论文部分内容阅读
利用免疫新西兰大白兔制备Cry1C毒素多克隆抗体,建立间接竞争时间分辨荧光免疫分析(CI-TRFIA)方法,用于稻米中Cry1C毒素的残留检测。通过4次免疫,采集Cry1C毒素多克隆抗体血清,并用饱和硫酸铵沉淀和Protein A柱纯化,测定抗体效价;以制备Eu-N1标记的羊抗兔IgG作为检测抗体,建立Cry1C毒素的CI-TRFIA检测方法,并进行抗原类似物的特异性分析和Cry1C毒素在稻米中的添加回收试验。结果表明:获得的多克隆抗体质量浓度为3.86 mg·mL-1,效价为1∶48 000;建立的CI-TRFIA检测方法,其灵敏度(IC10)为0.074 ng·mL-1,中抑制浓度(IC50)为60.16 ng·mL-1,线性检测范围(IC20~IC80)为0.96~1 633.60 ng·mL-1;对Cry1B、Cry1Ab和Cry1Ac均有一定的交叉反应;添加回收试验的批内、批间回收率为84.53%~107.12%,变异系数为6.05%~11.24%。结论:该检测方法稳定性和重复性较好,可以满足Cry1C毒素的检测要求。
Cry1C toxin polyclonal antibody was prepared by immunizing New Zealand white rabbits to establish Indirect Competitive Time-resolved Fluorescence Immunoassay (CI-TRFIA) for the detection of Cry1C toxin residues in rice. After four immunizations, the serum of Cry1C toxin polyclonal antibody was collected and purified by saturated ammonium sulfate precipitation and Protein A column to measure the antibody titer. Eu-N1 labeled goat anti-rabbit IgG was used as detection antibody to establish Cry1C toxin CI- TRFIA detection method, and specific analysis of antigen analogs and Cry1C toxin in rice recycling test. The results showed that the concentration of polyclonal antibody was 3.86 mg · mL-1 and the titer was 1:48 000. The established method of CI-TRFIA assay showed that the sensitivity (IC10) was 0.074 ng · mL-1, the inhibitory concentration (IC50) of 60.16 ng · mL-1, linearity detection range (IC20 ~ IC80) of 0.96 ~ 1633.60 ng · mL-1; some cross-reactions were observed on Cry1B, Cry1Ab and Cry1Ac; The recoveries between batches ranged from 84.53% to 107.12% with coefficients of variation from 6.05% to 11.24%. Conclusion: The detection method is stable and reproducible, which can meet the detection requirements of Cry1C toxin.