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Clinical and Vaccine Immunology, September 2008, p. 1410-1413, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00082-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluation of a Multiplex Flow Immunoassay for Detection of Epstein-Barr Virus-Specific Antibodies

M. J. Binnicker,^* D. J. Jespersen, J. A. Harring, L. O. Rollins, and E. M. Beito

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, and Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Received 3 March 2008/ Returned for modification 22 April 2008/ Accepted 3 July 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Conventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immunofluorescence assay (IFA) and enzyme immunoassay (EIA). While sensitive and specific, these methods are labor-intensive and require separate assays for each analyte. This study evaluated the performance of a multiplex bead assay (BioPlex 2200; Bio-Rad Laboratories, Hercules, CA) for the simultaneous detection of immunoglobulin G (IgG) and IgM class antibodies to the EBV viral capsid antigen (VCA) and IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1). Serum specimens (n = 1,315) submitted for routine EBV-specific antibody testing by EIA (Grifols-Quest, Inc., Miami, FL) were also tested by the multiplex bead assay using the BioPlex 2200 automated analyzer. Specimens showing discordant results were tested by IFA. Following IFA resolution, the BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated 97.9%, 91.4%, and 96.9% agreement, respectively, with the results obtained by EIA. Furthermore, the BioPlex assays showed an overall agreement of 94.1% with the EIA when the specimens were categorized by disease state (susceptible, acute, or past infection) based on the EBV-specific antibody profiles. These findings indicate that the BioPlex EBV assays demonstrate a performance comparable to that of the conventional EIA, while allowing for a more rapid (2.3 h for 100 samples versus 4.5 h by the EIA) and higher-throughput (400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Epstein-Barr virus (EBV) is the primary agent of infectious mononucleosis (IM), a common syndrome characterized by fever, pharyngitis, and lymphadenopathy. Most individuals become infected during childhood, and it is estimated that nearly 95% of the adult population worldwide is seropositive for the virus (20). While the majority of infections result in either asymptomatic or mild disease, serious complications, including B- and T-cell lymphomas, nasopharyngeal carcinoma, and central nervous system involvement, may occur, especially in immunocompromised hosts (14).

The diagnosis of IM is made, in most cases, on the basis of characteristic clinical manifestations or the detection of heterophile antibodies (24). However, a determination of the EBV-specific antibody response may be required for young children (especially those <4 years old) (26) and for adults suspected of having heterophile-negative IM. Testing for immunoglobulin M (IgM) and IgG class antibodies to the viral capsid antigen (VCA) and for IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1) allows for a discrimination between recent and remote infection (8, 18, 21, 22). Levels of antibodies (IgM and/or IgG) to VCA are typically elevated during the acute phase of IM (19, 22, 26), with anti-VCA IgM levels showing a steady decline 4 to 6 weeks after symptom onset (16). In contrast, anti-VCA IgG persists indefinitely, and its detection along with that of anti-EBNA-1 IgG suggests past exposure to the virus (25). The conventional methods used to detect EBV-specific antibodies include an indirect immunofluorescence assay (IFA) and an enzyme immunoassay (EIA). While sensitive and specific, the IFA is subjective and labor-intensive. Furthermore, the IFA and EIA require separate assays to be performed for each EBV-specific analyte. This may potentially increase the volume of sample needed, as well as the associated technologist and instrument time required for testing.

Recently, multiplex flow immunoassay (MFI) technology emerged as a novel method for analyzing the EBV serologic response (1, 13, 18). This approach is similar to traditional EIA but allows for the simultaneous detection and identification of multiple analytes in a single reaction. MFI technology uses a liquid suspension array of up to 100 unique microspheres (5- to 6-µm-diameter beads), each conjugated with a different capture molecule (e.g., antibody, antigen, nucleic acid). Each capture analyte is detected and quantitated following the addition of a fluorescently labeled reporter molecule whose emission is measured by a flow-based detector. In 2007, Bio-Rad Laboratories (Hercules, CA) released FDA-cleared IgM and IgG EBV assays based on MFI technology. These assays were fully automated on the BioPlex 2200 automated analyzer (Bio-Rad Laboratories), allowing for a high-throughput analysis of the EBV-specific antibody response.

Due to increasing test volumes (80% in the past 5 years) and the limitations of conventional methods for EBV-specific antibody testing, we undertook a study to evaluate the BioPlex EBV assays for the detection of anti-VCA IgM, anti-VCA IgG, and anti-EBNA-1 IgG. The goal of this study was to compare the results of BioPlex and EIA testing, using IFA to resolve discordant results.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Study design. Serum specimens (n = 1,315) submitted to our reference laboratory for routine EBV-specific antibody testing by EIA were also tested by the Bio-Rad EBV assays on the BioPlex 2200 automated analyzer. Specimens showing discordant results after initial testing were repeat tested by both the EIA and the BioPlex assay, with further discrepancies being resolved by IFA. The study protocol was reviewed and approved by the institutional review board of the Mayo Clinic.

EIA. Testing by EIA was performed according to the manufacturer's instructions using the SeraQuest EBV VCA IgM, VCA IgG, and EBNA-1 IgG kits (Grifols-Quest, Inc., Miami, FL). The SeraQuest VCA IgM assay uses recombinant gp125 capsid peptide as the capture antigen, while the VCA IgG assay utilizes a combination of recombinant gp125 and whole-cell lysate from viral capsid-induced cells. Testing was completed on the Triturus automated EIA analyzer (Grifols S.A., Barcelona, Spain).

MFI. In addition to testing by EIA, each specimen was tested according to the manufacturer's instructions using the BioPlex 2200 EBV IgG and IgM kits on the BioPlex 2200 analyzer. The BioPlex EBV IgG kit consists of three distinct bead sets allowing for the multiplex detection of IgG class antibodies to VCA, EBNA-1, and the diffuse component of early antigen (EA). The BioPlex EBV IgM kit utilizes two different bead sets for the detection of IgM class antibodies to VCA and heterophile antigen. The BioPlex VCA IgG and IgM beads are coated with recombinant VCA p18 (40 kDa). During testing, the BioPlex assay combines an aliquot of patient specimen, bead reagent, and sample diluent into a reaction vessel. After incubation and washing, a phycoerythrin-conjugated reporter antibody is added to the reaction mixture. Following a second incubation and wash cycle, the beads are suspended in buffer and passed through a flow-based detector. The detector first identifies each bead based on the internal dye composition and subsequently determines the amount of antibody bound to the capture antigen by measuring the fluorescence emitted from the attached phycoerythrin. The data are initially calculated as the relative fluorescence intensity, which is then converted to a fluorescence ratio by using an internal standard bead. The fluorescence ratio is compared to an assay-specific calibration curve in order to determine the analyte concentration in antibody index units. Results are then classified according to their antibody index values as negative (0.8), equivocal (0.9 to 1.0), or positive (1.1). For quality control purposes, the BioPlex assay also monitors the signals from three control bead sets incorporated into each reaction mixture. These internal controls verify the addition of patient sample to the reaction mixture, the absence of nonspecific binding, and the performance of the detector.

IFA. Specimens showing discordant results after repeat testing by the EIA and BioPlex assay were analyzed by IFA. Testers conducting the IFA were blinded to the results of the EIA and BioPlex assay, and testing was performed according to the manufacturer's instructions for anti-VCA IgM and IgG IFAs (Zeus Scientific, Inc., Raritan, NJ) and anti-EBNA IgG anticomplement immunofluorescence (ACIF) (Bion Enterprises, Des Plaines, IL). The Zeus VCA IFAs employ EBV-infected substrate cells, while the Bion ACIF slides consist of fixed Raji cells, which express the EBV genome but do not produce VCA or EA. For the interpretation of results, the IFA and ACIF assay were screened for characteristic patterns of fluorescence at 1:10 and 1:5 dilutions, respectively.

Classification of disease state. The criteria used to categorize specimens by disease state (susceptible, acute, or past infection) were based on conventional EBV-specific antibody profiles described in the literature (2, 18, 25). In brief, specimens classified as "susceptible" were negative for VCA IgM, VCA IgG, and EBNA-1 IgG. Specimens classified as "acute" were positive for VCA IgM, VCA IgG, or both but negative for EBNA-1 IgG. Specimens showing evidence of "past" infection were positive for VCA IgG and EBNA-1 IgG, with or without concomitant detection of VCA IgM. Specimens that did not meet any of these criteria were classified as "inconclusive."

