苯巴比妥单克隆抗体的研制及竞争ELISA血药浓度监测方法的建立

 目的研制苯巴比妥单克隆抗体(PBmAb),建立PB血药浓度竞争ELISA监测方法。方法将PB改造成对氨基苯巴比妥(pAPB),用重氮化法合成免疫原BSA-pAPB并免疫Balb/c小鼠;细胞融合技术筛选PB mAb杂交瘤细胞株,体内诱生腹水法制备PB mAb;应用PB mAb研制PB血药浓度监测竞争ELISA试剂盒(PB-Kit),并测定其性能。结果BSA-pAPB偶联成功;筛选出4株杂交瘤细胞,应用其中最好的4E6株制备的PB mAb间接ELISA效价为1∶6.4×10⁵,亲和常数(K_a)为2.08×10¹⁰L·moL^-1,50%抑制浓度(IC₅₀)为9.84μg·L^-1,与巴比妥的交叉反应率(CR)为7.9%,与其他化合物无CR;PB-Kit的检测范围为1.0～128μg·L^-1,灵敏度为0.89μg·L^-1,检测限为1μg·L^-1,血清样添加回收率为94.7%,批内和批间变异系数均<10%;PB-Kit与差示分光光度法(DS)的符合率为100%。结论成功研制出高价、敏感、特异的PB mAb,建立了PB血药浓度竞争ELISA监测方法。

Development of Monoclonal Antibodies Against Phenobarbital and Monitor of Phenobarbital Concentration in Serum by Competitive ELISA

OBJECTIVE To generate monoclonal antibody against phenobarbital (PB mAb) and to establish competitive ELISA for monitoring PB concentration in serum. METHODS PB was modified chemically to form p-amidophenobarbital (pAPB), diazotization was used to conjugate pAPB to BSA and immunogen BSA-pAPB was obtained. Balb/c mice immunized with BSA-pAPB were chosen for cell fusion by indirect ELISA and blocking ELISA. The hybridoma lines secreting PB mAb were filtered with cell fusion and the immunological traits of PB mAb were characterized. A competitive ELISA kit for monitoring PB (PB-Kit) was developed with PB mAb and its traits were tested. RESULTS PB immunogen was synthesized successfully. The best one among four hybridoma lines was 4E6, which secreted PB mAb with indirect ELISA titers of 1∶6.4×10⁵ in ascites, a high affinity constant (Ka) of 2.08×10¹⁰ L·moL^-1, a good sensitivity with 50% inhibitive concentration (IC₅₀) of 9.84 μg·L^-1.The PB mAb had 7.9% cross-reactivity to Barbital and little or no cross-reactivity to other compounds. PB-Kit had the linear detection of 1.0 to 128 μg·L^-1, the sensitivity of 0.89 μg·L^-1 and the detection limit of 1.0 μg·L^-1. The recoveries of PB spiked in serum were 94.7%. The precision and accuracy of the assay determined by inter-assay and intra-assay coefficient variation were both below 10%. Its coincidence rate was 100% compared with differential spectrophotometry (DS). CONCLUSION PB mAb with high-titer, sensitivity and specificity has been generated successfully, a competitive ELISA has been established with PB mAb, which can be used to monitor PB concentration in serum.