抗重组EB病毒抗原双重抗体检测血清学诊断鼻咽癌的研究

背景与目的：在评估4种EB病毒抗原酶联免疫吸附法的基础上,探讨优化抗重组：EB病毒抗原双重抗体检测应用于血清学诊断鼻咽癌。方法：收集广州地区57例治疗前鼻咽癌患者和58例健康成人的血清。应用：EB病毒特异抗原(谷胱甘肽转移酶重组融合蛋白)为基础的4种免疫酶联吸附法,即：EBNA1-IgA,EBNA1-Igg,Zta-IgA和Zta-IgG检测血清中抗EB病毒的抗体水平。结果：EBNA1-IgA的灵敏度(O．9123)和阴性预测值(0．9074)是单独使用4种ELISA实验中最高的。Zta-IgA具有最高的正确率(π,0．8870)和Youden指数(J,0．7738)。当评估配对的ELISA时,EBNA1-IgA和Zta-IgA双重阳性的所有指标是4种双重阳性实验中最高的。5例：EBNA1-IgA阴性的鼻咽癌患者呈Zta-IgA阳性,而7例Zta-IgA阴性的鼻咽癌患者呈EBNA1-IgA阳性。结论：EBNA1-IgA酶联免疫吸附的单独检测在血清学诊断鼻咽癌时优于其他3项(EBNA1-IgG、Zta-IgA和Zta-IgG)单独酶联免疫吸附检测。EBNA1-IgA和Zta-IgA两项的组合应用在血清学诊断鼻咽癌时有互补作用,是血清学检测的合适组合。     

血清EB病毒抗体EBNA1 IgA检测临界值探讨

目的:通过对健康人群与鼻咽癌患者EB病毒抗体EBNA1 IgA水平的分析,探讨在鼻咽癌高发区应用该抗体作为鼻咽癌血清学指标时临界值的确定.方法:采用ELISA法检测780例健康人和104例鼻咽癌患者血清EB病毒EBNA1 IgA,根据灵敏度和特异度曲线分别选择灵敏度、特异度在95%所对应的rOD值作为阴性临界值和阳性临界值,根据该临界值将人群划分成3个不同等级,rOD≥1.85为阳性,1.85＞rOD≥1.10为可疑阳性,rOD＜1.10为阴性.结果:健康人群EBNA1 IgA的均值是0.850±0.637,鼻咽癌患者为2.241±0.875.健康人群中EBNA1 IgA阴性、可疑阳性和阳性人群分别占75.13%、17.44%和7.44%,而鼻咽癌患者则分别是4.81%,17.31%,77.88%.结论:鼻咽癌高发区人群血清EBNA1 IgA的rOD值离散程度较大.以EBNA1 IgA作为鼻咽癌血清学诊断指标,可根据灵敏度和特异度确定阳性、可疑阳性和阴性3个人群,以利于临床作出对鼻咽癌的辅助诊断.     

联合检测EBNA1-IgA和EA-IgG在鼻咽癌血清学诊断中的价值

目的　探讨联合检测EBNA1 IgA和EA IgG在鼻咽癌血清学诊断中的价值。方法　收集 5 6例未经治疗的鼻咽癌患者和 5 8例健康成年人血清 ,用酶联免疫吸附法 (ELISA)检测EBNA1 IgA和EA IgG ,比较其单独或联合检测的灵敏度、特异度、阳性预测值、正确率和优势比。结果　EBNA1 IgA的灵敏度 (91.0 7% )高于EA IgG(87.5 0 % ) ,EA IgG的特异度 (87.93% )高于EBNA1 IgA (84 .4 8% )。二者联合检测的特异度为 94 .83% ,阳性预测值为 0 .9375 ,阳性似然比为 15 .5 4 35 ,优势比为 75 .0 0 0 0。4 5例EBNA1 IgA阳性的鼻咽癌患者与EA IgG阳性结果相吻合 ;5例EBNA1 IgA阴性的鼻咽癌患者中 ,有 4例EA IgG阳性 ;7例EA IgG阴性的鼻咽癌患者中 ,有 6例EBNA1 IgA阳性。结论　EBNA1 IgA和EA IgG在血清学检测中有较高的灵敏度和特异度 ,二者联合检测有互补作用 ,可提高鼻咽癌血清学诊断的可靠性。大多数鼻咽癌患者为EBNA1 IgA和EA IgG双阳性     

