重组人源细胞珠蛋白单抗制备及检测方法建立

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。

Preparation of monoclonal antibodies against recombinant human cytoglobin and establishment of method for detection of the protein

Objective To prepare monoclone antibody against recombinant human Cytoglobin（rhCygb） and establish a double antibody sandwich ELISA for the investigation of pharmacokinetic of rhCygb.Methods BALB/c mice were immunized with rhCygb.Methyl cellulose semi-solid culture medium was used for the screening of the hybirdoma cell lines.The characteristics of these monoclonal antibodies were determined by ELISA.A double antibody sandwich ELISA was established.Results Cell lines secreting monoclonal antibody against rhCygb were obtained and mouse ascites of five hybridoma cell lines were prepared and purified.Purity of these antibodies was 90%.The specificity was confirmed by Western blotting and the antibodies could not bind to another PET28a-BL21 recombinant protein by indirect ELISA.The sensitivity of this assay was 1.25 ng/ml（range 10～1250 ng/ml,R2=0.9931）.Intra-assay and inter-assay CV was 6.2% and 10.92%,respectively.Conclusion Monoclonal antibodies against rhCygb were obtained.A double antibody sandwich ELISA was established with satisfactory sensitivity and specificity.This work laid the foundation for further study of pharmacokinetic of rhCygb.