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Background: The detection of the Epstein–Barr capsid antigen (VCA) immunoglobulin A (IgA) is widely used in the diagnosis of nasopharyngeal carcinoma (NPC), but a reference standard for evaluating the presence of VCA-IgA is not yet available. Therefore, a reference standard is urgently needed for a uniform and quantitative detection of VCA-IgA. Methods: A mixed reference serum from three NPC patients diluted with healthy subject serum was made as a potential first international standard for VCA-IgA. VCA-IgA was detected in twenty NPC patients by four ELISA kits and two chemiluminescent immunoassays kits using the reference as a calibration curve. The performance of these six kits was evaluated, and the quantitative results were compared. Results: Our results showed a good linearity of the reference in different kits. Without reference, the difference of the total coefficient of variation (from 3.98 to 43.11%) and Within-run coefficient of variation (from 2.47 to 19.66%) was large in the 6 kits. The positive and negative coincidence rate between the 6 kits and indirect immunofluorescence for NPC diagnosis was 75% overall agreement, but a difference among the six kits was found, ranging from 55 to 90%. The concentration of VCA-IgA in the 20 NPC samples led in the division into three categories such as negative, low, or medium/high positive, but these concentrations were significantly different within these three categories depending on the kit used of the 6 considered. However,a good correlation (R = 0.986) was observed between Antu and Beier ELISA kits. (Continued on next page) * Correspondence: email@example.com; firstname.lastname@example.org H. Chen, QiaohuaZhong and Xiaobin Wu contributed equally to this article. Department of clinical laboratory, The cancer hospital of Shantou University Medical college, The Key laboratory of Molecular Biology for high cancer incidence coastal Chaoshan area, Shantou University Medical college, number 22, Xinling road, Shantou, Guangdong 515041, People’s Republic of China State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong Key Laboratory of Nasopharyngeal Carcinoma Diagnosis and Therapy, Sun Yat-sen University Cancer Center, Guangzhou 510060, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 2 of 10 (Continued from previous page) Conclusions: The reference serum mightbe used as a reference standard for a better comparison of the results from different kits/laboratories. However, the quantitative results of some kits are still inconsistent due to the diversity of VCA antigens. Keywords: Epstein–Barr virus, Nasopharyngeal carcinoma, Virus capsid antigen, Reference, Enzyme-linked immunosorbent assay (ELISA) Introduction of discussion concerns the potential use of nature refer- Nasopharyngeal carcinoma (NPC) is highly endemic in ence preparations to calibrate these assays for quantita- South China. The ASIRW (age-standardized incidence tive testing. In addition, aproper cross-validation and rates of world) in South China (9.69/100,000) was 3.4 evaluation of the available different methods and kit times higher than that in Southwest China (2.85/100, brands are lacking. 000), which is the second in terms of incidence [1, 2]. So far, a reference standard for VCA-IgA has not yet Epstein–Barr virus (EBV) infection is one of the most been determined by the World Health Organization important factors causing NPC onset . EBV associated (WHO) International Laboratory and Collaborating Cen- antibodies, such as virus capsid antigen (VCA-IgA) and ters for Biological Standards. Most commercial ELISA Epstein-Barr virus nuclear antigen 1 (EBNA1-IgA), are kits generate only negative or positive results . Al- used in the screening and diagnosis of NPC  and the though our previous study confirmed that the rOD value former being one of the most widely used [5–8]. VCA- of ELISA shows a good correlation with the titer from IgA antibody is usually measured by indirect immuno- IEA , at present, VCA-IgA detected by ELISA can not fluorescence (IFA)  or enzyme-linked immunosorbent be used in the prognosis of NPC in place of that de- assay (ELISA)  with IFA considered as the “gold tected by IFA. Since VCA-IgA ELISA is widely used in standard”. Thanks to the presence of enzyme-labeled clinical laboratories, a calibrator is urgently needed. Re- antibodies instead of fluorescent antibodies, the immu- cently, a potential reference sample was proposed by noenzymatic assay (IEA) does not need a fluorescence Zhong He (Guangzhou, China) and Sun Yat-sen Univer- microscope to interpret the results, which are semi- sity Cancer Center (SYSUCC, Guangzhou, China) as a quantitatively reported by the titers and are widely used request of the Guangdong Provincial Anticancer Associ- in southern China [2, 3]. ation (http://www.gdaca.org.cn/), Southern China The VCA-IgA titer is closely related to the longterm Tumor Markers Standardization Alliance (Guangzhou, curative effect and prognosis of NPC [11, 12]. Low titer Guangdong, P.R.China). This sample was obtained from has a better therapeutic effect on NPC and prognosis, the blood serum of three NPC patients and mixed with while ascending EBV antibody titers are strongly associ- the blood serum of healthy subjects for dilution pur- ated with an increased risk of NPC [13, 14]. Although the poses. This sample might become the first international titer detected by traditional IEA or IFA has considerable VCA-IgA reference only if it fulfills specific criteria es- prognostic value, these two approaches are not suitable sential for such use. Therefore, this study was performed for mass NPC screening because of the need for manual in six clinical laboratories to evaluate the quantitative interpretation. ELISA-based assays have detection sensi- detection of VCA-IgA by different immunoassays to ver- tivity and specificity similar to IFA [4, 15] but they have ify whether the results could be improved by the use of the advantage of simpler standardization, they are more this reference. tolerant to interference, and less time-consuming when a large number of specimens need to be analyzed. The Material and methods chemiluminescent immunoassay (CLIA) has advantages Preparation of the reference serum similar to ELISA, it was developed in China, but no litera- The VCA-IgA original reference sample was prepared ture is yet available. using serum from three highly VCA-IgA positive NPC Thus, ELISA and CLIA are convenient and automated, patients whose diagnosis was histologically confirmed by but they use different peptide segments of VCA for their biopsy. Twenty milliliter blood was collected from each performance and standardization, thus, this difference is NPC patient and the serum was obtained by centrifuga- still a matter of debate and concern. Inaddition, while tion. The serum was also obtained from the blood of 50 new methodologies and new platforms (automated healthy donors and mixed to the pool of the NPC pa- vs.manual, ELISA vs. CLIA) for the detection of VCA- tients to dilute it. The potential presence of VCA-IgA in IgA are available, a proper comparisons among different the serum sample of the healthy donors was tested using assays has not yet been carried out. An important topic the Euroimmun IFA assay and all samples resulted as Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 3 of 10 VCA-IgA negative. All serum samples were also negative China; http://www.szyhlo.com/). Antigen peptides, sub- for the HIV 1 + 2, HbsAg and Hepatitis C Virus (HCV). strate, incubation time, volume and dilution of the CaCl was added to the serum of patients and healthy serum, type of conjugate, enzymatic substrate and donors to a final concentration of 18 mmol/L. Subse- cut-off level of the six kits were not exactly the same quently, the solution was incubated for 2 h at 37 °C and and are listed in Table 1. the insoluble material was removedby centrifugation (30 The VCA-IgA titer of 20 patients was also tested by min at 10000 rpm). Finally, the supernatant was filtered IFA considered as the gold standard method. using a 0.2 μm pore size nitrocellulose filter. The VCA-IgA reference standard was obtained by pre- Linearity, precision and comparability paring a series of dilutions of the serum from NPC pa- The reference serum was serially pre-diluted from 1:1 to tients and testing them by ELISA assay from 1:64 to measure the sample using the six kits. The linearity Euroimmun AG (Lubeck,Germany) and CLIA from New of the measurements was calculated by the mean of the Industries (Shenzhen, China). The concentration closer linear regression statistical analysis (slope value with 95% to the upper limit of