Abstract
We introduced a simple method of cDNA cloning for variable regions of monoclonal antibodies which consists of amino acid micro-sequencing with the PVDF membrane (Immobilon-PTM) and reverse transcriptase-PCR followed by subcloning into plasmid and double stranded sequencing. The PVDF membrane and PCR made it possible to skip the procedures for extraction of protein from the electrophoretic gel or the blotting membrane and for screening a cDNA library. The entire process can be performed within 2 weeks and may be compatible with clinical laboratory practice. This procedure will be of use for the analysis of primary structure of idiotope, and for producing V-region peptides of functional MAbs and chimeric antibodies.
