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Journal of Clinical Microbiology, August 1999, p. 2709-2714, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Novel Immunoblot Assay Using Four Recombinant
Antigens for Diagnosis of Epstein-Barr Virus Primary Infection
and Reactivation
M.
Buisson,1,*
B.
Fleurent,2
M.
Mak,3
P.
Morand,1
L.
Chan,4
A.
Ng,3
M.
Guan,3
D.
Chin,3 and
J. M.
Seigneurin1
Laboratoire de Virologie Médicale
Moléculaire, RHAP-CNRS, Faculté de Médecine, 38043 Grenoble, France1; Genelabs Diagnostics
S.A., Geneva, Switzerland2; and Genelabs
Diagnostics Pte. Ltd.,3 and
Bioprocessing Technology Center, National University of
Singapore,4 Singapore, Republic of Singapore
Received 28 September 1998/Returned for modification 20 January
1999/Accepted 22 April 1999
 |
ABSTRACT |
A new immunoblot assay, composed of four Epstein-Barr virus
(EBV)-encoded recombinant proteins (virus capsid antigen [VCA] p23,
early antigen [EA] p138, EA p54, and EBNA-1 p72), was compared with
an immunofluorescence assay on a total of 291 sera. The test was
accurate in 94.5% of cases of primary EBV infection, while an
immunoglobulin G anti-VCA p23 band with strong intensity correlated with reactivation.
| |
TEXT |
Epstein-Barr virus (EBV) has a
worldwide distribution, with over 90% of the adult population showing
evidence of past infection. The virus, acquired during childhood,
usually causes no symptoms. However, in Western societies, 10 to 20%
of adolescents and young adults develop acute infectious mononucleosis
(IM). In humans, EBV is also associated with cancer, in particular
Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and
immunoblastic lymphoma (12). To diagnose
heterophile-negative IM cases (20 to 30%) (3) and other
EBV-associated diseases, a determination of EBV-specific antibodies is
necessary. The seroepidemiology of EBV infection and EBV-associated
diseases has predominantly relied upon immunofluorescence (IF). This
method uses fixed cells derived from various immortalized lymphoid cell
lines (5, 10). Serological parameters include the detection
of immunoglobulin G (IgG), IgM, and occasionally IgA, directed against
EBV latent nuclear antigens (EBNAs) (9); early antigens
(EAs), divisible into EA-D and EA-R components (6); and
virus capsid antigens (VCAs). The typical antibody pattern of primary
EBV infection is characterized by the presence of both IgM and IgG
antibodies to VCA and EA and by the absence of IgG antibodies to EBNA.
Anti-VCA IgM antibodies disappear during convalescence, and thus their presence is diagnostic of acute EBV infection, whereas anti-VCA IgG
antibodies are maintained for life after recovery. The IgG response to
EBNA (mainly EBNA-1) is not usually detectable until convalescence and
then persists for life. Anti-EA IgG antibodies (most frequently
anti-EA-D) are detected by IF in about 70% of patients with acute IM
and disappear after recovery. During EBV reactivation, anti-EA IgG can
reappear, frequently with a rise in anti-VCA IgG and sometimes in the
presence of anti-VCA IgM.
For the serodiagnosis of EBV infection, the IF assay, often considered
the "gold standard," is still commonly used. However, IF is
time-consuming and unsuitable for examining large numbers of samples.
Commercial enzyme-linked immunosorbent assay (ELISA) test kits for the
detection of EBV-specific antibodies have been available since the end
of the 1980s. Recently, recombinant EBV antigens have been incorporated
into ELISA kits to improve the specificity of antibody detection.
However, recent studies have shown that there are differences in the
quality of current ELISA test kits, with only a few being suitable for
routine diagnosis (19, 20). Test results sometimes conflict
from one evaluation to another, and the use of a combination of markers
from different manufacturers may be necessary (15).
In this study, we compared the performance of the new EBV IgM/IgG Blot
3.0 assay (Genelabs Diagnostics, Singapore, Republic of Singapore) with
an in-house IF assay on a total of 291 sera, assigned to one of seven
panels depending on their serological status, previously established by
IF (Table 1). Characteristics of the
panels are described in Table 2.
EBV IgM/IgG immunoblot.
The EBV IgM/IgG Blot 3.0 assay allowed
the determination of IgG and IgM antibodies directed against specific
EBV-encoded recombinant antigens. The following antigens were
incorporated onto nitrocellulose strips: VCA p23 (BLRF2), EA-D p54
(BMRF1), EA p138 (BALF2), and EBNA-1 p72 (BKRF1). As a check for serum
addition and procedural accuracy, anti-human IgG and IgM controls were
included on the strips. After the incubation of individual
nitrocellulose strips with diluted sera (1:100), antibodies that bound
specifically to EBV proteins were visualized with goat anti-human IgG
or IgM conjugated with alkaline phosphatase and a
5-bromo-4-chloro-3-indoylphosphate-nitroblue tetrazolium (BCIP/NBT)
substrate. Rheumatoid-factor absorbent was used in the IgM test.
