Journal of Cancer 2020, Vol.

11 7176

Ivyspring
International Publisher
Journal of Cancer
2020; 11(24): 7176-7183. doi: 10.7150/jca.47260
Research Paper

Efficacy of Chemiluminescence Immunoassays on

VCA-IgA and EBNA1-IgA Antibodies of Epstein-Barr
Virus in Diagnosing Nasopharyngeal Carcinoma
Xia Yu1, Fugui Li1, Weimin Cheng1, Biaohua Wu1, Huiyun Fang1, Fuzhen Xia2, Yijun Gong2, Wenjing Yu3,
Pu Liao4, Youde Cao5, Fenghua Yang6, Hong Zhu7, Jiang Li8, Yajun Huang9, Liying Gan10, Lei Zhang11,
Yonggang Lou12, Mingfang Ji1
1. Cancer Research Institute of Zhongshan City, Zhongshan City People's Hospital, Zhongshan, Guangdong, China
2. Guangdong Provincial Engineering Technology Research Center for Autoimmune Laboratory Diagnostic Products, Shenzhen, Guangdong, China
3. Department of Clinical Laboratory, First Hospital of Jilin University, Changchun, Jilin, China
4. Department of Clinical Laboratory, Chongqing General Hospital, Chongqing, China
5. Department of Clinical Laboratory, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, Hunan,
China
6. Department of Clinical Laboratory, Fushun Central Hospital, Fushun, Liaoning, China
7. Department of Clinical Laboratory, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
8. Department of Clinical Laboratory, Shenzhen Sixth People's Hospital (Nanshan Hospital), Huazhong University of Science and Technology Union
Shenzhen Hospital, Shenzhen, Guangdong, China
9. Department of Clinical Laboratory, The Second Affiliated Hospital of Fujian Medical University (Donghai Hospital District), Quanzhou, Fujian, China
10. Department of Clinical Laboratory, Guangxi International Zhuang Medicine Hospital, Nanning, Guangxi, China
11. Department of Clinical Laboratory, Capital Medical University Daxing Teaching Hospital, Beijing, China
12. Department of Clinical Laboratory, Dongyang People's Hospital, Dongyang, Zhejiang, China

 Corresponding author: Mingfang Ji ([email protected])

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.

Received: 2020.04.21; Accepted: 2020.10.03; Published: 2020.10.18

Abstract
Background: IgA antibodies against Epstein-Barr virus (EBV) capsid antigen (VCA) and nuclear antigen
1 (EBNA1) have been proposed to facilitate the diagnosis and early detection of nasopharyngeal
carcinoma (NPC) in high-incidence regions. However, while new methodologies and new platforms for
the detection of VCA-IgA and EBNA1-IgA have become available, proper interassay simultaneous
comparisons have not been carried out. The study was to compare the performance of the
chemiluminescent immunoassays (CLIA) and enzyme-linked immunosorbent assay (ELISA) for VCA-IgA
and EBNA1-IgA antibodies, and to evaluate the levels of EBV antibodies in healthy population from
different areas of China.
Methods: CLIA and ELISA for VCA-IgA and EBNA1-IgA were performed in NPC and healthy
populations from high-incidence areas of NPC in South China (N=555), medium-incidence areas of NPC
in Central China (N=318) and low-incidence areas of NPC in North China (N=379), and the results were
compared and analyzed.
Results: (1) The highest sensitivity in total, early and advanced NPC were 91.5% (CLIA for VCA-IgA),
86.4% (CLIA and ELISA-2 for EBNA1-IgA) and 93.6% (CLIA for VCA-IgA). However, the specificity of
EBV-IgA measured by CLIA was relatively lower than ELISA. The top three seromarkers with the largest
AUC was CLIA for VCA-IgA (AUC: 0.929, 95% CI: 0.905–0.953), ELISA-2 for EBNA1-IgA (AUC: 0.922,
95% CI: 0.896–0.947) and CLIA for EBNA1-IgA (AUC:0.919, 95% CI: 0.893–0.945), respectively. The
positive and negative coincidence rates of the two EBNA1-IgA kits were 69.5% and 91.9%, respectively.
However, the coincidence rates of VCA-IgA were relatively low. CLIA kits had good repeatability
between different laboratories. (2) The positive rates of EBV-IgA antibodies were relatively high in
high-incidence areas of NPC (P < 0.017), while there was no significant difference in the antibody positive
rates between medium-incidence areas and low-incidence areas of NPC (P > 0.05).

