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目的:制备抗硫氧还蛋白-1(TRX-1)的单克隆抗体(mAb)并进行特性鉴定。方法:以原核表达的TRX-1为抗原免疫BALB/c小鼠,按常规方法进行细胞融合。采用有限稀释法和间接ELISA法克隆和筛选阳性杂交瘤细胞株,用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析,用Westernblot法对mAb的特异性进行鉴定。结果:得到筛选出3株能稳定分泌抗TRX-1的杂交瘤细胞株B6、D5和E3,免疫球蛋白亚类均为IgG1(κ),腹水mAb效价均在106以上,3株抗体分别针对不同抗原表位。用mAb建立的竞争抑制ELISA灵敏度达1.22μg/L。结论:获得3株特异性好、亲和力高、能稳定分泌抗TRX-1的杂交瘤细胞株,为研究TRX-1在人类细胞、血液、组织中的表达及定位提供了可能,并为探索相关炎症、癌症发病机制及新治疗方法提供了强有力的工具。
OBJECTIVE: To prepare and characterize monoclonal antibodies (mAb) against thioredoxin-1 (TRX-1). Methods: BALB / c mice were immunized with TRX-1 expressed in prokaryotic cells and fused by conventional methods. The positive hybridoma cells were cloned and screened by limited dilution and indirect ELISA. The titer, relative affinity and epitope analysis of ascites were detected by ELISA. The specificity of mAb was identified by Western blot. Results: Three hybridoma cell lines B6, D5 and E3 that could stably secrete anti-TRX-1 were screened. The immunoglobulin subclasses were all IgG1 (κ). The titer of ascites mAb was above 106. Three antibodies Targeting different epitopes. Competitive inhibition of ELISA established with mAb was 1.22 μg / L. CONCLUSION: Three hybridoma cell lines with good specificity and high affinity that can stably secrete anti-TRX-1 were obtained, which provided a possibility for studying the expression and localization of TRX-1 in human cells, blood and tissues. Inflammation, cancer pathogenesis and new treatment methods provide a powerful tool.