Statistics. Statistical analyses were performed using JMP software, version 7 (SAS Institute, Inc., Cary, NC). In addition to percentages of agreement, kappa coefficients were calculated as a secondary measure of agreement. Levels of agreement of results by kappa values are categorized as near-perfect (0.81 to 1.0), substantial (0.61 to 0.8), moderate (0.41 to 0.6), fair (0.21 to 0.4), slight (0 to 0.2), or poor (<0) (15). Equivocal results by the BioPlex assay were considered negative for sensitivity calculations and positive for specificity calculations.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Agreement between the EIA and the BioPlex assay. To measure agreement, the results obtained by the EIA and BioPlex assay were compared following testing of 1,315 serum specimens. The BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated agreements of 96.2%, 89.4%, and 92.4%, respectively, with the results obtained by EIA (Table 1). Kappa coefficients showed substantial agreement for the VCA IgM ( = 0.8) and IgG ( = 0.74) assays and near-perfect agreement for the EBNA-1 IgG assay ( = 0.84) (Table 1). Specimens showing discordant results after repeat testing were analyzed by IFA. Among the specimens showing discordant VCA IgM results, 18/39 (46.2%) were resolved in favor of the BioPlex assay by IFA analysis. For specimens with discordant VCA IgG or EBNA-1 IgG results, IFA resolved 19/123 (15.4%) and 45/81 (55.6%), respectively, in favor of the BioPlex assay (Table 1). Following IFA resolution, the BioPlex assays showed overall adjusted agreements of 97.9% for VCA IgM, 91.4% for VCA IgG, and 96.9% for EBNA-1 IgG.

View this table: [in this window] [in a new window]

TABLE 1. Comparison of the Bio-Rad BioPlex assay and SeraQuest EIA for the detection of EBV-specific antibodies in prospective serum specimens (n = 1,315)

Patient specimens were then categorized by disease state (susceptible, acute, or past infection) based on conventional EBV-specific antibody profiles described in the literature (2, 18, 25). Specimens showing results that were not consistent with accepted antibody profiles were categorized as inconclusive. The BioPlex serologic profiles showed an overall agreement of 88.1% with those obtained by EIA (Table 2). Following IFA resolution of discrepant results, the BioPlex assay demonstrated an adjusted overall agreement of 94.1% (Table 2).

View this table: [in this window] [in a new window]

TABLE 2. Correlation of disease state based on EBV-specific antibody profiles after repeat testing by the EIA and BioPlex assaya

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Serology remains the method of choice for the laboratory diagnosis of IM (11, 16). In most cases, the detection of heterophile antibodies by either rapid agglutination or immunochromatographic assays is sufficient to support a clinical diagnosis of acute EBV infection. Heterophile assays are effective for 80 to 85% of patients with IM (4, 17) and are commonly performed as point-of-care tests or in small hospital-based laboratories. However, heterophile antibodies may not be detected in as many as 20% of adults and more than 50% of children less than 13 years old (9, 26); in these cases, an evaluation of the EBV-specific antibody response may be required to confirm a clinical diagnosis.

Testing for EBV-specific antibodies is generally restricted to large hospital-based or reference laboratories. In 2007, our laboratory at the Mayo Clinic tested 45,104 specimens by EBV serology. Of these, 40,770 (90.4%) specimens were submitted for anti-VCA (IgM and IgG) and anti-EBNA-1 IgG testing. The remaining specimens included 3,200 (7.1%) submitted for EA, 877 (1.9%) for heterophile, and 257 (0.6%) for anti-VCA IgA testing. Due to these ordering patterns, our laboratory uses the profile results of anti-VCA (IgM and IgG) and anti-EBNA-1 IgG tests to aid in the discrimination between recent and remote EBV infection. Past reports have underscored the use of these three EBV-specific markers in characterizing the disease state following viral infection (2, 8, 18, 21, 22).

The results of our evaluation demonstrated that the BioPlex VCA (IgM and IgG) and EBNA-1 IgG assays showed substantial agreement ( > 0.61) with routine testing by EIA. Furthermore, the BioPlex assays showed 94.1% agreement with EIA and IFA when specimens were categorized as susceptible, acute, or past infection based on the antibody profile results. Our findings are similar to those described in a recent report by Klutts et al. (13), in which they assessed the prototype BioPlex EBV assays using 167 nonconsecutive serum samples. We have extended their observations by evaluating the recently FDA-cleared BioPlex EBV assays in a large, prospective study using IFA to resolve discordant results. Our evaluation showed higher concordance between the BioPlex assay and the EIA for VCA IgM (96.2%) but lower concordance for EBNA-1 (92.4%) and VCA IgG (89.4%) than the concordance values of 92.0%, 97.0%, and 92.0%, respectively, reported in the previous study (13). Furthermore, we observed that the BioPlex assay had a higher sensitivity for VCA IgM (94.1%) but a lower specificity for EBNA-1 IgG (84.0%) than the values of 85.7% and 96.3%, respectively, reported by Klutts et al. (13). These differences are likely due to the increased number of specimens tested in our evaluation and the different EIAs used as the comparative method in these studies.