detection was considered as the CI, and adjusted R ) using the original reference, and best dilution, and corresponding to a target value of 16, seven pre-dilutions of the reference serum were obtained 000 U/ml. and further diluted according to the manufacturer’sproto- The bulk reference standard material was obtained by colofeach ofthe 6commercialkits (Table 1). diluting the VCA-IgA–positive NPC serum with the The precision of the kits was evaluated according to pooled serum from the blood of healthy donors, using the EP15-A2 protocol of the Clinical and Laboratory the optimal dilution factor as determined above. Finally, Standards Institute (CLSI) . The original reference 1 liter VCA-IgA bulk reference serum was obtained. and the 1:16 pre-dilution were analyzed three times per Subsequently, the prepared reference serum was divided day during a period of five consecutive working days(n = into 0.2 ml aliquots and stored at-80 °C. 15 per level). The precision of each kit was evaluated as the coefficient of variation (CV), which was calculated Patients from the mean and standard deviation of the data series. The diagnosis of the twenty NPC patients was confirmed To evaluate the efficacy of harmonization of the anti- by biopsy and consequent histological assay and further body levels,the sera from 20 NPC patients with different tests, including head and neck MRI and chest X-rays. VCA-IgA concentrations (13 positive and 7 negative) as Negativity, low, and medium/high positivity was defined measured by the IFA were analyzed using 6 commercial as the result of VCA-IgA titer detected by VCA-IgA kits. The VCA-IgA concentration was calculated by the Euroimmun IFA. Seven samples were negative (titer< 1: calibration curve obtained by the original and seven di- 20), seven were low positive (1:20 ≤ titer≤1:80), and six lutions of the reference serum. A concentration of 16, were medium/high positive (titer> 1:80) for VCA-IgA. 000 units (U/ml) was arbitrarily assigned to the original During the initial visit all patients were informed that reference serum. the samples remaining after the routine test might be used for medical research, without involving additional Statistical analysis costs and suffering and their written informed consent Statistical analysis was performed using IBM SPSS was obtained. In addition, the ethics committees of the Statistics version 19.0 (IBM Corp.). Scatter diagram and SYSUCC approved this procedure. linear regression analysis were performed by MedCalc version 184.108.40.206. The VCA-IgA concentration of each of VCA-IgA measurement the 20 patients detected by the 6 kits divided into three Six commercial kits were used to analyze the reference categories (negative, low and medium/high positive) was and sera from 20 patients with NPC to evaluate the VCA- compared via randomized block design ANOVA. Then, IgA level. Four ELISA commercial kits produced by the LSD test was used to perform multiple comparisons be- following manufacturers were used: Euroimmun AG tween the groups. Quantitative results were correlated (Lubeck, Germany; http://www.oumeng.com.cn/), Tarci- by the non-parametric Spearman test. A value of p < neBioMed (Beijing, China; http://www.tarcine.com.cn/), 0.05 was considered statistically significant. Antubio (Zhengzhou, China; http://www.autobio.com.cn/), Beier (Beijing, China; http://www.beierbio.com/). More- Results over, 2 CLIA kits produced by the following manufacturers Linearity and tested by matching detection systems were used: New The linear regression analysis was performed to evaluate Industries Biomedical Engineering (Maglumi 4000 Plus the linearity of the quantitative data. The graphs and System, Shenzhen, China; http://www.snibe.com/)and correspondent statistical values (slope with 95% CI and YHLO Biotech (iFlash 3000 CLIA System,Shenzhen, adjusted R ) are shown in Fig. 1 and Table S1. The Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 4 of 10 Table 1 Characteristics of the 7 commercial kits for detecting VCA-IgA antibodies Manufacturer Method Antigen Chromogenic / luminescence reaction system Sample dilution Testing laboratory Euroimmun IFA P3HR1 FITC 1:10 SYSUCC ELISA gp125 HRP/TMB 1:100 SAHGUCM Beier ELISA p18 HRP/TMB 1:10 CHSUMC Tarcine ELISA p18 HRP/TMB 1:10 PHJ Antu ELISA gp125,p18 HRP/TMB 1:10 ATHZU New Industries CLIA p18,p23 ABEI 1:10 