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Journal of Cancer 2020, Vol. 11 7177

Conclusions: The performance of EBV-IgA antibodies measured by CLIA has good repeatability, higher
sensitivity and similar specificity. The higher EBV-IgA positive rate in healthy subjects by CLIA raises
concern about its suitability for NPC-risk screening and requires further analysis.
Key words: nasopharyngeal carcinoma, VCA-IgA, EBNA1-IgA, CLIA, ELISA

Introduction
Nasopharyngeal carcinoma (NPC) is a common 3.19:1. The mean age was 49.18 ± 12.23 years with the
malignant tumor in the Southern China, which is median age 48 years. NPC staging was based on the
closely related to Epstein-Barr virus (EBV) infection [1, 8th edition of UICC staging, including 44 patients of
2]. Individuals with elevated levels of antibody early (I + II) NPC and 157 advanced (III + IV) NPC.
responses against EBV antigens (particularly IgA A total of 1,051 healthy subjects, who were 20-69
responses) are at increased risk for development of years old, were randomly selected among healthy
NPC [3-6]. At present, enzyme-linked immunosorbent people who participated in physical examinations at
assay (ELISA) combined detection of IgA antibodies hospital from March 2019 to December 2019, and their
against EBV capsid antigen (VCA-IgA) and nuclear serum samples came from 11 hospitals in different
antigen 1 (EBNA1-IgA) has been proposed for general regions of China. We excluded samples from
population screening to triage individuals for further immunocompromised patients (e.g.,those with
clinical evaluation [7-10]. ELISA-based assays are easier cancer, organ transplant recipients, or those with
to be standardized, with greater false tolerance to other infectious diseases). The classification criteria
interference, and are also more labor-saving when for different incidence areas of NPC is based on the
analyzing a large number of specimens. Chemilumi- 2018 China Cancer Registry Annual Report[12]. The
nescent immunoassay (CLIA) has advantages similar population came from the following three areas: (1)
to ELISA [11], but few literatures of CLIA testing High-incidence areas of NPC: Zhongshan and
EBV-IgA has been reported yet. In addition, while Shenzhen in Guangdong, Nanning in Guangxi, with
new methodologies and new platforms (ELISA 354 people, including 171 males and 183 females, with
vs.CLIA) for the detection of VCA-IgA have become male: female = 0.93: 1. The mean age was 43.52 ± 14.19
available, proper interassay simultaneous years with the median age 43 years. (2)
comparisons have not been carried out. Here, CLIA Medium-incidence areas of NPC were Changsha in
was employed to detect VCA-IgA and EBNA1-IgA for Hunan, Quanzhou in Fujian, Dongyang in Zhejiang,
the first time in this study, and compared with several and Chongqing, with 318 people in total, including
ELISA kits widely marketed for diagnostic 165 males and 153 females, with male : female =1.08 :
performance analysis of NPC. Meanwhile, the 1. The mean age was 45.10 ± 13.08 years, with the
difference of EBV antibody positive rate among median age 46 years. (3) Low-incidence areas of NPC
healthy population in high-incidence areas, included Beijing, Changchun in Jilin, Dalian and
medium-incidence areas and low-incidence areas of Fushun in Liaoning, with 379 people in total,
NPC in China was compared in order to provide including 190 males and 189 females, with male:
relevant scientific basis for clinical application of kit. female = 1.01:1. The mean age was 44.70 ± 14.14 years
with the median age was 46 years. There was no
Materials and Methods significant difference in sex and age among the three
groups of healthy population (χ2 =0. 861, P=0. 650,
Subjects
P>0.05; F=2. 338, P=0. 311, P>0.05).
The subjects were divided into NPC group and
healthy group. All subjects collected 2-4 mL fasting Reagents and Methods
venous blood. After centrifugation, the serum 1.2.1 CLIA reagent VCA-IgA and EBNA1-IgA
samples were stored at 4°C for use within one month, kits are manufactured by Shenzhen YHLO Biotech
or stored at -80°C for longer periods. The inclusion Co., Ltd. The iFlash 3000 chemiluminescence
criteria of NPC group included the following: being immunoanalyzer and matching reagent (Acridine
aged 20-69 years, pathological examination confirmed Ester Direct Chemiluminescence) were employed.
undifferentiated non-keratinized carcinoma and Following the manufacturer's instruments, reagents
untreated. NPC group were continuously collected and standard operating procedure (SOP), the two test
from 201 patients with NPC hospitalized in results are expressed by COI value with COI ≥ 1.1
Zhongshan City People's Hospital from March 2019 to stands for reacted (positive).
December 2019. Among them, 153 were males and 48 1.2.2 ELISA reagent for VCA-IgA kit is
were females, and the ratio of males to females was manufactured by Euroimmun Medizinische

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 Journal of Cancer 2020, Vol. 11 7178

