remote evidence of EBV infection are positive for these

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1 JOURNAL OF CLINICAL MICROBIOLOGY, May 1989, p /89/ $02.00/0 Copyright 1989, American Society for Microbiology Vol. 27, No. 5 Rapid Diagnosis of Acute Epstein-Barr Virus Infection by an Indirect Enzyme-Linked Immunosorbent Assay for Specific Immunoglobulin M (IgM) Antibody without Rheumatoid Factor and Specific IgG Interference DAVID W. T. HO, PETER R. FIELD,* AND ANTHONY L. CUNNINGHAM Virology Department, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales 2145, Australia Received 3 October 1988/Accepted 2 February 1989 An indirect enzyme-linked immunosorbent assay (ELISA) for detection of Epstein-Barr virus-specific immunoglobulin M (IgM) antibody was developed with commercial reagents. Sera containing rheumatoid factor (RF) (as little as 0.5 IU/ml) coupled with specific IgG resulted in false-positives in the ELISA. This interference was eliminated by the use of anti-human IgG antibodies to remove RF and IgG. Thus, pathogen-specific IgG complexes to which IgM-RF could be bound during the subsequent test were inhibited, and competition between specific IgG and IgM was also prevented. Of the 1,672 serum specimens tested, 353 were found to be Epstein-Barr virus IgM antibody positive by indirect immunofluorescence (IF). Compared with the IF test, the ELISA showed 96.6% sensitivity, 99.7% specificity, and 99% accuracy. Further evidence indicated that most of the 12 ELISA false-negatives were IF false-positives. There was a linear correlation between mean ELISA values and increasing IF titers (r = 0.96). However, the IF test has the disadvantages that it lacks automated reading and requires considerable technical expertise, both of which restrict the range of laboratories performing the test. The indirect ELISA has the advantages that it is simple and rapid and can be automated. Ali the reagents used in this assay are commercially available, have been prestandardized, and are stable. Epstein-Barr virus (EBV) eventually infects most humans, causing infections ranging from primary asymptomatic infection of infants to infectious mononucleosis (IM) (7). Furthermore, EBV has been strongly implicated as the cause of endemic Burkitt's lymphoma (1) and nasopharyngeal carcinoma (14). After primary infection the virus usually persists for life in the body as a latent infection of B lymphocytes and epithelial cells possessing the C3d receptor CR2 (4). Latent EBV in carriers periodically reactivates, as indicated by virus excretion and specific antibody response (9). Therefore, detection of virus in the oropharynx is of little use for diagnosing primary infection, and serological tests are the most useful tests for routine laboratory diagnosis of symptomatic primary EBV infection (10). EBV-specific serodiagnostic tests for a current primary infection are usually based upon the presence of immunoglobulin M (IgM) antibodies to viral capsid antigen (VCA); however, high titers of VCA-specific IgG antibodies, detection of antibodies to early antigen (EA), and no titer or a low titer (<10) of antibodies to EBV nuclear antigen (EBNA) may also be indicative of an acute infection (10). EBV VCA (EB-VCA) IgG antibodies are usually of little help in establishing a diagnosis of IM, because the demonstration of a significant rise in antibodies is rarely possible by the time the patient presents and because high titers can be found in patients with long-past EBV infection or immunodepressed patients, pregnant women, and persons of advanced age (18). Anti-EA antibodies like anti-vca IgM are acute transient serological markers but only occur in about two-thirds of patients and may not be present at the onset of symptoms (6, 28). In addition, up to 20% of patients with * Corresponding author. remote evidence of EBV infection are positive for these antibodies (12, 18). Antibodies to EBNA are the last EBV antibodies to develop in patients with IM and are of little use in the diagnosis of acute EBV infection. In contrast to anti-vca and anti-ea IgG antibodies, anti-vca IgM antibodies are relatively transient. They appear early in the acute phase of the disease, being