SYSUCC YHLO CLIA p18,p23 Acridinium ester/ H2O2 1:10 LMCHCH TMB Tetramethylbenzidine, ABEI N-(4-Aminobutyl)-N-ethylisoluminol, SYSUCC Sun Yat-sen University Cancer Center, SAHGUCM Second Affiliated Hospital of Guangzhou University of Chinese Medicine, CHSUMC The cancer hospital of shantou University Medical college, PHJ People’s Hospital of Jieyang, ATHZU Affiliated Tumor Hospital of Zhengzhou University, LMCHCH Liuzhou Maternity and Child Health Care Hospital Fig. 1 The regression curves obtained with each of the 6 commercial VCA-IgA methods. Values of the ordinate are given in the units used by the assay kit, while the abscissa shows the arbitrary units of the reference sample Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 5 of 10 coefficient of regression as a measure of linearity of the units of the Euroimmun kit, thus resulting in a very wide diluted reference serum tested with each of the 6 com- dispersion of the quantitative data. mercial kits ranged from 0.00007181 to 11.276 (Table The VCA-IgA concentration in each patient obtained S1). Except for the Tarcine BioMed kit (R > 0.9367), the using different detecting kits is shown in a line chart regression analysis for the other 5 commercial kits (Fig. 2a,b,c). Within the three categories (negative,low achieved a good correlation coefficient (R > 0.96) over and medium/high positive) detected by the 6 kits when the entire tested range (Fig. 1,Table S1). the results were obtained by the use of the reference To verify whether the linearity was present even at low standard, the concentration of VCA-IgA was signifi- and high antibody concentration, one serum sample with cantly different among the six kits according to the LSD a VCA-IgA concentration and another one with a high test(p < 0.05) (Fig. 2d,e,f). The value of New Industries VCA-IgA concentration were diluted in the same way in and other kits were slightly different. New Industries which the reference sample was diluted (pre-dilutions gave a result different from the one from other kits in from the original concentration to 1:64 followed by the the medium/high positive categories by LSD test(p < dilution requested in each kit. The results showed that 0.05) (Fig. 2f). This last one was also different from the linearity was maintained even when the antibody Euroimmun, Beier and Antu kits in the negative cat- concentration was low, but it was not maintained when egory (Fig. 2d), and it was different from the Euroimmun the antibody concentration was high (Fig. S1). kit in the low positive category (Fig. 2e). Correlation among the six assays Precision The positive VCA-IgA rates in the 20 NPC patients The VCA-IgA concentration detected by Euroimmun obtained using the six kits were analyzed. The posi- and Tarcine kits was calculated using one calibration tive rates of each kits were analyzed in 20 NPC pa- provided by each kit. The VCA-IgA concentration of tients (Table 3). Euroimmun ELISA resulted inthe New Industries and YHLO kits was calculated by the highest positive rate in seven kits. The positive and calibration curve provided by each kit. Since the Antu negative rates obtained by the six kits and the IFA and Beier ELISA kits did not have a self-made calibrator, considered as the gold standard method are the same, the precision was calculated by the OD value of each de- but the differences among the six kits are remarkable tection when all the negative and positive controls met (ranging 55–90%) (Table 3).Furthermore,the positive the manufacturer requirements. In addition to Beier and negative coincidence rates of the four ELISA kits ELISA kit, other 5 kits resulted in an excellent within- (Euroimmun and Beier and Tarcine and Antubio) run CVs (CV < 8%) at both low and high VCA-IgA level. were more than 70%. In addition, Beier and New In- Except for Antu and Beier ELISA kits, other 4 kits also dustries resulted in the highest positive-negative coin- resulted in a good total CVs (CV < 10%) (Table 2). cidence rate, reaching 90%. Table 4 shows the Spearman correlation coefficients Comparability between the different kits. Almost all of them gave a cor- Table 2 shows the highest VCA-IgA concentrations in relation coefficient lower than 0.80. Although the quan- the reference serum obtained using the 6 commercial titative results among the different kits are not exactly kits, the