以双合成肽为抗原建立的ELISA试验在鼻咽癌血清学诊断中的应用价值

目的研究EBNA1和VCA-P18双多肽包被（EBNA1＋VCA-P18）的ELISA检测在鼻咽癌（NPC）血清学诊断中的价值。方法用合成肽EBNA1和VCA-P18建立ELISA法,检测37例病理确诊的鼻咽癌患者血清和33例健康人血清,并与国产EBVVCA/IgAELISA试剂盒比较。结果双多肽EBNA1＋VCA-P18/IgA灵敏度为91.9%,明显高于国产试剂盒的83.8%;二者特异度相同;EBNA1＋VCA-P18/IgA的阳性预测值、阴性预测值、阳性似然比及优势比均高于国产试剂盒。结论双多肽EBNA1＋VCA-P18/IgA相比商品化试剂盒在鼻咽癌血清学检测中有更高的灵敏度,适用于鼻咽癌早期诊断和人群筛查。     

EBVDNA和EBV抗体指标早期诊断鼻咽癌方案的筛选

目的:探讨EB病毒DNA及EB病毒抗体在鼻咽癌联合诊断中的应用价值.方法:对81例未经治疗的病理确诊鼻咽癌怠者和89例健康体检者分别采用实时荧光定量PCR测定EB病毒DNA,计算病毒拷贝数,采用间接免疫酶法测定EBV VCA/IgA和EMIgA,采用酶联吸附试验(ELISA法)检测EBNA1/IgG、EBNA1/IgA以及ZTA/IgG.通过ROC曲线评价各指标单独和联合诊断鼻咽癌的效果.结果:分析发现VCA/IgA、EBV DNA和EBNA1/IgA 3个指标具有较高的灵敏度,分别为95.06%、83.95%和81.63%.而EA/IgA、EBNA1/IgG和ZTA/IgG则具有较高的特异度,分别为94.38%、95.51%和89.89%.EBNA1/IgA与VCM/IgA并联时灵敏度为100%,特异度为84.27%.结论:EB-NA1/IgA与VCA/IgA并联时灵敏度和特异度都在比较高的水平,可作为鼻咽癌早期筛查的选择方案.     

以双合成肽为抗原建立的ELISA试验在鼻咽癌血清学诊断中的应用价值

目的研究EBNA1和VCA-P18双多肽包被(EBNA1+VCA-P18)的ELISA检测在鼻咽癌(NPC)血清学诊断中的价值。方法用合成肽EBNA1和VCA-P18建立ELISA法,检测37例病理确诊的鼻咽癌患者血清和33例健康人血清,并与国产EBVVCA/IgAELISA试剂盒比较。结果双多肽EBNA1+VCA-P18/IgA灵敏度为91.9%,明显高于国产试剂盒的83.8%;二者特异度相同;EBNA1+VCA-P18/IgA的阳性预测值、阴性预测值、阳性似然比及优势比均高于国产试剂盒。结论双多肽EBNA1+VCA-P18/IgA相比商品化试剂盒在鼻咽癌血清学检测中有更高的灵敏度,适用于鼻咽癌早期诊断和人群筛查。     

以血清EB病毒抗体谱评估患鼻咽癌危险度的研究

目的：比较鼻咽癌患者与健康人群血清EB病毒抗体水平,评估患鼻咽癌的危险度。方法：收集珠江口地区121例鼻咽癌患者和332例健康人血清。采用酶联吸附试验（ELISA）检测血清中EBNA 1/IgA、EBNA 1／IgG和zta/IgG,以免疫酶法或免疫荧光法检测VCA／IgA。结果：EBNA 1／IgA、EBNA 1／IgG和zta/IgG的敏感度分别为85％、83％和79％;三者组合的敏感度最高,为92％。EBNA 1/IgA、EBNA 1／IgG和zta/IgG的特异度分别为86％、86％和80％;三者组合的特异度最高,为93％。根据优势比水平,可将患鼻咽癌的危险度分为低危险、中危险和高危险3个层次。93％的健康人是低危险的,优势比为0．0－0．3;0．4％的健康人是高危险的,优势比为137．9。结论：ELISA在诊断鼻咽癌的结果判断上更具客观性,较传统免疫酶法好;EBNA 1／IgA、EBNA 1／IgG和zta/IgG的联合应用可以评估人群患鼻咽癌的危险度。     

