孔雀石绿抗原的合成及多克隆抗体的制备

Synthesis of Artificial Antigen and Preparation of Polyclonal Antibody for Malachite Green

通过研究孔雀石绿(MG)抗原的合成和多克隆抗体的制备,探索研究食品中孔雀石绿残留的检测方法。本实验先合成含有羧基的孔雀石绿半抗原(CMG),并用液-质联用法进行分析鉴定。半抗原经重氮化法处理后分别偶联两种载体蛋白BSA和OVA,并通过紫外分光光度法进行鉴定。与CMG标准品和两种载体蛋白的紫外吸收光谱相比,偶联物的吸收峰发生了明显的偏移,计算得免疫原的偶联比为10.6:1,说明偶联成功。通过免疫制备多克隆抗体,并通过间接ELISA法对CMG抗血清的效价进行检测,并进一步检测抗体的特异性,两只兔子的IC50值分别为8.1ng/mL和4.6ng/mL。

In order to determine malachite green（MG） residues in food,the synthesis of antigen and preparation of polyclonal antibody for MG were explored.In this study,carboxylic malachite green（CMG） hapten was first synthesized and analyzed by LC-MS.The hapten was coupled separately with the two carrier proteins BSA and OVA by diazotization method,and identified by UV-visible spectroscopy method.Compared with CMG standard,BSA and OVA,the absorption peaks CMG-BSA and CMG-OVA displayed a marked shift,indicating that the successful conjugation between CMG and BSA or OVA,and the coupling ratio of the immunogen CMG-BSA was calculated to be 10.6：1.Polyclonal antibody for MG was prepared by immunization with CMG-BSA and evaluated using the titer of anti-CMG antisera determined by indirect ELISA.Furthermore,two rabbits were used to evaluate antibody specificity with determined IC50 values of 8.1 ng/mL and 4.6 ng/mL,respectively.