抗苯巴比妥单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定

苯巴比妥(PB)经化学修饰在其苯环上引入活性基团氨基,合成半抗原对氨基苯巴比妥(pAPB),用重氮化法将pAPB偶联于BSA,合成人工抗原BSA-pAPB,并用红外(IR)、紫外(UV)、SDS-PAGE鉴定;用BSA-pAPB免疫Balb/C小鼠,细胞融合技术建立抗PB的单克隆抗体mAb(PB mAb)杂交瘤细胞株,体内诱生腹水法制备PB mAb,并鉴定其免疫学特性。结果表明,BSA-pAPB偶联成功,偶联率为1∶19;筛选出1B9、3C4、3F6、4E6共4株杂交瘤细胞,其中最好的4E6株间接ELISA效价细胞培养上清为1∶8.1×102,腹水为1∶6.4×105,同种型为IgG1/κ,亲和常数(Ka)为2.08×1010L/mol,对PB的半数抑制浓度(IC50)为11.35μg/L,与巴比妥的交叉反应率(CR%)为12.4%,与其他化合物无CR。本试验研制出高效价、敏感、特异的PB mAb,可用于PB残留检测的免疫学实验。

GENERATION AND IMMUNOLOGICAL TRAITS OF MONOCLONAL ANTIBODY FOR PHENOBARBITAL

The active group amido was introduced to phenyl of PB and formed p-amidophenobarbital(pAPB) which has hapten structure by chemical modification.Diazotization was used to conjugate pAPB to BSA and obtained artificial antigen BSA-pAPB identified by Infrared(IR),ultraviolet(UV) and SDS-PAGE.Balb/c mice were immunized with BSA-pAPB and hybridoma lines that secrete monoclonal antibody against PB(PB mAb) were generated with cell fusion and filtered by indirect ELISA and blocking ELISA,the immunological traits of PB mAb were characterized.The results showed that BSA-pAPB was synthesized successfully and its conjugation ratio of pAPB to BSA was about 19∶1.Four hybridoma lines of 1B9,3C4,3F6 and 4E6 were filtered and the best one was 4E6,which secrete PB mAb with indirect ELISA titers of 1∶8.1×10~2 in supernatant and 1∶6.4×10~5 in ascites,the isotype of the mAb was IgG_1/κ and its affinity constant(Ka) was 2.08×10~(10) L/mol.The mAb showed good sensitivity with an IC_(50) of 11.35μg/L to PB,12.4% cross-reactivity(CR%) to Barbital and little or no CR to other compounds.PB mAb of high-titer,sensitivity and specificity has been generated and made it possible to establish immunoassay of PB residues in animal food.