To interpret the results, serum addition control bands (anti-IgG and
anti-IgM) must be present in the IgG-assayed strips, and the IgM
control band must be present in the IgM-assayed strips. A result was
said to be negative if no reactivity to the EBV-specific antigen bands
was found or if the intensity of any EBV-specific band was weaker than
that of the serum control band (anti-IgG). A result was said to be
positive if the antigen band was visible and if its intensity was equal
to or greater than that of the serum control band (anti-IgG). This
immunoblot assay can be processed automatically, and interpretation of
results and data management can be fully computerized. A visual
determination of band intensity was performed first and then verified
with AutoScan software (version 2.0), giving an objective
interpretation of results.
IF assay.
Indirect and anticomplement IF assays were carried
out with in-house slides for the detection of anti-VCA IgG anti-EA IgG and EBNA antibodies, respectively. EBV-specific antibodies were determined on the basis of slides, fixed in cold acetone, and prepared
from antigen-producing P3HR1 (VCA) and chemically induced Raji (EA-R
and EA-D) cells (7). Antibodies against EBNA were detected
by anticomplement IF assay in nonproducing Raji cells fixed in a
mixture of cold acetone and methanol (10). The detection of
anti-VCA IgM was performed with a commercial test kit (Gull Laboratories, Salt Lake City, Utah) according to the manufacturer's instructions. For the detection of anti-VCA IgG, anti-EA IgG, and
anti-EBNA antibodies, sera were diluted fourfold, from 1:5 to 1:5,120.
The concentration of antibodies was expressed as a titer, with the
endpoint corresponding to the last dilution showing any fluorescence.
Sera with titers greater than or equal to 1:5 were considered positive.
In the case of VCA IgM, a single dilution was performed (1:10), which
was considered the cutoff value for positivity. An algorithm of
interpretation is given in Fig. 1.
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FIG. 1.
EBV IF interpretation algorithm established from routine
laboratory experience. *, if the VCA IgG titer is  1,280, there is
possible reactivation or chronic infection; **, if the EA IgG titer
is 80, there is possible reactivation.
|
|
In the primary infection panel, heterophile antibodies were detected
with a Monospot test (Meridian Diagnostics, Cincinnati, Ohio),
following manufacturer's recommendations.
The analysis of each antibody response (Table
3) allowed us to define an interpretation
algorithm for the establishment of EBV serological status (Fig.
2). Sensitivity and specificity results are shown in Table 4.
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TABLE 3.
Prevalence of the four markers (p23, p54, p138, and p72)
in various EBV serological groups determined with the EBV IgM/IgG Blot
3.0 kit and IF assaya
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|
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FIG. 2.
EBV blot interpretation algorithm established by this
study. *, if the VCA p23 IgG titer is 3+, there is possible
reactivation without EBNA. If there is a band intensity of 3+ or less,
refer to IgM control band intensity of IgM assay.
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|
View this table:
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TABLE 4.
Results of sensitivity, specificity, and positive and
negative predictive values according to the
different panelsa
|
|
The overall immunoblot results with the classical serological markers
(EBNA IgG, VCA IgG, and VCA IgM) were very good for IgG. The results
obtained with the EBNA p72 antigen showed good specificity and
sensitivity with the different panels. On the other hand, the overall
sensitivity of VCA p23 IgG (89.5%) did not reflect the value obtained
with each panel, as this marker was consistently excellent except with
panel D, which indicated that anti-VCA p23 IgG was not among the
earliest capsid antibodies elicited during primary EBV infection. EBV
p23 is a viral late complex associated with virion particles
(16) and consists of two gene products, BFRF3
(17) and BLRF2 (11, 13). Until now, the detection
of anti-VCA antibodies in most serological ELISA tests has relied on
the response to a 110- to 125-kDa protein, a major immunogen of VCA,
encoded by BALF4 (8). Previous studies (11, 16,
17) showed that all healthy EBV carriers exhibited both BLRF2 and
BFRF3 antibodies. We confirm these results, since IgG antibodies were
detected in all past-infection sera (panel B). These antibodies can
therefore be considered the major immunodominant markers of EBV
seropositivity. However, little is known about the immunological
response to the p23 protein during an acute primary infection. Although
it was initially reported (11, 16) that sera from most IM
patients contained detectable levels of IgG and IgM to p23 (either
BFRF3 or BLRF2), further studies have suggested that antibodies to
BFRF3 could be absent in the early stages of IM (14, 18). In
our study, 49% of individuals lacked IgG BLRF2-specific antibodies,
while only 5.5% lacked antibodies to whole VCA, as determined by IF.