cut-off values proposed by each manufacturer, the same, a good correlation was observed between Antu and their ratio. The VCA-IgA concentrations varied and Beier ELISA kit (0.986), and between Tarcine ELISA from 1.4 arbitrary units of the Beier kit to 13 arbitrary kit and New Industries CLIA kit (0.897) (Fig. 3). Table 2 Levels of VCA-IgA antibodies detected in the VCA-IgA antibody reference serum by the 6 commercial kits Manufacturer Cut-off value Reference serum Ratio† Within-run CV Total CV (arbitrary units)* (arbitrary units) High Low High Low Euroimmun ELISA 1.1 13.0 11.8 3.98% 6.11% 6.24% 6.52% Beier** ELISA 1.1 3.2 2.9 5.03% 19.66% 13.77% 43.11% Tarcine ELISA 0.15 1.4 9.3 4.95% 7.58% 8.27% 7.22% Antu** ELISA 0.15 3.5 23.3 4.62% 6.45% 13.74% 16.85% New Industries CLIA 4 12.0 3.0 2.47% 4.34% 5.95% 9.86% YHLO CLIA 1.1 5.6 5.1 4.76% 5.22% 3.98% 4.87% *VCA-IgA antibody levels detected in the reference serum at the highest concentration tested in each kit †Ratio was calculated as reference serum arbitrary units divided by manufacturer cutoff value for each kit **Precision was calculated by OD value of each test when all the negative and positive controls met manufacturer requirements Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 6 of 10 Fig. 2 VCA-IgA results of 20 patients calibrated by the reference according three sample categories detected by 6 kits. Line chart of VCA-IgA results detected by each of 6 kits in three sample categories a negative; b low positive; c, medium/high positive. The mean VCA-IgA concentration of each patient by 6 kits divided into three sample categories were used LSD test to make multiple comparisons between the groups. d, negative; e low positive; f, medium/high positive. It has been marked out, when the difference between the two groups is significant (p < 0.05). * New Industries group was different from other kits group in medium/high positive categories by LSD test (p < 0.05) Discussion Standardization Alliance. The main objective of this IFA, ELISA and CLIA are three methods for the detec- study was to evaluate the candidate serum reference tion of the VCA-IgA antibody used in clinical laborator- standard and compare the performance of different ies and now available. Numerous manufacturers use commercial ELISA kits and CLIA for the quantitative different peptides as VCA antigens and different assays, detection of VCA-IgA. all greatly contributing to the NPC screening. However, Our results showed that the reference serum gave the comparison of the results obtained using different positive results in all the commercial VCA-IgA kits used. kits is necessary to evaluate the best performance and The linear analysis by the reference standard curve ob- the standardization of the assay is a priority. To this end, tained in all the 6 kits showed that the reference sample a frozen reference sample was prepared and proposed as could be used as a calibrator in different assays using a candidate serum reference standard forVCA-IgA different antigenic substrates. Residual differences may detection to the Southern China Tumor Markers be due to different assay reagents/procedure (including Table 3 Performance and coincidence rate of the different assays Assay Positive Coincidence rate of positive and negative(%) rate(%) Beier Tarcine Antubio New Industries YHLO IFA Euroimmun 90 70 75 70 85 75 75 Beier 60 75 80 65 55 75 Tarcine 65 85 90 60 75 Antubio 70 75 55 75 New Industries 70 70 75 YHLO 70 75 IFA 65 Positive rate of assay in 20 patients Coincidence rate of positive and negative in 20 patients Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 7 of 10 Table 4 Spearman correlations coefficients in the six assays Manufacturer Euroimmun Beier Tarcine Antubio New Industries YHLO Euroimmun 1 0.494 0.465 0.497 0.440 0.535 Beier 1 0.695 0.986 0.623 0.641 Tarcine 1 0.635 0.897 0.546 Antu 1 0.542 0.617 New Industries 1 0.447 YHLO 1 Bold represent results > 0.9 dilution buffer, antigenic peptides, and sample dilution). quantitative results using their own units. Thus, up to Since the linearity of the method could not be main- now, no systematic evaluation is available on the quanti- tained at a higher VCA-IgA concentration and the line- tative and standardized research of all the VCA-IgA arity(R ) did not reach 0.99, it is recommended the use methods. Previous studies on