Abstract:

In order to exploit the polycyclic aromatic hydrocarbons (PAHs) ELISA test kit for rapidly detecting PAHs in different environmental samples, the polyclonal antibody (pAb) against pyrene was prepared. The hapten (1\|Pyrenebutyric acid, 1\|PBA) was conjugated covalently to the carrier proteins including bovine serum albumin (BSA) and ovalbumin (OVA)to prepare immunogen and coating antigen via amide bonds by the active ester method. Antiserum was raised against the conjugate PBA\|BSA by stimulating immune responses in rabbits. The specific pAb against PAHs was obtained by affinity purification and developing an indirectly competitive enzyme\|linked immunosorbent assay (ic\|ELISA). A standard curve for pyrene was established to detect the specificity of antibody. The titer of compound antiserum was 1∶102 400. The average IC50 value was 0.06 mg/L, and the detection limit at 10% inhibition (IC90) was 0.01 mg/L. The pAb had the highest affinity with 4~6 ring PAHs by determining cross\|reactivity of 16 PAHs. Further, the additive of less than 40% methanol in the test sample has no significant effect on ELISA. The PAHs polyclonal antibody and its characterization are necessary for technical reserves in future development and improvement of the PAHs ELISA test kit.

Key words: PAHs；pyrene；polyclonal antibody； ELISA

摘要：

为实现环境中多环芳烃（PAHs）污染物的快速检测，制备了抗多环芳烃多克隆抗体，用于多环芳烃免疫学检测试剂盒的研制。采用活性酯法将半抗原芘丁酸（1\|PBA）分别于牛血清白蛋白（BSA）和卵清白蛋白（OVA）进行偶联，获得PBA\|BSA和PBA\|OVA偶联物，以PBA\|BSA偶联物免疫新西兰大白兔，获得针对多环芳烃的抗血清。以PBA\|OVA偶联物为包被原，间接ELISA法检测抗血清，效价为102 400，经纯化后得到多克隆抗体。采用间接竞争ELISA法绘制了针对芘的标准曲线，得到该方法的IC50值为0.06 mg/L,检出限为001 mg/L。与16种多环芳烃的交叉反应实验结果表明该抗体对高环PAHs的亲和力较高。反应体系中添加低于40%的甲醇对ELISA结果无影响。该抗体的制备及特性鉴定对后续多环芳烃酶联免疫试剂盒的开发奠定了技术基础。

关键词: 多环芳烃；芘；多克隆抗体；酶联免疫吸附分析法