抗氯霉素单克隆抗体杂交瘤细胞株的筛选及ciELISA试剂盒的研制

Filtration of Hybridoma Lines of Monoclonal Antibody and Development of ciELISA Kit for Chloramphenicol

将氯霉素（CAP）化学修饰引入羧基活性基团，形成具有半抗原结构特征的氯霉素半琥珀酸酯（CAP—HS），用混合酸酐法制备免疫原BSA-CAP—HS和包被原OVA—CAP—HS，用UV，SDS—PAGE进行鉴定；BSA—CAP—HS免疫BALB／C小鼠，细胞融合技术筛选抗氯霉素单克隆抗体（CAPmAb）杂交瘤细胞株，体内诱生腹水法制备CAPmAb，并鉴定其免疫学特性；应用CAPmAb研制CAP残留快速检测ciELISA试剂盒（CAP—Kit），并测定其性能．结果表明，BSA—CAP—HS偶联成功，分子结合比为1：15．6；筛选出3株杂交瘤，其中最好的4F0株间接ELISA效价细胞上清为1：1．28×10^3，腹水为156．4×10^5，亲和常数（Ka）为1．84×10^10L·mol^-1，半数抑制浓度（IC50）为2．4μg·L^-1，与氯霉素琥珀酸钠的交叉反应（CR）为150％，与其它酰胺醇类和抗菌素药物无交叉反应；CAP—Kit的标准曲线呈典型的S型，符合4参数logit曲线拟合，线性范围为0．1～128μg·L^-1，灵敏度为0．053μg·L^-1检测限为0．1μg·L^-1，奶样、肉样的平均添加回收率为88．5％，82．7％，平均批内和批间变异系数〈15％．CAP—Kit在4oC可保存180d．

The active group carboxyl was introduced to Chloramphenicol（ CAP） by chemical modification and formed CAP half succinaate （CAP-HS） which has hapten structure. Mixed anhydride （MA） was used to conjugate CAP-HS to BSA and OVA obtained immunogen BSA-CAP-HS and coating anti- gen OVA-CAP-HS , UV and SDS-PAGE were used to identify BSA-CAP-HS. BALB/C mice were immunized with BSA-CAP-HS and hybridoma lines that secrete CAP monoclonal antibody （CAP mAb） were filtered using cell fusion and the immunological traits of CAP mAb were characterized. A ciELISA kit for detection CAP（CAP-Kit）was developed with CAP mAb and its traits were tested. The results showed that CAP immunogen was synthesized successfully and its conjugation ratio of CAP-HS to BSA was about 15.6： 1. Three hybridoma lines were filtered and the best one was 4F9, which secretes CAP mAb with indirect ELISA titers of 1：1.28 × 10^3 in supernatant and 1：6.4 × 10^5 in ascites. CAP mAb has a high affinity constant（Ka） with 1.84 × 10^l0 L · mol^-1, a good sensitivity with an IC50 of 2.4 μg · L^-1 to CAP, 150% cross-reactivity to CAP succinate sodium and little or no cross-reactivity to other compounds. The calibration curve of CAP-Kit was typical sigmoid curve fitted to the four parame- ter logistic equation with the linear detection of 0.1 to 128 μg · L^-1, the sensitivity of 0. 053 μg · L^-1 and the detection limit of 0. 1 μg · L^-1 The recoveries of CAP spiked in milk were 88.5% and in pork were 82.7%. The precision and accuracy of the assay as determined by inter-assay and intra-as- say coefficient variation were below 15% The validity of CAP-Kit in 4 ℃ was 180 d