D. Ramana et al., LIMITATIONS IN THE USE OF HORSERADISH-PEROXIDASE AS AN ENZYME PROBE IN THE DEVELOPMENT OF A HOMOGENEOUS IMMUNOASSAY FOR AFLATOXIN B-1, Fresenius' journal of analytical chemistry, 352(1-2), 1995, pp. 43-48
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
.