Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia

Nasopharyngeal carcinoma (NPC) in Tunisia is characterized by its bimodal age distribution involving juvenile patients of 10-24 years and adult patients of 40-60 years. Three serological techniques were compared for primary diagnosis (N = 117) and post-treatment monitoring (N = 21) of NPC patients separated in two age groups. Immunofluorescence assay (IFA) was used as the "gold standard" for detection of IgG and IgA antibodies reactive with Epstein-Barr virus (EBV) early (EA) and viral capsid (VCA) antigens. Results were compared with ELISA measuring IgG and IgA antibody reactivity to defined EBNA1, EA, and VCA antigens. Immunoblot was used to reveal the molecular diversity underlying the anti-EBV IgG and IgA antibody responses. The results indicate that young NPC patients have significantly more restricted anti-EBV IgG and IgA antibody responses with aberrant IgG VCA/EA levels in 78% compared to 91.7% in elder patients. IgA VCA/EA was detected in 50% of young patients versus 89.4% for the elder group (P < 0.001). Immunoblot revealed a reduced overall diversity of EBV antigen recognition for both IgG and IgA in young patients. A good concordance was observed between ELISA and IFA for primary NPC diagnosis with 81-91% overall agreement. Even better agreement (95-100%) was found for antibody changes during follow-up monitoring, showing declining reactivity in patients in remission and increasing reactivity in patients with persistent disease or relapse. ELISA for IgA anti-VCA-p18 and immunoblot proved most sensitive for predicting tumor relapse. VCA-p18 IgA ELISA seems suitable for routine diagnosis and early detection of NPC complication.     

Serological diagnosis of Epstein-Barr virus infection by novel ELISAs based on recombinant capsid antigens p23 and p18

A new pair of Epstein-Barr virus ELISAs (Biotest Anti-EBV VCA IgG and VCA IgM ELISA) was evaluated for usefulness for routine diagnosis of acute EBV infections. The ELISAs are based on two viral capsid antigens (VCA), p23 (BLRF2, full-length) and p18 (BFRF3, carboxy-half), that are combined by autologous gene fusion. In total, 179 sera were tested in direct comparison with classical VCA immunofluorescence assays (IFA). With the help of clinical data and additional reference serology, i.e., heterophile antibodies, anti-EA IgG (IFA) and anti-EBNA-1 IgG (ELISA), the patients were divided into the following categories: seronegatives (46), acute primary infections (67), previous infections (39), suspected reactivations (20) and constellations with intermediate serological patterns (7). The VCA IgG and VCA IgM ELISAs showed overall agreement to IFA of 95.0% and 94.4%, respectively. The calculated analytical performance (sensitivity; specificity) of VCA IgG and VCA IgM was 94.0%; 97.8% and 97.1%; 96.5%, respectively. A certain delay in seroconversion of anti-p23-p18 IgG may account for a significant difference in sensitivity of the VCA IgG ELISA between primary (88.4%) and previous infections (100%). In summary, the new recombinant VCA ELISAs yielded good correlation to VCA IFA and in combination with EBNA-1 IgG allow rapid, sensitive, and specific diagnosis of infectious mononucleosis or EBV immune status in general.     

应用基于Logistic回归的ROC曲线评价鼻咽癌EB病毒抗体联合检测

目的 以基于Logistic回归的受试者工作特征(ROC)曲线分析方法,评价VCA/IgA、EA/IgA、Rta/IgG和EBNA1/IgA等EB病毒抗体不同组合在鼻咽癌诊断中的价值.方法 收集211例初治鼻咽癌和203例相似症状的非鼻咽癌病例的血清,采用免疫酶法检测VCA/IgA及EA/IgA,酶联免疫吸附法检测Rta/lgG和EBNA1/IgA.对各种的抗体组合建立Logistic回归模型,以预测概率为分析指标,应用ROC曲线分析,评价不同组合对鼻咽癌的诊断价值.结果 单一指标评价,VCA/IgA敏感度最高(98.1%),EA/IgA特异度最高(98.5%).以基于Logistic同归的ROC曲线分析各项组合,敏感度和特异度均有所提高.双指标组合中,VCA/IgA+Rta/lgG组合的ROC曲线下面积(AUC)为0.991,诊断效能最高,敏感度、特异度及约登指数分别为94.8%、98.0%及0.928.VCA/IgA+Rta/IgG+EBNA1/IgA组合和4项指标组合的敏感度、特异度及约登指数分别为94.8%、98.5%、0.933和96.7%、97.0%、0.937;这两种多指标组合的AUC与VCA/IgA+Rta/IgG组合比较差异均无统计学意义(P＞0.05).结论 基于Logistic回归的ROC曲线分析方法可以为多指标联合诊断试验提供更客观的综合分析,VCA/IgA和Rta/IgG联合检测具有互补作用,是鼻咽癌血清学诊断的合适组合.     

