Preparation And Identification Of Antibody Against HMIC-1

Objective:To make and identify the antibody against hMIC-1 for application in immnnoassay and study of gene functions.Methods:BALB/c mice were immunized with the recombinant hMIC-1 protein expressed in Pichia Pastoris,and monoclonal antibody(McAb) against hMIC-1 was produced by the classical hybridoma technique.Polyclonal antibody against hMIC-1 was obtained from New Zealand White rabbits immunized by multi-sites and multi-time periods.These anti-hMIC-1 antibodies were characterized by ELISA,Western blot and immunohistochemical staining.Furthermore,a enzyme-linked immunosorbent assay for the quantification of serum hMIC-1 was developed.Results:A hybridoma line was successfully established to secrete the McAb against hMIC-1, which named 2G7.By Western blot,it was found that the McAb 2G7 recognized the reducing and noreducing hMIC-1 in colon cancers,and had no cross-reaction with Pichia Pastoris crude extract.The characterized McAb was applied for immunohistochemical staining in colon cancers and prostate cancer,and displayed strong specificity and moderate affinity compared with the commercial McAb.The results showed that MIC-1 staining distribution and intensity were consistent with that of the commercial antibody,and expression of MIC-1 mainly located in cytoplasm.The rabbit anti-hMIC-1 polyclonal antibody was able to recognize the hMIC-1 expressed in colon cancer,and the titer was 10~6.However,ELISA wasn't developed.Conclusion:The results indicated that it proved success for the development of anti-hMIC-1 monoclonal antibody raised by immunization of BALB/c mice with the purified hMIC-1.In this study,the McAb 2G7 as a detection antibody may be applied in Western blot. Objective:To evaluate serum macrophage inhibitory cytokine-1(MIC-1) as a novel marker of pancreatic cancer.Methods:Serum MIC-1 levels were determined by sandwich ELISA assay in 101 patients with pancreatic adenocarcinomas,in 10 patients with benign pancreatic tumors,and in 50 healthy control subjects.The diagnostic performance of serum MIC-1 as a marker of pancreatic cancer was compared with that of serum CA199.RESULTS:MIC-1 levels were significantly higher in patients with pancreatic adenocarcin -oma than in those with benign pancreatic neoplasms or healthy controls.Diagnostic acc -uracy of pancreatic adenocarcinomas with MIC-1(sensitivity 81.2%,specificity 94%,positive predictive value96.5%,negative predictive value 71.2%,AUC 0.92) was higher than CA199 (sensitivity,72.4%specificity 89.6%,positive predictive value 93.4%,negative predictive value 61.4%,AUC 0.86),and the combination of MIC-1 and CA199 significantly improved diagnostic sensitivity(91.1%).Conclusion:MIC-1 can become a novel tumor marker and aid in the diagnosis of pancreatic adenocarcinoma.