抗HLJ1单克隆抗体的制备及抗原检测方法的建立

Preparation of the anti-HLJ1 Monoclonal Antibodies and Establishment of Method for Detection of the Antigen

摘要:

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。

Abstract:

Monoclonal antibodies(McAbs) against human liver DnaJ-like protein(HLJ1) was produced by using lymphocyte- hybridoma technique and then one method for the detection of HLJ1 antigen was established. Two hybridoma cell lines which stably secreted monoclonal antibodies against HLJ1 were generated and named for A4C7 and C4C8. Subtypes of the two McAbs were both IgG1, and the antibodies showed high titer and good specificity. Using the prepared monoclonal antibody, human embryonic liver tissues were examined by immunohistochemistry. The results indicated that HLJ1 located in the cytoplasm of the human embryonic liver cell. A double antibodies sandwich ELISA was established by using C4C8 and HRP labeled A4C7. This assay had good specificity, and the lowest detection limit was 7.5 ng/mL and the linear range was 7.5~750 ng/mL. In conclusion, an immunohistochemistry method and a sensitive sandwich ELISA were established for the detection of HLJ1 protein.