Background: Serum Golgi protein 73 (GP73) as a novel and potential marker for diagnosing hepatocellular
carcinoma (HCC) have been found to be elevated in HCC patients and associated with clinical variables
representing 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 immunosorbent
assay (s-ELISA), which would be used in the detection of serum GP73 (sGP73) as well as in the diagnosis of
HCC. Materials and Methods: 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 against
GP73 were obtained by fusing mouse Sp2/0 myeloma cells with spleen cells from the immunized mice. The titers
of anti-GP73 mAb reached 1:243,000. Western blotting analysis and Immunohistochemistry staining revealed
that anti-GP73 mAb could recognize GP73 protein. The double-antibody s-ELISA was successfully established
and validated by 119 HCC and 103 normal serum samples. Results: showed that the detection limit of this method
could reach 1.56 ng/ml, and sGP73 levels in HCC group (mean=190.6 ng/ml) were much higher than those of
in healthy controls (mean=70.92 ng/ml). Conclusions: Results of our study not only showed that sGP73 levels
of HCC patients were significantly higher than those of healthy controls, but also indicated that the laboratory
homemade anti-GP73 mAbs could be the optimal tool used in evaluating sGP73 levels, which would provide a
solid foundation for subsequent clinical applications.