Evaluation of Epstein-Barr virus antigen-based immunoassays for serological diagnosis of nasopharyngeal carcinoma

BACKGROUND: Immunofluorescence (IF) assays based on Epstein-Barr virus (EBV)-encoded antigens have traditionally been the preferred approach for serological screening of nasopharyngeal carcinoma (NPC). OBJECTIVES: To compare the performance of two new commercial assays (indicated by COMM) using, respectively, the IF and enzyme-linked immunosorbent assay (ELISA) formats with an in-house IF assay (IFA). STUDY DESIGN: Sera from 163 patients with histologically confirmed NPC, and 98 healthy controls were tested with each of these assays and their results compared. RESULTS: The sensitivity, specificity, positive and negative predictive values, respectively, for the COMM VCA IgA ELISA were 92.6%, 94.9%, 96.8%, 88.6%; for the COMM VCA IgA IFA were 96.9%, 41.8%, 73.5%, 89.1%; for the in-house VCA IgA IFA were 98.2%, 72.4%, 85.6%, 95.9%; for the COMM EA IgA ELISA were 46.6%, 100%, 100%, 53.0%; for the COMM EA IgA IFA were 77.3%, 100%, 100%, 72.6%; and for the in-house EA IgA IFA were 77.9%, 99.0%, 99.2%, 72.9%. CONCLUSIONS: The receiver operating characteristic curves comparison showed a marginal superior accuracy for the COMM VCA IgA ELISA, suggesting this to be used as a high-throughput serological screening assay, with the more specific COMM EA IgA IFA as a follow-up confirmatory assay in this NPC-endemic area.     

Evaluation of Epstein-Barr virus antigen-based immunoassays for serological diagnosis of nasopharyngeal carcinoma

BackgroundImmunofluorescence (IF) assays based on Epstein-Barr virus (EBV)-encoded antigens have traditionally been the preferred approach for serological screening of nasopharyngeal carcinoma (NPC).ObjectivesTo compare the performance of two new commercial assays (indicated by COMM) using, respectively, the IF and enzyme-linked immunosorbent assay (ELISA) formats with an in-house IF assay (IFA).Study designSera from 163 patients with histologically confirmed NPC, and 98 healthy controls were tested with each of these assays and their results compared.ResultsThe sensitivity, specificity, positive and negative predictive values, respectively, for the COMM VCA IgA ELISA were 92.6%, 94.9%, 96.8%, 88.6%; for the COMM VCA IgA IFA were 96.9%, 41.8%, 73.5%, 89.1%; for the in-house VCA IgA IFA were 98.2%, 72.4%, 85.6%, 95.9%; for the COMM EA IgA ELISA were 46.6%, 100%, 100%, 53.0%; for the COMM EA IgA IFA were 77.3%, 100%, 100%, 72.6%; and for the in-house EA IgA IFA were 77.9%, 99.0%, 99.2%, 72.9%.ConclusionsThe receiver operating characteristic curves comparison showed a marginal superior accuracy for the COMM VCA IgA ELISA, suggesting this to be used as a high-throughput serological screening assay, with the more specific COMM EA IgA IFA as a follow-up confirmatory assay in this NPC-endemic area.     

Evaluation of a multiplex flow immunoassay for detection of epstein-barr virus-specific antibodies

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 ( approximately 400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response.     

