乙醛酸还原酶/羟基丙酮酸还原酶单克隆抗体的制备与鉴定

目的：制备鼠抗人乙醛酸还原酗羟基丙酮酸还原酶单克隆抗体（mAb）并进行初步鉴定。方法：将正常成人肝组织匀浆离心并分离出肝脏胞质总蛋白，用肝脏胞质总蛋白免疫BALB／c小鼠，采用杂交瘤技术制备mAb，并通过间接ELISA法、Western blot及免疫组化的方法对mAb进行特异性鉴定，通过免疫沉淀联合质谱和Uni-ZAP XR表达文库筛选鉴定抗原。结果：通过间接ELISA筛选获得杂交瘤细胞株ADB291，其分泌的mAb Ig亚类（型）为IgG1（κ），Western blot结果显示该mAb识别相对分子质量（Mr）为35000的蛋白；对免疫沉淀获得的相应抗原回收、酶切、质谱鉴定为GRH—PR。同时应用mAb ADB291对人肝脏cDNA表达文库（Uni-ZAP XR）进行筛选，筛选所获得的阳性噬菌斑测序结果显示2个阳性克隆插入序列均为GRHPR；阳性克隆转化表达后的Western blot结果确认该抗体识别肘，35000的表达蛋白。结论：mAb ADB291特异识别的抗原为GRHPR，该mAb在GRHPR的功能研究和二型原发性尿草酸盐过多遗传疾病的临床诊断等方面具有应用价值。

Generation and characterization of monoclonal antibody against GRHPR

AIM: To generate monoclonal antibodies (mAb) against glyoxylate reductase/hydroxypyruvate reductase (GRHPR). METHODS: Normal human liver tissues were homogenized, and human liver cytosolic proteins were isolated by centrifugation. The total human liver cytosolic proteins were used to immunize BALB/c mice to prepare mAb by hybridoma technique. The mAbs were detected by ELISA, Western blot, and immunohistochemistry. The antibody specificities were identified by mass spectrometry (MS) following immunoprecipitation (IP) and confirmed by Uni-ZAP expression library screening. RESULTS: One hybridoma cell line, ADB291, secreting specific mAb against GRHPR was established. The Ig subclass of the mAb was IgG1(kappa). Data from immunohistochemistry showed that ADB291 can recognize hepatocyte cytoplasm. ADB291 mAb was used to isolate its protein antigen by IP. Proteins captured by the mAb were loaded to SDS-PAGE and subjected to Western blot and MALDI-TOF MS analysis. lambda expression Uni-ZAP XR pre-made liver cDNA library was screened with ADB291 hybridoma supernatants. All of our data demonstrated that ADB291 mAb specially reacted with GRHPR. CONCLUSION: A hybridoma cell line stably secreting specific mAb against GRHPR is established. The specific mAb against GRHPR would be very useful for the studies of GRHPR functions and distribution.