Epstein–Barr virus-associated antibody patterns in carcinoma of the post-nasal space

Sera from African patients with tumours of the post-nasal space, Hong Kong Chinese patients with carcinomas of the post-nasal space and from Indian donors with buccal, oro- and hypopharyngeal carcinomas, as well as African control sera from healthy persons and individuals with various non-neoplastic and neoplastic diseases were tested for anti-Epstein–Barr virus titres and for antibodies capable of blocking the direct membrane immunofluorescence reaction obtained between Epstein-Barr virus carrying lymphoblastoid cell lines and two different fluorescein conjugated reference sera; one from a patient with Burkitt lymphoma (F-Mutua conjugate) and the other from a patient with carcinoma of the post-nasal space (F-Kipkoech conjugate). Ninety per cent or more of the African and Chinese patients with carcinoma of the post-nasal space gave at least one high test, and 60% were high in all three assays. In contrast, African patients with post-nasal space tumours other than carcinomas, African controls and Indian buccal, oro- and hypopharyngeal carcinomas gave high reactions in all three assays in less than 20%.     

Rheumatoid factor: a cause of fals positive histoplasmin latex agglutination.

Ten of thirteen patients with positive histolatex agglutination titers of 1:32 or greater had no evidence of acute histoplasmosis.Three of these false positives had rheumatoid arthritis. A fourth had a rising mycoplasma complement fixation titer, and the fifth had a high titer of cold agglutinins. All of these are associated with abnormal immunoglobulin M production. To evaluate the role of rheumatoid factor in producing false positive histolatex agglutination, the histolatex test was performed on sera from 32 patients having rheumatoid factor at a titer of 1:40 or greater. Four of these sera agglutinated the histoplasmin-coated latex particles at titers of 1:32 or greater. Review of clinical records suggests the this reactivity is nonspecific. It is our purpose to call attention to rheumatoic factor as a cause of false positive histolatex agglutination.     

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.     

Epstein–Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti‐DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities – anti‐dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two‐thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two‐thirds of SLE patients reacted to a large spectrum of self‐molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein–Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.     

Role of IgM antibodies versus B cells in influenza virus-specific immunity

We have compared the role of IgM antibodies with the role of B cells in control of primary influenza virus infection. Mice deficient in IgM (IgM(-/-)), but capable of producing other Igisotypes, exhibited increased pulmonary virus titers compared to wild-type mice. However, IgM(-/-) mice were less susceptible compared to B cell-deficient micro MT) mice. CD4(+) T cells from spleen and lung draining lymph nodes of infected micro MT mice showed reduced proliferation upon virus re-stimulation in vitro. Furthermore, numbers of IFN-gamma-producing CD4(+) effector T cells were reduced in the alveolar lavage (BAL) of micro MT mice but not IgM(-/-) mice. In contrast, total number of virus-specific CTL was almost comparable in BAL of micro MT and wild-type mice. Pulmonary recruitment of inflammatory macrophages and neutrophils occurred normally in both micro MT and IgM(-/-) mice. Interestingly, virus-specific IgG2a and IgG2b antibody responses were affected locally in the BAL and in the serum of IgM(-/-) mice, while IgG1 responses remained largely normal. Taken together, our data suggest a role for B cells to promote effector T cell responses and a role of both IgM and IgG antibodies in the defense against acute influenza virus infection.     

Absence of Epstein—Barr viral RNA in lymphomatoid papulosis

Epstein-Barr virus (EBV) recently has been implicated in the pathogenesis of Hodgkin's disease (HD). Lymphomatoid papulosis (LyP) is a premalignant cutaneous lymphoproliferative disorder which shares several characteristics with HD. The hypothesis has been made that EBV may be associated with the pathogenesis of LyP. We therefore examined 17 skin biopsy specimens and two lymph nodes from nine patients with LyP for EBV RNA using the highly sensitive and specific EBER method. In all specimens, the large atypical cells were negative for EBV while poly T studies confirmed the presence of adequate RNA for detection of EBER. The negative EBER results were confirmed in seven LyP patients whose biopsies were also stained for latent membrane protein (LMP-1). Interestingly, one patient with clonally related LyP and HD had no EBV RNA detected in any specimen. We conclude that EBV is unlikely to be an important aetiological agent in LyP. If confirmed in other patients, HD associated with LyP may have a different aetiology from HD arising de novo.     

