Determination of azadirachtin in agricultural matrixes and commercial formulations by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza m olecule. Rabbits were immunized with either bovine serum albumin (BSA)-azad irachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antise ra by antibody capture assay showed that the antibody titer of antisera rai sed against OA-aza was 1:30 000. An indirect competitive ELISA was develope d with BSA-azadirachtin as coating antigen and aza-specific antibodies rais ed against OA-aza immunogen. The immunoassay showed an inhibitory concentra tion (IC50) value of 75 ppb, with a range of detection from 0.5 to 1000 ppb for azadirachtin [based on regression analysis, y = 85.87 (-18.89x); r(2) = -0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23 -dihydro-23 beta -methoxy azadirachtin and 3-tigloylazadirachtol) was 33 an d 29%, respectively. The indirect competitive ELISA was validated and evalu ated by quantitating aza in spiked agricultural commodities and from neem f ormulations. Azadirachtin was spiked into 5 different agricultural commodit ies: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1000 ppb and recovered at 62-100%. In samples drawn from 6 lots, the aza content in neem -seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2000 ppm. The method developed may be applied t o environmental monitoring of aza and quality assurance studies of aza-base d commercial formulations.