Comparison between inhibitory indirect ELISA and HPLC methods to quantify free and adducted aflatoxins in human urine.

Abstract

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis.