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Avidity of EBV VCA-specific IgG antibodies: distinction between recent primary infection, past infection and reactivation
Journal of Krological Methods ELSEVIER
Journal of VirologicaI
Methods 52 (1995) 95-104
Avidity of EBV VCA-specific IgG antibodies: distinction between recent primary infection, past infection and reactivation J.J. Gray Clinical Microbiology
and Public Health Laboratory, Addenbrooke’s Accepted
Hospital, Cambridge CB2 2QW, UK
5 October 1994
Abstract A commercial Epstein-Barr virus (EBV) antivirus capsid antigen (VCA) IgG antibody ELISA and an ‘in-house’ EBV VCA IgG immunofluorescence antibody assay (IFA) were used to detect EBV VCA IgG antibodies in 100 serum samples collected from organ transplant recipients and immunocompetent individuals. The avidity of EBV VCA IgG antibodies was determined in the IFA and ELISA using the mild reducing agent 8 M urea to remove low avidity antibodies. The samples were collected from patients who had previously been identified with a primary EBV infection, a reactivation of latent infection or evidence of a past EBV infection by means of EBV-specific serology. Using the ELISA, the antibody avidity was low in samples collected from patients with recent EBV infection and high in samples collected from patients with a past infection or a reactivation. There was a statistically significant difference of means (P < 0.001) of percentage reduction in optical density values, measured in the presence of 8 M urea, obtained with samples collected from patients with recent infection compared with samples from patients with a past infection or a reactivation of latent infection. Keywords: Epstein-Barr virus; Antivirus immunosorbent assay; Antibody avidity
capsid antigen; Immunofluorescence
antibody
assay; Enzyme-linked
1. Introduction Epstein-Barr virus (EBV) is ubiquitous with 80-90% of adults worldwide showing serological evidence of past infection (Crawford and Edwards, 1987). EBV is associated with infectious mononucleosis (Purtilo, 1987), Burkitt’s lymphoma (Epstein et al., 1964), nasopharyngeal carcinoma (Zur Hausen et al., 1970) and lymphoproliferative 0166-0934/95/$09.50 6 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00147-2
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disease in immunocompromised individuals (Hanto et al., 1985; Purtilo et al., 19851, but the majority of infections with EBV are asymptomatic (De The, 1982). EBV, in common with the other human herpes viruses, establishes a latent infection which may be reactivated in the immunocompromised host (Henle and Henle, 1981). The laboratory diagnosis of EBV infection is based on serological tests to detect both specific antibodies to EBV antigens (Henle and Henle, 1981, 1982; De The, 1982; Purtilo et al., 1985) and heterophile antibodies (Paul and Bunnell, 1932; Davidsohn and Lee, 1969). At the onset of symptoms, the humoral response to EBV infection is characterized by the presence of circulating antivirus capsid antigen (VCA) IgM antibodies and an almost concurrent rise in anti-VCA IgG antibodies (Okano et al., 1988). Antibodies to the diffuse early antigen component of EBV (anti-EA-D) are transient and can be detected in 80% of infected individuals; antibodies to the restricted EA component (anti-EA-R) are rare in acute infectious mononucleosis, but may be detected transiently during convalescence (Okano et al., 1988). IgM antibodies to the EBV nuclear antigen (EBNA) may be detected during primary infection, but EBNA IgG antibodies are not usually detected until several weeks after the onset of symptoms (Henle and Henle, 1981). Although heterophile antibodies may be detected in up to 85% of adults with primary EBV infection, they are absent in the majority of children under 4 years of age (Sumaya, 19861. Serological diagnosis of EBV infection is often based on the results obtained from testing a sample from a symptomatic patient against a panel of EBV antigens (VCA, EA-D and EBNA) combined with those obtained in the Paul-Bunnell test for heterophile antibodies. The humoral response to EBV infection or the reactivation of a past infection in an immunocompromised host may be atypical with elevated titres of anti-VCA and anti-EA-D with or without a concomitant rise in anti-EBNA (Okano et al., 1988). EBV VCA IgG antibodies are a consistent marker of EBV infection and are present in infectious mononucleosis, asymptomatic infection, past infection, reactivation, Burkitt’s lymphoma and nasopharyngeal carcinoma (Okano et al., 1988). The early virus-specific IgG antibody response after primary infection is made up primarily of low avidity antibodies, with avidity increasing in a few weeks to several months after infection (Inouye et al., 1984; Kocks and Rajewsky, 1988). This property of the humoral IgG response has been used in serological assays to distinguish between primary rubella virus infection and reinfection (Thomas and Morgan-Capner, 19881, primary cytomegalovirus (CMV) infection and reactivation of latent infection (Lutz et al., 1994), and recent and past parvovirus B19 infection (Gray et al., 1993). Gray and Wreghitt (1989) and de Ory et al. (1993) described modifications to the EBV VCA IFA assay which enabled the determination of the avidity of anti-VCA IgG antibodies. These modified assays were able to differentiate between primary EBV infection and a reactivation of latent infection in organ transplant recipients and between recent and past infection in immunocompetent individuals. In this study, a commercial EBV VCA IgG ELISA (Sigma Chemical Company Ltd., Dorset, UK) with purified EBV-infected cell lysate supplemented with purified EBV gp125 as antigens attached to the solid phase, was used to determine the presence of EBV VCA IgG antibodies and their avidity in samples collected from immunosuppressed organ transplant recipients and immunocompetent individuals. The value of this data as a ‘serological marker’ in
J.J. Gray/Journal
of Viological Methods 52 (I 995) 95-I 04
97
the diagnosis of a recent EBV infection, past EBV infection and reactivation of past infection was assessed and compared with the previous data (Gray and Wreghitt, 1989; de Ory et al., 1993).
2. Materials and methods 2.1. Serum samples One hundred samples of serum were investigated: 54 sera were collected from 13 organ transplant recipients with evidence of primary EBV infection or reactivation of past infection, and 46 from immunocompetent individuals with symptoms suggesting EBV infection. Twenty eight had evidence of recent EBV infection and 18 of past infection. The samples had previously been tested for EBV VCA IgG and IgM in IFAs, for EBV VCA IgM in an ELISA (Sigma Chemical Co. Ltd., Dorset, UK) and for heterophile antibodies in the Paul-Bunnell test. After transplantation, the most recent samples collected from the organ transplant recipients were tested for the presence of EBNA IgG. The avidity of EBV VCA IgG antibodies was determined in 36 of the samples collected from the transplant recipients (i.e. those samples in which EBV VCA IgG was detected using the ELISA) and in the 46 samples from immunocompetent individuals. 2.2. EBV VCA antibody ELZSAs The EBV VCA IgG ELISA (Sigma Chemical Co. Ltd., Dorset, UK) and the EBNA IgG ELISA (Biotest Diagnostics, Dreieich, Germany) were performed according to the manufacturers’ instructions. EBV VCA IgG antibody avidity was determined in the EBV VCA IgG ELISA (Sigma) with the first wash step modified to include two 5-min washes with 8 M urea incorporated in the wash fluid. A sample was considered as containing a significant proportion of low avidity antibody if there was a 2 50% reduction in optical density (OD) in the presence of 8 M urea. 2.3. Paul-Bunnell
test
The Paul-Bunnell test was performed as described by Gray et al. (1992) and the antibody titre was expressed as the reciprocal of the highest serum dilution to show macroscopic agglutination. 2.4. Indirect immunojluorescence
The EBV incorporating (1989). The EBV incorporating
antibody assay
VCA IgG indirect IFA and the IgG antibody avidity determination, 8 M urea in the IFA, were performed as described by Gray and Wreghitt VCA antibody titres obtained with the conventional IFA and the IFA 8 M urea were compared and the sample was deemed to contain a
J.J. Gray /Journal
98 Table 1 EBV serology Patient
in heart and heart/lung
Days postetx
of Virological Methods 52 (1995) 95-104
transplant
recipients Other EBV serology
VCA IgG ELISA EBV VCA IgG IFA titre < 10
EBV VCA IgG ELISA (AU/ml)
Result d
% Reduction in OD with 8 M urea
+ + +
n.a. n.a. 55.6 68.5 76.6 35.4
_ _ _ _ +
n.a. n.a. n.a. n.a. 27.5
92 119 160 224 931
< 10 640 2560 2560 2560
10.0 18.1 100.7 357.8 599.1 1149.6
52 79 116 177 914
< 10 < 10 < 10 160 2560
0.0 0.0 35.1 64.9 1171.9
52 100 190 371 800
< 10 < 10 40 2560 10240
0.0 0.0 29.8 666.5 1149.6
-2 25 110 171 388
< 10 < 10 2560 2560 > 10240
0.0 X8.9 224.2 605.5 1235.2
-1 19 55 82 140 193 817
< 10 10 < 10 < 10 160 640 2560
0.0 83.0 0.0 35.6 148.1 206.7 1321.8
+ + +
“.a. n.a. n.a. n.a. 57.1 60.5 17.5
39 58 86 184 402
< 10 < 10 < 10 640 2560
0.0 0.0 129.9 312.7 540.5
+ + f
n.a. n.a. 54.7 64.1 61.3
0 28 84 176 736
< 10 < 10 > 10240 > 10240 > 10240
0.0 0.0 145.8 534.7 1245.1
+ + +
n.a. n.a. 62.7 68.0 19.3
77 169 364
640 2560 2560
848.6 826.9 1040.1
+ + +
60.2 55.6 40.8
57
_
_ _ + + _ + + c
n.a. n.a. n.a. 64.8 50.2 n.a. n.a. 56.7 68.5 21.6
Antibody avidity b
EBV VCA IgM ELISA Sigma
EBNA-IgG ELISA Biotest
High
_ -
n.t. n.t. nt. n.t. n.t.
High
_ -
n.t. n.t. n.t. n.t.
LOW
+
LOW LOW LOW
LOW
LOW LOW
High
-
LOW LOW
High
LOW LOW LOW
LOW LOW
High LOW LOW
High
-
-
n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. _ + _
-
n.t. n.t. n.t. n.t.
_ _
n.t. n.t. n.t. n.t.
_ _ _ -
n.t. n.t. n.t. n.t.
+ + =
+ n.t. n.t.
_
_
J.J. Gray /Journal
99
of Virological Methods 52 (I 995) 95-l 04
Table 1 (continued) Patient
Days postmtx
Other EBV serology
VCA IgG ELISA EBV VCA IgG IFA titre
EBV VCA IgG ELISA (AU/ml)
Result a
% Reduction in OD with 8 M urea
Antibody avidity b
84 365 137
640 640 10240
699.8 924.7 1191.2
+ + +
39.3 33.5 17.8
High High High
10
104 239 335
640 2560 2560
380.6 762.5 665.3
+ + +
38.3 33.7 40.0
11
105 193 414
640 2560 2560
593.8 195.3 1140.9
+ + +
12
180 389
160 2560
912.4 1246.3
285 364
2560 > 10240
775.4 892.5
9
13
EBV VCA IgM ELISA Sigma
-
EBNA-IgG ELISA Biotest
+
-
n.t. n.t.
High High High
-
+ n.t. n.t.
42.0 38.5 19.2
High High High
+ -
+ n.t. n.t.
+ +
25.9 17.2
High
+
+ n.t.
+ +
29.7 22.8
High High
-
n.t.
+
n.a., not applicable; nt., not tested a - , negative; + , positive; = , equivalent. b Avidity: low, > 50% reduction; high, < 50% reduction.
significant proportion of low avidity antibody if there was an 2 B-fold reduction in antibody titre in the presence of 8 M urea.
3. Results 3.1. EBV infection in heart and heart/lung
transplant recipients
Seroconversion (> 4-fold increase in titre) was detected in serial samples collected from patients l-7 (Table 1) in both the EBV VCA IgG IFA and the EBV VCA IgG ELISA. EBNA IgG antibody was not detected in samples collected from these transplant recipients before seroconversion of EBV VCA IgG indicating primary EBV infection. Patient 8 (Table 1) had detectable EBV VCA IgG and EBNA IgG antibodies in a sample collected 77 days post-transplant. On the basis of this result it was difficult to determine whether this patient had experienced a primary EBV infection in the preceding 77 days (see below) or was experiencing a reactivation of a past infection. In comparing the two test procedures, infection was detected earlier by the IFA in patients 2 and 3 and earlier by ELISA in patient 6 (Table 1). EBV VCA IgM was detected in only one out of the 8 transplant recipients experiencing primary EBV infection (Table 1). Low avidity IgG was detected in samples from patients 1, 3, 4, 5, 6, 7 and 8, but not in patient 2. The presence of low avidity antibody in samples collected from patient 8 suggested a primary EBV infection. Low avidity EBV VCA IgG antibodies were
100
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of Virological Methods 52 (1995) 95-l 04
detected for up to 6 months in recipients 4, 5, 7 and 8 and for longer in recipients 1, 3 and 6 (Table 1). Passive antibody was detected by EBV VCA IgG IFA and EBNA IgG ELISA in a sample collected from patient 5, 19 days post-transplant and although there was a concomitant increase in the concentration of antibody detected in the ELISA (0.0-83.0 AU/ml), this was below the positive range of the assay. A subsequent sample collected from this patient on day 55 was EBV VCA IgG negative and EBNA IgG negative and the concentration of antibody detected in the ELISA had returned to its previous level (Table 1). Increasing concentrations of EBV VCA IgG were detected, using the ELISA, in samples from 5 organ transplant recipients (9-13) with evidence of reactivation, and from recipients (3-6) during reactivation of infection following a primary infection after transplant. EBNA IgG antibodies were detected in samples collected from patients 9-13 before a significant rise in EBV VCA IgG was detected, confirming reactivation of latent EBV infections in these patients. The increases in antibody concentration ranged from 1.2 to 6.4 times the concentration found in the previous sample. Increasing EBV VCA IgG antibody concentrations (1.3 to 3.7 times) were also found in recipients whose samples were shown to have stable titres when tested in the IFA. Results obtained in the ELISA but not in the IFA indicated that recipients 1, 7 and 8, who had evidence of primary EBV infection, experienced a subsequent reactivation of infection and recipient 11 experienced a reactivation of EBV infection on two occasions. EBV VCA IgM was detected in only two of the 9 transplant recipients experiencing a reactivation of EBV infection. The avidity of EBV VCA IgG antibodies was high in all samples collected from these recipients at the time of EBV reactivation (Table 1). 3.2. Immunocompetent
individuals
with evidence
of recent
or past infection
All 48 samples collected from immunocompetent individuals had detectable VCA IgG antibodies when tested by the ELISA. The 28 samples collected from patients with recent EBV infection contained significant concentrations of low avidity antibodies when tested by the modified ELISA. The mean percentage reduction in OD in the presence of 8 M urea was 70.9% (range 51.6-85.2%) compared with a mean percentage reduction of 30.8% (range 7.2-42.1%) obtained with the 18 samples collected from patients with evidence of past EBV infection (Table 2). Fig. 1 shows the difference in the percentage reduction in OD in the presence of 8 M urea in samples collected from patients (immunocompromised and immunocompetent)
Table 2 EBV VCA IgG antibody avidity in samples collected recent mimarv or east EBV infection
from immunocompetent
individuals
with evidence
Patients
Number of samples
Mean % reduction in OD in the presence of 8 M urea (range)
Recent primary infection Past infection
28 18
70.9 (51.6-85.2) 30.8 (7.2-42.1)
of
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of Virological Methods 52 (1995) 95-104
101
EBV VCA IgG avidtty
1-9
lo-19
20-29
30-39
40-49
% reduction m
Fig. 1. Percentage
reduction
past
infection
50-59
60-69
70-79
60-69
in OD 11101 recent
infection
in OD values obtained in the EBV VCA IgG ELISA in the presence of 8 M urea.
