Improved affinity results from small structural changes within the variable domains.

    PubMed

    Valjakka, Jarkko; Hemminki, Ari; Niemi, Seija; Söderlund, Hans; Takkinen, Kristiina; Rouvinen, Juha

    2002-11-15

    A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C(4)F(5)). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K(d) = 3 x 10(-10) m) when compared with the wild-type (3-C(4)F(5)) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 A) and without testosterone (2.10 A) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.

  13. Severely exacerbated neuromyelitis optica rat model with extensive astrocytopathy by high affinity anti-aquaporin-4 monoclonal antibody.

    PubMed

    Kurosawa, Kazuhiro; Misu, Tatsuro; Takai, Yoshiki; Sato, Douglas Kazutoshi; Takahashi, Toshiyuki; Abe, Yoichiro; Iwanari, Hiroko; Ogawa, Ryo; Nakashima, Ichiro; Fujihara, Kazuo; Hamakubo, Takao; Yasui, Masato; Aoki, Masashi

    2015-12-04

    Neuromyelitis optica (NMO), an autoimmune astrocytopathic disease associated with anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions preferentially involving the optic nerves and spinal cord. However, previous in-vivo experimental models injecting human anti-AQP4 antibodies only resulted in mild spinal cord lesions compared to NMO autopsied cases. Here, we investigated whether the formation of severe NMO-like lesions occurs in Lewis rats in the context of experimental autoimmune encephalomyelitis (EAE), intraperitoneally injecting incremental doses of purified human immunoglobulin-G from a NMO patient (hIgGNMO) or a high affinity anti-AQP4 monoclonal antibody (E5415A), recognizing extracellular domain of AQP4 made by baculovirus display method. NMO-like lesions were observed in the spinal cord, brainstem, and optic chiasm of EAE-rats with injection of pathogenic IgG (hIgGNMO and E5415A), but not in control EAE. Only in higher dose E5415A rats, there were acute and significantly severer clinical exacerbations (tetraparesis or moribund) compared with controls, within half day after the injection of pathogenic IgG. Loss of AQP4 was observed both in EAE rats receiving hIgGNMO and E5415A in a dose dependent manner, but the ratio of AQP4 loss in spinal sections became significantly larger in those receiving high dose E5415A up to about 50 % than those receiving low-dose E5415A or hIgGNMO less than 3 %. These lesions were also characterized by extensive loss of glial fibrillary acidic protein but relatively preserved myelin sheaths with perivascular deposition of IgG and C5b-9, which is compatible with post mortem NMO pathology. In high dose E5415A rats, massive neutrophil infiltration was observed especially at the lesion edge, and such lesions were highly vacuolated with partial demyelination and axonal damage. In contrast, such changes were absent in EAE rats receiving low-dose E5415A and hIgGNMO. In the present study, we established a

  14. 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

  15. 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.

  16. A monoclonal rat anti-mouse EMAP II antibody that functionally neutralizes pro- and mature-EMAP II in vitro.

    PubMed

    Rajashekhar, Gangaraju; Mitnacht-Kraus, Rita; Ispe, Ute; Garrison, Jana; Hou, Yonghao; Taylor, Brian; Petrache, Irina; Vestweber, Dietmar; Clauss, Matthias

    2009-10-31

    EMAP II is an endothelial cell and monocyte activating proinflammatory cytokine, which has been demonstrated to induce endothelial cell apoptosis. In order to analyze its role in disease models linked to inflammation and endothelial cell death, we aimed to develop a neutralizing antibody against mouse EMAP II. Therefore, we generated rat monoclonal anti-mouse EMAP II antibodies by immunization with recombinant full length, mouse pro-EMAP II protein. We could identify by ELISA, hybridoma clones from fusion with mouse myeloma SP2/0 cells which produced antibodies recognizing both full length and mature EMAP II. We further characterized one antibody, M7/1 and demonstrated its ability to detect both EMAP II forms in Western blotting and to neutralize EMAP II directed migration of human peripheral blood monocytes as well as EMAP II induced apoptosis of tumor and endothelial cells. We conclude that this antibody can be useful to both target and analyze murine disease models, in which EMAP II may be involved.

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

    PubMed

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

    2008-01-01

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

  18. 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.

  19. F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse

    PubMed Central

    Comrie, William A.; Babich, Alexander

    2015-01-01

    Integrin-dependent interactions between T cells and antigen-presenting cells are vital for proper T cell activation, effector function, and memory. Regulation of integrin function occurs via conformational change, which modulates ligand affinity, and receptor clustering, which modulates valency. Here, we show that conformational intermediates of leukocyte functional antigen 1 (LFA-1) form a concentric array at the immunological synapse. Using an inhibitor cocktail to arrest F-actin dynamics, we show that organization of this array depends on F-actin flow and ligand mobility. Furthermore, F-actin flow is critical for maintaining the high affinity conformation of LFA-1, for increasing valency by recruiting LFA-1 to the immunological synapse, and ultimately for promoting intracellular cell adhesion molecule 1 (ICAM-1) binding. Finally, we show that F-actin forces are opposed by immobilized ICAM-1, which triggers LFA-1 activation through a combination of induced fit and tension-based mechanisms. Our data provide direct support for a model in which the T cell actin network generates mechanical forces that regulate LFA-1 activity at the immunological synapse. PMID:25666810

  20. 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.

  1. Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries

    PubMed Central

    Harvey, Barrett R.; Georgiou, George; Hayhurst, Andrew; Jeong, Ki Jun; Iverson, Brent L.; Rogers, Geoffrey K.

    2004-01-01

    Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final KD of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning. PMID:15197275

  2. 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

  3. Monoclonal antibody TSd-1 is specific to elongating and matured spermatids in testis of common tree shrew (Tupaia glis).

    PubMed

    Maeda, S; Nakamuta, N; Nam, S Y; Kimura, J; Endo, H; Rerkamnuaychoke, W; Kurohmaru, M; Yamada, J; Hayashi, Y; Nishida, T

    1998-03-01

    The monoclonal antibody (MAb), named TSd-1, specific to spermatogenic cells of the common tree shrew (Tupaia glis) was established and characterized using immunohistochemistry and immunoblotting. MAb TSd-1 reacted with elongating and matured spermatids in a stage-dependent manner. TSd-1 recognized a 94 kilodalton (kDa) peptide in the plasma membrane and cytosol. Additionally, an extremely weak 107 kDa band was detected only in the cytosol. The reactions were not detected in round spermatids. In elongating Stage VI spermatids, the plasma membrane and the granular structure within the cytoplasm were intensely positive, and most intense after the appearance of new round spermatids in the lower layer (Stage I). The reactions were observed neither in the other organs of the common tree shrew nor in the testes of other animals, indicating that TSd-1 antigen is specific to the spermatogenic cells of the common tree shrew, and may act on elongating or matured spermatids.

  4. 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.

  5. An in vitro Comparison of Microdialysis Relative Recovery of Met- and Leu-Enkephalin Using Cyclodextrins and Antibodies as Affinity Agents

    PubMed Central

    Fletcher, Heidi J.; Stenken, Julie A.

    2008-01-01

    Cyclodextrins and antibodies have been used as affinity agents to improve relative recovery during microdialysis sampling. Two neuropeptides, methionine-enkephalin (ME) and leucine-enkephalin (LE), were chosen to compare the use of cyclodextrins and antibodies as possible affinity agents for improving their relative recovery across polycarbonate and polyethersulfone membranes during in vitro sampling. Cyclodextrins (CD) including β-CD, 2-hydroxypropyl-β-cyclodextrin (2HPβ-CD), and γ-CD gave improvements of relative recovery for both peptides of less than 2-fold as compared to controls. Comparisons of relative recovery between tyrosine-glycine-glycine, tyrosine, and phenylalanine using different cyclodextrins in the perfusion fluid were also obtained. Inclusion of an antibody against met-enkephalin in the microdialysis perfusion fluid resulted in relative recovery increases of up to 2.5-fold. These results show that using antibodies as affinity agents during microdialysis sampling may be more effective agents to improve the relative recovery of these opioid neuropeptides. PMID:18558138

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

    PubMed

    Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

    2017-08-08

    Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

  11. 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

  12. Affinity-matured recombinant immunotoxin targeting gangliosides 3′-isoLM1 and 3′,6'-isoLD1 on malignant gliomas

    PubMed Central

    Piao, Hailan; Kuan, Chien-Tsun; Chandramohan, Vidya; Keir, Stephen T; Pegram, Charles N; Bao, Xuhui; Månsson, Jan-Eric; Pastan, Ira H; Bigner, Darell D

    2013-01-01

    About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10−9M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and

  13. Affinity-matured recombinant immunotoxin targeting gangliosides 3'-isoLM1 and 3',6'-isoLD1 on malignant gliomas.

    PubMed

    Piao, Hailan; Kuan, Chien-Tsun; Chandramohan, Vidya; Keir, Stephen T; Pegram, Charles N; Bao, Xuhui; Månsson, Jan-Eric; Pastan, Ira H; Bigner, Darell D

    2013-01-01

    About 60 percent of glioblastomas highly express the gangliosides 3'-isoLM1 and 3',6'-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3'-isoLM1 and 3',6'-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3'-isoLM1 and 3',6'-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3'-isoLM1 and 3',6'-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the KD of DmAb14m-IT for gangliosides 3'-isoLM1 and 3',6'-isoLD1 was 2.6 × 10(-9)M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3'-isoLM1 and 3',6'-isoLD1. The DmAb14m-IT IC 50 was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3'-isoLM1 and 3',6'-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3'-isoLM1 and 3',6'-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.

  14. 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

  15. Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

    PubMed

    Matheson, Nicholas J; Peden, Andrew A; Lehner, Paul J

    2014-01-01

    Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing.

  16. 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.

  17. Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment.

    PubMed

    Stewart, Alex; Harrison, Joseph S; Regula, Lauren K; Lai, Jonathan R

    2013-04-08

    Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464-5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1-3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel information about this high-affinity

  18. Side chain requirements for affinity and specificity in D5, an HIV-1 antibody derived from the VH1-69 germline segment

    PubMed Central

    2013-01-01

    Background Analysis of factors contributing to high affinity antibody-protein interactions provides insight into natural antibody evolution, and guides the design of antibodies with new or enhanced function. We previously studied the interaction between antibody D5 and its target, a designed protein based on HIV-1 gp41 known as 5-Helix, as a model system [Da Silva, G. F.; Harrison, J. S.; Lai, J. R., Biochemistry, 2010, 49, 5464–5472]. Antibody D5 represents an interesting case study because it is derived from the VH1-69 germline segment; this germline segment is characterized by a hydrophobic second heavy chain complementarity determining region (HCDR2) that constitutes the major functional paratope in D5 and several antibodies derived from the same progenitor. Results Here we explore side chain requirements for affinity and specificity in D5 using phage display. Two D5-based libraries were prepared that contained diversity in all three light chain complementarity determining regions (LCDRs 1–3), and in the third HCDR (HCDR3). The first library allowed residues to vary among a restricted set of six amino acids (Tyr/Ala/Asp/Ser/His/Pro; D5-Lib-I). The second library was designed based on a survey of existing VH1-69 antibody structures (D5-Lib-II). Both libraries were subjected to multiple rounds of selection against 5-Helix, and individual clones characterized. We found that selectants from D5-Lib-I generally had moderate affinity and specificity, while many clones from D5-Lib-II exhibited D5-like properties. Additional analysis of the D5-Lib-II functional population revealed position-specific biases for particular amino acids, many that differed from the identity of those side chains in D5. Conclusions Together these results suggest that there is some permissiveness for alternative side chains in the LCDRs and HCDR3 of D5, but that replacement with a minimal set of residues is not tolerated in this scaffold for 5-Helix recognition. This work provides novel

  19. 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

  20. 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

  1. 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.

  2. Simulation of B Cell Affinity Maturation Explains Enhanced Antibody Cross-Reactivity Induced by the Polyvalent Malaria Vaccine AMA1

    DTIC Science & Technology

    2014-07-01

    which is critical to modeling vaccine efficacy for multiple-strain or multiple-serotype pathogens, such as Plasmodium falciparum or dengue virus...apical membrane antigen-1 (AMA1). The malaria vaccine candidate AMA1 is a subunit-based vaccine against P. falciparum that induces protection (37), but...F. Anders, and M. Foley. 2006. The most polymorphic residue on Plasmodium falciparum apical membrane antigen 1 determines binding of an invasion

  3. Treatment with anti-LFA-1 alpha monoclonal antibody selectively interferes with the maturation of CD4- 8+ thymocytes.

    PubMed

    Revilla, C; González, A L; Conde, C; López-Hoyos, M; Merino, J

    1997-04-01

    Maturation of T lymphocytes in the thymus is driven by signals provided by soluble factors and by the direct interaction between thymocytes and stromal cells. Although the interaction between T-cell receptor (TCR) and major histocompalibility complex (MHC) molecules on stromal cells is crucial for T-cell development, other accessory molecules seem to play a role in this process. In order to better understand the role of lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) molecules in thymocyte maturation, mice were treated from birth with saturating doses of non-cytolytic-specific monoclonal antibodies. The effect of this treatment on thymocyte subpopulations and the expression of CD3 and TCR-alpha beta by these cells was investigated by flow cytometry. Our data demonstrated that the effective saturation of LFA-1 alpha chain in the thymus, but not ICAM-I or LFA-I beta chain, selectively interfered with the maturation of CD8+ T cells, as manifested by a marked reduction in the frequency of CD4-8+ thymocytes expressing high levels of CD3 and TCR-alpha beta. This selective reduction was also observed in peripheral blood mononuclear cells and spleen cells. The analysis of the frequencies of various V beta TCR showed that CD4-8+ thymocytes were globally affected by the treatment. These results underline the importance of the interaction between LFA-1 and its ligands in the maturation of CD8+ T cells and document the existence of different molecular requirements for the differentiation of CD4+ and CD8+ T cells.

