[Preparation and characterization of antibodies against clusterin].

AIM

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.

CONCLUSION

pAb and mAbs against human CLU were successfully prepared, which will provide efficient tools for functional studies of CLU expressed in human tumors.