【摘要】 将氯霉素(CAP)中的羟基(-OH)与琥珀酸酐反应引入活性基团羧基形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BSA和OVA偶联合成人工免疫原BSA-CAP-HS和包被抗原OVA-CAP-HS,采用红外(IR)、紫外(UV)、凝胶电泳(SDS-PAGE)进行鉴定;用BSA-CAP-HS免疫BALB/C小鼠,间接ELISA和阻断ELISA选择细胞融合备用鼠;应用杂交瘤技术制备氯霉素单克隆抗体(CAPmAb),并对其效价、亲和性和特异性等进行鉴定。结果表明,BSA与CAP-HS偶联成功,分子结合比为1∶15.6;筛选出1A10、2B5、4F9共3株敏感特异的杂交瘤细胞,间接ELISA效价细胞培养上清分别为1∶6.4×102、1∶3.2×102、1∶1.28×103,腹水分别为1∶1.6×105、1∶8×104、1∶6.4×105,同种型分别为IgG1/κ、IgG2a/λ、IgG2a/κ,亲和常数(Ka)分别为2.14×108、1.68×109、1.84×1010(L/mol);亲和力最高的4F9株对CAP的IC50为7.54ng/ml,经WestGold鉴定与CAP特异性结合,与氯霉素琥珀酸钠的交叉反应分别为150%,与其它CAP类药物和抗菌素无交叉反应性。通过试验获得了抗CAP高价、敏感、特异的mAb,可用于CAP残留检测的免疫学试验。更多还原

【Abstract】 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. UV and SDS-PAGE were used to identify BSA-CAP-HS. BALB/C mice were immunized with BSA-CAP-HS, the titre of monoclonal antibody (mAb) was detected by indirect ELISA and blocking ELISA. The hybridoma lines that secrete CAP mAb were established with using hybridoma technology. The immunological traits such as titer, affinity, and specificity of the mAb were characterized. The results showed that CAP artificial immunogen was synthesized successfully and its the conjugation ratio of CAP-HS to BSA was about 15.6∶1. Three hybridoma cell lines of 1A10?2B5 and 4F9 were screened for specificity to CAP,the indirect ELISA titer of the mAb were 1∶6.4×102?1∶3.2×102?1∶1.28×103 in supernatant,1∶1.6×105, 1∶8×104, 1∶6.4×105 in ascites, the isotypes of the mAb were IgG2a/κ?IgG2a/λ?IgG1/κ and the affinity constant(Ka) was 2.14×108, 1.68×109, 1.84×1010(L/mol) respectively,the mAb of 4F9 showed good sensitivity with an IC50 of 7.54ng/ml to CAP,specificity integrated with BSA-CAP-HS that determined by WestGold assay,150% cross-reactivity to CAP succinate sodium and little or no cress-reactivity to other compounds. CAP mAb of high-titer, sensitivity and specificity had been generated and made it possible to establish immunoassay of CAP residues in feed and animal food.更多还原