Expression of the Epstein-Barr virus DNA polymerase in Escherichia coli for use as antigen for the diagnosis of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) encoded DNA polymerase (POL) was cloned and over-expressed in Escherichia coli. Western blot analysis confirmed the presence of antibody to this POL protein in sera from nasopharyngeal carcinoma (NPC) patients. By Western blot analysis, moderate to high concentration of IgG POL-specific antibodies were present in 43 of 48 NPC sera and only 4 of 48 healthy, seropositive controls. The POL-specific IgG antibodies appear as early as stage I of NPC, suggesting that the recombinant POL protein can be a useful diagnostic marker for early diagnosis of the disease. It was also found that human sera containing high titer of cytomegalovirus (CMV) antibodies or herpes simplex virus type 1 (HSV-1) antibodies did not cross-react with the recombinant EBV POL, despite the homology shared by DNA polymerase proteins of these viruses. © 1995 Wiiey-Liss, inc.     

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.     

Antibody to Epstein-Barr virus-specific DNase as a marker for the early detection of nasopharyngeal carcinoma

We have previously shown that high levels of antibody to Epstein-Barr virus (EBV)-specific DNase may be a useful marker for the early diagnosis of nasopharyngeal carcinoma (NPC). Sera from 3,368 males in an area with a high risk for the development of NPC were examined for the presence of antibody to EBV-specific DNase activity. Significant levels of the antibody were found in 430 of these sera. As a result, 32 individuals have attended the out-patient department of otolaryngology for clinical examination. Among them, one individual was found to have a stage II nasopharyngeal carcinoma. This result supports the value of determination of antibody to EBV-specific DNase as a marker for the early detection of NPC.     

Antibody to Epstein-Barr virus-specific DNase as a marker for field survey of patients with nasopharyngeal carcinoma in Taiwan

A serological survey using antibody to Epstein-Barr virus (EBV)-specific DNase activity as a marker for the identification of patients with nasopharyngeal carcinoma (NPC) has been carried out on healthy subjects who visited Government Employees' Clinic Center (GECC) for routine health examination and on individuals residing in NPC high-risk areas (HRA) in Taiwan. During a 3-year prospective study, 22,596 and 9,869 sera were collected from the GECC and HRA groups, respectively. Taking neutralization of 2 or more units of EBV DNase activity as a positive response, the positivity rates in the GECC and HRA groups were 5.4% and 11.92%, respectively. Among the antibody-positive individuals, three cases of NPC were found in the GECC group (detection rate 0.63%) and 11 in the HRA group (detection rate 1.32%). A further patient at stage III of the disease was found in the first year of following up of 1,005 antibody-positive individuals. Among the 12 NPC patients in the HRA, five were newly diagnosed as having stage II (three patients) and stage III (two patients) NPC. These results support the hypothesis that antibody against EBV-specific DNase activity may be a useful marker for detection of patients with NPC, and they imply that individuals having high levels of antibody to EBV DNase activity may have an increased risk of development of NPC.     

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-specific DNase in patients with nasopharyngeal carcinoma and control groups

Serum samples from 154 patients with nasopharyngeal carcinoma (NPC), 374 with other cancers, 1,000 normal controls from Government Employees' Clinic Center (GECC), and 3,642 individuals of various ethnic-dialect groups living in high-risk areas for NPC were collected and the concentration of antibodies to Epstein-Barr virus (EBV)-specific DNase activity was determined. Taking a serum sample where 1 ml will neutralize two or more units of the DNase activity as positive, 2-4 units as low level, 4-6 units as medium level, and more than 6 units as a high level of antibody, 90.3% of the NPC patients contained significant amounts of antibodies to EBV-specific DNase activity and most of those had high levels of the antibody. In contrast, only 11% of sera from patients with cancers other than NPC contained antibodies to EBV-specific DNase activity, and high levels were very rare (2.1%). The difference in positive rates between these two groups is highly significant according to the chi 2 test (P less than 0.001). The positive rate of this antibody in the control group (GECC) was 5.3% with 0.0%, 0.8%, and 4.5% having high, medium, and low levels of antibodies, respectively. Again, the difference in positive rates between the GECC group and the NPC group is statistically significant (P less than 0.001). Taken separately, the positive rates of anti-EBV DNase activity in the three high-risk groups were 11.7%, 13.0%, and 13.1%. No significant difference in age distribution for the levels of this antibody was observed in the control GECC group or the three high-risk groups. However, the positive rates of the three high-risk groups are more than twice those of the GECC group (11.7% approximately 13.1% vs 5.3%). This ratio coincides with the ratio of the probability of developing NPC in high-risk groups compared to that of the GECC group (also more than two times). The significance of this coincidence is discussed.     

