- Volume 16 Issue 5
DOI QR Code
Preparation and Characterization of Anti-GP73 Monoclonal Antibodies and Development of Double-antibody Sandwich ELISA
- Li, Qi-Wen (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Chen, Hong-Bing (Department of Laboratory Medicine, Nanjing Children's Hospital, Nanjing Medical University) ;
- Li, Zhi-Yang (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Shen, Peng (Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, The Second Affiliated Hospital, Nanjing Medical University) ;
- Qu, Li-Li (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Gong, Lai-Ling (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Xu, Hong-Pan (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Pang, Lu (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University) ;
- Si, Jin (Department of Laboratory Medicine, The Second Affiliated Hospital, Nanjing Medical University)
- Published : 2015.03.18
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.
GP73;HCC;monoclonal antibody;serum marker;ELISA
Supported by : National Natural Science Foundation of China
- Al-Kubaisy WA, Obaid KJ, Noor NA, et al (2014). Hepatitis C virus prevalence and genotyping among hepatocellular carcinoma patients in Baghdad. Asian Pac J Cancer Prev, 15, 7725-30. https://doi.org/10.7314/APJCP.2014.15.18.7725
- Block TM, Marrero J, Gish RG, et al (2008). The degree of readiness of selected biomarkers for the early detection of hepatocellular carcinoma: Notes from a recent workshop. Cancer Biomark, 4, 19-33.
- Bao YX, Cao Q, Yang Y, et al (2013). Expression and prognostic significance of golgiglycoprotein73 (GP73) with epithelialmesenchymal transition (EMT) related molecules in hepatocellular carcinoma (HCC). Diagn Pathol, 8, 197. https://doi.org/10.1186/1746-1596-8-197
- Fimmel CJ, Wright L (2009). Golgi protein73 as a biomarker of hepatocellular cancer: development of a quantitative serum assay and expression studies in hepatic and extrahepatic malignancies. Hepatology, 49, 1421-3. https://doi.org/10.1002/hep.22994
- Ferlay J, Shin HR, Bray F, et al (2010). Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer, 127, 2893-917. https://doi.org/10.1002/ijc.25516
- Gu Y, Chen W, Zhao Y, et al (2009). Quantitative analysis of elevated serum golgi protein-73 expression in patients with liver diseases. Ann Clin Biochem, 46, 38-43. https://doi.org/10.1258/acb.2008.008088
- Hu JS, Wu DW, Liang S, Miao XY (2010). GP73, a resident golgi glycoprotein, is sensibility and specificity for hepatocellular carcinoma of diagnosis in a hepatitis B-endemic Asian population. Med Oncol, 27, 339-45. https://doi.org/10.1007/s12032-009-9215-y
- Hu B, Tian X, Sun J, et al (2013). Evaluation of individual and combined applications of serum biomarkers for diagnosis of hepatocellular carcinoma: a meta-analysis. Int J Mol Sci, 14, 23559-80. https://doi.org/10.3390/ijms141223559
- Hou SC, Xiao MB, Ni RZ, et al (2013). Serum GP73 is complementary to AFP and GGT-II for the diagnosis of hepatocellular carcinoma. Oncol Lett, 6, 1152-8.
- Kladney RD, Bulla GA, Guo L, et al (2000). GP73, a novel golgi-localized protein upregulated by viral infection. Gene, 249, 53-65. https://doi.org/10.1016/S0378-1119(00)00136-0
- Lok AS, Sterling RK, Everhart JE, et al (2010). Des-gammacarboxy prothrombin and alpha-fetoprotein as biomarkers for the early detection of hepatocellular carcinoma. Gastroenterology, 138, 493-502. https://doi.org/10.1053/j.gastro.2009.10.031
- Liu X, Wan X, Li Z, et al (2011). Golgi protein 73 (GP73), a useful serum marker in liver diseases. Clin Chem Lab Med, 49, 1311-6.
- Li X, Zhong X, Chen ZH, et al (2014). Hepatitis B virus DNA negativity acts as a favorable prognostic factor in hepatocellular carcinoma patients. Asian Pac J Cancer Prev, 15, 9635-41. https://doi.org/10.7314/APJCP.2014.15.22.9635
- Marrero JA, Feng Z, Wang Y, et al (2009). Alpha-fetoprotein, des-gamma carboxyprothrombin, and lectin-bound alpha-fetoprotein in early hepatocellular carcinoma. Gastroenterology, 137, 110-8. https://doi.org/10.1053/j.gastro.2009.04.005
- Mao Y, Yang H, Xu H, et al (2010). Golgi protein 73 (GOLPH2) is a valuable serum marker for hepatocellular carcinoma. Gut, 59, 1687-93. https://doi.org/10.1136/gut.2010.214916
- Masuzaki R, Karp SJ, Omata M (2012). New serum markers of hepatocellular carcinoma. Semin Oncol, 39, 434-39. https://doi.org/10.1053/j.seminoncol.2012.05.009
- Munaf A, Memon MS, Kumar P, et al (2014). Comparison of viral hepatitis-associated hepatocellular carcinoma due to HBV and HCV-cohort from liver clinics in Pakistan. Asian Pac J Cancer Prev, 15, 7563-7. https://doi.org/10.7314/APJCP.2014.15.18.7563
- Ozkan H, Erdal H, Tutkak H, et al (2011). Diagnostic and prognostic validity of golgi protein 73 in hepatocellular carcinoma. Digestion, 83, 83-8. https://doi.org/10.1159/000320379
- Qiao Y, Chen J, Li X, et al (2014). Serum GP73 is also a biomarker for diagnosing cirrhosis in population with chronic HBV infection. Clin Biochem, 47, 216-22.