  4. Human germline antibody gene segments encode polyspecific antibodies.

    PubMed

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

    2013-04-01

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

  5. Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

    PubMed Central

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

    2013-01-01

    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. PMID:23637590

  6. Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

    PubMed Central

    Wykes, Michelle N; Beattie, Lynette; MacPherson, Gordon G; Hart, Derek N

    2004-01-01

    CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b− DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs. PMID:15500618

  7. Novel anti–B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma

    PubMed Central

    Mayes, Patrick A.; Acharya, Chirag; Zhong, Mike Y.; Cea, Michele; Cagnetta, Antonia; Craigen, Jenny; Yates, John; Gliddon, Louise; Fieles, William; Hoang, Bao; Tunstead, James; Christie, Amanda L.; Kung, Andrew L.; Richardson, Paul; Munshi, Nikhil C.; Anderson, Kenneth C.

    2014-01-01

    B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3–dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer. PMID:24569262

  8. 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.

  9. Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41.

    PubMed

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

    2013-01-01

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

  10. Specific Human Astrocyte Subtype Revealed by Affinity Purified GFAP+1 Antibody; Unpurified Serum Cross-Reacts with Neurofilament-L in Alzheimer

    PubMed Central

    Middeldorp, Jinte; van den Berge, Simone A.; Aronica, Eleonora; Speijer, Dave; Hol, Elly M.

    2009-01-01

    The human GFAP splice variants GFAPΔ164 and GFAPΔexon6 both result in a GFAP protein isoform with a unique out-of-frame carboxy-terminus that can be detected by the GFAP+1 antibody. We previously reported that GFAP+1 was expressed in astrocytes and in degenerating neurons in Alzheimer's disease brains. In this study we aimed at further investigating the neuronal GFAP+1 expression and we started by affinity purifying the GFAP+1 antibody. The purified antibody resulted in a loss of neuronal GFAP+1 signal, although other antibodies directed against the amino- and carboxy-terminus of GFAPα still revealed GFAP-immunopositive neurons, as described before. With an in-depth analysis of a western blot, followed by mass spectrometry we discovered that the previously detected neuronal GFAP+1 expression was due to cross-reactivity of the antibody with neurofilament-L (NF-L). This was confirmed by double-label fluorescent immunohistochemistry and western blotting with the unpurified GFAP+1 antibody and an antibody against NF-L. Our data imply that NF-L can accumulate in some tangle-like structures in Alzheimer brains. More importantly, the purified GFAP+1 antibody clearly revealed a specific subtype of astrocytes in the adult human brain. These large astrocytes are present throughout the brain, e.g., along the subventricular zone, in the hippocampus, in the striatum and in the spinal cord of controls, Alzheimer, and Parkinson patients. The presence of a specific GFAP-isoform suggests a specialized function of these astrocytes. PMID:19888461

  11. Maturation of secreted HCV particles by incorporation of secreted ApoE protects from antibodies by enhancing infectivity.

    PubMed

    Bankwitz, Dorothea; Doepke, Mandy; Hueging, Kathrin; Weller, Romy; Bruening, Janina; Behrendt, Patrick; Lee, Ji-Young; Vondran, Florian W R; Manns, Michael P; Bartenschlager, Ralf; Pietschmann, Thomas

    2017-09-01

    Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles. ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted. Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs). Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of

  12. Investigation of ferulate deposition in endosperm cell walls of mature and developing wheat grains by using a polyclonal antibody.

    PubMed

    Philippe, Sully; Tranquet, Olivier; Utille, Jean-Pierre; Saulnier, Luc; Guillon, Fabienne

    2007-04-01

    A polyclonal antibody has been raised against ferulic acid ester linked to arabinoxylans (AX). 5-O-feruloyl-alpha-L-arabinofuranosyl(1-->4)-beta-D-xylopyranosyl was obtained by chemical synthesis, and was coupled to bovine serum albumin for the immunization of rabbit. The polyclonal antibody designated 5-O-Fer-Ara was highly specific for 5-O-(trans-feruloyl)-L-arabinose (5-O-Fer-Ara) structure that is a structural feature of cell wall AX of plants belonging to the family of Gramineae. The antibody has been used to study the location and deposition of feruloylated AX in walls of aleurone and starchy endosperm of wheat grain. 5-O-Fer-Ara began to accumulate early in aleurone cell wall development (beginning of grain filling, 13 days after anthesis, DAA) and continued to accumulate until the aleurone cells were firmly fixed between the starchy endosperm and the nucelus epidermis (19 DAA). From 26 DAA to maturity, the aleurone cell walls changed little in appearance. The concentration of 5-O-Fer-Ara is high in both peri- and anticlinal aleurone cell walls with the highest accumulation of 5-O-Fer-Ara at the cell junctions at the seed coat interface. The situation is quite different in the starchy endosperm: whatever the stage of development, a low amount of 5-O-Fer-Ara epitope was detected. Contrary to what was observed for aleurone cell walls, no peak of accumulation of feruloylated AX was noticed between 13 and 19 DAA. Visualization of labelled Golgi vesicles suggested that the feruloylation of AX is intracellular. The distribution of (5-O-Fer-Ara) epitope is further discussed in relation to the role of ferulic acid and its dehydrodimers in cell wall structure and tissue organization of wheat grain.

  13. 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.

  14. 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.

  15. Detection of low-affinity anti-drug antibodies and improved drug tolerance in immunogenicity testing by Octet(®) biolayer interferometry.

    PubMed

    Li, Jian; Schantz, Allen; Schwegler, Maureen; Shankar, Gopi

    2011-01-25

    We assessed the utility of the FortéBio Octet(®) system for detection of anti-drug antibodies (ADAs) against an investigational therapeutic human IgG1 monoclonal antibody (mAb), CNTO X. To understand the relative merits of this technology, key performance requirements were compared with two popularly accepted ADA detection methods, a step-wise bridging ELISA and a Meso Scale Discovery (MSD) homogeneous (single step binding) bridging ECLIA. When used to detect 13 monoclonal ADAs of varying affinities and one polyclonal ADA, all three methods demonstrated their greatest apparent sensitivity to the polyclonal sample (1, 6, and 130 ng/mL, respectively for ECLIA, ELISA, and Octet). Sensitivity to monoclonal ADAs tended to vary in accordance with their affinities, however, the sensitivity of the Octet method varied much less between ADAs. As a result, the above ranking became reversed such that Octet was the most and ELISA least sensitive for detection of low-affinity ADAs. With regard to drug tolerance, the presence of CNTO X could lead to false-negative assay results, although each method was affected to a different degree, with the Octet method tolerating up to 10 times more drug than the ECLIA method, which in turn tolerated up to 10 times more than the ELISA. Finally, the ECLIA and Octet methods were applied to the bioanalysis of cynomolgus monkey sera from a pre-clinical multiple dose study of CNTO X. Octet indicated 3 positive animals developed ADA as early as day 15 of the dosing phase while drug was present at nearly 1mg/mL. ECLIA detected only one of these, and only in a day 57 recovery sample after drug had cleared from circulation. We conclude that the Octet is a promising platform for detection of lower affinity ADAs and is particularly suitable for ADA detection when drug persists at levels that negatively impact bridging immunoassays. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Tumor targeting properties of monoclonal antibodies with different affinity for target antigen CD44V6 in nude mice bearing head-and-neck cancer xenografts.

    PubMed

    Verel, Iris; Heider, Karl-Heinz; Siegmund, Miranda; Ostermann, Elínborg; Patzelt, Erik; Sproll, Marlies; Snow, Gordon B; Adolf, Günther R; van Dongen, Guus A M S

    2002-05-20

    The CD44 protein family consists of isoforms with tissue-specific expression, which are encoded by standard exons and up to 9 alternatively spliced variant exons (v2-v10) of the same gene. The murine MAbs U36 and BIWA-1, directed against overlapping epitopes within the v6 region of CD44, have previously been shown to efficiently target HNSCC. We herein report on the construction of 1 chimeric (BIWA-2) and 2 humanized (BIWA-4 and BIWA-8) derivatives of BIWA-1. Together with U36 and BIWA-1, these new antibodies were evaluated for affinity to the antigen in vitro as well as for biodistribution and efficacy in RIT using nude mice bearing the HNSCC xenograft line HNX-OE. As determined by surface plasmon resonance, the MAbs bound to CD44v6 with an up to 46-fold difference in affinity (K(d) ranging from 1.1 x 10(-8) to 2.4 x 10(-10) M) with the following ranking: mMAb U36 < hMAb BIWA-4 < hMAb BIWA-8 < mMAb BIWA-1 approximately cMAb BIWA-2. To evaluate their in vivo tumor-targeting properties, 2 MAbs with identical murine or human isotype were labeled with either (131)I or (125)I and administered simultaneously (50 microg/10 microCi each) as pairs showing a stepwise decrease in the difference in affinity: U36 vs. BIWA-1 (35.0-fold difference), BIWA-4 vs. BIWA-2 (14.0-fold) and BIWA-4 vs. BIWA-8 (4.0-fold). Biodistribution was assessed at 1, 2, 3 or 4 and 7 days after injection. Remarkably, for all 3 MAb pairs tested, the lower-affinity MAb showed a higher degree and specificity of tumor localization. The difference in tumor localization was more pronounced when the difference in affinity was larger. For example, 3 days after injection, the lower-affinity mMAb U36 showed a 50% higher tumor uptake than the higher-affinity mMAb BIWA-1, while blood levels and uptake in organs were similar. After labeling with (186)Re (300 or 400 microCi), the same MAb pairs showed RIT efficacy consistent with the biodistribution data: (186)Re-U36 was more effective than (186)Re-BIWA-1, (186)Re

  17. 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.

  18. MATURATION OF THE IMMUNE RESPONSE IN VITRO

    PubMed Central

    Macario, Alberto J. L.; de Macario, Everly Conway; Franceschi, Claudio; Celada, Franco

    1972-01-01

    We have cultivated lymph node microfragments from β-D-galactosidase (Escherichia coli) primed rabbits and have measured their secondary response directed towards the whole molecule (precipitating antibodies) and to a single determinant (activating antibodies) of the antigen. By decreasing the size of the fragments to 105 cells, we began to observe heterogeneity among identical cultures in terms of positivity of response, antibody specificity, and titers. The affinity of "early" activating antibodies was inversely proportional to the dose of challenge. While no maturation was seen in low and excessive challenge, in all cultures receiving intermediate doses the association constant was raised several orders of magnitude within periods of 20 days. The relevance of these data to the mechanism of affinity selection of antigen-sensitive cells is discussed. PMID:4557772

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

    PubMed

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

    1983-01-01

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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. [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.

  6. Blocking Allergic Reaction through Targeting Surface-Bound IgE with Low-Affinity Anti-IgE Antibodies.

    PubMed

    Zhang, Ke; Liu, Jeffrey; Truong, Thao; Zukin, Elyssa; Chen, Wendy; Saxon, Andrew

    2017-05-15

    Allergic disorders have now become a major worldwide public health issue, but the effective treatment options remain limited. We report a novel approach to block allergic reactivity by targeting the surface-bound IgE of the allergic effector cells via low-affinity anti-human IgE Abs with dissociation constants in the 10(-6) to 10(-8) M range. We demonstrated that these low-affinity anti-IgE mAbs bind to the cell surface-bound IgE without triggering anaphylactic degranulation even at high concentration, albeit they would weakly upregulate CD203c expression on basophils. This is in contrast to the high-affinity anti-IgE mAbs that trigger anaphylactic degranulation at low concentration. Instead, the low-affinity anti-IgE mAbs profoundly block human peanut- and cat-allergic IgE-mediated basophil CD63 induction indicative of anaphylactic degranulation; suppress peanut-, cat-, and dansyl-specific IgE-mediated passive cutaneous anaphylaxis; and attenuate dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mouse model. Mechanistic studies reveal that the ability of allergic reaction blockade by the low-affinity anti-IgE mAbs was correlated with their capacity to downregulate the surface IgE and FcεRI level on human basophils and the human FcεRIα transgenic mouse bone marrow-derived mast cells via driving internalization of the IgE/FcεRI complex. Our studies demonstrate that targeting surface-bound IgE with low-affinity anti-IgE Abs is capable of suppressing allergic reactivity while displaying an excellent safety profile, indicating that use of low-affinity anti-IgE mAbs holds promise as a novel therapeutic approach for IgE-mediated allergic diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen.