VCA-IgA detection gener- of multi-point curves for each kit in the future. ally used ELISA and IFA,  CLIA is only related to The precision of each kit is quite different due to the VCA IgG or IgM antibody. This study is the first includ- lack of a reference. At present, the VCA-IgA kits can be ing CLIA in VCA-IgA research [18, 19]. Our first study classified into three categories according to the kit cali- shows that CLIA have better Within-run CV and Total brator. The first category that includes Beier and Antu CV. This may be due to the advantages of methodology VCA-IgA ELISA kits does not have any calibration ma- and matching testing system. terial, but only negative and positive quality controls. A good agreement with IFA results was obtained using The results are acceptable when the negative and posi- both ELISA and CLIA. The concordance among the 6 tive controls are acceptable within a certain range. kits and IFA for primary NPC diagnosis was 75% overall Therefore, the results within the batch (Within-run CV) agreement (Table 3). This result was similar to the one may not be very different, but the results of each batch obtained by Karray, who observed 81–91% overall agree- (Total CV) are quite different. No comparison is made ment between ELISA (IgA anti-VCA-p18) and IFA for among the quantitative results of different test batches. primary NPC diagnosis. They also found a declining re- The second category that includes Euroimmun and Tar- activity in patients in remission and increasing reactivity cine VCA-IgA ELISA kits usesa critical value calibrator in patients with persistent disease or relapse during the with its own. They have satisfactory within-run CV and follow-up by monitoring the presence of the antibody by total CV, but the disadvantage of a single point calibra- both ELISA and IFA . Our previous study also found tor is that rODs (OD/cut-off OD) is difficult to accur- an excellent correlation and a high degree of concord- ately express all values because of the imperfect linearity ance between IgA titers (IEA) and rOD levels (ELISA) (R2 = 0.9779 and R2 = 0.9367). The third category that . Many studies also confirmed that changes in VCA- includes New Industries and YHLO VCA-IgA CLIA kits IgA antibody level can be used for NPC prognosis and used they own calibration curves and provide screening [13, 20–22]. Pretreatment serum EBV-VCA/ Fig. 3 The correlations of VCA-IgA concentrations between different kits. a Antu and Beier ELISA kits. b Tarcine ELISA kit and New Industries CLIA kit. The low correlation (R < 0.8) between other kits was not shown Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 8 of 10 IgA titer may be used as an independent prognostic Although shortcomings were still present in the quan- marker of NPC . Liuet al. found that the geometric titative results among different kits, the improvement of mean titer of anti-EBV/VCA IgA antibodies before and the results obtained in this study was evident. The refer- after radiotherapy was significantly different . People ence used could reduce the difference among different with ascending VCA-IgA antibody titers tend to have batches with the same kit. Furthermore, the quantitative higher risks and shorter time to develop NPC, compared results by reference standard curves were more accurate to those with a descending pattern . However, Yao than the semi-quantitative results of IFA in monitoring et al. did not confirm the role of VCA-IgA as a prognos- efficacy and risk. IFA/IEA as the “gold standard” for the tic biomarker among patients with NPC and undetect- detection of IgA to VCA, are laborious techniques, since able EBV DNA . A possible explanation for these they are not sufficiently automated to achieve a good contradictory results may be that the previous study ob- level of output, and a certain degree of subjectivity is still tained the positive results by IFA, although Yao et al. present in interpreting the results. The ELISA allows the uses ELISA (Beier kit). In the absence of calibrators, quantitative detection through the calibration curve, is inter-assay differences among ELISA kits may affect the easy to performand detect quickly a large number of outcome, as explained in the limitations of this article samples. Finally, the results of different manufacturers’  and was confirmed in our study (Total CV = kits were compared by the use of a reference standard. 