| Autoantibodies to tumor associated antigen can be a kind of tumor markers with high specificity and low sensitivity in the early stage of Hepatocellular carcinoma.The aims of this article were researched from three parts in the following: Part ?: Detection of Autoantibodies in Primary Liver Cancer and other Non-Autoimmune Liver DiseaseThis part was based on the clinic data form our hospital.The two kinds of autoantibodies of clinical commonly used were detected by linear immunoblotting in the total 525 patient's serum which includes Non-autoimmune hepatitis(NAIH,75),Non-autoimmune cirrhosis(NAIC,118)and Primary Liver Cancer(PLC,332)were collected in hospital from 2012-2014 to observe the positive rate of these disease and to explore its clinical value in the diagnosis of PLC.The result shows that the expression of autoantibodies with high positive rate in non-autoimmune liver disease were anti-AMA-M2 antibody(5.3%,10.2%,6.6%),anti-SSA antibody(4%,8.5%,7.2%)and anti-CENP-B antibody(4%,5.9%,3%).However,there were no significant difference among these groups(P>0.05).There were significant difference in the positive rate of anti-CENP-B,anti-AMA-M2,anti-SSA,anti-SSB and anti-SLA/LP in the group of negative HBsAg PLC compared with those in positive group.The positive rate of anti-CENP-B antibody in the PLC group with lower lever AFP(<20mg/l)was significant higher than the group with high lever AFP(?20mg/l)(P<0.05).Through Spearman correlation analysis,we found that anti-AMA-M2 and anti-CENP-B antibody were negatively correlated with AFP;Anti-AMA-M2 antibody was positively correlated with alkaline phosphatase,prealbumin and hemoglobin;Anti-SSA antibody and anti-CENP-B antibody were positively correlated with age;Anti-SSA antibody was positively correlated with serum albumin and hemoglobin(P<0.05).It showed that combined detections of anti-AMA-M2,anti-SSA and anti-CENP-B could be used as one of the independent risk factors for PLC(OR=2.187,P<0.05,95%CI 1.133-4.222)by multivariate regression analysis.Therefore,this study demonstrated there was no significant difference in the positive rate of 15 autoantibodies among PLC,NAIC and NAIH patients.It was higher positive rate of these autoantibodies in PLC group with negative AFP and HBsAg than positive.The combined detections of Anti-AMA-M2,anti-CENP-B and anti-SSA antibody could be used as an independent risk factor for PLC,but the related mechanism of them needs further study.Part ?:Screening of Autoantibodies for the Early Hepatocellular Carcinoma by Using HuProtTM High Throughput Protein ChipIn this part,we used high throughput protein microarray technology to conduct the research which was divided into two steps.The first step was selecting the 70 significant autoantibodies form the 18 serum samples which contain 9 HCC patients and 9 normal health persons which used the 19364 proteins' microarray.The second step was detecting the 70 autoantibodies in the 72 HCC patients and the 60 controls which used thecustomized microarray.The cut-off was set into 95 percent of all the controls result titers.We found 18 autoantibodies was high than 10%,and 5 form them was higher than 15%,respectively were SULT2A1(18.06%),HN1(16.67%),AAMP(16.67%),CRCP(15.28%),ANKRD45(15.28%).Further research on the co-expression of autoantibodies in the same HCC patients is necessary.The highest co-expression autoantibodies were anti-SULT2A1 and anti-CRCP(7,the number of the co-patients),which the Kappa coefficient was 0.500.the most other autoantibodies' Kappa coefficient were range from-0.2 to 0.2.then we got the best combination of two,three,four,five and six autoantibodies in the diagnosis of the early stage of Hepatocellular carcinoma.The yonder' index of best six performance autoantibodies' combination was 0.383,and their sensitivity and specificity respectively were 66.7% and 71.6%.Anti-SULT2A1,anti-HN1,antiANKRD45,anti-VCL and anti-AAMP was including in the best six performance combination.The two stages in this part were not only opened up a new way for screening the clinical autoantibodies for the early cancer,and provided many titps for the combination of autoantibodies in the diagnosis of the HCC.Part ? :Establishing the Method of ELISA for Detecting Autoantibodies in the Hepatocellular CarcinomaAccording to the result of the second part above,we mainly selected three Related autoantibodies for the remained researches in HCC patients.In this part,we focus on the potent of how to establish the stable ELISA method for detecting the autoantibodies in serum.the part mainly includes two aspects: the first,optimize the key parameter of the type of diluent in each stage of the ELISA method(serum dilution,coated solution and HRP enzyme labeled antibody IgG dilution);the second: optimizing the best concentration of different antibodies and coated protein.In previous basis design of ELISA,we established the stable method to detect the autoantibodies in the serum at the end of time.We collected 204 patients,included144 HCC patients,20 LC patients,20 CHB patients and 20 normal people.After detection,by setting the 95% of the result of the control group as the cut-off,the positive of three autoantibodies in the HCC group respectively were 9.72%(P>0.05),4.86%(p>0.05),24.31%(p<0.01).Spearman correlation analysis revealed that anti GAB1 autoantibodies was related anti-CRCP autoantibodies(r=0.377,p<0.001).Meanwhile,both the anti-GAB1 and anti-CRCP autoantibodies were correlated with tumor size(r=0.229,0.284,p<0.01),but the concrete mechanism needs further research.In this study,we made an improvement to choose the high-response or low-response as positive and negative autoantibodies to increase the reliability of ELISA method.However,the three selected autoantibodies above have a little diagnostic value for HCC patients.More research will be need to confirm the potential value of autoantibodies as biomarkers for HCC. |
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