Logistic回归模型在早期鼻咽癌多指标联合辅助诊断实验的应用

背景与目的:目前用于鼻咽癌辅助诊断的指标较多,但大部分早期诊断的效果较差.如何联合多个指标进行辅助诊断是近年研究的重点.本研究旨在评价鼻咽癌常用诊断指标VCA/IgA、EA/IgA、EBV DNA、EBNA1/IgA、EBNAl/IgG和ZTA/IgG的诊断价值.并采用logistic回归筛选优化的联合诊断组合.方法:对8l例未经治疗的病理确诊鼻咽癌患者和89例健康体检者分别采用实时荧光定量PCR法测定EB病毒DNA,计算病毒拷贝数,采用间接免疫酶法测定EBV、VCA/IgA和EA/IgA,采用酶联吸附试验(ELISA法)检测EBNA1/IgG、EBNA1/IgA以及ZTA/IgG.通过ROC曲线评价各指标单独和联合诊断鼻咽癌的效果.用logistic回归综合两指标信息,根据ROC曲线确定诊断界值,以增加鼻咽癌诊断的准确性.结果:logistic回归方法的诊断敏感度和特异度比常规的并联实验均有提高.筛选的优化组合方案为EBV DNA+EBNA1/IgA和VCA/IgA+EBNAI/IgA,两组合的并联试验敏感度和特异度分别为0.96、0.82和1.00、0.84,而logistic回归预测概率为诊断指标.ROC曲线确定界值时.两组合的灵敏度和特异度分别为1.00、0.87和0.98、0.88.结论:多指标联合建立logistic回归模型,利用预测概率进行辅助诊断能够达到同时提高灵敏度和特异度的效果,可供临床参考.     

鼻咽癌高发区健康成人六项EB病毒抗体检测结果的分析

背景与目的：鼻咽癌有明显地域倾向性，而鼻咽癌发病又与EBV密切相关。本研究通过检测来自不同地域的人群抗EBV抗体的水平和阳性率．以探讨EBV感染与鼻咽癌的地域倾向性之间的关系。方法：从鼻咽癌高发区广东省中山市的健康成人中收集303例中山原住居民的血清和92例来自非NPC高发区的外省移民的血清，用ELISA法检测血清中的EBNA1-IgA、EBNA1-IgG、VCA-p18-IgA、VCA-p18-IgG、Zta—IgA和Zta—IgG等六种抗体水平，以校正相对吸光度（ArA）值表示，逐一比较两个人群样本六项抗EBV抗体水平及阳性率的差别。结果：中山原住居民的Zta—IgA（0．84±0．03）和VCA—p18-IgA的ArA（0．96±0．05）明显高于外省移民（0．42±0．04和0．40±0．05，P〈0．05）。另外，中山原住居民30-年龄段组和50～年龄段组的Zta—IgA阳性率分别是29．27％和48．28％，均明显高于外省移民同一年龄段组的3．03％和6．67％（P〈0．05）。中山原住居民30～年龄段组和50-年龄段组的VCA—p18．IgA分别是28．46％和43．10％，均明显高于外省移民同一年龄段组的9．09％和13．33％（P〈0．05）。结论：中山原住居民针对EBV溶解期的抗体Zta-IgA和VCA—p18-IgA的水平和阳性率偏高．反映其感染的EBV更多地处于再激活状态，这提示NPC高发区中山的原住居民罹患鼻咽癌的风险比来自非NPC高发区的外省移民更高。     

EBV 抗体和EBV-DNA 在鼻咽癌诊断及分期的研究*

目的:评估EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA对不同分期鼻咽癌的诊断效能,探讨各指标阳性率与鼻咽癌分期的关系。方法:收集2010年3 月至2015年9 月中山大学附属中山医院收治的初诊鼻咽癌患者152 例,健康体检者675 例。采用酶联免疫吸附法（ELISA）检测血清EBNA1/IgA 、Zta/IgA 和VCA/IgA 抗体ROD 值,荧光定量PCR （fluorescence quantitative PCR,FQ-PCR ）检测血浆EBV-DNA水平。比较单独和联合应用EBV 标记物对各期鼻咽癌的诊断效能,同时分析各指标阳性率与鼻咽癌分期的关系。结果:鼻咽癌患者EBNA1/IgA 、Zta/IgA 、VCA/IgA 和EBV-DNA阳性率显著高于健康体检者（P < 0.01）。 EBNA1/IgA 在早期鼻咽癌表达相对较高,灵敏度为77.8% ,而EBV-DNA在晚期鼻咽癌的灵敏度最高为88.8% ,两者特异度均在96% 以上。联合检测中EBNA1/IgA 并联EBV-DNA检测的灵敏度为92.1%（早期为82.5% 、晚期为98.9%）,特异度为96.9% 。EBV-DNA阳性率与鼻咽癌临床分期和N 分期呈正相关,Zta/IgA 阳性率与N 分期呈正相关（P < 0.01）。 结论:在无症状人群中进行鼻咽癌筛查,单项指标首选EBNA1/IgA 。晚期患者的辅助诊断则推荐EBV-DNA。两者并联检测可进一步提高鼻咽癌诊断效能。EBV-DNA是鼻咽癌分期和病情监测的重要指标,Zta/IgA 可间接反映淋巴结转移情况,有望对患者病情评估起到参考作用。      

