Detection Of Polypeptide ELISA And Its Application Of Relative Research Of Early Diagnosis Of Hepatocellular Carcinoma

Hepatocellular carcinoma is one of the most frequently diagnosed disease and one ofmalignant tumor whose prognosis effect is poorer worldwide.It is closely linked to chronichepatitis B virus(HBV) and Liver cirrhosis. Therefore,early diagnosis technology of HCChas special research value and social significance on the prevention and diagnosis of HCCand liver disease’s developmental process. AFP,the most utilized surveillance biomarker,isused limitly in diagnosis of tumor under3cm in diameter which has bad sensitivity andspecificity for testing.Currently, many researchers have found serum peptidome play animportant role as an important source of tumor biomarkers,which have great researchsignificance in early diagnosis for tumor.Pep5is one of potential tumor biomarker that hadbeen reported. In this paper, a method of ELISA is established to detect serum peptidemarkers Pep5which is used in the analysis of serum peptide from patient with HCC toevaluate the diagnostic value of liver cancer and clinical significance.Firstly,we prepared three cell lines of Pep5monoclonal antibodies,2F6,4F6and7G7,using the traditional method of monoclonal antibody.The sensitivity of supernatantantibody is10ng/ml.41ml ascites antibody are prepared and its titer is1:2000000. Atthe same time to purify and Enzyme Labeling, total28ml purification antibody weregotten, the titer of purified antibody is0.5ng/ml(2F6and7F7) and5ng/ml(4F6).Purified antibody with high specificity and sensitivity supply an effective and abundantantibody sources for the further research.Moreover, we choose three methods of ELISA to detect Pep5, direct method, sandwichmethod and competitive ELISA method. The factors that affect the performance ofELISA were optimized for high sensitivity. Direct ELISA: concentration of HRP-2F6is1.25μg/ml, serum is1:20dilution; sandwich ELISA:7G7is1:2000dilution, concentration of HRP-2F6is5μg/ml and serum is1:10dilution; competitive ELISA:Pep5is0.5ng,HRP-2F6is1:20000dilution, serum is1:5dilution. Using these three methods to test some samples,direct ELISA method has the best optimizational effect(P<10-6)ondistinguishing liver cancer group from normal group and its sensitivity is93.3%andspecificity is83.3%。we evalued the optimized direct ELISA on linear range, limit of detection,precision andrecovery.The linear range of the method is1.5-20ng/ml with a detection limit of1.24mg/ml and correlation coefficient is0.9959. three different concentration Synthesisantigen which is9、12、15ng/ml were taken respectively to be tested.The intra-andinter-assay coefficients of variation(CV)is1.44%-6.17%,the cv between within-day andday to day is2.89%-4.89%.The recovery is98.98%,99.61%,101.58%respectively.Evaluation results show that the optimized method is suitable for the detection of serumpeptides.Finally, We detected420serum using optimized method of direct ELISA.The controlgroup and HCC group have significant discriminant analysis adjusted (P values <0.001).When the cut-off of Pep5is7.48ng/ml, sensitivity is80.8%and specificity is96.2%of diagnosis of HCC.We tested94HCC serum,the positive rate of Pep5(90.4%)is higher than the positive rate of AFP (63.8%).The detection rate of HCC is increasedto94.7%by combined detection of AFP with Pep5.The results show that the detectionof Pep5by this method may have important significance for the diagnosis of HCC.