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Sample records for antibody affinity maturation

  1. Visualizing antibody affinity maturation in germinal centers.

    PubMed

    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

    2016-03-01

    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 nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  2. Strategies to guide the antibody affinity maturation process.

    PubMed

    Doria-Rose, Nicole A; Joyce, M Gordon

    2015-04-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  3. Strategies to guide the antibody affinity maturation process

    PubMed Central

    Doria-Rose, Nicole A.; Joyce, M. Gordon

    2015-01-01

    Antibodies with protective activity are critical for vaccine efficacy. Affinity maturation increases antibody activity through multiple rounds of somatic hypermutation and selection in the germinal center. Identification of HIV-1 specific and influenza-specific antibody developmental pathways, as well as characterization of B cell and virus co-evolution in patients, has informed our understanding of antibody development. In order to counteract HIV-1 and Influenza viral diversity, broadly neutralizing antibodies precisely target specific sites of vulnerability and require high levels of affinity maturation. We present immunization strategies that attempt to recapitulate these natural processes and guide the affinity maturation process. PMID:25913818

  4. 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

  5. In vitro affinity maturation of a natural human antibody overcomes a barrier to in vivo affinity maturation

    PubMed Central

    Li, Bing; Fouts, Ashley E; Stengel, Katharina; Luan, Peng; Dillon, Michael; Liang, Wei-Ching; Feierbach, Becket; Kelley, Robert F; Hötzel, Isidro

    2014-01-01

    Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD < 10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid

  6. Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

    PubMed

    Briney, Bryan; Sok, Devin; Jardine, Joseph G; Kulp, Daniel W; Skog, Patrick; Menis, Sergey; Jacak, Ronald; Kalyuzhniy, Oleksandr; de Val, Natalia; Sesterhenn, Fabian; Le, Khoa M; Ramos, Alejandra; Jones, Meaghan; Saye-Francisco, Karen L; Blane, Tanya R; Spencer, Skye; Georgeson, Erik; Hu, Xiaozhen; Ozorowski, Gabriel; Adachi, Yumiko; Kubitz, Michael; Sarkar, Anita; Wilson, Ian A; Ward, Andrew B; Nemazee, David; Burton, Dennis R; Schief, William R

    2016-09-01

    Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses. PMID:27610570

  7. Antibody response and antibody affinity maturation in cats with experimental proliferative immune complex glomerulonephritis.

    PubMed

    Bishop, S A; Bailey, M; Lucke, V M; Stokes, C R

    1992-07-01

    An experimental model of proliferative glomerulonephritis (GN) in the cat, which closely resembles human proliferative forms of GN, has been used to study the role of antibody and antibody affinity in the development of immune complex-mediated renal disease. The serum IgG and IgM antibody response to antigen, average antibody affinity (avidity) and affinity heterogeneity of the IgG and IgM populations was assessed at varying times after commencement of chronic immunization with the antigen, human serum albumin (HSA), by enzyme immunoassay. Cats could be classified according to whether they were "low", "intermediate" or "high" IgG responders, by quantification of serum IgG values. Cats with the lowest serum IgG values failed to develop glomerulonephritis. However, there was no relationship between actual IgG values and the severity of the induced disease. In contrast to IgG, there was no division of cats into low or high IgM anti-HSA responders. Again, cats with the lowest IgM values failed to develop GN, but, more interestingly, a late, marked increase in serum IgM anti-HSA occurred only in cats that developed clinical signs of GN (anterior uveitis and nephrotic syndrome). Maturation of average, functional IgG affinity (avidity) for HSA following chronic immunization was clearly demonstrated for all cats. At the end of the experiment, all cats had IgG of high affinity for HSA and the average affinity heterogeneity of the IgG populations was less than in measurements taken earlier. Values of IgG affinity at the end of the experiment were very similar both in cats which developed GN and in those which remained clinically, biochemically and pathologically normal. In contrast to IgG antibody, some cats developed IgM of increased affinity, whilst others produced antibody of reduced affinity, following chronic immunization. There was no correlation between the development of disease and the production of either low or high affinity IgM antibody. Data indicated that an

  8. 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

  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. CD4+ T Cells Promote Antibody Production but Not Sustained Affinity Maturation during Borrelia burgdorferi Infection

    PubMed Central

    Elsner, Rebecca A.; Hastey, Christine J.

    2014-01-01

    CD4 T cells are crucial for enhancing B cell-mediated immunity, supporting the induction of high-affinity, class-switched antibody responses, long-lived plasma cells, and memory B cells. Previous studies showed that the immune response to Borrelia burgdorferi appears to lack robust T-dependent B cell responses, as neither long-lived plasma cells nor memory B cells form for months after infection, and nonswitched IgM antibodies are produced continuously during this chronic disease. These data prompted us to evaluate the induction and functionality of B. burgdorferi infection-induced CD4 TFH cells. We report that CD4 T cells were effectively primed and TFH cells induced after B. burgdorferi infection. These CD4 T cells contributed to the control of B. burgdorferi burden and supported the induction of B. burgdorferi-specific IgG responses. However, while affinity maturation of antibodies against a prototypic T-dependent B. burgdorferi protein, Arthritis-related protein (Arp), were initiated, these increases were reversed later, coinciding with the previously observed involution of germinal centers. The cessation of affinity maturation was not due to the appearance of inhibitory or exhausted CD4 T cells or a strong induction of regulatory T cells. In vitro T-B cocultures demonstrated that T cells isolated from B. burgdorferi-infected but not B. burgdorferi-immunized mice supported the rapid differentiation of B cells into antibody-secreting plasma cells rather than continued proliferation, mirroring the induction of rapid short-lived instead of long-lived T-dependent antibody responses in vivo. The data further suggest that B. burgdorferi infection drives the humoral response away from protective, high-affinity, and long-lived antibody responses and toward the rapid induction of strongly induced, short-lived antibodies of limited efficacy. PMID:25312948

  11. 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. PMID:23527922

  12. 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. PMID:27142297

  13. Affinity maturation of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies accompanies a modulation of antigen specificity.

    PubMed

    Oda, Masayuki; Azuma, Takachika

    2016-02-01

    Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation. PMID:26688069

  14. Combining somatic mutations present in different in vivo affinity-matured antibodies isolated from immunized Lama glama yields ultra-potent antibody therapeutics.

    PubMed

    Klarenbeek, Alex; Blanchetot, Christophe; Schragel, Georg; Sadi, Ava S; Ongenae, Nico; Hemrika, Wieger; Wijdenes, John; Spinelli, Silvia; Desmyter, Aline; Cambillau, Christian; Hultberg, Anna; Kretz-Rommel, Anke; Dreier, Torsten; De Haard, Hans J W; Roovers, Rob C

    2016-04-01

    Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody. PMID:26945588

  15. Affinity Maturation of a Potent Family of HIV Antibodies Is Primarily Focused on Accommodating or Avoiding Glycans.

    PubMed

    Garces, Fernando; Lee, Jeong Hyun; de Val, Natalia; de la Pena, Alba Torrents; Kong, Leopold; Puchades, Cristina; Hua, Yuanzi; Stanfield, Robyn L; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Ward, Andrew B; Wilson, Ian A

    2015-12-15

    The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates. PMID:26682982

  16. Affinity maturation of T-cell receptor-like antibodies for Wilms tumor 1 peptide greatly enhances therapeutic potential

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Tassev, Dimiter V.; Hasan, Aisha; Kuo, Tzu-Yun; Guo, Hong-fen; O’Reilly, Richard J.; Cheung, Nai-Kong V.

    2016-01-01

    WT1126 (RMFPNAPYL) is a human leukocyte antigen-A2 (HLA-A2) restricted peptide derived from Wilms tumor protein (WT1), which is widely expressed in a broad spectrum of leukemias, lymphomas and solid tumors. A novel T-cell-receptor (TCR)-like single chain variable fragment (scFv) antibody specific for the T cell epitope consisting of the WT1/HLA-A2 complex was isolated from a human scFv phage library. This scFv was affinity-matured by mutagenesis combined with yeast display, and structurally analyzed using a homology model. This monovalent scFv showed a 100-fold affinity improvement (dissociation constant [KD]= 3nM) and exquisite specificity towards its targeted epitope or HLA-A2+/WT1+ tumor cells. Bivalent scFv-huIgG1-Fc fusion protein demonstrated an even higher avidity (KD = 2pM) binding to the T cell epitope and to tumor targets, and was capable of mediating antibody-dependent cell-mediated cytotoxicity or tumor lysis by chimeric antigen receptor (CAR)-expressing human T or NK-92-MI transfected cells. This antibody demonstrated specific and potent cytotoxicity in vivo towards WT1-positive leukemia xenograft that was HLA-A2 restricted. In summary, T cell epitopes can provide novel targets for antibody-based therapeutics. By combining phage and yeast displays and scFv-Fc fusion platforms, a strategy for developing high affinity TCR-like antibodies could be rapidly explored for potential clinical development. PMID:25987253

  17. What limits affinity maturation of antibodies in Xenopus--the rate of somatic mutation or the ability to select mutants?

    PubMed Central

    Wilson, M; Hsu, E; Marcuz, A; Courtet, M; Du Pasquier, L; Steinberg, C

    1992-01-01

    Although the Xenopus immunoglobulin heavy chain locus is structurally and functionally similar to mammalian IgH loci, Xenopus antibodies are limited in heterogeneity, and they mature only slightly in affinity during immune responses. During the antibody response of isogenic frogs to DNP-KLH, mu and upsilon cDNA sequences using elements of the VH1 family were cloned, sequenced and compared with germline counterparts. There were zero to four mutations per sequence, mostly single base substitutions, in the framework and CDRs 1 and 2 of VH. No mutations were found in JH. Since the point mutation rate was only 4- to 7-fold lower than that calculated for mice, affinity maturation does not seem to be limited by mutant availability. Because of a relatively low ratio of replacement to silent mutations in the CDRs and a very high ratio of GC to AT base pairs altered by mutation, it is suggested that the problem results from the absence of an effective mechanism for selecting mutants, which in turn might be related to the absence of germinal centers in Xenopus. Images PMID:1425571

  18. 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

  19. 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

    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

    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

  20. 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

  1. Affinity purification of antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

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

  3. 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

  4. 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. PMID:26242599

  5. Aptamer Affinity Maturation by Resampling and Microarray Selection.

    PubMed

    Kinghorn, Andrew B; Dirkzwager, Roderick M; Liang, Shaolin; Cheung, Yee-Wai; Fraser, Lewis A; Shiu, Simon Chi-Chin; Tang, Marco S L; Tanner, Julian A

    2016-07-19

    Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents. PMID:27346322

  6. Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers

    PubMed Central

    Eisen, Ellen A.; Chakraborty, Arup K.

    2015-01-01

    Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens). However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964). Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal. PMID:26444899

  7. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    PubMed Central

    Xiao, Xiaodong; Chen, Weizao; Feng, Yang; Dimitrov, Dimiter S.

    2009-01-01

    Several human monoclonal antibodies (hmAbs) and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG) lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM) affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i) antibodies in HIV-1-infected patients (X5 is a CD4i antibody) as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and intermediate antibodies

  8. 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

  9. Measuring an antibody affinity distribution molecule by molecule

    SciTech Connect

    Bradbury, Andrew M; Werner, James H; Temirov, Jamshid

    2008-01-01

    Single molecule fluorescence mIcroscopy was used to observe the binding and unbinding of hapten decorated quantum dots with individual surface immobilized antibodies. The fluorescence time history from an individual antibody site can be used to calculate its binding affinity. While quantum dot blinking occurs during these measurements, we describe a simple empirical method to correct the apparent/observed affinity to account for the blinking contribution. The combination of many single molecule affinity measurements from different antibodies yields not only the average affinity, it directly measures the full shape and character of the surface affinity distribution function.

  10. 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

  11. Oral Priming with Replicating Adenovirus Serotype 4 Followed by Subunit H5N1 Vaccine Boost Promotes Antibody Affinity Maturation and Expands H5N1 Cross-Clade Neutralization

    PubMed Central

    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. PMID:25629161

  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. PMID:25629161

  13. 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. PMID:27367467

  14. Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

    PubMed

    Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

    2014-07-15

    Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule. PMID:24982157

  15. Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains

    PubMed Central

    Fera, Daniela; Schmidt, Aaron G.; Haynes, Barton F.; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B.; Harrison, Stephen C.

    2014-01-01

    Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus−antibody “arms race.” Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus−antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule. PMID:24982157

  16. Development and maturation of norovirus antibodies in childhood.

    PubMed

    Blazevic, Vesna; Malm, Maria; Honkanen, Hanna; Knip, Mikael; Hyöty, Heikki; Vesikari, Timo

    2016-04-01

    The burden of norovirus (NoV) gastroenteritis is substantial in young children. Maternal antibodies are thought to protect a child from NoV infection in early infancy but subsequent development of NoV-specific protective immunity in children is still largely unexplored. We have determined NoV-specific antibody seroconversion to GII.4 virus-like particles as an indicator of NoV infection in two children prospectively followed from birth to eight years of age. Blocking activity and affinity maturation of maternal and serum IgG antibodies were evaluated. Our results show that multiple infections occur in children up to eight years of age. The titer, blocking activity and avidity of maternal antibodies determined susceptibility of an infant to NoV infection. NoV GII.4-specific antibodies with high blocking potential and avidity were developed at two to three years of age and were retained throughout the follow-up. Subsequent NoV infections may have contributed to the duration of protective NoV-specific immune responses that lasted for several years. This study adds to current understanding of the duration of passive protection by maternal antibodies and the duration and quality of acquired immunity following primary and subsequent NoV infections in infants and young children, who are the main target group for NoV vaccine development. PMID:26724451

  17. 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. PMID:26187411

  18. On the Meaning of Affinity Limits in B-Cell Epitope Prediction for Antipeptide Antibody-Mediated Immunity

    PubMed Central

    Caoili, Salvador Eugenio C.

    2012-01-01

    B-cell epitope prediction aims to aid the design of peptide-based immunogens (e.g., vaccines) for eliciting antipeptide antibodies that protect against disease, but such antibodies fail to confer protection and even promote disease if they bind with low affinity. Hence, the Immune Epitope Database (IEDB) was searched to obtain published thermodynamic and kinetic data on binding interactions of antipeptide antibodies. The data suggest that the affinity of the antibodies for their immunizing peptides appears to be limited in a manner consistent with previously proposed kinetic constraints on affinity maturation in vivo and that cross-reaction of the antibodies with proteins tends to occur with lower affinity than the corresponding reaction of the antibodies with their immunizing peptides. These observations better inform B-cell epitope prediction to avoid overestimating the affinity for both active and passive immunization; whereas active immunization is subject to limitations of affinity maturation in vivo and of the capacity to accumulate endogenous antibodies, passive immunization may transcend such limitations, possibly with the aid of artificial affinity-selection processes and of protein engineering. Additionally, protein disorder warrants further investigation as a possible supplementary criterion for B-cell epitope prediction, where such disorder obviates thermodynamically unfavorable protein structural adjustments in cross-reactions between antipeptide antibodies and proteins. PMID:23209458

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

    PubMed

    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

  20. 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. PMID:23906643

  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. PMID:22841960

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

    PubMed

    Asti, Lorenzo; Uguzzoni, Guido; Marcatili, Paolo; Pagnani, Andrea

    2016-04-01

    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

  3. 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

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

    PubMed Central

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

    2006-01-01

    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. PMID:16446445

  5. Affinity and Avidity in Antibody-Based Tumor Targeting

    PubMed Central

    Rudnick, Stephen I.

    2009-01-01

    Summation Many factors contribute to successful tumor targeting by antibodies. Besides properties of the tumor tissue and general antibody pharmacology, a relationship exists between an antibody and its antigen that can shape penetration, catabolism, specificity, and efficacy. The affinity and avidity of the binding interactions play critical roles in these dynamics. In this work, we review the principles that guide models predicting tumor penetration and cellular internalization while providing a critical overview of studies aimed at experimentally determining the specific role of affinity and avidity in these processes. One should gain the perspective that binding affinity can, in part, dictate the localization of antibodies in tumors, leading to high concentrations in the perivascular space or low concentrations diffused throughout the tumor. These patterns can be simply due to the diminution of available dose by binding antigen and are complicated by internalization and degradation stemming from slow rates of dissociation. As opposed to the trend of simply increasing affinity to increase efficacy, novel strategies that increase avidity and broaden specificity have made significant progress in tumor targeting. PMID:19409036

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

    PubMed

    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

    2009-04-01

    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-1(157-165) 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-1(157-165) 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

  7. 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

  8. Molecular modeling of the affinity chromatography of monoclonal antibodies.

    PubMed

    Paloni, Matteo; Cavallotti, Carlo

    2015-01-01

    Molecular modeling is a methodology that offers the possibility of studying complex systems such as protein-ligand complexes from an atomistic point of view, making available information that can be difficultly obtained from experimental studies. Here, a protocol for the construction of molecular models of the interaction between antibodies and ligands that can be used for an affinity chromatography process is presented. The outlined methodology focuses mostly on the description of a procedure that may be adopted to determine the structure and free energy of interaction between the antibody and the affinity ligand. A procedure to extend the proposed methodology to include the effect of the environment (buffer solution, spacer, support matrix) is also briefly outlined. PMID:25749965

  9. 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.

  10. 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.

  11. Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

    SciTech Connect

    Sawutz, D.G.; Koury, R.; Homcy, C.J.

    1987-08-25

    The authors previously described the production of four monoclonal antibodies to the ..beta..-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes (/sup 125/I)iodocyanopinodolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and T9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9, consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site. This would allow increased contact of the ligand with the idiotype-anti-idiotype complex and result in an enhanced affinity of the ligand interaction.

  12. Generation of acetyllysine antibodies and affinity enrichment of acetylated peptides

    PubMed Central

    Guan, Kun-Liang; Yu, Wei; Lin, Yan; Xiong, Yue; Zhao, Shimin

    2016-01-01

    Lysine acetylation has emerged as one of the major post-translational modifications, as indicated by its roles in chromatin remodeling, activation of transcription factors and, most recently, regulation of metabolic enzymes. Identification of acetylation sites in a protein is the first essential step for functional characterization of acetylation in physiological regulation. However, the study of the acetylome is hindered by the lack of suitable physical and biochemical properties of the acetyl group and existence of high-abundance acetylated histones in the cell, and needs a robust method to overcome these problems. Here we present protocols for (i) using chemically acetylated ovalbumin and synthetic acetylated peptide to generate a pan-acetyllysine antibody and a site-specific antibody to Lys288-acetylated argininosuccinate lyase, respectively; (ii) using subcellular fractionation to reduce highly abundant acetylated histones; and (iii) using acetyllysine antibody affinity purification and mass spectrometry to characterize acetylome of human liver tissue. The entire characterization procedure takes ~2–3 d to complete. PMID:21085124

  13. 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

  14. 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. PMID:15542181

  15. 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. PMID:24832237

  16. Affinity measurement of single chain antibodies: a mathematical method facilitated by statistical software SigmaPlot.

    PubMed

    Safdari, Yaghoub; Farajnia, Safar; Asgharzadeh, Mohammad; Khalili, Masoumeh; Jaliani, Hossein Zarei

    2014-02-01

    Because they are monovalent for antigen, single chain antibodies display a different antibody-antigen interaction pattern from that of full-length antibodies. Using the law of mass action and considering the antibody-antigen binding pattern at OD-100% and OD-50% points, we introduced a formula for estimating single chain antibody affinity. Sigmoid curves of optical density values versus antibody concentrations were drawn and used to determine antibody concentrations at OD-50% points using statistical software SigmaPlot. The OD-50% points were then used to calculate the affinity via the mathematical formula. A software-adapted format of the equation is also presented for further facilitation of the calculation process. The accuracy of this method for affinity calculation was proved by surface plasma resonance. This method offers a precise evaluation of antibody affinity without requiring special material or apparatus, making it possible to be performed in any biological laboratory with minimum facilities. PMID:24555931

  17. The effect of immunological adjuvants on the relative affinity of anti-protein antibodies.

    PubMed Central

    Petty, R E; Steward, M W

    1977-01-01

    Inbred mice of a strain (B1OD2 new) known to produce either no detectable antibody or antibody of low affinity to two protein antigens administered in saline, were immunized with human serum transferrin (HST) in one of nine adjuvants. Such immunization increases the level and relative affinity of anti-HST antibody. The adjuvants used varied in the degree to which they augmented these parameters of the antibody response--that is, FCA and FIA were capable of inducing high levels of high affinity antibody, whereas other adjuvants elicited lower levels of high affinity antibody. The possibility is discussed that substances with adjuvant activity may effect antibody production at two stages: (1) at the stage of antigen selection of cells for proliferation and (2) at the stage or proliferation of antibody producing cell precursors. PMID:844888

  18. 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

  19. A novel high affinity human monoclonal antibody to mesothelin

    PubMed Central

    Ho, Mitchell; Feng, Mingqian; Fisher, Robert J.; Rader, Christoph; Pastan, Ira

    2010-01-01

    Mesothelin is a glycosylphosphatidylinisotol-anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here we report the identification of a novel human mAb to mesothelin. HN1, a human single chain Fv specific for mesothelin, was isolated from a naïve human scFv phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain, and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody-dependent cell-mediated cytotoxicity. HN1 binds a conformation-sensitive epitope in human mesothelin with high affinity (KD = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface-associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin-expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin-expressing cancer treatment and diagnosis. PMID:20635390

  20. 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

  1. Quantifying evolutionary constraints on B-cell affinity maturation.

    PubMed

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

    2015-09-01

    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

  2. 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. PMID:24862193

  3. 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

  4. Designing and optimizing library selection strategies for generating high-affinity antibodies.

    PubMed

    Hoogenboom, H R

    1997-02-01

    Since its invention at the beginning of the 1990s, antibody phage display has revolutionized the generation of monoclonal antibodies and their engineering. It is now possible to create antibodies binding to any chosen target antigen without the use of laboratory animals or hybridomas, in a system that completely by-passes the immune system. Making antibodies from single-pot phage libraries, and improving their affinity up to the picomolar range if necessary, has never appeared easier. In this review, a variety of phage library-based strategies for the isolation of high-affinity antibodies are presented. PMID:9081300

  5. Antibody purification using affinity chromatography: a case study with a monoclonal antibody to ractopamine.

    PubMed

    Wang, Zhanhui; Liang, Qi; Wen, Kai; Zhang, Suxia; Shen, Jianzhong

    2014-11-15

    The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA). PMID:25261834

  6. Isolation and optimization for affinity and biophysical characteristics of anti-CCL17 antibodies from the VH1-69 germline gene.

    PubMed

    Kehoe, John W; Whitaker, Brian; Bethea, Deidra; Lacy, Eilyn R; Boakye, Ken; Santulli-Marotto, Sandra; Ryan, Mary H; Feng, Yiqing; Wheeler, John C

    2014-06-01

    CCL17 is a homeostatic chemokine associated with several human inflammatory pathologies. This makes CCL17 a potential point of intervention in inflammatory diseases. Using a Fab-pIX phage display system we were able to select antibodies that specifically bind to CCL17 and neutralize CCL17-mediated signaling. Many of the selected antibodies belong to the VH1-69 germline gene family. The VH1-69 germline gene is represented at a high frequency in the human antibody repertoire and is seen in the early immune response to a variety of pathogens. The heavy chain CDR2 of this germline gene is notably hydrophobic and can insert into hydrophobic pockets of antigens, providing much of the binding energy for these antibodies. Affinity maturation of our primary binders by light chain mutagenesis produced antibodies with sub-nanomolar affinities, with affinity improvements up to 100-fold. These were screened for non-specific protein-protein interactions as a filter for solubility. All of our high affinity antibodies were found to have high levels of non-specific protein-protein interactions. We speculated that this was due to the hydrophobicity within the germline heavy chain CDR1 and CDR2. To ameliorate this problem, we generated a phage display library for one of the clones, where the surface-exposed residues within H-CDR1 and H-CDR2 were randomized. High stringency panning of this library against human CCL17 resulted in further affinity improvement, along with reduction in protein-protein interaction in some new variants. In addition, we improved the cross-reactivity to cynomolgus CCL17. We demonstrate that affinity maturation through targeted libraries in the VH1-69 germline gene can improve both affinity and biophysical characteristics of antibodies derived from this gene scaffold. PMID:24742503

  7. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation

    PubMed Central

    Mouquet, Hugo; Scheid, Johannes F.; Zoller, Markus J.; Krogsgaard, Michelle; Ott, Rene G.; Shukair, Shetha; Artyomov, Maxim N.; Pietzsch, John; Connors, Mark; Pereyra, Florencia; Walker, Bruce D.; Ho, David D.; Wilson, Patrick C.; Seaman, Michael S.; Eisen, Herman N.; Chakraborty, Arup K.; Hope, Thomas J.; Ravetch, Jeffrey V.; Wardemann, Hedda; Nussenzweig, Michel C.

    2013-01-01

    During immune responses, antibodies are selected for their ability to bind to foreign antigens with high affinity, in part by their ability to undergo homotypic bivalent binding. However, this type of binding is not always possible. For example, the small number of gp140 glycoprotein spikes displayed on the surface of the human immunodeficiency virus (HIV disfavours homotypic bivalent antibody binding1–3. Here we show that during the human antibody response to HIV, somatic mutations that increase antibody affinity also increase breadth and neutralizing potency. Surprisingly, the responding naive and memory B cells produce polyreactive antibodies, which are capable of bivalent heteroligation between one high-affinity anti-HIV-gp140 combining site and a second low-affinity site on another molecular structure on HIV. Although cross-reactivity to self-antigens or polyreactivity is strongly selected against during B-cell development4, it is a common serologic feature of certain infections in humans, including HIV, Epstein-Barr virus and hepatitis C virus. Seventy-five per cent of the 134 monoclonal anti-HIV-gp140 antibodies cloned from six patients5 with high titres of neutralizing antibodies are polyreactive. Despite the low affinity of the polyreactive combining site, heteroligation demonstrably increases the apparent affinity of polyreactive antibodies to HIV. PMID:20882016

  8. 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.

  9. Natural antibodies sustain differentiation and maturation of human dendritic cells

    PubMed Central

    Bayry, Jagadeesh; Lacroix-Desmazes, Sébastien; Donkova-Petrini, Vladimira; Carbonneil, Cédric; Misra, Namita; Lepelletier, Yves; Delignat, Sandrine; Varambally, Sooryanarayana; Oksenhendler, Eric; Lévy, Yves; Debré, Marianne; Kazatchkine, Michel D.; Hermine, Olivier; Kaveri, Srini V.

    2004-01-01

    The differentiation and maturation of dendritic cells (DCs) is governed by various signals in the microenvironment. Monocytes and DCs circulate in peripheral blood, which contains high levels of natural antibodies (NAbs). NAbs are germ-line-encoded and occur in the absence of deliberate immunization or microbial aggression. To assess the importance of NAbs in the milieu on DC development, we examined the status of DCs in patients with X-linked agammaglobulinemia, a disease characterized by paucity of B cells and circulating antibodies. We demonstrate that the in vitro differentiation of DCs is severely impaired in these patients, at least in part because of low levels of circulating NAbs. We identified NAbs reactive with the CD40 molecule as an important component that participates in the development of DCs. CD40-reactive NAbs restored normal phenotypes of DCs in patients. The maturation process induced by CD40-reactive NAbs was accompanied by an increased IL-10 and decreased IL-12 production. The transcription factor analysis revealed distinct signaling pathways operated by CD40-reactive NAbs compared to those by CD40 ligand. These results suggest that B cells promote bystander DC development through NAbs and the interaction between NAbs and DCs may play a role in steady-state migration of DCs. PMID:15381781

  10. Maturational characteristics of HIV-specific antibodies in viremic individuals

    PubMed Central

    Meffre, Eric; Louie, Aaron; Bannock, Jason; Kim, Leo J.Y.; Ho, Jason; Frear, Cody C.; Kardava, Lela; Wang, Wei; Buckner, Clarisa M.; Wang, Yimeng; Fankuchen, Olivia R.; Gittens, Kathleen R.; Chun, Tae-Wook; Li, Yuxing; Fauci, Anthony S.; Moir, Susan

    2016-01-01

    Despite the rare appearance of potent HIV-neutralizing mAbs in infected individuals requiring prolonged affinity maturation, little is known regarding this process in the majority of viremic individuals. HIV-infected individuals with chronic HIV viremia have elevated numbers of nonconventional tissue-like memory (TLM) B cells that predominate in blood over conventional resting memory (RM) B cells. Accordingly, we investigated affinity maturation in these 2 memory B cell populations. Analysis of IgG-expressing TLM B cells revealed a higher number of cell divisions compared with RM B cells; however, TLM B cells paradoxically displayed significantly lower frequencies of somatic hypermutation (SHM). To assess Ab reactivity in TLM and RM B cells, single-cell cloning was performed on HIV envelope CD4–binding site–sorted (CD4bs-sorted) B cells from 3 individuals with chronic HIV viremia. Several clonal families were present among the 127 cloned recombinant mAbs, with evidence of crosstalk between TLM and RM B cell populations that was largely restricted to non-VH4 families. Despite evidence of common origins, SHM frequencies were significantly decreased in TLM-derived mAbs compared with SHM frequencies in RM-derived mAbs. However, both cell populations had lower frequencies of SHMs than did broadly neutralizing CD4bs–specific mAbs. There was a significant correlation between SHM frequencies and the HIV-neutralizing capacities of the mAbs. Furthermore, HIV neutralization was significantly higher in the RM-derived mAbs compared with that seen in the TLM-derived mAbs, and both SHM frequencies and neutralizing capacity were lowest in TLM-derived mAbs with high polyreactivity. Thus, deficiencies in memory B cells that arise during chronic HIV viremia provide insight into the inadequacy of the Ab response in viremic individuals. PMID:27152362

  11. 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

  12. Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

    PubMed Central

    2009-01-01

    Background Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates. PMID:19878581

  13. A high-affinity human antibody that targets tumoral blood vessels.

    PubMed

    Tarli, L; Balza, E; Viti, F; Borsi, L; Castellani, P; Berndorff, D; Dinkelborg, L; Neri, D; Zardi, L

    1999-07-01

    Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 microg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of

  14. 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. PMID:23244324

  15. 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. PMID:26386257

  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. 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.

