氯霉素多克隆抗体的制备及其免疫学特性鉴定

Generation and Characterization of Immunological Traits of Polyclonal Antibody against Chloramphenicol

将氯霉素(CAP)进行化学修饰引入羧基活性基团,合成具有半抗原结构特征的氯霉素半琥珀酸酯(CAP-HS);采用混合酸酐(MA)法将CAP-HS与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成人工免疫原BSA-CAP-HS和包被原OVA-CAP-HS,用红外(IR)、紫外(UV)、凝胶电泳(SDS-PAGE)进行鉴定;用BSA-CAP-HS免疫新西兰白兔和SPF鸡制备多克隆抗体(pAb),间接ELISA测定其效价,阻断ELISA鉴定其敏感性,琼脂双扩散试验、WestGold膜杂交试验和交叉反应试验鉴定其特异性。结果表明,BSA-CAP-HS偶联成功,其分子结合比为15.6∶1,获得了高价、敏感、特异的CAPpAb,为CAP残留免疫学检测方法的建立奠定了基础。

The active group carboxyl was introduced to Chloramphenicol （CAP） and formed CAP half succinaate （CAP-HS） which has hapten structure by chemical modification. The method of mixed anhydride （MA） was used to conjugate CAP-HS to BSA and obtained artificial immunogen BSA-CAP-HS, the coating antigen OVA-CAP-HS was obtained in the same way. IR, UV and SDS-PAGE were used to identify BSA- CAP-HS. New Zealand white rabbits and SPF chickens were immunized with BSA-CAP-HS, the titre of polyclonal antibody （pAb） was detected by indirect ELISA, the sensitivity of pAb was identified by blocking ELISA and the specificity of pAb was identified by test of agar bidirectional pervasion, WestGold immunochromatography and cross-reaction. The results showed that BSA-CAP-HS had been synthesized successfully and its conjugation ratio of CAP-HS to BSA was about 15.6：1, the high-titer, sensitive and specific CAP pAb had been produced in sera of immunized New Zealand white rabbits and SPF chickens, which made it possible to establish immunoassay of CAP residues in feed and animal food.