抗氯吡脲单克隆抗体的制备及其间接竞争ELISA方法的建立

Preparation of Anti-CPPU Monoclonal Antibody and Development of an Indirect Competitive ELISA Method

在获得高特异性抗CPPU单克隆抗体的基础上,采用活性酯法将CPPU-H1与BSA、CPPU-H2与OVA偶联制备得到免疫抗原CPPU-H1-BSA和包被抗原CPPU-H2-OVA;用CPPU-H1-BSA对小鼠进行免疫,通过杂交瘤技术筛选获得了1株抗CPPU单克隆抗体的杂交瘤细胞株B7B6 24。经过体内诱生腹水法制备抗体,测定抗体亚类为Ig G1,效价为1∶32 000。通过对试验条件的优化,建立了一种检测CPPU的方法 ,该方法 IC50值为3.89 ng/m L;该抗体与四螨嗪、赤霉素、噻苯隆几乎不存在交叉反应;利用所建立的间接竞争ELISA法对葡萄样品进行检测,平均加标回收率为91.4%-112.4%,变异系数为5.9%-14.4%。本研究所建立的间接竞争ELISA方法具有低成本、高准确性和高灵敏度的特点,可以用来对葡萄中的CPPU残留情况进行检测。

A method to rapidly detect forchlorfenuron（CPPU） was developed by Indirect competitive ELISA on the basis of obtaining the Anti-CPPU Monoclonal Antibody with high specificity. CPPU-H1-BSA（immunogenic antigen） and CPPU-H2-OVA（coating antigen） were prepared by coupling with CPPU-H1 and CPPU-H2 with carrier proteins using active ester method,respectively. Through a hybridoma cell line,a strain of cells B7B6 24 which can produce anti-CPPU on monoclonal antibody was obtained. Ascites was produced by mice vivo culcure,the monoclonal antibody characterized by Ig G1 isotype,and the ELISA titer of ascites was 1∶32 000. A indirect competitive ELISA was developed for the detection of CPPU by optimizing experimental conditions. The IC50 value was 3.89 ng / m L,it did not react with clofentezine, gibberellic acid,thidiazuron. The average recovery rate for CPPU from grape samples were ranged from 91.4% to 112.4% and the variation coefficient were between 5.9% -14.4%. The indirect competitive ELISA allows for accurate,sensitive,and low-cost for determination of CPPU residues in grape.