Antibody response to Epstein-Barr-virus-specific DNase in 13 patients with nasopharyngeal carcinoma in Taiwan: a retrospective study

Serum samples obtained from 13 individuals who were found to have a previous history of or to be suffering from nasopharyngeal carcinoma (NPC) were examined for the presence of antibody to Epstein-Barr virus (EBV) DNase activity. Significant to high levels of antibody to EBV DNase activity were detected in most serum samples obtained from four patients prior to the diagnosis of NPC. The samples from the other nine patients showed variable levels of antibody. The majority of the samples collected at the remission stage of the disease, especially those from long-term survivors, contained little or no antibody to the DNase activity. Data presented here suggest that antibody to EBV DNase activity may be a useful marker for the early diagnosis as well as the prognosis of NPC.     

用基因工程表达的抗原早期诊断鼻咽癌

目的为了建立鼻咽癌（ＮＰＣ）早期诊断方法。方法以基因工程表达的、经纯化的ＥＢ病毒（Ｅｐｓｔｅｉｎ－Ｂａｒｖｉｒｕｓ，ＥＢＶ）早期抗原（ＥＡ）成分ＥＡ－Ｄ和ＥＡ－Ｒ作为诊断抗原，建立了酶联免疫吸附试验（ＥＬＩＳＡ），检查３０例ＮＰＣ病人及４９例正常人血清中的ＥＡ／ＩｇＡ抗体。结果用ＥＬＩＳＡ检测抗体较用细胞涂片免疫酶方法（ＩＥ）敏感。ＥＬＩＳＡ检测ＮＰＣ病人血清中ＥＡ／ｌｇＡ抗体，阳性率为１００％，ＥＡ／ｌｇＡ抗体效价均≥１∶１００。而用ＩＥ法，平行检测３０例ＮＰＣ病人血清中ＥＡ／ｌｇＡ抗体效价，结果６例为阴性（＜１∶１０），抗体阳性率为７０％。ＥＬＩＳＡ明显地提高了ＮＰＣ的检出率。以ｐ１３８（ＥＡ－Ｒ）和ｐ５４（ＥＡ－Ｄ）分别或混合包被，检测对ＥＢＶ特异的ＥＡ－Ｄ和ＥＡ－Ｒ的抗体。结论表明在ＮＰＣ病人血清中存在对ＥＡ两种抗原的抗体，对ＥＡ－Ｄ的抗体滴度高于对ＥＡ－Ｒ的抗体。因此，以两种抗原混合包被作为诊断抗原建立的ＥＬＩＳＡ方法，为ＮＰＣ的早期诊断提供更敏感、特异和简便的手段。     

Smooth muscle antibody in Burkitt''s lymphoma and in nasopharyngeal carcinoma.

Smooth muscle antibodies (SMA) with specificity for actin, were found with a higher frequency in sera from Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) patients than in sera from matched controls. No correlation could be found between SMA and anti-Epstein-Barr virus (EBV) antibody titres. There was no parallelism, in individual sera, between the finding of SMA and the occurrence of cold lymphocytotoxins, aother antibody activity found with an abnormally high frequency among BL and NPC patients. The reason why actin, a weak antigen in experimental animals, may become immunogenic in humans remains unexplained.     

Detection of IgA antibody to EBV membrane antigen using Staphylococcus aureus preabsorbed sera is closely associated with nasopharyngeal carcinoma

The presence of IgA antibody to membrane antigen (MA) of Epstein-Barr virus (EBV) was tested in sera from 48 nasopharyngeal carcinoma (NPC) patients, 40 patients with tumors other than NPC and 46 normal individuals. The sera were preabsorbed with Staphylococcus aureus (SPA) (strain no. 1800) prior to their use in the indirect immunofluorescence test. One hundred percent of the NPC patients had the IgA/MA antibody with a GMT of 1:141. In patients with tumors other than NPC or normal individuals, IgA/MA antibodies were not detectable. The IgA/MA antibodies have been demonstrated in 6 NPC patients lacking detectable antibody levels in the indirect immunofluorescence test using nonabsorbed sera. Our data indicate that preabsorbtion of sera with SPA renders the diagnostic test significantly more sensitive for the detection of the nasopharyngeal carcinoma and can be used for trials on the prognosis of patients.     