    PubMed

    Rutgers, A; Meyers, K E; Canziani, G; Kalluri, R; Lin, J; Madaio, M P

    2000-07-01

    Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation

  12. Multi-donor Analysis Reveals Structural Elements, Genetic Determinants, and Maturation Pathway for Effective HIV-1 Neutralization by VRCO1-class Antibodies

    PubMed Central

    Zhou, Tongqing; Zhu, Jiang; Wu, Xueling; Moquin, Stephanie; Zhang, Baoshan; Acharya, Priyamvada; Georgiev, Ivelin S.; Altae-Tran, Han R.; Chuang, Gwo-Yu; Joyce, M. Gordon; Kwon, Young Do; Longo, Nancy S.; Louder, Mark K.; Luongo, Timothy; McKee, Krisha; Schramm, Chaim A.; Skinner, Jeff; Yang, Yongping; Yang, Zhongjia; Zhang, Zhenhai; Zheng, Anqi; Bonsignori, Mattia; Haynes, Barton F.; Scheid, Johannes F.; Nussenzweig, Michel C.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Mullikin, James C.; Connors, Mark; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D.

    2014-01-01

    Summary Antibodies of the VRC01 class neutralize HIV-1, arise in diverse HIV-1-infected donors, and are potential templates for an effective HIV-1 vaccine. However, the stochastic processes that generate repertoires in each individual of >1012 antibodies make elicitation of specific antibodies uncertain. Here we determine the ontogeny of the VRC01 class by crystallography and next-generation sequencing. Despite antibody-sequence differences exceeding 50%, antibody-gp120 cocrystal structures reveal VRC01-class recognition to be remarkably similar. B cell transcripts indicate that VRC01-class antibodies require few specific genetic elements, suggesting that naive-B cells with VRC01-class features are generated regularly by recombination. Virtually all of these fail to mature, however, with only a few—likely one—ancestor B cell expanding to form a VRC01-class lineage in each donor. Developmental similarities in multiple donors thus reveal the generation of VRC01-class antibodies to be reproducible in principle, thereby providing a framework for attempts to elicit similar antibodies in the general population. PMID:23911655

  13. Monoclonal antibodies against Olea europaea major allergen: allergenic activity of affinity-purified allergen and depleted extract and development of a radioimmunoassay for the quantitation of the allergen.

    PubMed

    Lombardero, M; Quirce, S; Duffort, O; Barber, D; Carpizo, J; Chamorro, M J; Lezaun, A; Carreira, J

    1992-04-01

    Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.

  14. Diversity Against Adversity: How Adaptive Immune System Evolves Potent Antibodies

    NASA Astrophysics Data System (ADS)

    Heo, Muyoung; Zeldovich, Konstantin B.; Shakhnovich, Eugene I.

    2011-07-01

    Adaptive immunity is an amazing mechanism, whereby new protein functions—affinity of antibodies (Immunoglobulins) to new antigens—evolve through mutation and selection in a matter of a few days. Despite numerous experimental studies, the fundamental physical principles underlying immune response are still poorly understood. In considerable departure from past approaches, here, we propose a microscopic multiscale model of adaptive immune response, which consists of three essential players: The host cells, viruses, and B-cells in Germinal Centers (GC). Each moiety carries a genome, which encodes proteins whose stability and interactions are determined from their sequences using laws of Statistical Mechanics, providing an exact relationship between genomic sequences and strength of interactions between pathogens and antibodies and antibodies and host proteins (autoimmunity). We find that evolution of potent antibodies (the process known as Affinity Maturation (AM)) is a delicate balancing act, which has to reconcile the conflicting requirements of protein stability, lack of autoimmunity, and high affinity of antibodies to incoming antigens. This becomes possible only when antibody producing B cells elevate their mutation rates (process known as Somatic Hypermutation (SHM)) to fall into a certain range—not too low to find potency increasing mutations but not too high to destroy stable Immunoglobulins and/or already achieved affinity. Potent antibodies develop through clonal expansion of initial B cells expressing marginally potent antibodies followed by their subsequent affinity maturation through mutation and selection. As a result, in each GC the population of mature potent Immunoglobulins is monoclonal being ancestors of a single cell from initial (germline) pool. We developed a simple analytical theory, which provides further rationale to our findings. The model and theory reveal the molecular factors that determine the efficiency of affinity maturation

  15. 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 ...

  16. Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

    PubMed Central

    Tungatt, Katie; Bianchi, Valentina; Crowther, Michael D.; Powell, Wendy E.; Schauenburg, Andrea J.; Trimby, Andrew; Donia, Marco; Miles, John J.; Holland, Christopher J.; Cole, David K.; Godkin, Andrew J.; Peakman, Mark; Straten, Per Thor; Svane, Inge Marie; Dolton, Garry

    2015-01-01

    Fluorochrome-conjugated peptide–MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II–restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR–pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents. PMID:25452566

  17. Myeloperoxidase anti-neutrophil cytoplasmic antibody affinity is associated with the formation of neutrophil extracellular traps in the kidney and vasculitis activity in myeloperoxidase anti-neutrophil cytoplasmic antibody-associated microscopic polyangiitis.

    PubMed

    Yoshida, Masaharu; Yamada, Muneharu; Sudo, Yasuyo; Kojima, Tadasu; Tomiyasu, Tomohiro; Yoshikawa, Noriko; Oda, Takashi; Yamada, Michiyuki

    2016-07-01

    Anti-neutrophil cytoplasmic antibody (ANCA) is associated with small-vessel vasculitis particularly in the kidneys and can induce the formation of neutrophil extracellular traps (NETs) from primed neutrophils. Recently we have reported that the induction of NETs correlates with ANCA affinity for myeloperoxidase (MPO) and disease activity in patients with MPO-ANCA-associated microscopic polyangiitis. To investigate whether MPO-ANCA affinity is associated with the formation of NETs in vivo, we examined the occurrence of NETs in the renal tissues of patients with MPO-ANCA-associated microscopic polyangiitis and ANCA affinity by double immunofluorescence staining for NET components of citrullinated histone, MPO and PAD4 and by ELISA competition with MPO, respectively. We divided 30 MPO-ANCA-associated microscopic polyangiitis patients into 2 groups based on their ANCA affinity levels (IC50 for the high: 0.11 ± 0.04 µg/mL (Group1) and IC50 for the low: 0.66 ± 0.24 µg/mL (Group2)). Group1 showed a higher Birmingham vasculitis activity score (15.6 ± 5.7) and 73% of the patients presented clinically with rapidly progressive glomerulonephritis and histologically with focal/crescentic glomerulonephritis (GN). Group 2 showed a lower Birmingham vasculitis activity score (9.2 ± 4.9) and 73% of the patients presented clinically with chronic renal failure and histologically with mixed/sclerotic GN. Group 1 showed a much higher occurrence of NETs than Group 2. Our findings indicate that ANCA affinity was associated with the in vivo formation of NETs, which might be involved in the pathophysiology of patients with MPO-ANCA-associated microscopic polyangiitis. © 2016 The Authors Nephrology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Nephrology.

  18. 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

  19. 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.

  20. 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

  1. 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

  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. A general approach to antibody thermostabilization

    PubMed Central

    McConnell, Audrey D; Zhang, Xue; Macomber, John L; Chau, Betty; Sheffer, Joseph C; Rahmanian, Sorena; Hare, Eric; Spasojevic, Vladimir; Horlick, Robert A; King, David J; Bowers, Peter M

    2014-01-01

    Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications. PMID:25517312

  4. Humanization of Antibodies Using Heavy Chain Complementarity-determining Region 3 Grafting Coupled with in Vitro Somatic Hypermutation*

    PubMed Central

    Bowers, Peter M.; Neben, Tamlyn Y.; Tomlinson, Geoffery L.; Dalton, Jennifer L.; Altobell, Larry; Zhang, Xue; Macomber, John L.; Wu, Betty F.; Toobian, Rachelle M.; McConnell, Audrey D.; Verdino, Petra; Chau, Betty; Horlick, Robert A.; King, David J.

    2013-01-01

    A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. PMID:23355464

  5. Persistence and avidity maturation of antibodies to A(H1N1)pdm09 in healthcare workers following repeated annual vaccinations.

    PubMed

    Eidem, Synnøve; Tete, Sarah M; Jul-Larsen, Åsne; Hoschler, Katja; Montomoli, Emanuele; Brokstad, Karl A; Cox, Rebecca J

    2015-08-07

    Healthcare workers are at increased risk of influenza infection through direct patient care, particularly during the early stages of a pandemic. Although influenza vaccination is widely recommended in Healthcare workers, data on long-term immunogenicity of vaccination in healthcare workers are lacking. The present study was designed to assess the persistence of the humoral response after pandemic vaccination as well as the impact of repeated annual vaccination in healthcare workers (n=24). Pandemic influenza vaccination resulted in a significant increase in haemagglutination inhibition (HI) antibody titers with 93-100% of subjects achieving protective titers 21-days post each of the three annual vaccinations. Seroprotective antibodies measured by HI, microneutralization and single radial hemolysis assays were present in 77-94% of healthcare workers 6 months post-vaccination. Repeated vaccination resulted in an increased duration of seroprotective antibodies with seroprotective titers increasing from 35-62% 12 months after 2009 pandemic vaccination to 50-75% 12 months after 2010 vaccination. Furthermore, repeated annual vaccination augmented the avidity of influenza-specific IgG antibodies. In conclusion, we have shown that A(H1N1)pdm09 vaccination induces high seroprotective titers that persist for at least 6 months. We demonstrate that repeated vaccination is beneficial to healthcare workers and results in further avidity maturation of vaccine-induced antibodies.

  6. Fusion of C3d molecule with neutralization epitope(s) of hepatitis E virus enhances antibody avidity maturation and neutralizing activity following DNA immunization.

    PubMed

    Yang, Shucai; Wang, Chunling; Fang, Xuefeng; Zhai, Lijie; Dong, Chen; Ding, Lei; Meng, Jihong; Wang, Lixin

    2010-08-01

    Previous studies have identified that a hepatits E virus peptide (HEV-p179), spanning amino acids (aa) 439-617 in the 660-aa protein encoded by open reading frame 2(ORF2) of the Chinese epidemic strain (genotype 4), is the minimal size fragment of conformation-dependent neutralization epitope(s). We report here the successful immunization of mice with DNA vaccines expressing the secreted form of HEV-p179 (fused with a human tissue plasminogen activator (tPA) signal sequence) and the tPA-p179-C3d fusion protein (fused with three tandem copies of the murine complement C3d). Analysis of antibody responses in vaccinated mice revealed that immunizations with tPA-p179-C3d3 DNA vaccine dramatically increased both the level and avidity maturation of antibodies against HEV-p179 compared to p179 and tPA-p179 DNA vaccines. In addition, this increased antibody response correlated with neutralizing titers in a PCR-based cell culture neutralization assay. These results indicate that vaccination with C3d conjugated p179 DNA vaccine enhances antibody responses to HEV, and this approach may be applied to overcome the poor immunogenicity of DNA vaccines to generate HEV neutralizing antibodies. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Comparison of orthogonal chromatographic and lectin-affinity microarray methods for glycan profiling of a therapeutic monoclonal antibody.

    PubMed

    Cook, Matthew C; Kaldas, Sherif J; Muradia, Gauri; Rosu-Myles, Michael; Kunkel, Jeremy P

    2015-08-01

    The N-linked glycosylation of four lots of a marketed human therapeutic monoclonal antibody (mAb) was assessed by three orthogonal chromatographic methods and a commercial lectin microarray. For chromatography, the N-glycans were removed enzymatically from the mAbs using PNGase F. Native glycans were determined by HPAEC-PAD using a panel of 21 N-glycan standards and a multi-stage linear gradient eluent profile for sequential analyses of typical neutral and sialylated glycans in one chromatographic run. The monosaccharide contents of these glycans following acid hydrolysis were confirmed by HPAEC-PAD with monosaccharide standards. Glycosylation analysis by HILIC-FD after stoichiometric labelling with two different fluorescent tags (2-AA and 2-AB) enabled direct quantitation. The 2-AA- and 2-AB-labelled versions of the same glycan standard panel yielded distinctive separation profiles suitable for orthogonal identification of mAb glycans. Glycan profiling with the lectin microarray required partial denaturation of the intact mAbs to expose the sequestered Fc N-glycans. Glycosylation fingerprints were obtained using a fluorescently labelled antibody directed against human IgG Fc. Fluorescence intensities from the fingerprints were deconvoluted with a proprietary algorithm to obtain semi-quantitative "glycan structural class" information. Glycosylation analyses of the four mAb lots by these four methods, which separate and detect oligosaccharides according to different principles, provided complementary and corroboratory qualitative and quantitative information. The predominant N-linked structures were core-fucosylated asialo diantennary structures with varying galactosylation. There were also trace amounts of afucosyl and bisected glycans, but no detectable sialylation by any of the four methods. The therapeutic mAb demonstrated a high degree of consistency in the types and amounts of N-linked glycans in the four lots (<6% CV), and between all four analysis methods

  8. Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4–HLA-DR1 complex

    PubMed Central

    Wang, Xin Xiang; Li, Yili; Yin, Yiyuan; Mo, Min; Wang, Qian; Gao, Wei; Wang, Lili; Mariuzza, Roy A.

