抗AGR2单克隆抗体的制备、鉴定及初步应用

Preparation,characterization and potential application of monoclonal antibody 18A4 against AGR2

目的:制备鼠抗AGR2单克隆抗体(mAb)并对其特性进行鉴定及其初步应用研究。方法:以AGR2-MBP融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备鼠抗AGR2的mAb,并用Protein-G亲和层析法纯化mAb。用间接ELISA法测定mAb的效价,以Western blot鉴定mAb的特异性。用免疫荧光、免疫沉淀和MTT法对mAb进行初步应用研究。结果:获得1株可持续、稳定分泌抗AGR2的mAb的杂交瘤细胞。杂交瘤细胞分泌的mAb的Ig亚类(型)为IgG1(κ),腹水mAb的效价达1×10-6。Western blot检测显示,在Mr为20000处有1条清晰的带。免疫荧光显示,该mAb可与乳腺癌细胞中的AGR2蛋白结合。免疫沉淀证实该mAb可与天然状态的AGR2有效结合。MTT实验显示该mAb对乳腺癌细胞MCF7的生长有抑制作用。结论:制备出的鼠抗AGR2的mAb效价高、特异性良好,可抑制细胞的生长,有一定的应用价值,为进一步研究AGR2的功能及其在乳腺癌、前列腺癌等癌症的诊断和治疗中的作用提供了工具。

AIM：To prepare mouse monoclonal antibodies（mAb）against AGR2（human homolog of xenopus anterior gradient 2）,to characterize these antibodies＇ properties,and to develop potential applications.METHODS：BALB/c mice were immunized with AGR2-MBP（maltose binding protein）fusion protein.The mAb was prepared by hybridoma technique and purified by protein G affinity chromatography.The titer and specificity of the mAb was determined by ELISA and Western blot respectively.The mAb was then further characterized by immunoprecipitation,immunofluorescent staining and tumor cell inhibition assay.RESULTS：One clone of hybridoma,18A4,secreting specific mAb against AGR2,was obtained.The Ig subclass of the mAb was IgG1（κ）.The titer of the mAb was 1×10^-6.Western blot analysis showed specific binding of 18A4 with both recombinant and native AGR2 in cell extract.Immunofluorescent staining using 18A4 mAb demonstrated specific staining of AGR2 in the cytoplasma of Breast cancer cell line MCF7.Immunoprecipitation as say confirmed that this mAb could specifically bind and effectively precipitate the native AGR2.mAb 18A4 could also inhibit the growth of breast cancer cell MCF7.CONCLUSION：The mAb anti-AGR2 with high titer and specificity has been obtained.This mAb,18A4,can be used in most molecular biology studies including Western blot,immunostaining,immunoprecipitation.It also inhibited the growth of cancer cells and therefore is a potential therapeutic starting point.It is a useful tool for the functional study of AGR2 and for the diagnosis and potential treatment of certain cancers.