抗二羰基/L-木酮糖还原酶单克隆抗体的制备与鉴定

目的制备抗人二羰基/L-木酮糖还原酶(Dicarbon-yl/L-xylulosereductase,DCXR)的单克隆抗体(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线粒体,用线粒体总蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb,并通过间接ELISA法、Westernblot及免疫组化的方法对mAb进行特性鉴定,通过免疫沉淀联合质谱、Uni-ZAPXR表达文库筛选鉴定抗原。结果获得1株可稳定分泌抗人DCXRmAb的杂交瘤细胞系。mAb的Ig亚类(型)为IgG1(κ),该mAb可用于ELISA检测、Westernblot、免疫组化和免疫沉淀实验。结论成功制备了抗人DCXR的mAb,为DCXR的研究提供了有力的工具。

Preparation and characterization of monoclonal antibody against DCXR

AIM: To prepare monoclonal antibodies (mAbs) against Dicarbonyl/L-xylulose reductase(DCXR). METHODS: Normal human liver tissues were homogenized, and mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAbs by routine 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 clone of hybridoma AFF091 secreting specific mAb against DCXR was obtained. The Ig subclass of the mAb was IgG_1 and it can be used in ELISA, Western blot, immunohistochemistry, and immunoprecipitation. CONCLUSION: A hybridoma cell line stably secreting specific mAb against DCXR was established. The specific mAb against DCXR would be very useful for investigation of DCXR functions and distribution.