Detection Of MUC1 Protein In Peripheral Blood Of Adenocarcinoma Patients By Indirect Sandwich ELISA

MUC1 is one of the tumor markers for adenocarcinoma diagnosis.Analysis of MUC1 protein in peripheral blood has very important clinical application values to the diagnoses and prognosis of many adenocarcinomas. At present,the methods to detect the soluble MUC1 protein in peripheral blood include flow cytometry, immunocytochemical staining,enzyme-linked immunosorbent assay (ELISA) and Real Time RT-PCR.The sensitivity of flow cytometry and immunocytochemical staining is low.Even though the sensitivity of Real Time RT-PCR is high, because of the interference of normal tissues and blood cell genes,the false positive rate is about 10%, and it is rarely applied to clinical diagnosis due to the complicated procedures. ELISA is commonly used to detect the soluble MUC1 protein in peripheral blood.Two kinds of monoclonal antibodies were used to prepare the commercial ELISA kits.Tumors expose different antigen epitopes in different individuals,even though application of monoclonal antibodies can greatly improve the specificity of antigen recognition,it only can bind to a certain epitope, therefore,the detection ratio is decreased.We used anti-rat polyclonal antibodies and anti-rabbit polyclonal antibodies to establish the indirect sandwish ELISA.As it could react to different epitopes,so it improved the sensivity of detection of MUC1 protein in peripheral blood.In this study,firstly we optimized conditions forming the sandwich ELISA kit,then used it to detect MUC1 protein of many adenocarcinomas.We compared the the sensitivity of sandwich ELISA and CA15-3 kit through detecting breast cancer patients,benign breast disease and healthy individuals.Methods and Results:1. A large pGEX-MUC1/ BL21 cultured in LB medium were broken by sonicate and removed supernatant containing fusion protein.MUC1-GST protein was purified by Glutathione Sepharose 4B affinity chromatography and further purified by non-denaturing PAGE.2. A large pMAL- MUC1/DH5a cultured in LB medium were broken by sonicate and removed supernatant containing fusion protein.MUC1-MBP protein was purified by amylose resin affinity chromatography.3. The rabbit and rats were immunized by MUC1-GST and MUC1-MBP fusion protein respectively.We detected the titers of rabbit anti-MUC1 polyclonal antibody and rat anti-MUC1 polyclonal antibody. The titers of two antibodies were 320000 and 32000±4200 respectively.4. We purified rabbit anti-MUC1 and rat anti-MUC1 polyclonal antibodies with saturated (NH4)2SO4, Protein A/G, anti-GST antibody and removed other proteins.5. Preparation of MUC1 standard we digested MUC1-GST fusion protein with thrombin to obtain MUC1 standard.6. The optimum experimental conditions of indirect sandwich ELISA coating antibody: rabbit anti-MUC1 antibody,0.25μg∕well;detection antibody:rat anti-MUC1 antibody,140μg∕ml ;secondary antibody:goat peroxidase-conjugated anti-rat IgG, 1.6μg∕ml,the sensitivity of the indirect sandwich ELISA is 2 ng∕ml.7. We detect MUC1 protein of many adenocarcinoma patients by the indirect sandwich ELISA.The specificity of 120 healthy individuals is 100%;the sensitivity of breast cancer patients is 100% and the specificity of benign breast diseases is 78%;the sensitivity of gastric cancer patients is 88.2% and the specificity of gastric benign patients is 93%;the sensitivity of lung cancer patients is 87.5% and the specificity of lung benign patients is 100%;the sensitivity of gynecologic cancers is 83% and the specificity of gynecologic benign patients is 92.5%;the sensitivity of liver cancer patients is 36.4% and the specificity of liver benign patients is 93%.8. We detected breast cancer patients, benign breast disease and healthy individuals using the sandwich ELISA and CA15-3 kit respectively.ROC curves were drawn to compare the diagnostic accuracy of the two methods and the results suggested the accuracy of diagnosis of breast cancer was significantly higher in the sandwich ELISA method than CA15-3 kit.The established anti-MUC1 polyclonal antibody indirect sandwich ELISA kit has higher sensitivity in diagnoses of breast cancer,gastrointestinal cancer,lung cancer,and gynecologic cancers and it is expected to be used as a conventional ELISA kit in clinical diagnosis.Especially it can be applied to breast cancer mass screening and early diagnosis.