with a history of past infection compared to those with evidence of a recent infection. The mean reduction of OD in samples collected from all patients with a recent EBV infection was 67.6% (range 50.2-85.2%) and in patients with evidence of a past infection was 30.1% (range 7.2-42.1%). There was a statistically significant difference (P < 0.001 (Student’s t-test for differences of means)) between the mean percentage reduction in OD obtained with samples collected from patients with recent infection compared to samples collected from patients with past infection. 4. Discussion For the serological diagnosis of EBV infection it is necessary to test for specific antibodies against a range of antigens as, on many occasions, no single test can be relied upon to provide sufficient information for a definitive diagnosis. Anti-EA-D antibodies are transient and only detected in 80% of infected individuals and anti-EA-R antibodies are rarely present in the acute phase of infectious mononucleosis. Although EBNA IgM antibodies can indicate a recent infectidil, non-specific reactivity leading to false-positive results have been reported in assays for detecting EBNA IgM antibodies (Matheson et al., 1990; Gray et al., 1992). EBNA IgG antibodies may not be detected for several weeks after the onset of symptoms and reactivation of a latent EBV infection in the immunocompromised host may not elicit a rise in anti-EBNA antibodies. A negative result by the Paul-Bunnell test does not exclude a recent EBV infection as heterophile antibodies may not be present or detectable in the majority of children and some adults. The presence of EBV VCA IgM and anti&&D antibodies in the absence of antibodies to EBNA is often used as the criterion for the laboratory diagnosis of primary EBV infection. In this study, only one out of 8 transplant recipients who experienced a primary EBV infection had detectable EBV VCA IgM antibodies by the ELISA. This
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of Virological Methods 52 (1995) 95-104
may be due to the presence of EBV VCA IgG in the samples, even after adsorption with anti-IgG, competing with the virus-specific IgM for the available binding sites in the EBV VCA IgM indirect ELISA (Henle et al., 1974a,b). Wiedbrauk and Bassin (1993) reported, in an evaluation of 5 commercial ELISAs for detecting EBV VCA IgM, that the Sigma VCA IgM assay had a sensitivity of 78% and a specificity of 86% when compared with the IFA. Although the inability to detect EBV VCA IgM antibodies in the ELISA may reflect atypical immune responses to EBV, a well recognized phenomenon, associated with infection in the immunocompromised host (Okano et al., 1988>, EBV VCA IgM antibodies were detected using an EBV VCA IgM IFA in all 8 transplant recipients with primary EBV infection (data not shown). The usefulness of determining the avidity of EBV VCA IgG antibodies in immunocompromised and immunocompetent individuals using IFAs has been demonstrated (Gray and Wreghitt, 1989; G ray et al., 1992; de Ory et al., 1993). Recent infections were characterized by the presence of low avidity EBV VCA IgG antibodies and past infections by high avidity antibodies. Although these assays were satisfactory they were prone to the problems inherent in all IFAs. They were time consuming, labour intensive, prone to batch-to-batch variation, and their subjective nature made end-point determination difficult. In this study, the suitability of a semiquantitative EBV VCA IgG ELISA (Sigma) as an alternative assay to immunofluorescence and its use in determining IgG antibody avidity was evaluated. There was a very good correlation (P < 0.001) between the log IFA titres and log ELISA antibody concentrations (AU/ ml), and the ELISA was able to detect all the seroconversions in the transplant recipients previously detected in the IFA. The absence of guidelines in the ELISA protocol, indicating what constituted a significant rise in antibody concentration in a patient with pre-existing EBV VCA IgG antibodies, casts doubt on the suitability of the ELISA to identify a reactivation of past infection. Increases in antibody concentration were detected both in organ transplant recipients with significant rises in antibody titre in the IFA and in recipients with stable titres in the IFA. This may indicate a lack of reproducibility in this assay which had previously been shown to be the least variable of the commercial assays in day-to-day testing (Wiedbrauk and Bassin, 1993). It is possible that the ELISA could detect reactivations of past infection better, and these results may confirm the increased sensitivity of EBV-specific antibody ELISAs, when compared with the IFAs, as reported by Luka et al. (1984) who used purified EBV antigen in an ELISA and DSlken et al. (1984) who utilized EBV-infected cell lysate antigen. In this study the microtitre plate wells were coated with purified EBV-infected cell lysate antigen supplemented with purified EBV gp125, a major immunoreactive protein of EBV (Luka et al., 1984). The ELISA results are consistent with the atypical EBV serological profiles seen in many immunocompromised individuals, whether genetic or iatrogenic. (Henle and Henle, 1981; Henle and Henle, 1982; Purtilo et al., 1985; Purtilo, 1987). The antibody avidity of EBV VCA IgG antibodies detected by ELISA was low (characterized by a > 50% reduction of OD in the presence of 8 M urea) in samples collected from patients with recent EBV infection and high ( < 50% reduction in OD) in those collected from patients with a past infection or reactivation of a past infection. This was true for both the immunocompromised and immunocompetent patients. There
J.J. Gray /Journal
of Virological Methods 52 (1995) 95-l 04
103
