基因工程表达的EB病毒抗原在鼻咽癌及高危人群诊断中的应用

196 7年Ｈｅｎｌｅ证明在鼻咽癌病人的血清中存在ＥＢ病毒特异性的ＩｇＡ抗体 ,近 30年来 ,检测ＥＢ病毒壳抗原的ＩｇＡ抗体是鼻咽癌血清学诊断和高危人群筛查的主要方法 ,广泛地用于临床诊断和前瞻性血清流行病学研究 ,在早期发现、早期治疗、提高生存率上起了重要作用( 2 ) 。但是 ,至今仍使用Ｂ95 8细胞涂片 ,以间接免疫荧光或免疫酶方法检查血清中的抗体。检测方法繁琐 ,细胞中ＥＢ病毒其他相关抗原的干扰 ,影响检测的特异性和敏感性。Ｌｕｋａ等人1984年建立了ＥＬＩＳＡ检测血清中抗体 ,其敏感性较细胞涂片检测方法提高 10 0～ 10 0 …     

ELISA法测定EB病毒IgG/EA抗体在血清学诊断鼻咽癌中的意义

近年研究表明 ,鼻咽癌 (ＮＰＣ)的发展与ＥＢ病毒感染密切相关[1] 。本室应用酶联免疫吸附法 (ＥＬＩＳＡ)测定血清抗ＥＢ病毒早期抗原 (ＥＡ)的ＩｇＧ抗体 (ＩｇＧ ＥＡ)水平 ,与间接免疫酶染色法测定ＥＢ病毒壳抗原(ＶＣＡ)ＩｇＡ抗体进行比较 ,研究结果报道如下 :1　材料与方法1 1　血清样品来源　 32 6例 2 7～ 6 0岁健康人为我院体检者 ,其中男性 2 33人 ,女性 93人。 134例为入院初诊鼻咽癌患者 ,年龄 31～ 5 5岁 ,其中男性 10 4人 ,女性 30人。1 2　血清ＩｇＡ ＶＣＡ滴度测定　采用上海生物制品研究所的ＩｇＡ ＶＣＡ免疫酶染色法试…     

鼻咽癌组织EB病毒膜抗原和EB病毒核酸观察

目的 观察分析EB病毒膜抗原和EB病毒核酸的存在及其在鼻咽癌诊断中的作用。方法 应用抗EB病毒膜抗原（EBV／MA）的单克隆抗体检测51例病理确诊的鼻咽癌（NPC）活检标本的冰冻切片,以Southern杂交法检查20例鼻咽癌活检组织中EB病毒核酸（EBV／DNA）。结果 其中49例有EBV／MA的表达,经细胞形态学分析首次发现鼻咽癌细胞,增生上皮细胞以及正常鼻咽部上皮细胞都有BV／MA。而12例慢性炎症病例仅1例阳性,20例低分化鼻咽癌中17例存在与细胞转化有关的K片段。证实鼻咽癌组织中不仅有EB病毒核酸的重复序列W片段,还有与细胞转化有关的K片段的序列存在。结论 进一步表明EB病毒在鼻咽癌的发展中并非“过客”,而可能是其病原重要因素之一。     

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.     

EB病毒衣壳抗原IgA抗体胶体金快速检测法的建立及应用

目的建立了一种快速检测人血清中EB病毒衣壳抗原(VCA)IgA抗体的胶体金免疫层析(GICA)方法。方法预先将EB病毒衣壳抗原(EB-VCA)包被在硝酸纤维素膜上,将鼠抗人IgA抗体免疫金固化在金标垫上。检测时,样本通过层析作用向上流动,样本中的IgA抗体首先被胶体金标记的鼠抗人IgA抗体捕获,其中的EB-VCAIgA抗体再与包被在硝酸纤维素膜上的EB-VCA特异性结合,最后形成包被固相上的EB-VCA与EB-VCAIgA抗体及胶体金标记鼠抗人IgA抗体的三元复合物,通过检测线胶体金沉淀颜色的变化来定性判定样本中是否存在EB-VCAIgA抗体。结果采用自制的GICA试纸条进行了1028份临床标本的盲法检测,并与酶联免疫吸附试验的EB-VCAIgA抗体诊断试剂盒进行比对,结果表明两种试剂检测的总符合率达到94.56%,阳性符合率为93.40%,阴性符合率为95.17%,Kappa值为0.873,两种诊断试剂具备一致的诊断价值。结论用于检测血清中的EB-VCAIgA抗体的GICA试纸条的制备条件已基本建立,其检测结果特异性强、稳定性好、操作方法简便快捷且无需特殊仪器设备。     

