SMD残留检测的ELISA方法的建立和初步应用

The establishment and primary application of ELISA protocol for detecting SMD residue

用戊二醛法将SMD与载体蛋白BSA偶联 ,制备合成抗原SMD BSA作免疫原 ,同法合成包被抗原SMD OVA ,免疫健康家兔获得抗血清。分别用双向琼脂扩散试验和ELISA试验对抗血清进行定性定量测定 ,结果表明所获抗血清特异性针对SMD。用所制备的抗血清建立间接竞争ELISA方法。优化了ELISA的工作条件 ,方阵测定确定了包被抗原最佳浓度 (50 μg/mL) ,抗血清最佳稀释度 (1∶1 0 0 ) ,酶标抗体的最适工作稀释度 (1∶50 0 ) ,并建立了ELISA标准工作曲线。工作曲线表明在 1 0～ 2 0 0 0 μg/L浓度范围内呈良好的线性关系。该法检测底限为 63μg/L,低于国际规定残留限量 (1 0 0 μg/kg)和国内规定残留限量 (30 0 μg/kg)的要求。

Bovine serum albumin (BSA) and ovalbumin (OVA) were used as two protein carriers respectively to couple with semiantigen SMD by glutaraldehyde method in this paper. The complete angtigens SMD BSA and SMD OVA were then prepared and acted as immunoantigen and coating antigen respectively in ELISA protocol. The property of this antiserum was determined with two direction agar diffusion test and ELISA test, which showed that antiserum was specific to SMD. Indirect competitive ELISA (icELISA) was established with this antiserum. The most appropriate concentration of coating antigen was 50 μg/mL, and optimal dilution 1∶500 and 1∶100 correspondingly. The standard curve of icELISA was also established and the curve indicated that the lowest detection limit was 63 μg/L, which was under the demanded detection limit of 100 μg/kg (EU) and 300 μg/kg (domestic). The curve had a favorable linear relation within the concentration range of 10～2 000 μg/L. The recovery ratio was favorable to accord with anticipation. The content of SMD in serum of the test samples was obtained through the established ELISA protocol.