| BackgroundHepatitis C caused by Hepatitis C virus(HCV)and syphilis caused by Treponema Pallidum(TP)are common blood-borne diseases,mainly through blood transfusions,blood products and sexual transmission.HCV infection can cause acute or chronic hepatitis and will develop into cirhosis or liver cancer.TP infection can damage multi-organs,even threaten life?It can also threaten health of the next generation by mother-to-child transmission.The World Health Organization(WHO)reports that the incidence of hepatitis C and syphilis increase year by year and is a serious public health problem at present.There are curently no effective vaccines to prevent HCV and TP infection.These two diseases easily lead to misdiagnosis and delay treatment because the early infection has no obvious clinical symptoms and signs.Taken laboratory test results as the main clinical diagnostic indicators,the accurate results are of great significance to effectively treat and block disease spread.In the past decade,enzyme-linked immunosorbence assay(ELISA)and chemiluminescent immunoassay(CLIA)have been widely used in asymptomatic screening and disease diagnosis with high-throughtput and high degree of automation.However,lots of studies have shown that Anti-HCV and Anti-TP screening tests in both ELIA and CLIA have different false positive rates,especially in low-prevalence epidemics.There is no consensus and direct evidence on the reasons that caused false-positive.The false positive rates of different detection methods are different,which may be associated with the reaction system,antigenic components and epitopes used in the assay.TP47 protein is the most abundant,antigenic and specific antigen of TP,which was often used in syphilis detection assay.However,studies have reported that a sequence of TP47 has a high homology with human fibronectin,which may be associated with the cross-reaction.Using bioinformatics technology to predict specific antigen epitopes may reduce the cross-reaction.It may be one of the strategies to solve the false positive reaction for improvement of the specificity based on high sensitivity.Objective1.The false-positives of CLIA in Anti-HCV and Anti-TP detection of the borderline specimens were investigated by Architect i2000 and HISCL 5000.It would provide information and reference for laboratory diagnosis and treatment of HCV and TP infection.2.In order to explore the strategy to solve the problem of false positive,the double-antigen sandwich ELISA using TP47 E antigen epitope or TP47 whole protein was developed to evaluate the sensitivity and specificity of Anti-TP detection.The feasibility of using the antigen epitope to solve the false positive problem was explored.Methods1.A total of 161 cases of Anti-HCV positive borderline specimens(1?S/CO?5)dectected by Architect i2000 were collected.All specimens were detected by HISCL 5000,highly sensitive HCV nucleic acid(RNA)quantitative test and recombinant immunoblot assay(RIBA).SPSS 21.0 statistical software was used to test single factor analysis of variance and LSD-t test.A value of P < 0.05 was considered statistically significant.2.A total of 334 cases of Anti-TP positive specimens(1?S/CO?10)detected by Architect i2000 were collected.All specimens were detected by HISCL 5000 and Treponema pallidum particle agglutination(TPPA).Recombinant immunoblot assay(RIBA)was used to detect the specimens with inconsistant results examined by two kinds of chemiluminescence assay.SPSS 21.0 statistical software was used to test single factor analysis of variance and LSD-t test.A value of P < 0.05 was considered statistically significant.3.B-cell epitopes were predicted using EMBOSS,Bepipred and IEDB softwares.The amino acid sequence located at 81-120 of TP47 protein was selected as the dominant antigen epitope.The pET28b-TP47 E recombinant vector constructed by genetic engineering technology was identified by restriction endonuclease digestion and sequencing.The recombinant protein was induced by IPTG,purified by nickel ion affinity chromatography and identified by Western Blotting(WB).4.The recombinant protein was labeled with the method of sodium periodate.Double-antigen sandwich ELISA assays were developed using TP47 