  18. 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

    2016-01-01

    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

  19. 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

  20. A strategy of designing the ligand of antibody affinity chromatography based on molecular dynamics simulation.

    PubMed

    Dai, Lu; Li, Weikang; Sun, Fei; Li, Baizhi; Li, Hongrui; Zhang, Hongxing; Zheng, Qingchuan; Liang, Chongyang

    2016-09-01

    Designing affinity ligands has always been the development focus of affinity chromatography. Previous antibody affinity ligand designs were mostly based on the crystal structure of protein A (UniProt code number: P38507), and the antibody-binding domains were modified according to the properties of amino acid residues. Currently, more effective bioinformatic prediction and experimental validation has been used to improve the design of antibody affinity ligands. In the present study, the complex crystal structure (the domain D of protein A and the Fab segment of IgM, PDB code: 1DEE) was used as the model. The vital site that inhibits the binding between domain D and IgM was estimated by means of molecular dynamics (MD) simulation, then MM-GBSA calculations were used to design a mutant of domain D (K46E) for improving affinity on the above vital site. The binding analysis using Biacore showed the association and dissociation parameters of K46E mutant that were optimized with IgM. The affinity increase of K46E mutant preferred for IgM, the affinity order is K46E tetramer (KD=6.02×10(-9)M)>K46E mutant (KD=6.66×10(-8)M)>domain D (KD=2.17×10(-7)M). Similar results were obtained when the optimized ligands were immobilized to the chromatography medium. A complete designing strategy was validated in this study, which will provide a novel insight into designing new ligands of antibody affinity chromatography media. PMID:27524303

  1. 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.

  2. 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.; Angeletti, Davide; 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

  3. Systematic fractionation of serum antibodies using multiple antigen homologous peptides as affinity ligands.

    PubMed

    Tribbick, G; Triantafyllou, B; Lauricella, R; Rodda, S J; Mason, T J; Geysen, H M

    1991-06-01

    The fractionation of polyclonal antibodies on multiple peptide ligands is described. The method is an application of a procedure for the synthesis of large numbers of peptides on individual polyethylene pins (Geysen et al., 1987). In this application, each pin-bound peptide is used as an affinity support. Antibodies bound to the peptides are then eluted, using buffers of either high or low pH. Each eluted antibody is then tested for specific binding to peptides or proteins, using ELISA procedures. A rabbit antiserum raised to gonococcal pilin was fractionated on a complete set of octapeptides homologous with the sequence of the pilin protein. Antibodies eluted from some of the peptides bound to pilin in solution. In a second example three hyperimmune sera raised to three different potyviruses were fractionated on their respective homologous peptide sequences. Testing the eluted antibodies on the three virus coat proteins revealed peptides which bound cross-reacting antibodies. Thus the method can be used to confirm direct peptide binding evidence for sequential epitopes. These peptides can then be used in affinity chromatography to increase the specificity of polyclonal sera. This can be achieved either by elution of the specific antibody from the peptide or by removal of cross-reacting antibodies from the whole serum by absorption on peptide. PMID:1904463

  4. Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo

    PubMed Central

    Botkjaer, Kenneth A.; Kwok, Hang Fai; Terp, Mikkel G.; Karatt-Vellatt, Aneesh; Santamaria, Salvatore; McCafferty, John; Andreasen, Peter A.; Itoh, Yoshifumi; Ditzel, Henrik J.; Murphy, Gillian

    2016-01-01

    The membrane-associated matrix metalloproteinase-14, MT1-MMP, has been implicated in pericellular proteolysis with an important role in cellular invasion of collagenous tissues. It is substantially upregulated in various cancers and rheumatoid arthritis, and has been considered as a potential therapeutic target. Here, we report the identification of antibody fragments to MT1-MMP that potently and specifically inhibit its cell surface functions. Lead antibody clones displayed inhibitory activity towards pro-MMP-2 activation, collagen-film degradation and gelatin-film degradation, and were shown to bind to the MT1-MMP catalytic domain outside the active site cleft, inhibiting binding to triple helical collagen. Affinity maturation using CDR3 randomization created a second generation of antibody fragments with dissociation constants down to 0.11 nM, corresponding to an improved affinity of 332-fold with the ability to interfere with cell-surface MT1-MMP functions, displaying IC50 values down to 5 nM. Importantly, the new inhibitors were able to inhibit collagen invasion by tumor-cells in vitro and in vivo primary tumor growth and metastasis of MDA-MB-231 cells in a mouse orthotopic xenograft model. Herein is the first demonstration that an inhibitory antibody targeting sites outside the catalytic cleft of MT1-MMP can effectively abrogate its in vivo activity during tumorigenesis and metastasis. PMID:26934448

  5. Low-affinity IgM antibodies lacking somatic hypermutations are produced in the secondary response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl hapten.

    PubMed

    Murakami, Akikazu; Moriyama, Hayato; Osako-Kabasawa, Mina; Endo, Kanako; Nishimura, Miyuki; Udaka, Keiko; Muramatsu, Masamichi; Honjo, Tasuku; Azuma, Takachika; Shimizu, Takeyuki

    2014-04-01

    Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response. PMID:24285827

  6. Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies

    PubMed Central

    Hu, Dongmei; Hu, Siyi; Wan, Wen; Xu, Man; Du, Ruikai; Zhao, Wei; Gao, Xiaolian; Liu, Jing; Liu, Haiyan; Hong, Jiong

    2015-01-01

    Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs) of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 106 DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules. PMID:26046845

  7. High throughput solution-based measurement of antibody-antigen affinity and epitope binning.

    PubMed

    Estep, Patricia; Reid, Felicia; Nauman, Claire; Liu, Yuqi; Sun, Tingwan; Sun, Joanne; Xu, Yingda

    2013-01-01

    Advances in human antibody discovery have allowed for the selection of hundreds of high affinity antibodies against many therapeutically relevant targets. This has necessitated the development of reproducible, high throughput analytical techniques to characterize the output from these selections. Among these characterizations, epitopic coverage and affinity are among the most critical properties for lead identification. Biolayer interferometry (BLI) is an attractive technique for epitope binning due to its speed and low antigen consumption. While surface-based methods such as BLI and surface plasmon resonance (SPR) are commonly used for affinity determinations, sensor chemistry and surface related artifacts can limit the accuracy of high affinity measurements. When comparing BLI and solution equilibrium based kinetic exclusion assays, significant differences in measured affinity (10-fold and above) were observed. KinExA direct association (k(a)) rate constant measurements suggest that this is mainly caused by inaccurate k(a) measurements associated with BLI related surface phenomena. Based on the kinetic exclusion assay principle used for KinExA, we developed a high throughput 96-well plate format assay, using a Meso Scale Discovery (MSD) instrument, to measure solution equilibrium affinity. This improved method combines the accuracy of solution-based methods with the throughput formerly only achievable with surface-based methods. PMID:23575269

  8. 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.

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

    PubMed

    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

  10. 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

  11. Engineering protein therapeutics: predictive performances of a structure-based virtual affinity maturation protocol.

    PubMed

    Oberlin, Michael; Kroemer, Romano; Mikol, Vincent; Minoux, Hervé; Tastan, Erdogan; Baurin, Nicolas

    2012-08-27

    The implementation of a structure based virtual affinity maturation protocol and evaluation of its predictivity are presented. The in silico protocol is based on conformational sampling of the interface residues (using the Dead End Elimination/A* algorithm), followed by the estimation of the change of free energy of binding due to a point mutation, applying MM/PBSA calculations. Several implementations of the protocol have been evaluated for 173 mutations in 7 different protein complexes for which experimental data were available: the use of the Boltzamnn averaged predictor based on the free energy of binding (ΔΔG(*)) combined with the one based on its polar component only (ΔΔE(pol*)) led to the proposal of a subset of mutations out of which 45% would have successfully enhanced the binding. When focusing on those mutations that are less likely to be introduced by natural in vivo maturation methods (99 mutations with at least two base changes in the codon), the success rate is increased to 63%. In another evaluation, focusing on 56 alanine scanning mutations, the in silico protocol was able to detect 89% of the hot-spots. PMID:22788756

  12. Analysis of monoclonal antibody heterogeneity by post-capillary affinity detection for capillary electrophoresis

    SciTech Connect

    Kelly, J.A.; Lee, Cheng S.

    1997-12-31

    Production of monoclonal antibodies seldom results in a single uniform product. Although the use of hybridomas yields antibodies with a homogeneous amino acid sequence, differences exist in degree of glycosylation. Oligosaccharide variation is known to vary with culture conditions as well as proliferation state. Glycosylation is significant biologically, particularly agalactosyl glycoforms of IgG which can be pathogenic. This suggests a need for rapid analysis of antibody heterogeneity, including glycosylation, during production to optimize quality and yield. Post-capillary affinity detection for capillary electrophoresis is a novel bioanalytical tool which analyzes protein microheterogeneity without interference from complex sample matrices. Mouse monoclonal antibody samples from cell culture media are selectively analyzed by post-capillary affinity detection. Separation of IgG variants is accomplished by capillary zone electrophoresis (CZE) prior to on-line affinity detection with fragment B of Protein A lagged with fluorescein (BF). IgG isoforms are observed while serum proteins and cell culture media are discriminated against.

  13. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8*

    PubMed Central

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y.; Cheung, Nai-Kong V.

    2015-01-01

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. PMID:25851904

  14. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

    PubMed

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

    2015-05-22

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. PMID:25851904

  15. High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires.

    PubMed

    Gene, Robert W; Kumaran, Jyothi; Aroche, Cristina; van Faassen, Henk; Hall, J Christopher; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies. PMID:25450000

  16. 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. PMID:26473627

  17. HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation.

    PubMed

    Dai, Kaifan; Khan, Salar N; Wang, Yimeng; He, Linling; Guenaga, Javier; Ingale, Jidnyasa; Sundling, Christopher; O'Dell, Sijy; McKee, Krisha; Phad, Ganesh; Corcoran, Martin; Wilson, Richard; Mascola, John R; Zhu, Jiang; Li, Yuxing; Karlsson Hedestam, Gunilla B; Wyatt, Richard T

    2016-01-01

    The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs "knock out" trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities for the majority of the igL mAbs to gp120 and of Fabs to gp140, which, as expected, increased when the antibodies 'matured' following vaccination. Affinity increases were associated with slower off-rates as well as with acquisition of neutralizing capacity. These data reveal in vitro binding properties associated with in vivo activation that result in functional archiving of antigen-specific B cells elicited by a complex glycoprotein antigen following immunization. PMID:26879974

  18. Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

    PubMed Central

    Kaplinsky, Joseph; Li, Anthony; Sun, Amy; Coffre, Maryaline; Koralov, Sergei B.; Arnaout, Ramy

    2014-01-01

    Antibody repertoires are known to be shaped by selection for antigen binding. Unexpectedly, we now show that selection also acts on a non–antigen-binding antibody region: the heavy-chain variable (VH)–encoded “elbow” between variable and constant domains. By sequencing 2.8 million recombined heavy-chain genes from immature and mature B-cell subsets in mice, we demonstrate a striking gradient in VH gene use as pre-B cells mature into follicular and then into marginal zone B cells. Cells whose antibodies use VH genes that encode a more flexible elbow are more likely to mature. This effect is distinct from, and exceeds in magnitude, previously described maturation-associated changes in heavy-chain complementarity determining region 3, a key antigen-binding region, which arise from junctional diversity rather than differential VH gene use. Thus, deep sequencing reveals a previously unidentified mode of B-cell selection. PMID:24927543

  19. 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. PMID:25569629

  20. Purification of infective bluetongue virus particles by immuno-affinity chromatography using anti-core antibody.

    PubMed

    Chand, Karam; Biswas, Sanchay K; Mondal, Bimalendu

    2016-03-01

    An immuno-affinity chromatography technique for purification of infective bluetongue virus (BTV) has been descried using anti-core antibodies. BTV anti-core antibodies (prepared in guinea pig) were mixed with cell culture-grown BTV-1 and then the mixture was added to the cyanogens bromide-activated protein-A Sepharose column. Protein A binds to the antibody which in turn binds to the antigen (i.e. BTV). After thorough washing, antigen-antibody and antibody-protein A couplings were dissociated with 4M MgCl2, pH6.5. Antibody molecules were removed by dialysis and virus particles were concentrated by spin column ultrafiltration. Dialyzed and concentrated material was tested positive for BTV antigen by a sandwich ELISA and the infectivity of the chromatography-purified virus was demonstrated in cell culture. This method was applied for selective capture of BTV from a mixture of other viruses. As group-specific antibodies (against BTV core) were used to capture the virus, it is expected that virus of all BTV serotypes could be purified by this method. This method will be helpful for selective capture and enrichment of BTV from concurrently infected blood or tissue samples for efficient isolation in cell culture. Further, this method can be used for small scale purification of BTV avoiding ultracentrifugation. PMID:26925450

  1. 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.

  2. High-throughput kinetic screening of hybridomas to identify high-affinity antibodies using bio-layer interferometry.

    PubMed

    Lad, Latesh; Clancy, Sheila; Kovalenko, Maria; Liu, Chian; Hui, Terence; Smith, Victoria; Pagratis, Nikos

    2015-04-01

    Kinetic analysis of antibodies is crucial in both clone selection and characterization. Historically, antibodies in supernatants from hybridomas are selected based on a solid-phase enzyme-linked immunosorbent assay (ELISA) in which the antigen is immobilized on the assay plate. ELISA selects clones based on a combination of antibody concentration in the supernatant and affinity. The antibody concentration in the supernatant can vary significantly and is typically unknown. Using the ELISA method, clones that express high levels of a low-affinity antibody can give an equivalent signal as clones that express low levels of a high-affinity antibody. As a consequence, using the ELISA method, superior clones can be overshadowed by inferior clones. In this study, we have applied Bio-Layer Interferometry to screen hybridoma clones based on disassociation rates using the OctetRED 384 platform. Using the OctetRED platform, we were able to screen 2000 clones within 24 hours and select clones containing high-affinity antibodies for further expansion and subsequent characterization. Using this method, we were able to identify several clones producing high-affinity antibodies that were missed by ELISA. PMID:25425568

  3. High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

    PubMed Central

    2015-01-01

    High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

  4. A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

    PubMed Central

    Liu, Keyi; Park, Minha; DeChene, Neal; Stephenson, Robert; Tenorio, Edgar; Ellsworth, Stote L.; Tabata, Takako; Petitt, Matthew; Tsuge, Mitsuru; Fang-Hoover, June; Adler, Stuart P.; Cui, Xiaohong; McVoy, Michael A.; Pereira, Lenore

    2014-01-01

    Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation. PMID:25534746

  5. 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

  6. Optimal fusion of antibody binding domains resulted in higher affinity and wider specificity.

    PubMed

    Dong, Jinhua; Kojima, Tomoki; Ohashi, Hiroyuki; Ueda, Hiroshi

    2015-11-01

    Antibody is a very important protein in biotechnological and biomedical fields because of its high affinity and specificity to various antigens. Due to the rise of human antibody therapeutics, its cost-effective purification is an urgent issue for bio-industry. In this study, we made novel fusion proteins PAxPG with a flexible (DDAKK)n linker between the two Ig binding domains derived from Staphylococcus protein A and Streptococcus protein G. The fusion proteins bound human and mouse IgGs and their fragments with up to 58-times higher affinity and wider specificity than the parental binding domains. Interestingly, the optimal linker for human Fab fragment was n = 4, which was close to the modeled distance between the termini of domains bound to heavy chain, implying increased avidity as a possible mechanism. For binding to Fc, the longest n=6 linker gave the highest affinity, implying longer interchain distance between the two binding sites. The novel fusion protein with optimized interdomain linker length will be a useful tool for the purification and detection of various IgGs including mouse IgG1 that binds only weakly to natural protein A. PMID:25910963

  7. Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

    PubMed

    O'Shaughnessy, Colette M; Micoli, Francesca; Gavini, Massimiliano; Goodall, Margaret; Cobbold, Mark; Saul, Allan; Maclennan, Calman A

    2013-01-31

    Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella

  8. HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation

    PubMed Central

    Dai, Kaifan; Khan, Salar N; Wang, Yimeng; He, Linling; Guenaga, Javier; Ingale, Jidnyasa; Sundling, Christopher; O’Dell, Sijy; McKee, Krisha; Phad, Ganesh; Corcoran, Martin; Wilson, Richard; Mascola, John R; Zhu, Jiang; Li, Yuxing; Hedestam, Gunilla B Karlsson; Wyatt, Richard T

    2016-01-01

    The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs “knock out” trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities for the majority of the igL mAbs to gp120 and of Fabs to gp140, which, as expected, increased when the antibodies ‘matured’ following vaccination. Affinity increases were associated with slower off-rates as well as with acquisition of neutralizing capacity. These data reveal in vitro binding properties associated with in vivo activation that result in functional archiving of antigen-specific B cells elicited by a complex glycoprotein antigen following immunization. PMID:26879974

  9. 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

  10. Theoretical analysis of antibody targeting of tumor spheroids: importance of dosage for penetration, and affinity for retention.

    PubMed

    Graff, Christilyn P; Wittrup, K Dane

    2003-03-15

    The interplay among antibody/antigen binding kinetics, antibody diffusion, and antigen metabolic turnover together determines the depth of penetration of antitumor antibodies into prevascular tumor spheroid cell clumps. A sharp boundary between an outer shell of bound high-affinity antibody and an inner antibody-free core has been previously observed and mathematically modeled and was termed the "binding site barrier." We show here that this process is well described by a simplified shrinking core model wherein binding equilibration is much more rapid than diffusion. This analysis provides the following experimentally testable predictions: (a) the binding site barrier is a moving boundary whose velocity is proportional to the time integral of antibody concentration at the spheroid surface (i.e. plasma antibody AUC); (b) the velocity of this moving boundary is independent of binding affinity, if the affinity is sufficiently high to strongly favor antibody/antigen complex formation at prevailing antibody concentrations; and (c) maximum tumor retention is achieved when the antibody/antigen dissociation rate approaches the rate of antigen metabolic turnover. The consistency of these predictions with published experimental results is demonstrated. The shrinking core model provides a simple analytic relationship predicting the effects of altered antibody pharmacokinetics, antibody molecular weight, antigen turnover rate, antigen expression level, and micrometastasis size on antibody penetration and retention. For example, a formula is provided for predicting the bolus dose necessary to accomplish tumor saturation as a function of antibody and tumor properties. Furthermore, this analysis indicates certain attributes necessary for an optimal tumor targeting agent. PMID:12649189

  11. Preparation and Affinity-Purification of Supervillin Isoform 4 (SV4) Specific Polyclonal Antibodies.

    PubMed

    Chen, Xueran; Li, Hao; Wang, Hongzhi; Yang, Haoran; Ye, Fang; Liang, Chaozhao; Fang, Zhiyou

    2016-04-01

    Human Supervillin isoform 4 (SV4), a bigger splicing isoform of Supervillin, contains extra coding exons 3, 4 and 5 (E345), compared to Supervillin isoform 1. Although previous studies have shown that SV4 associated with membrane and cytoskeleton, regulated cell migration and cell survival, its functions are still largely unknown. To broaden our understanding, SV4 specific antibody is important for further study in signaling pathway. The His-SV4 (E345) and GST-SV4 (E345) fusion proteins, which contained SV4 specific domain E345, were purified from bacteria. The His-SV4 (E345) proteins were injected in rabbits as immunogen to produce anti-SV4 serum, and SV4 antibodies were purified by GST-SV4 (E345) proteins cross-linked to affinity resins. SV4 antibodies exclusively recognized SV4 protein both in vitro and in vivo through multi-step testing by ELISA, western blot, immunoprecipitation, and immunofluorescence. Taken together, our data demonstrate a novel SV4-specific polyclonal antibody which will provide a useful tool for further characterization of SV4 function. PMID:27015936

  12. 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.

  13. 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.

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

    PubMed

    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

  16. 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

  17. 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

    2016-04-01

    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. PMID:26949186

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

    DOE PAGESBeta

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M.  Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A.; Wiehe, Kevin; et al

    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

  19. In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody.

    PubMed

    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

  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. Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine

    PubMed Central

    Li, Lei; Honda-Okubo, Yoshikazu; Li, Connie; Sajkov, Dimitar; Petrovsky, Nikolai

    2015-01-01

    There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation. Trial Registration Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709 PMID:26177480

  2. 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. PMID:26041237

  3. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    SciTech Connect

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. We have studied the binding of 125I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind (formula; see text). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10(8) M-1 are not likely to be useful for drug targeting or tumor imaging.

  4. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development. PMID:27224530

  5. 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

  6. Determination of Mother Centriole Maturation in CPAP-Depleted Cells Using the Ninein Antibody

    PubMed Central

    Lee, Miseon

    2015-01-01

    Background Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. Methods We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. Results Ninein signals were significantly impaired in CPAP-depleted cells. Conclusion The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells. PMID:25827458

  7. 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

    2015-01-01

    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. PMID:25996440

  8. New High Affinity Monoclonal Antibodies Recognize Non-Overlapping Epitopes On Mesothelin For Monitoring And Treating Mesothelioma

    PubMed Central

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

    2015-01-01

    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. PMID:25996440

  9. 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

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

    PubMed

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

    2013-01-01

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

  11. “Velcro” Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein α (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis*

    PubMed Central

    Ho, Chia Chi M.; Guo, Nan; Sockolosky, Jonathan T.; Ring, Aaron M.; Weiskopf, Kipp; Özkan, Engin; Mori, Yasuo; Weissman, Irving L.; Garcia, K. Christopher

    2015-01-01

    CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the “don't-eat-me” signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined “Velcro” engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that “Velcro” engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy

  12. Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta.

    PubMed

    Nagai, Taku; Furusho, Yuko; Li, Hua; Hasui, Kazuhisa; Matsukita, Sumika; Sueyoshi, Kazunobu; Yanagi, Masakazu; Hatae, Masaki; Takao, Sonshin; Matsuyama, Takami

    2015-06-01

    Folate receptors α (FRα) and β (FRβ) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRα is rare in normal cells and elevated in cancer cells. Thus, FRα-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRβ and that these cells can enhance tumor growth. Although FRα and FRβ share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRα-expressing cancer cells and FRβ-expressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRα or anti-FRβ MAbs. In this study, we developed a MAb that recognizes both FRα and FRβ (anti-FRαβ). The anti-FRαβ specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRαβ bound to soluble forms of the FRα and FRβ proteins with high affinity (KD=6.26×10(-9) M and 4.33×10(-9) M, respectively). In vitro functional analysis of the anti-FRαβ showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRα-expressing and FRβ-expressing cell lines. The anti-FRαβ MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression. PMID:26090596

  13. High-Affinity Self-Reactive Human Antibodies by Design and Selection: Targeting the Integrin Ligand Binding Site

    NASA Astrophysics Data System (ADS)

    Barbas, Carlos F., III; Languino, Lucia R.; Smith, Jeffrey W.

    1993-11-01

    A strategy for the design and selection of human antibodies that bind receptors is described. We have demonstrated the validity of the approach by producing semisynthetic human antibodies that bind integrins α_vβ_3 and αIIbβ_3 with high affinity (10-10 M). The selected antibodies mimic the integrins' natural ligands as demonstrated by their ability to compete with these ligands and Arg-Gly-Asp (RGD)-containing peptides for binding to the integrins. Furthermore, the antibodies bind in a cation-dependent fashion and are functional in cell adhesion assays. Antibodies that are high-affinity inhibitors of cell adhesion receptors should be of use in assessing receptor function and dissecting mechanisms of adhesion. Semisynthetic human antibodies that target integrins are potential therapeutic agents for the treatment of a number of diseases including thrombosis and metastasis. Furthermore, antibodies that are optimized to bind by a single complementarity determining region may be important lead compounds for the design of small molecule pharmaceuticals.

  14. Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics.

    PubMed

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2016-09-01

    The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a "fit-for-purpose" approach in instrument selection for biosensor studies. PMID:27365220

  15. 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. PMID:27095060

  16. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    SciTech Connect

    Miles, L.A.; Plow, E.F.

    1986-11-04

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound (/sup 125/I)EDP I, (/sup 125/I)Glu-plasminogen, and (/sup 125/I)Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of (/sup 125/I)EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 ..mu..M, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. ..cap alpha../sub 2/-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of (/sup 125/I)EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor.

  17. 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. PMID:24583603

  18. Impact of Intranasal Insulin on Insulin Antibody Affinity and Isotypes in Young Children With HLA-Conferred Susceptibility to Type 1 Diabetes

    PubMed Central

    Ryhänen, Samppa J.; Härkönen, Taina; Siljander, Heli; Näntö-Salonen, Kirsti; Simell, Tuula; Hyöty, Heikki; Ilonen, Jorma; Veijola, Riitta; Simell, Olli; Knip, Mikael

    2011-01-01

    OBJECTIVE Despite promising results from studies on mouse models, intranasal insulin failed to prevent or delay the development of type 1 diabetes in autoantibody-positive children with HLA-conferred disease susceptibility. To analyze whether the insulin dose was inadequate to elicit an immunomodulatory response, we compared the changes observed in insulin antibody (IA) affinity and isotypes after treatment with nasal insulin or placebo. RESEARCH DESIGN AND METHODS Ninety-five children (47 in the placebo group and 48 in the insulin group of the total of 224 children randomized for the trial) with HLA-conferred susceptibility to type 1 diabetes derived from the intervention arm of the Finnish Type 1 Diabetes Prediction and Prevention study were included in these analyses. Blood samples drawn before or at the beginning of the treatment and after treatment for 3 and 6 months were analyzed for IA affinity and isotype-specific IAs (IgG1–4, IgA, IgM, and IgE). RESULTS IgG3- and IgA-IA levels (P = 0.031 and 0.015, respectively) and the number of IgG3-IA–positive subjects (P = 0.022) were significantly higher at 6 months after the initiation of the treatment in the insulin group. No significant differences were observed between the two groups in IA affinity or other IA isotypes. CONCLUSIONS The insulin dose administered induced a modest change in the IA isotype profile. The lack of impact of nasal insulin on IA affinity implies that the immune response of study subjects was already mature at the beginning of the intervention. PMID:21515841

  19. 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

  20. The Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor Targeting of Monoclonal Antibodies.

    PubMed

    Glatt, Dylan M; Beckford Vera, Denis R; Parrott, Matthew C; Luft, J Christopher; Benhabbour, S Rahima; Mumper, Russell J

    2016-06-01

    Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells in vitro, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones in vivo. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice. PMID:27079967

  1. 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.

  2. 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

  3. 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

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

    PubMed

    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

  5. Direct binding of radioiodinated monoclonal antibody to tumor cells: significance of antibody purity and affinity for drug targeting or tumor imaging

    SciTech Connect

    Kennel, S.J.; Foote, L.J.; Lankford, P.K.; Johnson, M.; Mitchell, T.; Braslawsky, G.R.

    1983-01-01

    For MoAb to be used efficiently for drug targeting and tumor imaging, the fraction of antibody binding to tumor cells must be maximized. The authors have studied the binding of /sup 125/I MoAb in three different tumor systems. The fraction of antibody that could be bound to the cell surface was directly proportional to the antibody purity. The affinity constant also limits the fraction of antibody that can bind to cells at a given antigen concentration. Rearrangement of the standard expression for univalent equilibrium binding between two reactants shows that in antigen excess, the maximum fraction of antibody that can bind =Ka(Ag total)/1 + Ka(Ag total). Binding data using four different MoAb with three cell systems confirm this relationship. Estimates for reasonable concentrations of tumor antigens in vivo indicate that antibodies with binding constants less than 10/sup 8/ M/sup -1/ are not likely to be useful for drug targeting or tumor imaging.

