[Generation and characterization of monoclonal antibody against GRHPR].

ABSTRACT

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.