环境激素邻苯二甲酸二丁酯免疫检测Ⅱ——抗体的制备

Immunological Assaying of Environmental Hormone Dibutyl Phthalate Ⅱ——Preparation of Antibody

邻苯二甲酸二丁酯(DBP)半抗原衍生物通过偶氮键与载体蛋白BSA偶合后作为免疫原,免疫新西兰大白兔,得到合格的DBP抗血清,双向琼脂扩散方法测定血清效价为1∶32～1∶64,间接荧光免疫法检测效价为1∶6 400～1∶12 800。通过兔颈部采血、抗血清纯化,得到的抗邻苯二甲酸二丁酯多克隆抗体IgG冻干后,分装在-20℃低温保存。建立了间接荧光免疫法检测纯化抗体的方法。间接竞争荧光免疫法考察交叉反应试验结果显示,纯化抗邻苯二甲酸二丁酯抗体与邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丙酯、邻苯二甲酸二环己酯、邻苯二甲酸二戊酯、邻苯二甲酸二(2-乙基己基)酯等邻苯二甲酸酯类环境激素的交叉反应均小于10%。制备的抗体可应用于环境激素DBP的荧光免疫分析。

The haptene of dibutyl phthalate （DBP） was conjugated to bovine serum by azo coupling. The conjugate was used as an immunogen. High titer polyclonal antibodies against dibutyl phthalate were produced from New Zealand white rabbits immunized with the immunogen. The final sera had titers against DBP with dilutions of 1：32 -1：64 with double immunodiffusion, and 1：6 400 -1：12 800 with indirect competitive fluorescence immunoassay. After an acceptable antibody titer was observed, the animals were exsanguinated and antiserum was purified. The purified IgG fractions were lyophilized aliquoted into vials, and stored at -20 ℃. An indirect fluorescence immunoassay to detect antibody was developed. The analysis experiment of anti-DBP by indirect competitive fluorescence immunoassay showed that anti-DBP antibody was cross-reacted with other similar phthalate compounds, such as dimethyl phthalate （DMP）, diethyl phthalate （DEP） dipropyl phthalate （ DPrP）, dicyclohexyl phthalate （ DCHP）, di-n-pentyl phthalate （DPP） and diethylhexyl phthalate（ DEHP）, and the cross reactivities were less than 10%. Therefore, this anti-DBP antibody could be interesting for developing the fluorescence immunoassay of DBP.