Fresenius J Anal Chem (1995) 352:43-48 Fresenius' Journal of © Springer-Verlag 1995 Limitations in the use of horseradish peroxidase as an enyzme probe in the development of a homogeneous immunoassay for aflatoxin B1 D. Ramana l, A. K. Capoor 1, R. B. Sashidhar 1, Ramesh V. Bhat 2 1 Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, India 2Food and Drug Toxicology Research Centre, National Institute of Nutrition, Hyderabad-500 007, India Received: 25 July 1994/Accepted: 24 November 1994 Abstract. Horseradish peroxidase (HRPO) was used as a probe to quantitate aflatoxin B 1 by a homogeneous im- munoassay. The conjugation of AFB~ 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 AFB~. The homo- geneous assay carried out with free AFB~ and HRPO- AFBI 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 AFB~ conjugation were 6-8. The 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 mole- cule 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 dilu- tion for the detection of AFB1 by ELISA indicating that antibodies per se were not the limiting factor. Introduction Among the fungal toxins, aflatoxins produced by the As- pergillusflavus and Aspergillus parasiticus group of fungi have received world wide attention, due to their deleteri- ous effects on human and animal health and their impor- tance in international trade. The toxicological properties of aflatoxins include carcinogenic, mutagenic, teratogenic and immunosuppressive effects [1-4]. Earlier, various an- alytical methods have been developed for screening and detection of aflatoxins in food, feed and biological fluids. These methods include physico-chemical methods such as Correspondence to. R. B. Sashidhar TLC, HPLC, GLC and HPTLC and immunoanalytical methods like RIA and ELISA for AFB~ [5-9]. In the recent past, methods based on ELISA have also been collaboratively evaluated for aflatoxin (AFB~) analy- sis [10]. So far, all the enzyme immunoassays developed for AFB1 quantitation have been based on solid-phase im- munoassay systems. However, no enzyme-mediated im- munoassay for AFB~ based on a separation-free system has been reported. The objective of the present communi- cation was to develop a homogeneous (separation-free) immunoassay for the detection of AFB~ based on an en- zyme-modulator mediated assay using AFB1 specific poly- clonal antibodies, with horseradish peroxidase (HRPO) as the enzyme probe. Materials and methods Reagents Aflatoxin B~ and carboxymethoxylamine hemichloride were obtained from Aldrich Chemical Co., USA. Bovine serum albumin (fatty acid free), poly-D-lysine, horseradish peroxidase type VI (EC 1.11.1.7.), tributylamine, isobutyl chloroformate, thimerosal, 1-ethyl-3-(3-dimethyl-amino- propyl) carbodiimide hydrochloride, o-phenylene diamine and anti-rabbit IgG (raised in goat) conjugated to alkaline phosphatase were purchased from Sigma Chemical Co., USA. N-hydroxy succinimide and dicyclohexyl carbodi- imide were obtained from Spectrochem, India. Freund's complete and incomplete adjuvants were obtained from Difco Laboratories, USA. Polystyrene microtiter ELISA plates were procured from Nunc., Denmark. All other chemicals and buffer salts were of analytical grade. Preparation of antigen." BSA-AFB~ conjugate Aflatoxin B1 was activated by the addition of a carboxyl group to the molecule by the methods of Langone and Van Vunakis [11]. Briefly, 5 mg (16 ~tmol) of AFB~ and 7.6