A novel strategy for generation of monoclonal antibodies from single B cells using rt-PCR technique and in vitro expression.
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.
Jiang
XiuPing
X
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Japan.
Suzuki
Hirotatsu
H
Hanai
Yuko
Y
Wada
Fumitaka
F
Hitomi
Kiyotaka
K
Yamane
Tsuneo
T
Nakano
Hideo
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D001483:Base Sequence; D002469:Cell Separation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains