In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis.
Monoclonal antibodies (mAbs) are an effective tool in therapeutics and diagnostics. A novel approach called the single-cell RT-PCR-linked in vitro expression system (SICREX) enables the high-throughput generation and screening of mAbs from single B cells. In this paper, instead of using B cells, cDNAs were synthesized from single plasma cells of an immunized mouse spleen. The light chain (Lc) and the Fd portion of the heavy chain (Hc) genes of each cell were amplified separately and followed by overlapping PCR to add a T7 promoter, a ribosome-binding site, and a T7 terminator. The paired Lc and Hc genes were simultaneously expressed by an Escherichia coli in vitro transcription and translation system followed by ELISA to measure their affinity for the antigen. A Fab fragment with affinity against the antigen was obtained from plasma cells of an immunized mouse with hepatitis B surface antigen (HBsAg).
Nagoya University, Furo-cho, Chikusa-ku, Japan.
Yunita
Y
Sabrina
Muhamad
M
Ali
Hideo
H
Nakano
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000235:genetics*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010950:Plasma Cells / Q000276:immunology*; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007140:Immunoglobulin Fab Fragments