EVALUATION OF COMBINED EBNA1—IgA AND EA—IgG ASSAYS IN SEROLOGICAL DIAGNOSIS OF NASOPHARYNGEAL CARCINOMA

To evaluate the value of combined assays of serum EBNA1-IgA and EA-IgG for serological diagnosis of nasopharyngeal carcinoma(NPC).Methods:The serum EBNA1-IgA and EA-IgG were tested by use of ELISA for 56 patients with NPC and 58 healthy adults.The sensitivity,specificity,positive predictive value,accuracy rate and odds ratio of the 2 tests used singly or in combination were compared with each other.Results:The sensitivity of EBNA1-IgA test(91.09%)was higher than that (87.50%)of EA-IgG test.The specificity of EA-IgG test(87.93%)was higher than that (84.48%) of EBNA1-IgA test.The combined usage of EBNA1-IgA and EA-IgG could enhance the specificity(94.83%),predictive value of a positive test(0.9375),likelihood ratio of a positive test(15.5435) and odds ratio(75.0)for serological diagnosis of NPC.Forty-five NPC patients showed positivity to EBNA1-IgA and EA-IgG concurrently.A positive EA-IgG reaction was demonstrated in 4 out of 5 NPC patients with negative EBNA1-IgA reaction in 6 out of 7 NPC patients with negative EA-IgG result as well.Conclusion:Though high sensitivity and specificity could be obtained by EBNA1-IgA and EA-IgG test,respectively,the combined use of these 2 tests is able to enhancing the reliability of serological diagnosis of NPC.The majority of NPC patients showed positivity to ENBA1-IgA and EA-IgG concurrently.There is a complementary effect through using EBNA1-IgA and EA-IgG for serological diagnosis of NPC.     

Establishment of VCA and EBNA1 IgA-based combination by enzyme-linked immunosorbent assay as preferred screening method for nasopharyngeal carcinoma: a two-stage design with a preliminary performance study and a mass screening in southern China

A two-stage study was conducted in southern China to determine and validate an optimal combination of Epstein-Barr virus (EBV)-related seromarkers for nasopharyngeal carcinoma (NPC) screening. In the first stage, six seromarkers [VCA-IgA, EA-IgA, Epstein-Barr virus nuclear antigen 1 (EBNA1-IgA), EBNA1-IgG, Zta-IgA and Rta-IgG] were detected by enzyme-linked immunosorbent assay (ELISA) and two traditional NPC screening seromarkers (VCA-IgA and EA-IgA) were detected by immunofluorescence assay (IFA) in serum samples from 191 NPC patients and 337 controls. An optimal combination of seromarkers for NPC diagnosis was selected using logistic regression models. Results showed that the diagnostic performances of VCA-IgA and EA-IgA tested by ELISA were superior to the performances of the same seromarkers by IFA. VCA-IgA combined with EBNA1-IgA by ELISA was identified as the optimal combination, with an area under the receiver operating characteristic (ROC) curve (AUC) up to 0.97, a sensitivity of 95.3% and a specificity of 94.1% for classification of NPCs vs. controls. In the second stage, 5,481 participants aged 30-59 years and without clinical evidence of NPC were recruited into a population-based NPC screening program from May 2008 to February 2009 in Sihui City, China. Their sera were tested simultaneously by both the new and the traditional screening schemes and eight early stage NPC patients were subsequently histopathologically confirmed. The traditional and the new screening schemes had comparable specificity (estimated as 98.5%), but the sensitivity of the new scheme (75.0%) was significantly higher than that of the traditional one (25.0%). The combination of VCA-IgA and EBNA1-IgA by ELISA outperforms the traditional NPC screening scheme and could become the preferred serodiagnostic strategy for NPC screening in high-incidence areas.     

