| In this study, In order to remedy the defects of the rapid method for detecting Clenbuterol hydrochloride on the market, especially long operation cycle and high specialized specification basing on monoclonal antibody, antiserum containing antibody against CLB was prepared by immuning rabbits with artificial antigen BSA-CLB,and titer identification of antibody against CLB was performed by indirect ELISA(iELISA). Then the method of ELISA for detecting CLB was established. This study would lay a foundation for market inspection and quarantine.First, by orthogonal experiments, the optimization of conjugation conditions for preparing complete antigen based on diazotization was given, and the influence of every factors on coupling ratio was further discussed. Under the optimum conditions, high coupling ratio immunogen BSA-CLB and coatigen OVA-CLB was prepared and identified with UV scanning. Through calculation by improved method, the coupling ratio of immunogen and coatigen was 52.3:1 and 29:1 respectively.Immunization of rabbits with BSA-CLB is carried out by hypodermic injection. During the period, serum titer was identified by iELISA with coating OVA-CLB, and bloodletting time was determined according to the result. After four months, the serum titer was up to 1:640000.In the last part, the suitable working dilution of coatigen, antiserum, second antibody was established by iELISA. Then according to the standard curve and sensitivity, the pH, ionic strength and reaction time in detection system was optimized by using indirect compete ELISA(ciELISA). Finally, the method for detecting CLB by ciELISA with coating OVA-CLB was initially established with the sensitivity 0.013μg/mL. The imitation degree of standard curve in linear regression model was high, and the good stability and repeatability show this study has wide application prospect.
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