Isolation of monoclonal antibodies with predetermined conformational epitope specificity |
| |
Authors: | Sholukh Anton M Mukhtar Muhammad M Humbert Michael Essono Sosthène S Watkins Jennifer D Vyas Hemant K Shanmuganathan Vivekanandan Hemashettar Girish Kahn Maria Hu Shiu-Lok Montefiori David C Polonis Victoria R Schur Peter H Ruprecht Ruth M |
| |
Affiliation: | Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America. |
| |
Abstract: | Existing 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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|