Synthesis of Bifunctional Antibodies for Immunoassays

The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fabʹ fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(abʹ)2 fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(abʹ)2 fragments thus produced did not contain any IgG impurities. Fabʹ fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.