With the IgM class, reactivity against the VCA p23 component was found
in 75.6% of subjects. Thus, consistent with previous data concerning
BFRF3 antibodies, this evaluation highlighted a delay in the detection
of BLRF2 antibodies in patients undergoing a primary infection,
although IgM-specific antibodies were detected earlier than IgG
antibodies (18). As previously described (2, 4,
9), the predominant response during IM is directed against early
antigens p54 and p138. Indeed, anti-p138 IgM antibodies seem to
characterize the primary infection, since they were present in 100% of
acute cases while generally undetected during EBV reactivation. The
difference in EA antibody detection observed between immunoblotting and
IF during primary infection could be explained by the lack of p138 protein in the induced Raji cell line used.
From to the results obtained with the panels, a prevalence analysis of
these four IgG and IgM antibodies allowed us to define an algorithm for
interpretation. The serological profiles obtained with the immunoblot
with this decision-making tree showed a very good correlation with the
profiles obtained by IF. This blot test identifies almost all
IF-seronegative subjects and all patients with a history of past
infection and a normal positive serology. We observed very limited
interference in the IgM test, unlike some ELISAs (1). In
fact, we observed only a weak reactivity in a single sample that
corresponded to a primary infection with herpes simplex virus type 1. Thus, it seems that the serological status of patients either not
infected by EBV or presenting a past infection with anti-EBNA
antibodies does not constitute a problem with the EBV IgM/IgG Blot 3.0 assay. For subjects with past EBV infections without anti-EBNA IgG, a
state observed in certain immunosuppressed patients and elderly
individuals, the prevalence of anti-p54 IgG and anti-p138 IgG was lower
than in subjects with primary infections and reactivations. In contrast to subjects with primary infections, who all had anti-p138 antibodies, only 17.6% of the subjects with past infections showed IgM reactivity. Analysis of the different interpretations given by the algorithm showed
a 38.2% difference between IF and the new assay, due mainly to
reactivations not diagnosed by IF.
The immunoblot assay is accurate for the diagnosis of primary EBV
infection in most cases. In this assay, the detection of antibodies to
early antigens compensates for the delay observed in VCA p23 IgG-IgM
detection during the early phase of infection. There was no correlation
between the presence of heterophile antibodies and anti-p23 IgM
antibodies. IF-diagnosed serological reactivations were well detected
by immunoblotting with a very strong intensity (3+) of VCA p23 IgG
and/or EA p54 IgG, which has not been observed during primary
infection. A comparative study of the kinetics of antibodies in five
organ transplant recipients (Table 5)
revealed that, in three of the five recipients, serological
reactivation may be characterized by different immunoblot profiles. In
the other two, discrepancies between the IF and immunoblot methods were
obtained. These discrepancies show that the serological results obtained in immunosuppressed patients must be interpreted with caution.
In fact, serology is often not informative when latent EBV is
reactivated, especially in conjunction with lymphoproliferative disorders. A diagnosis of EBV reactivation should be evaluated at the
molecular level by the quantitative determination of viral load and
correlated with clinical manifestations.
Initially based on purified antigens, ELISAs developed by a number of
companies now use recombinant proteins, but a minimum of three to five
markers are required for their use. Furthermore, serological tests are
generally not suitable for the diagnosis of reactivated infection,
since there is rarely a good correlation between IF titers and ELISA
absorbance or values (1). Our present evaluation shows that
this novel immunoblot assay has diagnostic capabilities comparable to
those of the IF assay and therefore provides a new alternative for the
screening and diagnosis of EBV infection.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, CHU/Faculté de médecine, BP 217, 38043 Grenoble,
France. Phone: 334 76 76 56 04. Fax: 334 76 76 52 28. E-mail:
Marlyse.Buisson{at}ujf-grenoble.fr.
| |
REFERENCES |
| 1.
|
Debyser, Z.,
M. Reynders,
P. Goubau, and J. Desmyter.
1997.
Comparative evaluation of three ELISA techniques and an indirect immunofluorescence assay for the serological diagnosis of Epstein-Barr virus infection.
Clin. Diagn. Virol.
8:71-81[Medline].
|
| 2.
|
Färber, I.,
P. Wutzler,
P. Wohlrabe,
H. Wolf,
W. Hinderer, and H.-H. Sonneborn.
1993.
Serological diagnosis of infectious mononucleosis using three anti-Epstein-Barr virus recombinant ELISAs.
J. Virol. Methods
42:301-308[Medline].
|
| 3.
|
Fleisher, G.,
E. T. Lennette,
G. Henle, and W. Henle.
1979.
Incidence of heterophil antibody responses in children with infectious mononucleosis.