  6. A Lentiviral Vector Allowing Physiologically Regulated Membrane-anchored and Secreted Antibody Expression Depending on B-cell Maturation Status.

    PubMed

    Fusil, Floriane; Calattini, Sara; Amirache, Fouzia; Mancip, Jimmy; Costa, Caroline; Robbins, Justin B; Douam, Florian; Lavillette, Dimitri; Law, Mansun; Defrance, Thierry; Verhoeyen, Els; Cosset, François-Loïc

    2015-11-01

    The development of lentiviral vectors (LVs) for expression of a specific antibody can be achieved through the transduction of mature B-cells. This approach would provide a versatile tool for active immunotherapy strategies for infectious diseases or cancer, as well as for protein engineering. Here, we created a lentiviral expression system mimicking the natural production of these two distinct immunoglobulin isoforms. We designed a LV (FAM2-LV) expressing an anti-HCV-E2 surface glycoprotein antibody (AR3A) as a membrane-anchored Ig form or a soluble Ig form, depending on the B-cell maturation status. FAM2-LV induced high-level and functional membrane expression of the transgenic antibody in a nonsecretory B-cell line. In contrast, a plasma cell (PC) line transduced with FAM2-LV preferentially produced the secreted transgenic antibody. Similar results were obtained with primary B-cells transduced ex vivo. Most importantly, FAM2-LV transduced primary B-cells efficiently differentiated into PCs, which secreted the neutralizing anti-HCV E2 antibody upon adoptive transfer into immunodeficient NSG (NOD/SCIDγc(-/-)) recipient mice. Altogether, these results demonstrate that the conditional FAM2-LV allows preferential expression of the membrane-anchored form of an antiviral neutralizing antibody in B-cells and permits secretion of a soluble antibody following B-cell maturation into PCs in vivo. PMID:26281898

  7. Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against. cap alpha. -transforming growth factor

    SciTech Connect

    Hazarika, P.; Pardue, R.L.; Earls, R.; Dedman, J.R.

    1987-04-07

    Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human ..cap alpha..-transforming growth factor (..cap alpha..-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting ..cap alpha..-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native ..cap alpha..-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of ..cap alpha..-TCF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of ..cap alpha..-TGF has a cellular role beyond that of an autocrine growth factor.

  8. mCSM-AB: a web server for predicting antibody-antigen affinity changes upon mutation with graph-based signatures.

    PubMed

    Pires, Douglas E V; Ascher, David B

    2016-07-01

    Computational methods have traditionally struggled to predict the effect of mutations in antibody-antigen complexes on binding affinity. This has limited their usefulness during antibody engineering and development, and their ability to predict biologically relevant escape mutations. Here we present mCSM-AB, a user-friendly web server for accurately predicting antibody-antigen affinity changes upon mutation which relies on graph-based signatures. We show that mCSM-AB performs better than comparable methods that have been previously used for antibody engineering. mCSM-AB web server is available at http://structure.bioc.cam.ac.uk/mcsm_ab. PMID:27216816

  9. 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

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

    PubMed

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

    2016-01-01

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

  11. 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. PMID:27391930

  12. 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

  13. Affinity maturation of anti-TNF-alpha scFv with somatic hypermutation in non-B cells.

    PubMed

    Chen, Shaopeng; Qiu, Junkang; Chen, Chuan; Liu, Chunchun; Liu, Yuheng; An, Lili; Jia, Junying; Tang, Jie; Wu, Lijun; Hang, Haiying

    2012-06-01

    Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient. PMID:22467272

  14. Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

    PubMed Central

    2014-01-01

    Background The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies. Results We have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication. Conclusions Together, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef. PMID:24620746

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

    PubMed Central

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

    2011-01-01

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

  16. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    PubMed

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  17. A comparison of binding surfaces for SPR biosensing using an antibody-antigen system and affinity distribution analysis

    PubMed Central

    Zhao, Huaying; Gorshkova, Inna I.; Fu, Gregory L.; Schuck, Peter

    2013-01-01

    The application of optical biosensors in the study of macromolecular interactions requires immobilization of one binding partner to the surface. It is often highly desirable that the immobilization is uniform and does not affect the thermodynamic and kinetic binding parameters to soluble ligands. To achieve this goal, a variety of sensor surfaces, coupling strategies and surface chemistries are available. Previously, we have introduced a technique for increasing the level of detail on the immobilized sites beyond an average affinity by determining the distribution of affinities and kinetic rate constants from families of binding and dissociation traces acquired at different concentrations of soluble ligand. In the present work, we explore how this affinity distribution analysis can be useful in the assessment and optimization of surface immobilization. With this goal, using an antibody-antigen interaction as a model system, we study the activity, thermodynamic and kinetic binding parameters, and heterogeneity of surface sites produced with different commonly used sensor surfaces, at different total surface densities and with direct immobilization or affinity capture. PMID:23270815

  18. 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

    2016-04-01

    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. PMID:26952369

  19. 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

    2015-09-01

    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. TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

    PubMed

    Foss, Stian; Watkinson, Ruth E; Grevys, Algirdas; McAdam, Martin B; Bern, Malin; Høydahl, Lene Stokken; Dalhus, Bjørn; Michaelsen, Terje E; Sandlie, Inger; James, Leo C; Andersen, Jan Terje

    2016-04-15

    Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation. In this article, we show how Ab affinity modulates TRIM21 immune function. We constructed a humanized monoclonal IgG1 against human adenovirus type 5 (AdV5) and a panel of Fc-engineered variants with a wide range of affinities for TRIM21. We found that IgG1-coated viral particles were neutralized via TRIM21, even when affinity was reduced by as much as 100-fold. In contrast, induction of NF-κB signaling was more sensitive to reduced affinity between TRIM21 and the Ab variants. Thus, TRIM21 mediates neutralization under suboptimal conditions, whereas induction of immune signaling is balanced according to the functional affinity for the incoming immune stimuli. Our findings have implications for engineering of antiviral IgG therapeutics with tailored effector functions. PMID:26962230

  1. Discovery of highly soluble antibodies prior to purification using affinity-capture self-interaction nanoparticle spectroscopy.

    PubMed

    Wu, Jiemin; Schultz, Jason S; Weldon, Caroline L; Sule, Shantanu V; Chai, Qing; Geng, Steven B; Dickinson, Craig D; Tessier, Peter M

    2015-10-01

    Self-association of monoclonal antibodies (mAbs) at high concentrations can result in developability challenges such as poor solubility, aggregation, opalescence and high viscosity. There is a significant unmet need for methods that can evaluate self-association propensities of concentrated mAbs at the earliest stages in antibody discovery to avoid downstream issues. We have previously developed a method (affinity-capture self-interaction nanoparticle spectroscopy, AC-SINS) that is capable of detecting weak antibody self-interactions using unusually dilute mAb solutions (tens of µg/ml). Here we optimize and implement this assay for characterization of unpurified and highly dilute mAbs directly in cell culture media. This assay was applied to screen 87 mAbs obtained via immunization. Our measurements reveal a wide range of self-associative propensities for mAbs that bind to the same antigen and which differ mainly in their complementarity-determining regions. The least associative mAbs identified by AC-SINS were confirmed to be highly soluble when purified and concentrated by three to five orders of magnitude. This approach represents a key advance in screening mAb variants using unpurified antibody samples, and it holds significant potential to both improve initial candidate selection as well as to guide protein engineering efforts to improve the properties of specific mAb candidates. PMID:26363633

  2. Single domain antibody-alkaline phosphatase fusion proteins for antigen detection--analysis of affinity and thermal stability of single domain antibody.

    PubMed

    Liu, Jinny L; Zabetakis, Dan; Lee, Audrey Brozozog; Goldman, Ellen R; Anderson, George P

    2013-07-31

    Single domain antibody (sdAb)-alkaline phosphatase (AP) fusion proteins have been demonstrated to be useful immunodiagnostic reagents for bio-threat agent detection. The bivalent nature of sdAb-AP fusion proteins significantly increases effective affinity and thus the sensitivity of detection, but the thermal stability of the fusion protein had not been explored. This property is critical for the development of immunoassays for use in austere environments. In this study four sdAbs with specificity for MS2 phage coat protein (CP) were expressed as fusions with AP in order to evaluate the thermal stability and affinity of the resulting constructs. The melting temperature (Tm) of the sdAb and sdAb-AP fusion proteins was measured by a combination of Circular Dichroism (CD), differential scanning calorimetry (DSC) and Fluorescence-based Thermal Shift assay. Binding kinetics were assessed using surface plasmon resonance (SPR). Our results indicated that the AP fusion protein did not increase the Tm or enhance thermal stability of the sdAb, but did provide the expected increase in binding affinity as compared to the original sdAb. PMID:23570946

  3. 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. PMID:27236551

  4. Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

    PubMed Central

    Koduvayur, Sujatha P.; Gussin, Hélène A.; Parthasarathy, Rajni; Hao, Zengping; Kay, Brian K.; Pepperberg, David R.

    2014-01-01

    The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure. PMID:24586298

  5. 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

  6. Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

    PubMed Central

    Sørensen, Morten Dræby; Agerholm, Inge Errebo; Christensen, Britta; Kølvraa, Steen; Kristensen, Peter

    2010-01-01

    Abstract Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non-invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV-irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific. PMID:20726925

  7. Preparation and characterization of novel IgG affinity resin coupling anti-Fc camelid single-domain antibodies.

    PubMed

    Tu, Zhui; Xu, Yang; Fu, Jinheng; Huang, Zhibing; Wang, Yao; Liu, Bin; Tao, Yong

    2015-03-01

    This work aimed to evaluate novel affinity resin used to purify immunoglobulin G (IgG) with a variable domain of the heavy chain of the heavy-chain antibody (VHH) as an affinity ligand. The VHH, isolated from a naïve camelid single-domain phage display library, exhibits not only affinity to the fragment crystallizable (Fc) region of IgG but also high thermal stability. This anti-Fc VHH (AFV) was expressed as a soluble protein in Escherichia coli and purified using a simple heat treatment procedure. The effects of pH and NaCl concentrations on the capacity of AFV resin were also investigated. Results showed a robust property of the AFV resin. It could bind IgGs at various pH conditions (from 6.0 to 9.0) and NaCl concentrations. The static binding capacities of AFV resin ranged from 3.40±0.53mg/ml to 15.04±0.37mg/ml measured using rabbit, mouse, and human IgGs. The bound IgGs can be efficiently eluted at pH 5.0, which is conducive to acid-sensitive IgGs and prevents the aggregation of IgGs. After 10 purification cycles or a 7-day period of storage at 37°C, recovery did not decrease. These findings suggested that VHHs from non-immunized library could also be robust and functional reagent as an affinity purification ligand. PMID:25614967

  8. 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

  9. High-Resolution Longitudinal Study of HIV-1 Env Vaccine-Elicited B Cell Responses to the Virus Primary Receptor Binding Site Reveals Affinity Maturation and Clonal Persistence.

    PubMed

    Wang, Yimeng; Sundling, Christopher; Wilson, Richard; O'Dell, Sijy; Chen, Yajing; Dai, Kaifan; Phad, Ganesh E; Zhu, Jiang; Xiao, Yongli; Mascola, John R; Karlsson Hedestam, Gunilla B; Wyatt, Richard T; Li, Yuxing

    2016-05-01

    Because of the genetic variability of the HIV-1 envelope glycoproteins (Env), the elicitation of neutralizing Abs to conserved neutralization determinants including the primary receptor binding site, CD4 binding site (CD4bs), is a major focus of vaccine development. To gain insight into the evolution of Env-elicited Ab responses, we used single B cell analysis to interrogate the memory B cell Ig repertoires from two rhesus macaques after five serial immunizations with Env/adjuvant. We observed that the CD4bs-specific repertoire displayed unique features in the third CDR of Ig H chains with minor alterations along the immunization course. Progressive affinity maturation occurred as evidenced by elevated levels of somatic hypermutation (SHM) in Ab sequences isolated at the late immunization time point compared with the early time point. Abs with higher SHM were associated with increased binding affinity and virus neutralization capacity. Moreover, a notable portion of the CD4bs-specific repertoire was maintained between early and late immunization time points, suggesting that persistent clonal lineages were induced by Env vaccination. Furthermore, we found that the predominant persistent CD4bs-specific clonal lineages had larger population sizes and higher affinities than that from the rest of the repertoires, underscoring the critical role of Ag affinity selection in Ab maturation and clonal expansion. Genetic and functional analyses revealed that the accumulation of SHM in both framework regions and CDRs contributed to the clonal affinity and antigenicity evolution. Our longitudinal study provides high-resolution understanding of the dynamically evolving CD4bs-specific B cell response after Env immunization in primates. PMID:27001953

  10. 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. PMID:1517325

  11. Applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes - relevance for vaccine design.

    PubMed

    Denisova, Galina F; Denisov, Dimitri A; Bramson, Jonathan L

    2010-01-01

    To properly characterize protective polyclonal antibody responses, it is necessary to examine epitope specificity. Most antibody epitopes are conformational in nature and, thus, cannot be identified using synthetic linear peptides. Cyclic peptides can function as mimetics of conformational epitopes (termed mimotopes), thereby providing targets, which can be selected by immunoaffinity purification. However, the management of large collections of random cyclic peptides is cumbersome. Filamentous bacteriophage provides a useful scaffold for the expression of random peptides (termed phage display) facilitating both the production and manipulation of complex peptide libraries. Immunoaffinity selection of phage displaying random cyclic peptides is an effective strategy for isolating mimotopes with specificity for a given antiserum. Further epitope prediction based on mimotope sequence is not trivial since mimotopes generally display only small homologies with the target protein. Large numbers of unique mimotopes are required to provide sufficient sequence coverage to elucidate the target epitope. We have developed a method based on pattern recognition theory to deal with the complexity of large collections of conformational mimotopes. The analysis consists of two phases: 1) The learning phase where a large collection of epitope-specific mimotopes is analyzed to identify epitope specific "signs" and 2) The identification phase where immunoaffinity-selected mimotopes are interrogated for the presence of the epitope specific "signs" and assigned to specific epitopes. We are currently using computational methods to define epitope "signs" without the need for prior knowledge of specific mimotopes. This technology provides an important tool for characterizing the breadth of antibody specificities within polyclonal antisera. PMID:21067548

  12. Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies

    PubMed Central

    Bhiman, Jinal N.; Anthony, Colin; Doria-Rose, Nicole A.; Karimanzira, Owen; Schramm, Chaim A.; Khoza, Thandeka; Kitchin, Dale; Botha, Gordon; Gorman, Jason; Garrett, Nigel J.; Abdool Karim, Salim S.; Shapiro, Lawrence; Williamson, Carolyn; Kwong, Peter D.; Mascola, John R.; Morris, Lynn; Moore, Penny L.

    2015-01-01

    The elicitation of broadly neutralizing antibodies (bNAbs) is likely to be essential for a preventative HIV-1 vaccine, but this has not yet been achieved by immunization. In contrast some HIV-1-infected individuals naturally mount bNAb responses during chronic infection, suggesting that years of maturation are required for breadth1-6. Recent studies have shown that viral diversification precedes the emergence of bNAbs but the significance of this observation is unknown7,8. Here, we delineate the key viral events that drove neutralization breadth within the CAP256-VRC26 family of 33 monoclonal antibodies (mAbs) isolated from a superinfected individual. First, we identified minority viral variants that were distinct from both transmitted/founder (T/F) viruses and efficiently engaged the bNAb precursor, termed bNAb-initiating envelopes. Second, deep sequencing revealed a pool of diverse epitope variants (immunotypes) that were preferentially neutralized by broader members of the antibody lineage. In contrast, a “dead-end” antibody sublineage unable to neutralize these immunotypes showed limited evolution and failed to develop breadth. Thus, early viral escape at key antibody-virus contact sites selects for sublineages that can tolerate these changes, providing a new mechanism for the generation of neutralization breadth within a developing antibody lineage. PMID:26457756

  13. 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. PMID:25865483

  14. Arginine as an eluent overcomes the hindrance of monoclonal antibody quantification by dextran sulfate in protein A affinity chromatography.

    PubMed

    Kim, Bong Gyun; Park, Hong Woo

    2015-01-01

    Analytical chromatography using protein A affinity columns was employed for the fast and simple quantitative analysis of monoclonal antibodies (mAb) from suspension cultures of recombinant Chinese hamster ovary (rCHO) cells. Reliable results could not be obtained from analysis of rCHO cell culture supernatants containing dextran sulfate using elution buffers such as phosphate, glycine, or MgCl2 . These problems increased as the number of analysis and the concentration of dextran sulfate in samples increased. Arginine was identified as an alternative eluent to overcome the hindrance by dextran sulfate. When the samples contain dextran sulfate up to 100 mg/L, the elution buffer containing 0.6-1.0 M arginine at pH 3.0-3.8 is useful for the effective analysis. Reproducible results in the mAb quantification could be obtained by this developed arginine elution buffer from rCHO cell culture supernatants containing dextran sulfate. PMID:26363185

  15. 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. PMID:26187320

  16. A high-affinity anti-salbutamol monoclonal antibody: key to a robust lateral-flow immunochromatographic assay.

    PubMed

    Xie, Chun-hua; Chen, Fa-ju; Yang, Tang-bin

    2012-07-15

    Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose-response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL-BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5 min, with the visual detection limit of 1 ngml(-1) in phosphate-buffered saline. Cross-reactions with other β-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine. PMID:22507376

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

    PubMed

    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. Graphical Abstract ᅟ. PMID:27067900

  18. 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.

  19. 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

    2016-04-01

    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.

  20. Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.

    PubMed

    Taylor, M A; Baker, K C; Briggs, G S; Connerton, I F; Cummings, N J; Pratt, K A; Revell, D F; Freedman, R B; Goodenough, P W

    1995-01-01

    Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes. PMID:7770454

  1. Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins.

    PubMed

    Xu, Jingjing; Ambrosini, Serena; Tamahkar, Emel; Rossi, Claire; Haupt, Karsten; Tse Sum Bui, Bernadette

    2016-01-11

    We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins. PMID:26644006

  2. 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

  3. 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

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

    PubMed

    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

  5. 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

  6. Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

    PubMed Central

    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. PMID:25360777

  7. Preliminary study of the metal binding site of an anti-DTPA-indium antibody by equilibrium binding immunoassays and immobilized metal ion affinity chromatography.

    PubMed

    Boden, V; Colin, C; Barbet, J; Le Doussal, J M; Vijayalakshmi, M

    1995-01-01

    Creating metal coordination sites by modifying an existing enzyme or by eliciting antibodies against metal chelate haptens is of great interest in biotechnology to create enzyme catalysts with novel specificities. Here, we investigate the metal binding potential of a monoclonal antibody raised against a DTPA-In(III) hapten (mAb 734). We study its relative binding efficiency to metals of biological relevance by equilibrium binding immunoassays and immobilized metal ion affinity chromatography, two approaches which can give complementary information regarding composition and/or structure of the metal binding site(s). Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(II) binding was compared to In(III). All of them were shown to displace indium, but their affinity for mAb 734 decreased by 100-fold compared to indium. Competitive metal binding immunoassays between Zn(II) and In(III) revealed an unusual behavior by Zn(II) which remains to be explained. Moreover, IMAC allowed us to predict the metal binding amino acids involved in the antibody paratope. The antibody metal binding site was shown to contain at least two histidine residues in a cluster, and the presence of aspartic and glutamic acid as well as cysteine residues could not be excluded. Thus, simple competition studies allows us to obtain some partial information on the metal binding structural features of this anti-metal chelate antibody and to guide our screening of its catalytic potential. PMID:7578356

  8. 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

  9. 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

  10. Antibody raised to AKAAAKAAAKA sequence on tropoelastin recognizes tropoelastin but not mature crosslinked elastin: A new tool in metabolic and structural studies of elastogenesis.

    PubMed

    Starcher, B; Conrad, N; Hinek, A; Hill, C H

    1999-01-01

    Tropoelastin, which is secreted from the cell in a soluble form, contains specific alanine rich repeat domains that are destined to form covalent desmosine and isodesmosine crosslinks in mature insoluble elastin. We raised a monospecific polyclonal antibody to a AKAAAKAAAKA synthetic peptide (AKA) which represents this alanine rich region of tropoelastin. The antibody was reactive with the original peptide antigen and purified tropoelastin, but not with mature crosslinked elastin isolated from several animal species. Conditioned media from chick aorta smooth muscle cells in culture reacted in an ELISA with the AKA antibody, but only in the presence of BAPN to block the conversion of the epsilon-amino groups to aldehydes. Immunofluorescence demonstrated that the AKA antibody decorated newly deposited tropoelastin assembled in fine fibrils in matrix produced by cultured human skin fibroblasts. EM-immunogold specifically localized this antibody to the immature elastic fibers present in fetal sheep ductus arteriosus. Moreover, immunohistochemistry demonstrated that the antibody recognized nonpolymerized tropoelastin assembled on the periphery of elastic fibers in the aorta of chicks raised on copper deficient and BAPN containing diets. These studies demonstrate that this new anti-tropoelastin antibody can be used as a useful tool to investigate elastin metabolism where it is important to distinguish between tropoelastin and mature crosslinked elastin. PMID:10757115

  11. 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

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

    PubMed

    Tai, Yu-Tzu; 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

    2014-05-15

    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

  13. Characterization of the Native and Denatured Herceptin by ELISA and QCM using a High-Affinity Single Chain Fragment Variable (scFv) Recombinant Antibody

    PubMed Central

    Shang, Yuqin; Mernaugh, Ray

    2012-01-01

    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 an scFv (designated 2B4) to a linear synthetic peptide representing Herceptin’s heavy chain CDR3. 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 amount 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 × 1013 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 non-specific binding events in assays. The potential theoretical and clinical implications of these results and the advantages of using QCM to characterize human therapeutic antibodies in samples are also discussed. PMID:22934911

  14. 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. PMID:20869832

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

    PubMed

    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

  16. 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

  17. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity.

    PubMed

    Lu, Dan; Shen, Juqun; Vil, Marie D; Zhang, Haifan; Jimenez, Xenia; Bohlen, Peter; Witte, Larry; Zhu, Zhenping

    2003-10-31

    Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original naïve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides

  18. 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

  19. Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

    PubMed Central

    Grate, Jay W.; Tyler, Abby; Ozanich, Richard M.; Miller, Keith D.; Lou, Jianlong; Marks, James D.; Bruckner-Lea, Cynthia J.

    2009-01-01

    A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum neurotoxin serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) 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 using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. Detection to 31 pM with a total incubation time of 3 hours was demonstrated. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. The beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell equipped with a fiber optic system for fluorescence measurements. In PBS buffer, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach. PMID:19643593

  20. 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.

  1. 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

  2. FYWHCLDE-based affinity chromatography of IgG: effect of ligand density and purifications of human IgG and monoclonal antibody.

    PubMed

    Zhao, Wei-Wei; Shi, Qing-Hong; Sun, Yan

    2014-08-15

    This work reports the development of an octapeptide-based affinity adsorbent for the purification of human IgG (hIgG) and monoclonal antibody (mAb). The octapeptide was FYWHCLDE selected earlier by the biomimetic design of affinity peptide ligands for hIgG. The ligand was coupled to Sepharose gel at four densities from 10.4 to 31.0μmol/mL, and the effect of peptide density on the adsorption of hIgG and bovine serum albumin (BSA) was first investigated. The binding capacity of hIgG increased from 104.2 to 176.4mg/mL within the ligand density range, and the binding affinity (dissociation constant) kept at 2.4-3.7μM. Batch adsorption revealed that the selectivity of FYWHCLDE-Sepharose for IgG was 30-40 times over BSA. The effective pore diffusivity of IgG decreased somewhat with increasing ligand density, but the dynamic binding capacity at 10% breakthrough, measured by using 10-fold diluted human serum as feedstock, doubled with increasing ligand density from 10.4 to 31.0μmol/mL due to the remarkable increase of static binding capacity. By using the affinity column with a ligand density of 23.9μmol/mL, hIgG and humanized mAb purifications from human serum and cell culture supernatant, respectively, were achieved at high purities and recovery yields. Finally, the robustness of the peptide gel was demonstrated by recycled use of the affinity column in 20 breakthrough cycles. PMID:24947889

  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. 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 ...

  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. PMID:15969005

  6. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities

    PubMed 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

  7. Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2*

    PubMed Central

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle; van Agthoven, Jan; Raynal, Bertrand; Hoos, Sylviane; Kragelund, Birthe B.; Kelly, Paul A.; Ducruix, Arnaud; England, Patrick; Goffin, Vincent

    2010-01-01

    We report the first crystal structure of a 1:2 hormone·receptor complex that involves prolactin (PRL) as the ligand, at 3.8-Å resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL·PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 (“three-pin plug”): the conserved glycine 129 of helix α3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL·PRLR2 structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer. PMID:20053995

  8. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    PubMed Central

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  9. Molecular engineering of high affinity single-chain antibody fragment for endothelial targeting of proteins and nanocarriers in rodents and humans.

    PubMed

    Greineder, Colin F; Hood, Elizabeth D; Yao, Anning; Khoshnejad, Makan; Brenner, Jake S; Johnston, Ian H; Poncz, Mortimer; Gottstein, Claudia; Muzykantov, Vladimir R

    2016-03-28

    Endothelial cells (EC) represent an important target for pharmacologic intervention, given their central role in a wide variety of human pathophysiologic processes. Studies in lab animal species have established that conjugation of drugs and carriers with antibodies directed to surface targets like the Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, a highly expressed endothelial transmembrane protein) help to achieve specific therapeutic interventions in ECs. To translate such "vascular immunotargeting" to clinical practice, it is necessary to replace antibodies by advanced ligands that are more amenable to use in humans. We report the molecular design of a single chain variable antibody fragment (scFv) that binds with high affinity to human PECAM-1 and cross-reacts with its counterpart in rats and other animal species, allowing parallel testing in vivo and in human endothelial cells in microfluidic model. Site-specific modification of the scFv allows conjugation of protein cargo and liposomes, enabling their endothelial targeting in these models. This study provides a template for molecular engineering of ligands, enabling studies of drug targeting in animal species and subsequent use in humans. PMID:26855052

  10. 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

  11. 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

  12. Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking.

    PubMed

    Li, Qian; Wang, Jing; Yang, Lingjian; Gao, Xiaokang; Chen, Hongwei; Zhao, Xinfeng; Bian, Liujiao; Zheng, Xiaohui

    2015-07-01

    Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10(-10) M and (1.39 ± 0.12) × 10(9) M(-1). Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. PMID:25727342

  13. STRUCTURE OF A HIGH-AFFINITY “MIMOTOPE” PEPTIDE BOUND TO HIV-1-NEUTRALIZING ANTIBODY b12 EXPLAINS ITS INABILITY TO ELICIT gp120 CROSS-REACTIVE ANTIBODIES

    PubMed Central

    Saphire, Erica Ollmann; Montero, Marinieve; Menendez, Alfredo; van Houten, Nienke E.; Irving, Melita B.; Pantophlet, Ralph; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Scott, Jamie K.; Wilson, Ian A.

    2007-01-01

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary HIV-1 isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N-terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Å resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing

  14. Substitution of Heavy Complementarity Determining Region 3 (CDR-H3) Residues Can Synergistically Enhance Functional Activity of Antibody and Its Binding Affinity to HER2 Antigen

    PubMed Central

    Moon, Seung Kee; Park, So Ra; Park, Ami; Oh, Hyun Mi; Shin, Hyun Jung; Jeon, Eun Ju; Kim, Seiwhan; Park, Hyun June; Yeon, Young Joo; Yoo, Young Je

    2016-01-01

    To generate a biobetter that has improved therapeutic activity, we constructed scFv libraries via random mutagenesis of several residues of CDR-H3 and -L3 of hu4D5. The scFv clones were isolated from the phage display libraries by stringent panning, and their anti-proliferative activity against HER2-positive cancer cells was evaluated as a primary selection criterion. Consequently, we selected AH06 as a biobetter antibody that had a 7.2-fold increase in anti-proliferative activity (IC50: 0.81 nM) against the gastric cancer cell line NCI-N87 and a 7.4-fold increase in binding affinity (KD: 60 pM) to HER2 compared to hu4D5. The binding energy calculation and molecular modeling suggest that the substitution of residues of CDR-H3 to W98, F100c, A101 and L102 could stabilize binding of the antibody to HER2 and there could be direct hydrophobic interactions between the aromatic ring of W98 and the aliphatic group of I613 within HER2 domain IV as well as the heavy and light chain hydrophobic interactions by residues F100c, A101 and L102 of CDR-H3. Therefore, we speculate that two such interactions were exerted by the residues W98 and F100c. A101 and L102 may have a synergistic effect on the increase in the binding affinity to HER2. AH06 specifically binds to domain IV of HER2, and it decreased the phosphorylation level of HER2 and AKT. Above all, it highly increased the overall level of p27 compared to hu4D5 in the gastric cancer cell line NCI-N82, suggesting that AH06 could potentially be a more efficient therapeutic agent than hu4D5. PMID:26743905

  15. Tn Antigen Mimics Based on sp(2)-Iminosugars with Affinity for an anti-MUC1 Antibody.

    PubMed

    Fernández, Elena M Sánchez; Navo, Claudio D; Martínez-Sáez, Nuria; Gonçalves-Pereira, Rita; Somovilla, Víctor J; Avenoza, Alberto; Busto, Jesús H; Bernardes, Gonçalo J L; Jiménez-Osés, Gonzalo; Corzana, Francisco; Fernández, José M García; Mellet, Carmen Ortiz; Peregrina, Jesús M

    2016-08-01

    The first examples of amino acid (Ser/Thr)-sp(2)-iminosugar glycomimetic conjugates featuring an α-O-linked pseudoanomeric linkage are reported. The key synthetic step involves the completely diastereoselective α-glycosylation of Ser/Thr due to strong stereoelectronic and conformational bias imposed by the bicyclic sp(2)-iminosugar scaffold. Mucin-related glycopeptides incorporating these motifs were recognized by the monoclonal antibody (mAb) scFv-SM3, with activities depending on both the hydroxylation pattern (Glc/Gal/GlcNAc/GalNAc) of the sp(2)-iminosugar and the peptide aglycone structure (Ser/Thr). PMID:27453399

  16. Germinal center B cells govern their own fate via antibody feedback

    PubMed Central

    Zhang, Yang; Meyer-Hermann, Michael; George, Laura A.; Figge, Marc Thilo; Khan, Mahmood; Goodall, Margaret; Young, Stephen P.; Reynolds, Adam; Falciani, Francesco; Waisman, Ari; Notley, Clare A.; Ehrenstein, Michael R.; Kosco-Vilbois, Marie

    2013-01-01

    Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells’ own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates. PMID:23420879

  17. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    PubMed Central

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  18. Mechanism-Based Competitive Binding Model to Investigate the Effect of Neonatal Fc Receptor Binding Affinity on the Pharmacokinetic of Humanized Anti-VEGF Monoclonal IgG1 Antibody in Cynomolgus Monkey.

