The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a coupling agent in the preparation of enzyme-antibody conjugates.

The present study was undertaken to develop a novel method for the enzyme labeling of antibodies. Goat anti-rabbit IgG was used as a prototype and coupled to beta-D-galactosidase (Gal) with a new heterobifunctional cross-linking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM). The antibody was first azo-coupled with DPEM to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were purified by DEAE-cellulose column chromatography, being completely separated from non-reacted antibodies but remaining mixed with free Gal, and showing approximately 30% enzyme activity bound to antibody. This method is simple, reproducible and so mild that almost full enzyme and antibody activity can be retained. The conjugates were used as a label in an immunoassay and were able to detect first antibody at concentrations as low as 2 ng/tube. Furthermore, the present method was compared with the method using N-(gamma-maleimidobutyryloxy)succinimide (GMBS), and it was found that both conjugates produced comparable results.