Add time:08/17/2019         Source:sciencedirect.com

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 β-D-galactosidase (Gal) with a new heterobifunctional cross-linking agent N-[β-(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-(γ-maleimidobutryloxy)succinimide (GMBS), and it was found that both conjugates produced comparable results.

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