    PubMed

    Ng, Chee M; Fielder, Paul J; Jin, Jin; Deng, Rong

    2016-07-01

    The quantitative relationship between neonatal Fc receptor (FcRn) binding affinity at both acidic and physiological pH and the pharmacokinetics of protein engineered FcRn IgG1 variants has not yet been reported. Our objective was to develop a quantitatively mechanism-based competitive binding model to describe the effects of FcRn binding affinity at acidic and physiological pH on the pharmacokinetics of anti-VEGF IgG1 antibodies when both endogenous and exogenous antibodies are competing for the same FcRn. Pharmacokinetic (PK) and FcRn binding data from five Fc variants of humanized anti-VEGF IgG1 monoclonal antibodies with wide range of FcRn binding affinity were used for the analysis. Sixty-seven anti-VEGF IgG1 antibody-treated animals and 25 control animals with simulated endogenous IgG levels were used to develop the final model. A hybrid iterative two stages and Monte Carlo parametric expectation-maximization method was used to obtain the final model parameters estimates. The final model well described the observed PK data. Quantitative FcRn binding affinity-pharmacokinetics relationships was constructed to provide important biological insights in better understanding of the FcRn binding effect on pharmacokinetics of anti-VEGF IgG1 antibodies in cynomolgus monkeys and served as an important model-based drug discovery platform to guide the design and development of the future generation of anti-VEGF or other therapeutic IgG1 antibodies. PMID:27075465

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

    PubMed

    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; Kauvar, Lawrence M

    2016-04-01

    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 observedin vitroat an antibody concentration of 1.2 μg/ml over 12 h. The effect of TRL1068in vivowas evaluated in a murine tissue cage infection model in which a biofilm is formed by infection with methicillin-resistantStaphylococcus 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

  20. 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

  1. Early Antibody Lineage Diversification and Independent Limb Maturation Lead to Broad HIV-1 Neutralization Targeting the Env High-Mannose Patch.

    PubMed

    MacLeod, Daniel T; Choi, Nancy M; Briney, Bryan; Garces, Fernando; Ver, Lorena S; Landais, Elise; Murrell, Ben; Wrin, Terri; Kilembe, William; Liang, Chi-Hui; Ramos, Alejandra; Bian, Chaoran B; Wickramasinghe, Lalinda; Kong, Leopold; Eren, Kemal; Wu, Chung-Yi; Wong, Chi-Huey; Kosakovsky Pond, Sergei L; Wilson, Ian A; Burton, Dennis R; Poignard, Pascal

    2016-05-17

    The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ∼11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways. PMID:27192579

  2. Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy.

    PubMed

    Wang, Taia T; Maamary, Jad; Tan, Gene S; Bournazos, Stylianos; Davis, Carl W; Krammer, Florian; Schlesinger, Sarah J; Palese, Peter; Ahmed, Rafi; Ravetch, Jeffrey V

    2015-07-01

    Protective vaccines elicit high-affinity, neutralizing antibodies by selection of somatically hypermutated B cell antigen receptors (BCR) on immune complexes (ICs). This implicates Fc-Fc receptor (FcR) interactions in affinity maturation, which, in turn, are determined by IgG subclass and Fc glycan composition within ICs. Trivalent influenza virus vaccination elicited regulation of anti-hemagglutinin (HA) IgG subclass and Fc glycans, with abundance of sialylated Fc glycans (sFc) predicting quality of vaccine response. We show that sFcs drive BCR affinity selection by binding the Type-II FcR CD23, thus upregulating the inhibitory FcγRIIB on activated B cells. This elevates the threshold requirement for BCR signaling, resulting in B cell selection for higher affinity BCR. Immunization with sFc HA ICs elicited protective, high-affinity IgGs against the conserved stalk of the HA. These results reveal a novel, endogenous pathway for affinity maturation that can be exploited for eliciting high-affinity, broadly neutralizing antibodies through immunization with sialylated immune complexes. PMID:26140596

  3. 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.

  4. 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.

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

    DOE PAGESBeta

    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

  6. 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.

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:26563652

  8. 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

  9. Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display

    PubMed Central

    Plittersdorf, Hanna; Hesse, Oliver; Scheidig, Andreas; Strerath, Michael; Gritzan, Uwe; Pellengahr, Klaus; Scholz, Peter; Eicker, Andrea; Myszka, David; Haupts, Ulrich

    2012-01-01

    In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts. PMID:22531438

  10. 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

  11. AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

    PubMed

    Meyer, Steffen; Woodward, Martin; Hertel, Christina; Vlaicu, Philip; Haque, Yasmin; Kärner, Jaanika; Macagno, Annalisa; Onuoha, Shimobi C; Fishman, Dmytro; Peterson, Hedi; Metsküla, Kaja; Uibo, Raivo; Jäntti, Kirsi; Hokynar, Kati; Wolff, Anette S B; Krohn, Kai; Ranki, Annamari; Peterson, Pärt; Kisand, Kai; Hayday, Adrian

    2016-07-28

    APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility. PMID:27426947

  12. Affinity Chromatography.

    ERIC Educational Resources Information Center

    Gray, Gary R.

    1980-01-01

    Presents selected recent advances in immobilization chemistry which have important connections to affinity chromatography. Discusses ligand immobilization and support modification. Cites 51 references. (CS)

  13. Novel Human Three-Domain Antibody Fragments Against sTNFα as Well as tmTNFα with High Affinity Generated by the Combination of Ribosome Display and E. coli Expression System.

    PubMed

    Zhao, X-L; Tian, L-F; Zhang, S-J; Li, J-M; Feng, H; Wang, L-M; Wang, S; Wang, J; Wang, T; Chen, W-Q

    2016-04-01

    Human tumour necrosis factor α (hTNFα) has been proved to be a validated therapeutic target in a number of immune-mediated inflammatory diseases (IMIDs). Fully human monoclonal antibodies (mAbs) that can neutralize soluble hTNFα (sTNFα) as well as transmembrane hTNFα (tmTNFα) are more desirable hTNFα antagonists. Here, we report that novel anti-hTNFα human low-molecular-weight MAbs have been selected and identified using both sTNFα and tmTNFα as target antigens by the combination of ribosome display and E. coli expression system for the first time. As a newly born engineering small molecular antibody, three-domain antibody fragment (VH /κ) provides an alternative promising molecular principle to generate biological agents for TNFα-dependent IMIDs. In this study, a panel of novel human VH /κs (F09, F21, F49 and F409) with high affinity (10(-10) -10(-9) mol/l) to neutralize sTNFα as well as tmTNFα was generated by the combination of ribosome display and E. coli expression system. Among the four clones, F21 and F409 could reduce cytotoxicity on L929 cells induced by sTNFα as well as tmTNFα effectively, and both of them had great potential to inhibit hTNFα-mediated NF-κB activation. Soluble F21 and F409 were also able to inhibit the binding of hTNFα to TNFR1 and TNFR2. The new human antibodies described here have desirable capability to neutralize sTNFα as well as tmTNFα effectively with high affinity and reasonable stability; this may provide an alternative approach for patients who are not responding adequately to currently available anti-TNFα agents. PMID:26860639

  14. Natural monoclonal antibodies and cancer.

    PubMed

    Vollmers, Peter H; Brändlein, Stephanie

    2008-06-01

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

  15. 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

  16. A Yeast Glycoprotein Shows High-Affinity Binding to the Broadly Neutralizing Human Immunodeficiency Virus Antibody 2G12 and Inhibits gp120 Interactions with 2G12 and DC-SIGN▿

    PubMed Central

    Luallen, Robert J.; Fu, Hu; Agrawal-Gamse, Caroline; Mboudjeka, Innocent; Huang, Wei; Lee, Fang-Hua; Wang, Lai-Xi; Doms, Robert W.; Geng, Yu

    2009-01-01

    The human immunodeficiency virus type 1 (HIV-1) envelope (Env) protein contains numerous N-linked carbohydrates that shield conserved peptide epitopes and promote trans infection by dendritic cells via binding to cell surface lectins. The potent and broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose-type oligosaccharides on the gp120 subunit of Env, revealing a conserved and highly exposed epitope on the glycan shield. To find an effective antigen for eliciting 2G12-like antibodies, we searched for endogenous yeast proteins that could bind to 2G12 in a panel of Saccharomyces cerevisiae glycosylation knockouts and discovered one protein that bound weakly in a Δpmr1 strain deficient in hyperglycosylation. 2G12 binding to this protein, identified as Pst1, was enhanced by adding the Δmnn1 deletion to the Δpmr1 background, ensuring the exposure of terminal α1,2-linked mannose residues on the D1 and D3 arms of high-mannose glycans. However, optimum 2G12 antigenicity was found when Pst1, a heavily N-glycosylated protein, was expressed with homogenous Man8GlcNAc2 structures in Δoch1 Δmnn1 Δmnn4 yeast. Surface plasmon resonance analysis of this form of Pst1 showed high affinity for 2G12, which translated into Pst1 efficiently inhibiting gp120 interactions with 2G12 and DC-SIGN and blocking 2G12-mediated neutralization of HIV-1 pseudoviruses. The high affinity of the yeast glycoprotein Pst1 for 2G12 highlights its potential as a novel antigen to induce 2G12-like antibodies. PMID:19264785

  17. Generation of novel recombinant antibodies against nitrotyrosine by antibody phage display.

    PubMed

    Hof, Danielle; Cooksley-Decasper, Seraina; Moergeli, Sandra; von Eckardstein, Arnold

    2011-01-01

    Nitrotyrosine is a posttranslational protein modification that occurs under oxidative and nitrosative stress, and plays an important role in numerous pathological conditions. To analyse nitrotyrosine formation several commercial monoclonal and polyclonal antibodies reacting with 3-nitrotyrosine have been developed which however do not work properly in all required assays. Here, antibody phage display was used to select recombinant antibodies that specifically react with nitrotyrosine in various protein contexts. Nine initial selections were carried out, using synthetic peptides, peroxynitrite-modified proteins and conjugated proteins as antigens. Four antibodies were isolated that each exhibited a characteristic binding reactivity that greatly depended on the antigens that were used for their selections. In general, the selections using small, synthetic and biotinylated peptides were the most successful approach. Subsequently, antibody 11B1 was affinity matured by error prone mutagenesis, resulting in the isolation of two antibodies, designated 47A7 and 47B1. Competition ELISA and immunoblotting after treatment with sodium dithionite further demonstrated the specificity of antibody 47B1 for nitrotyrosine. The results presented here demonstrate that antibody phage display is a useful method to isolate antibodies against posttranslational modifications, which are powerful tools in the proteomic era. PMID:21558620

  18. Quantitative specificity-based display library screening identifies determinants of antibody-epitope binding specificity

    PubMed Central

    Hall, Sejal S; Daugherty, Patrick S

    2009-01-01

    Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications. PMID:19610073

  19. Generation of a panel of high affinity antibodies and development of a biosensor-based immunoassay for the detection of okadaic acid in shellfish.

    PubMed

    Le Berre, Marie; Kilcoyne, Michelle; Kane, Marian

    2015-09-01

    Okadaic acid (OA) and its derivatives, DTX-1 and DTX-2, are marine biotoxins associated with diarrhetic shellfish poisoning. Routine monitoring of these toxins relies on the mouse bioassay. However, due to the technical unreliability and animal usage of this bioassay, there is always a need for convenient and reliable alternative assay methods. A panel of monoclonal antibodies against OA was generated and the most suitable was selected for biosensor-based assay development using surface plasmon resonance. The cross reactivity of the selected antibody with DTX-1 was found to be 73%, confirming the antibody suitability for both OA and DTX detection. The OA and derivative assay was designed as an inhibition assay covering the concentrations 1-75 ng/ml, with a sensitivity of 22.4 ng/ml. The assay was highly reproducible and preliminary validation showed no matrix interference from mussel extracts and good recovery of added standard in mussel extracts, with %CV of <9.3%. This assay could provide a useful and convenient screening tool for OA and its derivatives with a comprehensive extraction protocol for shellfish monitoring programmes. PMID:26169671

  20. Semi-quantitative Measurement of a Specific Glycoform Using a DNA-tagged Antibody and Lectin Affinity Chromatography for Glyco-biomarker Development*

    PubMed Central

    Lee, Ju Hee; Cho, Chang Hee; Kim, Sun Hee; Kang, Jeong Gu; Yoo, Jong Shin; Chang, Chulhun Ludgerus; Ko, Jeong-Heon; Kim, Yong-Sam

    2015-01-01

    Aberrant glycosylation-targeted disease biomarker development is based on cumulative evidence that certain glycoforms are mass-produced in a disease-specific manner. However, the development process has been hampered by the absence of an efficient validation method based on a sensitive and multiplexed platform. In particular, ELISA-based analytical tools are not adequate for this purpose, mainly because of the presence of a pair of N-glycans of IgG-type antibodies. To overcome the associated hurdles in this study, antibodies were tagged with oligonucleotides with T7 promoter and then allowed to form a complex with corresponding antigens. An antibody-bound specific glycoform was isolated by lectin chromatography and quantitatively measured on a DNA microarray chip following production of fluorescent RNA by T7-trascription. This tool ensured measurement of targeted glycoforms of multiple biomarkers with high sensitivity and multiplexity. This analytical method was applied to an in vitro diagnostic multivariate index assay where a panel of hepatocellular carcinoma (HCC) biomarkers comprising alpha-fetoprotein, hemopexin, and alpha-2-macroglobulin (A2M) was examined in terms of the serum level and their fuco-fractions. The results indicated that the tests using the multiplexed fuco-biomarkers provided improved discriminatory power between non- hepatocellular carcinoma and hepatocellular carcinoma subjects compared with the alpha-fetoprotein level or fuco-alpha-fetoprotein test alone. The developed method is expected to facilitate the validation of disease-specific glycan biomarker candidates. PMID:25525205

  1. Molecular description of flexibility in an antibody combining site.

    PubMed

    Zimmermann, Jörg; Romesberg, Floyd E; Brooks, Charles L; Thorpe, Ian F

    2010-06-01

    Mature antibodies (Abs) that are exquisitely specific for virtually any foreign molecule may be produced by affinity maturation of naïve (or germline) Abs. However, the finite number of germline Abs available suggests that, in contrast to mature Abs, germline Abs must be broadly polyspecific so that they are able to recognize a wide range of ligands. Thus, affinity maturation must play a role in mediating Ab specificity. One biophysical property that distinguishes polyspecificity from specificity is protein flexibility; a flexible combining site is able to adopt different conformations that recognize different foreign molecules (or antigens), while a rigid combining site is locked into a conformation that is specific for a given antigen. Recent studies (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8821-8826) have examined, at the atomic level, the structural properties that mediate changes in flexibility at four stages of affinity maturation in the 4-4-20 Ab. These studies employed molecular dynamics simulations to reveal a network of residue interactions that mediate the flexibility changes accompanying maturation. The flexibility of the Ab combining sites in these molecular systems was originally measured using three-pulse photon echo spectroscopy (3PEPS). The present investigation extends this work by providing a concrete link between structural properties of the Ab molecules and features of the spectroscopic measurements used to characterize their flexibility. Results obtained from the simulations are in good qualitative agreement with the experimental measurements and indicate that the spectroscopic signal is sensitive to protein dynamics distributed throughout the entire combining site. Thus, the simulations provide a molecular-level interpretation of the changes induced by affinity maturation of the Ab. The results suggest that 3PEPS spectroscopy in combination with molecular dynamics simulations can provide a detailed description of protein dynamics and, in

  2. 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

  3. 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. PMID:25970341

  4. 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

  5. On-line coupling of surface plasmon resonance optical sensing to size-exclusion chromatography for affinity assessment of antibody samples.

    PubMed

    Lakayan, Dina; Haselberg, Rob; Niessen, Wilfried M A; Somsen, Govert W; Kool, Jeroen

    2016-06-24

    Surface plasmon resonance (SPR) is an optical technique that measures biomolecular interactions. Stand-alone SPR cannot distinguish different binding components present in one sample. Moreover, sample matrix components may show non-specific binding to the sensor surface, leading to detection interferences. This study describes the development of coupled size-exclusion chromatography (SEC) SPR sensing for the separation of sample components prior to their on-line bio-interaction analysis. A heterogeneous polyclonal human serum albumin antibody (anti-HSA) sample, which was characterized by proteomics analysis, was used as test sample. The proposed SEC-SPR coupling was optimized by studying system parameters, such as injection volume, flow rate and sample concentration, using immobilized HSA on the sensor chip. Automated switch valves were used for on-line regeneration of the SPR sensor chip in between injections and for potential chromatographic heart cutting experiments, allowing SPR detection of individual components. The performance of the SEC-SPR system was evaluated by the analysis of papain-digested anti-HSA sampled at different incubation time points. The new on-line SEC-SPR methodology allows specific label-free analysis of real-time interactions of eluting antibody sample constituents towards their antigenic target. PMID:27215465

  6. Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

    PubMed Central

    Griffiths, Amy E.; Wang, Wensheng; Hagen, Fred K.; Fay, Philip J.

    2011-01-01

    Summary Background Neutralizing factor (F) VIII antibodies develop in ~30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein. Methods Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined following immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS). Results Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of <12 residues. Conclusions IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions. PMID:21668738

  7. 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

  8. Anti-myelin antibodies modulate clinical expression of childhood multiple sclerosis.

    PubMed

    O'Connor, K C; Lopez-Amaya, C; Gagne, D; Lovato, L; Moore-Odom, N H; Kennedy, J; Krupp, L; Tenembaum, S; Ness, J; Belman, A; Boyko, A; Bykova, O; Mah, J K; Stoian, C A; Waubant, E; Kremenchutzky, M; Ruggieri, M; Bardini, M R; Rensel, M; Hahn, J; Weinstock-Guttman, B; Yeh, E A; Farrell, K; Freedman, M S; Iivanainen, M; Bhan, V; Dilenge, M; Hancock, M A; Gano, D; Fattahie, R; Kopel, L; Fournier, A E; Moscarello, M; Banwell, B; Bar-Or, A

    2010-06-01

    Anti-myelin basic protein (MBP) antibodies in pediatric-onset MS and controls were characterized. Serum samples were obtained from 94 children with MS and 106 controls. Paired CSF and serum were obtained from 25 children with MS at time of their initial episode of acute demyelinating syndrome (ADS). Complementary assays were applied across samples to evaluate the presence, and the physical binding properties, of anti-MBP antibodies. While the prevalence and titers of serum anti-MBP antibodies against both immature and mature forms of MBP were similar in children with MS and in controls, binding characteristics and formal Surface Plasmon Resonance (SPR) studies indicated surprisingly high binding affinities of all pediatric anti-MBP antibodies. Serum levels of anti-MBP antibodies correlated significantly with their CSF levels, and their presence in children with MS was associated with significantly increased risk of an acute disseminated encephalomyelitis-like initial clinical presentation. While antibodies to both immature and mature forms of MBP can be present as part of the normal pediatric humoral repertoire, these anti-myelin antibodies are of surprisingly high affinity, can access the CNS during inflammation, and have the capacity to modulate disease expression. Our findings identify an immune mechanism that could contribute to the observed heterogeneity in spectrum of clinical presentations in early-onset MS. PMID:20381173

  9. Natural antibodies and the host immune responses to xenografts.

    PubMed

    Cramer, D V

    2000-05-01

    Natural antibodies are present in the serum of individuals in the absence of known antigenic stimulation. These antibodies are primarily IgM, polyreactive, and encoded by immunoglobulin V genes in germline configuration. Natural antibodies are produced by B-1 lymphocytes, cells that form the primary cell of the fetal and newborn B cell repertoire and may represent the basic foundation upon which the adult repertoire of B cell antibodies is based. Natural antibodies react with a variety of endogenous and exogenous antigens, including xenoantigens expressed by tissues between unrelated species. These antibodies are capable of causing the immediate rejection of grafts exchanged across species barriers. One of the central issues related to our understanding of the immunopathologic mechanisms responsible for rejection of xenografts is whether pre-formed natural antibodies and new antibodies induced following xenotransplantation are produced by the same pathways of B cell antibody production. We have established in studies conducted in rodents and humans that the initial phases of antibody production xenogeneic tissues involves the use of a restricted population of Ig germline genes to encode xenoantibody binding. As the humoral xenoantibody response matures, the same closely-related groups of Ig V genes are used to encode antibody binding and there is evidence for an isotype switch to IgG antibody production and the appearance of somatic mutations consistent with antigen-driven affinity maturation. Our findings in both rodent and human studies form the basis for our proposal that the xenograft response reflects the use of B cell natural antibody repertoires originally intended to provide protection against infection. The host humoral response is inadvertently recruited to mount antibody responses against foreign grafts because they display carbohydrate antigens that are shared by common environmental microbes. This model of xenoantibody responses is being tested in our

  10. Structural Analysis and Optimization of Context-Independent Anti-Hypusine Antibodies.

    PubMed

    Zhai, Qianting; He, Meng; Song, Aimin; Deshayes, Kurt; Dixit, Vishva M; Carter, Paul J

    2016-02-13

    Context-independent anti-hypusine antibodies that bind to the post-translational modification (PTM), hypusine, with minimal dependence on flanking amino acid sequences, were identified. The antibodies bind to both hypusine and deoxyhypusine or selectively to hypusine but not to deoxyhypusine. Phage display was used to further enhance the affinity of the antibodies. Affinity maturation of these anti-hypusine antibodies improved their performance in affinity capture of the only currently known hypusinated protein, eukaryotic translation initiation factor 5A. These anti-hypusine antibodies may have utility in the identification of novel hypusinated proteins. Crystal structures of the corresponding Fab fragments were determined in complex with hypusine- or deoxyhypusine-containing peptides. The hypusine or deoxyhypusine moiety was found to reside in a deep pocket formed between VH and VL domains of the Fab fragments. Interaction between the antibodies and hypusine includes an extensive hydrogen bond network. These are, to our knowledge, the first reported structures of context-independent anti-PTM antibodies in complex with the corresponding PTM. PMID:26778617

  11. Attachment of antibody to biotinylated red blood cells: immuno-red blood cells display high affinity to immobilized antigen and normal biodistribution in rats.

    PubMed

    Muzykantov, V R; Murciano, J C

    1996-08-01

    Streptavidin-mediated attachment of biotinylated antibodies (b-Ab) to biotinylated red blood cells (b-RBC) is useful for preparation of immuno-red blood cells, a prospective vehicle for drug targeting. However, streptavidin (SA) induces lysis of extensively biotinylated RBC by complement due to cross-linking and inactivation of RBC complement regulators. To reduce cross-linking of RBC membrane proteins, we utilized mild biotinylation of RBC with 20 microM biotin ester (b20-RBC). SA effectively binds to rat b20-RBC (10(5) SA molecules/cell) and provides for following attachment of 5 x 10(4) molecules of b-IgG/SA per b20-RBC. By in vitro assay, b-Ab/SA/b20-RBC were stable in fresh rat serum. Serum-stable immuno-red blood cells (b-Ab/SA/b20-RBC) specifically bound to antigen-coated surfaces, but not to BSA-coated surfaces. Biodistribution of 51Cr-labelled b-Ab/SA/b20-RBC in rats was similar to that of control RBC, with no indication of lysis in vivo. These results suggest b-Ab/SA/b20-RBC may be explored as a vehicle for drug targeting. PMID:8756393

  12. Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients.

    PubMed

    Kosugi, S; Ban, T; Akamizu, T; Kohn, L D

    1991-10-31

    Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site. PMID:1719963

  13. 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. PMID:23098305

  14. Monoclonal antibodies to equine CD23 identify the low-affinity receptor for IgE on subpopulations of IgM+ and IgG1+ B-cells in horses.

    PubMed

    Wagner, Bettina; Hillegas, Julia M; Babasyan, Susanna

    2012-04-15

    CD23, also called FcεRII, is the low-affinity receptor for IgE and has first been described as a major receptor regulating IgE responses. In addition, CD23 also binds to CD21, integrins and MHC class II molecules and thus has a much wider functional role in immune regulation ranging from involvement in antigen-presentation to multiple cytokine-like functions of soluble CD23. The role of CD23 during immune responses of the horse is less well understood. Here, we expressed equine CD23 in mammalian cells using a novel IL-4 expression system. Expression resulted in high yield of recombinant IL-4/CD23 fusion protein which was purified and used for the generation of monoclonal antibodies (mAbs) to equine CD23. Seven anti-CD23 mAbs were further characterized. The expression of the low-affinity IgE receptor on equine peripheral blood mononuclear cells was analyzed by flow cytometric analysis. Cell surface staining showed that CD23 is mainly expressed by a subpopulation of equine B-cells. Only a very few equine T-cells or monocytes expressed CD23. CD23(+) B-cells were either IgM(+) or IgG1(+) cells. All CD23(+) cells were also positive for cell surface IgE staining suggesting in vivo IgE binding by the receptor. Two of the CD23 mAbs detected either the complete extracellular region of CD23 or a 22kDa cleavage product of CD23 by Western blotting. The new anti-CD23 mAbs provide valuable reagents to further analyze the roles of CD23 during immune responses of the horse in health and disease. PMID:22405681

  15. 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. PMID:26970185

  16. 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

  17. Protein Complex Purification by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Affinity capture has become a powerful technique for consistently purifying endogenous protein complexes, facilitating biochemical and biophysical assays on otherwise inaccessible biological assemblies, and enabling broader interactomic exploration. For this procedure, cells are broken and their contents separated and extracted into a solvent, permitting access to target macromolecular complexes thus released in solution. The complexes are specifically enriched from the extract onto a solid medium coupled with an affinity reagent-usually an antibody-that recognizes the target either directly or through an appended affinity tag, allowing subsequent characterization of the complex. Here, we discuss approaches and considerations for purifying endogenous yeast protein complexes by affinity capture. PMID:27371601

  18. Virus-like particles from Escherichia Coli-derived untagged papaya ringspot virus capsid protein purified by immobilized metal affinity chromatography enhance the antibody response against a soluble antigen.

    PubMed

    Guerrero-Rodríguez, Jesús; Manuel-Cabrera, Carlos Alberto; Palomino-Hermosillo, Y Apatzingan; Delgado-Guzmán, Paola Guadalupe; Escoto-Delgadillo, Martha; Silva-Rosales, Laura; Herrera-Rodríguez, Sara Elisa; Sánchez-Hernández, Carla; Gutiérrez-Ortega, Abel

    2014-12-01

    There is a growing interest in using virus-like particles (VLPs) as scaffolds for the presentation of antigens of choice to the immune system. In this work, VLPs from papaya ringspot virus capsid protein expressed in Escherichia coli were evaluated as enhancers of antibody response against a soluble antigen. Interestingly, although the capsid protein lacks a histidine tag, its purification by immobilized metal affinity chromatography was achieved. The formation of VLPs was demonstrated by electron microscopy for the first time for this capsid protein. VLPs were enriched by polyethylene glycol precipitation. Additionally, these VLPs were chemically coupled to green fluorescent protein in order to evaluate them as antigen carriers; however, bioconjugate instability was observed. Nonetheless, the adjuvant effect of these VLPs on BALB/c mice was evaluated, using GFP as antigen, resulting in a significant increase in anti-GFP IgG response, particularly, IgG1 class, demonstrating that the VLPs enhance the immune response against the antigen chosen in this study. PMID:25119647

  19. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    PubMed Central

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

    2012-01-01

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

  20. Characterization of germline antibody libraries from human umbilical cord blood and selection of monoclonal antibodies to viral envelope glycoproteins: Implications for mechanisms of immune evasion and design of vaccine immunogens.

