Preparation and Characterization of Anti-GP73 Monoclonal Antibodies and Development of Double-antibody Sandwich ELISA

Abstract

<b>Background:</b> Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellularcarcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variablesrepresenting tumor growth and invasiveness. The aim of this study was to prepare a pair of monoclonal antibodys(mAbs) against GP73 and develop a newly designed double-antibody sandwich enzyme-linked immunosorbentassay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis ofHCC. Materials and <br/><b>Methods</b>: Produced by prokaryotic expression, the purified recombinant GP73 (rGP73),produced by prokaryotic expression, was used to immunize the Balb/c mice. Two hybridoma cell lines againstGP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titersof anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealedthat anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully establishedand validated by 119 HCC and 103 normal serum samples. <br/><b>Results</b>: showed that the detection limit of this methodcould reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those ofin healthy controls (mean=70.92 ng/ml). <br/><b>Conclusions</b>: Results of our study not only showed that sGP73 levelsof HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratoryhomemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide asolid foundation for subsequent clinical applications.