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A Novel Strategy for Generation of Monoclonal Antibodies from Single B Cells Using RT‐PCR Technique and in Vitro Expression
Author(s) -
Jiang XiuPing,
Suzuki Hirotatsu,
Hanai Yuko,
Wada Fumitaka,
Hitomi Kiyotaka,
Yamane Tsuneo,
Nakano Hideo
Publication year - 2006
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060092h
Subject(s) - microbiology and biotechnology , monoclonal antibody , antigen , in vitro , b cell , antibody , immunoglobulin light chain , biology , cd19 , epitope , chemistry , biochemistry , flow cytometry , immunology
Monoclonal antibodies (Mabs) are important biomolecules in immunology and have widespread applications in prognosis, diagnosis, and therapeutics. Here, we describe a novel approach called single‐cell RT‐PCR‐linked in vitro expression (SICREX), which enables the high‐throughput generation and screening of Mabs. This approach entails the isolation of B cells from immunized mouse spleen or human peripheral blood using magnetic microbeads conjugated with a B‐cell‐selective marker, anti‐CD19. The light chain (Lc) and Fd portion of heavy‐chain (Hc) genes of each cell are separately amplified by RT‐PCR and then combined with the sequences of a T7 promoter, a ribosome binding site (rbs), and a T7 terminator by an overlapping PCR technique. The paired full‐length DNA fragments of Lc and Hc genes from single B cells are simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by an enzyme‐linked immunosorbent assay to find positive fragments possessing the affinity for the antigen. From spleen cells of an immunized mouse with calcium binding protein 40, a Fab fragment with K d of 1.6 (± 0.3) × 10 −8 against the antigen was obtained. From human peripheral blood, Fab fragments against a blood group B‐BSA were obtained in a similar manner. The SICREX approach is simple, rapid and versatile, allowing the high‐throughput generation of naturally paired Lc and Hc with antigen‐binding activity from various animal sources.