Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity
Essono, Sosthène S.
Montefiori, David C.
Polonis, Victoria R.
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CitationSholukh, Anton M., Muhammad M. Mukhtar, Michael Humbert, Sosthène S. Essono, Jennifer D. Watkins, Hemant K. Vyas, Vivekanandan Shanmuganathan, Girish Hemashettar, Maria Kahn, Shiu-Lok Hu, David C. Montefiori, Victoria R. Polonis, Peter H. Schur, and Ruth M. Ruprecht. 2012. Isolation of monoclonal antibodies with predetermined conformational epitope specificity. PLoS ONE 7(6): e38943.
AbstractExisting technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.
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