E or TP47 antigen,respectively.The protein coating concentration,blocking condition,serum dilution,serum reactive time,concentration of labeled antigen,labeled antigen reactive time and substrate reactive time were optimized.46 cases of Anti-TP negative serum of TPPA and 91 cases of Anti-TP positive borderline specimens(1?S/CO?10)of Architect i2000 were detected by double-antigen sandwich ELISA.The sensitivity and specificity of ELISA using TP47 E and TP47 protein were evaluated.Results1.A total of 23 specimens were found Anti-HCV positive and 138 were showed negative by HISCL 5000.All specimens were negative by highly sensitive HCV RNA quantitative test.A total of 13 cases were positive,45 cases were indeterminate,and 104 cases were negative by RIBA.Taken RIBA as the standard,the coincidence rate of Architect i2000 and RIBA was 8.1%(13/161)and the coincidence rate of HISCL 5000 and RIBA was 69.6%(112/161).The false positive specimens of Architect accounted for 91.9%(148/161)of the total positive samples and the false positive specimens of HISCL accounted for 52.2%(12/23)of the total positive specimens.The average COI values of the RIBA positive group,the indeterminate group and the negative group in HISCL 5000 were 3.0,1.0 and 0.2,respectively.The average S/CO values of the RIBA positive group,the indeterminate group and the negative group in Architect i2000 were 2.8,2.2 and 2.0,respectively.There was statistically significant different among groups in HISCL 5000 and Architect i2000(P=0.000<0.01 and P=0.027<0.05).2.A total of 209 specimens were found Anti-TP positive and 125 showed negative by HISCL 5000.The coincidence rate of the Anti-TP antibody results of two CLIA was 62.6%(209/334).Taken TPPA as the standard,the coincidence rate of Architect i2000 and HISCL 5000 with TPPA was 61.7%(206/334)and 85.3%(285/334),respectively.The false positive specimens of Architect accounted for 38.3%(128/334)of the total positive samples and the false positive specimens of HISCL accounted for 12.4%(26/209)of the total positive specimens.The average S/CO values of Architect i2000 and the average COI values of HISCL 5000 of TPPA positive group and negative group were 4.8 and 2.5,3.5 and 0.8,respectively.There was statistically significant different in S/CO value or COI value between the two groups(all P =0.000<0.05).The specimens with inconsistent results of Architect i2000 were detected by RIBA.6.4% of the specimens were positive(8/125),67.2% were negative(84/125),26.4% were indeterminate(33/125).3.According to the prediction results of B-cell epitopes,the amino acid sequence located at 81-120 was the dominant antigen epitope,named as TP47 E.Its amino acid sequence was AFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWWWETET.The TP47 E protein gene was amplified by polymerase chain reaction(PCR)and was consistent with the record in the GenBank.The recombinant protein could react with syphilis positive serum demonstrated by WB.4.The recombinant protein was labeled with horseradish peroxidase(HRP).The optimal protein coating concentration,blocking condition,serum dilution,serum reactive time,concentration of labeled antigen and substrate reactive time of double-antigen sandwich ELISA established by TP47 E and TP47,was 1?g/m L and 0.5?g/mL,1 % BSA at 4? overnight,serum,90 min,1?g/m L,30 min and 10 min,respectively.The sensitivity and specificity of the ELISA method using TP47 E and TP47 were 81.3%(47/58)and 94.0%(31/33),93.2%(55/59)and 78.8%(26/33),respectively.The coincidence rate of the two methods is 85.7%(78/91).Conclusions1.High false-positive rates were found for the detection of Anti-HCV and Anti-TP in the borderline specimens by Architect i2000 and HISCL 5000.There is an urgent need to develop more specific screening method of Anti-HCV and Anti-TP detection.2.Double-antigen sandwich ELISA methods using TP47 E antigen epitope and TP47 whole protein have developed successfully.The specificity of assay used TP47 E is higher than that used TP47.Maybe it is a good strategy to solve the false positive reaction.It would lay the foundation for further study of syphilis diagnosis assay with high specificity. |
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