呋喃唑酮代谢物单克隆抗体和胶体金免疫层析试纸条的研制

Preparation of Monoclonal Antibody and Colloidal Gold Immunochromatographic Test Strip against Furazolidone Metabolites

摘要:

本研究旨在建立一种快速检测呋喃唑酮代谢物残留的方法。试验用呋喃唑酮代谢物的衍生物（CPAOZ）与牛血清白蛋白（BSA）的偶联物免疫小鼠，利用单克隆抗体技术制备杂交瘤细胞，用间接ELISA和间接竞争ELISA法对阳性克隆进行筛选。单克隆细胞株诱生腹水后，经纯化即为单克隆抗体，用于胶体金标记，制备呋喃唑酮代谢物胶体金免疫层析试纸条。融合后得到2株稳定分泌抗体的杂交瘤细胞株，其中4G3纯化后的抗体效价达到1:100万，对CPAOZ的50%抑制质量浓度（IC50）为1.7 μg/L，亲和常数Ka=1.6×109 L/mol。该抗体制备的胶体金试纸条的检测限为4 μg/L，与其他3种硝基呋喃代谢物的衍生物CPAHD、CPSEM和CPAMOZ均不存在交叉反应，对样品的检测与高效液相色谱结果一致。本研究制备了抗呋喃唑酮代谢物特异性单克隆抗体，并研制了以单抗为基础的胶体金免疫层析试纸条，能够实现呋喃唑酮代谢物残留的快速、灵敏的检测。

Abstract:

The aim of the study was to establish a method for the rapid detection of the furazolidone metabolites. A conjugate of the derivatives of furazolidone metabolites and bovine serum albumin (BSA) was used to immunize mice. The cells were prepared by monoclonal antibody technology, and the positive clones were screened by indirect ELISA and icELISA. Monoclonal antibodies purified from ascitic fluid were used in the colloidal gold tag and for the preparation of the colloidal gold immunochromatographic test strip. Two cell types were obtained, which could stably secrete antibodies. The titer of the purified antibody named 4G3 was 1:106, and the median inhibitory concentration (IC50) was 1.7 μg/L with affinity constant Ka=1.6×109 L/mol. The results showed that the detection using the test strips was limited to four μg/L, there were no cross reactions between CPAHD, CPSEM, and CPAMOZ, and the test results of the samples were consistent with the results of HPLC. In this study, specific monoclonal antibodies against CPAOZ were prepared, and colloidal gold immunochromatographic test strips were developed based on the antibodies, which could achieve rapid and sensitive detection of furazolidone metabolite residues.