Labordiagnostika AG (represented as ELISA-1). Statistical Analysis

ELISA reagent VCA-IgA and EBNA1-IgA kits are SPSS19.0 software was used for analysis, and the
manufactured by Zhongshan Bioengineering Co., Ltd counting data were expressed as percentages, and
(represented as ELISA-2). The levels of these chi-square test was used for comparison of data
seromarkers were assessed by photometric between groups. The variables were tested for
measurement, according to manufacturer's normality, and the skewness distribution data were
instructions, and standardized by calculating the ratio tested under rank sum of independent samples. The
of the optical density (OD) of the sample over that of area under curve (AUC) was calculated by the
the reference control (rOD). If the specific rOD was thereceiver operating characteristic curve (ROC) to
greater than 1, the sample was regarded as positive. evaluate the diagnostic efficacy of EBV in NPC. The
1.2.3 VCA-IgA of ELISA-1 was purified VCA level of significant test was α = 0.05, with P＜ 0.05
gp125 from P3HR1 cells, while ELISA-2 and CLIA kits deemed significant.
used recombinant VCA protein components (p18 and
p23). EBNA1-IgA of ELISA-2 and CLIA were Results
produced with purified recombinant peptides
specified by EBV BKRF1 (72kDa). All the serological Analysis of Positive Rate of EBV Antibody
tests were conducted by the same technicians (sample Detected by CLIA and ELISA in Healthy
information was blinded) .Serial dilutions of quality Population
control sera and negative control were applied to each CLIA and ELISA were used to detect VCA-IgA
assay for the evaluationg of intra-variability. and EBNA1-IgA antibodies in healthy population in
high-incidence areas, medium-incidence areas and
Evaluation of Detecting Performance low-incidence areas of NPC, respectively, and the
Sensitivity= number of people with positive positive rates of healthy population were calculated
antibody tests in NPC/total number of NPC; respectively, detailed in Table 1. The positive rates of
Specific= number of people with negative antibody CLIA, ELISA-1 and ELISA-2 for VCA-IgA varied
tests in healthy population/total number of healthy between three kits, and the positive rates of total
population; Positive coincidence rate= a / (a + b + c), population were 5.6%, 2.5% and 7.3%, respectively.
negative coincidence rate = d / (b + c + d). Where a However, the positive rates of CLIA and ELISA-2
represents the number of positive detection results of reagents for EBNA1-IgA were similar with 5.6% and
both detection systems; b indicates the number of 5.2%, respectively. In healthy population, the positive
positive cases detected by the X detection system but rate of EBV-IgA measured by CLIA was higher than
negative by the Y detection system; c indicates the ELISA.
number of negative cases detected by the X detection EBNA1-IgA by CLIA and ELISA-2, and
system but positive by the Y detection system; d VCA-IgA by ELISA-1 revealed that there were
represents the number of negative tests by both significant differences in the three positive rates of
detection systems. Parallel detection: If one of the two EBV antibody in three different areas (P< 0.05). The
indicators is positive, it will be determined as positive, results showed that the EBNA1-IgA positive rates of
and if all indicators are negative, it will be determined CLIA and ELISA-2 were 8.5% and 8.8%, respectively,
as negative. Laboratories in different hospitals were and the VCA-IgA positive rate by ELISA-1 was 4.8%
randomly selected to use CLIA kits to detect in healthy people in high-incidence areas, and the
EBNA1-IgA and VCA-IgA, and to evaluate the antibody positive rate was higher in healthy
consistency of tests results between different population in high-incidence areas (P < 0.017), while
laboratories. there were no significant differences in the antibody
positive rates between medium-incidence areas and
low-incidence areas of NPC (P > 0.05).

Table 1. Analysis of Positive Rate of EBV Antibody Detected by CLIA and ELISA in Healthy Population
Antibody Kit Total population (n=1051) high-incidence areas (n=354) medium-incidence areas (n=318) low-incidence areas (n=379) Χ2 P value#
VCA-IgA CLIA 59 (5.6%) 24 (6.8%) 15 (4.7%) 20 (5.3%) 1.472 0.479
ELISA-2 77 (7.3%) 32 (9.0%) 18 (5.7%) 27 (7.1%) 2.853 0.240
ELISA-1 27 (2.5%) 17 (4.8%)* 6 (1.9%) 4 (1.1%) 11.11 0.004
EBNA1-IgA CLIA 59 (5.6%) 30 (8.5%)* 11 (3.5%) 18 (4.7%) 8.789 0.012
ELISA-2 55 (5.2%) 31 (8.8%)* 11 (3.5%) 13 (3.4%) 13.37 0.001
Note: The values outside brackets were positive numbers and values in brackets were positive rates. “*” indicated high positive rate. “#” indicated the comparison of EBV
antibody positive rate in high-incidence, medium-incidence and low-incidence areas of NPC.

