[Preparation, characterization and application of polyclonal antibody against human carboxylesterases-II].
OBJECTIVE
To construct a prokaryotic plasmid expressing human carboxyles-terases-II (hCE-II ), purify the recombinant protein and investigate the rabbit polyclonal antibody against hCE-II .


METHODS
A recombinant plasmid expressing pGEX-4T-1-hCE-II (hCE-II -GST) and pET-32a- hCE-II (hCE-II -His) was constructed and then it was expressed in E.coli BL21 induced by IPTG. The polyclonal antibody was prepared by immunizing the rabbits with the purified recombinant protein. The sensitivity and specificity of the antibody was detected using enzyme -linked immunosorbent assay, immunohistochem ical staining and Western blot analysis.


RESULTS
The polyclonal antibody against hCE-II was successfully prepared. Western blot analysis showed that the antibody specifically reacted with the recombinant protein and natural human liver microsomal proteins. Immunohistochemis result demonstrated that the pAb could combine with liver cytoplasm protein but not vascular smooth muscle cell protein.


CONCLUSIONS
The successful preparation of the pAb against hCE-II with high titer and specificity lays a foundation for the investigation of diagnosis kit of liver cancer.
Wu
Chun-ling
CL
Biopharmaceutical Department, College of Pharmacy, Key Lab of Biopharmaceutical Engineering of Heilongjiang Province, Key Laboratory of Drug and Endogenous Substance Metabolism of Higher Learning Institution of Heilongjiang Province, Harbin, China. dlutwcl@yahoo.com.cn
Bao
Hong-xia
HX
Tang
Xiao-bo
XB
Zhu
Da-ling
DL
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases