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.     

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.     

Evaluation of a multiplex fluorescent microsphere immunoassay for the determination of epstein-barr virus serologic status

Epstein-Barr virus (EBV), a human herpesvirus, affects up to 95% of adults. Diagnosis of acute EBV infection can be challenging and often relies on the serologic antibody pattern to 3 distinct antigens, most often determined by indirect fluorescent antibody (IFA), enzyme-linked immunosorbent assays (ELISAs), and, more recently, multiplex assays. We compared a multiplex assay for the simultaneous detection of antibodies to viral capsid (VCA), nuclear (EBNA), and early (EA) EBV antigens with ELISAs using IFA for discrepancy resolution. Concordance of the multiplex assay was good for all 4 antigens: VCA IgM, 86.6% vs ELISA and 92.9% vs IFA; VCA IgG, 92.8% vs ELISA and 98.0% vs IFA; EBNA IgG, 90.3% vs ELISA and 98.1% vs IFA; and EA IgG, 83.8% vs ELISA and 92.8% vs IFA. After IFA resolution, correlation between the multiplex assay and ELISA for serologic disease stage, based on the antibody profile of all 4 analytes, was 90%. The multiplex assay showed good correlation with an established ELISA and even better correlation with the "gold standard" IFA. Advantages of the multiplex assay over traditional methods include multiple results per assay, inclusion of internal controls for each assay, and well-to-well monitoring of assay drift.     

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.     

EBV4型IgA/VCA IgA/EA IgG/EA IgG/ZEBRA抗体在鼻咽癌普查和早期诊断中的应用

目的摸索以疱疹病毒4型（EBV）IgG／ZEBRA为捕捉抗原的间接酶联免疫吸附试验（ELISA）条件,为大量人群普查奠定基础。方法将纯化的ZEBRA抗原用于对鼻咽癌（NPC）患者血清及健康人血清IgG／ZEBRA抗体的ELISA检测。结果检测NPC患者血清288份,其中ELISA实验显示阳性262份,敏感度91％,检测正常人血清96份,其中阳性5份,特异度94．8％。其结果显示NPC组的阳性率与健康对照组的数据之间差异有统计学意义（P〈0．001）。本研究在此基础上对广东惠州5463份和广西桂平2017份血清进行检测,检出早期鼻咽癌患者5例。并将结果与免疫酶法检测IgA／VCA、IgA/EA、IgG／EA比较。结论以EBV早期抗原ZEBRA为捕捉抗原的间接ELISA方法具有较高的特异性和敏感性,可以用于大量人群的NPC早期筛查和早期诊断。     

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.     

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.     

Epstein-Barr病毒壳抗原gp125的纯化和应用

目的研制ｅｐｓｔｅｉｎ－Ｂａｒ（ＥＢ）病毒诊断试剂。方法将重组痘苗病毒表达的Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）壳抗原（ＶＣＡ）主要多肽ｇｐ１２５纯化，作为诊断抗原建立了酶联免疫吸附试验（ＥＬＩＳＡ），检测了４８份鼻咽癌（ＮＰＣ）病人血清及１０份正常人血清中的ＶＣＡ／ＩｇＡ抗体。结果该方法与免疫荧光（ＩＦ）检测结果一致，但ＥＬＩＳＡ的平均几何滴度（ＧＭＴ）是ＩＦ的１２倍。结论以纯化的ＥＢ病毒壳抗原主要多肽ｇｐ１２５作为诊断抗原建立的检测方法，更适合于ＥＢＶ相关疾病的血清学诊断和血清流行病学调查。     

Analyses of the prognostic significance of the Epstein-Barr virus transactivator ZEBRA protein and diagnostic value of its two synthetic peptides in nasopharyngeal carcinoma.

BACKGROUND: Although numerous serological studies have determined the diagnostic and prognostic values of Epstein-Barr virus (EBV) antibodies in adult patients with nasopharyngeal carcinoma (NPC), little data about the anti-EBV immune response in children with NPC is available. OBJECTIVES: To examine the diagnostic value of IgG antibodies against BamHI Z Epstein-Barr replication activator (ZEBRA) protein and two related synthetic peptides (Zp125 and Zp130). To compare the prognostic value of IgA antibodies against early antigens (EA) and viral capsid antigen (VCA), and IgG antibodies against ZEBRA protein, of Moroccan children treated for NPC with their prognostic value for young and adult NPC patients. STUDY DESIGN: Sera were collected from 255 newly diagnosed Moroccan NPC patients and 226 healthy donors. IgA antibody against VCA and EA was measured by immunofluorescence assays. IgG antibody against ZEBRA, Zp125, and Zp130 was measured by ELISA. RESULTS: No significant difference in the detection of IgG-Zp125 and Zp130 antibodies was observed in children with NPC. IgG-Zp130 were detected less frequently than IgG-Zp125 in young and adult patients, as compared to children. High specificity of IgG-Zp125 and -Zp130 antibodies was found in the three age groups. A decrease in IgG-ZEBRA was observed in patients with NPC in clinical remission, whereas patients with NPC who died or developed metastases maintained or had an increase in these titers. CONCLUSION: IgG-ZEBRA is a better diagnostic and post-therapeutic prognostic marker in children with NPC, who showed very low titers of IgA -VCA and -EA.     

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.     

Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.     

Evaluation of multiple antibodies to Epstein-Barr virus as markers for detecting patients with nasopharyngeal carcinoma

Five serological tests were assessed for their sensitivity for screening and early detection of nasopharyngeal carcinoma (NPC). The tests included the detection of antibodies to various gene products of EBV: viral capsid antigen (VCA) using an indirect immunofluorescence assay (FA), DNase using an activity neutralisation test (NT), DNase using an enzyme-linked immunosorbent assay (ELISA), DNA polymerase (DP) using NT, and major DNA binding protein (MDBP) by ELISA. Sera from 100 NPC outpatients and 20 NPC patients, who were detected in a prospective study, were examined. The results showed that levels of antibody to DNase detected by ELISA and to DP detected by NT and the positivity rate for VCA by FA increased with NPC stage. More species of EBV antibody became detectable as NPC progressed. The detection of anti-MDBP antibody by ELISA was suitable for screening for NPC. Anti-DP antibody detected by NT was a valuable marker both for early detection and prognosis of NPC. Detection of anti-DNase antibody by ELISA was the most sensitive method for detection of NPC. No single test was sufficient to detect all the NPC patients and a combination of anti-DNase by ELISA with other tests are recommended to identify NPC patients. J. Med. Virol. 52:262–269, 1997. © 1997 Wiley-Liss, Inc.     

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.     

IgA directed against early antigen of Epstein-Barr virus is no specific marker for the diagnosis of nasopharyngeal carcinoma

The aim of this study was to evaluate the significance and specificity of IgA directed against Epstein-Barr virus (EBV)-specific early antigens (EA) for the unequivocal diagnosis of nasopharyngeal carcinoma (NPC). Therefore, sera from patients with diseases other than NPC, selected on the basis of elevated antibody titres against EBV antigens, were compared to sera from NPC patients with regard to the presence of IgA directed against EBV viral capsid antigen (VCA-IgA) and IgA directed against EA (EA-IgA). Four hundred forty-seven out of 7,508 non-NPC sera tested showed high titres (>512) of IgG directed against Epstein Barr viral capsid antigen (VCA-IgG) and positive VCA-lgA (?32). Two hundred twenty-seven of these sera were compared to 51 VCA-IgA-positive sera from NPC patients regarding the titre of EA-lgA. 60.7% of VCA-lgA-positive NPC sera showed positive EA-lgA, however 33% of VCA-IgA-positive non-NPC patients also exhibited EA-lgA. This result demonstrates that EA-lgA is not specific for NPC and does not allow an unequivocal serological diagnosis of NPC in individual cases. It seems therefore to be of questionable use for screening programs in NPC low-risk areas. The data do not contradict the usefulness of this marker for monitoring of patients treated for NPC and for screening programmes in high-risk areas. © 1994 Wiley-Liss, Inc.     

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.     

Epstein-Barr virus serology in the diagnosis of nasopharyngeal carcinoma

The antibody levels to viral capsid antigen (VCA) and early antigen (EA) of Epstein-Barr virus (EBV) in 164 nasopharyngeal carcinoma (NPC) patients from Sarawak, East Malaysia were significantly higher than those in 147 sex, age and ethnically matched healthy controls. As diagnostic markers of NPC, IgG/VCA at reciprocal titers > or =160 was the most sensitive (89%, with 98% specificity), while IgA/EA at > or =5 was the most specific (100%) but the least sensitive (75%). The sensitivity and specificity of IgA/VCA at reciprocal titers > or =10 were 84% and 97%. IgA/VCA has an advantage over IgG/VCA despite the slightly lower sensitivity due to its consistently more distinct fluorescence reaction. The sensitivity and specificity can be marginally improved by a combination of two tests.     

Two-Step Epstein-Barr Virus Immunoglobulin A Enzyme-Linked Immunosorbent Assay System for Serological Screening and Confirmation of Nasopharyngeal Carcinoma

Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.Nasopharyngeal carcinoma (NPC) is a common cancer in China and Southeast Asia and closely associated with Epstein-Barr Virus (EBV) (26). In Indonesia, especially in the southern part of central Java, undifferentiated carcinoma (WHO type III) is the most common head and neck cancer and among the five most prevalent cancers overall. Due to unspecific symptoms and the hidden localization of the primary tumor at the early stage, more than 80% of the patients come to the hospital at a late stage (III or IV), when they already have metastasis in the cervical lymph node. Whereas late-stage disease has a poor prognosis and requires combined chemo-radiotherapy, early-stage NPC may reach complete remission by radiotherapy only (17). Therefore, screening for early-stage NPC among the population is important and clinically relevant. For developing countries, such an approach should be economical, employing standardization methods suited for mass screening.Patients with NPC have high-level broad-spectrum anti-EBV antibodies, especially immunoglobulin A (IgA), compared to regional healthy carriers and patients with other head and neck diseases (13, 14). Our group recently demonstrated that the molecular diversity underlying anti-EBV IgG and IgA responses in NPC patients was different, requiring multiple EBV antigens for complete serological coverage (7). Prior studies in China and Taiwan have shown the feasibility of using IgA serology for population screening (2, 15, 27). However, in these studies laborious and poorly standardized cell-based serological techniques were used. Nevertheless, these studies revealed the appearance of serological abnormalities, i.e., positive EBV IgA responses 2 to 3 years prior to onset of NPC (2, 15), which clearly demonstrated the opportunity of using EBV serology for early-stage detection of NPC. This particularly applies for screening in high-risk groups, such as family members of NPC patients and patients with suspicious head and neck symptoms (18, 21).For NPC serodiagnosis, cell-based indirect immunofluorescent assay (IFA) methods are still widely considered the gold standard. IFA involves the separate analysis of antibody responses to viral capsid antigen (VCA), early antigen (EA), and nuclear antigens (EBNA), each comprising multiple proteins and requiring different cell lines for specific analysis (10, 12, 13). However, this method shows considerable variation among laboratories and is time-consuming, subjective, and not suitable for large-scale automatic handling. Enzyme-linked immunosorbent assay (ELISA) techniques are increasingly used and have shown a better sensitivity and specificity compared to IFA and are suitable for large-scale application (4, 10, 11, 16, 20, 21).Recently, we developed an EBV IgA ELISA based on a combination of VCA p18- and EBNA1-derived synthetic peptides which is routinely used as an NPC diagnostic test in our local hospital (Sardjito Hospital, Yogyakarta, Indonesia). This EBV IgA ELISA combines the separate features of IgA VCA and IgA EBNA1, each of which has its value in NPC diagnosis. The combination of these markers in a single assay provided sensitivity and specificity of 85.4% and 90.1%, respectively (8). The presence of NPC-related serological abnormalities can be confirmed by immunoblotting to reveal the spectrum of antibody responses, which has diagnostic value by itself (5, 7, 16, 25). The combined EBV IgA ELISA and immunoblot assay showed increased sensitivity and specificity and positive predictive value (PPV) and negative predictive value (NPV) of more than 95% (7, 8). Because immunoblot studies revealed a diagnostic value of multiple EBV proteins, in particular certain EBV-EA markers, we recently developed a separate IgA EA ELISA using native EA proteins (22). In addition to their role in primary diagnosis, anti-EBV IgA responses, in particular the IgA EA response, also have a distinct role for posttreatment follow-up monitoring, as declining responses correlate with a good prognosis and increasing responses are related to persistence of relapsing tumor (6, 16, 24). The availability of two distinct and biochemically well-defined EBV IgA ELISA systems addressing responses to different EBV antigens for NPC-specific serology may add to further standardization. In this study we evaluate the combination of these two tests for primary diagnosis of NPC in a high-incidence population in Indonesia.     

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.     

Immune responses to Epstein-Barr virus lytic proteins in patients with nasopharyngeal carcinoma

Immune responses to three Epstein-Barr virus (EBV) lytic proteins, DNase, thymidine kinase (TK), and BMRF-1 gene products (50/52 kDa diffused early antigen, EA-D complex) were determined in EBV-infected control individuals and patients with nasopharyngeal carcinoma (NPC). Immunofluorescence assays (IFA) were used to detect their humoral immune responses using recombinant EBV lytic proteins expressed in a baculovirus system as antigens. Cell proliferation assays were performed to evaluate their cellular immune responses by monitoring 3H-thymidine incorporation. Seventy patients with NPC and 32 non-cancer controls were analyzed. The results of IFA showed antibody titers to all three EBV lytic proteins to be higher in the patients with NPC especially for the IgA class. Positivity rates of the three IgA antibodies also were higher in the patients with NPC population. Furthermore, the profiles of the IgA antibodies correlated with those to total early antigens (EA) expressed in the early phase and viral capsid antigen (VCA) expressed in the late phase, of EBV replication. The most interesting finding was that antibody titers to the three EBV lytic proteins were associated significantly with metastases of cervical lymph nodes in patients with NPC. As for cellular immunity to the EA-D complex and DNase, weak responses were observed in the cell proliferation assays. Peripheral blood cells from most individuals could not be stimulated to proliferate, except for a few patients with NPC whose antibody titers against the EA-D complex and DNase also were very high.