    2011-01-01

    Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4–MHC class II interaction is its exceptionally low affinity, which ranges from KD = ∼200 μM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell–cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with KD = 8.8 μM, compared with >400 μM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and β2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development. PMID:21900604

  9. Microbiota-Driven Immune Cellular Maturation Is Essential for Antibody-Mediated Adaptive Immunity to Staphylococcus aureus Infection in the Eye

    PubMed Central

    Zaidi, Tanweer; Zaidi, Tauqeer; Cywes-Bentley, Colette; Lu, Roger; Priebe, Gregory P.

    2014-01-01

    As an immune-privileged site, the eye, and particularly the outer corneal surface, lacks resident mature immune effector cells. Physical barriers and innate mediators are the best-described effectors of immunity in the cornea. When the barriers are breached, infection can result in rapid tissue destruction, leading to loss of visual acuity and frank blindness. To determine the cellular and molecular components needed for effective adaptive immunity on the corneal surface, we investigated which immune system effectors were required for protection against Staphylococcus aureus corneal infections in mice, which are a serious cause of human eye infections. Both systemically injected and topically applied antibodies to the conserved cell surface polysaccharide poly-N-acetylglucosamine (PNAG) were effective at mediating reductions in corneal pathology and bacterial levels. Additional host factors impacting protection included intercellular adhesion molecule 1 (ICAM-1)-dependent polymorphonuclear leukocyte (PMN) recruitment, functional CD4+ T cells, signaling via the interleukin-17 (IL-17) receptor, and IL-22 production. In germfree mice, there was no protective efficacy of antibody to PNAG due to the lack of LY6G+ inflammatory cell coeffector recruitment to the cornea. Protection was manifest after 3 weeks of exposure to conventional mice and acquisition of a resident microbiota. We conclude that in the anterior eye, ICAM-1-mediated PMN recruitment to the infected cornea along with endogenous microbiota-matured CD4+ T cells producing both IL-17 and IL-22 is required for antibody to PNAG to protect against S. aureus infection. PMID:24914214

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

    PubMed Central

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

    2012-01-01

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

  11. Monoclonal antibody affinity purification of a Leishmania membrane glycoprotein and its inhibition of leishmania-macrophage binding.

    PubMed Central

    Chang, C S; Chang, K P

    1986-01-01

    Specific monoclonal antibody coupled to Affi-Gel 10 was used to purify a major membrane glycoprotein of Leishmania mexicana amazonensis, one of a group of parasitic protozoa that specifically infect mammalian macrophages. Immobilized antigen was eluted at a 34% efficiency with buffers at either pH 2.5 or 11 or with MgCl2, but only the antigen eluted under basic conditions could be readsorbed to the immunobeads. Sephacryl S-300 gel filtration of the purified antigen gave a single peak of protein estimated to have a molecular mass of 400 kDa. However, NaDodSO4/polyacrylamide gel electrophoresis showed a single band of this protein with an apparent molecular mass of 63 kDa. The antigen is an N-linked glycoprotein, as indicated by its increase in electrophoretic mobility after treatment with endoglycosidase H and by its binding to lentil lectin-Sepharose, elutable with methyl alpha-D-mannoside and methyl alpha-D-glucoside. Purified antigen inhibits the binding of leishmania cells to macrophages by 50%, suggesting that it may play a role in the process of infection. Images PMID:3079902

  12. Chronic treatment of (+)-methamphetamine-induced locomotor effects in rats using one or a combination of two high affinity anti-methamphetamine monoclonal antibodies

    PubMed Central

    Hambuchen, Michael D.; Rüedi-Bettschen, Daniela; Gunnell, Melinda G.; Hendrickson, Howard; Owens, S. Michael

    2016-01-01

    ABSTRACT We hypothesized that treatment of methamphetamine (METH) effects with a mixture of 2 high affinity anti-METH monoclonal antibodies (mAb) with differing molecular recognition for METH-like structures could increase efficacy compared to treatment with a single mAb. The antibodies studied were mAb7F9 (METH and amphetamine [AMP] KD = 7.7 and 270 nM) and mAb4G9 (16 nM and 110 nM, respectively) in a 50:50 mixture. Adult male Sprague Dawley Rats were treated with iv saline or a loading dose of mAb7F9-mAb4G9 (141 mg/kg of each mAb) followed by 2 weekly doses (70.5 mg/kg total) on days 7 and 14. METH challenge doses (0.56 mg/kg) were administered 4 hrs and 3 days after each mAb7F9-mAb4G9 treatment, and 7 days after the final treatment (day 21). Locomotor activity (0–4 hrs) and serum METH and AMP concentrations (at 5 hrs) were measured after each METH challenge. MAb7F9-mAb4G9 treatment significantly reduced the duration of locomotor activity after 6 of the 7 METH doses (P < 0.05) and significantly increased serum METH and AMP concentrations. Administering three-fold higher METH doses (1.68 mg/kg) on days 24 and 28 showed mAb7F9-mAb4G9 treatment had negligible effects on the duration of METH-induced locomotor activity. These data were then compared to previous monotherapy data. While mAb7F9-mAb4G9 therapy inhibited the effects of multiple METH challenge doses, the inhibition was not as profound or as long lasting as the effects of mAb7F9 treatment alone. These data demonstrate the importance of both mAb affinity and specificity in the production of effective, long-lasting anti-METH mAb therapies. PMID:27163775

  13. Therapeutic antibodies: Discovery and development using the ProteOn XPR36 biosensor interaction array system.

    PubMed

    Bronner, Vered; Denkberg, Galit; Peled, Mira; Elbaz, Yael; Zahavi, Efrat; Kasoto, Harel; Reiter, Yoram; Notcovich, Ariel; Bravman, Tsafrir

    2010-11-15

    Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection. Copyright 2010 Elsevier Inc. All rights reserved.

  14. 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

  15. Requirement for both H and L chain V regions, VH and VK joining amino acids, and the unique H chain D region for the high affinity binding of an anti-phosphotyrosine antibody.

    PubMed

    Ruff-Jamison, S; Glenney, J R

    1993-04-15

    Sequence analysis of a panel of antibodies to phosphotyrosine revealed predominant H and L chain V regions in the immune response and a unique D segment in the Py20 mAb, which exhibits a high affinity for phosphotyrosine. In order to determine the influence of somatic diversity on the high affinity binding of Py20, H and L chain V regions were expressed in Escherichia coli as an Fv dimer. Whereas the H or L chain V regions of Py20 alone were unable to bind phosphotyrosine, the Fv binds phosphotyrosine with an affinity comparable with the intact IgG as determined by fluorescence quenching experiments (1.55 x 10(-7) M vs 1.25 x 10(-7) M, respectively). Substitution of the Py20 V regions with other IgG V regions that differed greatly in sequence abolished binding. A high affinity Py20-combining site was dependent on the presence of the unique D-D segment. Replacement of the Py20 D-D region with a single homologous D region resulted in a decrease in affinity (5.9 x 10(-7) M). Substitution of this D-D region for the D region of another anti-phosphotyrosine antibody that is known to bind phosphotyrosine weakly (1 x 10(-3) M) conferred high affinity binding. Removal of three tyrosines from the first of the two D regions was accompanied by a fivefold reduction in affinity for phosphotyrosine. In addition, changing the VK and VH junctional amino acids resulted in a complete loss of binding. Therefore, the formation of the high affinity Py20 combining site requires both a H and L chain that are similar in sequence to those of Py20 including the unique D region and the junctional amino acids.

  16. Involvement of spleen components of mature fowl in the primary and secondary humoral antibody response following experimental EDS'76 virus infection.

    PubMed

    van Eck, J H

    1986-04-01

    Cellular changes in spleens of mature fowl in relation to both the primary and secondary humoral antibody response following experimental EDS'76 virus infection were studied. The influence of splenectomy on humoral antibody response was also examined. Experimental fowl had been naturally infected with fowl adenovirus (FAV) but did not possess precipitins to these viruses at the time of EDS'76 virus infection. Since EDS'76 infection provokes a recall of the group antibody to FAV, this infection simultaneously induces a primary response against EDS'76 virus and a secondary response due to the recall of the group antibody to FAV. HI and precipitating antibody to EDS'76 virus (primary response) were first detected at 6 and 8 days p.i. respectively. Curves of HI, precipitating and neutralising antibody titres were biphasic; the first peak (IgM peak) occurred at 10-11 days p.i., the second (IgG peak) at 16-28 days p.i. Precipitating antibodies to FAV (secondary response) were demonstrated from 4 days p.i. The curve of these antibody titres was also biphasic, with peaks at the same times as in the primary response. Based on HI and AGP testing of primary and secondary immune response in both splenectomised and non-splenectomised fowl it is concluded that in the primary response the spleen of the adult fowl is involved significantly in only IgM secretion, while in the secondary response it is likely that both IgM and IgG are secreted in considerable amounts. Clusters of lymphoblasts and plasmablasts were observed at 3 days p.i. in the red pulp. It is very likely that antigen-antibody complexes are formed from that time and circulate bound to the surface of lymphocytes. These antigen-loaded lymphocytes are 'picked up' from the blood stream by -red pulp macrophages, leading to enhanced formation of lymphoblasts in the red pulp. Great numbers of these cells (which are very probably IgM secreting cells) were present on days 6 and 7 p.i., but were no longer detectable after day

  17. Efficacy, but not antibody titer or affinity, of a heroin hapten conjugate vaccine correlates with increasing hapten densities on tetanus toxoid, but not on CRM197 carriers.

    PubMed

    Jalah, Rashmi; Torres, Oscar B; Mayorov, Alexander V; Li, Fuying; Antoline, Joshua F G; Jacobson, Arthur E; Rice, Kenner C; Deschamps, Jeffrey R; Beck, Zoltan; Alving, Carl R; Matyas, Gary R

    2015-06-17

    Vaccines against drugs of abuse have induced antibodies in animals that blocked the biological effects of the drug by sequestering the drug in the blood and preventing it from crossing the blood-brain barrier. Drugs of abuse are too small to induce antibodies and, therefore, require conjugation of drug hapten analogs to a carrier protein. The efficacy of these conjugate vaccines depends on several factors including hapten design, coupling strategy, hapten density, carrier protein selection, and vaccine adjuvant. Previously, we have shown that 1 (MorHap), a heroin/morphine hapten, conjugated to tetanus toxoid (TT) and mixed with liposomes containing monophosphoryl lipid A [L(MPLA)] as adjuvant, partially blocked the antinociceptive effects of heroin in mice. Herein, we extended those findings, demonstrating greatly improved vaccine induced antinociceptive effects up to 3% mean maximal potential effect (%MPE). This was obtained by evaluating the effects of vaccine efficacy of hapten 1 vaccine conjugates with varying hapten densities using two different commonly used carrier proteins, TT and cross-reactive material 197 (CRM197). Immunization of mice with these conjugates mixed with L(MPLA) induced very high anti-1 IgG peak levels of 400-1500 μg/mL that bound to both heroin and its metabolites, 6-acetylmorphine and morphine. Except for the lowest hapten density for each carrier, the antibody titers and affinity were independent of hapten density. The TT carrier based vaccines induced long-lived inhibition of heroin-induced antinociception that correlated with increasing hapten density. The best formulation contained TT with the highest hapten density of ≥30 haptens/TT molecule and induced %MPE of approximately 3% after heroin challenge. In contrast, the best formulation using CRM197 was with intermediate 1 densities (10-15 haptens/CRM197 molecule), but the %MPE was approximately 13%. In addition, the chemical synthesis of 1, the optimization of the conjugation

  18. Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA.

    PubMed

    Stanker, Larry H; Merrill, Paul; Scotcher, Miles C; Cheng, Luisa W

    2008-07-20

    Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The 'gold standard' for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD(50)) of approximately 10 pg mL(-1). Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDS-PAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDS-PAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay.

  19. Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy.