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 Journal of Cancer 2020, Vol. 11 7179

Comparison of Efficacy of CLIA and ELISA in ELISA-2 reached 86.4%. Among five kits, the highest
Detecting EBV Antibody in Diagnosis of NPC specificity was ELISA-1, as shown in Table 2.
Taking NPC and healthy people as research
CLIA and ELISA were used to detect VCA-IgA objects, ROC curves were made (Figures 1, 2). The
and EBNA1-IgA antibodies in NPC and healthy AUC of VCA-IgA in diagnosis of NPC by CLIA,
population, and the sensitivity and specificity of ELISA-1 and ELISA-2 were 0.929 (95% CI:
various EBV antibodies in diagnosing NPC were 0.905-0.953), 0.814 (95% CI: 0.774-0.854) and 0.906
calculated respectively. In total NPC and advanced (95% CI: 0.879-0.933), respectively. The order efficacy
NPC, CLIA detected the highest VCA-IgA sensitivity of VCA-IgA in diagnosing NPC was CLIA, ELISA-2
with 91.5% and 93.6% respectively. However, in early and ELISA-1.
NPC, the EBNA1-IgA sensitivity of CLIA and

Table 2. Sensitivity and Specificity of CLIA and ELISA Detection of EBV Antibody in NPC
Antibodys Kit Sensitivity (95%CI) Specificity (95%CI)
Total NPC (n=201) Early NPC (n=44) Advanced NPC (n=157)
VCA-IgA CLIA 91.5(86.6-94.8) 84.1(69.3-92.8) 93.6(88.3-96.7) 94.4(92.8-95.7)
ELISA-2 88.6(83.1-92.5) 84.1(69.3-92.8) 89.8(83.7-93.9) 92.6(90.8-94.1)
ELISA-1 67.7(60.7-74.0) 54.5(39.0-69.3) 73.9(66.2-80.4) 97.4(96.2-98.3)
EBNA1-IgA CLIA 89.6(84.3-92.3) 86.4(72.0-94.3) 90.4(84.5-94.4) 94.4(92.8-95.7)
ELISA-2 89.6(84.3-92.3) 86.4(72.0-94.3) 90.4(84.5-94.4) 94.8(93.2-96.0)
Note: Value shown as percentage.

Fig. 1. ROC of VCA-IgA in diagnosis of NPC

Fig. 2. ROC of EBNA1-IgA in diagnosis of NPC

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Journal of Cancer 2020, Vol. 11 7180

Table 3. Results Consistency Analysis of EBV Antibody Detection Kit

Antibody Kit Positive Coincidence Rate (%)/Negative Coincidence Rate (%)
ELISA-1 (Reference) ELISA-2 (Reference)
VCA-IgA CLIA 50.5/87.2 61.0/88.6
ELISA-1 49.2/86.3
EBNA1-IgA CLIA 69.5/91.9