Titers of Epstein-Barr virus-related antibodies in nasopharyngeal carcinoma in Japan

It is thought that nonkeratinizing or undifferentiated squamous cell carcinoma in the nasopharynx (NPC) is intimately correlated with Epstein-Barr Virus (EBV). Twenty-one patients with NPC were followed in Kyoto University Hospital and 4 in Osaka Red Cross Hospital during the past 2 years from 1980 to 1981. These patients were classified histopathologically according to the WHO classification in 1978 and staged with the TNM classification in Union Internationale Contre le Cancer (UICC) in 1978. The incidence rate of NPC among the head and neck tumors was 5.6% in the authors' university from 1980 to 1981. The sex ratio of male to female was nearly equal. The mean age of NPC patients was 56.7 years. Sera from these 25 patients with nasopharyngeal carcinoma were collected at intervals of 3 to 8 months over a 2-year period, and were examined for their spectra and titers of antibodies of EBV-related antigens. They were titrated for IgG, IgA and IgM antibodies to EB viral capsid antigen (VCA), for IgG and IgA antibodies to early antigen-DR component (EA) and for antibodies to EBV-associated nuclear antigen (EBNA). All of these patients were primarily treated with radiation, while a few who did not respond to this therapy were subsequently treated with surgery or chemotherapy. EBV antibodies of VCA-IgG, -IgA, EA(DR)-IgG, and -IgA and EBNA were elevated in 73% and 90% of the nonkeratinizing and undifferentiated NPC patients, respectively. The VCA-IgM was elevated in almost none of the cases. In contrast to this, these values were all in a normal range in the NPC patients with keratinizing squamous cell carcinoma and malignant lymphoma. Also 9% and 10% of nonkeratinizing and undifferentiated carcinomas showed the normal ranges of EBV antibodies, possibly indicating a nonassociation with EBV. When NPC disappeared with radiation therapy, EBV antibodies became normal for 6-18 months. However, those whose NPC did not respond to the combined therapy with radiation, surgery and chemotherapy maintained high titers of EBV antibodies. The prognosis was the poorest in the patients with undifferentiated carcinoma, 40% of whom died within 4 years after diagnosis.     

Assessing the risk of nasopharyngeal carcinoma on the basis of EBV antibody spectrum.

We have evaluated the performance of 3 new EBV ELISA for the diagnosis of nasopharyngeal carcinoma (NPC). The tests were specific for EBNA 1 IgA, EBNA 1 IgG and zta IgG, respectively. Their distinct antigenic specificity permits these assays to be used in concert in an approach that differentiates patients and apparently healthy subjects on the basis of their antibody spectrum. By so exploiting a distinguishing feature of NPC first described by Lloyd Olds and his group (Olds et al., Proc Nat Acad Sci 1966;56:1699-1704) [corrected] that the patients sustain high levels of a broad spectrum of serum EBV antibodies, this approach achieved a sensitivity of 92% and a specificity of 93%, surpassing the performance of each of these assays individually. The enhanced performance is especially useful in population screening. It was shown that relative risk of NPC sustained by apparently healthy subjects residing in a high incidence area for NPC in the Pearl River estuary in Southern China may vary according to EBV antibody spectrum. The risk of the cancer was markedly reduced with odds ratios of 0.009 for 59% of those who had low level of all 3 antibodies. The risk was increased as antibody spectrum broadens and the risk was the highest with an odds ratio of 138 for 0.4% of those who had high levels of all 3 antibodies. Thus, EBV antibody spectrum may serve to guide follow-up measures for early detection of the cancer and/or risk counseling according to level of the risk of the cancer sustained by the screened individuals.     

Diagnostic Significance of Combined Detection of Epstein-Barr Virus Antibodies,VCA/IgA,EA/IgA,Rta/IgG and EBNA1/IgA for Nasopharyngeal Carcinoma

The objective of this study was to investigate the diagnostic significance of EBV antibody combined detectionfor nasopharyngeal carcinoma (NPC) in a high incidence region of southern China. Two hundred and elevenuntreated NPC patients, 203 non-NPC ENT patients, and 210 healthy controls were recruited for the study. Thetiters of VCA/IgA and EA/IgA were assessed by immunoenzyme assay, and the levels of Rta/IgG and EBNA1/IgAwere determined by enzyme-linked immunosorbent assay. The levels of VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA demonstrated no association with gender or age (p>0.05). The receiver operating characteristic curve andthe area under the curve were used to evaluate the diagnostic value. The sensitivity of VCA/IgA (98.1%) andthe specificity of EA/IgA (98.5%) were the highest. When a logistic regression model was used to combine theresults from multiple antibodies to increase the accuracy, the combination of VCA/IgA+Rta/IgG, whose areaunder the curve was 0.99, had the highest diagnostic efficiency, and its sensitivity, specificity and Youden indexwere 94.8%, 98.0% and 0.93 respectively. The data suggest that the combination of VCA/IgA+Rta/IgG may bemost suitable for NPC serodiagnosis.