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Preparation and characterization of anti-GP73 monoclonal antibodies and development of double-antibody sandwich ELISA.
- Source :
-
Asian Pacific journal of cancer prevention : APJCP [Asian Pac J Cancer Prev] 2015; Vol. 16 (5), pp. 2043-9. - Publication Year :
- 2015
-
Abstract
- 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.<br />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.<br />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).<br />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.
- Subjects :
- Animals
Biomarkers, Tumor blood
Carcinoma, Hepatocellular blood
Cell Line, Tumor
Female
Hep G2 Cells
Humans
Immunohistochemistry methods
Liver Neoplasms blood
Membrane Proteins blood
Mice, Inbred BALB C
Antibodies, Monoclonal immunology
Carcinoma, Hepatocellular diagnosis
Enzyme-Linked Immunosorbent Assay methods
Liver Neoplasms diagnosis
Membrane Proteins immunology
Subjects
Details
- Language :
- English
- ISSN :
- 2476-762X
- Volume :
- 16
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Asian Pacific journal of cancer prevention : APJCP
- Publication Type :
- Academic Journal
- Accession number :
- 25773808
- Full Text :
- https://doi.org/10.7314/apjcp.2015.16.5.2043