| In this study, Zeranol(ZER) was coupled to carrier protein bovine serumalbumin(BSA) and ovalbumin(OVA) using the mixed anhydride method. The ZER-BSA was used as immunogen and the ZER-OVA used as coating antigen. The coupling ratio of ZER to BSA was 10:1 and to OVA was 8:1. BALB/c mice were immuned by the immunogen and the titer of anti-serum was 1:8000 by two-dimensional titration method.Two hybridomas producing anti-ZER monoclonal antibodies were obtained by limited dilution, named 1C 12 and 9B4. The characteristics of monoclonal antibody was performed by EL1SA test, chromosomal counting and SDS-PAGE electrophoresis. The results indicated that the subclass of the McAb was IgGl, the titer of ascitic fluid was 1: 3.2×105, the molecular weight was 151.8KD .the chromosomal numbers were 95-102. The cross-reactivities of McAb to zeranol, β-zearalanol,α-zearalenol, β-zearalenol, zearalanone, zearalenone were 100%, 35.5%, 47.4%, 7.1%, 73.0%, 56.3% respectively, and to diethylstilbestrol, 17 β-estradiol, testosterone, clenbuterol were all below 0.1% . The indirect competitive Enzyme Linked Immunosorbent Assay(ELISA) for ZER was developed. The best concentration of coating antigen was 0.5ug/ml, the optional working dilution of the McAb and enzyme -conjugate were all 1:5000 , The linear detecting range of calibration curves to detect ZER was 0.5ng/ml~100ng/ml , The formular was y = -66.844x+424.15, R2 = 0.996, the 50% inhibition concentration(IC50) was 3.0ng/ml. The limit of detection was 0.6ng/ml.The enzyme immunoassay kit for the quantitative analysis of zeranol was developed with McAb against ZER. The lower detection limit of ZER test was 0.60ng/ml. According to the sample preparation protocol, the detection limit was 7.74ng/ml in bovine urine. When the spiked concentration of ZER were 10ng/ml, 21ng/ml and 35ng/ml, the recovery range was all from 70.0% to 116.0%, the coefficient range of variation was from 6.0% to 15.9%. The ELISA kit could be reserved for 6 months at 2~8℃.
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