[Preparation, characterization and application of monoclonal antibody against CPS1].
OBJECTIVE
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.


CONCLUSIONS
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.
Ju
Yan-fang
YF
General Hospital of Beijing, Beijing 100853, China. yanfangju@yahoo.com.cn
Liu
Rong
R
Yang
Jin-ju
JJ
Liu
Xiao-lan
XL
Gao
Jian-en
JE
Sun
Qi-hong
QH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000276:immunology*; D020165:Carbamoyl-Phosphate Synthase I Deficiency Disease / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)