两阶段EB病毒酶联免疫吸附法筛查鼻咽癌的探讨

目的：采用两阶段EB病毒血清学筛查方法，进行人群鼻咽癌的筛查。方法：用ELISA检测血清EB病毒抗体，首先以EBNAlIgA作为人群鼻咽癌的初筛指标，第二阶段的检查是在EBNAlIgA阳性人群中再进一步检测EBNAlIgG和ZtaIgG两种抗体。结果：EBNAlIgA的灵敏度和特异度分别为91．9％和91．4％，均高于EBNAlIgG和ZtaIgG；第二阶段检查可以将鼻咽癌筛查的特异度提高到96．5％，同时可将人群划分高、中、低三个不同危险层次，其中高危险人群仅占0．39％。结论：实行两阶段筛查法，使鼻咽癌筛查既能达到良好的灵敏度，又能提高检测的特异性，同时还可以将筛查人群划分不同的危险层次。     

用EBER-1作探针原位杂交检测鼻咽癌组织中的EB病毒

ｐｓｔｅｉｎＢａｒ病毒（ＥＢＶ）与鼻咽癌（ｎａｓｏｐｈａｒｙｎｇｅａｌｃａｒｃｉｎｏｍａ,ＮＰＣ）有非常密切的关系［１］。各家检测ＥＢＶＤＮＡ的方法均不完全相同,灵敏度也存在差异［１４］。原位杂交是比较传统的方法,但用ＥＢＶＤＮＡ片段作探针原...     

EB病毒抗体与DNA载量联合检测在儿童传染性单核细胞增多症中的诊断价值

目的 探讨EB病毒IgG、IgA和IgM抗体及DNA载量联合检测在儿童传染性单核细胞增多症中的诊断价值.方法 收集2012年1月至2015年3月间我院收治的170例儿童传染性单核细胞增多症患者作为观察组进行回顾性分析,另取130例健康体检儿童作为对照组,对其EB病毒抗体检测情况和DNA载量检测情况进行观察对比.结果 观察组患者EB-DNA阳性率及载量,VCA-IgG及VCA-IgM阳性率均明显高于对照组,差异有统计学意义(P＜0.05);两组儿童VCA-IgA阳性率比较差异无统计学意义(P＞0.05).3～6岁患儿人数最多,达到全部患儿的49.41％,且随年龄增长,EB-DNA阳性率及载量、VCA-IgG阳性率均有明显增高趋势,差异有统计学意义(P＜0.05),而VCA-IgA及VCA-IgM则无明显差异(P＞0.05).各单项检测中,VCA-IgM具有最佳的诊断价值,与其他指标相比差异有统计学意义(P＜0.05).采用VCA-IgM联合EB-DNA检测可有效提高灵敏度、特异度、准确度、阳性预测值及阴性预测值等各项指标,差异有统计学意义(P＜0.05).结论 EB病毒VCA-IgG、VCA-IgA和VCA-IgM三种抗体在对儿童传染性单核细胞增多症的诊断中,以IgM具有最佳的敏感度、特异度及阳性预测值,采用VCA-IgM联合EB病毒DNA载量检测对于儿童传染性单核细胞增多症有良好的诊断价值,值得临床推广应用.     

Eostein—Barr病毒IgA／VCA抗体变动规律和鼻咽癌发…

对苍梧县Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒抗体阳性的９３１人每年进行观察，连续１０年追踪发现他们中２９４人抗体转阴，６６人抗体波动，１２０人抗体下降，这些人中未出现鼻咽癌病人。在３７５例抗体无明显改变者中出现６例病人，检出率为１．６％，在抗体上升的７６人中出现了１７例病人，检出北为２２．４％，抗体持续阳性特别是滴度升高者是鼻咽癌的高危险人群。     