a statistically significant (P < 0.001) difference between the means of the percentage reduction in OD values in the two groups represented by patients with recent infection compared with patients with past infection or a reactivation of a latent infection. Plotting the frequency of the percentage reduction of OD values in samples collected from patients with recent and past infection suggests the possibility of an overlap between the two groups (Fig. 1). This indicates the need for an equivocal zone to take account of similar percentage reductions in OD values that may be detected in samples from either recent infections or past infections. An equivocal zone can be created by determining the standard deviation (S.D.) of the means of the percentage reductions in OD values in samples from patients with recent infection and patients with past infection. The equivocal zone would be the values between the mean percentage reduction value (OD f 3 S.D.) in past infection and in recent infection. In this study, the equivocal zone would include all percentage reductions in OD values obtained in the presence of 8 M urea between 41.47 and 57.4%. None of results obtained with the samples from patients with a history of past infection were in the equivocal zone, but 8 samples from patients with recent infection were. All 8 had > 50% reduction of OD values in the presence of 8 M urea. The time taken for the antibody response to mature from low to high avidity in patients experiencing a primary EBV infection was variable in the immunocompromised organ transplant recipients. This confirms previous reports that immunosuppression may delay the maturation of the humoral immune response to infection (Gray and Wreghitt, 1989; Lutz et al., 1994; Ward et al., 1994). The EBV VCA IgG antibody avidity assay by ELISA can be a useful diagnostic tool for the serological diagnosis of EBV infection. It is a suitable alternative to the more labour intensive and subjective EBV VCA IFAs and is able to differentiate primary from past infection using only one EBV serological marker (EBV VCA IgG). The correlation between recent primary EBV infection and the presence of significant concentrations of low avidity antibodies will allow the diagnosis of recent primary EBV infection using a single serum sample collected during the acute phase of the illness. was
References Crawford, D.H. and Edwards, J.M.B. (1987) Epstein-Barr virus. In: A.J. Zuckerman, J.E. Banatvala and J.R. Pattison (Eds.), Principles and Practice of Clinical Virology. John Wiley and Sons, London, pp. 111-133. Davidsohn, I. and Lee, C.L. (1969) The clinical serology of infectious mononucleosis. In: R.L. Carter and H.G. Penman (Eds.), Infectious Mononucleosis. Blackwell Scientific Publications, Ltd., Oxford, pp. 177-200. de Ory, F., Antonaya, J., Fernandez, M.V. and Echevarria, J.M. (1993) Application of low-avidity immunoglobulin G studies to diagnosis of Epstein-Barr virus infectious mononucleosis. J. Clin. Microbial. 31, 1669-1671. De-The, G. (1982) Epidemiology of Epstein-Barr virus and associated diseases in man. In: B. Roizman (Ed.), The Herpesviruses, Vol. 1. Plenum Publishing Corp., New York, pp. 25-103. DBlken, G., Weitzmann, U., Boldt, C., Bitzer, M., Brugger, W. and Lohr, G. (1984) Enzyme-linked immunosorbent assay for IgG antibodies to Epstein-Barr virus-associated early antigens and viral capsid antigen. J. Immunol. Methods 67, 225-233.
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Epstein, M.A., Achong, B.G. and Barr, Y.M. (1964) Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet i, 702-703. Gray, J.J. and Wreghitt, T.G. (1989) Immunoglobulin G avidity in Epstein-Barr virus infections in organ transplant recipients. Serodiagn. Immunother. Infect. Dis. 3, 389-393. Gray, J.J., Caldwell, J. and Sillis, M. (1992) The rapid serological diagnosis of infectious mononucleosis. J. Infect. 25, 39-46. Gray, J.J., Cohen, B.J. and Desselberger, U. (1993) Detection of human parvovirus B19-specific IgM and IgG antibodies using a recombinant viral VP1 antigen expressed in insect cells and estimation of time of infection by testing for antibody avidity. J. Virol. Methods 44, 11-24. Hanto, D.W., Frizzera, G., Gajl-Peczalska and Simmons, R.L. (1985) Epstein-Barr virus, immunodeficiency, and B cell lymphoproliferation. Transplantation 39, 461-472. HenIe, W. and HenIe, G. (1981) Epstein-Barr virus specific serology in immunologically compromised individuals. Cancer Res. 41, 4222-4225. HenIe, W. and Henle, G. (1982) Immunology of Epstein-Barr virus, In: B. Roizman (Ed.), The Herpesviruses, Vol. 1. Plenum Publishing Corp., New York, pp. 209-252. Henle, G., Henle, W. and Horwitz, C.A. (1974a) Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J. Infect. Dis. 130, 231-239. Henle, W., Henle, G. and Horowitz, CA. (1974b) Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. Hum. Pathol. 5, 551-565. Inouye, S., Hasegawa, A., Matsuno, S. and Katwo, S. (1984) Changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed. J. Clin. Microbial. 20, 525-529. Kocks, C. and Rajewsky, K. (1988) Stepwise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Acad. Sci. U.S.A. 85, 8206-8210. Luka, J., Chase, R.C. and Pearson, G.R. (1984) A sensitive enzyme-linked immunosorbent assay (ELISAl against the major EBV-associated antigens. I. Correlation between ELISA and immunofluorescence titers using purified antigens. J. Immunol. Methods 67, 145-156. Lutz, E., Ward, K.N. and Gray, J.J. (1994) Maturation of antibody avidity after primary human cytomegalovirus infection is delayed in immunosuppressed solid organ transplant patients. J. Med. Virol., accepted. Matheson, B.A., Chisholm, S.M. and Ho-Yen, D.O. (1990) Assessment of rapid ELISA test for detection of Epstein-Barr virus infection. J. Clin. Pathol. 43, 691-693. Okano, M., Thiele, G.M., Davis, J.R., Grierson, H.L. and Purtilo, D.T. (1988) Epstein-Barr virus and human diseases: recent advances in diagnosis. Clin. Microbial. Rev. 1, 300-312. Paul, J.R. and Bunnell, W.W. (1932) The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 183, 90-104. Purtilo, D.T. (1987) Epstein-Barr virus: the spectrum of its manifestations in humans. South Med. J. 80, 943-947. Purtilo, D.T., Tatsumi, E., Manolov, G., Manolova, Y., Harada, S., Lipscomb, H. and Krueger, G. (1985) Epstein-Barr virus as an etiological agent in the pathogenisis of lymphoproliferative and aproliferative disease in immune deficient patients, Int. Rev. Exp. Pathol. 27, 113-183. Sumaya, C.V. (1986) Infectious mononucleosis and other EBV infections: diagnostic factors. Lab. Manag. 24, 37-45. Thomas, H.I.J. and Morgan-Capner, P. (1988) Rubella-specific IgG subclass avidity and its role in differentiation between primary rubella and rubella reinfection. Epidemiol. Infect. 101, 591-598. Ward, K.N., Dhaliwal, W., Ashworth, K.L., Clutterbuck, E.J. and Teo, C.G. (1994) Measurement of antibody avidity for hepatitis C virus distinguishes primary antibody responses from passively acquired antibody. J. Med. Virol., in press. Wiedbrauk, D.L. and Bassin, S. (1993) Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus virus capsid antigens. J. Clin. Microbial. 31, 1339-1341. Zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P. and Santesson, L. (1970) EBV DNA in biopsies of Burkitt’s tumors and anaplastic carcinoma of the nasopharynx. Nature 228, 1056-1058.
Journal of VirologicaI
Methods 52 (1995) 95-104
Avidity of EBV VCA-specific IgG antibodies: distinction between recent primary infection, past infection and reactivation J.J. Gray Clinical Microbiology
and Public Health Laboratory, Addenbrooke’s Accepted
Hospital, Cambridge CB2 2QW, UK
5 October 1994
Abstract A commercial Epstein-Barr virus (EBV) antivirus capsid antigen (VCA) IgG antibody ELISA and an ‘in-house’ EBV VCA IgG immunofluorescence antibody assay (IFA) were used to detect EBV VCA IgG antibodies in 100 serum samples collected from organ transplant recipients and immunocompetent individuals. The avidity of EBV VCA IgG antibodies was determined in the IFA and ELISA using the mild reducing agent 8 M urea to remove low avidity antibodies. The samples were collected from patients who had previously been identified with a primary EBV infection, a reactivation of latent infection or evidence of a past EBV infection by means of EBV-specific serology. Using the ELISA, the antibody avidity was low in samples collected from patients with recent EBV infection and high in samples collected from patients with a past infection or a reactivation. There was a statistically significant difference of means (P < 0.001) of percentage reduction in optical density values, measured in the presence of 8 M urea, obtained with samples collected from patients with recent infection compared with samples from patients with a past infection or a reactivation of latent infection. Keywords: Epstein-Barr virus; Antivirus immunosorbent assay; Antibody avidity
capsid antigen; Immunofluorescence
antibody
assay; Enzyme-linked
1. Introduction Epstein-Barr virus (EBV) is ubiquitous with 80-90% of adults worldwide showing serological evidence of past infection (Crawford and Edwards, 1987). EBV is associated with infectious mononucleosis (Purtilo, 1987), Burkitt’s lymphoma (Epstein et al., 1964), nasopharyngeal carcinoma (Zur Hausen et al., 1970) and lymphoproliferative 0166-0934/95/$09.50 6 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00147-2
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disease in immunocompromised individuals (Hanto et al., 1985; Purtilo et al., 19851, but the majority of infections with EBV are asymptomatic (De The, 1982). EBV, in common with the other human herpes viruses, establishes a latent infection which may be reactivated in the immunocompromised host (Henle and Henle, 1981). The laboratory diagnosis of EBV infection is based on serological tests to detect both specific antibodies to EBV antigens (Henle and Henle, 1981, 1982; De The, 1982; Purtilo et al., 1985) and heterophile antibodies (Paul and Bunnell, 1932; Davidsohn and Lee, 1969). At the onset of symptoms, the humoral response to EBV infection is characterized by the presence of circulating antivirus capsid antigen (VCA) IgM antibodies and an almost concurrent rise in anti-VCA IgG antibodies (Okano et al., 1988). Antibodies to the diffuse early antigen component of EBV (anti-EA-D) are transient and can be detected in 80% of infected individuals; antibodies to the restricted EA component (anti-EA-R) are rare in acute infectious mononucleosis, but may be detected transiently during convalescence (Okano et al., 1988). IgM antibodies to the EBV nuclear antigen (EBNA) may be detected during primary infection, but EBNA IgG antibodies are not usually detected until several weeks after the onset of symptoms (Henle and Henle, 1981). Although heterophile antibodies may be detected in up to 85% of adults with primary EBV infection, they are absent in the majority of children under 4 years of age (Sumaya, 19861. Serological diagnosis of EBV infection is often based on the results obtained from testing a sample from a symptomatic patient against a panel of EBV antigens (VCA, EA-D and EBNA) combined with those obtained in the Paul-Bunnell test for heterophile antibodies. The humoral response to EBV infection or the reactivation of a past infection in an immunocompromised host may be atypical with elevated titres of anti-VCA and anti-EA-D with or without a concomitant rise in anti-EBNA (Okano et al., 1988). EBV VCA IgG antibodies are a consistent marker of EBV infection and are present in infectious mononucleosis, asymptomatic infection, past infection, reactivation, Burkitt’s lymphoma and nasopharyngeal carcinoma (Okano et al., 1988). The early virus-specific IgG antibody response after primary infection is made up primarily of low avidity antibodies, with avidity increasing in a few weeks to several months after infection (Inouye et al., 1984; Kocks and Rajewsky, 1988). This property of the humoral IgG response has been used in serological assays to distinguish between primary rubella virus infection and reinfection (Thomas and Morgan-Capner, 19881, primary cytomegalovirus (CMV) infection and reactivation of latent infection (Lutz et al., 1994), and recent and past parvovirus B19 infection (Gray et al., 1993). Gray and Wreghitt (1989) and de Ory et al. (1993) described modifications to the EBV VCA IFA assay which enabled the determination of the avidity of anti-VCA IgG antibodies. These modified assays were able to differentiate between primary EBV infection and a reactivation of latent infection in organ transplant recipients and between recent and past infection in immunocompetent individuals. In this study, a commercial EBV VCA IgG ELISA (Sigma Chemical Company Ltd., Dorset, UK) with purified EBV-infected cell lysate supplemented with purified EBV gp125 as antigens attached to the solid phase, was used to determine the presence of EBV VCA IgG antibodies and their avidity in samples collected from immunosuppressed organ transplant recipients and immunocompetent individuals. The value of this data as a ‘serological marker’ in
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97
the diagnosis of a recent EBV infection, past EBV infection and reactivation of past infection was assessed and compared with the previous data (Gray and Wreghitt, 1989; de Ory et al., 1993).
2. Materials and methods 2.1. Serum samples One hundred samples of serum were investigated: 54 sera were collected from 13 organ transplant recipients with evidence of primary EBV infection or reactivation of past infection, and 46 from immunocompetent individuals with symptoms suggesting EBV infection. Twenty eight had evidence of recent EBV infection and 18 of past infection. The samples had previously been tested for EBV VCA IgG and IgM in IFAs, for EBV VCA IgM in an ELISA (Sigma Chemical Co. Ltd., Dorset, UK) and for heterophile antibodies in the Paul-Bunnell test. After transplantation, the most recent samples collected from the organ transplant recipients were tested for the presence of EBNA IgG. The avidity of EBV VCA IgG antibodies was determined in 36 of the samples collected from the transplant recipients (i.e. those samples in which EBV VCA IgG was detected using the ELISA) and in the 46 samples from immunocompetent individuals. 2.2. EBV VCA antibody ELZSAs The EBV VCA IgG ELISA (Sigma Chemical Co. Ltd., Dorset, UK) and the EBNA IgG ELISA (Biotest Diagnostics, Dreieich, Germany) were performed according to the manufacturers’ instructions. EBV VCA IgG antibody avidity was determined in the EBV VCA IgG ELISA (Sigma) with the first wash step modified to include two 5-min washes with 8 M urea incorporated in the wash fluid. A sample was considered as containing a significant proportion of low avidity antibody if there was a 2 50% reduction in optical density (OD) in the presence of 8 M urea. 2.3. Paul-Bunnell
test
The Paul-Bunnell test was performed as described by Gray et al. (1992) and the antibody titre was expressed as the reciprocal of the highest serum dilution to show macroscopic agglutination. 2.4. Indirect immunojluorescence
The EBV incorporating (1989). The EBV incorporating
antibody assay
VCA IgG indirect IFA and the IgG antibody avidity determination, 8 M urea in the IFA, were performed as described by Gray and Wreghitt VCA antibody titres obtained with the conventional IFA and the IFA 8 M urea were compared and the sample was deemed to contain a
J.J. Gray /Journal
98 Table 1 EBV serology Patient
in heart and heart/lung
Days postetx
of Virological Methods 52 (1995) 95-104
transplant
recipients Other EBV serology
VCA IgG ELISA EBV VCA IgG IFA titre < 10
EBV VCA IgG ELISA (AU/ml)
Result d
% Reduction in OD with 8 M urea
+ + +
n.a. n.a. 55.6 68.5 76.6 35.4
_ _ _ _ +
n.a. n.a. n.a. n.a. 27.5
92 119 160 224 931
< 10 640 2560 2560 2560
10.0 18.1 100.7 357.8 599.1 1149.6
52 79 116 177 914
< 10 < 10 < 10 160 2560
0.0 0.0 35.1 64.9 1171.9
52 100 190 371 800
< 10 < 10 40 2560 10240
0.0 0.0 29.8 666.5 1149.6
-2 25 110 171 388
< 10 < 10 2560 2560 > 10240
0.0 X8.9 224.2 605.5 1235.2
-1 19 55 82 140 193 817
< 10 10 < 10 < 10 160 640 2560
0.0 83.0 0.0 35.6 148.1 206.7 1321.8
+ + +
“.a. n.a. n.a. n.a. 57.1 60.5 17.5
39 58 86 184 402
< 10 < 10 < 10 640 2560
0.0 0.0 129.9 312.7 540.5
+ + f
n.a. n.a. 54.7 64.1 61.3
0 28 84 176 736
< 10 < 10 > 10240 > 10240 > 10240
0.0 0.0 145.8 534.7 1245.1
+ + +
n.a. n.a. 62.7 68.0 19.3
77 169 364
640 2560 2560
848.6 826.9 1040.1
+ + +
60.2 55.6 40.8
57
_
_ _ + + _ + + c
n.a. n.a. n.a. 64.8 50.2 n.a. n.a. 56.7 68.5 21.6
Antibody avidity b
EBV VCA IgM ELISA Sigma
EBNA-IgG ELISA Biotest
High
_ -
n.t. n.t. nt. n.t. n.t.
High
_ -
n.t. n.t. n.t. n.t.
LOW
+
LOW LOW LOW
LOW
LOW LOW
High
-
LOW LOW
High
LOW LOW LOW
LOW LOW
High LOW LOW
High
-
-
n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. _ + _
-
n.t. n.t. n.t. n.t.
_ _
n.t. n.t. n.t. n.t.
_ _ _ -
n.t. n.t. n.t. n.t.
+ + =
+ n.t. n.t.
_
_
J.J. Gray /Journal
99
of Virological Methods 52 (I 995) 95-l 04
Table 1 (continued) Patient
Days postmtx
Other EBV serology
VCA IgG ELISA EBV VCA IgG IFA titre
EBV VCA IgG ELISA (AU/ml)
Result a
% Reduction in OD with 8 M urea
Antibody avidity b
84 365 137
640 640 10240
699.8 924.7 1191.2
+ + +
39.3 33.5 17.8
High High High
10
104 239 335
640 2560 2560
380.6 762.5 665.3
+ + +
38.3 33.7 40.0
11
105 193 414
640 2560 2560
593.8 195.3 1140.9
+ + +
12
180 389
160 2560
912.4 1246.3
285 364
2560 > 10240
775.4 892.5
9
13
EBV VCA IgM ELISA Sigma
-
EBNA-IgG ELISA Biotest
+
-
n.t. n.t.
High High High
-
+ n.t. n.t.
42.0 38.5 19.2
High High High
+ -
+ n.t. n.t.
+ +
25.9 17.2
High
+
+ n.t.
+ +
29.7 22.8
High High
-
n.t.
+
n.a., not applicable; nt., not tested a - , negative; + , positive; = , equivalent. b Avidity: low, > 50% reduction; high, < 50% reduction.
significant proportion of low avidity antibody if there was an 2 B-fold reduction in antibody titre in the presence of 8 M urea.
3. Results 3.1. EBV infection in heart and heart/lung
transplant recipients
Seroconversion (> 4-fold increase in titre) was detected in serial samples collected from patients l-7 (Table 1) in both the EBV VCA IgG IFA and the EBV VCA IgG ELISA. EBNA IgG antibody was not detected in samples collected from these transplant recipients before seroconversion of EBV VCA IgG indicating primary EBV infection. Patient 8 (Table 1) had detectable EBV VCA IgG and EBNA IgG antibodies in a sample collected 77 days post-transplant. On the basis of this result it was difficult to determine whether this patient had experienced a primary EBV infection in the preceding 77 days (see below) or was experiencing a reactivation of a past infection. In comparing the two test procedures, infection was detected earlier by the IFA in patients 2 and 3 and earlier by ELISA in patient 6 (Table 1). EBV VCA IgM was detected in only one out of the 8 transplant recipients experiencing primary EBV infection (Table 1). Low avidity IgG was detected in samples from patients 1, 3, 4, 5, 6, 7 and 8, but not in patient 2. The presence of low avidity antibody in samples collected from patient 8 suggested a primary EBV infection. Low avidity EBV VCA IgG antibodies were
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of Virological Methods 52 (1995) 95-l 04
detected for up to 6 months in recipients 4, 5, 7 and 8 and for longer in recipients 1, 3 and 6 (Table 1). Passive antibody was detected by EBV VCA IgG IFA and EBNA IgG ELISA in a sample collected from patient 5, 19 days post-transplant and although there was a concomitant increase in the concentration of antibody detected in the ELISA (0.0-83.0 AU/ml), this was below the positive range of the assay. A subsequent sample collected from this patient on day 55 was EBV VCA IgG negative and EBNA IgG negative and the concentration of antibody detected in the ELISA had returned to its previous level (Table 1). Increasing concentrations of EBV VCA IgG were detected, using the ELISA, in samples from 5 organ transplant recipients (9-13) with evidence of reactivation, and from recipients (3-6) during reactivation of infection following a primary infection after transplant. EBNA IgG antibodies were detected in samples collected from patients 9-13 before a significant rise in EBV VCA IgG was detected, confirming reactivation of latent EBV infections in these patients. The increases in antibody concentration ranged from 1.2 to 6.4 times the concentration found in the previous sample. Increasing EBV VCA IgG antibody concentrations (1.3 to 3.7 times) were also found in recipients whose samples were shown to have stable titres when tested in the IFA. Results obtained in the ELISA but not in the IFA indicated that recipients 1, 7 and 8, who had evidence of primary EBV infection, experienced a subsequent reactivation of infection and recipient 11 experienced a reactivation of EBV infection on two occasions. EBV VCA IgM was detected in only two of the 9 transplant recipients experiencing a reactivation of EBV infection. The avidity of EBV VCA IgG antibodies was high in all samples collected from these recipients at the time of EBV reactivation (Table 1). 3.2. Immunocompetent
individuals
with evidence
of recent
or past infection
All 48 samples collected from immunocompetent individuals had detectable VCA IgG antibodies when tested by the ELISA. The 28 samples collected from patients with recent EBV infection contained significant concentrations of low avidity antibodies when tested by the modified ELISA. The mean percentage reduction in OD in the presence of 8 M urea was 70.9% (range 51.6-85.2%) compared with a mean percentage reduction of 30.8% (range 7.2-42.1%) obtained with the 18 samples collected from patients with evidence of past EBV infection (Table 2). Fig. 1 shows the difference in the percentage reduction in OD in the presence of 8 M urea in samples collected from patients (immunocompromised and immunocompetent)
Table 2 EBV VCA IgG antibody avidity in samples collected recent mimarv or east EBV infection
from immunocompetent
individuals
with evidence
Patients
Number of samples
Mean % reduction in OD in the presence of 8 M urea (range)
Recent primary infection Past infection
28 18
70.9 (51.6-85.2) 30.8 (7.2-42.1)
of
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of Virological Methods 52 (1995) 95-104
101
EBV VCA IgG avidtty
1-9
lo-19
20-29
30-39
40-49
% reduction m
Fig. 1. Percentage
reduction
past
infection
50-59
60-69
70-79
60-69
in OD 11101 recent
infection
in OD values obtained in the EBV VCA IgG ELISA in the presence of 8 M urea.
