Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.     

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.     

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.     

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.     

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.     

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.     

Dried-blood sampling for epstein-barr virus immunoglobulin G (IgG) and IgA serology in nasopharyngeal carcinoma screening

Dried-blood (DB) samples on filter paper are considered clinical specimens for diagnostic use because of the ease of collection, storage, and transport. We recently developed a synthetic-peptide-based immunoglobulin A (IgA) (EBNA1 plus viral capsid antigen [VCA]-p18) enzyme-linked immunosorbent assay (ELISA) for nasopharyngeal carcinoma (NPC) screening. Here, we evaluate the use of two filter papers for DB sampling, i.e., Schleicher & Schuell (S&S) no. 903 and Whatman no. 3; the DB samples were either taken directly from a finger prick or spotted from a Vacutainer blood collector. The elution of DB samples on filter paper was optimized and tested for IgG and IgA reactivity by ELISA (EBNA1 plus VCA-p18) and compared to simultaneously collected plasma samples. The results showed that both types of filter paper can be used for sample collection in NPC diagnosis by using either finger prick or blood spot sampling. Both DB sampling methods produced comparable ELISA (EBNA1 plus VCA-p18) results for IgG and IgA reactivity in 1:100-diluted plasma samples. DB samples of whole blood or finger prick blood show correlation coefficients (r(2)) of 0.825 to 0.954 for IgA on S&S no. 903 filter paper, 0.9133 to 0.946 for IgA on Whatman no. 3 filter paper, 0.807 to 0.886 for IgG on S&S no. 903 filter paper, and 0.819 to 0.934 for IgG on Whatman no. 3 filter paper. Using plasma IgA as a reference, DB sampling showed sensitivities and specificities of 75.0 to 96.0% and 93.5 to 100%, respectively. DB samples could be stored at 37 degrees C for 1 to 4 weeks on S&S no. 903 filter paper and 1 to 6 weeks on Whatman no. 3 filter paper without a significant loss of reactivity, with provision of transport options for tropical conditions. IgA proved to be more stable than IgG. Whatman no. 3 filter paper is a more economical yet diagnostically comparable alternative to S&S no. 903 filter paper. Finger prick DB sampling is proposed for NPC diagnosis, particularly for remote hospitals and field screening studies.     

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.     

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。     

中山市健康成人外周血EB病毒抗体谱的调查研究

目的探讨来自不同地域的人群感染EB病毒的血清学抗体谱是否存在差异。方法从鼻咽癌高发区中山市的健康成人中收集348例中山籍贯原住居民的血清和81例外省籍贯居民的血清,用ELISA法检测血清中的EBNA1-IgA、EBNA1-IgG、VCA-p18-IgA、VCA-p18-IgG、Zta-IgA和Zta-IgG等6种抗体水平,比较不同抗体组合时两类人群样本的抗EB病毒抗体谱的差别。结果当三项IgA类抗体皆为阴性时,外省籍贯阴性率远远大于中山籍贯样本（P=0）;当EBNA1-IgA单阳时,外省籍贯样本阳性率远远高于中山籍贯样本（P=0.001）;当Zta-IgA单项阳性时（P=0.008）,或者Zta-IgA和VCA-p18-IgA同时阳性时（P=0.001）,中山籍贯样本阳性率明显高于外省籍贯样本;而三项IgG类抗体的任意组合,中山籍贯和外省籍贯都无统计学意义。结论中山籍贯原住居民感染的EB病毒常常处于激活状态;相反,外省籍贯的中山居民感染的EB病毒处于潜伏期时表达的EBNA1水平更高。提示中山籍贯原住居民比外省籍贯居民具有更高的风险罹患鼻咽癌。     

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.     

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.     

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.     

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.     

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.     

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。     

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。     

Significance of specific Epstein-Barr virus IgA and elevated IgG antibodies to viral capsid antigens in nasopharyngeal carcinoma patients

The feasibility of using elevated Epstein-Barr virus (EBV) specific-IgG antiviral capsid antigen (VCA) and IgA anti-VCA antibody levels as an aid in diagnosis of nasopharyngeal carcinoma (NPC) was analyzed by determination of serum antibody titers to EBV in 54 NPC patients, 114 healthy blood donors, and 40 family members by the immunoperoxidase assay (IPA). No significant difference was found in the prevalence rate of EBV IgG anti-VCA antibodies (titer greater than or equal to 20) between the patient group and the control and family groups (100% vs 92% and 90%, respectively). The prevalence rate of elevated EBV IgG anti-VCA titers (greater than or equal to 80, greater than or equal to 160, greater than or equal to 320, greater than or equal to 640) was significantly higher in the NPC patients than in controls. For example, at an IgG titer of greater than or equal to 320, the prevalence rate was 82% in the NPC patient group and 1.7% in the controls (P less than 0.0001). The prevalence of EBV IgA anti-VCA antibodies (greater than or equal to 10) was significantly higher in the NPC patients than in control and family groups (82% vs 6.1% and 0%, respectively). The prevalence rate for elevated EBV IgA anti-VCA (greater than or equal to 20) was found to be significantly higher (P less than 0.0001) in NPC patients than in the control group (70% vs. 1.7%). A significantly high proportion (P = 0.0004) of NPC patients who had serum EBV IgA anti-VCA titers of less than 20 had elevated IgG titers to VCA greater than or equal to 320 (21% vs 1.7% among controls). It appears that testing for IgG antibodies at a serum dilution of 1:320 and for IgA antibodies at a dilution of 1:20 by the IPA technique comprises the best combination for the differentiation between NPC patients and health controls (91% vs 3.4%), and it is suggested that these be used as screening markers for NPC patients.     

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.     

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.