抗苯巴比妥单克隆抗体杂交瘤细胞株的筛选及ciELISA试剂盒的研制

用BSA-pAPB免疫Balb/c小鼠，用细胞融合技术制备并用间接ELISA和阻断ELISA筛选抗苯巴比妥单克隆抗体(PB mAb)杂交瘤细胞株，体内诱生腹水法生产PB mAb，应用PB mAb研制PB残留竞争ELISA(ciELISA)快速检测试剂盒(PB-Kit)，并测定其性能。结果表明，筛选出3株杂交瘤细胞，最好的3F6-C4株的PB mAb间接ELISA效价为1∶6.4×105，亲和常数(Ka)为1.96×1010 L/moL，半数抑制浓度(IC50)为5.7 μg/L，与巴比妥的交叉反应率(CR%)12.4%，与其它化合物无CR；PB-Kit的线性检测范围1.0～81 μg/L，灵敏度0.75 μg/L，检测限1 μg/L；饲料样和猪尿样的平均添加回收率85.8%和91.3%，平均批内和批间变异系数均<15%。PB-Kit具有快速、敏感、特异、简便等特点，适合PB残留快速检测的推广应用。

Screening of Hybridoma Lines of Monoclonal Antibody and Development of ciELISA Kit for Rapid Detection of Phenobarbital

Balb/c mice were immunized with BSA-pAPB and hybridoma lines secreted monoclonal antibody against phenobarbital (PB mAb) were generated with cell fusion and filtered by indirect ELISA and blocking ELISA. A ciELISA kit for detection PB (PB-Kit) was developed with PB mAb and its traits were tested. The results showed that three hybridoma lines were filtered and the best one was 3F6-C4, which secreted PB mAb with indirect ELISA titers of 1∶6.4×105 in ascites. PB mAb had a high affinity constant(Ka) with 1.96×1010 L/mol, a good sensitivity with an IC50 of 5.7 μg/L to PB, 12.4% cross-reactivity to barbiturate sodium and little or no cross-reactivity to other compounds. The PB-Kit had the linear detection range of 1.0 to 81 μg/L, the sensitivity of 0.75 μg/L and the detection limit of 1.0 μg/L. The recoveries of PB spiked in feed and swine urine were 85.8% and 91.3% respectively. The precision and accuracy of the assay determined by inter-assay and intra-assay coefficient variation was both below 15%. The PB-Kit possesses rapidity, sensitivity, specificity and briefness. That is proved to be used for the rapid detection of PB residues in animal food.