Development Of Immunoassay Techniques For Zeranol And Milbemycin Oxime

Immunoassay is an important tool for analysis and detection of trace microbial metablites and its derivatives in complex matrix. According to current-research focus on veterinary residues analysis and natural products screening, we have developed immunological assays for zeranol (ZER) and milbemycin oxime (MILO). In this work, two haptens were designed for ZER and MILO, respectively. And then they were conjugated with the carrier protein to synthesize the immunogens. CI-ELISA were established with corresponding polyclonal antibodies, the results were described as below:1. To produce a novel type of immunogen susceptible to raise antibodies, zeranol was converted to zerenol-16-carboxy propyl ether and conjugated to bovine serum albumin (BSA) by the mixed-anhydried method. Three New Zealand white rabbits were immunized with ZER-BSA and the titers of antiserum had reached 1:102400. A ZER-OVA₁ -based CI-ELISA was conducted with the antiserum, the response range for zeranol was between 18.39—1744.70 ng/mL and the detection limit was 8.61ng/mL. Cross reactivity of the antibodies with zearalenone was 1.35%. In distilled water, the zeranol recovery is in the range of 90%¹15% and the coefficient of variation 1.75%⁷.02%.2. The 5-o-Succinoyhnilbemycin-oxime was obtained after the reaction of milbemycin withsuccinic anhydride in anhydrous pyridine, and then the hapten were bound to BSA by the carbodiimide activation. Two New Zealand white rabbits were immunized with MILO-BSA and the titers of antiserum had reached 1:12800. A MELO-OVA -based CI-ELISA was conducted with the antiserum, the linear range for milbemycin oxime was between 16.00⁴394.18 ng/mL and the limit of detection was 6.28ng/mL. The cross reactivities for avermectin B₁ and ivermectin B₁ were both less than 0.1%.The results showed that the antibodies for zeranol could be used for ZER residues analysis and MILO antibodies were potentially useful in construction of antibody models in high -throughput screening for MIL-producing strains.