detectable in 90 to 94% of patients at presentation (6, 15), develop in all cases of EBV infection, and decline rapidly to undetectable levels in 8 to 12 weeks (19). The presence of anti-vca IgM is diagnostic of acute or recent infection with EBV (20, 27). In addition, approximately 90 to 96% of adult patients show heterophile antibodies of the IgM class which reach peak titers usually in week 2 or 3 of the illness. However, the test for Paul-Bunnell-type heterophile antibodies is not EBV specific (18, 21), and about 50% or more of young children, usually under 4 or 5 years old, do not develop detectable heterophile antibodies (5, 25, 29). The detection of IgM antibody to EBV by indirect immunofluorescence (IF) is currently the standard method for the diagnosis of acute EBV infections (10). However, the IF test is time-consuming and labor-intensive and requires considerable technical expertise, all of which limit its use in certain laboratories. This report describes the development of an enzymelinked immunosorbent assay (ELISA) for the detection of specific IgM antibody to EB-VCA in human serum and a comparison of this ELISA with the standard IF test. MATERIALS AND METHODS Clinical specimens. We studied 716 serum specimens retrospectively and 956 serum specimens prospectively from 952

2 VOL. 27, 1989 INDIRECT ELISA FOR DIAGNOSIS OF ACUTE EBV INFECTION 953 patients with clinical histories suggestive of a recent EBV infection. The sera tested were submitted mainly by hospitals, pathologists, and medical practitioners in New South Wales for the detection of EBV IgM antibody. The ages of patients ranged from 3 to 63 years. Retrospective study. The sera were initially tested by IF and then stored at -20 C for no longer than 12 months until subsequent testing with the ELISA developed in our laboratory. The retrospective study comprised the following groups: (A) 424 serum samples from patients with no detectable specific IgM to EB-VCA in the IF test; (B) 292 serum samples from patients with detectable specific IgM to EB- VCA in the IF test (IgM titers ranged from 8 to 128); (C) 10 serum samples from patients with specific IgM to varicellazoster virus confirmed by the IF test; (D) 10 serum samples from patients with specific IgM to herpes simplex virus confirmed by the IF test; (E) 31 serum samples from patients with specific IgM to cytomegalovirus (CMV) confirmed by the IF test; and (F) 55 serum specimens from patients with no detectable specific IgM to EB-VCA but with IgG to EB-VCA and rheumatoid factor (RF). This group (F) was divided into two subgroups: (i) whole serum and (ii) serum depleted of IgG. Both subgroups were tested for IgM to EB-VCA by the ELISA. Prospective study. The 956 fresh serum specimens received in the laboratory were stored at 4 C and tested within 2 days of receipt for IgM to EB-VCA by the IF test and the developed ELISA. Of the 956 serum specimens tested, 61 were found to contain specific IgM to EB-VCA by the IF test and 895 had no detectable specific IgM to EB-VCA in the IF test. IF test. This procedure used the Burkitt's lymphomaderived cell line, Gorotala, for EB-VCA and the Raji cell line for the control antigen (Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia). At least four dilutions of untreated patient sera were reacted with antigen for 3.5 h at 37 C in a humidified chamber. After washing, fluorescein-labeled goat anti-human IgM-F(ab')2 fragment conjugate (Kallestad Laboratories, Inc.) was reacted with the serum-antigen complex for 0.5 h at 37 C. The slides were mounted in buffered glycerol (ph 8.5) and examined in a Zeiss standard microscope with an IVFL incident-light fluorescence condenser (Carl Zeiss, Sydney, New South Wales, Australia); an incident-light source (HBO 50W) with a broad-band interference exciter filter (BP ) and a chromatic beam splitter (FT 510) were used. Sera showing RF activity by reaction with IgG-coated latex particles (Rheuma-Wellcotest; Wellcome Diagnostics, Rosebery, New South Wales, Australia) or nonspecific, atypical, or weak reactions were retested for EBV IgM after removal of IgG by quaternary aminoethyl-sephadex A-50 ion-exchange column chromatography (Quik-Sep System I IgM Isolation