Evaluation of four commercial systems for the diagnosis of Epstein-Barr virus primary infections

To compare the performance of four diagnostic commercial systems for Epstein-Barr virus (EBV) serology (for IgM and IgG virus capsid antigen [VCA] and EBV nuclear antigen [EBNA] antibodies), a collection of 125 samples from clinically suspected infectious mononucleosis cases was studied. Indirect immunofluorescence (IIF) for VCA IgM and IgG antibodies and anticomplement immunofluorescence for EBNA antibodies (Meridian Bioscience Inc.) were used as reference methods. By these methods, the cases were classified EBV primary infection (presence of IgM to VCA or IgG to VCA in the absence of EBNA antibodies; n = 82), EBV past infection (presence of VCA IgG and EBNA antibodies in the absence of VCA IgM; n = 26), or no infection (negative for the three markers; n = 17). The following systems were tested: two chemiluminescent immunoassays (CLIAs; the Liason [CLIA-L; DiaSorin] and the Immulite 2000 [CLIA-I; Siemens]), immunofiltration (IF; All.Diag), and an enzyme-linked immunosorbent assay (ELISA; DiaSorin). In the IgM assays, sensitivities ranged from 67.1% (ELISA) to 92.2% (CLIA-L) and specificities ranged from 93.8% (CLIA-L) to 100% (IF). In the VCA IgG assays, sensitivities varied from 79.4% (IF) to 94.4% (CLIA-I) and specificities varied from 94.4% (IF and CLIA-L) to 100% (CLIA-I and ELISA). In EBNA assays, sensitivities ranged from 78.1% (IF) to 93.8% (CLIA-I) and specificities ranged from 32.3% (CLIA-L) to 91.4% (IF). In relation to EBV profiles, the corresponding figures for sensitivity (in detecting primary infection) for IF, CLIA-L, CLIA-I, and ELISA were 92.7%, 93.8%, 89%, and 89.6%, respectively, and those for specificity (to exclude primary recent infection) were 90.7%, 94.6%, 97.7%, and 95.2%, respectively. Although there were limitations in some individual markers, especially CLIA-L for EBNA IgG, the systems evaluated appear to be useful for diagnosis of EBV infection.     

Performance of Two Commercially Available Automated Immunoassays for the Determination of Epstein-Barr Virus Serological Status

This study evaluated the performance of two automated Vidas (V) and Liaison (L) immunoassays for Epstein-Barr virus (EBV) serology. The detection of the viral capsid antigen (VCA) IgM, the VCA/early antigen (VCA/EA) IgG, and the Epstein-Barr nuclear antigen (EBNA) IgG was assessed on 526 sera collected for routine EBV testing in immunocompetent subjects. The determination of expected EBV status (186 EBV primary infections, 183 past EBV infections, and 157 EBV-seronegative individuals) was based on results of routine laboratory enzyme immunoassays (EIAs) together with clinical data. The sensitivity and specificity of each individual marker were determined in comparison to the expected EBV status. The agreement between the V and L profiles and the expected EBV status was established through the interpretation of combinations of the different EBV markers. Statistically significant differences between the two tests were found for the specificity of the VCA IgM marker (96.2% for V versus 93.2% for L), the sensitivity of the VCA/EA IgG marker (89% for V versus 94% for L), and the specificity of the EBNA IgG marker (96.5% for V versus 74.2% for L). The results determined for the two assays with respect to overall agreement with the established expected EBV status were not significantly different (89.7% for V versus 88.2% for L), with discrepancies mainly observed in sera referenced as primary infections. These findings demonstrated the similar performances of the Vidas and the Liaison assays for the establishment of an EBV serological status using the VCA, EA, and EBNA markers.     

Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.     

Synthetic peptides deduced from the amino acid sequence of Epstein-Barr virus nuclear antigen 6 (EBNA 6): Antigenic properties,production of monoreactive reagents,and analysis of antibody responses in man