    PubMed

    Unkauf, Tobias; Miethe, Sebastian; Fühner, Viola; Schirrmann, Thomas; Frenzel, André; Hust, Michael

    2016-01-01

    Antibody phage display is an in vitro technology to generate recombinant antibodies. In particular for pathogens like viruses or toxins, antibody phage display is an alternative to hybridoma technology, since it circumvents the limitations of the immune system. Phage display allows the generation of human antibodies from naive antibody gene libraries when either immunized patients are not available or immunization is not ethically feasible. This technology also allows the construction of immune libraries to select in vivo affinity matured antibodies if immunized patients or animals are available.In this review, we describe the generation of human and human-like antibodies from naive antibody gene libraries and antibodies from immune antibody gene libraries. Furthermore, we give an overview about phage display derived recombinant antibodies against viruses and toxins for diagnostics and therapy.

  20. Microbiota-driven immune cellular maturation is essential for antibody-mediated adaptive immunity to Staphylococcus aureus infection in the eye.

    PubMed

    Zaidi, Tanweer; Zaidi, Tauqeer; Cywes-Bentley, Colette; Lu, Roger; Priebe, Gregory P; Pier, Gerald B

    2014-08-01

    As an immune-privileged site, the eye, and particularly the outer corneal surface, lacks resident mature immune effector cells. Physical barriers and innate mediators are the best-described effectors of immunity in the cornea. When the barriers are breached, infection can result in rapid tissue destruction, leading to loss of visual acuity and frank blindness. To determine the cellular and molecular components needed for effective adaptive immunity on the corneal surface, we investigated which immune system effectors were required for protection against Staphylococcus aureus corneal infections in mice, which are a serious cause of human eye infections. Both systemically injected and topically applied antibodies to the conserved cell surface polysaccharide poly-N-acetylglucosamine (PNAG) were effective at mediating reductions in corneal pathology and bacterial levels. Additional host factors impacting protection included intercellular adhesion molecule 1 (ICAM-1)-dependent polymorphonuclear leukocyte (PMN) recruitment, functional CD4(+) T cells, signaling via the interleukin-17 (IL-17) receptor, and IL-22 production. In germfree mice, there was no protective efficacy of antibody to PNAG due to the lack of LY6G(+) inflammatory cell coeffector recruitment to the cornea. Protection was manifest after 3 weeks of exposure to conventional mice and acquisition of a resident microbiota. We conclude that in the anterior eye, ICAM-1-mediated PMN recruitment to the infected cornea along with endogenous microbiota-matured CD4(+) T cells producing both IL-17 and IL-22 is required for antibody to PNAG to protect against S. aureus infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. 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.

  2. The low affinity IgG receptor Fc gamma RIIB contributes to the binding of the mast cell specific antibody, mAb BGD6.

    PubMed

    Guiraldelli, Michel F; Berenstein, Elsa H; Grodzki, Ana Cristina G; Siraganian, Reuben P; Jamur, Maria Celia; Oliver, Constance

    2008-04-01

    The mast cell specific monoclonal antibody, mAb BGD6, is a mast cell lineage marker [Jamur, M.C., Grodzki, A.C., Berenstein, E.H., Hamawy, M.M., Siraganian, R.P., Oliver, C., 2005. Identification and characterization of undifferentiated mast cells in mouse bone marrow. Blood 105, 4282-4289]. In rat basophilic leukemia (RBL-2H3) cells, mAb BGD6 precipitates cell-surface proteins of approximately 110 and 40-60 kDa. An expression cloning strategy was used to identify proteins that interact with mAb BGD6. A RBL-2H3 cDNA library in plasmids was transfected into PEAK cells, which do not bind mAb BGD6, and positive cells were selected with mAb BGD6. The plasmids recovered from the positive cells were amplified; retransfected into PEAK cells and after several screening cycles a positive clone was identified. This clone showed almost complete identity to Fc gamma RIIB (CD32), the low affinity IgG receptor. However, in contrast to the sequence in GenBank, this clone had an insert of 141 bp which codes for a longer isoform of this molecule with an extra 47 aa in its cytoplasmic domain. In RBL-2H3 cells both isoforms were expressed, with higher expression of the shorter form. The mechanism of binding of mAB BGD6 on both RBL-2H3 and CD32 transfected PEAK cells was then examined. Intact mAb BGD6 bound to both RBL-2H3 and CD32 expressing PEAK cells, but F(ab')(2) fragments bound only to RBL-2H3 cells demonstrating that mAb BGD6 binds to Fc gamma RIIB only through its Fc portion. On RBL-2H3 cells, the Fab of an anti-CD32 mAb partially inhibited the binding of intact mAb BGD6. The binding pattern of mAb BGD6 inhibited with anti-CD32 resembled that of the F(ab')(2) fragment of the antibody suggesting that the Fc portion of mAb BGD6 contributes to its binding on cells that have Fc gamma RIIB. These results are consistent with a model where mAb BGD6 binds through its Fab portion to a approximately 110 kDa protein and the Fc tail interacts with Fc gamma RIIB (CD32).

  3. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms.

    PubMed

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M

    2016-01-01

    Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies' potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.

  4. Interleukin-15 Increases Vaccine Efficacy through a Mechanism Linked to Dendritic Cell Maturation and Enhanced Antibody Titers

    DTIC Science & Technology

    2007-11-26

    Enhanced Antibody Titers Kamal U. Saikh,* Teri L. Kissner, Steven Nystrom, Gordon Ruthel, and Robert G. Ulrich Department of Immunology, Army Medical...Katsikis. 2005. Interleukin-15 increases effector memory CD8 T cells and NK cells in simian immunodeficiency virus -infected macaques. J. Virol. 79: 4877...vaccinia viruses express- ing IL-15 but not IL-2 induces long-lasting cellular immunity. Proc. Natl. Acad. Sci. USA 100:3392–3397. 24. Ohteki, T. 2002

  5. Developing recombinant antibodies for biomarker detection

    SciTech Connect

    Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.

    2010-10-01

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

  6. OptMAVEn – A New Framework for the de novo Design of Antibody Variable Region Models Targeting Specific Antigen Epitopes

    PubMed Central

    Li, Tong; Pantazes, Robert J.; Maranas, Costas D.

    2014-01-01

    Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn). OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced immunogenicity. PMID

  7. Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

    PubMed

    Fennell, B J; Darmanin-Sheehan, A; Hufton, S E; Calabro, V; Wu, L; Müller, M R; Cao, W; Gill, D; Cunningham, O; Finlay, W J J

    2010-07-09

    The shark antigen-binding V(NAR) domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V(NAR) shares many of the properties of the well-characterised single-domain camelid V(H)H but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V(NAR) 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V(NAR) domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K(D) of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K(D) yet reported for V(NAR)-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V(NAR) paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V(NAR) database. This study allowed us to identify, for the first time, both V(NAR)-specific and V(NAR)/Ig V(L)/TCR V(alpha) overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V(NAR)-specific hallmarks align precisely with previously defined mutational 'cold spots' in

  8. Anti-citrullinated protein antibodies promote apoptosis of mature human Saos-2 osteoblasts via cell-surface binding to citrullinated heat shock protein 60.

    PubMed

    Lu, Ming-Chi; Yu, Chia-Li; Yu, Hui-Chun; Huang, Hsien-Bin; Koo, Malcolm; Lai, Ning-Sheng

    2016-01-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) react with osteoblast surface citrullinated proteins and affect cell function, leading to joint damage in patients with rheumatoid arthritis (RA). First, we purified ACPAs by cyclic citrullinated peptide (CCP)-conjugated affinity column chromatography. The cognate antigens of ACPAs on Saos-2 cells, a sarcoma osteogenic cell line generated from human osteoblasts, were probed by ACPAs, and the reactive bands were analyzed using proteomic analyses. We found that ACPAs bind to Saos-2 cell membrane, and several protein candidates, including HSP60, were identified. We then cloned and purified recombinant heat shock protein 60 (HSP60) and citrullinated HSP60 (citHSP60) and investigated the effect of ACPAs on Saos-2 cell. We confirmed that HSP60 obtained from Saos-2 cell membrane were citrullinated and reacted with ACPAs, which induces Saos-2 cells apoptosis via binding to surface-expressed citHSP60 through Toll-like receptor 4 signaling. ACPAs promoted interleukin (IL)-6 and IL-8 expression in Saos-2 cells. Finally, sera from patients with RA and healthy controls were examined for their titers of anti-HSP60 and anti-citHSP60 antibodies using an enzyme-linked immunosorbent assay. The radiographic change in patients with RA was evaluated using the Genant-modified Sharp scoring system. Patients with RA showed higher sera titers of anti-citHSP60, but not anti-HSP60, antibodies when compared with controls. In addition, the anti-citHSP60 level was positively associated with increased joint damage in patients with RA. In conclusion, Saos-2 cell apoptosis was mediated by ACPAs via binding to cell surface-expressed citHSP60 and the titer of anti-citHSP60 in patients with RA positively associated with joint damage. Copyright © 2015 Elsevier GmbH. All rights reserved.

  9. A study of the `termination' of tolerance to BSA with DNP—BSA in rabbits: relative affinities of the antibodies for the immunizing and the paralysing antigens

    PubMed Central

    Paul, W. E.; Siskind, G. W.; Benacerraf, B.

    1967-01-01

    Rabbits rendered immunologically tolerant to BSA produced antibodies capable of precipitating BSA following immunization with DNP—BSA. Antigen binding studies using purified `anti-BSA' antibodies produced by tolerant animals indicated that these antibodies bound DNP—BSA with far greater avidity than BSA. It was concluded that a true termination of tolerance had not occurred. The implications of the results with regard to induction of antibody formation and of tolerance are discussed. PMID:4952101

  10. The challenges of modelling antibody repertoire dynamics in HIV infection

    DOE PAGES

    Luo, Shishi; Perelson, Alan S.

    2015-07-20

    Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selectionmore » and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.« less

  11. The challenges of modelling antibody repertoire dynamics in HIV infection

    SciTech Connect

    Luo, Shishi; Perelson, Alan S.

    2015-07-20

    Antibody affinity maturation by somatic hypermutation of B-cell immunoglobulin variable region genes has been studied for decades in various model systems using well-defined antigens. While much is known about the molecular details of the process, our understanding of the selective forces that generate affinity maturation are less well developed, particularly in the case of a co-evolving pathogen such as HIV. Despite this gap in understanding, high-throughput antibody sequence data are increasingly being collected to investigate the evolutionary trajectories of antibody lineages in HIV-infected individuals. Here, we review what is known in controlled experimental systems about the mechanisms underlying antibody selection and compare this to the observed temporal patterns of antibody evolution in HIV infection. In addition, we describe how our current understanding of antibody selection mechanisms leaves questions about antibody dynamics in HIV infection unanswered. Without a mechanistic understanding of antibody selection in the context of a co-evolving viral population, modelling and analysis of antibody sequences in HIV-infected individuals will be limited in their interpretation and predictive ability.

  12. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  13. Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions.

    PubMed

    Ramírez, Juan C; Tapia, Esther; Esteban, Mariano

    2002-05-01

    The WHO smallpox eradication program was concluded 21 years ago and the non-vaccinated population is now at risk of poxvirus infections, either by contact with monkeypox or through bioterrorism. Since drugs specific against poxvirus infections are limited, neutralizing monoclonal antibodies (mAbs) that are effective in vivo may be an important tool in controlling poxvirus infections. To this end, we studied the efficacy of the mAb C3, reactive against the trimeric 14-kDa protein of vaccinia virus (VV) localized in the membrane of the intracellular form of mature virus, for its ability to neutralize VV infection in mice. The results show that prophylactic as well as therapeutic administration of mAb C3 can be an effective means of control of VV replication within the host. The interval of antibody efficacy following a single administration, before and after VV inoculation, has been defined. This study reinforces the notion that neutralizing mAbs should be developed to control health-related human infections by poxviruses.

  14. Spiramycin treatment of Toxoplasma gondii infection in pregnant women impairs the production and the avidity maturation of T. gondii-specific immunoglobulin G antibodies.

    PubMed

    Meroni, V; Genco, F; Tinelli, C; Lanzarini, P; Bollani, L; Stronati, M; Petersen, E

    2009-10-01

    The aim of the study was to evaluate the influence of treatment with spiramycin on the increase of immunoglobulin G (IgG) titers and IgG avidity indexes (AI) in pregnant women with seroconversion from the beginning of therapy until delivery and after delivery. This group was compared with adult patients with recently acquired untreated toxoplasmosis. One hundred four samples from 32 pregnant women with seroconversion for toxoplasmosis and/or very low IgG AI were followed from the beginning of therapy with spiramycin until delivery. Twenty-nine women were further followed some months after delivery and interruption of therapy. Thirty-eight samples from 16 untreated, nonpregnant patients were evaluated as the control group. The Toxoplasma gondii-specific IgG antibody and the T. gondii-specific IgG AI were significantly delayed in pregnant women receiving therapy compared to nonpregnant, untreated controls, and the findings were consistent with the results of assays from two different manufacturers. The T. gondii-specific IgG AI increased in pregnant women after they gave birth. Avidity maturation is delayed during pregnancy and treatment, and low-avidity antibodies in pregnant women within 3 to 4 months cannot be taken as a sign of infection.