Serodiagnosis of Lyme disease by ELISA using Borrelia burgdorferi flagellum antigen

Antibodies to a 41,000 (41 kD) polypeptide in flagella of Borrelia burgdorferi were measured in patients with Lyme disease in Japan by flagellum ELISA. The IgG and IgM Classes of antibodies to a flagellum antigens were detected in the sera as early as 0.5 months after infection. The IgG antibodies continued to exist in their sera for more than one year, while the IgM antibodies quickly faded out from their sera. With respect to a diagnostic specificity of the flagellum ELISA, false positive reactions showing more than 10% were observed in sera with high levels of IgG or IgM, and with anti-syphilis antibody. This method, however, was unaffected by sera with high levels of IgA, rheumatoid factor or anti-nuclear antibody. In three cases of patients with erythema migrans preceded by tick-bite, and treated with antibiotics, seronegative results were observed by a immunoperoxidase (IP) test. Since two of them showed the positive level of IgM antibody by the flagellum ELISA, this method seems to be more sensitive and useful than the IP test for serodiagnosis of the Lyme disease.     

Specific IgA antibody to Epstein-Barr viral capsid antigen: a better marker for screening nasopharyngeal carcinoma than EBV-DNA detection by polymerase chain reaction

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. To assess whether EBV DNA detection by polymerase chain reaction (PCR) or presence of specific serum antibody to viral capsid antigen (VCA) was a better marker for screening NPC, nasopharyngeal tissues and blood samples from 58 NPC patients and 24 non-NPC patients (23 with laryngotracheal stenosis and 1 with chronic tonsillitis) were tested for the presence of EBV DNA and serum specific VCA antibodies, respectively. EBV DNA was detected in 56 (96.5%) of NPC patients and 15 (62.5%) of non-NPC controls, with predominantly EBV type A in both groups. On the other hand, specific VCA IgA antibody was detected in the majority of NPC patients: 52 (89.7%) while only 4 (16.7%) were detected in non-NPC controls. Therefore, specific VCA IgA antibody may serve as a better marker for screening NPC than EBV DNA detected by PCR.     

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。     

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。     

Oligomeric immunoglobulin A antibody response to rubella virus infection.

Pooled sera from rubella patients in the early convalescent stage, containing a high titer of hemagglutination-inhibiting (HI) antibody, were treated with protein A-conjugated gel to reduce immunoglobulin G (IgG) antibody and then centrifuged in sucrose gradients. This treatment resulted in the detection of an HI activity peak sedimenting at a rate intermediate between 7S and 19S. In contrast to the 19S antibody, the HI activity of this peak was not abolished by 2-mercaptoethanol, but sedimented at 7S after this treatment. The activity was considered to consist of IgA oligomers, since it was removed by anti-IgA immunosorbent. The appearance of the oligomeric IgA antibody after the infection was then studied using serum samples collected sequentially from five rubella patients. Shortly after the onset of the disease, the HI activity appeared at high titer and thereafter gradually decreased in titer until it could no longer be detected in the sera. The time of its disappearance varied with each patient.     

Dynamics of Epstein-Barr virus DNA levels in serum during EBV-associated disease

A real-time polymerase chain reaction assay for quantitation of Epstein-Barr virus (EBV) DNA in serum was developed. This assay detected EBV DNA in 24 (89%) of 27 sera from patients with infectious mononucleosis, but only in 9 (18%) of 51 sera from EBV carriers (P < 0.001) and in none of the sera from 32 EBV-seronegative individuals. EBV DNA levels were higher in sera from infectious mononucleosis (median 8,000, range 1833-150,069 copies/ml) than from carriers (median < 2, range < 2-2980; P < 0.001). In sera of 36 children with infectious mononucleosis followed prospectively, EBV DNA levels correlated inversely with the duration of symptoms. Among 18 children with tumors including Hodgkin's disease (n = 7), non-Hodgkin's lymphoma (n = 6), Burkitt's lymphoma (n = 1), lymphoproliferative disorder (n = 4), and osteosarcoma (n = 1), EBV DNA was detected in serum from those 9 (100%) expressing EBV in the tumor (Hodgkin's disease, 3; non-Hodgkin's lymphoma, 2; lymphoproliferative disorder, 4), the levels peaking at diagnosis and correlating with disease activity. Quantitation of EBV DNA in serum may offer a simple means of monitoring patients at risk of EBV-associated lymphoproliferation.     