System; Isolab Inc.) (13). The limit of detection of the latex agglutination slide test for RF is approximately 0.75 IU/ml. Antigen. EB-VCA-coated 96-well microdilution plates were obtained from Biotech Research Laboratories (the antigen was purified with monoclonal antibodies). ELISA. The indirect ELISA incorporated pretreatment of all sera tested with RF Absorbent (anti-human IgG; Behringwerke AG, Marburg, Federal Republic ofgermany). Briefly, each well of an EB-VCA-coated plate was filled with 250,ul of phosphate-buffered saline (ph 7.2) containing 2% (wt/vol) casein (Sigma Chemical Co., St. Louis, Mo.) and incubated at 37 C for 30 min in a moist chamber to block nonspecific protein-binding sites. The plates were washed five times with phosphate-buffered saline-1% (vol/vol) Tween 20 washing solution (Behringwerke). A Titertek Microplate washer (model 120; Flow Laboratories, North Ryde, New South Wales, Australia) was used for all washing. RF Absorbenttreated serum (100,ul) was added to appropriate wells of the plates (treatment of serum with RF Absorbent was done in accordance with manufacturer instructions). After incubation for 1 h at 37 C in a moist chamber, the plates were washed five times with washing solution, 50 pil of rabbit anti-human IgM(,u)-alkaline phosphatase conjugate (Behringwerke) was added to each well, and the plates were incubated at 37 C for 1 h. The plates were washed five times with washing solution, and 100,ul of p-nitrophenol phosphate substrate (Behringwerke) was added to each well. After 30 min of incubation at room temperature, the reaction was stopped by the addition of 50 pil of 2 N NaOH. The A405 was determined with a reference wavelength of 492 nm against a substrate blank within 30 min by a Titertek Multiskan MCC plate reader (Flow Laboratories). Three pools of sera with high, medium, and low EBV IgM titers in the IF test and one pool of sera with no detectable EBV IgM in the IF test were used as controls in each run of tests. To test for the removal of IgG and RF, we included a serum pool containing RF and a high level of IgG but no detectable EBV IgM in each run. RF absorption with latex-igg. The nine discrepant sera containing RF in the retrospective group (see Table 1) were absorbed with polystyrene latex particles coated with human IgG in an independent reference laboratory by a method similar to that described by Henle et al. (8). IgG-coated latex particles were suspended in 1 ml of a 1:10 dilution of test serum. After absorption at room temperature for 1 h, the latex particles were deposited by centrifugation and the supernatant was retested for RF activity and EBV lgm antibody. The absorption procedure was repeated if RF was not removed. Discrepant and equivocal results. All patient serum specimens showing discrepant results were tested at least twice until consistent results were obtained. Serum specimens giving values ± 0.05 absorbance unit of the cutoff point were retested in duplicate. RESULTS Determination of the optimal test serum dilution for the EBV IgM ELISA. Serial dilutions of five serum pools with positive IF EBV IgM titers ranging from 8 to 128 as well as a negative serum pool (IF EBV IgM titer <8) were tested by the ELISA to determine the absorbance response with dilution. The serum dilution showing the clearest distinction between positive and negative samples was adopted as the optimal dilution for the ELISA procedure. The dilution chosen for this purpose was 1:40 (Fig. 1). Titration of serum samples plotted against absorbance values for each serum dilution gave sigmoid-shaped curves. The curves were linear and roughly parallel over a broad range of serum dilutions, suggesting that the ELISA antibody titer of each serum sample could be predicted from the absorbance of a single dilution of that serum sample over the linear part of the curve. Determination of the cutoff absorbance for distinguishing positive and negative samples. To determine a cutoff absorbance value for distinguishing negative and positive samples, we tested a total of 424 serum samples with no detectable IF EBV IgM antibody (IF titer, <8) by the ELISA. The distribution of negative sera was skewed to the right and