The AUC of EBNA1-IgA by CLIA and ELISA-2 relative deviation and absolute deviation for
were 0.919 (95% CI: 0.893-0.945) and 0.922 (95% CI: VCA-IgA and EBNA1-IgA were 12 and 7, 22 and 27,
0.896-0.947), respectively. The CLIA and ELISA-2 respectively, and the results between hospital
reagents of EBNA1-IgA have similar diagnostic laboratories met the requirements, as shown in Table
efficacy in NPC. 5.
Analysis of Consistency of CLIA and ELISA in
Detection of EBV Antibody Table 4. Sensitivity Analysis of Different Kits for Alloantibody in
Parallel Examination in Diagnosis of NPC
The consistency analysis of the detection results
Different Reagent VCA-IgA (n) EBNA1-IgA
of EBV antibody kits was shown in Table 3. Generally, Combination (n)
the negative coincidence rate among kits was higher CLIA vs CLIA vs ELISA-1 vs CLIA vs
ELISA-2 ELISA-1 ELISA-2 ELISA-2
than the positive coincidence rate. The positive and + + 168 136 136 166
negative coincidence rates of EBNA1-IgA kits were + － 16 48 0 14
69.5% and 91.9%, respectively. The negative － + 10 0 42 14
7 17 23 7
coincidence rate of VCA-IgA kits was more than 85%, － －
Parallel sensitivity 96.5% 91.5% 88.6% 96.5%
but the positive coincidence rate was relatively low.
The positive coincidence rates of CLIA with ELISA-1
and ELISA-2 of VCA-IgA were 50.5% and 61.0% Discussion
respectively, while the positive coincidence rate of Zhongshan City is one of the high-incidence
ELISA-1 and ELISA-2 of VCA-IgA was the lowest, areas of NPC [13], and is also the project site of
only 49.2%. National Early Diagnosis and Early Treatment of
Sensitivity Analysis of Different Kits for NPC. EBV serological detection has been the most
Parallel Detection of Alloantibody in Diagnosis promising tool used for NPC screening. Since the
NPC 1980s, EBV antibody detection methods have
successively gone through indirect immunoenzyma-
The positive coincidence rates of different EBV
ticassay (IEA), immunofluorescence assay (IFA) and
antibody kits were low. Further analysis of the
ELISA. The first two assays have been presently rarely
combined detection results of EBV antibodies of
used, while ELISA is currently the most commonly
different kits in NPC manifested that the kits were
used. However, the sensitivity and specificity of the
with a good complementary relationship. Pairwise
assay need to be improved, and the detection
parallel showed that the sensitivity was improved,
performance of kits from different manufacturers
especially the parallel detection of CLIA and ELISA-2
varies [9,14,15]. CLIA is a novel technique of ELISA,
for VCA-IgA, CLIA and ELISA-2 for EBNA1-IgA. The
which replaces the traditional color substrate with
sensitivity of EBV-IgA antibody in diagnosing NPC
chemiluminescence substrate and characterized by
was increased to 96.5%, as shown in Table 4.
enhancing the luminescence signal by enzyme, and
Compliance Analysis of Results Between stabilizing and prolonging the luminescence signal
Different Laboratories with CLIA Kit time. It not only boosts high sensitivity of luminescent
reaction, but also holds specificity of immune
Laboratories at four hospitals were randomly
reaction. Previous studies have shown that CLIA
selected, and VCA-IgA and EBNA1-IgA detection of
embraced satisfactory sensitivity and specificity for
34 samples were repeated by CLIA. The test results
detecting EBV IgM and IgG antibodies[16-18]. To further
were analyzed as follows: If the concentration was
screen and optimize the detection of EBV-IgA
greater than 0.9CI, the relative deviation was
antibody, we selected people from high-incidence
calculated, and the deviation was required to be
areas, medium-incidence areas and low-incidence
within 15%. If the concentration was less than 0.9CI,
areas of NPC in China to compare the diagnostic
the absolute deviation was calculated, and the
efficacy of CLIA and ELISA for NPC, and to compare
difference between the highest and lowest values was
EBV antibody level in different areas.
less than 0.25. The results showed the numbers of