  15. MicroRNA-155 controls affinity-based selection by protecting c-MYC+ B cells from apoptosis

    PubMed Central

    Nakagawa, Rinako; Leyland, Rebecca; Meyer-Hermann, Michael; Lu, Dong; Turner, Martin; Arbore, Giuseppina; Phan, Tri Giang; Brink, Robert; Vigorito, Elena

    2015-01-01

    The production of high-affinity antibodies by B cells is essential for pathogen clearance. Antibody affinity for antigen is increased through the affinity maturation in germinal centers (GCs). This is an iterative process in which B cells cycle between proliferation coupled with the acquisition of mutations and antigen-based positive selection, resulting in retention of the highest-affinity B cell clones. The posttranscriptional regulator microRNA-155 (miR-155) is critical for efficient affinity maturation and the maintenance of the GCs; however, the cellular and molecular mechanism by which miR-155 regulates GC responses is not well understood. Here, we utilized a miR-155 reporter mouse strain and showed that miR-155 is coexpressed with the proto-oncogene encoding c-MYC in positively selected B cells. Functionally, miR-155 protected positively selected c-MYC+ B cells from apoptosis, allowing clonal expansion of this population, providing an explanation as to why Mir155 deletion impairs affinity maturation and promotes the premature collapse of GCs. We determined that miR-155 directly inhibits the Jumonji family member JARID2, which enhances B cell apoptosis when overexpressed, and thereby promotes GC B cell survival. Our findings also suggest that there is cooperation between c-MYC and miR-155 during the normal GC response, a cooperation that may explain how c-MYC and miR-155 can collaboratively function as oncogenes. PMID:26657861

  16. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    SciTech Connect

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R.

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  17. Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

    PubMed

    Oren, Ravit; Hod-Marco, Moran; Haus-Cohen, Maya; Thomas, Sharyn; Blat, Dan; Duvshani, Nerri; Denkberg, Galit; Elbaz, Yael; Benchetrit, Fabrice; Eshhar, Zelig; Stauss, Hans; Reiter, Yoram

    2014-12-01

    Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αβ-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αβ-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αβ-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

  18. Folding, assembly, and intracellular trafficking of the human immunodeficiency virus type 1 envelope glycoprotein analyzed with monoclonal antibodies recognizing maturational intermediates.

    PubMed Central

    Otteken, A; Earl, P L; Moss, B

    1996-01-01

    Monoclonal antibodies (MAbs) that bind linear or conformational epitopes on monomeric or oligomeric human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins were screened for their recognition of maturational intermediates. On the basis of reactivities with gp160 at different times after pulse-labeling, the MAbs were sorted into groups that exhibited binding which was immediate and constant, immediate but transient, delayed, late, or very late. This grouping was consistent with the selectivity of the MAbs for structural features of gp160. Thus, a MAb to the V3 loop reacted with envelope proteins at all times, in accord with the relative conformational independence and accessibility of the epitope. Several MAbs that preferentially react with monomeric gp160 exhibited diminished binding after the pulse. A 10-min tag occurred before gp160 reacted with conformational MAbs that inhibited CD4 binding. The availability of epitopes for other conformational MAbs, including some that react equally with monomeric and oligomeric gp160 and some that react better with oligomeric forms, was half-maximal in 30 min and closely followed the kinetics of gp160 oligomerization. Remarkably, there was a 1- to 2-h delay before gp160 reacted with stringent oligomer-specific MAbs. After 4 h, approximately 20% of the gp160 was recognized by these MAbs. Epitopes recognized by monomerspecific or CD4-blocking MAbs but not by oligomer-dependent MAbs were present on gp160 molecules associated with the molecular chaperone BiP/GRP78. MAbs with a preference for monomers reacted with recombinant or HIV-1 envelope proteins in the endoplasmic reticulum, whereas the oligomer-specific MAbs recognized them in the Golgi complex. Additional information regarding gp160 maturation and intracellular trafficking was obtained by using brefeldin A, dithiothreitol, and a low temperature. PMID:8648672

  19. Anti-IgD antibody attenuates collagen-induced arthritis by selectively depleting mature B-cells and promoting immune tolerance.

    PubMed

    Nguyen, Tue G; Little, Christopher B; Yenson, Vanessa M; Jackson, Christopher J; McCracken, Sharon A; Warning, Julia; Stevens, Veronica; Gallery, Eileen G; Morris, Jonathan M

    2010-08-01

    Membrane (m)IgD forms a major part of B-cell receptor complexes. Its wider role in the immune system has been enigmatic. Stimulation of mIgD with an antibody (anti-IgD) can activate B-cells and elicit a broad immune response in vivo. Given the role of B-cells in autoimmune diseases and the profound impact of anti-IgD on B-cells, the potential effects of anti-IgD on autoimmune conditions are intriguing and yet to be explored. Here we report a novel therapeutic effect of anti-IgD in the collagen-induced arthritis (CIA) mouse model. Administration of anti-IgD at the onset of early clinical symptoms as a therapeutic intervention, but not as a prophylactic treatment, significantly ameliorates disease severity and joint pathology. Anti-IgD treatment selectively depletes mature B cells while it spares regulatory B-cell subsets. This results in a significant reduction of autoantibody levels but does not affect antibody responses to a T-cell-dependent antigen. Therapeutic treatment with anti-IgD increases the numbers of regulatory B-cells and regulatory T-cells whilst it augments adaptive Th1/Th2 responses in vivo. In human PBMC samples, anti-IgD also promotes adaptive Th1/Th2 responses and modulates the innate responses toward an anti-inflammatory Th2-biased response. Collectively, anti-IgD treatment may offer a selective approach to B-cell depletion that also promotes immune tolerance and anti-inflammatory tendencies without compromising the general adaptive B-cell and T-cell responses. The multiple mechanisms of action by anti-IgD treatment suggest a wider clinical application for a number of chronic inflammatory and autoimmune conditions.

  20. High affinity anti-BSEP antibodies after liver transplantation for PFIC-2 - Successful treatment with immunoadsorption and B-cell depletion.

    PubMed

    Kubitz, Ralf; Dröge, Carola; Kluge, Stefanie; Stindt, Jan; Stross, Claudia; Häussinger, Dieter; Flechtenmacher, Christa; Wenning, Daniel; Teufel, Ulrike; Schmitt, Claus Peter; Engelmann, Guido

    2016-11-01

    PFIC due to BSEP mutations (PFIC type 2) often necessitates OLT. It has recently been recognized that some PFIC-2 patients develop phenotypic disease recurrence post-OLT due to the appearance of anti-BSEP antibodies. Here, we describe a boy who became cholestatic four yr after OLT during modification of immunosuppression. Canalicular antibody deposits were detected in biopsies of the transplant and antibodies specifically reacting with BSEP were identified at high titers in his serum. These antibodies bound extracellular epitopes of BSEP and inhibited BS transport and were assumed to cause disease recurrence. Consequently, anti-BSEP antibody depletion was pursued by IA and B-cell depletion by anti-CD20 antibodies (rituximab) along with a switch of immunosuppression. This treatment resulted in prolonged relief of symptoms. Depletion of pathogenic anti-BSEP antibodies causing AIBD after OLT in PFIC-2 patients should be considered as a central therapeutic goal. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments

    PubMed Central

    Li, Tong; Tracka, Malgorzata B.; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J.; Livesay, Dennis R.

    2015-01-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation. PMID:26132144

  2. Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments.

    PubMed

    Li, Tong; Tracka, Malgorzata B; Uddin, Shahid; Casas-Finet, Jose; Jacobs, Donald J; Livesay, Dennis R

    2015-07-01

    The effects of somatic mutations that transform polyspecific germline (GL) antibodies to affinity mature (AM) antibodies with monospecificity are compared among three GL-AM Fab pairs. In particular, changes in conformational flexibility are assessed using a Distance Constraint Model (DCM). We have previously established that the DCM can be robustly applied across a series of antibody fragments (VL to Fab), and subsequently, the DCM was combined with molecular dynamics (MD) simulations to similarly characterize five thermostabilizing scFv mutants. The DCM is an ensemble based statistical mechanical approach that accounts for enthalpy/entropy compensation due to network rigidity, which has been quite successful in elucidating conformational flexibility and Quantitative Stability/Flexibility Relationships (QSFR) in proteins. Applied to three disparate antibody systems changes in QSFR quantities indicate that the VH domain is typically rigidified, whereas the VL domain and CDR L2 loop become more flexible during affinity maturation. The increase in CDR H3 loop rigidity is consistent with other studies in the literature. The redistribution of conformational flexibility is largely controlled by nonspecific changes in the H-bond network, although certain Arg to Asp salt bridges create highly localized rigidity increases. Taken together, these results reveal an intricate flexibility/rigidity response that accompanies affinity maturation.

  3. 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

  4. 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.

  5. Experimental and theoretical investigation of effect of spacer arm and support matrix of synthetic affinity chromatographic materials for the purification of monoclonal antibodies.

    PubMed

    Zamolo, Laura; Salvalaglio, Matteo; Cavallotti, Carlo; Galarza, Benedict; Sadler, Chris; Williams, Sharon; Hofer, Stefan; Horak, Jeannie; Lindner, Wolfgang

    2010-07-29

    The aim of this study was to elucidate the influence of each material component-the support, the spacer, and the surface chemistry-on the overall material performance of an affinity type purification media for the capture of immunoglobulin G (IgG). Material properties were investigated in terms of an experimental evaluation using affinity chromatography as well as computer modeling. The biomimetic triazine-based A2P affinity ligand was chosen as a fixed point, while spacer and support were varied. The investigated spacers were 1-2-diaminoethane (2LP), 1,3-propanedithiol (SS3), 3,6-dioxo-1,8-octanedithiol (DES), and a 1,4-substituted [1,2,3]-triazole spacer (TRZ). The support media considered were the agarose (AG) resins, PuraBead, the polyvinylether, Fractoprep, the polymethacrylate, Fractogel, and the porous silica, Fractosil. All materials were tested with pure IgG standard solution, with a mock feed solution as well as real cell culture supernatant. The interaction between IgG and A2P linked through the investigated spacers to AG was studied using molecular dynamics. The effect of a modification of the support chemical structure or of the protein-ligand binding site on the material performances was studied through target oriented simulations. Dynamic binding experiments (DBC) revealed that the performances of materials containing 2LP spacers were significantly decreased in the presence of Pluronic F68. The simulations indicated that this is probably determined by the establishment of intermolecular interactions between the 2LP charged amino group and the ether oxygen of Pluronic F68. The spacer giving the highest IgG dynamic binding capacity when Pluronic F68 was present in the feed was TRZ. The simulations showed that, among the investigated spacers, TRZ is the only one that prevents the adsorption of A2P on the support surface, thus suggesting that the mobility and lack of interaction of the ligand with the support is an important property for an affinity

  6. A dynamic mathematical model for monoclonal antibody N-linked glycosylation and nucleotide sugar donor transport within a maturing Golgi apparatus.

    PubMed

    Jimenez del Val, Ioscani; Nagy, Judit M; Kontoravdi, Cleo

    2011-01-01

    Monoclonal antibodies (mAbs) are one of the most important products of the biopharmaceutical industry. Their therapeutic efficacy depends on the post-translational process of glycosylation, which is influenced by manufacturing process conditions. Herein, we present a dynamic mathematical model for mAb glycosylation that considers cisternal maturation by approximating the Golgi apparatus to a plug flow reactor and by including recycling of Golgi-resident proteins (glycosylation enzymes and transport proteins [TPs]). The glycosylation reaction rate expressions were derived based on the reported kinetic mechanisms for each enzyme, and transport of nucleotide sugar donors [NSDs] from the cytosol to the Golgi lumen was modeled to serve as a link between glycosylation and cellular metabolism. Optimization-based methodologies were developed for estimating unknown enzyme and TP concentration profile parameters. The resulting model is capable of reproducing glycosylation profiles of commercial mAbs. It can further reproduce the effect gene silencing of the FucT glycosylation enzyme and cytosolic NSD depletion have on the mAb oligosaccharide profile. All novel elements of our model are based on biological evidence and generate more accurate results than previous reports. We therefore believe that the improvements contribute to a more detailed representation of the N-linked glycosylation process. The overall results show the potential of our model toward evaluating cell engineering strategies that yield desired glycosylation profiles. Additionally, when coupled to cellular metabolism, this model could be used to assess the effect of process conditions on glycosylation and aid in the design, control, and optimization of biopharmaceutical manufacturing processes.

  7. How to assess the binding strength of antibodies elicited by vaccination against HIV and other viruses

    PubMed Central

    Klasse, P.J.