3 954 HO ET AL. J. CLIN. MICROBIOL Cut-off_\ F reciprocal serum dilution FIG. 1. Relationship between antibody concentration and absorbance values for five serum pools known to contain EBV IgM antibody (IF titers: A, 128; B, 64; C, 32; D, 16; E, 8) and one serum pool with no detectable EBV IgM antibody (IF titer: F, <8). appeared log normal (Fig. 2). In this study, a 99.9% confidence limit was used for the determination of the cutoff value. Statistical analysis showed that the mean absorbance of the negative sera was 0.057, with a standard deviation of A cutoff absorbance of 0.17 was established by determining the mean value plus 3 standard deviations. The distribution curve of the 424 EBV IgM-negative and 292 EBV IgM-positive (IF titer, 8 to 128) sera showed a clear distinction between negative and positive populations totalling 716 specimens (Fig. 3). Therefore, the probability of erroneously classifying a negative serum sample as positive or vice versa was less than Comparison of endpoint titers determined for EBV IgM by the ELISA and the IF test. To compare the endpoint titers determined for EBV IgM by the ELISA and the IF test, we e4- le d z H F0F1[1n n n Absorbance FIG. 2. Distribution of absorbance values of sera with no detectable EBV IgM antibody (IF titer: <8). tested serial dilutions of the five serum pools with IF EBV IgM titers ranging from 8 to 128 and the negative serum pool by the ELISA. The titers determined by the ELISA were, on the average, at least 10- to 20-fold higher than those determined by the IF test, demonstrating the markedly increased sensitivity of the ELISA procedure (Fig. 1). Specificity of the EBV IgM ELISA. With the increase in sensitivity demonstrated by the ELISA over the IF test, it was obviously necessary to assess the specificity of the procedure. To assess the specificity, we treated 16 serum e o z cut-of f level 424 negative seraa 292 positive sera /,d f\ / \, 1og10 Absorbance FIG. 3. Distribution of absorbance values obtained by testing 424 serum samples without detectable IF EBV IgM antibody (IF titer: <8) and 292 serum samples with IF EBV IgM antibody (IF titers: 8 to 128) in the indirect ELISA.

4 VOL. 27, 1989 INDIRECT ELISA FOR DIAGNOSIS OF ACUTE EBV INFECTION 955 I s I 1. t1.8 m 8. as %à *te.. m je a t i t t M 8* a. 0.1 T ~1 of-o* T (a 424) specimens with IF EBV IgM titers ranging from 10 to >64 and with positive ELISA values with an equal volume of 0.5 M 2-mercaptoethanol in phosphate-buffered saline at 37 C for 1 h and then retested them in parallel with untreated serum specimens in the ELISA. There was a highly significant reduction in the ELISA values ranging from 0.22 to 1.80 before treatment to less than 0.10 in all cases after treatment, as determined by the Wilcoxon rank sum test (P < 0.001). There was no significant reduction in absorbance following 2-mercaptoethanol treatment in 16 serum specimens without detectable EBV IgM (IF titers, <8) (data not shown). Interference of RF and specific IgG in the EBV IgM ELISA. A total of 55 serum samples positive for RF and EBV IgG and negative for EBV IgM were tested by the ELISA for EBV-specific IgM antibody (Fig. 4). In group Fi, which represented whole serum samples tested by the ELISA for EBV IgM, 36 of 55 serum samples (56.45%) showed falsepositive results, whereas all group F serum samples which had been pretreated with RF Absorbent showed true-negative results (group Fii; Fig. 4). Concentrations of RF and specific IgG interfering in the EBV IgM ELISA. To determine the effect of concentrations of RF coupled with specific IgG interfering with the detection of EBV IgM antibody, we tested serial dilutions of a c D E (A-292) (ou10) (mal) FIG. 4. EBV IgM ELISA reactivity for groups A to F. (nm3 1) F (1) <s-gb) F (I) (<05S) serum sample which was positive for EBV IgG (320) and negative for EBV IgM (<8) in the IF test and positive for RF (40 IU/ml) in the ELISA. An RF concentration as low as 0.5 IU/ml gave a false-positive EBV IgM result. No false cut-of gg depleted 0.0 fzà-im- srum RF (IWUmL) FIG. 5. Interference of RF in the indirect ELISA for EBV IgM. The serum sample tested was positive for EBV IgG (320) and RF (40 lu/ml) and negative for EBV IgM (<8).