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Table 5. Compliance Analysis of Results Between Different Laboratories with CLIA Kit
Number VCA-IgA EBNA1-IgA
COI-1 COI-2 COI-3 COI-4 average relative/absolute deviation COI-1 COI-2 COI-3 COI-4 average relative /absolute deviation
1 2.52 2.53 2.29 2.36 2.43 4.91%* 0.13 0.16 0.35 0.11 0.19 0.24
2 3.83 3.82 3.43 3.48 3.64 5.9%* 0.16 0.2 0.21 0.18 0.19 0.05
3 0.13 0.13 0.14 0.11 0.13 0.03 0.09 0.14 0.12 0.09 0.11 0.05
4 0.26 0.28 0.26 0.28 0.27 0.02 1.01 1.09 1.05 1 1.04 3.96%*
5 0.5 0.5 0.41 0.51 0.48 0.1 1.99 2.13 2.08 2.09 2.07 2.85%*
6 1.29 1.33 1.19 1.26 1.27 4.66%* 5.17 5.5 5.3 5.2 5.29 2.82%*
7 0.21 0.22 0.22 0.2 0.21 0.02 0.14 0.22 0.3 0.12 0.2 0.18
8 0.21 0.22 0.19 0.19 0.2 0.03 0.38 0.45 0.39 0.4 0.41 0.07
9 1.65 1.59 1.37 1.55 1.54 7.83%* 0.06 0.1 0.1 0.07 0.08 0.04
10 13.1 13.6 12.7 12.6 13 3.50%* 0.15 0.21 0.2 0.13 0.17 0.08
11 1.83 1.88 1.7 1.82 1.81 4.22%* 0.1 0.17 0.21 0.12 0.15 0.11
12 0.62 0.63 0.56 0.57 0.6 0.07 0.09 0.14 0.12 0.08 0.11 0.06
13 5.17 5.06 4.24 4.86 4.83 8.59%* 27.4 28.6 26.3 26.6 27.23 3.77%*
14 9.34 9.97 8.79 9.12 9.31 5.35%* 0.06 0.07 0.18 0.07 0.1 0.12
15 15.6 15.7 14.2 14.6 15.03 4.93% 0.12 0.14 0.16 0.13 0.14 0.04
16 0.07 0.07 0.06 0.07 0.07 0.01 0.25 0.25 0.25 0.24 0.25 0.01
17 0.06 0.07 0.06 0.06 0.06 0.01 0.13 0.17 0.17 0.13 0.15 0.04
18 6.27 6.42 5.91 6.12 6.18 3.52%* 0.39 0.43 0.42 0.39 0.41 0.04
19 0.07 0.04 0.03 0.04 0.05 0.04 0.12 0.12 0.12 0.09 0.11 0.03
20 0.24 0.28 0.22 0.24 0.25 0.06 1.16 1.3 1.14 1.12 1.18 6.92%*
21 8.02 8.16 7.32 7.63 7.78 4.90%* 0.09 0.09 0.12 0.06 0.09 0.06
22 0.23 0.26 0.23 0.24 0.24 0.03 1.3 1.51 1.42 1.35 1.4 6.53%*
23 4.08 4.13 3.31 3.9 3.86 9.77%* 19.9 20.9 19.3 20.3 20.1 3.35%*
24 0.79 0.82 0.73 0.78 0.78 0.09 0.61 0.72 0.7 0.57 0.65 0.15
25 0.1 0.11 0.1 0.1 0.1 0.01 0.22 0.29 0.2 0.21 0.23 0.09
26 0.03 0.03 0.02 0.03 0.03 0.01 0.06 0.08 0.08 0.05 0.07 0.03
27 0.16 0.16 0.15 0.14 0.15 0.02 0.04 0.05 0.09 0.05 0.06 0.05
28 0.02 0.03 0.03 0.03 0.03 0.01 0.09 0.13 0.13 0.08 0.11 0.05
29 0.04 0.05 0.04 0.04 0.04 0.01 0.16 0.19 0.16 0.15 0.17 0.04
30 0.04 0.05 0.04 0.04 0.04 0.01 0.11 0.13 0.15 0.11 0.13 0.04
31 0.04 0.03 0.07 0.04 0.05 0.04 0.1 0.1 0.21 0.09 0.13 0.12
32 0.08 0.09 0.07 0.08 0.08 0.02 0.09 0.14 0.1 0.08 0.1 0.06
33 0.1 0.1 0.09 0.09 0.1 0.01 0.29 0.36 0.32 0.28 0.31 0.08
34 0.05 0.08 0.05 0.07 0.06 0.03 0.18 0.27 0.23 0.18 0.22 0.09
Note: “*”indicated relative deviation

In this study, the positive rates of EBV antibody reached 91.5% and 93.6% respectively in total NPC
in healthy population in different regions showed that and advanced NPC. ROC curve analysis also showed
the positive rates of CLIA, ELISA-1 and ELISA-2 that the diagnostic efficacy of VCA-IgA for CLIA was
reagents of VCA-IgA were quite different, with the higher than that of the other two ELISA kits, while the
total positive rates of 5.6%, 2.5% and 7.3%, diagnostic efficacy of EBNA1-IgA for CLIA was
respectively. However, the positive rates of CLIA and similar to that of ELISA-2 kit. A domestic ELISA
ELISA-2 reagents for EBNA1-IgA were similar (5.6% compared EBV antibodies and reached a similar
and 5.2%). In healthy population, the positive rate of conclusion [22].
EBV-IgA measured by CLIA was higher than ELISA. Consistency analysis of EBV antibody kits
Considering that the NPC incidence rate among 20-69 showed that the two kits of EBNA1-IgA had relatively
year old individuals does not exceed 50 cases per excellent consistency, while the kits of VCA-IgA,
100,000 person-years even in an endemic area, a especially the positive coincidence rate was low. This
higher specificity is required for mass screening. was consistent with Liu et al’s research [23]. The
Whether CLIA reagent for EBV-IgA is suitable for consistency of VCA-IgA of ELISA-1 with domestic
NPC screening needs further study. Previous studies kits was low, which may be due to the different
have shown that ELISA for VCA-IgA has poor coating antigens used in different kits. VCA of EBV is
sensitivity in diagnosing NPC, while EBNA1-IgA has a complex containing BcLF1 (P160), BFRF3
relatively high sensitivity, especially for the diagnosis (VCA-P18), BdRF1 (VCA-p40), BLRF2 and BALF4 etc.
of early NPC [19-21]. This study also explored that in Various VCA components contain different immune
early NPC, CLIA and ELISA-2 had relatively high dominant domains, resulting in different levels of
sensitivity to detect EBNA1-IgA, both reaching 86.4%. antibody reactions. ELISA-1 employs natural proteins
The sensitivity of ELISA-1 and ELISA-2 for purified from VCA lysated from EBV infected cells
diagnosing VCA-IgA was not satisfactory, but the (VCA gp125), while domestic kits may use
sensitivity of CLIA for the detection of VCA-IgA can recombinant VCA protein components (VCAp18 and
be further improved. The sensitivity of this kit p23), which are less than purified virus natural

