Study On Enzyme Immunoassay For Detection Of Medroxyprogesterone Acetate Residuals In Food

This thesis was to develop an ELISA kit to detect MPA. MPA was derived, and then coupled to bovine serum albumin (BSA) and ovalbumin (OVA) to synthesize immunogen MPA-BSA and coating antigen MPA-OVA respectively through mixed anhydride. According to the immune protocol, four New Zealand white rabbits were immunized for five times by MPA-BSA during four months, and it was found that one of the four antiserum titres reached 6400. Based on this antiserum, the immunoreagent conditions, such as dilution reagent, the pH and incubation time and so on, were optimized, and then a competitive indirect ELISA was developed.This ELISA method exhibited that the value of IC50 was 4.95ng/mL, and the range of linearity was 0-100ng/mL. Preliminary evaluation of assay was performed through following items: sensitivity, precision, accuracy and speciality. The LOD range was from 0.1 to 0.3ng/mL, and the cross-reactivity was 4.2% for megestrol acetate , while the other similarities were≤0.1%. The coefficients of variation of the standards and the samples were < 15%, and they were < 20% between different batches. Two different concentrations (1ng/g and 5ng/g) of the MPA were put into the samples to detect the recoveries. The samples of urine and milk should be centrifugated fist and then diluted before detection, while the muscle tissue should be extracted by the acetic ester fist and then saponificated. The result of the recoveries were between 40% and 110% ,and the coefficients of variation were <15%.Finally, this ELISA kit was compared with the one from the Euro-diagnostica company in Holland, and found that the recoveries and the coefficients of variation were nearly the same, and also found that the detection result was nearly to the GC-MS. So it could be shown that all of the parameters of the ELISA kit we developed had reached the level of foreign standards, and it could be applied into practice.