5 956 HO ET AL. J. CLIN. MICROBIOL * untreated * treated Ar- t untreated 1 EBV-IgG EBV-IgM c a.ô 0.40 a serum dilution FIG. 6. Effect of RF Absorbent on the removal of EBV IgM and EBV IgG, as determined by the indirect ELISA. positive results were observed in corresponding serum dilutions pretreated with RF Absorbent (Fig. 5). Specificity of RF Absorbent for the removal of IgG. To exclude the possibility that pretreatment of the serum with RF Absorbent had a negative influence on the EBV IgM test, we tested serial dilutions of serum with an IF EBV IgM titer of 8, an IF EBV IgG titer of 320, and an RF level of 40 IU/ml. The RF Absorbent-treated serum showed only 1 log2 decline in the ELISA IgM antibody titer from 640 to 320. Furthermore, at the optimal dilution chosen for testing (1:40) and at each subsequent dilution up to a titer of 320, only a marginal decline in IgM absorbance was observed (Fig. 6), indicating that RF Absorbent, although removing specific IgG and RF, does not significantly reduce the amount of specific IgM. The specificity of RF Absorbent for the removal of IgG was shown by the significantly reduced EBV IgG absorbance levels after treatment with RF Absorbent. Effect of a blocking agent (2% casein) on the EBV IgM ELISA. High background or false-positive results may be due to IgM attaching nonspecifically to the solid phase. To reduce this effect, we used 2% casein-phosphate-buffered saline to block the nonspecific protein-binding sites on the solid phase before testing serum samples. When a total of seven serum samples containing EBV IgG and lacking RF and EBV IgM were tested in the EBV IgM ELISA, the absorbance values ranging from 0.16 to 0.24 were decreased significantly with a casein blocking step by 0.05 to 0.15 absorbance unit to below the cutoff point (P < 0.001; Wilcoxon rank sum test) (data not shown). Determination of EBV IgM antibody in patients with IgM to other herpes viruses. The differences in EBV IgM ELISA values obtained in the control groups and a proven-infection group are shown in Fig. 4. Of 31 CMV IgM-positive serum samples, 2 (6.45%) reacted in the EBV IgM ELISA. These two serum samples were obtained from kidney transplantation patients. None of the varicella-zoster virus IgM-positive sera or herpes simplex virus IgM-positive sera cross-reacted in the EBV IgM ELISA. Precision of the EBV IgM ELISA. Serum pools with high, medium, and low IF EBV IgM antibody titers (128, 32, and 8, respectively) were tested by the ELISA to determine the mean coefficients of variation for interassay and intraassay precision. The interassay precision was determined by testing 2 duplicates each of three controls on each of eight runs; the intraassay precision was determined by testing 16 duplicates each of three controls on each of three consecutive days. The mean interassay coefficients of variation were 10.3% for high, 8.1% for medium, and 9.8% for low positive controls. The mean intraassay coefficients of variation were 5.6% for high, 6.2% for medium, and 5.0% for low positive controls. The results showed a high degree of reproducibility Ċomparison of the ELISA and the IF test for EBV IgM. A total of 1,672 serum specimens were tested by both the ELISA and the IF test. Of these, 716 serum specimens were studied retrospectively and 956 serum specimens were studied prospectively. The performance characteristics for both the retrospective and prospective groups and for the com- TABLE 1. Comparison of the ELISA with the standard IF test for EBV IgM in 716 serum specimens studied retrospectively, 956 serum specimens studied prospectively, and the combined retrospective and prospective groups (1,672 serum specimens) No. of specimens % Agree- Group and with indicated % Sensi- % Speci- ment IF result ELISA result tivity of ficity of between IFresult ELISA" ELISA' ELISA and Positive Negative IF test Retrospective Positive Negative Prospective Positive 58 3 Negative Both Positive Negative 4 1,315 ` Calculated as 100 x [(total number of specimens positive in the IF test - number of specimens negative in the ELISA but positive in the IF test)/total number of specimens positive in the IF test]. ' Calculated as 100 x [(total number of specimens negative in the IF test - number of specimens positive in the ELISA but negative in the IF test)/total number of specimens negative in the IF test].

