[Preparation and identification of monoclonal antibody against human GCRG213].
OBJECTIVE
To prepare and characterize the monoclonal antibody against human GCRG213.


METHODS
The HIS-GCRG213 fusion protein was expressed in E.coli. Mice were immunized with the purified HIS-GCRG213 protein. Hybridoma cell lines secreting monoclonal antibodies against GCRG213 were screened by regular cell fusion and subcloning approach. The titer and specificity of the antibody was characterized by ELISA and Western blot,  respectively. The expression of GCRG213 was determined using immunohistochemistry technique on paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer.


RESULTS
The HIS-GCRG213 fusion protein with relative molecular mass  of   20 800 was over expressed in E.coli. Two hybridoma cell lines which secreted monoclonal antibody specifically against human GCRG213 fusion protein were successfully obtained. The ascite titers of this monoclonal antibody reached 1:10(6);. Western blot analysis showed that the monoclonal antibody could bind to the recombinant HIS-GCRG213 protein specifically.The immunohistochemistry showed that GCRG213 were expressed higher in gastric cancer tissues than in normal ones.


CONCLUSIONS
The monoclonal antibody against human GCRG213 with high titer and specificity has been successfully prepared,  which could be utilized as a useful reagent for further studying the biological function of the GCRG213.
Department of Gastroenterology, South Building, General Hospital of PLA, Beijing 100853, China. wuyq56@hotmail.com
Yin-qiao
YQ
Wu
Ben-yan
BY
Wu
Gang-shi
GS
Wang
Wei-di
WD
You
Wei-hua
WH
Wang
Meng-wei
MW
Wang
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D036361:Peptide Hormones / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D013274:Stomach Neoplasms / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; C477000:GKN1 protein, human; D036361:Peptide Hormones; D011993:Recombinant Fusion Proteins