血浆EBV DNA检测对鼻咽癌的诊断作用

目的探讨血浆EBV DNA检测对鼻咽癌的诊断作用。方法将2006年12月至2007年3月在我科就诊、病理确诊的65例鼻咽癌作为治疗前组,经我院放疗科治疗后,分别于放疗结束后3、5、8个月来我科系统复查的病人作为治疗后组,将同期行健康体检的29例作为对照组,共3组。采用荧光定量PCR方法对3组的空腹血浆EBV DNA进行检测。结果3组间血浆EBV DNA阳性率比较有统计学意义（P〈0．05）;治疗后组的阳性病例全部有鼻咽癌复发,阴性病例没有复发;有、无淋巴结转移组间阳性率比较差异有统计学意义（P=0．00）。结论血浆EBV DNA是鼻咽癌诊断的重要分子标记,其阳性的病例有较高淋巴结侵袭发生率。     

Epstein-Barr virus serology in bone marrow transplantations: a one-year retrospective study with detection of EBV IgM-VCA-specific antibodies

The specific antibody response to Epstein-Barr virus (EBV) antigens of 41 bone marrow transplant recipients with leukemia or aplastic anemia was examined retrospectively by immunofluorescence test (IF) over 1 year. We observed high titers (greater than 640) of IgG-viral capsid antigen (VCA) with emergence of IgG-early antigen (EA) and frequent absence or low levels of Epstein-Barr nuclear antigen (EBNA) antibodies. After absorption to remove rheumatoid factor (RF), five of the 41 recipients had IgM-VCA antibody to EBV, which appeared between weeks 26 and 48 after BMT and persisted for 1-4 months. No heterophil antibodies were detected in these sera, and none of the five recipients had a history of infectious mononucleosis.     

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.     

Seroreactivity against Epstein-Barr virus (EBV) among first-degree relatives of sporadic EBV-associated nasopharyngeal carcinoma in Indonesia

Epstein–Barr virus (EBV) infection and family history are significant risk factors associated with undifferentiated nasopharyngeal carcinoma. The presence of aberrant immunoglobulin A (IgA) antibodies against specific EBV antigens in healthy individuals can be predictive of the disease. Very limited reports explored the EBV IgA antibody presence within families of sporadic cases of nasopharyngeal carcinoma. This study aimed to determine whether EBV IgA was observed more frequently among family members of sporadic cases of nasopharyngeal carcinoma compared to community controls and evaluated the non‐viral factors as determinants of antibody level. First‐degree relatives of nasopharyngeal carcinoma patients (n = 520) and case‐matched community controls (n = 86) were recruited. Sera from all individuals were tested in standardized peptide‐based EBV IgA ELISA. Data on demographic variables and other exogenous factors were collected using a questionnaire through face‐to‐face interviews. A similar frequency of EBV IgA (cut‐off value/CoV 0.354) was observed in the first‐degree relatives of cases and in community controls (41.2% vs. 39.5%, P = 0.770). However, with a higher antibody level (OD₄₅₀ = 1.000; about three times standard CoV), the relatives showed significantly higher frequency (36.9% vs. 14.7%, P = 0.011). When adjusted for all exogenous factors, the strongest factors associated with seropositivity are being a father (odds ratio/OR = 4.36; 95% confidence interval/CI = 1.56–12.21) or a sibling (OR = 1.89; 95% CI = 1.06–3.38) of a case of nasopharyngeal carcinoma. The higher level of EBV IgA seroreactivity in first‐degree relatives of sporadic cases of nasopharyngeal carcinoma compared to the general population supports the use of EBV IgA ELISA for screening among family members. J. Med. Virol. 84:768–776, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Expression of the Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in EBV-associated nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) is associated with virtually all cases of undifferentiated nasopharyngeal carcinoma (NPC) and has been classified as a group I carcinogen. In addition to its potential role in the pathogenesis of NPC, EBV also provides a possible target for immunotherapy of NPC, since a limited number of viral genes are expressed in the neoplastic cells. The EBV-encoded latent membrane protein 2A (LMP2A) is considered a promising target since it provides epitopes recognized by EBV-specific T-cells. Using immunohistochemistry, the present study shows that LMP2A is expressed at the protein level in the neoplastic cells of 16 of 35 (45.7%) NPC biopsies. This finding provides further evidence suggesting that NPC tumour cells may be susceptible to lysis by cytotoxic T-cells directed against LMP2A and should encourage efforts to develop immunotherapeutic approaches for the treatment of NPC.     

Upregulation of LMP1 Expression by Histone Deacetylase Inhibitors in an EBV Carrying NPC Cell Line

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.