    PubMed

    Chen, Weizao; Streaker, Emily D; Russ, Daniel E; Feng, Yang; Prabakaran, Ponraj; Dimitrov, Dimiter S

    2012-01-27

    We have previously observed that all known HIV-1 broadly neutralizing antibodies (bnAbs) are highly divergent from germline antibodies in contrast to bnAbs against Hendra virus, Nipah virus and SARS coronavirus (SARS CoV). We have hypothesized that because the germline antibodies are so different from the mature HIV-1-specific bnAbs they may not bind the epitopes of the mature antibodies and provided the first evidence to support this hypothesis by using individual putative germline-like predecessor antibodies. To further validate the hypothesis and understand initial immune responses to different viruses, two phage-displayed human cord blood-derived IgM libraries were constructed which contained mostly germline antibodies or antibodies with very low level of somatic hypermutations. They were panned against different HIV-1 envelope glycoproteins (Envs), SARS CoV protein receptor-binding domain (RBD), and soluble Hendra virus G protein (sG). Despite a high sequence and combinatorial diversity observed in the cord blood-derived IgM antibody repertoire, no enrichment for binders of Envs was observed in contrast to considerable specific enrichments produced with panning against RBD and sG; one of the selected monoclonal antibodies (against the RBD) was of high (nM) affinity with only few somatic mutations. These results further support and expand our initial hypothesis for fundamental differences in immune responses leading to elicitation of bnAbs against HIV-1 compared to SARS CoV and Hendra virus. HIV-1 uses a strategy to minimize or eliminate strong binding of germline antibodies to its Env; in contrast, SARS CoV and Hendra virus, and perhaps other viruses causing acute infections, can bind germline antibody or minimally somatically mutated antibodies with relatively high affinity which could be one of the reasons for the success of sG and RBD as vaccine immunogens. PMID:22226962

  1. A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

    PubMed Central

    Goh, Angeline XH; Bertin-Maghit, Sebastien; Ping Yeo, Siok; Ho, Adrian; Derks, Heidi; Mortellaro, Alessandra; Wang, Cheng-I

    2014-01-01

    The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal. PMID:24671001

  2. Highly sensitive and unbiased approach for elucidating antibody repertoires.

    PubMed

    Lin, Sherry G; Ba, Zhaoqing; Du, Zhou; Zhang, Yu; Hu, Jiazhi; Alt, Frederick W

    2016-07-12

    Developing B lymphocytes undergo V(D)J recombination to assemble germ-line V, D, and J gene segments into exons that encode the antigen-binding variable region of Ig heavy (H) and light (L) chains. IgH and IgL chains associate to form the B-cell receptor (BCR), which, upon antigen binding, activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. The ability of V(D)J recombination to generate vast primary B-cell repertoires results from a combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B-cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as high-throughput genome-wide translocation sequencing-adapted repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or nonproductive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions, and also for following antibody affinity maturation processes. PMID:27354528

  3. Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

    PubMed

    Lyon, J A; Haynes, J D; Diggs, C L; Chulay, J D; Pratt-Rossiter, J M

    1986-03-15

    Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to

  4. Affinity improvement by fine tuning of single-chain variable fragment against aflatoxin B1.

    PubMed

    Min, Won-Ki; Na, Kang-In; Yoon, Jung-Hyun; Heo, Yoon-Jee; Lee, Daesang; Kim, Sung-Gun; Seo, Jin-Ho

    2016-10-15

    Aflatoxin B1 (AFB1) produced in Aspergillus flavus is a major hepatocarcinogen found in foods and feed. For effective immunological detection of AFB1 at low concentrations, the development of high affinity antibody for AFB1 is required. Previously, an affinity-maturated single-chain variable fragment containing 6 mutations (scFv-M37) was isolated from an artificial mutagenic library, which showed a 9-fold higher affinity than its wild type scFv. In this study, the effect of the 6 mutated residues on the affinity improvement was characterized using surface plasmon resonance analysis, which identified a deleterious mutation (VH-A110T) located on a framework region of the scFv-M37. The back mutation of VH-A110T resulted in a 3.2-fold affinity improvement, which was attributed to decrease of dissociation rate constant (kd) in interaction between AFB1 and the back mutant scFv. The biophysical analyses using circular dichroism and gel filtration revealed that the back mutation of VH-A110T caused a subtle conformational change of the scFv toward tighter binding to AFB1. PMID:27173568

  5. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

    PubMed Central

    Whitehead, Timothy A; Chevalier, Aaron; Song, Yifan; Dreyfus, Cyrille; Fleishman, Sarel J; De Mattos, Cecilia; Myers, Chris A; Kamisetty, Hetunandan; Blair, Patrick; Wilson, Ian A; Baker, David

    2013-01-01

    We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility. PMID:22634563

  6. Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing

    SciTech Connect

    Whitehead, Timothy A.; Chevalier, Aaron; Song, Yifan; Dreyfus, Cyrille; Fleishman, Sarel J.; De Mattos, Cecilia; Myers, Chris A.; Kamisetty, Hetunandan; Blair, Patrick; Wilson, Ian A.; Baker, David

    2012-06-19

    We show that comprehensive sequence-function maps obtained by deep sequencing can be used to reprogram interaction specificity and to leapfrog over bottlenecks in affinity maturation by combining many individually small contributions not detectable in conventional approaches. We use this approach to optimize two computationally designed inhibitors against H1N1 influenza hemagglutinin and, in both cases, obtain variants with subnanomolar binding affinity. The most potent of these, a 51-residue protein, is broadly cross-reactive against all influenza group 1 hemagglutinins, including human H2, and neutralizes H1N1 viruses with a potency that rivals that of several human monoclonal antibodies, demonstrating that computational design followed by comprehensive energy landscape mapping can generate proteins with potential therapeutic utility.

  7. Viral receptor-binding site antibodies with diverse germline origins

    PubMed Central

    Schmidt, Aaron G.; Therkelsen, Matthew D.; Stewart, Shaun; Kepler, Thomas B.; Liao, Hua-Xin; Moody, M. Anthony; Haynes, Barton F.; Harrison, Stephen C.

    2015-01-01

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by eleven different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B-cell targets. PMID:25959776

  8. Viral receptor-binding site antibodies with diverse germline origins.

    PubMed

    Schmidt, Aaron G; Therkelsen, Matthew D; Stewart, Shaun; Kepler, Thomas B; Liao, Hua-Xin; Moody, M Anthony; Haynes, Barton F; Harrison, Stephen C

    2015-05-21

    Vaccines for rapidly evolving pathogens will confer lasting immunity if they elicit antibodies recognizing conserved epitopes, such as a receptor-binding site (RBS). From characteristics of an influenza-virus RBS-directed antibody, we devised a signature motif to search for similar antibodies. We identified, from three vaccinees, over 100 candidates encoded by 11 different VH genes. Crystal structures show that antibodies in this class engage the hemagglutinin RBS and mimic binding of the receptor, sialic acid, by supplying a critical dipeptide on their projecting, heavy-chain third complementarity determining region. They share contacts with conserved, receptor-binding residues but contact different residues on the RBS periphery, limiting the likelihood of viral escape when several such antibodies are present. These data show that related modes of RBS recognition can arise from different germline origins and mature through diverse affinity maturation pathways. Immunogens focused on an RBS-directed response will thus have a broad range of B cell targets. PMID:25959776

  9. Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design.

    PubMed

    Joyce, Joseph G; Hurni, William M; Bogusky, Michael J; Garsky, Victor M; Liang, Xiaoping; Citron, Michael P; Danzeisen, Renee C; Miller, Michael D; Shiver, John W; Keller, Paul M

    2002-11-29

    The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation. We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope. Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response. PMID:12237296

  10. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    PubMed

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  11. Ablation of the cellular prion protein, PrPC, specifically on follicular dendritic cells has no effect on their maturation or function.

    PubMed

    McCulloch, Laura; Brown, Karen L; Mabbott, Neil A

    2013-03-01

    Follicular dendritic cells (FDC) are situated in the primary follicles of lymphoid tissues where they maintain the structural integrity of the B-lymphocyte follicle, and help to drive immunoglobulin class-switch recombination, somatic hypermutation and affinity maturation during the germinal centre response. FDC can also provide a reservoir for pathogens that infect germinal centres including HIV and prions. FDC express high levels of the normal cellular form of the prion protein (PrP(C) ), which makes them susceptible to prion infection. The function of PrP(C) is uncertain and it is not known why FDC require such high levels of expression of a protein that is found mainly on cells of the central nervous system. In this study, the function of FDC was assessed in mice that had PrP(C) ablated specifically in their FDC. In mice with FDC-specific PrP(C) ablation, our analysis revealed no observable deficits in lymphoid follicle microarchitecture and FDC status. No effects on FDC ability to trap immune complexes or drive antigen-specific antibody responses and affinity maturation in B lymphocytes were observed. These data clearly demonstrate that PrP(C) expression is dispensable for the functional maturation of FDC and their ability to maintain antigen-specific antibody responses and affinity maturation. PMID:23121447

  12. Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Walters, Rodney R.

    1985-01-01

    Supports, affinity ligands, immobilization, elution methods, and a number of applications are among the topics considered in this discussion of affinity chromatography. An outline of the basic principles of affinity chromatography is included. (JN)

  13. Ligand-inducible dimeric antibody for selecting antibodies against a membrane protein based on mammalian cell proliferation.

    PubMed

    Miura, Tomohiro; Nagamune, Teruyuki; Kawahara, Masahiro

    2016-05-01

    A method for selecting antibodies against a membrane protein is important for attaining a variety of antibody-based diagnostics and therapies. In this study, we propose a novel system to select specific antibodies against a membrane protein based on mammalian cell proliferation as a readout. The system employs a chimeric membrane protein in which a target membrane protein of interest is fused to the intracellular signaling domain of a cytokine receptor. The chimeric membrane protein transduces a cell proliferation signal through dimerization when co-expressed with a specific single-chain Fv fused with a mutant of FK-binding protein 12 (scFv-Fk) that can be conditionally dimerized by a synthetic ligand AP20187. To demonstrate this system, ErbB2 and gp130 were chosen as the target membrane protein and cytokine receptor, respectively. Consequently, co-expression of the ErbB2/gp130 chimera and ErbB2-specific scFv-Fk rendered the cells proliferative in response to AP20187. The system also allowed selection of high-affinity binders from a mixture composed of dominant low-affinity binders. This system may be extended to affinity maturation of scFvs by modulating AP20187 concentration in the selection process. Biotechnol. Bioeng. 2016;113: 1113-1123. © 2015 Wiley Periodicals, Inc. PMID:26479395

  14. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    SciTech Connect

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J.

    2012-10-16

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

  15. Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

    PubMed

    Zhang, Xiao; Sun, Chunyun; Xiao, Xiangqian; Pang, Lin; Shen, Sisi; Zhang, Jie; Cen, Shan; Yang, Burton B; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed cross-neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display-derived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization. PMID:26073737

  16. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    SciTech Connect

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D.

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  17. A human monoclonal antibody specific to placental alkaline phosphatase, a marker of ovarian cancer

    PubMed Central

    Ravenni, Niccolò; Weber, Marcel; Neri, Dario

    2014-01-01

    Placental alkaline phosphatase (PLAP) is a promising ovarian cancer biomarker. Here, we describe the isolation, affinity-maturation and characterization of two fully human monoclonal antibodies (termed B10 and D9) able to bind to human PLAP with a dissociation constant (Kd) of 10 and 30 nM, respectively. The ability of B10 and D9 antibodies to recognize the native antigen was confirmed by Biacore analysis, FACS and immunofluorescence studies using ovarian cancer cell lines and freshly-frozen human tissues. A quantitative biodistribution study in nude mice revealed that the B10 antibody preferentially localizes to A431 tumors, following intravenous administration. Anti-PLAP antibodies may serve as a modular building blocks for the development of targeted therapeutic products, armed with cytotoxic drugs, radionuclides or cytokines as payloads. PMID:24247025

  18. REAL-Select: Full-Length Antibody Display and Library Screening by Surface Capture on Yeast Cells

    PubMed Central

    Günther, Ralf; Becker, Stefan; Kolmar, Harald; Hock, Björn

    2014-01-01

    We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1∶1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells. PMID:25501029

  19. Affinity Proteomics in the mountains: Alpbach 2015.

    PubMed

    Taussig, Michael J

    2016-09-25

    The 2015 Alpbach Workshop on Affinity Proteomics, organised by the EU AFFINOMICS consortium, was the 7th workshop in this series. As in previous years, the focus of the event was the current state of affinity methods for proteome analysis, including complementarity with mass spectrometry, progress in recombinant binder production methods, alternatives to classical antibodies as affinity reagents, analysis of proteome targets, industry focus on biomarkers, and diagnostic and clinical applications. The combination of excellent science with Austrian mountain scenery and winter sports engender an atmosphere that makes this series of workshops exceptional. The articles in this Special Issue represent a cross-section of the presentations at the 2015 meeting. PMID:27118167

  20. Optimized Affinity Capture of Yeast Protein Complexes.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Hakhverdyan, Zhanna; Rout, Michael P

    2016-01-01

    Here, we describe an affinity isolation protocol. It uses cryomilled yeast cell powder for producing cell extracts and antibody-conjugated paramagnetic beads for affinity capture. Guidelines for determining the optimal extraction solvent composition are provided. Captured proteins are eluted in a denaturing solvent (sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer) for gel-based proteomic analyses. Although the procedures can be modified to use other sources of cell extract and other forms of affinity media, to date we have consistently obtained the best results with the method presented. PMID:27371596

  1. Aptamers in Affinity Separations: Stationary Separation

    NASA Astrophysics Data System (ADS)

    Ravelet, Corinne; Peyrin, Eric

    The use of DNA or RNA aptamers as tools in analytical chemistry is a very promising field of research because of their capabilities to bind specifically the target molecules with an affinity similar to that of antibodies. Notably, they appear to be of great interest as target-specific ligands for the separation and capture of various analytes in affinity chromatography and related affinity-based methods such as magnetic bead technology. In this chapter, the recent developments of these aptamer-based separation/capture approaches are addressed.

  2. Antibody-specific model of amino acid substitution for immunological inferences from alignments of antibody sequences.

    PubMed

    Mirsky, Alexander; Kazandjian, Linda; Anisimova, Maria

    2015-03-01

    Antibodies are glycoproteins produced by the immune system as a dynamically adaptive line of defense against invading pathogens. Very elegant and specific mutational mechanisms allow B lymphocytes to produce a large and diversified repertoire of antibodies, which is modified and enhanced throughout all adulthood. One of these mechanisms is somatic hypermutation, which stochastically mutates nucleotides in the antibody genes, forming new sequences with different properties and, eventually, higher affinity and selectivity to the pathogenic target. As somatic hypermutation involves fast mutation of antibody sequences, this process can be described using a Markov substitution model of molecular evolution. Here, using large sets of antibody sequences from mice and humans, we infer an empirical amino acid substitution model AB, which is specific to antibody sequences. Compared with existing general amino acid models, we show that the AB model provides significantly better description for the somatic evolution of mice and human antibody sequences, as demonstrated on large next generation sequencing (NGS) antibody data. General amino acid models are reflective of conservation at the protein level due to functional constraints, with most frequent amino acids exchanges taking place between residues with the same or similar physicochemical properties. In contrast, within the variable part of antibody sequences we observed an elevated frequency of exchanges between amino acids with distinct physicochemical properties. This is indicative of a sui generis mutational mechanism, specific to antibody somatic hypermutation. We illustrate this property of antibody sequences by a comparative analysis of the network modularity implied by the AB model and general amino acid substitution models. We recommend using the new model for computational studies of antibody sequence maturation, including inference of alignments and phylogenetic trees describing antibody somatic hypermutation in

  3. The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

    PubMed Central

    Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

    2016-01-01

    The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

  4. The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6.

    PubMed

    Boschert, V; Frisch, C; Back, J W; van Pee, K; Weidauer, S E; Muth, E-M; Schmieder, P; Beerbaum, M; Knappik, A; Timmerman, P; Mueller, T D

    2016-08-01

    The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure-function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

  5. Cloning single-chain antibody fragments (ScFv) from hyrbidoma cells.

    PubMed

    Toleikis, Lars; Frenzel, André

    2012-01-01

    Despite the rising impact of the generation of antibodies by phage display and other technologies, hybridoma technology still provides a valuable tool for the generation of high-affinity binders against different targets. But there exist several limitations of using hybridoma-derived antibodies. The source of the hybridoma clones are mostly rat or mouse B-lymphocytes. Therefore a human-anti-mouse or human-anti-rat antibody response may result in immunogenicity of these antibodies. This leads to the necessity of humanization of these antibodies where the knowledge of the amino acid sequence of the proteins is inalienable. Furthermore, additional in vitro modifications, e.g., affinity maturation or fusion to other proteins, are dependent on cloning of the antigen-binding domains.Here we describe the isolation of RNA from hybridoma cells and the primers that can be used for the amplification of VL and VH as well as the cloning of the antibody in scFv format and its expression in Escherichia coli. PMID:22907345

  6. Selection of recombinant anti-SH3 domain antibodies by high-throughput phage display.

    PubMed

    Huang, Haiming; Economopoulos, Nicolas O; Liu, Bernard A; Uetrecht, Andrea; Gu, Jun; Jarvik, Nick; Nadeem, Vincent; Pawson, Tony; Moffat, Jason; Miersch, Shane; Sidhu, Sachdev S

    2015-11-01

    Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale. PMID:26332758

  7. Selection and characterization of human antibody fragments specific for psoriasin - a cancer associated protein.

    PubMed

    Cyranka-Czaja, Anna; Wulhfard, Sarah; Neri, Dario; Otlewski, Jacek

    2012-03-01

    S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvβ6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvβ6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer. PMID:22342672

  8. Polyreactive Antibodies: Function and Quantification

    PubMed Central

    Gunti, Sreenivasulu; Notkins, Abner Louis

    2015-01-01

    Polyreactive antibodies, a major component of the natural antibody repertoire, bind with low affinity to a variety of structurally unrelated antigens. Many of these antibodies are germline or near germline in sequence. Little is known, however, about the function of these antibodies. In the present mini-review we show: (1) that the broad antibacterial activity of the natural antibody repertoire is largely due to polyreactive antibodies, which in the presence of complement lyse bacteria and enhance phagocytosis; (2) that polyreactive antibodies bind to UV- or human immunodeficiency virus-induced apoptotic cells and with complement enhance the phagocytosis of these cells by macrophages; and (3) that dinitrophenol can be used as a surrogate for quantitating the level of polyreactive antibodies in serum. We conclude that polyreactive antibodies protect the host against both foreign invaders and its own damaged/apoptotic cells. PMID:26116731

  9. Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

    PubMed

    Luginbühl, Béatrice; Kanyo, Zoltan; Jones, R Mark; Fletterick, Robert J; Prusiner, Stanley B; Cohen, Fred E; Williamson, R Anthony; Burton, Dennis R; Plückthun, Andreas

    2006-10-13

    Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations. PMID:16962610

  10. Constant Domain-regulated Antibody Catalysis*

    PubMed Central

    Sapparapu, Gopal; Planque, Stephanie; Mitsuda, Yukie; McLean, Gary; Nishiyama, Yasuhiro; Paul, Sudhir

    2012-01-01

    Some antibodies contain variable (V) domain catalytic sites. We report the superior amide and peptide bond-hydrolyzing activity of the same heavy and light chain V domains expressed in the IgM constant domain scaffold compared with the IgG scaffold. The superior catalytic activity of recombinant IgM was evident using two substrates, a small model peptide that is hydrolyzed without involvement of high affinity epitope binding, and HIV gp120, which is recognized specifically by noncovalent means prior to the hydrolytic reaction. The catalytic activity was inhibited by an electrophilic phosphonate diester, consistent with a nucleophilic catalytic mechanism. All 13 monoclonal IgMs tested displayed robust hydrolytic activities varying over a 91-fold range, consistent with expression of the catalytic functions at distinct levels by different V domains. The catalytic activity of polyclonal IgM was superior to polyclonal IgG from the same sera, indicating that on average IgMs express the catalytic function at levels greater than IgGs. The findings indicate a favorable effect of the remote IgM constant domain scaffold on the integrity of the V-domain catalytic site and provide a structural basis for conceiving antibody catalysis as a first line immune function expressed at high levels prior to development of mature IgG class antibodies. PMID:22948159

  11. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  12. Immunogenic stimulus for germline precursors of antibodies that engage the influenza hemagglutinin receptor-binding site

    PubMed Central

    Schmidt, Aaron G.; Do, Khoi T.; McCarthy, Kevin R.; Kepler, Thomas B.; Liao, Hua-Xin; Moody, M. Anthony; Haynes, Barton F.; Harrison, Stephen C.

    2015-01-01

    SUMMARY Influenza-virus antigenicity evolves to escape host immune protection. Antibody lineages within individuals evolve in turn to increase affinity and hence potency. Strategies for a “universal” influenza vaccine to elicit lineages that escape this evolutionary arms race and protect against seasonal variation and novel, pandemic viruses will require directing B-cell ontogeny to focus the humoral response on conserved epitopes on the viral hemagglutinin (HA). The unmutated common ancestors (UCAs) of six distinct, broadly-neutralizing antibody lineages from one individual bind the HA of a virus circulating at the time the participant was born. HAs of viruses circulating more than five years later no longer bind the UCAs, but mature antibodies in the lineages bind strains from the entire 18-year lifetime of the participant. The analysis shows how immunological memory shaped the response to subsequent influenza exposures and suggests that early imprinting by a suitable influenza antigen may enhance likelihood of later breadth. PMID:26711348

  13. Structural evolution of glycan recognition by a family of potent HIV antibodies

    PubMed Central

    Garces, Fernando; Sok, Devin; Kong, Leopold; McBride, Ryan; Kim, Helen J.; Saye-Francisco, Karen F.; Julien, Jean-Philippe; Hua, Yuanzi; Cupo, Albert; Moore, John P.; Paulson, James C.; Ward, Andrew B.; Burton, Dennis R.; Wilson, Ian A.

    2014-01-01

    SUMMARY The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g. Ab PGT124) or engage multiple glycans (e.g. Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans. PMID:25259921

  14. Structural evolution of glycan recognition by a family of potent HIV antibodies.

    PubMed

    Garces, Fernando; Sok, Devin; Kong, Leopold; McBride, Ryan; Kim, Helen J; Saye-Francisco, Karen F; Julien, Jean-Philippe; Hua, Yuanzi; Cupo, Albert; Moore, John P; Paulson, James C; Ward, Andrew B; Burton, Dennis R; Wilson, Ian A

    2014-09-25

    The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans. PMID:25259921

  15. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  16. Structural basis for germline antibody recognition of HIV-1 immunogens

    PubMed Central

    Scharf, Louise; West, Anthony P; Sievers, Stuart A; Chen, Courtney; Jiang, Siduo; Gao, Han; Gray, Matthew D; McGuire, Andrew T; Scheid, Johannes F; Nussenzweig, Michel C; Stamatatos, Leonidas; Bjorkman, Pamela J

    2016-01-01

    Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design. DOI: http://dx.doi.org/10.7554/eLife.13783.001 PMID:26997349

  17. Surface plasmon resonance measurements of plasma antibody avidity during primary and secondary responses to anthrax protective antigen

    PubMed Central

    Lynch, Heather E.; Stewart, Shelley M.; Kepler, Thomas B.; Sempowski, Gregory D.; Alam, S. Munir

    2014-01-01

    Establishment of humoral immunity against pathogens is dependent on events that occur in the germinal center and the subsequent induction of high-affinity neutralizing antibodies. Quantitative assays that allow monitoring of affinity maturation and duration of antibody responses can provide useful information regarding the efficacy of vaccines and adjuvants. Using an anthrax protective antigen (rPA) and alum model antigen/adjuvant system, we describe a methodology for monitoring antigen-specific serum antibody concentration and avidity by surface plasmon resonance during primary and secondary immune responses. Our analyses showed that following a priming dose in mice, rPA-specific antibody concentration and avidity increases over time and reaches a maximal response in about six weeks, but gradually declines in the absence of antigenic boost. Germinal center reactions were observed early with maximal development achieved during the primary response, which coincided with peak antibody avidity responses to primary immunization. Boosting with antigen resulted in a rapid increase in rPA-specific antibody concentration and five-fold increase in avidity, which was not dependent on sustained GC development. The described methodology couples surface plasmon resonance-based plasma avidity measurements with germinal center analysis and provides a novel way to monitor humoral responses that can play a role in facilitating vaccine and adjuvant development. PMID:24316020

  18. Changes in Avidity and Level of Immunoglobulin G Antibodies to Mycobacterium tuberculosis in Sera of Patients Undergoing Treatment for Pulmonary Tuberculosis

    PubMed Central

    Pereira Arias-Bouda, Lenka M.; Kuijper, Sjoukje; Van Der Werf, Anouk; Nguyen, Lan N.; Jansen, Henk M.; Kolk, Arend H. J.

    2003-01-01

    Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity of antibodies to Mycobacterium tuberculosis in patients with pulmonary tuberculosis, to explore the value of avidity determination for the diagnosis of tuberculosis, and to study changes in levels of antibodies and their avidity during treatment. Antibody avidity was measured by an enzyme-linked immunosorbent assay with thiocyanate elution. Avidity indices and serum levels of immunoglobulin G to M. tuberculosis were determined for 22 patients with pulmonary tuberculosis before and during treatment and for 24 patients with other pulmonary diseases. Antibody levels and avidity were both significantly higher in untreated tuberculosis patients than in the controls. Avidity determination had more diagnostic potential than determination of the antibody levels. Tuberculosis patients with a long duration of symptoms had higher antibody avidity than those with a recent onset of symptoms, indicating affinity maturation of specific antibodies during active disease. In the early phase of treatment, a decrease in antibody avidity was observed for 73% of all tuberculosis patients, accompanied by an initial increase in antibody levels in 36% of these patients. These phenomena could be explained by an intense stimulation of the humoral response by antigens released from killed bacteria, reflecting early bactericidal activity of antituberculous drugs leading to the production of low-affinity antibodies against these released antigens. PMID:12853408

  19. Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

    PubMed

    Sato, Yusui; Tanaka, Yusuke; Inaba, Satomi; Sekiguchi, Hiroshi; Maruno, Takahiro; Sasaki, Yuji C; Fukada, Harumi; Kobayashi, Yuji; Azuma, Takachika; Oda, Masayuki

    2016-10-01

    Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events. PMID:27222286

  20. Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

    NASA Astrophysics Data System (ADS)

    Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  1. Antibody Request - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  2. Adoptive transfer of natural antibodies to non-immunized chickens affects subsequent antigen-specific humoral and cellular immune responses.

    PubMed

    Lammers, Aart; Klomp, Marcel E V; Nieuwland, Mike G B; Savelkoul, Huub F J; Parmentier, Henk K

    2004-01-01

    To determine a regulatory function of natural antibodies in the immune response of chickens, pooled plasma obtained from non-immunized (naïve) 15 months old hens was subjected to keyhole limpet hemocyanin (KLH) antigen-affinity chromatography. Purified KLH-binding antibodies were adoptively transferred intravenously to 5 weeks-old cocks that were subsequently immunized subcutaneously 24 h later with KLH. Control groups consisted of birds that were either adoptively transferred with KLH-binding antibodies purified from plasma of KLH-immunized chickens, or PBS, or a salt precipitated total immunoglobulin fraction obtained from the corresponding pooled nai;ve chicken plasma, respectively.Total, IgM and IgY antibody titers to KLH in the plasma of recipients adoptively transferred with KLH-NAb, but not in the plasma of the groups transferred with salt precipitate or KLH-binding specific antibodies, were significantly enhanced as compared to the non-treated, KLH immunized group. Titers of IgA antibodies binding KLH were decreased in the plasma of the group that received specific KLH-binding antibodies, but not in the plasma of the other groups. Proliferation from peripheral blood leucocytes in whole blood from the KLH-NAb treated group, the group treated with KLH-binding specific antibodies and the group treated with salt precipitate, respectively, to both concanavalin A and KLH were significantly decreased as compared to the group receiving PBS. Our data show that antigen-specific antibodies can be isolated from plasma obtained from non-immunized chickens. Such antibodies that resemble natural antibodies as described in mammals may perform an important role in the enhancement of subsequent antigen-specific antibody responses or the maturation of the immune system, which may differ from the role of specific antibodies. PMID:12962982

  3. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  4. The use of C1q, conglutinin and low affinity rabbit IgM antibody to human Fc in a ligand coctail radioassay for detecting and characterizing immune complexes in pathological sera.

