A novel strategy for generation of monoclonal antibodies from single B cells using rt-PCR technique and in vitro expression.
Biotechnol Prog
; 22(4): 979-88, 2006.
Article
em En
| MEDLINE
| ID: mdl-16889373
ABSTRACT
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) x 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.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Linfócitos B
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Anticorpos Monoclonais
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article