| Malignant tumors are the most dangerous diseases because of high mortality and fatality rate. The best strategy to combat cancer is by early diagnosis and effective treatment strategies. Tumor markers play important roles in tumor early diagnosis, prognosis, monitoring of therapy, and prediction of therapeutic response. Accumulated evidences suggested that GP73 and HDGF might be the potential promising biomarkers in tumor diagnosis and prognosis. The aims of this study were to develop the monoclonal and polyclonal antibodies against GP73 and HDGF, to establish sandwich ELISA methods for measuring GP73 and HDGF in serum samples; and to validate their clinical application.We have cloned GP73 and HDGF gene from the frozen tissue of an HCC specimen by RT-PCR. Human recombinant GP73 and HDGF expression plasmid were constructed and expressed in E. coli strain. High purity recombinant human GP73 and HDGF protein were purified by affinity chromatography under optimized conditions, and identified by Western blot and mass spectrum.We have raised polyclonal anti-GP73 and anti-HDGF antibodies by immunizing rabbits with purified GP73 and HDGF protein respectively. We have also generated anti-GP73 and anti-HDGF monoclonal antibody, designated GP73-6A2 mAb and HDGF-2F12 mAb, by using hybridoma technology. The specificity and subcellular localization of GP73-6A2 mAb or HDGF-2F12 mAb were characterized by Western blot, immunohistochemistry (IHC), immunofluorescent staining (IF) and immunoprecipitation (IP). The results showed that both mAb not only bind with the GP73 or HDGF protein specifically, but also can be used on multi-purpose immunotests described above.To quantitatively analyze GP73 or HDGF in serum samples, we established double antibody sandwich ELISA method using mouse GP73-6A2 mAb or HDGF-2F12 mAb as capture and rabbit anti-GP73 pAb or anti-HDGF pAb as tracer antibody. AP conjugate anti-rabbit IgG acted as detection antibody and recombinant GP73 or HDGF protein served as protein standard. The linear range of GP73 is 0.25～4ng/ml, and the detection sensitivity is 0.123 ng/ml. The linear range of HDGF is 0.5-16.0ng/ml, and the detection sensitivity is 0.193ng/ml. Three independent standard curve was performed at different time, demonstrating that these two systems are stable, reliable and reproducible. In this study, GP73 protein could be detected in the serum samples from both healthy volunteers and liver cancer patients by sandwich ELISA. After statistical analysis, we found that the serum concentration of GP73 in liver cancer patients was significantly higher than that of healthy control (p=0.0036). Also, the significant difference of GP73 concentration was found between lung cancer patients and normal people (p = 0.0245). To validate the application of HDGF sandwich ELISA in clinical serum samples, 95 healthy volunteers and 86 lung cancer patients were tested. The serum concentration of HDGF in lung cancer was significantly higher than that of healthy control, 9.43±6.13ng/ml vs. 4.36±2.50ng/ml (p=1.12E-10).In addition, our IHC staining result showed that HDGF is also overexpressed in a variety of cancers including gliomas, breast cancer, prostate cancer and lyotheosarcoma which have not been reported up to date, demonstrating that HDGF could be a broader biomarker for cancer diagnosis and prognosis.In summary, high purity recombinant human GP73 and HDGF protein were expressed and purified. The GP73 and HDGF monoclonal, polyclonal antibodies were generated and then characterized by Western blot, IP, IF and IHC. We have developed a double antibody sandwich ELISA assay by using mAb as capture and pAb as tracer antibody. The application of the GP73 and HDGF sandwich ELISA assay were validated in the normal serum, liver cancer and lung cancer serum samples. An increased serum GP73 level in liver cancer and lung caner, or elevated HDGF level in lung cancer patients suggesting that these two systems have potential clinical application on cancer diagnosis and prognosis. |
PDF Full Text Request