Disappearance of the Epstein–Barr virus in a relapse of Hodgkin's disease

Hodgkin's disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case of nodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negative in the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This finding implies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not required to maintain neoplastic growth of HD tumour cells. © 1997 John Wiley & Sons, Ltd.     

High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Synthesis of measles virus-specific IgM antibodies and IgM-class rheumatoid factor in relation to clinical onset of subacute sclerosing panencephalitis

Thirty-seven serum and 37 cerebrospinal fluid (CSF) specimens from 27 patients with subacute sclerosing panencephalitis (SSPE) were tested for measles virus (MV) IgM antibodies and IgM-class rheumatoid factor (RF) to determine if a temporal relationship exists between SSPE onset and these immunoglobulins. Sensitive solid-phase radioimmunoassays were used for immunoglobulin detection. Five of six MV IgM-positive CSF, both MV IgM-positive sera and six out of eight sera with elevated RF levels were collected within 1 yr of SSPE onset. Also collected during this time were six other sera having high binding in the MV IgM assay, which was not due to MV-specific IgM antibodies. Two of these sera had as high or higher binding of IgM to a Vero cell control antigen, suggesting involvement of an IgM-class autoantibody. The other four sera had false-positive MV IgM assay results due to RF interference. RF interference was dependent on both the titer and avidity of the MV IgG antibodies involved. Three conclusions can be drawn from these results. First, MV IgM antibodies and elevated RF levels are not markers for acute SSPE, despite the tendency for their synthesis at this stage of the disease. Second, immunoassay analysis of viral IgM antibodies must employ an appropriate control antigen to account for background IgM binding. Finally, even if RF levels are normal or near normal, a false-positive IgM immunoassay result can still occur if antigen-specific IgG antibodies in the same sample have the right combination of titer and avidity.     

Characterization of ELISA detection of broad‐spectrum anti‐Epstein–Barr virus antibodies associated with nasopharyngeal carcinoma

Epstein–Barr virus (EBV) infection is associated with undifferentiated nasopharyngeal carcinomas (NPC). A distinct seroreactivity pattern to EBV is predictive of subsequent risk of sporadic and familial nasopharyngeal carcinomas. There are currently no accepted screening tools for guiding the clinical management of individuals at high‐risk for nasopharyngeal carcinomas, particularly unaffected relatives from nasopharyngeal carcinoma multiplex families. Therefore, the reproducibility of a panel of largely synthetic peptide‐based anti‐EBV antibody ELISAs was evaluated and their ability to distinguish nasopharyngeal carcinoma cases from controls was explored. IgG and IgA antibodies against 6 different EBV antigens (10 assays, total) were tested on sera from 97 individuals representing the full spectrum of anti‐EBV seroprevalence (i.e., healthy individuals with no known EBV seroreactivity, healthy individuals with known EBV seroreactivity, and nasopharyngeal carcinoma cases). Each specimen was tested in triplicate to assess within‐batch and across‐batch variation, and the triplicate testing was repeated on two separate days. Reproducibility was assessed by the coefficients of variation (CVs) and intraclass correlation coefficients (ICCs). All markers were detectable in 17% or more of samples. For all but one marker, the overall, within‐batch, and across‐batch CVs were below 15%, and the ICCs were above 70% for all but three markers. Sensitivity of these markers to detect prevalent nasopharyngeal carcinomas ranged from 22% to 100%, and among unaffected controls, most distinguished those with and without known seropositivity. In conclusion, a large number of EBV markers can be measured reliably in serum samples using peptide‐based anti‐EBV ELISAs. J. Med. Virol. 85:524–529, 2013. Puiblished 2012. This is a US government work, and, as such, is in the public domain of The United States of America.     

Enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus-specific IgM antibodies

Herpes simplex virus (HSV)-specific IgM in human serum could be detected by a microplate enzyme-linked immunosorbent assay, using extracts of HSV-infected cells as antigen. Peroxidase-conjugated anti-human IgM was used to detect human IgM bound to viral antigen. Pretreatment of sera with protein A-bearing staphylococcus or with aggregated human IgG was necessary to eliminate false-positive results caused by the presence of rheumatoid factor. Specificity controls included sera of patients with other herpes group virus infections.     

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.