J. Pediatr.
94:723-728[Medline].
|
| 4.
|
Gorgievski-Hrisoho, M.,
W. Hinderer,
H. Nebel-Schickel,
J. Horn,
R. Vornhagen,
H.-H. Sonneborn,
H. Wolf, and G. Siegl.
1990.
Serodiagnosis of infectious mononucleosis by using recombinant Epstein-Barr virus antigens and enzyme-linked immunosorbent assay technology.
J. Clin. Microbiol.
28:2305-2311[Abstract/Free Full Text].
|
| 5.
|
Henle, G., and W. Henle.
1966.
Immunofluorescence in cells derived from Burkitt's lymphoma.
J. Bacteriol.
91:1248-1256[Abstract/Free Full Text].
|
| 6.
|
Henle, W.,
G. Henle,
B. A. Zajak,
G. Pearson,
R. Waubke, and M. Scriba.
1970.
Differential reactivity of human serums with early antigens induced by Epstein-Barr virus.
Science
169:188-190[Abstract/Free Full Text].
|
| 7.
|
Henle, W.,
G. Henle, and C. A. Horwitz.
1974.
Epstein-Barr virus specific diagnostic test in infectious mononuclosis.
Hum. Pathol.
5:551-565[Medline].
|
| 8.
|
Luca, J.,
R. C. Chase, and G. R. Pearson.
1984.
A sensitive enzyme-linked immunosorbent assay (ELISA) against the major EBV-associated antigens. 1. Correlation between ELISA and immunofluorescence titers against purified antigens.
J. Immunol. Methods
67:145-156[Medline].
|
| 9.
|
Middeldorp, J. M., and P. Herbrink.
1988.
Epstein-Barr virus specific marker molecules for early diagnosis of infectious mononucleosis.
J. Virol. Methods
21:133-146[Medline].
|
| 10.
|
Reedman, B. M., and G. Klein.
1973.
Cellular localisation of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines.
Int. J. Cancer
11:599-620.
|
| 11.
|
Reischl, U.,
C. Gerdes,
M. Motz, and H. Wolf.
1996.
Expression and purification of an Epstein-Barr virus encoded 23-kDa protein and characterization of its immunological properties.
J. Virol. Methods
57:71-85[Medline].
|
| 12.
|
Rickinson, A. B., and E. Kieff.
1996.
Epstein-Barr virus, p. 2397-2446.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Virology, 3rd ed. Raven Press, New York, N.Y.
|
| 13.
|
Serio, T. R.,
A. Angeloni,
J. L. Kolman,
L. Gradoville,
R. Sun,
D. A. Katz,
W. M. J. van Grunsven,
J. M. Middeldorp, and G. Miller.
1996.
Two 21-kilodalton components of the Epstein-Barr virus capsid antigen complex and their relationship to ZEBRA-associated protein p21 (ZAP21).
J. Virol.
70:8047-8054[Abstract].
|
| 14.
|
Shedd, D.,
A. Angeloni,
J. Niederman, and G. Miller.
1995.
Detection of human serum antibodies to the BFRF3 Epstein-Barr virus capsid component by means of a DNA-binding assay.
J. Infect. Dis.
172:1367-1370[Medline].
|
| 15.
|
Svahn, A.,
M. Magnusson,
L. Jägdahl,
L. Schloss,
G. Kahlmeter, and A. Linde.
1997.
Evaluation of three commercial enzyme-linked immunosorbent assays and two latex agglutination assays for diagnosis of primary Epstein-Barr virus infection.
J. Clin. Microbiol.
35:2728-2732[Abstract].
|
| 16.
|
van Grunsven, W. M. J.,
A. Nabbe, and J. M. Middeldorp.
1993.
Identification and molecular characterization of two diagnostically relevant marker proteins of the Epstein-Barr virus capsid antigen complex.
J. Med. Virol.
40:161-169[Medline].
|
| 17.
|
van Grunsven, W. M. J.,
E. C. Van Heerde,
H. J. W. De Haard,
W. J. M. Spaan, and J. M. Middeldorp.
1993.
Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins.
J. Virol.
67:3908-3916[Abstract/Free Full Text].
|
| 18.
|
van Grunsven, W. M. J.,
W. J. M. Spaan, and J. M. Middeldorp.
1994.
Localization and diagnostic application of immunodominant domains of the BFRF3-encoded Epstein-Barr virus capsid protein.
J. Infect. Dis.
170:13-19[Medline].
|
| 19.
|
Weber, B.,
M. Brunner,
W. Preiser, and H. W. Doerr.
1996.
Evaluation of 11 enzyme immunoassays for the detection of immunoglobulin M antibodies to Epstein-Barr virus.
J. Virol. Methods
57:87-93[Medline].
|
| 20.
|
Wiedbrauk, D. L., and S. Bassin.
1993.
Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus viral capsid antigens.
J. Clin. Microbiol.
31:1339-1341[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, August 1999, p. 2709-2714, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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