[Preparation and characterization of monoclonal antibody against human carboxylesterases-II].
OBJECTIVE
To prepare monoclonal antibody(mAb) against human carboxylesterases-II (hCE-II) and characterize its properties.


METHODS
BALB/c mice were immunized with human liver microsome protein which contained hCE-II. The mAb was prepared by hybridoma technique and purified by protein-G affinity chromatography. The titer and specificity of mAb was detected by ELISA and Western blot respectively. Tissue localization of antigen was detected by immunohistochemical staining. Antigen was appraised by peptide mass fingerprint (PMF) matched with Mascot human protein database. PMF was obtained by immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).


RESULTS
One clone of hybridoma secreting specific mAb against hCE-II was obtained. The Ig subclass of the mAb was IgG1(kappa). The titer of the mAb was 1 x 10(-7). Western blot analysis showed one clear belt in the Mr of 62,000. Immunohistochemistry demonstrated that the mAb had special combination with the liver cytoplasm protein, but not with the vascular smooth muscle cell protein. Immunoprecipitation showed one clear band in the Mr of 62,000, which was in conformity with the Mr of hCE-II and the antigen was confirmed to be hCE-II after being analyzed with mass spectrometry.


CONCLUSIONS
The mAb against hCE-II with high titer and specificity has been obtained, which lays the foundation for investigation of hCE-II function and diagnosis and therapy of liver cancer.
Wu
Zheng-hua
ZH
Biopharmacy Department of Pharmacy College, Harbin Medical University, Harbin 150086, China. wuzhenghua_81@yahoo.com.cn
Tang
Xiao-bo
XB
Wang
Zhi-gang
ZG
Ji
Hong-yu
HY
Yu
Lei
L
Liu
Hui-wen
HW
Zhu
Da-ling
DL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D030562:Databases, Protein; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D002265:Carboxylic Ester Hydrolases