Preliminary Rearch On Joint Detection Of Serum Peptides Pep4and Related Peptides For Tumor Diagnosis

Abstract:Objective:To develop new serum peptide biomarkers and its detecting technologies is significant for diagnosis of tumor. The joint detection of different serum peptide was practically important. During early research, Peptide Pep4and the relative peptide Pep9、Pep15were proved to act as an potential biomarkers for tumor diagnosis. But research on detection method of the three peptides and the application value of them has not proceeded. In this paper, we aimed to establish a method of ELSIA for detection of peptide biomarker Pep4, and the method for joint detection of HKP09、HKP15. The content of biomarkers in different tumors was investigated, and the potential value of Pep4and the relative peptide act as an biomarker was assessed. Further more, we discussed the significant promoting effect of joint detection for tumor diagnosis. Methods:The Carbodiimide coupling method was adopted for preparation of immunogen of Pep4HKP09and HKP15. And the mouse monoclonal antibodies were prepared by immune of animals with the peptide coupling immunoge. The antibodies were purified via affinity chromatography and were labeled with HRP. The ELISA method was established for detection of target peptide, and key factors were optimized. The ELSIA method optimized was applied to detection of clinical tumor sample(the amount of serum sample was192, altogether), and the content of Pep4, HKP09and HKP15in different tumor was analyzed via a statistical software named Medcalc. Results：Finally, we obtained3antibodies of peptide Pep4. And titer of the purified antibody2B7、7D8、5A7was1:320000、1:160000.1:160000. we obtained2antibodies (2B7、3D11) of peptide HKP09、 HKP15. The titer of the purified antibody3D11was1:2000、1:4000apparently to HKP09. HKP15.3antibodies of Pep4and the relative peptide antibody3D11was labeled with HRP. Compared with the purity antibodies, titer of the labeled antibodies didn’t changed so much. We established3different type of ELISA method for detection of Pep4, direct method, sandwich method and indirect method. All the3method was used to test clinical samples, and the indirect ELSIA method was proved to be the suitable method for detection of serum peptide Pep4. And the ratio of dilution for antibody2B7was optimized to be1:20000, the range of linearity was80-1000ng/mL, r=0.9946. The serum of6different type of tumor could be distinguished notably. And the P value was less than0.05. The specificity and sensitivity of diagnosis for liver cancer and breast cancer were both greater than90%. We established a sandwich ELISA method for detection of Pep9and Pep15of "combination of Protein-peptide". The coat quality of antibody IgG-HKa was optimized to be100ng, and HRP-IgG-peptide was diluted as1:1000. The range of linearity was6-120ng/mL (r=0.9997), with a detection limit of1.7ng/mL. The serum of6different type of tumor could be distinguished notably. And the P value was less than0.05. The specificity and sensitivity of diagnosis for sausage cancer was the highest. By contrasting and analyzing the on detecting results of pep4and Pep9, Pep15, we found that the biomarkers agreed with each other very well in diagnosis of esophagus cancer (Kappa=0.55). The result of jointing detection shows good consistency with clinical diagnosis (Kappa>0.6), especially for diagnosis of breast cancer. Conclusions:The cell lines of monoclonal antibodies of pep4and Pep9, Pep15presented high specificity and sensitivity. The cell lines of monoclonal antibodies also provided qualified and sufficient matetial for the establishment of ELISA method and the joint detection of different biomarkers. Different type of ELSIA method was established for detection of different biomarkers. The indirect ELSIA method was more perfect for detection of Pep4, and the sandwich ELSIA method of "protein-peptide" was suitable for joint detection of the relative peptides. Application value of Pep4and the relative peptides was proved by detecting different serum of tumors. Pep4and the relative peptides could act as biomarkers for diagnosis of different tumor, and the role they played are relatively complemented. There are24figures,12tables and45literatures in this paper.