苯巴比妥人工免疫原合成及抗体特性研究

Study on Synthesis of Artificial Immunogen and Characteristics of Polyclonal Antibody of Phenobarbital

将苯巴比妥(PB)经硝化和还原两步化学修饰后,在其苯环上引入活性基团氨基,合成具有半抗原结构特征的对氨基苯巴比妥(pAPB);用重氮化法将pAPB偶联于载体蛋白BSA和OVA上,合成人工免疫原BSA-pAPB和包被抗原OVA-pAPB,用红外(IR)、紫外(UV)、SDS-PAGE进行鉴定,推算分子结合比;用BSA-pAPB免疫新西兰白兔和SPF雏鸡,间接ELISA测定多克隆抗体(pAb)效价,阻断ELISA鉴定其敏感性,WestGold层析试验和交叉反应试验鉴定其特异性。结果表明,BSA-pAPB偶联成功,分子结合比为1∶19;获得了高价、敏感、特异的pAb,为PB残留免疫学检测方法的建立奠定了基础。

The active group amido was introduced into phenyl of PB and formed p-amidophenobarbital （pAPB） which had hapten structure by chemical modification of nitration and deoxidization. Diazotization was used to conjugate pAPB to carrier protein BSA and obtained artificial immunogen BSA-pAPB, the coating antigen OVA-pAPB was obtained in the same way. IR, UV and SDS-PAGE were used to identify PB artificial immunogen. New Zealand white rabbits and SPF chickens were immunized with BSA-pAPB, the titre of polyclonal antibody （pAb） was measured by indirect ELISA, the sensitivity of the pAb was identified by blocking ELISA and the specificity of pAb was identified by experiments of WestGold immuno chromatography and cross-reaction. The results showed that PB artificial immunogen was synthesized successfully and its conjugation ratio of pAPB to BSA was about 19：1, the high-titer, sensitive and specific PB pAb were produced in sera of Balb/C mice immunized with BSA-pAPB, which made it possible to establish immunoassay of PB residues in animal food.