Studies on the antibody responses to various Epstein-Barr virus (EBV) antigens have been instrumental in the understanding of the seroepidemiology and diagnosis of this viral infection and the subsequent carrier state. While antibodies to the viral capsid antigen (VCA), early antigen (EA), and nuclear antigens 1 and 2 (EBNA 1 and 2) have been well characterized, the antibody response to the other nuclear antigens is not well understood. EBNA 6 is expressed by lymphoblasts during acute EBV infection and may be an important antigen for diagnosis and evaluation of the immune response. In order to analyze the antibody response to EBNA 6, ten peptides (20–21 amino acids), deduced from the EBNA 6 coding region, were synthesized and evaluated for antigenicity by ELISA. One peptide (p-63; PA-PQAPYQGYQEPPAPQAPY) derived from the amino acid repeats showed the highest specific reactivity with human sera. This peptide was evaluated further for detection of human EBNA 6-reactive antibodies. Forty-two of forty-nine (86%) EBV-seropositive healthy donors had p-63- specific IgG reactivity, while none of 50 EBV seronegative patients reacted with the p-63 peptide. Twenty-two of fifty-one (43%) patients with ongoing primary EBV infection had detectable p-63-specific IgG. Serum samples drawn sequentially from patients during and after primary EBV infection revealed an increase in p-63-reactive IgG over time. A similar pattern was found for reactivity with an EBNA I-specific peptide (p-107), in contrast to the EBNA 2 (polyproline) response, which decreased over time. Some EBV seropositive individuals who had no detectable IgG against peptide p-63 did have antibodies against the native EBNA 6 by anticomplement immunofluorescence to EBNA 6 transfected cells. Rabbit antiserum raised against p-63 reacted specifically with native EBNA 6 by an immunofluorescence assay and by immunoblotting, indicating the EBNA 6-specific antigenicity of the peptide. Thus, the peptide p-63 derived from the amino acid repeats of the EBNA 6 coding region constitutes a predominant, although not exclusive, epitope in the EBNA 6 antibody response. © 1995 Wiley-Liss, Inc.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Evaluation of a multianalyte profiling assay and an enzyme-linked immunosorbent assay for serological examination of Epstein-Barr virus-specific antibody responses in diagnosis of nasopharyngeal carcinoma

Assessment of antibody responses to Epstein-Barr virus (EBV) antigens has been used to assist in nasopharyngeal carcinoma (NPC) diagnosis by several methods. In this study, we evaluated an in-house Luminex multianalyte profiling (xMAP) technology and commercial enzyme-linked immunosorbent assay (ELISA) kits for serological examination of EBV-specific antibody responses in 135 NPC patients and 130 healthy controls. Four EBV biomarkers were measured: immunoglobulin A (IgA) against viral capsid antigen (VCA), EBV nuclear antigen 1 (EBNA1), diffused early antigen (EA-D), and IgG against EA-D. The sensitivities and specificities of the four markers ranged between 71.5 and 90% for xMAP assays and 80 and 92% for ELISA. Logistic regression analysis revealed that the combined markers in the xMAP assay had overall sensitivity and specificity values of 82% and 92%, respectively. The correlation coefficient (r) values for the xMAP assay and ELISA were lowest for IgA-VCA (0.468) and highest for IgA-EBNA1 (0.846); for IgA-EA-D and IgG-EA-D, the r values were 0.719 and 0.798, respectively. The concordances of the two methods for NPC discrimination were good (79 to 88%). Our results suggest that both the xMAP assay and ELISA are satisfactory for EBV antibody evaluation when multiple antigens are included.     

Evaluation of 12 Commercial Tests for Detection of Epstein-Barr Virus-Specific and Heterophile Antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc.), Clearview IM (Unipath Ltd.), and Cards±OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards±OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Detection of Epstein-Barr virus antigens and antibodies by peroxidase-labeled specific immunoglobulins.

Detection of the Epstein-Barr (EBV) antigens, early antigen (EA), viral capsid antigen (VCA), and nuclear antigen (EBNA) by the indirect immunoperoxidase technique was highly sensitive. Antibody titers to EBNA, EA, and VCA were determined in more than 25 sera of patients with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC), or normal persons. A good correlation between the titers of these antigens was obtained by the immunoperoxidase and immunofluorescence methods. The indirect (anti-IgG) immunoperoxidase technique for the detection of EBNA is, in contrast to the indirect immunofluorescence method, highly sensitive. EBNA was associated with the chromosomes in cells arrested in the metaphase with colchicine.     