    2016-01-01

    Summary Vaccines that protect against viral infections generally induce neutralizing antibodies. When vaccines are evaluated, the need arises to assess the affinity maturation of the antibody responses. Binding titers of polyclonal sera depend not only on the affinities of the constituent antibodies but also on their individual concentrations, which are difficult to ascertain. Therefore an assay based on chaotrope disruption of antibody-antigen complexes was designed for measuring binding strength. This assay works well with many viral antigens but gives differential results depending on the conformational dependence of epitopes on complex antigens such as the envelope glycoprotein of HIV-1. Kinetic binding assays might offer alternatives, since they can measure average off-rate constants for polyclonal antibodies in a serum. Here, potentials and fallacies of these techniques are discussed. PMID:26641943

  8. Extraction of BoNT/A, /B, /E, and /F with a Single, High Affinity Monoclonal Antibody for Detection of Botulinum Neurotoxin by Endopep-MS

    PubMed Central

    Kalb, Suzanne R.; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Baudys, Jakub; Smith, Theresa J.; Marks, James D.; Smith, Leonard A.; Pirkle, James L.; Barr, John R.

    2010-01-01

    Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies. PMID:20808925

  9. An asymmetric antibody repertoire is shaped between plasmablasts and plasma cells after secondary immunization with (4-hydroxy-3-nitrophenyl)acetyl chicken γ-globulin.

    PubMed

    Tashiro, Yasuyuki; Murakami, Akikazu; Goizuka, Ryo; Shimizu, Takeyuki; Kishimoto, Hidehiro; Azuma, Takachika

    2015-12-01

    Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.

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

    PubMed

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

    2013-10-01

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

  11. Structural insights into the evolution of an antibody combining site

    SciTech Connect

    Wedemayer, G.J.; Patten, P.A.; Wang, L.H.; Schultz, P.G.; Stevens, R.C. |

    1997-06-13

    The crystal structures of a germline antibody Fab fragment and its complex with hapten have been solved at 2.1 {angstrom} resolution. These structures are compared with the corresponding crystal structures of the affinity-matured antibody, 48G7, which has a 30,000 times higher affinity for hapten as a result of nine replacement somatic mutations. Significant changes in the configuration of the combining site occur upon binding of hapten to the germline antibody, whereas hapten binds to the mature antibody by a lock-and-key fit mechanism. The reorganization of the combining site that was nucleated by hapten binding is further optimized by somatic mutations that occur up to 15 {angstrom} from bound hapten. These results suggest that the binding potential of the primary antibody repertoire may be significantly expanded by the ability of germline antibodies to adopt more than one combining-site configuration, with both antigen binding and somatic mutation stabilizing the configuration with optimal hapten complementarily.

  12. 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.

  13. Integrated bio-affinity nano-platform into a microfluidic immunosensor based on monoclonal bispecific trifunctional antibodies for the electrochemical determination of epithelial cancer biomarker.

    PubMed

    Bravo, Karina; Ortega, Francisco G; Messina, Germán A; Sanz, María I; Fernández-Baldo, Martín A; Raba, Julio

    2017-01-01

    The epithelial cell adhesion molecule (EpCAM) is a biomarker that is highly overexpressed on the surface of epithelial carcinoma cells. In this study, silver nanoparticles covered with polyvinyl alcohol (AgNPs-PVA) were synthesized, characterized and used in a microfluidic immunosensor based on the use of anti-EpCAM recombinant antibodies as a trapping agent. The concentration of trapped EpCAM is then electrochemically quantified by HRP-conjugated anti-EpCAM-antibody. HRP reacted with its enzymatic substrate in a redox process which resulted in the appearance of a current whose magnitude (at a working voltage as low as -0.10V) is directly proportional to the concentration of EpCAM. Under optimized conditions, the detection limits for the microfluidic immunosensor and a commercial ELISA were 0.8 and 13.9pg/L, respectively. The within-assay and between-assay coefficients of variation are below 6.5% for the proposed method. The immunosensor was validated by analyzing patient samples, and a good correlation with a commercial ELISA was obtained. The good analytical performance is attributed to the efficient immobilization of the anti-EpCAM recombinant antibodies on the AgNPs-PVA, and its high specificity for EpCAM. This microfluidic immunosensor is intended for use in diagnosis and prognosis of epithelial cancer, to monitor the disease, and to assess therapeutic efficacy. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. The T cell response to allelic determinants on Igh-1-encoded IgG2a antibodies: dual recognition examined by using antigenic antibodies with binding affinity for Ia molecules.

    PubMed

    Hedrick, S M; Schwartz, R H

    1983-04-01

    In this report we examine the effects of binding antigen directly to Ia molecules during immunization and stimulation of T cells in culture. To accomplish this we characterize the T cell response to Igh-1j-encoded IgG2a antibodies in C57BL/10 H-2 congenic mice. In contrast to the response to the closely related Igh-1a-encoded IgG2a antibodies, high responder mice were (C57BL/10 X B10.A)F1 hybrids, and the important alleles appeared to map to I-Ab and I-Ak. C57BL/10 and B10.A(5R) strains were low responders (I-Ab), and B10.A and B10.A(4R) strains were nonresponders (I-Ak). The interest in using monoclonal IgG2a antibodies as T cell antigens was that different V-region-encoded binding specificities could be associated with the same IgG2a antigenic determinant. In our interpretation of the Langman and Cohn dual binding site model of the T cell receptor, the response to anti-Ia antibody should have some of the same characteristics as that to an allogeneic Ia molecule; in particular, it should be non-H-2-restricted. Although the response to anti-Ia antibody had several interesting properties, no difference in the restriction specificity was found for the T cell response to anti-Ia antibodies relative to unreactive IgG2a antibodies. Our conclusion is that T cell receptors must bind both Ia and antigen molecules for activation to occur. In a separate set of experiments, the T cell response resulting from immunization with anti-Ia antibody was examined. Because the antigen was shown to be associated with the Ia molecule during immunization, experiments were designed to detect an alteration or lack of H-2 restriction in the resulting T cell population. A long-term T cell line induced after such immunization showed no qualitative differences from a T cell line produced with unreactive IgG2a antibody. These results demonstrate that simply attaching a foreign antigen to a restriction element is not sufficient to lead to unrestricted T cell activation. This in turn suggests the

  15. 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

  16. Phase 1/2 study of ocaratuzumab, an Fc-engineered humanized anti-CD20 monoclonal antibody, in low-affinity FcγRIIIa patients with previously treated follicular lymphoma.

    PubMed

    Ganjoo, Kristen N; de Vos, Sven; Pohlman, Brad L; Flinn, Ian W; Forero-Torres, Andres; Enas, Nathan H; Cronier, Damien M; Dang, Nam H; Foon, Kenneth A; Carpenter, Susan P; Slapak, Christopher A; Link, Brian K; Smith, Mitchell R; Mapara, Markus Y; Wooldridge, James E

    2015-01-01

    This phase 2 study assessed the safety and efficacy of ocaratuzumab, a humanized anti-CD20 monoclonal antibody. Fifty patients with previously treated follicular lymphoma (FL) and a low-affinity genotype of FcγRIIIa received ocaratuzumab 375 mg/m(2) weekly for 4 weeks. Grade 3/4/5 adverse events (AEs) were reported in 11/1/1 patients, respectively. Serious AEs were reported by 11/50 patients, and three discontinued due to AEs. One patient died from aspiration pneumonia due to possibly drug-related nausea and vomiting. Investigator-assessed response rate was 30% (15/50), including four complete responses (CR), three CR unconfirmed (CRu) and eight partial responses (PR). Investigator-assessed median Progression-free survivial (PFS) was 38.3 weeks. Ocaratuzumab's pharmacokinetic profile was similar to that reported for rituximab. Lymphocyte subset analysis showed significant, selective reduction of B-cells during and after ocaratuzumab treatment. Ocaratuzumab at this dose and schedule is active and well tolerated in patients with previously treated FL with low affinity FcγRIIIa genotypes. ClinTrials registry number: NCT00354926.

  17. Antithyroglobulin antibody

    MedlinePlus

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

  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. Directed Evolution of a Yeast-Displayed HIV-1 SOSIP gp140 Spike Protein toward Improved Expression and Affinity for Conformational Antibodies

    PubMed Central

    Grimm, Sebastian K.; Battles, Michael B.; Ackerman, Margaret E.

    2015-01-01

    Design of an envelope-based immunogen capable of inducing a broadly neutralizing antibody response is thought to be key to the development of a protective HIV-1 vaccine. However, the broad diversity of viral variants and a limited ability to produce native envelope have hampered such design efforts. Here we describe adaptation of the yeast display system and use of a combinatorial protein engineering approach to permit directed evolution of HIV envelope variants. Because the intrinsic instability and complexity of this trimeric glycoprotein has greatly impeded the development of immunogens that properly represent the structure of native envelope, this platform addresses an essential need for methodologies with the capacity to rapidly engineer HIV spike proteins towards improved homogeneity, stability, and presentation of neutralizing epitopes. We report for the first time the display of a designed SOSIP gp140 on yeast, and the in vitro evolution of derivatives with greatly improved expression and binding to conformation-dependent antibodies. These efforts represent an initial and critical step toward the ability to rapidly engineer HIV-1 envelope immunogens via directed evolution. PMID:25688555

  1. 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.

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

    DOEpatents

    Jin, Hongjun; Zangar, Richard C.

    2017-01-17

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

  3. Somatic Hypermutation-Induced Changes in the Structure and Dynamics of HIV-1 Broadly Neutralizing Antibodies.

    PubMed

    Davenport, Thaddeus M; Gorman, Jason; Joyce, M Gordon; Zhou, Tongqing; Soto, Cinque; Guttman, Miklos; Moquin, Stephanie; Yang, Yongping; Zhang, Baoshan; Doria-Rose, Nicole A; Hu, Shiu-Lok; Mascola, John R; Kwong, Peter D; Lee, Kelly K

    2016-07-20

    Antibody somatic hypermutation (SHM) and affinity maturation enhance antigen recognition by modifying antibody paratope structure to improve its complementarity with the target epitope. SHM-induced changes in paratope dynamics may also contribute to antibody maturation, but direct evidence of this is limited. Here, we examine two classes of HIV-1 broadly neutralizing antibodies (bNAbs) for SHM-induced changes in structure and dynamics, and delineate the effects of these changes on interactions with the HIV-1 envelope glycoprotein (Env). In combination with new and existing structures of unmutated and affinity matured antibody Fab fragments, we used hydrogen/deuterium exchange with mass spectrometry to directly measure Fab structural dynamics. Changes in antibody structure and dynamics were positioned to improve complementarity with Env, with changes in dynamics primarily observed at the paratope peripheries. We conclude that SHM optimizes paratope complementarity to conserved HIV-1 epitopes and restricts the mobility of paratope-peripheral residues to minimize clashes with variable features on HIV-1 Env.

  4. 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.

  5. Efficacy of intravenous immunoglobulin (IVIG) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris.

    PubMed

    Mimouni, D; Blank, M; Payne, A S; Anhalt, G J; Avivi, C; Barshack, I; David, M; Shoenfeld, Y

    2010-12-01

    Pemphigus vulgaris is a rare life-threatening autoimmune bullous disease caused by immunoglobulin G (IgG) autoantibodies directed against desmogleins 1 and 3. Previously, we showed that intravenous immunoglobulin (IVIG) ameliorates anti-desmoglein-induced experimental pemphigus vulgaris in newborn naive mice. The aim of this study was to examine the efficacy of anti-anti-desmoglein-specific IVIG in a similar model. Pemphigus-vulgaris-specific IVIG (PV-sIVIG) was affinity-purified from IVIG on a column of single-chain variable fragment (scFv) anti-desmogleins 1 and 3. The anti-idiotypic activity of PV-sIVIG was confirmed by enzyme-linked immunosorbent assay, inhibition assay. After induction of pemphigus by injection of anti-desmogleins 1 and 3 scFv to newborn mice, the animals were treated with PV-sIVIG, IVIG (low or high dose) or IgG from a healthy donor (n = 10 each). The skin was examined 24-48 h later, and samples of affected areas were analysed by histology and immunofluorescence. In vitro study showed that PV-sIVIG significantly inhibited anti-desmogleins 1 and 3 scFv binding to recombinant desmoglein-3 in a dose-dependent manner. Specificity was confirmed by inhibition assay. In vivo analysis revealed cutaneous lesions of pemphigus vulgaris in mice injected with normal IgG (nine of 10 mice) or low-dose IVIG (nine of 10 mice), but not in mice treated with PV-sIVIG (none of 10) or high-dose IVIG (none of 10). On immunopathological study, PV-sIVIG and regular IVIG prevented the formation of acantholysis and deposition of IgG in intercellular spaces. In conclusion, the PV-sIVIG preparation is more effective than native IVIG in inhibiting anti-desmoglein-induced pemphigus vulgaris in mice and might serve as a future therapy in patients with the clinical disease.

