Primary Structure and Functional Expression of Heavy- and Light-Chain Variable Region Genes of a Monoclonal Antibody Specific for Human Fibrin

Abstract

The immunoglobulin heavy- and light-chain variable region (VH and VK) genes were isolated from 8E5 hybridoma cells, which secreted monoclonal antibody against human fibrin by RT—PCR. An expression vector pOPE51—8E5 was constructed for the recombinant VH—VK scFv expression. The primary sequence of the variable regions was determined. Expression product was found in the periplasmic space and inclusion bodies by SDS—PAGE and immunoblotting. It was a 30 KDa single chain fragment (scFv) with the antigen-binding specificity of the parental monoclonal antibody. A light chain shuffling with an unspecific VL did not result in a loss of fibrin binding specificity.