Identification Of Autoantigens For Biliary Atresia Using Human Proteome Microarrays

BackgroundBiliary atresia(BA)is a hepatobiliary disease in which cholangiocytes are damaged by inflammation and progressive fibrous obstruction of hepatic bile ducts.The disease progresses so rapidly that it can develop into cirrhosis or liver failure within a few weeks after birth.The pathogenesis of BA is unclear,and the diagnosis mainly depends on cholangiography,but there is still a lack of specific and effective early diagnostic markers in clinic.Many studies have shown that the etiology of BA is closely related to the inflammatory injury of bile duct caused by autoimmune response induced by perinatal virus infection,so it is important to find out the autoantigens related to its mechanism.Our previous study have found some autoantibodies which may be helpful for diagnosis and prognosis,therefore,we are going to screen and identify specific autoantigens by human proteome chip,which will provide a new serum marker for early diagnosis and prognosis for this disease,as well as new clues for elucidating the mechanism of immune injury in bile ducts.ObjectiveTo identify autoantigens of BA which make sense to its early diagnosis,disease progression and prognosis evaluation by using human proteome microarray,and to establish a newly method of enzyme-linked immunosorbent assay for measuring serum autoantibody in BA.MethodFirst of all,on the basis of antigen-antibody binding reaction,a human proteome microarray was used to screen the autoimmune target antigens in a cohorts(20 BA,5disease controls,5 healthy controls)by serum "mixed pool" method.Then we fabricated a BA-focused microarray with these newly identified candidates with a larger additional cohorts of 80 BA patients and 160 controls,to further verify the specific autoantigens for BA.Finally,we developed ELISA detecting serum antibodies to the specific antigens and tested its levels in sera from 120 BA patients and 233 controls,to analyze the potential values in diagnosis and prognosis for BA.Result1.A total of 281 candidate autoantigens including 105 Ig G and 176 Ig M were screened out in the patients with BA,in which 26 candidate autoantigens responsed to both of Ig G and Ig M simultaneously.Forty-nine BA-related differential autoantigens were identified by the screening criteria of fold change>2 and stable expression.GO functional analysis revealed that these differential autoantigens mainly involved in the regulation of cell development,proliferation,apoptosis and the formation of biliary tracts.The prevalence of antibodies against copine VI(CENP6),ubiquilin 3(UBQLN3),programmed cell death2(PDCD2),SPEG complex locus(SPEG),recombination signal binding protein for immunoglobulin kappa J region(RBPJ)in BA were significantly higher than that in control groups(100% vs.0%,P<0.05).2.According to the criteria of average signal fold change >1.2,discrimination ≥55%,AUC >0.5 and P value,the results of experiments with focused microarray showed that under the premise of ensuring 90% specificity,the sensitivity of the autoantigens such as RBPJ and canopy homologous protein 4(CNPY4)to distinguish NC and DC was 50%and 30%,respectively.After enlarging the sample size,the response to RBPJ in different structures still showed high reactivity,and its signal values were positively correlated with ALT,AST,ALP,γ-GT,TBIL,DBIL and TBA(P<0.001),but negatively correlated with age,Ig A and Ig M(P<0.05).Furthermore,the positive rate of antibodies to RBPJ(Q06330-2,Q06330-4,Q06330-6)did not correlate with status of CMV and EBV infections in BA patients.However,the response to RBPJ was up-regulated with the aggravation of liver inflammatory activity,and positively correlated to the degree of fibrosis,but the response did not correlate with cholangitis occurrence and jaundice regression within half a year after Kasai operation.3.An indirect ELISA method detecting serum anti-RBPJ autoantibody was successfully constructed using the purified recombinant RBPJ protein as the coating antigen.The levels of anti-RBPJ antibodies in sera of 120 patients with BA showed that the overall OD value of BA group was significantly higher than that of the control group(F=13.52,P<0.001).The AUC of BA and non-BA diagnosed by anti-RBPJ antibody was 0.694(P<0.001,95%CI: 0.633～0.754),with the Yoden index 0.339,sensitivity 55.8% and specificity of 78.1% at the optimal cut-off of 0.427.The levels of anti-RBPJ in BA patients were weakly positively related to ALT,AST,γ-GT,TBIL,DBIL,TBA,and weakly negatively correlated with age(P<0.001),but there was no correlation with ALP,Ig G,Ig M,C3,C4 and Ig E.Taking the OD value greater than mean+3SD of normal controls as the cut-off value,there was no significant correlation observed between the positive rate of antibody and the severity of fibrosis(P>0.05).77 children who underwent Kasai operation were followed up for 3-6 months.25.9% of the patients with positive anti-RBPJ occurred postoperative cholangitis,and 44.2% were unsatisfied with postoperative jaundice.The existence of anti-RBPJ before operation did not prognoses occurrence of cholangitis and jaundice regression(P>0.05).Conclusion1.RBPJ and CNPY4 have been screened as the specific autoantigens for biliary atresia using by human proteome microarray.2.An indirect ELISA method for detecting serum anti-RBPJ autoantibody has been successfully established.3.Anti-RBPJ antibody could be used as a novel serum biomarker to distinguish BA from non-BA.