Detection of IgA antibodies to Epstein-Barr virus-associated antigens by ELISA

The detection of IgA antibodies to the Epstein-Barr virus (EBV)-associated viral capsid antigen (VCA) and early antigens (EA) is of diagnostic and prognostic importance for patients with nasopharyngeal carcinoma (NPC). An ELISA for the determination of serum IgG antibodies to these antigens has been developed which uses the double antibody method. 136 sera obtained from healthy donors and patients with non-EBV related tumors and lymphomas were tested by ELISA; only 3 sera, from patients with chronic lymphatic leukemia, hairy cell leukemia and Burkitt-like lymphoma, contained antibodies of IgA class to VCA and EA. Ninety-five sera from patients suspected of having NPC were tested. IgA anti-VCA was found in 28 sera (29.5%), 12 of which also contained IgA anti-EA. The assays described are suitable for diagnosis and follow-up of patients with EBV-associated nasopharyngeal carcinoma. Furthermore, isolated EA components may be tested for their reactivity with IgA antibodies, as was shown for the 60 kDa polypeptide associated with the EA complex.     

A systematic study of Epstein-Barr virus serologic assays following acute infection.

We determined the presence of IgG and IgM antibody to viral capsid antigen (VCA-IgG, VCA-IgM) and IgG antibody to the Epstein-Barr virus nuclear antigen (EBNA) by indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) during the acute illness and at 1, 2, 6, and 48 months in a prospective population-based case series of 95 persons with an acute illness serologically confirmed as Epstein-Barr virus infection. The acute illness was characterized by the presence of VCA-IgG and VCA-IgM (by ELISA) and by the absence of EBNA in most, but not all, patients. During follow-up, VCA-IgG antibodies remained detectable in all patients, while the proportion with VCA-IgM declined and the number with detectable EBNA antibodies steadily increased. The primary differences between the 2 serologic test methods were the increased persistence of VCA-IgM during follow-up by ELISA and the earlier detection of EBNA by IFA. Clinicians should consider the illness stage and the laboratory technique to appropriately interpret serologic test results in suspected cases of mononucleosis caused by the Epstein-Barr virus.     

Evaluation of Four Commercially Available Epstein-Barr Virus Enzyme Immunoassays with an Immunofluorescence Assay as the Reference Method

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.     

Evaluation of four commercially available Epstein-Barr virus enzyme immunoassays with an immunofluorescence assay as the reference method

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.     

Evaluation of the Architect Epstein-Barr Virus (EBV) Viral Capsid Antigen (VCA) IgG,VCA IgM,and EBV Nuclear Antigen 1 IgG Chemiluminescent Immunoassays for Detection of EBV Antibodies and Categorization of EBV Infection Status Using Immunofluorescence Assays as the Reference Method

Commercial immunoassays for detecting IgG and IgM antibodies against Epstein-Barr virus (EBV), viral capsid antigens (VCA), and IgGs toward EBV nuclear antigen-1 (EBNA-1) are routinely used in combination to categorize EBV infection status. In this study, we evaluated the performances of the Architect EBV VCA IgG, VCA IgM, and EBNA-1 IgG chemiluminescent microparticle assays (CMIAs) in EBV serological analyses using indirect immunofluorescence assays and anticomplement immunofluorescence assays as the reference methods for VCA IgG, VCA IgM, and EBNA-1 IgG antibody detection, respectively. A total of 365 serum samples representing different EBV serological profiles were included in this study. The κ values (concordances between the results) obtained in the Architect CMIA and those in the reference assays were 0.905 (P < 0.0001) for VCA IgM, 0.889 (P < 0.0001) for VCA IgG, and 0.961 (P < 0.0001) for EBNA-1 IgG. The sensitivities and specificities were, respectively, 91.08% and 99.48% for VCA IgM, 99.23% and 86.27% for VCA IgG, and 96.77% and 99.16% for EBNA-1 IgG. The sensitivities and specificities of the Architect CMIA panel were, respectively, 99.15% and 98.6% for diagnosing a primary infection, 97.62% and 93.39% for diagnosing a past EBV infection, and 92.42% and 97.82% for diagnosing the absence of an EBV infection. In summary, we demonstrated that the Architect EBV antibody panel performs very well for EBV antibody detection and correctly categorizes clinically relevant EBV infection states.