Limitations in the use of horseradish peroxidase as an enyzme probe in the development of a homogeneous immunoassay for aflatoxin B1 |
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| Authors: | D Ramana A K Capoor R B Sashidhar and Ramesh V Bhat |
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| Institution: | (1) Department of Biochemistry, University College of Science, Osmania University, 500 007 Hyderabad, India;(2) Food and Drug Toxicology Research Centre, National Institute of Nutrition, 500 007 Hyderabad, India |
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| Abstract: | Horseradish peroxidase (HRPO) was used as a probe to quantitate aflatoxin B1 by a homogeneous immunoassay. The conjugation of AFB1 to HRPO resulted in 54% loss of enyzme activity. In the presence of AFB1 specific antibodies, the HRPO-AFB1 conjugate showed reversal of its lost enzyme activity by 12%. This positive modulatory effect of antibody on the enzyme activity was used as an analytical tool to quantitate AFB1. The homogeneous assay carried out with free AFB1 and HRPO-AFB1 conjugate in the presence of antibodies indicated poor linearity as compared to the heterogeneous assay. It was observed that the number of HRPO-lysine residues involved in AFB1 conjugation were 6–8. The low level of modulation of enzyme activity by antibody with respect to HRPO-AFB1 conjugate, could possibly be attributed to the limited number of lysine residues in the HRPO molecule and its proximity to the active site of the enzyme. Thus, HRPO was found to be limiting as an enzyme with respect to the homogeneous enzyme immunoassay for AFB1 analysis. The antibodies raised were specific for AFB1, and showed excellent linearity even at high dilution for the detection of AFB1 by ELISA indicating that antibodies per se were not the limiting factor. |
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