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Journal of Cancer 2020, Vol. 11 7182

proteins in the number of antigenic epitopes. the 20th National Science and Technology Support
However, EBNA1-IgA is merely encoded by a single Program of China (2014BAI09B10), the Sun Yat-sen
gene (BKRF1), and the singleness of antigen may be University Clinical Research 5010 Program (2013012),
one of the reasons responsible for the high consistency and the Early Detection of Cancer Project in China
between kits from different manufacturers. In the (2010-13).
future, the standards should be divided and unified
calibrators should be developed according to the Competing Interests
different peptide segments of the antigen protein to The authors have declared that no competing
achieve an accurate consistency of the results. It is interest exists.
urgent to create a nation-wide golden standard
serum/plasma pool in order to standardize EBV-IgA References
testing among laboratories and kit manufacturers [23]. 1. Tang LL, Chen WQ, Xue WQ, et al. Global trends in incidence and mortality of
nasopharyngeal carcinoma. Cancer Lett. 2016; 374: 22-30.
The positive coincidence rates of EBV antibody 2. Wei KR, Zheng RS, Zhang SW, et al. Nasopharyngeal carcinoma incidence and
detected by different kits relatively were low. Further mortality in China, 2013. Chin J Cancer. 2017; 36: 90.
3. Henle G, Henle W. Epstein-Barr Virus-specific IgA serum antibodies as an
analysis of the combined detection results of EBV outstanding feature of nasopharyngeal carcinoma. Int J Cancer. 1976; 17: 1-7.
antibodies of different kits for NPC manifested that 4. Ho HC, Ng MH, Kwan HC, et al. Epstein-Barr-Virus-specific IgA and IgG
serum antibodies in nasopharyngeal carcinoma. Br J Cancer. 1976; 34: 655-660.
the kits were with a good complementary relation- 5. Wong MM, Lye MS, Cheng HM, et al. Epstein-Barr Virus serology in the
ship. Pairwise parallel showed that the sensitivity was diagnosis of nasopharyngeal carcinoma. Asian Pac J Allergy Immunol. 2005;
23: 65–67.
improved, especially the parallel detection of 6. Dardari R, Hinderer W, Lang D, et al. Antibody responses to recombinant
VCA-IgA by CLIA and ELISA-2, EBNA1-IgA by CLIA Epstein-Barr Virus antigens in nasopharyngeal carcinoma patients:
complementary test of ZEBRA protein and early antigens p54 and p138. J Clin
and ELISA-2. The sensitivity of EBV-IgA antibody in Microbiol. 2001; 39: 3164–3170.
7. Ji MF, Sheng W, Cheng WM, et al. Incidence and mortality of nasopharyngeal
diagnosis of NPC was increased to 96.5%. Whether carcinoma: interim analysis of a cluster randomized controlled screening trial
the two different detection methods were (PRO-NPC-001) in southern China. Ann Oncol. 2019; 30: 1630-1637.
8. Fachiroh J, Paramita DK, Hariwiyanto B, et al. Single-assay combination of
complementary to each other, this topic will enlarge Epstein-Barr Virus (EBV) EBNA1-and viral capsid antigen-p18-derived
the sample size for further research. In addition, CLIA synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA
antibody levels in sera from nasopharyngeal carcinoma patients: options for
kit repeatability test found that the same specimens field screening. J Clin Microbiol. 2006; 44: 1459-1467.
tested for VCA-IgA and EBNA1-IgA in four hospital 9. Gao R, Wang L, Liu Q, et al. Evaluation of seven recombinant VCA-IgA ELISA
kits for the diagnosis of nasopharyngeal carcinoma in China: a case-control
laboratories, and the results showed that the differ- trial. BMJ Open. 2017; 7: e013211.
ences between laboratories met the requirements. 10. Liu Y, Huang QH, Liu WL, et al. Establishment of VCA and EBNA1 IgA-based
combination by enzyme-linked immunosorbent assay as preferred screening
This study also compared the positive rates of method for nasopharyngeal carcinoma: a two-stage design with a preliminary
EBV antibody in healthy population in different performance study and a mass screening in southern China. Int J Cancer. 2012;
131: 406-416.
regions. The results showed that the positive rate of 11. Ory F, Guisasola ME, Sanz JC, et al. Evaluation of four commercial systems for