43.11%), confirming once more the importance of the A good correlation (correlations coefficient = 0.986) reference in VCA-IgA ELISA detection. was obtained between Antu and Beier ELISA kits in the Using the same reference allows connecting different results obtained by standard curves using the reference methods or kits to analyze the results that were not pre- standard. viously comparable. This work showed that the same pa- tient had similar results with some different kits, or that Conclusion the values were different but the trends were the same In conclusion, the use of the serum as the reference might with different kits (Fig. 2). The results in the 20 patients reduce the differences among different kits and allow a obtained by the use of the kits calibrated on the refer- comparison among different kits. Our subsequent studies ence serum divided into three sample categories showed should focus on the diversity between different antigens acertain significant difference in the 6 kits compared via and the attempt to unify them to promote the further de- randomized block design ANOVA and further analysis velopment of VCA-related research. according to LSD test (Fig. 2). The New Industries kit was different from the other kits in medium/high posi- Supplementary information tive category by LSD test(p < 0.05). Moreover, New In- Supplementary information accompanies this paper at https://doi.org/10. 1186/s13027-020-00294-8. dustries and Euroimmun gave different results in the three groups. The coincidence rate of positive and nega- Additional file 1: Table S1. A summary of linear regression studies tive between the six kits were also quite different. (Table Figure S1. The regression curves obtained by diluted one more low and 3) This might be due to the different sources of antigens. high VCA-IgA serum concentrations with each of the 6 commercial methods. Values of the ordinate are givenin the units used by the assay This aspectis also supported by Gu . The EBV VCA kit, while the abscissa shows the arbitrary units of the reference sample. antigen consists of several proteins, such as gp125, p18, and p23. As regards the measurement of IgA, except Abbreviations Euroimmun that uses gp125, other assays use the p18 NPC: Nasopharyngeal carcinoma; VCA: Virus capsid antigen; peptide or other protein mixtures (Antu,New Industries, IgA: Immunoglobulin A; CLIA: Chemiluminescent immunoassays; EBNA1- YHLO). The results depend on the use of a range of dif- IgA: Epstein-Barr virus nuclear antigen 1; IFA: Indirect immunofluorescence; ELISA: Enzyme-linked immunosorbent assay; EBV: Epstein-Barr virus; EA: Early ferent antigens producing different serological responses. antigen; IEA: Immunoenzymatic assay; WHO: World Health Organization; Thus, the low correlation between the results obtained ASIRW: Age-standardized incidence rates of world; SYSUCC: Sun Yat-sen Uni- using Euroimmunand the other commercial ELISA kit versity Cancer Center for IgA-VCA might be due to the different antigens. Acknowledgments Apart of the use of the same antigen, other factors such We thank the following: all members of the Guangdong Provincial Anticancer as the different dilutions used for the analysis maybe a Association and Southern China Tumor Markers Standardization Alliance for their support and, in particular, the WL Liu and Hao Chen, for their initiative and source of differences . These differences may also efforts in preparing the reference sample. And we are very grateful to the lead to a different outcome in the therapeutic and prog- patients who participated in this study. nostic studies using different VCA-IgA kits. Therefore, in the future, the standards should be divided and uni- Authors’ contributions YWX and SLC and HC conceived and designed the experiments. HC and QC fied calibrators should be developed according to the dif- and XBW and NX and YLD performed theexperiments: HC and QHZ analyzed ferent peptide segments of the antigen protein to the data. HC and YWX wrote the paper. The authors read and approved the achieve anaccurate consistency of the results. final manuscript. Chen et al. Infectious Agents and Cancer (2020) 15:25 Page 9 of 10 Funding 6. Chien YC, Chen JY, Liu MY, Yang HI, Hsu MM, Chen CJ, et al. 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