- Riener MO, Stenner F, Liewen H, et al (2009). Golgi phosphoprotein 2 (GOLPH2) expression in liver tumors and its value as a serum marker in hepatocellular carcinomas. Hepatology, 49, 1602-9. https://doi.org/10.1002/hep.22843
- Stefaniuk P, Cianciara J, Wiercinska-Drapalo A (2010). Present and future possibilities for early diagnosis of hepatocellular carcinoma. World J Gastroenterol, 16, 418-24. https://doi.org/10.3748/wjg.v16.i4.418
- Sherman M (2010). Hepatocellular carcinoma: epidemiology, surveillance, and diagnosis. Semin Liver Dis, 30, 3-16. https://doi.org/10.1055/s-0030-1247128
- Sun Y, Yang H, Mao Y, et al (2011). Increased golgi protein 73 expression in hepatocellular carcinoma tissue correlates with tumor aggression but not survival. J Gastroenterol Hepatol, 26, 1207-12. https://doi.org/10.1111/j.1440-1746.2011.06733.x
- Shan SG, Gao YT, Xu YJ, et al (2013). Gradually increased golgi protein 73 expression in the progression of benign liver diseases to precancerous lesions and hepatocellular carcinoma correlates with prognosis of patients. Hepatol Res, 43, 1199-210. https://doi.org/10.1111/hepr.12078
- Somboon K, Siramolpiwat S, Vilaichone RK (2014). Epidemiology and survival of hepatocellular carcinoma in the central region of Thailand. Asian Pac J Cancer Prev, 15, 3567-70. https://doi.org/10.7314/APJCP.2014.15.8.3567
- Sangmala P, Chaikledkaew U, Tanwandee T, Pongchareonsuk P (2014). Economic evaluation and budget impact analysis of the surveillance program for hepatocellular carcinoma in Thai chronic hepatitis B patients. Asian Pac J Cancer Prev, 15, 8993-9004. https://doi.org/10.7314/APJCP.2014.15.20.8993
- Thun MJ, DeLancey JO, Center MM, et al (2010). The global burden of cancer: priorities for prevention. Carcinogenesis, 31, 100-10. Wang M, Long RE, Comunale MA, et al (2009). Novel fucosylated biomarkers for the early detection of hepatocellular carcinoma. Cancer Epidemiol Biomarkers Prev, 18, 1914-21. https://doi.org/10.1158/1055-9965.EPI-08-0980
- Witjes CD, van Aalten SM, Steyerberg EW, et al (2013). Recently introduced biomarkers for screening of hepatocellular carcinoma: a systematic review and meta-analysis. HepaInter, 7, 59-64.
- Wang NY, Wang C, Li W, et al (2014). Prognostic value of serum AFP, AFP-L3, and GP73 in monitoring short-term treatment response and recurrence of hepatocellular carcinoma after radiofrequency ablation. Asian Pac J Cancer Prev, 15, 1539-44. https://doi.org/10.7314/APJCP.2014.15.4.1539
Wang Y, Yang H, Xu H, et al (2014). Golgi protein 73, not Glypican-3, may be a tumor marker complementary to
$\alpha$-Fetoprotein for hepatocellular carcinoma diagnosis. J Gastroenterol Hepatol, 29, 597-602. https://doi.org/10.1111/jgh.12461
- Xu H, Tian YN, Dun BY, et al (2014). A novel monoclonal antibody induces cancer cell apoptosis and enhances the activity of chemotherapeutic drugs. Asian Pac J Cancer Prev, 15, 4423-8. https://doi.org/10.7314/APJCP.2014.15.11.4423
Yamashita T, Forgues M, Wang W, et al (2008). EpCAM and
$\alpha$-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res, 68, 1451-61. https://doi.org/10.1158/0008-5472.CAN-07-6013
- Yamamoto K, Imamura H, Matsuyama Y, et al (2010). AFP, AFP-L3, DCP, and GP73 as markers for monitoring treatment response and recurrence and as surrogate markers of clinicopathological variables of HCC. J Gastroenterol, 45, 1272-82. https://doi.org/10.1007/s00535-010-0278-5
- Zhao XY, Li N, Ding HG, Jiang FF (2010). Detection and evaluation of serum GP73, a resident golgi glycoprotein, as a marker in diagnosis of hepatocellular carcinoma. Chin J Oncol, 32, 943-6.
- Zhou Y, Yin X, Ying J, Zhang B (2012). Golgi protein 73 versus alpha-fetoprotein as a biomarker for hepatocellular carcinoma: a diagnostic meta-analysis. BMC Cancer, 12, 17. https://doi.org/10.1186/1471-2407-12-17
- Zhang A, Cao B (2012). Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA. J Biome Res, 26, 467-73. https://doi.org/10.7555/JBR.26.20120057
- Abnormal Expression of Golgi Protein 73 in Clinical Values and Their Role in HBV-Related Hepatocellular Carcinoma Diagnosis and Prognosis vol.15, pp.12, 2015, https://doi.org/10.5812/hepatmon.32918
- MMSA-1 expression pattern in multiple myeloma and its clinical significance vol.16, pp.4, 2016, https://doi.org/10.1007/s10238-015-0393-y
- A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection vol.7, pp.1, 2017, https://doi.org/10.1038/srep40090
- Golgi protein 73 and its diagnostic value in liver diseases pp.09607722, 2018, https://doi.org/10.1111/cpr.12538