Development and characterization of enzyme-linked immunosorbent assay for aflatoxin B1 measurement in urine sample using penicillinase as label

Abstract

A simple, sensitive, rapid and specific enzyme linked immunosorbent assay (ELISA) for quantitative measurement of aflatoxin B(1) (AFB(1)) in urine samples was developed in this study. Polyclonal antibodies were raised against a C(1)-carboxymethyl oxime (CMO) derivative of AFB(1) conjugated bovine serum albumin (BSA). AFB(1)-C(8)-penicillinase (AFB(1)-C(8)-P) and AFB(1)-C(1)-carboxymethyl oxime-penicillinase (AFB(1)-CMO-P) were prepared and used as tracer molecule. A heterologous combination of antibody and enzyme conjugates (AFB(1)-C(1)-CMO-BSA and AFB(1)-C(8)-P) proved to work better with respect to specificity and sensitivity. Ig purified antibody (4 microg/well) was coated onto the pre-coated (BSA) wells of microtiter plate. The assay procedure was completed within 3 hr and the sensitivity was calculated to be from 200 pg/ml. The standard curve was linear up to 10 ng/ml so was able to detect high concentration of AFB(1) in sample. Affinities were calculated for homologous and heterologous system in which the heterologous system showed better affinities (1.9 x 10(8) M(-1)). The antibody prepared in this study showed minimal cross-reaction with structurally related molecules being affected by homology and heterology of the assay system that is the site of conjugation of carrier protein for antibody production using the hapten BSA conjugate and the site of enzyme conjugated on the hapten molecule used as tracer as well as direct and indirect coating of antibody on the surface of microtiter plat. The results reported here indicated that the heterologous combination of antibody and enzyme conjugate performs better in assay qualities in general. More than 90% recovery of AFB(1) added to stripped urine samples were observed in this type of assay. Inter and intra-assay percent of coefficient of variations for ten successive assays were found to be 10.2 and 6.9% respectively. Logit -log transformation of standard curve and sample dilution with urine sample containing no AFB(1) in a serial manner exhibited parallel line with the slope of -1.03 and -1.03 respectively. A correlation of 0.90 was found between the ELISA reported in this study and radioimmunoassay (RIA) of AFB(1) in urine samples.