雷洛昔芬抗体的制备及ELISA方法的初步建立

Preparation of Raloxifene Antibody and Preliminary Establishment of Enzyme-Linked Immunosorbent Assay

本文主要研究了雷洛昔芬抗体的制备和酶联免疫吸附测定(enzyme-linked immuno sorbent assay,ELISA)方法的初步建立。实验中采用丁二酸酐对雷洛昔芬进行衍生,合成了雷洛昔芬半抗原。采用碳二亚胺法,将雷洛昔芬半抗原与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原和包被抗原,并通过紫外光谱进行鉴定,结果显示雷洛昔芬与载体蛋白偶联成功。免疫大白兔制备了雷洛昔芬抗体,抗体效价达1.28×10~5,半数抑制浓度(half inhibit concentration,IC50)15.4μg/L,与其他类似抗雌激素药物没有交叉反应,说明所制备的抗体特异性好。通过优化抗原抗体反应浓度初步建立了雷洛昔芬ELISA方法,结果显示最佳抗原浓度为300μg/L,最佳抗体工作浓度为1:1.0×10~5,标准曲线在0.4~102.4μg/L范围内线性关系好,R^2=0.9853,最低检测能力0.4μg/L。本研究为进一步开发雷洛昔芬快速检测试剂盒提供了技术参考。

The preparation of raloxifene antibody and the establishment of enzyme-linked immuno sorbent assay（ELISA） were mainly studied in this paper. Succinic anhydride was used for the raloxifene derivative and the raloxifene haptens were formed. The immunogen and inclusion antigen were synthesized by coupling the raloxifene hapten with bovine serum albumin（BSA） and ovalbumin（OVA） using the carbodiimide method, and then determined by UV spectroscopy. Results showed that raloxifene was successfully coupled with the carrier protein. Raloxifene-bovine serum albumin（Ra-BSA） was used to immune New Zealand white rabbits, and the antibodies showed a titer of 1.28×10~5 and a IC50 of 15.4 μg/L. There was no cross reaction with other anti-estrogens, indicating that the antibody specificity was good. The raloxifene ELISA method was initially established by optimizing the reaction concentration of antigen antibody, and the optimal conditions were as follows： antigen concentration, 300 μg/L, antibody concentration, 1：1.0×10~5. The standard curve was good（R^2=0.9853） in the range of 0.4~102.4 g/L with a lowest detection limit, 0.4 μg/L. This research provided a technical reference for further development of the rapid test kit for raloxifene.