    PubMed Central

    Harkiss, G D; Brown, D L

    1980-01-01

    A ligand radioassay for the detection of IC which utilizes C1q, bovine conglutinin and low affinity rabbit IgM anti-human Fc in a reagent coctail, is presented. IC are first isolated from serum by precipitation in polyethylene glycol, then analysed for their ability to react with the ligand coctail. Dual-label studies with 125I and 131I-tagged ligands, designed to determine whether the ligands bound independently to IC, indicate that the binding of each ligand to IC is not significantly affected by the presence of the other two ligands. The results of assaying pathological sera for IC by the ligand coctail radioassay correlate well with the results of three other assays. The assay system is also flexible enough to allow other low affinity IgM reagents to be used which could potentially cover the whole range of immunoglobulin classes occurring in pathological IC. PMID:7379330

  5. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  6. Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

    SciTech Connect

    Hirata, T.; Bitterman, P.B.; Mornex, J.; Crystal, R.G.

    1986-02-15

    The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, (/sup 125/I)-labeled human transferrin and a (/sup 32/P)-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (K/sub a/) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 x 10/sup 8/ M/sup -1/, and the number of receptors per cell was 4.4 +/- 1.2 x 10/sup 4/. In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, > 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.

  7. Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library.

    PubMed

    Xu, Yang; Xiong, Liang; Li, Yanping; Xiong, Yonghua; Tu, Zhui; Fu, Jinheng; Chen, Bo

    2015-07-01

    Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10% (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library. PMID:25910884

  8. Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential.

    PubMed

    Lohmueller, Jason J; Sato, Shuji; Popova, Lana; Chu, Isabel M; Tucker, Meghan A; Barberena, Roberto; Innocenti, Gregory M; Cudic, Mare; Ham, James D; Cheung, Wan Cheung; Polakiewicz, Roberto D; Finn, Olivera J

    2016-01-01

    MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1(+) target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

  9. Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential

    PubMed Central

    Lohmueller, Jason J.; Sato, Shuji; Popova, Lana; Chu, Isabel M.; Tucker, Meghan A.; Barberena, Roberto; Innocenti, Gregory M.; Cudic, Mare; Ham, James D.; Cheung, Wan Cheung; Polakiewicz, Roberto D.; Finn, Olivera J.

    2016-01-01

    MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

  10. Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10

    PubMed Central

    Finton, Kathryn A. K.; Friend, Della; Jaffe, James; Gewe, Mesfin; Holmes, Margaret A.; Larman, H. Benjamin; Stuart, Andrew; Larimore, Kevin; Greenberg, Philip D.; Elledge, Stephen J.; Stamatatos, Leonidas; Strong, Roland K.

    2014-01-01

    The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies. PMID:25254371

  11. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

    PubMed

    Doria-Rose, Nicole A; Schramm, Chaim A; Gorman, Jason; Moore, Penny L; Bhiman, Jinal N; DeKosky, Brandon J; Ernandes, Michael J; Georgiev, Ivelin S; Kim, Helen J; Pancera, Marie; Staupe, Ryan P; Altae-Tran, Han R; Bailer, Robert T; Crooks, Ema T; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J; Hoi, Kam H; Kong, Rui; Louder, Mark K; Longo, Nancy S; McKee, Krisha; Nonyane, Molati; O'Dell, Sijy; Roark, Ryan S; Rudicell, Rebecca S; Schmidt, Stephen D; Sheward, Daniel J; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C; Binley, James M; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Ward, Andrew B; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S; Morris, Lynn; Kwong, Peter D; Shapiro, Lawrence; Mascola, John R

    2014-05-01

    Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development. PMID:24590074

  12. Special Report: Affinity Chromatography.

    ERIC Educational Resources Information Center

    Parikh, Indu; Cuatrecasas, Pedro

    1985-01-01

    Describes the nature of affinity chromatography and its use in purifying enzymes, studying cell interactions, exploring hormone receptors, and other areas. The potential the technique may have in treating disease is also considered. (JN)

  13. 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 Central

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

    2011-01-01

    Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on 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 substratess, 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), was able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. 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 synthetic homogeneous Fc

  14. Alga-produced malaria transmission-blocking vaccine candidate Pfs25 formulated with a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes.

    PubMed

    Patra, Kailash P; Li, Fengwu; Carter, Darrick; Gregory, James A; Baga, Sheyenne; Reed, Steven G; Mayfield, Stephen P; Vinetz, Joseph M

    2015-05-01

    A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene-oil-in-water emulsion, and GLA plus squalene-oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene-oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene-oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration. PMID:25690099

  15. Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

    PubMed Central

    Patra, Kailash P.; Li, Fengwu; Carter, Darrick; Gregory, James A.; Baga, Sheyenne; Reed, Steven G.; Mayfield, Stephen P.

    2015-01-01

    A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate for Plasmodium falciparum transmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. Here Chlamydomonas reinhardtii microalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface of P. falciparum macrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration. PMID:25690099

  16. Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds 1

    PubMed Central

    Wu, Hua-Cheng; Poulton, Jonathan E.

    1991-01-01

    Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by α-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668338

  17. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    SciTech Connect

    X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

    2005-04-18

    canonical structures method detailed by Morea et al. (J. Mol. Biol. 275:269), and the participation of specific residues in antigen recognition was assessed using site-directed mutagenesis. Three amino acids in the light chain variable region, H39, Y54 and F103, were particularly important in antigen recognition. In a separate series of experiments, a recombinant phage-displayed antibody library has been prepared using RNA isolated from the spleens of sheep and rabbits immunized with specific metal-chelate complexes. Phage-display libraries produced from an immunized source are inclined to include variable genes specific for the immunized antigen(s), many of which are already affinity matured. An antibody fragment specific for the UO{sub 2}{sup 2+}-DCP complex was isolated from this combined phage display library. While the binding affinity of this antibody fragment for UO{sub 2}{sup 2+}-DCP was not as high as that of the 12F6 monoclonal antibody, the beauty of antibody phage display technology is that it allows for the potential manipulation and saturation of the antibody's binding affinity, which may drastically improve and ultimately surpass that of monoclonal antibodies.

  18. Trade-offs in antibody repertoires to complex antigens

    PubMed Central

    Childs, Lauren M.; Baskerville, Edward B.; Cobey, Sarah

    2015-01-01

    Pathogens vary in their antigenic complexity. While some pathogens such as measles present a few relatively invariant targets to the immune system, others such as malaria display considerable antigenic diversity. How the immune response copes in the presence of multiple antigens, and whether a trade-off exists between the breadth and efficacy of antibody (Ab)-mediated immune responses, are unsolved problems. We present a theoretical model of affinity maturation of B-cell receptors (BCRs) during a primary infection and examine how variation in the number of accessible antigenic sites alters the Ab repertoire. Naive B cells with randomly generated receptor sequences initiate the germinal centre (GC) reaction. The binding affinity of a BCR to an antigen is quantified via a genotype–phenotype map, based on a random energy landscape, that combines local and distant interactions between residues. In the presence of numerous antigens or epitopes, B-cell clones with different specificities compete for stimulation during rounds of mutation within GCs. We find that the availability of many epitopes reduces the affinity and relative breadth of the Ab repertoire. Despite the stochasticity of somatic hypermutation, patterns of immunodominance are strongly shaped by chance selection of naive B cells with specificities for particular epitopes. Our model provides a mechanistic basis for the diversity of Ab repertoires and the evolutionary advantage of antigenically complex pathogens. PMID:26194759

  19. Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

    SciTech Connect

    Pancera, Marie; McLellan, Jason S.; Wu, Xueling; Zhu, Jiang; Changela, Anita; Schmidt, Stephen D.; Yang, Yongping; Zhou, Tongqing; Phogat, Sanjay; Mascola, John R.; Kwong, Peter D.

    2010-11-03

    HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 {angstrom}, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming {approx}20 {angstrom} above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.

  20. Antibody Production with Synthetic Peptides.

    PubMed

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

    2016-01-01

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

  1. NK Cell and Ig Interplay in Defense against Herpes Simplex Virus Type 1: Epistatic Interaction of CD16A and IgG1 Allotypes of Variable Affinities Modulates Antibody-Dependent Cellular Cytotoxicity and Susceptibility to Clinical Reactivation.

    PubMed

    Moraru, Manuela; Black, Laurel E; Muntasell, Aura; Portero, Francisca; López-Botet, Miguel; Reyburn, Hugh T; Pandey, Janardan P; Vilches, Carlos

    2015-08-15

    HSV-1 latently infects most humans, causing a variable clinical picture that depends, in part, on host genetic factors. Both IgG and its cellular FcRs, CD16A and CD32A-C (encoded by FCGR3A and FCGR2A-C, respectively, on chromosome 1), display polymorphisms that could affect their defensive function. Of potential relevance are a FCGR3A dimorphism resulting in CD16A-valine/phenylalanine-158 allotypes with different IgG affinity, variations conditioning NK cell expression of CD32B or CD32C, and IgG1 H chain (IGHG1) and kappa-chain (IGKC) polymorphisms determining allotypes designated G1m and Km. In this study, we assessed the contribution of Ig genetic variations and their interaction with FcR polymorphism to HSV-1 susceptibility, as well as their impact on NK cell-mediated Ab-dependent cellular cytotoxicity (ADCC). Our results show an epistatic interaction between IGHG1 and FCGR3A such that the higher affinity CD16A-158V/V genotype associates with an asymptomatic course of HSV-1 infection only in homozygotes for G1m3. Furthermore, CD16A-158V and G1m3 allotypes enhanced ADCC against opsonized HSV-1-infected fibroblasts. Conversely, Km allotypes and CD32B or CD32C expression on NK cells did not significantly influence HSV-1 susceptibility or ADCC. NK cells degranulating against immune serum-opsonized HSV-1-infected fibroblasts had heterogeneous phenotypes. Yet, enhanced ADCC was observed among NK cells showing a differentiated, memory-like phenotype (NKG2C(bright)NKG2A(-)CD57(+)FcRγ(-)), which expand in response to human CMV. These results extend our knowledge on the importance of immunogenetic polymorphisms and NK cell-Ab interplay in the host response against HSV-1 and point to the relevance of interactions between immune responses elicited during chronic coinfection by multiple herpesviruses. PMID:26179905

  2. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen

    PubMed Central

    Jardine, Joseph G.; Ota, Takayuki; Sok, Devin; Pauthner, Matthias; Kulp, Daniel W.; Kalyuzhniy, Oleksandr; Skog, Patrick D.; Thinnes, Theresa C.; Bhullar, Deepika; Briney, Bryan; Menis, Sergey; Jones, Meaghan; Kubitz, Mike; Spencer, Skye; Adachi, Yumiko; Burton, Dennis R.; Schief, William R.; Nemazee, David

    2015-01-01

    A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01-class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short light chain complementarity determining region 3 (CDRL3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets. PMID:26089355

  3. Related Mechanisms of Antibody Somatic Hypermutation and Class Switch Recombination

    PubMed Central

    HWANG, JOYCE K.; ALT, FREDERICK W.; YEAP, LENG-SIEW

    2015-01-01

    The primary antibody repertoire is generated by mechanisms involving the assembly of the exons that encode the antigen-binding variable regions of immunoglobulin heavy (IgH) and light (IgL) chains during the early development of B lymphocytes. After antigen-dependent activation, mature B lymphocytes can further alter their IgH and IgL variable region exons by the process of somatic hypermutation (SHM), which allows the selection of B cells in which SHMs resulted in the production of antibodies with increased antigen affinity. In addition, during antigen-dependent activation, B cells can also change the constant region of their IgH chain through a DNA double-strand-break (DSB) dependent process referred to as IgH class switch recombination (CSR), which generates B cell progeny that produce antibodies with different IgH constant region effector functions that are best suited for a elimination of a particular pathogen or in a particular setting. Both the mutations that underlie SHM and the DSBs that underlie CSR are initiated in target genes by activation-induced cytidine deaminase (AID). This review describes in depth the processes of SHM and CSR with a focus on mechanisms that direct AID cytidine deamination in activated B cells and mechanisms that promote the differential outcomes of such cytidine deamination. PMID:26104555

  4. Expression and purification of recombinant antibody formats and antibody fusion proteins.

    PubMed

    Siegemund, Martin; Richter, Fabian; Seifert, Oliver; Unverdorben, Felix; Kontermann, Roland E

    2014-01-01

    In the laboratory-scale production of antibody fragments or antibody fusion proteins, it is often difficult to keep track on the most suitable affinity tags for protein purification from either prokaryotic or eukaryotic host systems. Here, we describe how such recombinant proteins derived from Escherichia coli lysates as well as HEK293 cell culture supernatants are purified by IMAC and by different affinity chromatography methods based on fusions to FLAG-tag, Strep-tag, and Fc domains. PMID:24515473

  5. Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

    PubMed

    Tian, Ming; Cheng, Cheng; Chen, Xuejun; Duan, Hongying; Cheng, Hwei-Ling; Dao, Mai; Sheng, Zizhang; Kimble, Michael; Wang, Lingshu; Lin, Sherry; Schmidt, Stephen D; Du, Zhou; Joyce, M Gordon; Chen, Yiwei; DeKosky, Brandon J; Chen, Yimin; Normandin, Erica; Cantor, Elizabeth; Chen, Rita E; Doria-Rose, Nicole A; Zhang, Yi; Shi, Wei; Kong, Wing-Pui; Choe, Misook; Henry, Amy R; Laboune, Farida; Georgiev, Ivelin S; Huang, Pei-Yi; Jain, Suvi; McGuire, Andrew T; Georgeson, Eric; Menis, Sergey; Douek, Daniel C; Schief, William R; Stamatatos, Leonidas; Kwong, Peter D; Shapiro, Lawrence; Haynes, Barton F; Mascola, John R; Alt, Frederick W

    2016-09-01

    The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages. PMID:27610571

  6. High affinity FRβ-specific CAR T cells eradicate AML and normal yeloid lineage without HSC toxicity

    PubMed Central

    Lynn, Rachel C; Feng, Yang; Schutsky, Keith; Poussin, Mathilde; Kalota, Anna; Dimitrov, Dimiter S; Powell, Daniel J

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  7. Multiplexed protein profiling by sequential affinity capture.

    PubMed

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-04-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  8. Engineered affinity proteins for tumour-targeting applications.

    PubMed

    Friedman, Mikaela; Ståhl, Stefan

    2009-05-01

    Targeting of tumour-associated antigens is an expanding treatment modality in clinical oncology as an alternative to, or in combination with, conventional treatments, such as chemotherapy, external-radiation therapy and surgery. Targeting of antigens that are unique or more highly expressed in tumours than in normal tissues can be used to increase the specificity and reduce the cytotoxic effect on normal tissues. Several targeting agents have been studied for clinical use, where monoclonal antibodies have been the ones most widely used. More than 20 monoclonal antibodies are approved for therapy today and the largest field is oncology. Advances in genetic engineering and in vitro selection technology has enabled the feasible high-throughput generation of monoclonal antibodies, antibody derivatives [e.g. scFvs, Fab molecules, dAbs (single-domain antibodies), diabodies and minibodies] and more recently also non-immunoglobulin scaffold proteins. Several of these affinity proteins have been investigated for both in vivo diagnostics and therapy. Affinity proteins in tumour-targeted therapy can affect tumour progression by altering signal transduction or by delivering a payload of toxin, drug or radionuclide. The ErbB receptor family has been extensively studied as biomarkers in tumour targeting, primarily for therapy using monoclonal antibodies. Two receptors in the ErbB family, EGFR (epidermal growth factor receptor) and HER2 (epidermal growth factor receptor 2), are overexpressed in various malignancies and associated with poor patient prognosis and are therefore interesting targets for solid tumours. In the present review, strategies are described for tumour targeting of solid tumours using affinity proteins to deliver radionuclides, either for molecular imaging or radiotherapy. Antibodies, antibody derivatives and non-immunoglobulin scaffold proteins are discussed with a certain focus on the affibody (Affibody) molecule. PMID:19341363

  9. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  10. The pathogenic role of virus-specific antibody-secreting cells in the central nervous system of rats with different susceptibility to coronavirus-induced demyelinating encephalitis.

    PubMed Central

    Schwender, S; Imrich, H; Dörries, R

    1991-01-01

    The humoral immune response in the central nervous system (CNS) of susceptible Lewis (LE) rats and resistant Brown Norway (BN) rats was analysed after intracerebral infection with the murine coronavirus JHM (MHV4). The subclinical course of the infection in BN rats was characterized by an early rise of neutralizing antibodies in the cerebrospinal fluid (CSF) 7 days post-infection. At this time in LE rats, neutralizing antibodies were not detectable in the CSF and the animals developed neurological signs of infection. Subsequently, LE rats recovered from disease. This process was accompanied by increasing titres of virus-neutralizing antibodies. Within the CNS parenchyma of both rat strains, equivalent numbers of IgM-secreting cells were detected. However, in BN rats, virus-specific IgG secreting cells appeared earlier and in higher numbers. Moreover, based on the size of zones of antibody secreted by single cells in the Spot-ELISA assay, it appeared that cells from BN rats secreted IgG antibody of higher affinity. These data suggest that early maturation of antiviral antibody responses in the resistant BN rat probably restricts the spread of viral infection to small foci within the CNS, resulting in a subclinical level of primary demyelination. In contrast, the absence of neutralizing antibodies in the susceptible LE rats favours spread of the virus throughout the CNS, resulting finally in severe neurological disease. Images Figure 1 Figure 2 Figure 3 PMID:1663078

  11. A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses.

    PubMed

    Hu, Hongxing; Voss, Jarrod; Zhang, Guoliang; Buchy, Philippi; Zuo, Teng; Wang, Lulan; Wang, Feng; Zhou, Fan; Wang, Guiqing; Tsai, Cheguo; Calder, Lesley; Gamblin, Steve J; Zhang, Linqi; Deubel, Vincent; Zhou, Boping; Skehel, John J; Zhou, Paul

    2012-03-01

    Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains. PMID:22238297

  12. High-affinity immobilization of proteins using biotin- and GST-based coupling strategies.

    PubMed

    Hutsell, Stephanie Q; Kimple, Randall J; Siderovski, David P; Willard, Francis S; Kimple, Adam J

    2010-01-01

    Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  13. High affinity immobilization of proteins using biotin- and GST-based coupling strategies

    PubMed Central

    Hutsell, Stephanie Q.; Kimple, Randall J.; Siderovski, David P.; Willard, Francis S.; Kimple, Adam J.

    2011-01-01

    Surface Plasmon Resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  14. Monoclonal Antibodies.

    ERIC Educational Resources Information Center

    Killington, R. A.; Powell, K. L.

    1984-01-01

    Monoclonal antibodies have provided an exciting addition to the "armory" of the molecular biologist and immunologist. This article discusses briefly the concept of, techniques available for, production of, and possible uses of monoclonal antibodies. (Author)

  15. Antimitochondrial antibody

    MedlinePlus

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

  16. NCI Requests Targets for Monoclonal Antibody Production and Characterization - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In an effort to provide well-characterized monoclonal antibodies to the scientific community, NCI's Antibody Characterization Program requests cancer-related protein targets for affinity production and distribution.

  17. Enhancement of Immune Effector Functions by Modulating IgG’s Intrinsic Affinity for Target Antigen

    PubMed Central

    Mazor, Yariv; Yang, Chunning; Borrok, M. Jack; Ayriss, Joanne; Aherne, Karen; Wu, Herren; Dall’Acqua, William F.

    2016-01-01

    Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics. PMID:27322177

  18. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  19. Local Affinity Release.

    PubMed

    Delplace, Vianney; Obermeyer, Jaclyn; Shoichet, Molly S

    2016-07-26

    The use of hydrogels for therapeutic delivery is a burgeoning area of investigation. These water-swollen polymer matrices are ideal platforms for localized drug delivery that can be further combined with specific ligands or nanotechnologies to advance the controlled release of small-molecule drugs and proteins. Due to the advantage of hydrophobic, electrostatic, or specific extracellular matrix interactions, affinity-based strategies can overcome burst release and challenges associated with encapsulation. Future studies will provide innovative binding tools, truly stimuli-responsive systems, and original combinations of emerging technologies to control the release of therapeutics spatially and temporally. Local drug delivery can be achieved by directly injecting a therapeutic to its site of action and is advantageous because off-target effects associated with systemic delivery can be minimized. For prolonged benefit, a vehicle that provides sustained drug release is required. Hydrogels are versatile platforms for localized drug release, owing to the large library of biocompatible building blocks from which they can be formed. Injectable hydrogel formulations that gel quickly in situ and provide sustained release of therapeutics are particularly advantageous to minimize invasiveness. The incorporation of polymers, ligands or nanoparticles that have an affinity for the therapeutic of interest improve control over the release of small-molecule drugs and proteins from hydrogels, enabling spatial and temporal control over the delivery. Such affinity-based strategies can overcome drug burst release and challenges associated with protein instability, allowing more effective therapeutic molecule delivery for a range of applications from therapeutic contact lenses to ischemic tissue regeneration. PMID:27403513

  20. Somatic hypermutations and isotype restricted exceptionally long CDR3H contribute to antibody diversification in cattle.

    PubMed

    Kaushik, Azad K; Kehrli, Marcus E; Kurtz, A; Ng, S; Koti, M; Shojaei, F; Saini, Surinder S

    2009-01-15

    Antibody diversification in IgM and IgG antibodies was analyzed in an 18-month old bovine (Bos taurus) suffering from naturally occurring chronic and recurrent infections due to bovine leukocyte adhesion deficiency (BLAD). The BLAD, involving impaired leukocyte beta2 integrin expression on leukocytes, develops due to a single point mutation in conserved region of the CD18 gene resulting in substitution of aspartic acid128 with glycine (D128G). Twenty four VDJCmu and 25 VDJCgamma recombinations from randomly constructed cDNA libraries, originating from peripheral blood lymphocytes, were examined for the variable-region structural characteristics in IgM and IgG antibody isotypes. These analyses led to conclude that: (a) expression of exceptionally long CDR3H is isotype restricted to cattle IgM antibody; (b) VDJ recombinations encoding IgM with exceptionally long CDR3H undergo clonal selection and affinity maturation via somatic mutations similar to conventional antibodies; (c) somatic mutations contribute significantly to both IgM and IgG antibody diversification but significant differences exist in the patterns of 'hot spot' in the FR1, FR3 and CDR1H and, also, position-dependant amino acid diversity; and (d) transition nucleotide substitutions predominate over transversions in both VDJCmu and VDJCgamma recombinations consistent with the evolutionary conservation of somatic mutation machinery. Overall, these studies suggest that both somatic mutations and exceptional CDR3H size generation contribute to IgM and IgG antibody diversification in cattle during the development of immune response to naturally occurring chronic and multiple microbial infections. PMID:19012969

  1. Genetic and Phenotypic Selection Affect Natural (Auto-) Antibody Reactivity of Chickens

    PubMed Central

    de Jong, Britt G.; Lammers, Aart; Oberendorf, Leonora A. A.; Nieuwland, Mike G. B.; Savelkoul, Huub F. J.; Parmentier, Henk K.

    2013-01-01

    Specificity, antibody isotype distribution and levels of natural antibodies (NAb) may be potential informative parameters for immune mediated natural disease resistance, immune modulation, and maintenance of physiological homeostasis. A large proportion of mammalian NAb have affinity for or are directed against self-antigens; so called natural auto antibodies (N(A)Ab). In the present study we showed the presence and typed levels and isotypes (total immunoglobulins, IgG and IgM) of N(A)Ab in plasma binding the ‘auto-antigen’ complex chicken liver cell lysate (CLL) of one-year old chickens from different genotype and phenotype backgrounds by ELISA and quantitative Western blotting. Higher levels of N(A)Ab binding CLL were found in plasma from chickens genetically selected for high specific antibody responses. In all birds, extensive staining patterns of plasma antibodies binding CLL were found for all isotypes, with IgG binding the highest number of CLL antigens and also showing the highest variation in staining patterns between individuals. Patterns of IgM antibodies binding CLL appeared to be more similar in all lines. Significant differences of binding patterns of N(A)Ab (antigen fragments of CLL and staining intensity) were detected between the different chicken lines, and lines could be clustered on the basis of their auto-antibody profile. In addition, also individual differences within lines were found. The present results indicate that analysis of the levels and the N(A)Ab repertoire of poultry like in mammals could provide a new way of distinguishing differences of immune competence and immune maturation between individuals, and could provide tools to select birds for health traits, or optimize hygiene and husbandry procedures. PMID:24039748

  2. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  3. Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response.

    PubMed

    Ehrenstein, M R; O'Keefe, T L; Davies, S L; Neuberger, M S

    1998-08-18

    Accelerated development of the secondary immune response may be attributable in part to the rapid delivery of antigen to lymphoid follicles by circulating antibody elicited on primary immunization. Here we provide evidence indicating that the nonspecific IgM present in naive mice (natural antibody) plays a role in the acceleration of the primary response. Targeted deletion of the Ig microseconds polyadenylation site by use of Cre recombinase allowed the creation of mice that, although harboring a normal number of B cells expressing surface IgM, completely lacked serum IgM while retaining the other Ig isotypes. These mice retained a broadly normal B lymphocyte distribution (although containing a somewhat expanded peritoneal B1a subset) but exhibited substantial delays in mounting affinity-matured IgG responses to T cell-dependent antigens. The T cell-independent response, however, was augmented. The data indicate that the IgM present before antigen challenge (as well, possibly, as that elicited immediately after immunization) accelerates maturation of the primary response, presumably by complexing with the antigen and facilitating lymphocyte activation and/or antigen trapping. PMID:9707605

  4. Morphometric affinities of gigantopithecus.

    PubMed

    Gelvin, B R

    1980-11-01

    Multivariate analyses, supplemented by univariate statistical methods, of measurements from mandibular tooth crown dimensions and the mandible of Gigantopithecus blacki, G. bilaspurensis, Plio-Plelstocene hominids, Homo erectus, and seven Neogene ape species from the genera Proconsul, Sivapithecus, Ouranopithecus, and Dryopithecus were used to assess the morphometric affinities of Gigantopithecus. The results show that Gigantopithecus displays affinities to Ouranopithecus and to the hominids, particularly the Plio-Plelstocene hominids, rather than to the apes. Ouranopithecus demonstrated dental resemblances to G. bilaspurensis and the Plio-Pleistocene hominids but mandibular similarities to the apes. Results of analyses of tooth and mandibular shape indices, combined with multivariate distance and temporal relationships, suggest that Ouranopithecus is a more likely candidate for Gigantopithecus ancestry than is Silvapithecus indicus. Shape and allometric differences between G. bilaspurensis and the robust australopithecines weaken the argument for an ancestral-descendant relationship between these groups. The results support the hypothesis that Gigantopithecus is an extinct side branch of the Hominidae. PMID:7468790

  5. Exceptional Antibodies Produced by Successive Immunizations.

    PubMed

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

    2015-12-01

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

  6. Single-domain antibodies for biomedical applications.

    PubMed

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

    2016-01-01

    Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development. PMID:26551147

  7. Adjuvant-specific regulation of long-term antibody responses by ZBTB20

    PubMed Central

    Wang, Yinan

    2014-01-01

    The duration of antibody production by long-lived plasma cells varies with the type of immunization, but the basis for these differences is unknown. We demonstrate that plasma cells formed in response to the same immunogen engage distinct survival programs depending on the adjuvant. After alum-adjuvanted immunization, antigen-specific bone marrow plasma cells deficient in the transcription factor ZBTB20 failed to accumulate over time, leading to a progressive loss of antibody production relative to wild-type controls. Fetal liver reconstitution experiments demonstrated that the requirement for ZBTB20 was B cell intrinsic. No defects were observed in germinal center numbers, affinity maturation, or plasma cell formation or proliferation in ZBTB20-deficient chimeras. However, ZBTB20-deficient plasma cells expressed reduced levels of MCL1 relative to wild-type controls, and transgenic expression of BCL2 increased serum antibody titers. These data indicate a role for ZBTB20 in promoting survival in plasma cells. Strikingly, adjuvants that activate TLR2 and TLR4 restored long-term antibody production in ZBTB20-deficient chimeras through the induction of compensatory survival programs in plasma cells. Thus, distinct lifespans are imprinted in plasma cells as they are formed, depending on the primary activation conditions. The durability of vaccines may accordingly be improved through the selection of appropriate adjuvants. PMID:24711582

  8. Complement receptors and the shaping of the natural antibody repertoire.

    PubMed

    Holers, V Michael

    2005-03-01

    Complement and complement receptors have been known for several decades to play important roles in immune effector mechanisms related to pathogen elimination and tissue inflammation. In addition, studies over the last 10 years have clearly demonstrated a key role for the complement C3d activation fragment receptor designated CR2 (complement receptor type 2) in the switched-isotype, high-affinity and memory humoral immune responses to T-dependent foreign antigens. More recent studies have extended those observations to include a key role for CR2 and C3d in the humoral immune response to T-independent foreign antigens. Conversely, as these studies have proceeded, a parallel series of analyses have linked defects in expression or function of complement C4 and other classical pathway activation pathway proteins, as well as CR2 and the closely related CR1, to the loss of self tolerance to nuclear antigens such as double-stranded DNA and chromatin in systemic lupus erythematosus. With regard to the topic of this issue, it is now becoming increasingly clear that CR2 also plays a major role in the development of the natural antibody repertoire. Specifically, in the absence of this receptor natural IgM and IgG develop in the naïve animal that demonstrate clearly altered recognition patterns for specific natural antibody targets. This repertoire change is important physiologically in at least one setting because these CR2-dependent natural antibodies are necessary for the recognition of ischemic self tissues. In addition, it is possible that certain of the phenotypes manifest by CR2-deficient mice may be strongly influenced not only by effects on later stages of B cell activation and maturation, as commonly thought, but also by alterations in the pre-existing pool of natural antibodies that are influenced by this receptor. This review will examine the evidence that has accumulated over the last few years supporting these hypotheses. PMID:15614507

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

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke

    2014-11-01

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

  10. Role of natural and immune IgM antibodies in immune responses.

    PubMed

    Boes, M

    2000-12-01

    IgM antibodies constitute the major component of the natural antibodies and is also the first class of antibodies produced during a primary antibody response. The IgM-type antibodies differ from other classes of antibodies in that they are predominantly produced by B1 cells, in the absence of apparent stimulation by specific antigens. In addition, IgM antibodies are mostly encoded by germline V gene segments and have low affinities but broad specificites to both foreign and self structures. New developments regarding the function of both immune IgM antibodies and natural IgM antibodies will be examined here. PMID:11451419

  11. Diacylglycerol kinase ζ limits B cell antigen receptor-dependent activation of ERK signaling to inhibit early antibody responses.