EBV-IgA antibody was relatively high in the diagnosis of Epstein-Barr Virus primary infections. Clin Vaccine Immunol.
2011; 18: 444-448.
high-incidence area of NPC, while there was no 12. Hao J, Wei WQ, Zhang SW, et al. 2018 China Cancer Registry Annual Report
[article in Chinese]. Beijing, China: People’ s Health Publishing House; 2019.
significant differences in the antibody positive rates 13. Li ZM, Liang ZH, Wei KR. Cancer incidence and mortality in Zhongshan City,
between medium-incidence areas and low-incidence Guangdong Province, 2014 [article in Chinese]. China Cancer, 2019; 28:
175-180.
areas of NPC. CLIA and ELISA-2 kits for EBNA1-IgA, 14. Park Y, Park BG, Ha J, et al. Diagnostic performance and comparative
ELISA-1 kit for VCA-IgA all supported the above evaluation of the architect, liaison, and platelia Epstein-Barr Virus antibody
assays. Ann Lab Med. 2018; 38: 458-465.
conclusions. This was consistent with the results of 15. Xia C, Zhu K, Zheng GX. Expression of EBV antibody EA-IgA, Rta-IgG and
another large-scale population study [24], but it was VCA-IgA and SA in serum and the implication of combined assay in
nasopharyngeal carcinoma diagnosis. Int J Clin Exp Pathol. 2015; 8:
different from the results of Yi Bing et al.[25], 16104-16110.
considering factors such as the different sex ratio of 16. Tang ZG, Li HH, Li YC, et al. Epstein-Barr Virus EA-IgA, VCA-IgA, and
EBVNA-IgG antibodies in a population of Wuhan, China. Curr Med Sci. 2020;
men and women in the study population. 40: 168-171.
To sum up, CLIA method has good repeatability, 17. Ory F, Minguito T, Balfagon P, et al. Comparison of chemiluminescent
immunoassay and ELISA for measles IgG and IgM. APMIS. 2015;123: 648-651.
higher sensitivity and similar specificity. The level of 18. Gorrales I, Gimenez E, Navarro D. Evaluation of the architect Epstein-Barr
EBV-IgA antibody in healthy population in Virus (EBV) viral capsid antigen (VCA) IgG, VCA IgM, and EBV nuclear
antigen 1 IgG chemiluminescent immunoassays for detection of EBV
high-incidence areas of NPC is highly expressed. antibodies and categorization of EBV infection status using
immunofluorescence assays as the reference method. Clin Vaccine Immunol.
Detection of EBV-IgA antibody by CLIA may has 2014; 21: 684-688.
good application prospect in diagnosing NPC in 19. Yu X, Ji MF, Cheng WM, et al. Assessment of EBV antibodies and EBV-DNA in
the diagnosis and stages of nasopharyngeal carcinoma [article in Chinese].
high-incidence areas, but further studies on Chin J Clin Oncol. 2016; 43: 650-654.
improving CLIA specificity are required. 20. Yu X, Ji MF, Cheng WM, et al. Assessment of the long-term diagnostic
performance of a new serological screening in large-scale nasopharyngeal
carcinoma screening. J Cancer. 2018; 9: 2093-2097.
Acknowledgements 21. Guo XY, Li TD, Li FG, et al. Internittentabortive reactivation of Epstein-Barr
Virus during the progression of nasopharyngeal cancer as indicated by
This research was supported by grants from the elevated antibody levels. Oral Oncology. 2019; 93: 85-90.
National Science Foundation of China (No. 81572062),

http://www.jcancer.org
 Journal of Cancer 2020, Vol. 11 7183
22. Shi DW, Wang PP, Ji MF, et al. Evaluation of commercial kits of detecting EBV
antibodies for nasopharyngeal carcinoma diagnosis [article in Chinese].
Labeled Immunoassays & Chin Med. 2014; 21: 310-314.
23. Liu ZW, Yu KJ, Coghill AE, et al. Multilaboratory assessment of Epstein-Barr
Virus serologic assays: the case for standardization. J Clin Microbiol. 2019; 57:
1107-1119.
24. He YQ, Xue WQ, Xu FH, et al. The Relationship between enviromental factors
and the profile of Epstein-Barr Virus antibodies in the lytic and latent infection
periods in healthy populations from endemic and non-endemic
nasopharyngeal carcinoma areas in China. EBioMedicine. 2018; 30: 184-191.
25. Yi B, Gu YL, Zong YS, et al. Six anti-Epstein-Barr Virus antibodies in healthy
adults in a high risk area of nasopharyngeal carcinoma [article in Chinese].
Chinese Journal of Cancer. 2009; 28: 822-826.

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