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目的制备重组人源细胞珠蛋白(recombinanthumanCytoglobin,rhCygb)单克隆抗体,并建立检测该蛋白双抗体夹心ELISA法,为下一步研究rhCygb药代动力学做准备。方法用纯化的rhCygb免疫BALB/c小鼠,采用甲基纤维素半固体培养基法获得抗rhCygb的单克隆抗体杂交瘤细胞,间接ELISA法筛选制备单克隆抗体,建立双抗体夹心ELISA法。结果筛选获取了稳定分泌单克隆抗体的杂交瘤细胞株,通过抗原表位相加法实验获得5株表位不同的细胞株,Western-blotting验证能与rhCygb特异性结合,间接ELISA法验证其不与本实验室制备的其它PET28a-BL21蛋白及BL21裂解液发生交叉反应。本方法灵敏度为1.25ng/ml,在浓度为10～1250ng/ml时,线性关系良好,相关性达0.9931,实验内和实验间平均变异系数分别为6.2%和10.92%。结论成功建立了灵敏度好、特异性高的双抗体夹心法,为下一步研究rhCygb药代动力学奠定了基础。
Objective To prepare recombinant human monoclonal antibody (rhCygb) for human rhGygb and to establish sandwich ELISA for the detection of rhCygb in order to study the pharmacokinetics of rhCygb. Methods BALB / c mice were immunized with purified rhCygb, and monoclonal antibodies against rhCygb were obtained by using methylcellulose semi-solid medium. The monoclonal antibodies were screened by indirect ELISA and the double antibody sandwich ELISA was established. Results The hybridoma cell lines secreting monoclonal antibodies were obtained by screening. Five different cell lines with different epitopes were obtained by the antigen epitope addition assay. Western blotting confirmed the specific binding to rhCygb. Other PET28a-BL21 proteins prepared by the laboratory cross-reacted with BL21 lysate. The sensitivity of the method was 1.25 ng / ml. The linearity was good at a concentration of 10 ~ 1250 ng / ml with a correlation of 0.9931. The average coefficient of variation (CV) was 6.2% and 10.92%, respectively. Conclusion The double antibody sandwich method with good sensitivity and specificity was successfully established, which laid the foundation for the further study of pharmacokinetics of rhCygb.