    PubMed

    Wheeler, Matthew L; Dong, Matthew B; Brink, Robert; Zhong, Xiao-Ping; DeFranco, Anthony L

    2013-10-15

    Signaling downstream of the B cell antigen receptor (BCR) is tightly regulated to enable cells to gauge the strength and duration of antigen-receptor interactions and to respond appropriately. We investigated whether metabolism of the second messenger diacylglycerol (DAG) by members of the family of DAG kinases (DGKs) played a role in modulating the magnitude of signaling by DAG downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling, measured as activation of the Ras-extracellular signal-regulated kinase (ERK) pathway, was markedly reduced in mature follicular B cells, which resulted in enhanced responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ limited the number of antibody-secreting cells that were generated early in response to T cell-independent type 2 antigens, as well as to T cell-dependent antigens. Furthermore, the effect of loss of DGKζ closely resembled the effect of increasing the affinity of the BCR for antigen during the T cell-dependent antibody response. These results suggest that the magnitude of DAG signaling is important for translating the affinity of the BCR for antigen into the amount of antibody produced during the early stages of an immune response. PMID:24129701

  12. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule

    PubMed Central

    Petters, Edyta; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2015-01-01

    Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer. PMID:26307975

  13. Selection and Characterization of Single Chain Antibody Fragments Specific for Hsp90 as a Potential Cancer Targeting Molecule.

    PubMed

    Petters, Edyta; Sokolowska-Wedzina, Aleksandra; Otlewski, Jacek

    2015-01-01

    Heat shock proteins play an essential role in facilitating malignant transformation and they have been recognized as important factors in human cancers. One of the key elements of the molecular chaperones machinery is Hsp90 and it has recently become a target for anticancer therapeutic approaches. The potential and importance of Hsp90-directed agents becomes apparent when one realizes that disruption of Hsp90 function may influence over 200 oncogenic client proteins. Here, we described the selection and characterization of Hsp90-specific antibody fragments from commercially available Tomlinson I and J phage display libraries. The affinities of Hsp90-binding scFv variants were measured using SPR method. Then, based on the best clone selected, we performed the affinity maturation procedure and obtained valuable Hsp90-specific clones. The selected binders were expressed and applied for immunostaining, ELISA and SPR analysis using model cancer cell lines. All performed experiments confirmed the ability of selected antibodies to interact with the Hsp90. Therefore, the presented Hsp90-specific scFv, might be a starting point for the development of a novel antibody-based strategy targeting cancer. PMID:26307975

  14. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    PubMed

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  15. Enhanced HIV-1 neutralization by antibody heteroligation

    PubMed Central

    Mouquet, Hugo; Warncke, Malte; Scheid, Johannes F.; Seaman, Michael S.; Nussenzweig, Michel C.

    2012-01-01

    Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti–HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation. PMID:22219363

  16. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  17. ENGINEERED ANTIBODIES FOR MONITORING OF POLYNUCLEAR AROMATIC HYDROCARBONS

    EPA Science Inventory

    The objective of this multidisciplinary project is to use molecular biological techniques to derive a set of antibodies with useful affinities and selectivities for recovery and detection of polynuclear aromatic hydrocarbons (PAHs) in environmental and biological samples. The lon...

  18. Toll-like Receptors and B-cell Receptors Synergize to Induce Immunoglobulin Class Switch DNA Recombination: Relevance to Microbial Antibody Responses

    PubMed Central

    Pone, Egest J.; Zan, Hong; Zhang, Jinsong; Al-Qahtani, Ahmed; Xu, Zhenming; Casali, Paolo

    2011-01-01

    Differentiation of naïve B cells, including immunoglobulin (Ig) class switch DNA recombination (CSR), is critical for the immune response and depends on the extensive integration of signals from the B cell receptor (BCR), tumor necrosis factor (TNF) receptor family members, Toll-like receptors (TLRs) and cytokine receptors. TLRs and BCR synergize to induce CSR in T cell-dependent and T cell-independent antibody responses to microbial pathogens. BCR triggering together with simultaneous endosomal TLR engagement leads to enhanced B cell differentiation and antibody responses. The requirement of both BCR and TLR engagement would ensure appropriate antigen-specific activation in an infection. Co-stimulation of TLRs and BCR likely plays a significant role in anti-microbial antibody responses to contain pathogen loads until the T cell-dependent antibody responses peak. Furthermore, the temporal sequence of different signals is also critical for optimal B cell responses, as exemplified by the activation of B cells by initial TLR engagement, leading to the upregulation of co-stimulatory CD80 and MHC-II receptors, which, in turn, result in more efficient interactions with T cells, thereby enhancing the germinal center (GC) reaction and antibody affinity maturation. Overall, BCR and TLR stimulation and the integration with signals from the pathogen or immune cells and their products, determine the ensuing B cell antibody response. PMID:20370617

  19. Structural analysis of the unmutated ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee☆

    PubMed Central

    Nicely, Nathan I.; Wiehe, Kevin; Kepler, Thomas B.; Jaeger, Frederick H.; Dennison, S. Moses; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Robb, Merlin L.; O'Connell, Robert J.; Michael, Nelson L.; Kim, Jerome H.; Liao, Hua-Xin; Munir Alam, S.; Hwang, Kwan-Ki; Bonsignori, Mattia; Haynes, Barton F.

    2015-01-01

    Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties. PMID:26288844

  20. Structural analysis of the unmutated ancestor of the HIV-1 envelope V2 region antibody CH58 isolated from an RV144 vaccine efficacy trial vaccinee.

    PubMed

    Nicely, Nathan I; Wiehe, Kevin; Kepler, Thomas B; Jaeger, Frederick H; Dennison, S Moses; Rerks-Ngarm, Supachai; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Robb, Merlin L; O'Connell, Robert J; Michael, Nelson L; Kim, Jerome H; Liao, Hua-Xin; Munir Alam, S; Hwang, Kwan-Ki; Bonsignori, Mattia; Haynes, Barton F

    2015-07-01

    Human monoclonal antibody CH58 isolated from an RV144 vaccinee binds at Lys169 of the HIV-1 Env gp120 V2 region, a site of vaccine-induced immune pressure. CH58 neutralizes HIV-1 CRF_01 AE strain 92TH023 and mediates ADCC against CD4 + T cell targets infected with CRF_01 AE tier 2 virus. CH58 and other antibodies that bind to a gp120 V2 epitope have a second light chain complementarity determining region (LCDR2) bearing a glutamic acid, aspartic acid (ED) motif involved in forming salt bridges with polar, basic side amino acid side chains in V2. In an effort to learn how V2 responses develop, we determined the crystal structures of the CH58-UA antibody unliganded and bound to V2 peptide. The structures showed an LCDR2 structurally pre-conformed from germline to interact with V2 residue Lys169. LCDR3 was subject to conformational selection through the affinity maturation process. Kinetic analyses demonstrate that only a few contacts were responsible for a 2000-fold increase in KD through maturation, and this effect was predominantly due to an improvement in off-rate. This study shows that preconformation and preconfiguration can work in concert to produce antibodies with desired immunogenic properties. PMID:26288844

  1. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... test is done to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also ...

  2. Cone Early Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hop cone early maturity is thought to be caused by diffuse infections of cone, just prior to harvest, by Podosphaera macularis. The disease is best managed by limiting the amount of leaf infection by P. macularis prior to bloom. The yield and quality reductions associated with Hop cone early matur...

  3. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  4. Selective high affinity polydentate ligands and methods of making such

    SciTech Connect

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  5. Affinity chromatography: a historical perspective.

    PubMed

    Hage, David S; Matsuda, Ryan

    2015-01-01

    Affinity chromatography is one of the most selective and versatile forms of liquid chromatography for the separation or analysis of chemicals in complex mixtures. This method makes use of a biologically related agent as the stationary phase, which provides an affinity column with the ability to bind selectively and reversibly to a given target in a sample. This review examines the early work in this method and various developments that have lead to the current status of this technique. The general principles of affinity chromatography are briefly described as part of this discussion. Past and recent efforts in the generation of new binding agents, supports, and immobilization methods for this method are considered. Various applications of affinity chromatography are also summarized, as well as the influence this field has played in the creation of other affinity-based separation or analysis methods. PMID:25749941

  6. Recent advances in affinity capillary electrophoresis for binding studies.

    PubMed

    Albishri, Hassan M; El Deeb, Sami; AlGarabli, Noura; AlAstal, Raghda; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Wätzig, Hermann

    2014-01-01

    The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry. PMID:25534793

  7. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. PMID:23743326

  8. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T-cells

    PubMed Central

    Hudecek, Michael; Lupo-Stanghellini, Maria-Teresa; Kosasih, Paula L.; Sommermeyer, Daniel; Jensen, Michael C.; Rader, Christoph; Riddell, Stanley R.

    2013-01-01

    Purpose The adoptive transfer of T-cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module has activity in advanced malignancies. ROR1 is a tumor-associated molecule expressed on prevalent B-lymphoid and epithelial cancers, and is absent on normal mature B-cells and vital tissues, making it a candidate for CAR T-cell therapy. Experimental Design We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T-cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma engrafted into immunodeficient mice. Results ROR1-CARs containing a short ‘Hinge-only’ extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared to CARs with long ‘Hinge-CH2-CH3’ spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation induced T-cell death. T-cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR modified T-cells in mediating regression of JeKo-1 mantle cell lymphoma in immunodeficient mice. Conclusions Our results demonstrate that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T-cells modified with an optimized ROR1-CAR have significant anti-tumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. PMID:23620405

  9. Novel antibodies as anticancer agents.

    PubMed

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

    2007-05-28

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

  10. Antithyroglobulin antibody

    MedlinePlus

    ... may be due to: Graves disease Hashimoto thyroiditis Hypothyroidism Systemic lupus erythematosus (SLE) Thyrotoxicosis Type 1 diabetes ... Antibody Chronic thyroiditis (Hashimoto disease) Graves disease Hyperthyroidism Hypothyroidism Systemic lupus erythematosus T3 test Update Date 5/ ...

  11. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin

    PubMed Central

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

    2016-01-01

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

  12. Postbooster Antibodies from Humans as Source of Diphtheria Antitoxin.

    PubMed

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

    2016-07-01

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

  13. Novel Antibody Vectors for Imaging

    PubMed Central

    Olafsen, Tove; Wu, Anna M.

    2010-01-01

    Non-invasive molecular imaging approaches include nuclear, optical, MRI, CT, ultrasound and photoacoustic imaging, which require accumulation of a signal delivered by a probe at the target site. Monoclonal antibodies (mAbs) are high affinity molecules that can be used for specific, high signal delivery to cell surface molecules. However, their long circulation time in blood makes them unsuitable as imaging probes. Efforts to improve antibodies pharmacokinetics without compromising affinity and specificity have been made through protein engineering. Antibody variants that differ in antigen binding sites and size have been generated and evaluated as imaging probes to target tissues of interest. Fast clearing fragments such as single-chain Fv (scFv; 25 kDa) with one antigen binding site (monovalent) demonstrated low accumulation in tumors due the low exposure time to the target. Using scFv as building block to produce larger, bivalent fragments such as scFv dimers (diabodies, 50 kDa) and scFv-fusion proteins (80 kDa minibodies and 105 kDa scFv-Fc) resulted in higher tumor accumulation due to their longer residence time in blood. Imaging studies with these fragments following radiolabeling have demonstrated excellent, high contrast images in gamma cameras and PET scanners. Several studies have also investigated antibody fragments conjugated to fluorescence (near infrared dyes), bioluminescence (luciferases) and quantum dots for optical imaging and iron oxides nanoparticles for MRI. However, these studies indicate that there are several factors that influence successful targeting and imaging. These include stability of the antibody fragment, the labeling chemistry (direct or indirect), whether critical residues are modified, the number of antigen expressed on the cell, and whether the target has a rapid recycling rate or internalizes upon binding. The preclinical data presented are compelling and it is evident that antibody-based molecular imaging tracers will play an

  14. Data Product Maturity

    Atmospheric Science Data Center

    2013-03-25

    ... document, maturity levels are provided separately for each scientific data set (SDS) included with the data files. The data product ... indiscriminate use of these data products as the basis for research findings, journal publications, and/or presentations.   ...

  15. Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection.

    PubMed

    Sheng, Zizhang; Schramm, Chaim A; Connors, Mark; Morris, Lynn; Mascola, John R; Kwong, Peter D; Shapiro, Lawrence

    2016-05-01

    Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages. PMID:27191167

  16. Effects of Darwinian Selection and Mutability on Rate of Broadly Neutralizing Antibody Evolution during HIV-1 Infection

    PubMed Central

    Sheng, Zizhang; Schramm, Chaim A.; Connors, Mark; Morris, Lynn; Mascola, John R.; Kwong, Peter D.; Shapiro, Lawrence

    2016-01-01

    Accumulation of somatic mutations in antibody variable regions is critical for antibody affinity maturation, with HIV-1 broadly neutralizing antibodies (bnAbs) generally requiring years to develop. We recently found that the rate at which mutations accumulate decreases over time, but the mechanism governing this slowing is unclear. In this study, we investigated whether natural selection and/or mutability of the antibody variable region contributed significantly to observed decrease in rate. We used longitudinally sampled sequences of immunoglobulin transcripts of single lineages from each of 3 donors, as determined by next generation sequencing. We estimated the evolutionary rates of the complementarity determining regions (CDRs), which are most significant for functional selection, and found they evolved about 1.5- to 2- fold faster than the framework regions. We also analyzed the presence of AID hotspots and coldspots at different points in lineage development and observed an average decrease in mutability of less than 10 percent over time. Altogether, the correlation between Darwinian selection strength and evolutionary rate trended toward significance, especially for CDRs, but cannot fully explain the observed changes in evolutionary rate. The mutability modulated by AID hotspots and coldspots changes correlated only weakly with evolutionary rates. The combined effects of Darwinian selection and mutability contribute substantially to, but do not fully explain, evolutionary rate change for HIV-1-targeting bnAb lineages. PMID:27191167

  17. Functional effects of anticardiolipin antibodies.

    PubMed

    Harris, E N; Pierangeli, S S

    1996-10-01

    The 'lupus anticoagulant' phenomenon is the best documented functional effect of antiphospholipid (aPL) antibodies, occurring either by inhibition of the prothrombinase and/or Factor X activation reactions. Understanding the mechanism by which aPL antibodies inhibit phospholipid dependent coagulation reactions may yield important clues about their 'thrombogenic effects' in vivo. We conducted a series of studies to determine the specificity, diversity, and mechanism by which aPL antibodies inhibit phospholipid dependent reactions. Results showed that purified immunoglobulins with lupus anticoagulant and anti-cardiolipin activities were absorbed by negatively charged phospholipids and both activities were recovered from the phospholipid-antibody precipitate. Purified aPL antibodies inhibited the prothrombinase reaction in a plasma free system in which beta 2-glycoprotein 1 (beta 2-GP1) was absent. Affinity purified aPL antibodies had 25-50 times the inhibitory activity of immunoglobulin preparations. The phospholipid binding proteins, beta 2-GPI and placental anticoagulant protein I (PAP I), independently inhibited the prothrombinase reaction, and when these proteins were combined with aPL, inhibition of the prothrombinase reaction was additive. Antibodies of syphilis had no inhibitory effect, partially accounted for by lack of specificity for phosphotidylserine (PS). Although aPL antibodies inhibited the protein C activation reaction, there was no correlation of these activities with inhibition of the prothrombinase reaction. Together, these results show that aPL exert their effects by interaction with negatively charged phospholipids, in particular phosphotidylserine, but lack of correlation between inhibition of the prothrombinase and protein C activation reactions, suggests that the nature of the coagulation protein is also important. PMID:8902763

  18. Generalized platform for antibody detection using the antibody catalyzed water oxidation pathway.

    PubMed

    Welch, M Elizabeth; Ritzert, Nicole L; Chen, Hongjun; Smith, Norah L; Tague, Michele E; Xu, Youyong; Baird, Barbara A; Abruña, Héctor D; Ober, Christopher K

    2014-02-01

    Infectious diseases, such as influenza, present a prominent global problem including the constant threat of pandemics that initiate in avian or other species and then pass to humans. We report a new sensor that can be specifically functionalized to detect antibodies associated with a wide range of infectious diseases in multiple species. This biosensor is based on electrochemical detection of hydrogen peroxide generated through the intrinsic catalytic activity of all antibodies: the antibody catalyzed water oxidation pathway (ACWOP). Our platform includes a polymer brush-modified surface where specific antibodies bind to conjugated haptens with high affinity and specificity. Hydrogen peroxide provides an electrochemical signal that is mediated by Resorufin/Amplex Red. We characterize the biosensor platform, using model anti-DNP antibodies, with the ultimate goal of designing a versatile device that is inexpensive, portable, reliable, and fast. We demonstrate detection of antibodies at concentrations that fall well within clinically relevant levels. PMID:24410628

  19. Novel trends in affinity biosensors: current challenges and perspectives

    NASA Astrophysics Data System (ADS)

    Arugula, Mary A.; Simonian, Aleksandr

    2014-03-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  20. Bispecific antibodies.

    PubMed

    Kontermann, Roland E; Brinkmann, Ulrich

    2015-07-01

    Bispecific antibodies (bsAbs) combine specificities of two antibodies and simultaneously address different antigens or epitopes. BsAbs with 'two-target' functionality can interfere with multiple surface receptors or ligands associated, for example with cancer, proliferation or inflammatory processes. BsAbs can also place targets into close proximity, either to support protein complex formation on one cell, or to trigger contacts between cells. Examples of 'forced-connection' functionalities are bsAbs that support protein complexation in the clotting cascade, or tumor-targeted immune cell recruiters and/or activators. Following years of research and development (R&D), the first bsAb was approved in 2009. Another bsAb entered the market in December 2014 and several more are in clinical trials. Here, we describe the potentials of bsAbs to become the next wave of antibody-based therapies, focusing on molecules in clinical development. PMID:25728220

  1. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  2. Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy

    PubMed Central

    Cheng, Ming; Ahmed, Mahiuddin; Xu, Hong; Cheung, Nai-Kong V.

    2015-01-01

    Antibody based immunotherapy has proven efficacy for patients with high risk neuroblastoma. However, despite being the most efficient tumoricidal effectors, T cells are underutilized because they lack Fc receptors. Using a monovalent single chain fragment (ScFv) platform, we engineered tandem scFv bispecific antibodies (BsAbs) that specifically target disialoganglioside (GD2) on tumor cells and CD3 on T cells. Structural variants of BsAbs were constructed and ranked based on binding to GD2, and on competency in inducing T cell mediated tumor cytotoxicity. In vitro thermal stability and binding measurements were used to characterize each of the constructs, and in silico molecular modeling was used to show how the orientation of the variable region heavy (VH) and light (VL) chains of the anti-GD2 ScFv could alter the conformations of key residues responsible for high affinity binding. We showed that the VH-VL orientation, the (GGGGS)3 linker, disulfide bond stabilization of scFv, when combined with an affinity matured mutation provided the most efficient BsAb to direct T cells to lyse GD2 positive tumor cells. In vivo, the optimized BsAb could efficiently inhibit melanoma and neuroblastoma xenograft growth. These findings provide preclinical validation of a structure-based method to assist in designing BsAb for T-cell mediated therapy. PMID:24895182

  3. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    PubMed Central

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  4. Development of humanized antibodies as cancer therapeutics.

    PubMed

    Qu, Zhengxing; Griffiths, Gary L; Wegener, William A; Chang, Chien-Hsing; Govindan, Serengulam V; Horak, Ivan D; Hansen, Hans J; Goldenberg, David M

    2005-05-01

    Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described. PMID:15848077

  5. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  6. Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics.

    PubMed

    Garnett, Graham A E; Starke, Melissa J; Shaurya, Alok; Li, Janessa; Hof, Fraser

    2016-04-01

    Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods. PMID:26973166

  7. Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

    PubMed Central

    Davey, Gayle M.; Schober, Sonya L.; Endrizzi, Bart T.; Dutcher, Angela K.; Jameson, Stephen C.; Hogquist, Kristin A.

    1998-01-01

    During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes. PMID:9815264

  8. The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies

    PubMed Central

    Vigneault, Francois; Julien, Jean-Philippe; Briney, Bryan; Ramos, Alejandra; Saye, Karen F.; Le, Khoa; Mahan, Alison; Wang, Shenshen; Kardar, Mehran; Yaari, Gur; Walker, Laura M.; Simen, Birgitte B.; St. John, Elizabeth P.; Chan-Hui, Po-Ying; Swiderek, Kristine; Kleinstein, Stephen H.; Alter, Galit; Seaman, Michael S.; Chakraborty, Arup K.; Koller, Daphne; Wilson, Ian A.; Church, George M.; Burton, Dennis R.; Poignard, Pascal

    2013-01-01

    Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121–134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121–134 but were still capable of neutralizing roughly 40–80% of PGT121–134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121–134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121–134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design. PMID:24278016

  9. Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation.

    PubMed

    Sakaguchi, N; Maeda, K

    2016-01-01

    Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells. PMID:27235683

  10. Jealousy and Moral Maturity.

    ERIC Educational Resources Information Center

    Mathes, Eugene W.; Deuger, Donna J.

    Jealousy may be perceived as either good or bad depending upon the moral maturity of the individual. To investigate this conclusion, a study was conducted testing two hypothesis: a positive relationship exists between conventional moral reasoning (reference to norms and laws) and the endorsement and level of jealousy; and a negative relationship…

  11. Development of high-affinity single chain Fv against foot-and-mouth disease virus.

    PubMed

    Jung, Joon-Goo; Jeong, Gu Min; Yim, Sung Sun; Jeong, Ki Jun

    2016-03-01

    Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV. PMID:26827774

  12. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Sondek, John

    2004-09-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, and for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:18429272

  13. Overview of affinity tags for protein purification.

    PubMed

    Kimple, Michelle E; Brill, Allison L; Pasker, Renee L

    2013-01-01

    Addition of an affinity tag is a useful method for differentiating recombinant proteins expressed in bacterial and eukaryotic expression systems from the background of total cellular proteins, as well as for detecting protein-protein interactions. This overview describes the historical basis for the development of affinity tags, affinity tags that are commonly used today, how to choose an appropriate affinity tag for a particular purpose, and several recently developed affinity tag technologies that may prove useful in the near future. PMID:24510596

  14. Antithyroid microsomal antibody

    MedlinePlus

    ... Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb Images Blood test References Guber HA, Faraq AF. Evaluation of endocrine function. In: McPherson RA, Pincus MR, eds. Henry's Clinical ...

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

    SciTech Connect

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

    2008-03-07

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

  16. Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

    NASA Astrophysics Data System (ADS)

    Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

    2008-05-01

    Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

  17. The Cellular Bases of Antibody Responses during Dengue Virus Infection

    PubMed Central

    Yam-Puc, Juan Carlos; Cedillo-Barrón, Leticia; Aguilar-Medina, Elsa Maribel; Ramos-Payán, Rosalío; Escobar-Gutiérrez, Alejandro; Flores-Romo, Leopoldo

    2016-01-01

    Dengue virus (DENV) is one of the most significant human viral pathogens transmitted by mosquitoes and can cause from an asymptomatic disease to mild undifferentiated fever, classical dengue, and severe dengue. Neutralizing memory antibody (Ab) responses are one of the most important mechanisms that counteract reinfections and are therefore the main aim of vaccination. However, it has also been proposed that in dengue, some of these class-switched (IgG) memory Abs might worsen the disease. Although these memory Abs derive from B cells by T-cell-dependent processes, we know rather little about the (acute, chronic, or memory) B cell responses and the complex cellular mechanisms generating these Abs during DENV infections. This review aims to provide an updated and comprehensive perspective of the B cell responses during DENV infection, starting since the very early events such as the cutaneous DENV entrance and the arrival into draining lymph nodes, to the putative B cell activation, proliferation, and germinal centers (GCs) formation (the source of affinity-matured class-switched memory Abs), till the outcome of GC reactions such as the generation of plasmablasts, Ab-secreting plasma cells, and memory B cells. We discuss topics very poorly explored such as the possibility of B cell infection by DENV or even activation-induced B cell death. The current information about the nature of the Ab responses to DENV is also illustrated. PMID:27375618

  18. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  19. Quantifying Affinity among Chinese Dialects.

    ERIC Educational Resources Information Center

    Cheng, Chin-Chuan

    A study of the relationships between Chinese dialects based on a quantitative measure of dialect affinity is summarized. First, tone values in all the dialect localities available in the early 1970s were used to calculate the dialectal differences in terms of tone height with respect to the "yin and yang" split. In the late 1970s, calculations of…

  20. Immune and endocrine responses of adult spring Chinook salmon during freshwater migration and sexual maturation

    USGS Publications Warehouse

    Maule, A.G.; Schrock, R.M.; Slater, C.; Fitzpatrick, M.S.; Schreck, C. B.

    1996-01-01

    The immune –endocrine responses in spring chinook salmon (Oncorhynchus tshawytscha) were examined during their freshwater migration and final maturation. In 1990, migrating fish had high plasma cortisol titres (means 200 ng ml−1) and generated relatively few antibody-producing cells (APC) from peripheral blood leukocytes (PBL) (100 –200 per culture). After three weeks acclimation in constant environmental conditions, plasma cortisol was reduced and APC increased. There were no changes in number or affinity of glucocorticoid receptors. Concentrations of several sex steroids correlated with APC in females, but there were no such correlations in males. In 1993, fish in a hatchery had significantly greater cortisol concentrations in primary circulation than in secondary circulation, but sex steroid concentrations did not differ between circulations. Mean lysozyme activity in the primary and secondary circulation did not differ in June. In August, activity in the primary circulation was significantly less than that of the secondary, perhaps the result of acute stress associated with sampling. While some sex steroids correlated with lysozyme activity, the fact that in both years all endocrine and immune variables that correlated with each other also correlated with the date of sample, raises the question as to whether or not these are cause-and-effect relations.