with a history of past infection compared to those with evidence of a recent infection. The mean reduction of OD in samples collected from all patients with a recent EBV infection was 67.6% (range 50.2-85.2%) and in patients with evidence of a past infection was 30.1% (range 7.2-42.1%). There was a statistically significant difference (P < 0.001 (Student’s t-test for differences of means)) between the mean percentage reduction in OD obtained with samples collected from patients with recent infection compared to samples collected from patients with past infection. 4. Discussion For the serological diagnosis of EBV infection it is necessary to test for specific antibodies against a range of antigens as, on many occasions, no single test can be relied upon to provide sufficient information for a definitive diagnosis. Anti-EA-D antibodies are transient and only detected in 80% of infected individuals and anti-EA-R antibodies are rarely present in the acute phase of infectious mononucleosis. Although EBNA IgM antibodies can indicate a recent infectidil, non-specific reactivity leading to false-positive results have been reported in assays for detecting EBNA IgM antibodies (Matheson et al., 1990; Gray et al., 1992). EBNA IgG antibodies may not be detected for several weeks after the onset of symptoms and reactivation of a latent EBV infection in the immunocompromised host may not elicit a rise in anti-EBNA antibodies. A negative result by the Paul-Bunnell test does not exclude a recent EBV infection as heterophile antibodies may not be present or detectable in the majority of children and some adults. The presence of EBV VCA IgM and anti&&D antibodies in the absence of antibodies to EBNA is often used as the criterion for the laboratory diagnosis of primary EBV infection. In this study, only one out of 8 transplant recipients who experienced a primary EBV infection had detectable EBV VCA IgM antibodies by the ELISA. This
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of Virological Methods 52 (1995) 95-104
may be due to the presence of EBV VCA IgG in the samples, even after adsorption with anti-IgG, competing with the virus-specific IgM for the available binding sites in the EBV VCA IgM indirect ELISA (Henle et al., 1974a,b). Wiedbrauk and Bassin (1993) reported, in an evaluation of 5 commercial ELISAs for detecting EBV VCA IgM, that the Sigma VCA IgM assay had a sensitivity of 78% and a specificity of 86% when compared with the IFA. Although the inability to detect EBV VCA IgM antibodies in the ELISA may reflect atypical immune responses to EBV, a well recognized phenomenon, associated with infection in the immunocompromised host (Okano et al., 1988>, EBV VCA IgM antibodies were detected using an EBV VCA IgM IFA in all 8 transplant recipients with primary EBV infection (data not shown). The usefulness of determining the avidity of EBV VCA IgG antibodies in immunocompromised and immunocompetent individuals using IFAs has been demonstrated (Gray and Wreghitt, 1989; G ray et al., 1992; de Ory et al., 1993). Recent infections were characterized by the presence of low avidity EBV VCA IgG antibodies and past infections by high avidity antibodies. Although these assays were satisfactory they were prone to the problems inherent in all IFAs. They were time consuming, labour intensive, prone to batch-to-batch variation, and their subjective nature made end-point determination difficult. In this study, the suitability of a semiquantitative EBV VCA IgG ELISA (Sigma) as an alternative assay to immunofluorescence and its use in determining IgG antibody avidity was evaluated. There was a very good correlation (P < 0.001) between the log IFA titres and log ELISA antibody concentrations (AU/ ml), and the ELISA was able to detect all the seroconversions in the transplant recipients previously detected in the IFA. The absence of guidelines in the ELISA protocol, indicating what constituted a significant rise in antibody concentration in a patient with pre-existing EBV VCA IgG antibodies, casts doubt on the suitability of the ELISA to identify a reactivation of past infection. Increases in antibody concentration were detected both in organ transplant recipients with significant rises in antibody titre in the IFA and in recipients with stable titres in the IFA. This may indicate a lack of reproducibility in this assay which had previously been shown to be the least variable of the commercial assays in day-to-day testing (Wiedbrauk and Bassin, 1993). It is possible that the ELISA could detect reactivations of past infection better, and these results may confirm the increased sensitivity of EBV-specific antibody ELISAs, when compared with the IFAs, as reported by Luka et al. (1984) who used purified EBV antigen in an ELISA and DSlken et al. (1984) who utilized EBV-infected cell lysate antigen. In this study the microtitre plate wells were coated with purified EBV-infected cell lysate antigen supplemented with purified EBV gp125, a major immunoreactive protein of EBV (Luka et al., 1984). The ELISA results are consistent with the atypical EBV serological profiles seen in many immunocompromised individuals, whether genetic or iatrogenic. (Henle and Henle, 1981; Henle and Henle, 1982; Purtilo et al., 1985; Purtilo, 1987). The antibody avidity of EBV VCA IgG antibodies detected by ELISA was low (characterized by a > 50% reduction of OD in the presence of 8 M urea) in samples collected from patients with recent EBV infection and high ( < 50% reduction in OD) in those collected from patients with a past infection or reactivation of a past infection. This was true for both the immunocompromised and immunocompetent patients. There
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of Virological Methods 52 (1995) 95-l 04
103