Despite substantial agreement between the BioPlex assay and the EIA, there are differences in the performance of the individual assays that may affect antibody profile results. In our evaluation, the major difference was in patients whose condition was categorized as "acute infection" by the EIA but "past infection" by the BioPlex assay (Table 2). This is most likely due to the increased detection of anti-EBNA-1 IgG by the BioPlex assay in comparison to the EIA. IFA analysis of specimens showing discordant anti-EBNA-1 IgG results revealed that 45/81 (55.5%) BioPlex assay-positive, EIA-negative specimens were truly positive for anti-EBNA-1 IgG (Table 1). This indicates that the BioPlex EBNA-1 IgG assay may be more sensitive than the EIA. A second notable difference was observed for patients classified as having "acute infection" by the EIA but as "susceptible" by the BioPlex assay (Table 2). This group was positive for anti-VCA IgG by the EIA but negative by the BioPlex assay and was negative for anti-EBNA-1 IgG by both tests. IFA analysis confirmed the presence of anti-VCA IgG in 19/24 (79.2%) of these patients (Table 2). These findings correlated with our overall observation that the BioPlex VCA IgG assay was less sensitive than the EIA (Table 1). This may be due, in part, to the different capture antigens used by the assays, with recombinant VCA p18 for the BioPlex assay and recombinant gp125 and whole-cell lysate for the EIA. We should also emphasize that the presence of VCA IgG alone was classified as "acute infection" in this study; however, this profile may be observed in subacute infection or in certain patients (e.g., immunocompromised hosts, transplant patients) with past infection who fail to develop detectable anti-EBNA-1 IgG (5, 23). Additional laboratory or clinical information may be required for these patients in order to accurately categorize the disease state. Despite the differences in performance between the BioPlex assay and the EIA, our evaluation showed closer correlation between these methods than past studies comparing conventional tests, such as the EIA and IFA (6-8, 22).

This study has several additional limitations. First, the conclusions that can be drawn regarding the clinical sensitivity and specificity of the BioPlex assay are limited by the lack of available clinical information. Second, this report does not describe the performance of the BioPlex heterophile and EA assays. As mentioned, heterophile tests are commonly performed as point-of-care tests and are infrequently carried out at large reference laboratories. Tests for EA may be useful in cases of acute IM, but the detection of antibodies to EA is often transient and variable (3, 12). Testing for anti-EA IgG is generally of significant benefit only under select clinical circumstances, including the differentiation of primary and reactivated viral infections (26) and the serologic evaluation of patients with EBV-associated malignancies (e.g., nasopharyngeal carcinoma) (10). It will be of interest in future studies to evaluate the BioPlex heterophile and EA assays in order to determine their use in these clinical settings and their performance in comparison to conventional methods.

In conclusion, we have demonstrated that the BioPlex EBV assays show a performance comparable to that of routine testing by EIA while offering several advantages. First, the BioPlex assay has the capacity to test for as many as five EBV serologic markers using only two aliquots of sample. This may reduce both sample volume requirements and aliquot errors. Second, the BioPlex assay incorporates three internal controls into each reaction, allowing for an assessment of specimen addition, nonspecific binding, and detector performance. Finally, the BioPlex assay allows for a more rapid (2.3 h for 100 samples, compared to 4.5 h by the EIA) and higher-throughput (400 samples per 9 h, compared to 200 samples by the EIA) analysis of the EBV serologic response. This may prove beneficial for high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for EBV serologic testing.

 
We thank the laboratory technologists and assistants at the Infectious Diseases Serology laboratory at Mayo Clinic Rochester, who provided excellent laboratory and technical support during this study. We also thank Thomas Smith for critical review of the manuscript. The kits and reagents used in this study were provided by Bio-Rad Laboratories.

 
* Corresponding author. Mailing address: Mayo Clinic, 200 First St. SW, Hilton 860A, Rochester, MN 55905. Phone: (507) 538-1640. Fax: (507) 284-4272. E-mail: binnicker.matthew{at}mayo.edu

Published ahead of print on 16 July 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Clinical and Vaccine Immunology, September 2008, p. 1410-1413, Vol. 15, No. 9
1071-412X/08/$08.00+0     doi:10.1128/CVI.00082-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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• Bravo, D., Munoz-Cobo, B., Costa, E., Clari, M. A., Tormo, N., Navarro, D. (2009). Evaluation of an Immunofiltration Assay That Detects Immunoglobulin M Antibodies against the ZEBRA Protein for the Diagnosis of Epstein-Barr Virus Infectious Mononucleosis in Immunocompetent Patients. CVI 16: 885-888 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Binnicker, M. J. Articles by Beito, E. M. Search for Related Content PubMed PubMed Citation Articles by Binnicker, M. J. Articles by Beito, E. M.