抗软骨藻酸单克隆抗体杂交瘤细胞株的构建

Preparation of hybridoma cell strains secreting monoclonal antibodies against domic acid

为了获得稳定、高效分泌抗软骨藻酸(DA)单克隆抗体的杂交瘤细胞株,采用活泼酯法制备软骨藻酸免疫抗原(DA-KLH)和包被抗原(DA-BSA),以DA-KLH免疫BALB/c小鼠,用细胞融合技术筛选抗软骨藻酸单克隆抗体杂交瘤细胞株,体内诱生腹水法制备大量单抗,捕获EL ISA法测定小鼠免疫球蛋白亚型,间接ELISA法测定腹水效价;用G蛋白亲和层析法来纯化腹水。结果显示,成功构建2株能稳定分泌DA单克隆抗体(McAb)的杂交瘤细胞株2B1和4C2,抗体亚类分别为IgG1和IgG2a,间接EL ISA检测小鼠腹水的抗体效价达1∶64 000,腹水纯化后蛋白浓度为1.27和0.675 mg/mL。研究结果表明,成功制备的抗DA单克隆抗体杂交瘤细胞株稳定、高效,为利用该单抗研制快速检测软骨藻酸的胶体金免疫层析试纸条打下基础。

To obtain stable and efficient hybridoma cell strains secreting monoclonal antibody（McAb） against domic acid（DA）,completed antigen（DA-KLH） and coating antigen（DA-BSA） were synthesized by active ester method.BALB/c mice were immunized with DA-KLH and hybridoma cell strains secreting McAb against DA were screened by cell fusion,and the immunological titers of DA-McAb were determined.Capture ELISA method was used to determine isotypes of the mouse monoclonal antibody and the indirect ELISA method was performed to determine the titers of McAbs.The protein G affinity chromatography protein purification method was used to purify ascites.Results showed that： two strains of hybridomas,2B1and 4C2,which could secret McAb against DA steadily were successfully prepared and the isotypes were IgG1 and IgG2a.The titer of purified McAb was 1∶64 000 and the protein concentration of purified ascites were 1.27 and 0.675 mg/mL respectively.It could be demonstrated that the successful establishment of hybridoma cell strains secreting McAb against DA supply a firm foundation for developing a gold immunochromatography assay that can detect DA as quickly,economically and simplely as possible in future research.