  6. 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

  7. Demystified...recombinant antibodies.

    PubMed

    Smith, K A; Nelson, P N; Warren, P; Astley, S J; Murray, P G; Greenman, J

    2004-09-01

    Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanized antibodies is given, together with details of the generation of antibody fragments--for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display methodology can also be used for the affinity enrichment of existing antibody fragments to provide a reagent with a higher affinity. Here, phage antibodies are demystified to provide a greater understanding of the potential of these reagents and to engage clinicians and biomedical scientists alike to think about potential applications in pathology and clinical settings.

  8. Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk*

    PubMed Central

    Huang, Bin; Yin, Yun; Lu, Lei; Ding, Hai; Wang, Lin; Yu, Ting; Zhu, Jia-jin; Zheng, Xiao-dong; Zhang, Yan-zhen

    2010-01-01

    A convenient competitive enzyme-linked immunosorbent assay (ELISA) for ciprofloxacin (CPFX) was developed by using rabbit monoclonal antibodies (RabMAbs) against a hapten-protein conjugate of CPFX-bovine serum albumin (BSA). The indirect competitive ELISA of CPFX had a concentration at 50% inhibition (IC50) of 1.47 ng/ml and a limit of detection (LOD) of 0.095 ng/ml. The mAb exhibited some cross-reactivity, however, not so high with enrofloxacin (28.8%), ofloxacin (13.1%), norfloxacin (11.0%), fleroxacin (22.6%), and pefloxacin (20.4%). And it showed almost no cross-reactivity with other antibiotics or sulfonamides evaluated in this study. The competitive ELISA kit developed here could be used as a screening tool to detect and control illegal addition of CPFX in food products. This kit had been applied to milk detection and the recovery rates from samples spiked by CPFX were in a range of 63.02%–84.60%, with coefficients of variation of less than 12.2%. PMID:20872990

  9. Comprehensive Characterization of Relationship Between Higher-Order Structure and FcRn Binding Affinity of Stress-Exposed Monoclonal Antibodies.

    PubMed

    Tsuchida, Daisuke; Yamazaki, Katsuyoshi; Akashi, Satoko

    2016-04-01

    In biopharmaceutical development, information regarding higher-order structure (HOS) is important to verify quality and characterize protein derivatives. In this study, we aimed to characterize the association between HOS and pharmacokinetic property of a stress-exposed monoclonal antibody (mAb). Purity, primary structure, thermal stability, and HOS were evaluated for mAbs exposed to heat, photo-irradiation, and chemical oxidation. To investigate conformation of stress-exposed mAbs, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was utilized. No distinct difference in secondary or tertiary structure between stress-exposed and non-stressed samples was found by conventional spectroscopic techniques. In binding activity with the neonatal Fc receptor (FcRn), however, a marked decline was observed for force-oxidized mAb and a slight decline was observed for heat- and photodegraded mAbs. Using differential scanning calorimetry, a change in thermal stability was observed in the CH2 domain for all the stress-exposed samples. Using HDX-MS analyses, individual regions with altered conformation could be identified for heat-degraded and force-oxidized samples. These findings indicate that comprehensive study is important for detecting conformational changes and helpful for predicting biophysical property, and that the evaluation of HOS using several analytical techniques is indispensable for confirming biopharmaceutical quality.

  10. Comparative analysis of binding affinities to epidermal growth factor receptor of monoclonal antibodies nimotuzumab and cetuximab using different experimental animal models.

    PubMed

    Ledón, N; Casacó, A; Casanova, E; Beausoleil, I

    2011-07-01

    Although pharmaco/toxicological studies have always been conducted in pharmacologically relevant species in which the test material is pharmacologically active, the very specificity of many biopharmaceuticals could present challenges in the identification of a relevant species for pharmaco/toxicological studies. Alternative approaches may improve the predictive value of preclinical assessments of species-specific biopharmaceuticals. This could lead to improved decision-making, reduce the number of experimental animals by eliminating non-relevant studies, and decrease the time and cost involved in the drug development process. As an alternative to utilizing traditional animal models, this study investigated the activity of human EGF and the anti-EGF receptor monoclonal antibodies nimotuzumab and cetuximab using the placenta microsomal fraction of different experimental animals. Ligand-receptor binding curves were obtained from the different experimental animal models, and binding constants were calculated based on the Scatchard plots. The constants for human and monkey EGF receptor expressed on the placental extract showed a K(a)<10(-8)M, while rabbits, mice and rats showed a K(a)>10(-8)M. The K(a) values obtained from animal placentas show that Macaca fascicularis and Cercopitecus aethiops monkeys are relevant species for studying the pharmaco/toxicological properties of nimotuzumab and cetuximab.

  11. Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies

    PubMed Central

    Dashivets, Tetyana; Stracke, Jan; Dengl, Stefan; Knaupp, Alexander; Pollmann, Jan; Buchner, Johannes; Schlothauer, Tilman

    2016-01-01

    ABSTRACT Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the VL domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation. PMID:27612038

  12. A simplified method to attach antibodies on liposomes by biotin-streptavidin affinity for rapid and economical screening of targeted liposomes.

    PubMed

    Papadia, Konstantina; Markoutsa, Eleni; Antimisiaris, Sophia G

    2014-05-01

    The biotin-Streptavidin (STREP) technique for attachment of monoclonal antibodies (mAbs) (or other ligand types) on liposome surface offers high attachment yield, however it is time consuming and expensive due to the number of steps used and the consumption of large quantities of STREP. Herein, a simplified, fast and economic technique, by incubating pre-mixed biotin-mAb/STREP with biotin-liposomes, at a 3:1:1 biotin-mAb/STREP/biotin-LIP ratio (mol/mol/mol) was evaluated. The physichochemical properties, final mAb attachment yield and targeting potential of liposomes decorated with an anti-transferrin receptor mAb (TfR-mAb), prepared by the simple method (SM) and the conventional method (CM), were compared. The vesicle uptake by hCMEC/D3 cells (known to overexpress TfR) were considered as a measure of liposome targeting capability. Results show that both targeted liposome types (SM and CM) have small size (mean diameters around 150 nm), low poly-dispersity (approx. 0.20) and similar mAb attachment yield (between 64-88%). However, the uptake of the SM-liposomes is slightly lower compared to CM-LIP (24-30% decrease), suggesting that the modulated conformation of mAbs on the liposome surface (triplets attached to one single STREP molecule) results in decreased targeting capability. Nevertheless, the simpler and faster one-step preparation procedure which has very high lipid recovery (> 95%) compared to the CM (50-60%) and 15-30 times lower consumption of STREP, may be a good alternative for initial screening of various mAbs as ligands for targeted liposomal or other nanotechnologies, during pre-clinical development.

  13. Therapeutic effects of antigen affinity-purified polyclonal anti-receptor of advanced glycation end-product (RAGE) antibodies on cholestasis-induced liver injury in rats.

    PubMed

    Xia, Peng; Deng, Qing; Gao, Jin; Yu, Xiaolan; Zhang, Yang; Li, Jingjing; Guan, Wen; Hu, Jianjun; Tan, Quanhui; Zhou, Liang; Han, Wei; Yuan, Yunsheng; Yu, Yan

    2016-05-15

    Cholestasis leads to acute hepatic injury, fibrosis/cirrhosis, inflammation, and duct proliferation. We investigated whether blocking receptor of advanced glycation end-products (RAGE) with polyclonal anti-RAGE antibodies (anti-RAGE) could regulate acute liver injury and fibrosis in a rat bile duct ligation (BDL) model. Male Wister rats received 0.5mg/kg rabbit anti-RAGE or an equal amount of rabbit IgG by subcutaneous injection twice a week after BDL. Samples of liver tissue and peripheral blood were collected at 14 days after BDL. Serum biochemistry and histology were used to analyze the degree of liver injury. Quantitative real-time PCR (qPCR) and immunohistochemical staining were used to further analyze liver injury. Anti-RAGE improved the gross appearance of the liver and the rat survival rate. Liver tissue histology and relevant serum biochemistry indicated that anti-RAGE attenuated liver necrosis, inflammation, liver fibrosis, and duct proliferation in the BDL model. qPCR and western blotting showed significant reductions in interleukin-1β expression levels in the liver by treatment with anti-RAGE. Anti-RAGE also significantly reduced the mRNA levels of α1(1) collagen (Col1α1) and cholesterol 7α-hydroxylase, and the ratio of tissue inhibitor of matrix metalloproteinase-1 to matrix metalloproteinases (MMPs) in the liver. In addition, anti-RAGE regulated the transcriptional level of Col1α1 and MMP-9 in transforming growth factor-β-induced activated LX-2 cells in vitro. Anti-RAGE was found to inhibit hepatic stellate cell proliferation in vivo and in vitro. Therefore, anti-RAGE can protect the liver from injury induced by BDL in rats. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor.

    PubMed

    Zou, Guozhang; Ochiai, Hirofumi; Huang, Wei; Yang, Qiang; Li, Cishan; Wang, Lai-Xi

    2011-11-23

    Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both FcγRIIIa and FcγRIIb. The

  15. Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.

    PubMed

    Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan

    2015-01-01

    Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.

  16. 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.

  17. A monoclonal antibody against leptin.

    PubMed

    Mahmoudian, Jafar; Jeddi-Tehrani, Mahmood; Bayat, Ali Ahmad; Mahmoudi, Ahmad Reza; Vojgani, Yasaman; Tavangar, Banafsheh; Hadavi, Reza; Zarei, Saeed

    2012-10-01

    Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.

  18. Sequence-Intrinsic Mechanisms that Target AID Mutational Outcomes on Antibody Genes

    PubMed Central

    Yeap, Leng-Siew; Hwang, Joyce K.; Du, Zhou; Meyers, Robin M.; Meng, Fei-Long; Jakubauskaitė, Agnė; Liu, Mengyuan; Mani, Vinidhra; Neuberg, Donna; Kepler, Thomas B.; Wang, Jing H.; Alt, Frederick W.

    2016-01-01

    Summary In activated B lymphocytes, AID initiates antibody variable (V) exon somatic hypermutation (SHM) for affinity maturation in germinal centers (GCs) and IgH switch (S) region DNA breaks (DSBs) for class-switch recombination (CSR). To resolve long-standing questions, we have developed an in vivo assay to study AID-targeting of passenger sequences replacing a V exon. First, we find AID targets SHM hotspots within V exon and S region passengers at similar frequencies and that the normal SHM process frequently generates deletions, indicating that SHM and CSR employ the same mechanism. Second, AID mutates targets in diverse non-Ig passengers in GC B cells at levels similar to those of V exons, definitively establishing the V exon location as "privileged" for SHM. Finally, Peyer's patch GC B cells generate a reservoir of V exons that are highly mutated before selection for affinity maturation. We discuss implications of these findings for harnessing antibody diversification mechanisms. PMID:26582132

  19. Development of an interleukin (IL)-33 sandwich ELISA kit specific for mature IL-33.

    PubMed

    Kim, Eunsom; Kwak, Areum; Jhun, Hyunjhung; Lee, Siyoung; Jo, Seunghyun; Lee, Jongho; Kang, Tae-Bong; Her, Erk; Bae, Suyoung; Lee, Youngmin; Kim, Soohyun

    2016-01-01

    Interleukin (IL)-33 is an inflammatory cytokine and belongs to the IL-1 family of cytokines. There are eleven members of the IL-1 family of cytokines and all have important roles in host defense against infections. Their levels are increased during infection and in various auto-inflammatory diseases. IL-33 is also associated with autoimmune diseases such as asthma, atopic dermatitis, rheumatoid arthritis, and atherosclerosis. IL-33 receptors consist of IL-1R4 and IL-1R3 to induce both Th1 and Th2 type immune response. Here we present the development of monoclonal antibodies (mAbs) against human mature IL-33. Recombinant human mature IL-33 protein was expressed in E. coli and purified by multi-step affinity chromatography. The human IL-33 activity was examined in HMC-1 and Raw 264.7 cells. Mice were immunized with the biologically active mature IL-33 to generate mAb against IL-33. The anti-IL-33 mAb (clone/4) was used as a capture antibody for a sandwich enzyme-linked immunosorbent assay (ELISA). This assay detects mature IL-33 with a high sensitivity (80 pg/mL) but does not recognize the biologically inactive precursor IL-33. This article describes the methods for a newly developed IL-33 ELISA kit that is specific for mature IL-33 and may be used to analyze bioactive mature IL-33 in various immunological diseases.

  20. 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

  1. Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure.

    PubMed

    Chapman, M D; Aalberse, R C; Brown, M J; Platts-Mills, T A

    1988-02-01

    Two mAb were used to develop new techniques for the purification and quantitation of the major feline salivary allergen, Felis domesticus allergen I (Fel d I). The allergen was purified from aqueous house dust extract with a high Fel d I content by affinity chromatography over a monoclonal immunosorbent and elution with 4 mM HCl, pH 2.5. This single step procedure gave 40 to 50% recovery of 90% pure allergen w