Multiple Tumor Markers Combined Detection In Colorectal Cancer And The Establishment Of Pro-Gastrin-Releasing-Peptide ELISA Assay

[Objective] To study the macrophages inhibitory cytokine-1(MIC-1) as a new tumor marker for early stage colorectal cancer, and explore the feasibility of the early detection by a combination of multiple biomarkers in colorectal cancer patients.[Methods] Four hundred and twenty-nine serum samples from the patients with different TNM phases of colorectal cancer and129samples from healthy controls were collected in the Clinic of Cancer Hospital, CAMS. The tumor markers MIC-1, CA19-9, CEA and TPS were detected by self-developed MIC-1double antibody sandwich ELISA Kit and Roche Cobas601ECL analyzer, respectively. The serum levels were evaluated in different stages of the patients and the relationship among the markers was also assessed.[Results] The serum level of MIC-1in patients with colorectal cancer is significantly higher than that of controls,(1045.88±892.67pg/ml,398.04±263.19pg/ml; P<0.001). A stepwise increase of MIC-1level was noted in patients with the progress of the disease,and positively correlated with the depth of tumor invasion(P=0.001),lymph node metastasis (P=0.007) and remote metastasis (P<0.001). The results also showed that the sensitivity of the combination of MIC-1with CEA and TPS was61.3%, which was significantly higher than that of CEA(25.6%), the one which was commonly used in clinical diagnosis.[Conclusions] MIC-1is a valuable biomarker for detection of colorectal cancer and the combination of MIC-1with CEA and TPS maybe have an important value for early detecting and screening of the colorectal cancer in high risk population. [Objective] To construct a Pro-GRP (31-98) peptide prokaryotic vector expressing system, establish monoclonal antibody hybridoma cell lines with stable secretion antibody against Pro-GRP (31-98), and set a detection system for Pro-GRP (31-98) in human serum.[Method] Total RNA was extracted from a patient suffer from small cell lung cancer(SCLC) and the Pro-GRP (31-98) gene sequence was cloned and linked into prokaryotic plasmid pGEX-4T-1.The Pro-GRP recombinant protein was expressed in E.coli BL21. The purified recombinant protein was used as antigen to immunize the mice and the hybridoma cell lines of secreting monoclonal antibody against Pro-GRP (31-98) were set up with the hybridoma technique. The Pro-GRP (31-98) monoclonal antibody and polyclonal antibody were marched to get a quantitative detection ELISA assay. Finally, sixteen serum samples from SCLC patients were tested..[Result] The Pro-GRP (31-98) recombinant protein was expressed efficiently in BL21E. Coli and twelve hybridoma cell lines secreting monoclonal antibody against Pro-GRP(31-98) were built succsesfully. Monoclonal antibody and polyclonal antibody were purified by affinity chromatography. A double-antibody sandwich ELISA was used (Polyclonal antibody was labeled with biotin as the detection antibody and monoclonal antibody was coated at a concentration of1ug/mL in96well plates) for detecting the serum samples from16SCLC patients. The average level of Pro-GRP in serum is576.87±514.06ng/L (Mean±SD) with a508.35ng/L median value, and the sensitivity of Pro-GRP for detecting SCLC is75%.[Conclusion] Monoclonal and polyclonal antibodies against Pro-GRP were produced successfully. ELISA assay was built and16serum samples of SCLC patients were tested successfully.