  1. New haptens and antibodies for ractopamine.

    PubMed

    Wang, Zhanhui; Liu, Meixuan; Shi, Weimin; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong

    2015-09-15

    In this work, three unreported immunizing haptens of ractopamine (RAC) were synthesized and used to produce highly sensitive and specific polyclonal antibody. The spacer arms of haptens for coupling to protein carrier were located on different position of RAC with different length. High affinity polyclonal antibodies were obtained and characterized in terms of titer and sensitivity by using enzyme-linked immunosorbent assay (ELISA). The best antibody employed in a heterologous competitive ELISA exhibited an IC50 value as low as 0.12ngmL(-1) and could not recognize other 10 β-agonists including clenbuterol and salbutamol. The heterologous competitive ELISA was preliminary applied to swine urine and the results showed the new antibody was sufficiently sensitive and specific, and potentially used for the detection of RAC at trace level in real samples. PMID:25863617

  2. Minute Time Scale Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope*

    PubMed Central

    Fassolari, Marisol; Chemes, Lucia B.; Gallo, Mariana; Smal, Clara; Sánchez, Ignacio E.; de Prat-Gay, Gonzalo

    2013-01-01

    Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a “hinge” region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t½ ∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 107 m−1 s−1, a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with KD = 1.2 × 10−7 m is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be “locked” in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation. PMID:23504368

  3. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems. PMID:21117653

  4. Construction of a Single Chain Variable Fragment Antibody (scFv) against Carbaryl and Its Interaction with Carbaryl.

    PubMed

    Xiuyuan, Zhang; Zhihong, Huang; Lixia, Wang; Xiaonan, Liu

    2015-05-01

    Carbaryl is a low molecular weight insecticide that inhibits cholinesterase. Residues of carbaryl in food and the environment have damaged human health. A high-specificity scFv that can identify carbaryl is still lacking. In the present study, an anti-carbaryl scFv gene was prepared by cloning VL and VH genes from hybridoma cells secreting monoclonal antibody, then VH and VL were fused together using splicing by overlap extension (SOE) PCR with a flexible polypeptide linker connector (Gly4Ser)3, and then the scFv-pET-26b recombinant plasmid was constructed and transformed into E. coli BL21 for expression using IPTG as an inducer. The expressed recombinant protein was identified by SDS-PAGE and ELISA. The three-dimensional structure of the anti-carbaryl scFv was constructed by computer modeling, and carbaryl was docked to the scFv model to obtain the structure of the binding complex. The binding site was composed of Ala51, Ser52, Ile51, Gly54, Ser56, Arg98, and Gly100. This helps to understand the mechanism of interaction between anti-carbaryl antibody and antigen. Furthermore, it provides guidance for in vitro affinity maturation of anti-carbaryl antibody. PMID:26071785

  5. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    PubMed Central

    Ullman, Christopher; Mathonet, Pascale; Oleksy, Arkadiusz; Diamandakis, Agata; Tomei, Licia; Demartis, Anna; Nardi, Chiara; Sambucini, Sonia; Missineo, Antonino; Alt, Karen; Hagemeyer, Christoph E.; Harris, Matt; Hedt, Amos; Weis, Roland; Gehlsen, Kurt R.

    2015-01-01

    Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model. PMID:26313909

  6. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future. PMID:26032605

  7. Antibody Engineering and Therapeutics

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  9. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

    PubMed

    Yonamine, Yusuke; Hoshino, Yu; Shea, Kenneth J

    2012-09-10

    Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules. PMID:22813352

  10. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  11. A Socioanalytic Model of Maturity

    ERIC Educational Resources Information Center

    Hogan, Robert; Roberts, Brent W.

    2004-01-01

    K0 describes a point of view on maturity that departs from earlier treatments in two ways. First, it rejects the popular assumption from humanistic psychology that maturity is a function of self-actualization and stipulates that maturity is related to certain performance capacities--namely, the ability to form lasting relationships and to achieve…

  12. Cannabinoids induce incomplete maturation of cultured human leukemia cells

    SciTech Connect

    Murison, G.; Chubb, C.B.H.; Maeda, S.; Gemmell, M.A.; Huberman, E.

    1987-08-01

    Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 ..mu..M ..delta../sup 9/-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody of the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 ..mu..M THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype. However, treatment of these incompletely matured cells with either phorbol 12-myristate 13-acetate of 1..cap alpha..,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced incomplete cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

  13. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells.

    PubMed

    Yu, Changming; Pike, Gennett M; Rinkoski, Tommy A; Correia, Cristina; Kaufmann, Scott H; Federspiel, Mark J

    2015-08-11

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates. PMID:26216971

  14. Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

    PubMed Central

    Yu, Changming; Pike, Gennett M.; Rinkoski, Tommy A.; Correia, Cristina; Kaufmann, Scott H.; Federspiel, Mark J.

    2015-01-01

    Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates. PMID:26216971

  15. Linker peptide and affinity tag for detection and purification of single-chain Fv fragments.

    PubMed

    Küttner, Gabriele; Giessmann, Elke; Wessner, Helga; Scholz, Christa; Reinhardt, Dina; Winkler, Karsten; Marx, Uwe; Höhne, Wolfgang

    2004-05-01

    The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. PMID:15152607

  16. 26-10 Fab-digoxin complex: affinity and specificity due to surface complementarity.

    PubMed Central

    Jeffrey, P D; Strong, R K; Sieker, L C; Chang, C Y; Campbell, R L; Petsko, G A; Haber, E; Margolies, M N; Sheriff, S

    1993-01-01

    We have determined the three-dimensional structures of the antigen-binding fragment of the anti-digoxin monoclonal antibody 26-10 in the uncomplexed state at 2.7 A resolution and as a complex with digoxin at 2.5 A resolution. Neither the antibody nor digoxin undergoes any significant conformational changes upon forming the complex. Digoxin interacts primarily with the antibody heavy chain and is oriented such that the carbohydrate groups are exposed to solvent and the lactone ring is buried in a deep pocket at the bottom of the combining site. Despite extensive interactions between antibody and antigen, no hydrogen bonds or salt links are formed between 26-10 and digoxin. Thus the 26-10-digoxin complex is unique among the known three-dimensional structures of antibody-antigen complexes in that specificity and high affinity arise primarily from shape complementarity. Images Fig. 1 PMID:8234291

  17. Monoclonal antibodies against tyrosyl-tRNA synthetase and its isolated cytokine-like domain.

    PubMed

    Kondratiuk, Iuliia; Khoruzenko, Antonina; Cherednyk, Olga; Filonenko, Valeriy; Kornelyuk, Aleksander

    2013-06-01

    Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization. PMID:23750478

  18. Lectin affinity chromatography of glycolipids

    SciTech Connect

    Torres, B.V.; Smith, D.F.

    1987-05-01

    Since glycolipids (GLs) are either insoluble or form mixed micelles in water, lectin affinity chromatography in aqueous systems has not been applied to their separation. They have overcome this problem by using tetrahydrofuran (THF) in the mobile phase during chromatography. Affinity columns prepared with the GalNAc-specific Helix pomatia agglutinin (HPA) and equilibrated in THF specifically bind the (/sup 3/H)oligosaccharide derived from Forssman GL indicating that the immobilized HPA retained its carbohydrate-binding specificity in this solvent. Intact Forssman GL was bound by the HPA-column equilibrated in THF and was specifically eluted with 0.1 mg/ml GalNAc in THF. Purification of the Forssman GL was achieved when a crude lipid extract of sheep erythrocyte membranes was applied to the HPA-column in THF. Non-specifically bound GLs were eluted from the column using a step gradient of aqueous buffer in THF, while the addition of GalNAc was required to elute the specifically bound GLs. Using this procedure the A-active GLs were purified from a crude lipid extract of type A human erythrocytes in a single chromatographic step. The use of solvents that maintain carbohydrate-binding specificity and lipid solubility will permit the application of affinity chromatography on immobilized carbohydrate-binding proteins to intact GLs.

  19. Monoclonal antibodies to human glycophorin a and cell lines for the production thereof

    SciTech Connect

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

    1988-06-21

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

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

    DOEpatents

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

    1988-01-01

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

  1. miRNA Tagging and Affinity-purification (miRAP)

    PubMed Central

    He, Miao

    2016-01-01

    MicroRNAs(miRNAs) are a group of endogenously expressed 20~23 nt small noncoding RNAs, which can directly regulate mRNA stability or translation in a sequence specific manner by incomplete base pairing at the 3′UTR of target mRNA, or indirectly affect transcriptional network by regulating transcription factors. As key regulators of gene expression, miRNAs are involved in the control of diverse developmental and physiological processes, including embryogenesis, differentiation, developmental timing, organogenesis, growth control, and programmed cell death. Aberrant miRNA expression profiles have been observed in many pathological conditions, including cancers, psychiatric diseases, virus infection, etc. However, the underlying mechanisms have been difficult to study in part due to the cellular heterogeneity of complex tissue. To systematically analyze miRNA expression in complex tissue, we present here a novel miRNA tagging and Affinity Purification method, miRAP, which can be applied to genetically defined cell types in any complex tissues in mice. This method is based on the fact that mature miRNAs are incorporated into RNA-induced silencing complex (RISC), in which the Argonaute protein AGO2 directly binds miRNAs and their mRNA targets. We demonstrate that epitope tagging of AGO2 protein allows direct purification of miRNAs from tissue homogenates using antibodies against the engineered molecular tag. We further established a Cre-loxP binary expression system to deliver epitope-tagged AGO2 (tAGO2) to genetically defined cell types.

  2. Development of monoclonal antibodies to pre-haptoglobin 2 and their use in an enzyme-linked immunosorbent assay (ELISA).

    PubMed

    Flanagan, J J; Arjomandi, A; Delanoy, M L; Du Paty, E; Galea, P; Laune, D; Rieunier, F; Walker, R P; Binder, S R

    2014-04-01

    Haptoglobins (HPs) are alpha 2-globulin proteins that bind free hemoglobin in plasma to prevent oxidative damage. HPs are produced as preproteins that are proteolytically cleaved in the ER into alpha and beta chains prior to forming mature, functional tetramers. Two alleles exist in humans (HP1 and HP2), therefore three genotypes are present in the population, i.e., HP1-1, HP2-1, and HP2-2. A biochemical role for nascent haptoglobin 2 (pre-haptoglobin 2 or pre-HP2) as the only known modulator of intestinal permeability has been established. In addition, elevated levels of serum pre-HP2 have been detected in multiple conditions including celiac disease and type I diabetes, which are believed to result in part through dysregulation of the intestinal barrier. In this study, we report the development of a monoclonal antibody that is specific for pre-HP2 with a binding affinity in the nanomolar range. Additional antibodies with specificities for preHP but not mature haptoglobin were also characterized. A sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated. The ELISA showed high specificity for pre-HP2 even in the presence of excess pre-HP1 or mature haptoglobins, and has excellent linearity and inter- and intra-assay reproducibility with a working range from 3.1ng/mL to 200ng/mL. Testing of sera from 76 healthy patients revealed a non-Gaussian distribution of pre-HP2 levels with a mean concentration of 221.2ng/mL (95% CI: 106.5-335.9ng/mL) and a median value of 23.9ng/mL. Compared to current approaches, this ELISA offers a validated, monoclonal-based method with high sensitivity and specificity for measuring pre-HP2 in human serum. PMID:24583194

  3. Vector-Mediated In Vivo Antibody Expression.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  4. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  5. Monoclonal antibodies.

    PubMed

    2009-01-01

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

  6. [Development of antibody medicines by bio-venture: lesson from license negotiations with mega pharmacies].

    PubMed

    Takada, Kenzo

    2013-01-01

    The current method of antibody production is mainly the hybridoma method, in which mice are immunized with an excess amount of antigen for a short period to promote activation and proliferation of B-lymphocytes producing the antibodies of interest. Because of the excess antigen, those producing low-affinity antibodies are activated. In contrast, human blood B-lymphocytes are activated through natural immune reactions, such as the reaction to infection. B-lymphocytes are stimulated repeatedly with a small amount of antigen, and thus only those producing high-affinity antibodies are activated. Consequently, the lymphocytes producing the high-affinity antibodies are accumulated in human blood. Therefore, human lymphocytes are an excellent source of high-affinity antibodies. Evec, Inc. has established a unique method to produce high-affinity antibodies from human lymphocytes using Epstein-Barr virus (EBV), which induces the proliferation of B-lymphocytes. The method first induces the proliferation of B-lymphocytes from human blood using EBV, and then isolates those producing the antibodies of interest. The key features of the Evec technique are: 1) development of a lymphocyte library consisting of 150 donors' lymphocytes from which donors suited to develop the antibodies of interest can be selected in 4 days; and 2) development of a sorting method and cell microarray method for selecting lymphocyte clones producing the target antibodies. Licensing agreements have been concluded with European and Japanese pharmaceutical companies for two types of antibody. This paper describes Evec's antibody technology and experience in license negotiations with Mega Pharmacies. PMID:23292021

  7. Immobilized metal ion affinity chromatography.

    PubMed

    Yip, T T; Hutchens, T W

    1992-01-01

    Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme

  8. Serum herpes simplex antibodies

    MedlinePlus

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

  9. Higher cytotoxicity of divalent antibody-toxins than monovalent antibody-toxins

    SciTech Connect

    Won, JaeSeon; Nam, PilWon; Lee, YongChan; Choe, MuHyeon

    2009-04-24

    Recombinant antibody-toxins are constructed via the fusion of a 'carcinoma-specific' antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G{sub 4}S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]{sub 2}) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.

  10. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  11. Antibody engineering for increased potency, breadth and half-life

    PubMed Central

    Sievers, Stuart A.; Scharf, Louise; West, Anthony P.; Bjorkman, Pamela J.

    2015-01-01

    Purpose of review This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies. Recent findings Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems. Summary Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed. PMID:25760931

  12. Profiling antibody responses by multiparametric analysis of primary B cells.

    PubMed

    Story, Craig M; Papa, Eliseo; Hu, Chih-Chi Andrew; Ronan, Jehnna L; Herlihy, Kara; Ploegh, Hidde L; Love, J Christopher

    2008-11-18

    Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (approximately 10(3)-10(4)). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases. PMID:19004776

  13. XGef Mediates Early CPEB Phosphorylation during Xenopus Oocyte Meiotic Maturation

    PubMed Central

    Martínez, Susana E.; Yuan, Lei; Lacza, Charlemagne; Ransom, Heather; Mahon, Gwendolyn M.; Whitehead, Ian P.; Hake, Laura E.

    2005-01-01

    Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process. PMID:15635100

  14. Molecular Evolution of Antibody Cross-Reactivity for Two Subtypes of Type a Botulinum Neurotoxin

    SciTech Connect

    Garcia-Rodriguez, C.; Levy, R.; Arndt, J.W.; Forsyth, C.M.; Razai, A.; Lou, J.; Geren, I.; Stevens, R.C.; Marks, J.D.; /UC, San Francisco /Scripps Res. Inst.

    2007-07-09

    Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

  15. Monoclonal antibody purification with hydroxyapatite.

    PubMed

    Gagnon, Pete

    2009-06-01

    Hydroxyapatite (HA) has been used for IgG purification since its introduction in the 1950s. Applications expanded to include IgA and IgM in the 1980s, along with elucidation of its primary binding mechanisms and the development of ceramic HA media. With the advent of recombinant monoclonal antibodies, HA was demonstrated to be effective for removal of antibody aggregates, as well as host cell proteins and leached protein A. HA's inherent abilities have been enhanced by the development of elution strategies that permit differential control of its primary binding mechanisms: calcium metal affinity and phosphoryl cation exchange. These strategies support reduction of antibody aggregate content from greater than 60% to less than 0.1%, in conjunction with enhanced removal of DNA, endotoxin, and virus. HA also has a history of discriminating various immunological constructs on the basis of differences in their variable regions, or discriminating Fab fragments from Fc contaminants in papain digests of purified monoclonal IgG. Continuing development of novel elution strategies, alternative forms of HA, and application of robotic high throughput screening systems promise to expand HA's utility in the field. PMID:19491046

  16. Single-Chain Antibody Library

    DOE Data Explorer

    Baird, Cheryl

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

  17. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  18. Indian craniometric variability and affinities.

    PubMed

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with "Caucasoid" populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  19. Indian Craniometric Variability and Affinities

    PubMed Central

    Raghavan, Pathmanathan; Bulbeck, David; Pathmanathan, Gayathiri; Rathee, Suresh Kanta

    2013-01-01

    Recently published craniometric and genetic studies indicate a predominantly indigenous ancestry of Indian populations. We address this issue with a fuller coverage of Indian craniometrics than any done before. We analyse metrical variability within Indian series, Indians' sexual dimorphism, differences between northern and southern Indians, index-based differences of Indian males from other series, and Indians' multivariate affinities. The relationship between a variable's magnitude and its variability is log-linear. This relationship is strengthened by excluding cranial fractions and series with a sample size less than 30. Male crania are typically larger than female crania, but there are also shape differences. Northern Indians differ from southern Indians in various features including narrower orbits and less pronounced medial protrusion of the orbits. Indians resemble Veddas in having small crania and similar cranial shape. Indians' wider geographic affinities lie with “Caucasoid” populations to the northwest, particularly affecting northern Indians. The latter finding is confirmed from shape-based Mahalanobis-D distances calculated for the best sampled male and female series. Demonstration of a distinctive South Asian craniometric profile and the intermediate status of northern Indians between southern Indians and populations northwest of India confirm the predominantly indigenous ancestry of northern and especially southern Indians. PMID:24455409

  20. Synthetic Antibodies for Reversible Cell Recognition

    NASA Astrophysics Data System (ADS)

    Zhou, Jing Zhou

    2011-12-01

    Antibody-mediated cell recognition plays a critical role in various biological and biomedical applications. However, strong antibody-cell interactions can lead to the difficulty of separating antibodies from the bound cells in a simple and non-destructive manner, which is often necessary to numerous applications such as cell sorting or separation. Thus, this thesis research is aimed to create an antibody-like nanomaterial with the function of reversible cell recognition It was hypothesized that nucleic acid aptamer and dendrimer could be used as fundamental structural components to develop an antibody-like nanomaterial. The aptamer functions as the binding site of an antibody; the dendrimer is used as a robust, defined nano-scaffold to support the aptamer and to carry small molecules (e.g., fluorophores). To test this hypothesis, a novel method was first developed to discover the essential nucleotides of full-length aptamers to mimic the binding sites of antibodies. The essential nucleotides were further conjugated with a dendrimer to synthesize a monovalent aptamer-dendrimer nanomaterial. The results clearly showed that the essential nucleotides could maintain high affinity and specificity after tethered on dendrimer surface. To further test the hypothesis that antibody-like nanomaterials can be rationally designed to acquire the capability of reversible cell recognition, an aptamer that was selected at 0 °C was used as a model to synthesize a "Y-shaped" nanomaterial by conjugating two aptamers to the same dendrimer. The results showed that the nanomaterial-cell interaction could be affected by the distance between two binding aptamers. In addition, the "Y-shaped" antibody-like nanomaterial could bind target cells more strongly than its monovalent control. Importantly, the strong cell-nanomaterial interaction could be rapidly reversed when the temperature was shifted from 0 °C to 37 °C. In summary, we developed a synthetic antibody that can not only mimic the

  1. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries

    PubMed Central

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-01-01

    Broadly neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information. PMID:26456860

  2. Construction of a reshaped HMFG1 antibody and comparison of its fine specificity with that of the parent mouse antibody.

    PubMed Central

    Verhoeyen, M E; Saunders, J A; Price, M R; Marugg, J D; Briggs, S; Broderick, E L; Eida, S J; Mooren, A T; Badley, R A

    1993-01-01

    A human antibody with milk mucin specificity was obtained by transferring the complementarity determining regions (CDR) of the mouse antibody HMFG1 onto carefully selected human framework regions. The resulting reshaped human antibody, HuHMFG1, showed no difference in relative affinity for its antigen compared with the parent mouse HMFG1. The minimum epitope recognized by both the mouse and reshaped antibodies was demonstrated by epitope mapping to be identical, and consists of the tetramer PDTR. In a replacement net analysis, in which each of the amino acids was replaced in turn with the 19 other residues, it was determined that mouse HMFG1 and HuHMFG1 reacted with this series of synthetic peptides in an equivalent manner, indicating retention of identical fine specificity in the HuHMFG1 antibody. In contrast to other published reports, this was achieved without involvement of any framework residues in the binding site transfer. These data demonstrate that if well-matching human framework regions are employed grafting the CDR only can be sufficient to confer desired specificities to human antibodies and can, indeed, provide human analogues of mouse antibodies with virtually indistinguishable affinities and fine specificities relative to the mouse parent antibodies. PMID:7682986

  3. MALDI immunoscreening (MiSCREEN): a method for selection of anti-peptide monoclonal antibodies for use in immunoproteomics.

    PubMed

    Razavi, Morteza; Pope, Matthew E; Soste, Martin V; Eyford, Brett A; Jackson, Angela M; Anderson, N Leigh; Pearson, Terry W

    2011-02-01

    A scalable method for screening and selection of peptide-specific monoclonal antibodies (mAbs) is described. To identify high affinity anti-peptide mAbs in hybridoma supernatants, antibodies were captured by magnetic affinity beads followed by binding of specific peptides from solution. After timed washing steps, the remaining bound peptides were eluted from the beads and detected by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). This allowed measurement of monovalent interactions of peptides with single antigen binding sites on the antibodies, thus reflecting antibody affinity rather than avidity. Antibodies that were able to bind target peptides from solution phase and retain them during washing for a minimum of 10 min were identified by the strength of the appropriate m/z peptide MS signals obtained. This wash time reflects the minimum peptide dissociation time required for use of these antibodies in several current immuno-mass spectrometry assays. Kinetic analysis of antibody-peptide binding by surface plasmon resonance (SPR) showed that the selected antibodies were of high affinity and, most importantly, had low dissociation constants. This method, called MALDI immunoscreening (MiSCREEN), thus enables rapid screening and selection of high affinity anti-peptide antibodies that are useful for a variety of immunoproteomics applications. To demonstrate their functional utility in immuno-mass spectrometry assays, we used the selected, purified RabMAbs to enrich natural (tryptic) peptides from digested human plasma. PMID:21078325

  4. Rational design of a low-affinity peptide for the detection of cystatin C in a fast homogeneous immunoassay.

    PubMed

    Dobslaff, Kristin; Zscharnack, Kristin; Kreisig, Thomas; Zuchner, Thole

    2016-02-01

    Immunoassays play an essential role in current research and diagnostics resulting in a variety of detection principles. Thereby, homogeneous assays are often used for a fast signal response as demanded for example in point-of-care diagnostics. These systems often rely on a competitive assay design where the sample analyte and the corresponding dye-labeled substance are competing for binding sites on an antibody present in limited amounts. Due to the similar affinities of the antibody towards the sample analyte and the competitor, both sensitivity and assay time are limited. As a consequence, a competitor with a slightly reduced affinity towards the antibody can potentially overcome these drawbacks. Here, we present the rational design of a low-affinity peptide (donor peptide) as a specific analyte competitor for a FRET-based homogeneous immunoassay for the analysis of the protein cystatin C. Thereby, the strategy of peptide-induced antibody generation was combined with the selective variation of the immunization sequence in order to achieve a lower affinity towards the antibody. We could show that shortened donor peptides improved the resulting quenching efficiency in the immunoassay. In addition, the substitution of small hydrophobic amino acids by those with a higher steric demand appeared to be the most promising strategy providing a fast assay response for cystatin C of only 90 s. PMID:26403846

  5. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  6. Antidrug Antibodies: B Cell Immunity Against Therapy.

    PubMed

    Fogdell-Hahn, A

    2015-09-01

    Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review. PMID:26098690

  7. Development and Evaluation of Single Domain Antibodies for Vaccinia and the L1 Antigen

    PubMed Central

    Walper, Scott A.; Liu, Jinny L.; Zabetakis, Daniel; Anderson, George P.; Goldman, Ellen R.

    2014-01-01

    There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies. PMID:25211488

  8. Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor.

    PubMed

    Kamio, M; Uchiyama, T; Arima, N; Itoh, K; Ishikawa, T; Hori, T; Uchino, H

    1990-01-01

    Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta

  9. Selection of antibodies from synthetic antibody libraries.

    PubMed

    Harel Inbar, Noa; Benhar, Itai

    2012-10-15

    More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science. PMID:22244834

  10. A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

    PubMed Central

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

    2013-01-01

    1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization. PMID:24278299

  11. Affine hypersurfaces with parallel difference tensor relative to affine α-connection

    NASA Astrophysics Data System (ADS)

    Li, Cece

    2014-12-01

    Li and Zhang (2014) studied affine hypersurfaces of R n + 1 with parallel difference tensor relative to the affine α-connection ∇ (α), and characterized the generalized Cayley hypersurfaces by K n - 1 ≠ 0 and ∇ (α) K = 0 for some nonzero constant α, where the affine α-connection ∇ (α) of information geometry was introduced on affine hypersurface. In this paper, by a slightly different method we continue to study affine hypersurfaces with ∇ (α) K = 0, if α = 0 we further assume that the Pick invariant vanishes and affine metric is of constant sectional curvature. It is proved that they are either hyperquadrics or improper affine hypersphere with flat indefinite affine metric, the latter can be locally given as a graph of a polynomial of at most degree n + 1 with constant Hessian determinant. In particular, if the affine metric is definite, Lorentzian, or its negative index is 2, we complete the classification of such hypersurfaces.

  12. Surface-modified magnetic colloids for affinity adsorption of immunoglobulins

    NASA Astrophysics Data System (ADS)

    Martins, Fernanda; Pinho, Samantha C.; Zollner, Terezinha C. A.; Zollner, Ricardo L.; de Cuyper, Marcel; Santana, Maria Helena A.

    This work describes the preparation, characterization and in vitro adsorption tests of surface-modified magnetoliposomes for affinity binding of (i) anticardiolipin (isotype G) antibodies and (ii) specific isotype E antibodies generated by hypersensitivity reactions in humans with respiratory allergy. In the first case, cardiolipin embedded in the bilayer of magnetoliposomes was used as specific ligand. In the second case, antigenic proteins present in an extract of Dermatophagoids pteronyssinus and Blomia tropicalis mites were covalently coupled on the surface of magnetoliposomes via a diglycolic spacer arm, and used as specific ligands for IgE. Antibody adsorption was performed in a high-gradient magnetophoresis system, using either sera of healthy individuals or a pool of sera from autoimmune or allergic patients. The selectivity and capacity of the system were quantified by a frontal analysis in a capillary column, and by constructing breakthrough curves. The results show that the highest yield and selectivity were obtained if the ligand was extended into the aqueous layer surrounding the magnetoliposome surface. A 100% selectivity was obtained for adsorption of specific IgE, and 8% for IgG. These results demonstrate the potentialities of both types of surface-modified magnetic biocolloids in the field of in vitro diagnosis tests for allergic or autoimmune conditions.

  13. "Diabodies": small bivalent and bispecific antibody fragments.

    PubMed Central

    Holliger, P; Prospero, T; Winter, G

    1993-01-01

    Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria. Images Fig. 1 Fig. 3 PMID:8341653

  14. The maximal affinity of ligands

    PubMed Central

    Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A.

    1999-01-01

    We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin. PMID:10468550

  15. The Mature Athlete

    PubMed Central

    McCarthy, Moira M.; Hannafin, Jo A.

    2014-01-01

    Context: Aging changes the biology, healing capacity, and biomechanical function of tendons and ligaments and results in common clinical pathologies that present to orthopedic surgeons, primary care physicians, physical therapists, and athletic trainers. A better understanding of the age-related changes in these connective tissues will allow better patient care. Evidence Acquisition: The PubMed database was searched in December 2012 for English-language articles pertaining to age-related changes in tendons and ligaments. Level of Evidence: Level 5. Results: The mature athlete faces challenges associated with age-dependent changes in the rotator cuff, Achilles tendon, lateral humeral epicondylar tendons, quadriceps tendon, and patellar tendon. The anterior cruciate ligament and the medial collateral ligament are the most studied intra-articular and extra-articular ligaments, and both are associated with age-dependent changes. Conclusion: Tendons and ligaments are highly arranged connective tissue structures that maintain joint motion and joint stability. These structures are subject to vascular and compositional changes with increasing age that alter their mechanotransduction, biology, healing capacity, and biomechanical function. Emerging research into the etiology of age-dependent changes will provide further information to help combat the age-related clinical complications associated with the injuries that occur to tendons and ligaments. PMID:24427441

  16. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  17.  De novo isolation of antibodies with pH-dependent binding properties.

    PubMed

    Bonvin, Pauline; Venet, Sophie; Fontaine, Gaëlle; Ravn, Ulla; Gueneau, Franck; Kosco-Vilbois, Marie; Proudfoot, Amanda Ei; Fischer, Nicolas

    2015-01-01

    pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies. PMID:25608219

  18. Indium-111 labeled anti-melanoma monoclonal antibodies

    DOEpatents

    Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

    1984-04-30

    A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

  19. Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining

    PubMed Central

    Wang, Bo; Kluwe, Christien A.; Lungu, Oana I.; DeKosky, Brandon J.; Kerr, Scott A.; Johnson, Erik L.; Jung, Jiwon; Rezigh, Alec B.; Carroll, Sean M.; Reyes, Ann N.; Bentz, Janelle R.; Villanueva, Itamar; Altman, Amy L.; Davey, Robert A.; Ellington, Andrew D.; Georgiou, George

    2015-01-01

    The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138+ antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported. PMID:26355042

  20. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  1. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  2. Compact noncontraction semigroups of affine operators

    NASA Astrophysics Data System (ADS)

    Voynov, A. S.; Protasov, V. Yu

    2015-07-01

    We analyze compact multiplicative semigroups of affine operators acting in a finite-dimensional space. The main result states that every such semigroup is either contracting, that is, contains elements of arbitrarily small operator norm, or all its operators share a common invariant affine subspace on which this semigroup is contracting. The proof uses functional difference equations with contraction of the argument. We look at applications to self-affine partitions of convex sets, the investigation of finite affine semigroups and the proof of a criterion of primitivity for nonnegative matrix families. Bibliography: 32 titles.

  3. Antibodies to myeloid precursor cells in autoimmune neutropenia.

    PubMed

    Hartman, K R; LaRussa, V F; Rothwell, S W; Atolagbe, T O; Ward, F T; Klipple, G

    1994-07-15

    Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further stud