Quantitative plasma/serum EBV DNA load by LMP2A determination in an Italian cohort of NPC patients.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is frequently associated with Epstein-Barr virus (EBV), but little is known about the EBV DNA prevalence on peripheral blood in Western Countries, where the tumour is not endemic and its incidence is low. OBJECTIVES: To set up and evaluate an internally controlled qualitative polymerase chain reaction (PCR) followed by quantitative competitive PCR for the detection of EBV DNA in clinical specimens. To investigate whether EBV DNA load in peripheral blood was a consistent feature of Italian NPC patients. MATERIALS AND METHODS: A PCR assay based on latent membrane protein 2A (LMP2A) sequence amplification was chosen. Best assay conditions, sensitivity and reproducibility were determined. Sixty-four sera and 63 plasma from an Italian cohort of 39 NPC patients were analyzed. Samples from 5 patients followed up after radiotherapy were also assayed. Qualitative and quantitative beta-globin amplification was performed in parallel in order to provide an independent control for amplification competence of DNA and to investigate whether EBV DNA levels could be due to intracellular EBV viral genomes from cells lysed during plasma/serum collection. RESULTS: Twenty-five patients had undifferentiated carcinoma (UC) and 14 squamous cell carcinoma (SCC). EBV DNA has been quantified in 58 and 9% of the UC and SCC cases, respectively. No statistically significative differences were observed between the EBV DNA levels (469 vs 750 copies/ml, P=0.16) and prevalence (64 vs 57%, chi2(1)=0.22, P=0.64) in plasma and serum samples. Increased EBV viremia was found in patients with considerable extension of the primary tumour (172 vs 2250 copies/ml, low vs high tumour burden). Three UC subjects, which had detectable pre-treatment EBV DNA levels, became negative after radiotherapy. Clinical examination revealed that all had complete tumour regression. CONCLUSIONS: These PCR procedures allow an accurate and reproducible estimation of plasma/serum EBV DNA load in NPC patients living in non endemic areas, being strictly associated with UC WHO III and with tumour severity.     

Enzyme immunoassay analysis of antibody specificities present in the circulating immune complexes of selected pathological sera

Using immobilized anti-C3 antibody and an enzyme immunoassay, sera from 26 patients (eight with systemic lupus erythematosus (SLE), four with Hashimoto's thyroiditis, eight haemophiliacs and six with post-hepatitis cirrhosis) containing high levels of circulating immune complexes (IC) were selected. The IC were precipitated with 2.5% polyethylene glycol, washed, treated with acid buffer, neutralized and tested using an enzyme immunoassay in parallel with the original sera for antibody activity against a panel of antigens: human myosin and thyroglobulin, mouse actin and tubulin, calf thymus DNA and trinitrophenyl coupled to bovine serum albumin (TNP/BSA). It was found that all the isolated IC may contain IgG, IgA and IgM antibodies reacting with actin tubulin and TNP/BSA and also, depending upon the disease, antibodies reacting with some of the other antigens of the panel. By comparison to the antibodies present in the original sera, higher titers of antibodies were found in the isolated IC while some antibody specificities not detected in a given serum were occasionally noted in the isolated IC. The antibodies present in the IC seem to possess characteristics similar to those of polyreactive human natural autoantibodies. It is concluded that natural autoantibodies participate actively in the formation of IC found in pathological sera.     

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.     

Neutralization and enhancement of HIV-1 infection by sera from HIV-1 infected individuals who progress to disease at different rates

We examined the neutralizing/enhancing activity in sera collected at an early and a later time point postinfection from 13 HIV-positive nonprogressors, 13 moderate progressors, and 13 rapid progressors to determine the relationship between neutralizing/enhancing activity and disease progression. Early sera from each group reduced virus replication at low dilutions (10(-1) to 10(-2)) when compared with negative sera. The reduction was statistically significant for moderate and rapid progressors at 10(-1) dilution (P = 0.02 and P = 0.02 respectively) but not for nonprogressors (P = 0.16). Late sera from nonprogressors and moderate progressors reduced virus replication at low dilution but late sera from rapid progressors lost neutralizing activity. These data suggest that an association exists between neutralizing activity in sera and nonprogression or slower progression to disease and that loss of neutralizing activity is associated with disease progression. At higher dilutions (10(-3) to 10(-6)), both early and late sera from each group increased virus replication over negative sera. The levels and frequency of enhancement were higher for sera from a subgroup of nonprogressors than a subgroup of rapid progressors who exhibited enhancement. This suggests that enhancement is not associated with disease progression. The neutralizing/enhancing activity observed in sera of these three groups of subjects suggest that enhancement levels may reflect the overall level of antibody response to HIV. The replication patterns observed for early and late sera from individuals in the different groups reflect changes in antibody activity that appear to be associated with protection or disease progression.