[Preparation and characterization of antibodies against clusterin].
OBJECTIVE
To prepare rabbit polyclonal antibodies (pAb) and mouse monoclonal antibodies (mAbs) against human clusterin(CLU) and characterize these antibodies' properties.


METHODS
CLU fragment was amplified from human liver cDNA library, and recombinant expression vectors pGEX-4T-1-CLU and PET-32a-CLU were constructed. GST-CLU fusion protein was expressed in E.coli and then used as the immunogen. Properties of antiserum against human CLU were identified by ELISA, Western blot, and the mAbs against human CLU was characterized by Western blot, indirect immunofluorescent staining and immunohistochemistry staining.


RESULTS
The GST-CLU fusion protein was highly expressed with a molecular weight of M(r)54,000. Western blot analysis proved that the rabbit pAb could specifically recognize 52,000 and 58,000 proteins in human liver total protein. All of the nine established mAbs recognized recombinant human CLU protein, two of which specifically bound to proteins in the cytoplasm of HepG2 cells and four of which specifically bound to proteins in the cytoplasm of adult liver tissue.


CONCLUSIONS
pAb and mAbs against human CLU were successfully prepared, which will provide efficient tools for functional studies of CLU expressed in human tumors.
Zhang
Ying-chun
YC
State Key Laboratory of Proteomics, Department of Antibody Engineering, Beijing Proteome Research Center, Beijing 100850, China. yingchun889@yahoo.com.cn
Liu
Ying
Y
Yang
Jin-ju
JJ
Liu
Xiao-lan
XL
Chi
Jun
J
Gao
Jian-en
JE
Tang
Xiao-bo
XB
Zhu
Da-ling
DL
Sun
Qi-hong
QH
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D051152:Clusterin / Q000276:immunology*; D006224:Cricetinae; D003412:Cricetulus; D056945:Hep G2 Cells / Q000276:immunology*; D006801:Humans; D007106:Immune Sera; D007150:Immunohistochemistry; D051379:Mice; D011817:Rabbits
D000906:Antibodies; D000911:Antibodies, Monoclonal; C495242:CLU protein, human; D051152:Clusterin; D007106:Immune Sera