Cloning and characterization of cDNAs encoding V-H and V-L of a monoclonal anti-CEA antibody (CEA 79) cross-reactive with NCA-95 and generation of a single-chain Fv molecule (scFv)

Abstract

We cloned complementary DNA (cDNA) encoding the variable regions of heavy chain (V-H) and of light chain (V-L,) of a monoclonal anti-carcinoembryonic antigen (CEA) antibody cross-reactive with nonspecific cross-reacting antigen-95 (NCA-95), which had been previously prepared and designated as CEA 79 (gamma(2a),,, kappa). From these cDNAs, a phagemid expression vector for the CEA79 single chain variable fragment (scFv) was generated. Enzyme-linked immunosorbent assay (ELISA), competitive ELISAs, and Western blotting confirmed that the scFv displayed on the surface of the bacteriophage had retained affinity for CEA and NCA-95. We then determined the nucleotide sequences of the cloned cDNAs for V-H, and V-L,. The sequence analysis revealed that V-H, and V-L, of the CEA 79 antibody represent new members of the mouse heavy chain subgroup "miscellaneous" and the kappa light chain subgroup "V", respectively.