DIRECT PRODUCTION OF THE FAB FRAGMENT DERIVED FROM THE SPERM IMMOBILIZING ANTIBODY USING POLYMERASE CHAIN-REACTION AND CDNA EXPRESSION VECTORS

PROBLEM: Sperm immobilizing antibodies cause infertility mainly throug h complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already establish ed cell lines that secrete IgM monoclonal antibody (MAb H6-3C4) and Ig G monoclonal antibody (MAb EnBCMGS). The latter was a class-switched r ecombinant IgG antibody that shares the same variable region as MAb H6 -3C4. The biological effects of the IgG antibody were also reported pr eviously to eliminate sperm immobilizing or sperm agglutinating activi ties, However, the method of chemical digestion of IgG had some disadv antage to prepare the purified Fab fragment stably and in large quanti ties. This time we report a unique method to obtain the recombinant Fa b fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cD NA expression vectors. METHOD: Two kinds of PCR primers were designed to make a truncated heavy chain (Fd) gene of MAb EnBCMGS. The amplifie d Ed gene and light chain gene were ligated into cDNA expression vecto rs and then transfected into mammalian cells. RESULTS: Expression of t he Ed gene and light chain gene were confirmed by Northern blotting. S ecretion of the recombinant Fab fragment from mammalian cells was also confirmed by Western blotting. The Fab fragment showed biological act ivity as is expected by FAGS analysis. CONCLUSION: This method enables the stable production of genuine Fab fragments of IgG in mammalian ce lls without any chemical treatment that may be time consuming and affe ct the quality of the Fab fragments.