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.     

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.     

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.     

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.     

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.     

Purified hexameric Epstein-Barr virus-encoded BARF1 protein for measuring anti-BARF1 antibody responses in nasopharyngeal carcinoma patients

WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n = 155) compared to healthy EBV-positive (n = 56) and EBV-negative (n = 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P = 0.015) than in regional Indonesian controls or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.     

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.     

基于2008分期标准的壮族鼻咽癌各期患者中EB病毒Zta/IgG等抗体阳性率的比较

探讨桂西地区壮族鼻咽癌患者EB病毒EBNA1/IgA、Zta/IgG及Rta/IgG抗体阳性率与鼻咽癌2008分期的关系。收集140例未经治疗的鼻咽癌患者的血清,用酶联免疫吸附法(ELISA)检测EBNA1/IgA、Zta/IgG及Rta/IgG抗体,按"2008分期法"进行分期,分别计算临床分期的各抗体阳性率并进行两两对比,进行统计学分析。鼻咽癌各临床分期组EB-NA1/IgA、Zta/IgG及Rta/IgG抗体阳性率均有统计学差异(P<0.05)。III期的各抗体阳性率明显低于IV期临床分期(P<0.05),Rta/IgG抗体有显著差异(P=0.001)。壮族鼻咽癌患者EB病毒EBNA1/IgA、Zta/IgG及Rta/IgG抗体阳性率与鼻咽癌临床分期有关。或许EBNA1/IgA、Zta/IgG及Rta/IgG抗体对于评估鼻咽癌临床分期有一定的参考价值。     

Humoral immune response in Epstein-Barr virus infections. I. Elevated serum concentration of the IgG1 subclass in infectious mononucleosis and nasopharyngeal carcinoma

Using radial immunodiffusion we measured IgG subclass concentrations and studied their distribution in serum samples from patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC), two Epstein-Barr virus (EBV)-associated diseases, in comparison with two control groups [completely anti-EBV negative persons and subjects carrying antibodies to the viral capsid antigen (VCA)]. Antibody titres to VCA and to the early antigen (EA) were determined by indirect immunofluorescence and revealed characteristic patterns for the respective diagnostic groups. Nephelometric assays served for quantitating total protein, albumin, total IgG, IgA and IgM in all the sera. In the IM and NPC groups the concentration of IgG1 was significantly elevated by more than 50% whereas the other three subclasses remained unchanged as compared with the controls. Correspondingly, we found a significant increase of total IgG in IM and NPC. In IM, the only disease where VCA-specific IgM antibodies have been reported to occur, IgM levels were markedly elevated. Our data suggest that the IgG1 subclass plays an important role in the humoral immune response to EBV-determined antigens and that it is possibly involved in the control of virus infection.     

五厂家EB病毒抗体检测试剂盒血清学诊断鼻咽癌的比较

目的 通过比较EB病毒抗体检测试剂盒血清学诊断鼻咽癌的准确性和检测结果的一致性,为试剂盒在临床上的使用选择和性能改进提供依据.方法 使用五厂家的EB病毒衣壳抗原IgA和IgG抗体检测试剂盒（VCA IgA和VCA IgG试剂盒）、核抗原I IgA和IgG抗体检测试剂盒（EBNA1 IgA和EBNA1IgG试剂盒）、早期抗原IgA和IgG抗体检测试剂盒（EA IgA和EA IgG试剂盒）以及Zta IgA抗体检测试剂盒,分别检测33例鼻咽癌患者（NPC）、30例健康体检者（HD）和41例非鼻咽癌的其他肿瘤患者（NNPC）血清或血浆样本.结果 A厂家的VCA IgA试剂盒灵敏度高于其他厂家同品种试剂盒,但对于NNPC特异度最低（36.6％）;而D厂家VCA IgA试剂盒的特异度最高（97.6％）,且对HD的特异度均大于90％.B和D厂家的EBNA1 IgA试剂盒间阳性、阴性符合率分别为92.1％和100.0％.A和E厂家的EA IgA试剂盒的灵敏度均较低而特异度高,试剂盒间阳性符合率低（39.4％）,阴性符合率高（98.6％）;而VCA IgG试剂盒的灵敏度高但特异度低.A和C厂家的EBNA1IgG试剂盒的灵敏度高（100.0％,97.0％）但特异度低（3.3％,13.3％）.C厂家EA IgG试剂盒检测所有样本结果均为阴性.结论 五个不同厂家VCA LgA、EA IgA试剂盒诊断鼻咽癌的准确性和检测结果的一致性存在差异,特别是A厂家和其他国内厂家同品种试剂间差异明显,需根据临床目的进行选择.三家国产VCA IgA试剂盒的灵敏度需进一步提高.相反,EBNA IgA试剂盒诊断鼻咽癌的准确性和结果一致性较好.单独使用VCA IgG和EBNA1 IgG试剂盒血清学诊断鼻咽癌的特异度差,其判读界值可能需根据检测且的进行调整.     

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.     

IgA antibodies against the Epstein‐Barr nuclear antigen1 as a valuable biomarker for the diagnosis of nasopharyngeal carcinoma in Tunisian patients

Serological tests for Epstein‐Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein‐Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)‐p138 and IgG EA‐p138 antibodies represent the most specific anti‐EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme‐linked immunosorbent assay (ELISA) was established using a GST‐EBNA1 protein expressed in bacteria, containing the P‐threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients. J. Med. Virol. 81:1412–1421, 2009. © 2009 Wiley‐Liss, Inc.     

Serum antibody response to lytic and latent Epstein-Barr virus antigens in undifferentiated nasopharyngeal carcinoma patients from an area of nonendemicity

Epstein-Barr virus (EBV)-associated undifferentiated carcinoma of the nasopharyngeal type (UCNT) is highly prevalent in southeast China, where immunoglobulin A (IgA) antibodies to viral capsid antigen and early antigen (EA) represent important markers, routinely used to assist in diagnosing this malignancy. Our study aimed at determining the EBV serological profiles of 78 UCNT patients from Italy, an area of nonendemicity for this tumor, using different assays specific for both lytic and latent EBV antigens. Serum IgA against both EA and EBNA1 and IgG and IgA to the latent membrane protein 1 (LMP1), to EA, and to the EBV transactivator ZEBRA protein were assessed. These serological responses were then evaluated according to the clinicopathologic parameters at diagnosis. The sensitivities of the IgG assays were 37.7% for LMP1, 73.6% for EA, and 61.0% for ZEBRA. EA/EBNA1 IgA reactivity was 84.4%, and a high association (odds ratio [OR], 2.6; 95% confidence interval [CI], 1.7 to 4.0) with UCNT was observed. When EBV serological reactivities were analyzed according to the tumor, node, and metastasis staging system (TNM), a statistically significant association was found between N stage and IgG antibody rates for EA (OR, 3.6; 95% CI, 1.2 to 10.9) and ZEBRA (OR, 2.6; 95% CI, 1.2 to 5.5) and between M stage and IgG antibody rates for ZEBRA (OR, 7.1; 95% CI, 3.2 to 16.0) and LMP1 (OR, 14.0; 95% CI, 1.8 to 110.9). Our results show that no single serological marker allows the detection of all UCNT cases. EA/EBNA1 IgA represents a reliable marker for diagnosis, with a high predictive value also in areas where UCNT is not endemic, such as Italy. The analysis of serological results according to TNM classification is consistent with a progressive impairment of humoral immune response to EBV as the disease advances and may be used to improve the accuracy of diagnosis.     

Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors.

The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine-alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex.     

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.     

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.     

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.     

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.     

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.     

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.