[Preparation, characterization and application of monoclonal antibody against CPS1]
Yan-fang Ju1, Rong Liu, Jin-ju Yang
1General Hospital of Beijing, Beijing 100853, China. yanfangju@yahoo.com.cn
Aim:
To prepare and characterize the monoclonal antibody (mAb) against human carbamyl phosphate synthetase I (CPSI) and make a study of its application.
Methods:
Normal human liver tissues were homogenized, and their mitochondria were isolated by differential centrifugation. The total mitochondrial proteins were used to immunize BALB/c mice to prepare mAb using the routine hybridoma technique. The mAb was detected by ELISA, Western blot immunohistochemistry and immunofluorecent staining. The specificity of mAb was identified by mass spectrometry (MS) and immunoprecipitation (IP) and then confirmed by Uni-ZAP expression library screening. The antibody was used to isolate potential enzymatic complexes by immunocapturing.
Results:
Three hybridoma cell lines BEH045, ACB271 and BFG021 secreting specific mAb against CPS1 were obtained. The Ig subclass of the mAb was IgG(1), which was used in ELISA, Western blot immunohistochemistry, immunoprecipitation, immunofluorecent staining and the isolation of potential enzymatic complexes.
Conclusion:
A hybridoma cell line which can secre specific mAb against CPSI stably has been established. The specific mAb against CPSI is of value to the research into the functions and distribution of CPSI.