a statistically significant (P < 0.001) difference between the means of the percentage reduction in OD values in the two groups represented by patients with recent infection compared with patients with past infection or a reactivation of a latent infection. Plotting the frequency of the percentage reduction of OD values in samples collected from patients with recent and past infection suggests the possibility of an overlap between the two groups (Fig. 1). This indicates the need for an equivocal zone to take account of similar percentage reductions in OD values that may be detected in samples from either recent infections or past infections. An equivocal zone can be created by determining the standard deviation (S.D.) of the means of the percentage reductions in OD values in samples from patients with recent infection and patients with past infection. The equivocal zone would be the values between the mean percentage reduction value (OD f 3 S.D.) in past infection and in recent infection. In this study, the equivocal zone would include all percentage reductions in OD values obtained in the presence of 8 M urea between 41.47 and 57.4%. None of results obtained with the samples from patients with a history of past infection were in the equivocal zone, but 8 samples from patients with recent infection were. All 8 had > 50% reduction of OD values in the presence of 8 M urea. The time taken for the antibody response to mature from low to high avidity in patients experiencing a primary EBV infection was variable in the immunocompromised organ transplant recipients. This confirms previous reports that immunosuppression may delay the maturation of the humoral immune response to infection (Gray and Wreghitt, 1989; Lutz et al., 1994; Ward et al., 1994). The EBV VCA IgG antibody avidity assay by ELISA can be a useful diagnostic tool for the serological diagnosis of EBV infection. It is a suitable alternative to the more labour intensive and subjective EBV VCA IFAs and is able to differentiate primary from past infection using only one EBV serological marker (EBV VCA IgG). The correlation between recent primary EBV infection and the presence of significant concentrations of low avidity antibodies will allow the diagnosis of recent primary EBV infection using a single serum sample collected during the acute phase of the illness. was
References Crawford, D.H. and Edwards, J.M.B. (1987) Epstein-Barr virus. In: A.J. Zuckerman, J.E. Banatvala and J.R. Pattison (Eds.), Principles and Practice of Clinical Virology. John Wiley and Sons, London, pp. 111-133. Davidsohn, I. and Lee, C.L. (1969) The clinical serology of infectious mononucleosis. In: R.L. Carter and H.G. Penman (Eds.), Infectious Mononucleosis. Blackwell Scientific Publications, Ltd., Oxford, pp. 177-200. de Ory, F., Antonaya, J., Fernandez, M.V. and Echevarria, J.M. (1993) Application of low-avidity immunoglobulin G studies to diagnosis of Epstein-Barr virus infectious mononucleosis. J. Clin. Microbial. 31, 1669-1671. De-The, G. (1982) Epidemiology of Epstein-Barr virus and associated diseases in man. In: B. Roizman (Ed.), The Herpesviruses, Vol. 1. Plenum Publishing Corp., New York, pp. 25-103. DBlken, G., Weitzmann, U., Boldt, C., Bitzer, M., Brugger, W. and Lohr, G. (1984) Enzyme-linked immunosorbent assay for IgG antibodies to Epstein-Barr virus-associated early antigens and viral capsid antigen. J. Immunol. Methods 67, 225-233.
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Epstein, M.A., Achong, B.G. and Barr, Y.M. (1964) Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet i, 702-703. Gray, J.J. and Wreghitt, T.G. (1989) Immunoglobulin G avidity in Epstein-Barr virus infections in organ transplant recipients. Serodiagn. Immunother. Infect. Dis. 3, 389-393. Gray, J.J., Caldwell, J. and Sillis, M. (1992) The rapid serological diagnosis of infectious mononucleosis. J. Infect. 25, 39-46. Gray, J.J., Cohen, B.J. and Desselberger, U. (1993) Detection of human parvovirus B19-specific IgM and IgG antibodies using a recombinant viral VP1 antigen expressed in insect cells and estimation of time of infection by testing for antibody avidity. J. Virol. Methods 44, 11-24. Hanto, D.W., Frizzera, G., Gajl-Peczalska and Simmons, R.L. (1985) Epstein-Barr virus, immunodeficiency, and B cell lymphoproliferation. Transplantation 39, 461-472. HenIe, W. and HenIe, G. (1981) Epstein-Barr virus specific serology in immunologically compromised individuals. Cancer Res. 41, 4222-4225. HenIe, W. and Henle, G. (1982) Immunology of Epstein-Barr virus, In: B. Roizman (Ed.), The Herpesviruses, Vol. 1. Plenum Publishing Corp., New York, pp. 209-252. Henle, G., Henle, W. and Horwitz, C.A. (1974a) Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J. Infect. Dis. 130, 231-239. Henle, W., Henle, G. and Horowitz, CA. (1974b) Epstein-Barr virus specific diagnostic tests in infectious mononucleosis. Hum. Pathol. 5, 551-565. Inouye, S., Hasegawa, A., Matsuno, S. and Katwo, S. (1984) Changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed. J. Clin. Microbial. 20, 525-529. Kocks, C. and Rajewsky, K. (1988) Stepwise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl. Acad. Sci. U.S.A. 85, 8206-8210. Luka, J., Chase, R.C. and Pearson, G.R. (1984) A sensitive enzyme-linked immunosorbent assay (ELISAl against the major EBV-associated antigens. I. Correlation between ELISA and immunofluorescence titers using purified antigens. J. Immunol. Methods 67, 145-156. Lutz, E., Ward, K.N. and Gray, J.J. (1994) Maturation of antibody avidity after primary human cytomegalovirus infection is delayed in immunosuppressed solid organ transplant patients. J. Med. Virol., accepted. Matheson, B.A., Chisholm, S.M. and Ho-Yen, D.O. (1990) Assessment of rapid ELISA test for detection of Epstein-Barr virus infection. J. Clin. Pathol. 43, 691-693. Okano, M., Thiele, G.M., Davis, J.R., Grierson, H.L. and Purtilo, D.T. (1988) Epstein-Barr virus and human diseases: recent advances in diagnosis. Clin. Microbial. Rev. 1, 300-312. Paul, J.R. and Bunnell, W.W. (1932) The presence of heterophile antibodies in infectious mononucleosis. Am. J. Med. Sci. 183, 90-104. Purtilo, D.T. (1987) Epstein-Barr virus: the spectrum of its manifestations in humans. South Med. J. 80, 943-947. Purtilo, D.T., Tatsumi, E., Manolov, G., Manolova, Y., Harada, S., Lipscomb, H. and Krueger, G. (1985) Epstein-Barr virus as an etiological agent in the pathogenisis of lymphoproliferative and aproliferative disease in immune deficient patients, Int. Rev. Exp. Pathol. 27, 113-183. Sumaya, C.V. (1986) Infectious mononucleosis and other EBV infections: diagnostic factors. Lab. Manag. 24, 37-45. Thomas, H.I.J. and Morgan-Capner, P. (1988) Rubella-specific IgG subclass avidity and its role in differentiation between primary rubella and rubella reinfection. Epidemiol. Infect. 101, 591-598. Ward, K.N., Dhaliwal, W., Ashworth, K.L., Clutterbuck, E.J. and Teo, C.G. (1994) Measurement of antibody avidity for hepatitis C virus distinguishes primary antibody responses from passively acquired antibody. J. Med. Virol., in press. Wiedbrauk, D.L. and Bassin, S. (1993) Evaluation of five enzyme immunoassays for detection of immunoglobulin M antibodies to Epstein-Barr virus virus capsid antigens. J. Clin. Microbial. 31, 1339-1341. Zur Hausen, H., Schulte-Holthausen, H., Klein, G., Henle, W., Henle, G., Clifford, P. and Santesson, L. (1970) EBV DNA in biopsies of Burkitt’s tumors and anaplastic carcinoma of the nasopharynx. Nature 228, 1056-1058.