Generation of monoclonal antibodies using simplified single-cell reverse transcription-polymerase chain reaction and cell-free protein synthesis.
The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from the cDNAs of a single cell was facilitated using a low concentration of cDNA-specific primers with 5' homotags in the presence of a homotag-specific primer. This method was found to be successful in generating a functional antibody with an antigen-binding activity and useful for the high-throughput generation or screening of monoclonal antibodies.
Ali
Muhamad
M
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan.
Hitomi
Kiyotaka
K
Nakano
Hideo
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry; D001709:Biotechnology / Q000379:methods*; D002474:Cell-Free System; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D000941:Antigens; D017931:DNA Primers; D018076:DNA, Complementary; D007074:Immunoglobulin G