Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.