6 VOL. 27, 1989 bined retrospective and prospective groups are shown in Table 1. As compared with the IF test, the ELISA for each group showed sensitivities of 97, 95.08, and 96.6%, respectively, while the respective specificities were 100, 99.55, and 99.7%. The ELISA showed excellent overall agreement with the IF test for each group (98.7, 99.27, and 99%, respectively). Discrepant results. Twelve discrepant results were obtained on initial testing (<2% of specimens) in the retrospective group. However, on repeated retesting the ELISA results remained unchanged and 3 of the 12 serum specimens became negative in the IF test. These results strongly suggest that the original low positive IF results were incorrect, most likely because of a technical error (Table 1). The results for the seven discrepant serum specimens in the prospective group remained unchanged on retesting. DISCUSSION In this report an ELISA for the detection of specific IgM antibody to EB-VCA was developed and compared with the standard IF test. As compared with the IF test, the ELISA showed, overall, 96.6% specificity, 99.7% sensitivity, and 99% agreement. The performance characteristics for both the retrospective and prospective comparative studies were similar, thus demonstrating the validity of the results obtained by the ELISA in relation to the IF test in an everyday working situation in a busy laboratory. Indeed, the agreement obtained in the prospective study was better than expected, especially since the prospective testing was divided between three technologists. The EBV IgM titers determined in the ELISA were, on the average, at least 10- to 20-fold higher than those determined in the IF test, showing the markedly increased sensitivity of the procedure. The specificity for EBV IgM was shown by the uniform highly significant reduction of absorbance with 2-mercaptoethanol. The overall sensitivity of the ELISA (96.6%) relative to the IF test was good, and there are several explanations for the 12 false-negative serum samples. These serum samples contained EBV IgG and RF and were repeatedly negative in the ELISA and weakly positive in the IF test. Following ion-exchange chromatography to remove IgG, the IF EBV IgM antibody titers dropped from >64 to 10 to 20 (data not shown). After ion-exchange column chromatography, the IgM fraction contained both IgM-RF and subclass IgG4 antibody. Residual IgG4 may have reacted with the solidphase antigen to form antigen-antibody complexes to which IgM-RF attached, causing false-positive results. Low titers of EB-VCA IgG4 were found in 15% of a population similar in composition to ours (16). Further evidence for possible false-positive reactivity following ion-exchange column chromatography was provided by an independent reference laboratory which obtained IF-negative results for EBV IgM with seven of the nine false-positive serum samples in the retrospective group after the removal of RF with IgG-coated latex particles. The consistently negative results obtained for these nine IFpositive serum samples by the ELISA were most likely true-negative values, as the RF Absorbent serum pretreatment used routinely in the ELISA removed both IgM-RF and more than 90% of IgG without lowering EBV IgM ELISA values (Fig. 6). In addition, elevated background readings in the ELISA which might have caused possible false-positive results were minimized by the use of EB-VCA INDIRECT ELISA FOR DIAGNOSIS OF ACUTE EBV INFECTION purified on an immunoaffinity column containing monoclonal antibodies (17) and the use of an excellent blocking agent (2% casein). Casein inhibited over 90% of nonspecific binding when solutions containing even as little as 400,ug of total protein were used to pretreat plastic wells (22, 31). Therefore, a control antigen reaction well was not considered necessary in this system because background reactions were extremely rare, as shown by the excellent specificity (99.7%). Some of the 12 discrepant false-positive IF results might have been caused by IgG binding to Fc receptors induced in