LIMITATIONS IN THE USE OF HORSERADISH-PEROXIDASE AS AN ENZYME PROBE IN THE DEVELOPMENT OF A HOMOGENEOUS IMMUNOASSAY FOR AFLATOXIN B-1

Horseradish peroxidase (HRPO) was used as a probe to quantitate aflato xin B-1 by a homogeneous im munoassay. The conjugation of AFB(1) to HR PO resulted in 54% loss of enyzme activity. In the presence of AFB(1) specific antibodies, the HRPO-AFB(1) conjugate showed reversal of its lost enzyme activity by 12%. This positive modulatory effect of antibo dy on the enzyme activity was used as an analytical tool to quantitate AFB(1). The homogeneous assay carried out with free AFB(1) and HRPO-A FB(1) conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. It was observed that the numb er of HRPO-lysine residues involved in AFB(1) conjugation were 6-8. Th e low level of modulation of enzyme activity by antibody with respect to HRPO-AFB(1) conjugate, could possibly be attributed to the limited number of lysine residues in the HRPO molecule and its proximity to th e active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme with respect to the homogeneous enzyme immunoassay for AFB(1) analysis. The antibodies raised were specific for AFB(1), and showed e xcellent linearity even at high dilution for the detection of AFB(1) b y ELISA indicating that antibodies per se were not the limiting factor .