the EBV-infected cells. Such results could have also been due to IgM binding nonspecifically to the cytoplasm of EBV-infected cells (23, 26) or to anticellular antibody belonging to the IgM class (24). The four serum specimens in the prospective group which were EBV IgM ELISA positive and IF negative had low ELISA absorbance values (0.22 to 0.24), just above the cutoff point (0.17). Three of these serum specimens, two of which were Monospot test (Ortho Diagnostic Systems Inc.) positive, were from patients in the acute stage of a mononucleosislike illness and probably reflected the greater sensitivity of the ELISA. Confirmatory tests for anti-ea and anti-ebna antibodies were not available at the time. Some investigators use protein A to remove IgG before testing for EBV-specific IgM (30). This method fails to remove subclass IgG3, which constitutes about 5% of total IgG. Virus-specific IgG3 has been found in 97% of patients with primary EBV infections and in all patients with symptoms suspected to be the result of EBV reactivations (16). Therefore, false-positive results may occur because of the presence of IgM-RF coupled with virus-specific IgG3 antibody. Protein A also has the further disadvantage that it removes up to 60% of IgM antibody (3). Because the presence of RF or specific IgG may cause false-positive (8) or false-negative results, respectively, the removal of IgG and RF is essential in any indirect ELISA for IgM antibody. This study confirms that sera containing as little as 0.5 IU of RF per ml coupled with specific IgG may show false-positive EBV IgM results unless IgG and RF are removed. Cross-reactivity caused by closely related viruses may cause ambiguous interpretations of the tests. In this study, no cross-reaction with serum samples positive for varicellazoster virus and herpes simplex virus IgM antibodies was found, but 2 of 31 CMV IgM-positive serum samples (from patients for whom later serum samples did not show a rise in CMV antibody levels) reacted in the EB-VCA ELISA. This result may have been due to antigens shared between EBV and CMV, reactivation of one after primary infection with the other, or dual infection with the two viruses. It is more likely that the two serum samples were from acute EBV infections because it has been reported that about 30% of all sera derived from patients with acute IM and with specific IgM antibody to EBV also show IgM antibody to CMV in the IF test (or in an enzyme immunoassay) (11, 24, 26). This cross-reactivity appears to occur in one direction only. Therefore, in view of the overlapping symptomatology observed in patients with CMV mononucleosis and IM, further testing for VCA IgG, EA IgG, and EBNA IgG antibodies may be necessary in cases in which both EBV IgM and CMV IgM are positive. Our EBV ELISA, when compared with the IF test, showed excellent sensitivity, specificity, accuracy, and reproducibility. Furthermore, all the reagents used in this assay are commercially available, have been prestandard- 957

7 958 HO ET AL. ized, and are stable, thus minimizing the chore of preparation and titration of reagents. The technical time has been shortened so that results can be provided within 3 h. The major disadvantages of the IF test are the greater technical expertise required for fluorescence reading, thus limiting the use of the IF test to more expert laboratories, the lack of automated reading, and the need to test several serum dilutions to obtain an endpoint titer. Automation of ELISAs makes them suitable for most diagnostic laboratories. Although the IF test for EBV IgM is 90 to 94% sensitive in diagnosing IM at presentation (6, 15), the sensitivity is lower (82%) for patients presenting in the first week of illness (2). Such patients with negative results should have an ELISA for IgM done 1 week later. ACKNOWLEDGMENT We thank P. W. Robertson, Serology Unit, Prince of Wales Hospital, Randwick, New South Wales, Australia, for the removal of RF with latex IgG in nine serum specimens. 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