Cryoimmunoglobulinaemia in patients with primary renal disease and systemic lupus erythematosus. I. IgG- and DNA-binding assessed by co-precipitation.

Sera were tested for cryoglobulin precipitates from 206 consecutive patients with renal disease, ninety-eight normals and sixteen patients with systemic lupus erythematosus (SLE) without evident renal disease. Cryoprecipitates were detected in 17% of test subjects overall and 2% of normals; the incidence was highest in patients with SLE, regardless of detectable renal disease. Cryoprecipitates usually were comprised of IgG and IgM or IgG, IgM, and IgA in thirty-six out of forty-two instances, although a single immunoglobulin class was detected in five patients. Co-precipitation experiments showed IgG-binding by virtually all sera forming cryoprecipitates; isolated cryoprecipitates bound radiolabelled homologous IgG, and Fc fragment and sometimes IgG subclass proteins preferentially. Freshly forming cryoprecipitates sometimes co-precipitated DNA, whereas all isolated cryoprecipitates co-precipitated DNA from dilute solutions. The data are compatible with the current hypothesis that cryoimmunoprecipitates are immune complexes that are insoluble in vitro in the cold, that they usually comprise mixed immunoglobulins with anti-IgG activity, and may contain a mixture of antigens and antibodies.     

IgM and IgG antibodies to hepatitis C virus in patients with mixed cryoglobulinaemia

To assess the relationship between hepatitis C virus (HCV) infection and essential mixed cryoglobulinaemia (EMC), sera from 23 patients with EMC were tested for IgG and IgM antibodies to HCV antigens and for HCV RNA. Quantitative HCV antibody studies were performed on scrum and purified cryoglobulin fractions. HCV antibodies of both IgG and IgM class were found in 22 (96%) patients. Ten of these were also HCV-RNA positives. Higher litres of anti-HCV IgM were present in the 11 patients with evidence of liver damage. Anti-HCV IgG antibodies were shown to be concentrated in the IgG fraction of cryoglobulins in all eight patients studied. These results strongly suggest a role for HCV in the pathogenesis of EMC.     

Epstein-Barr virus infections in childhood.

IgM and IgG antibodies against Epstein-Barr virus (EBV) capsid antigen and antibodies against EBV nuclear antigen and heterophil antibodies were investigated in 115 paired sera of children with acute infections and in 100 sera of healthy controls of the same age and sex. EBV-specific IgM antibodies could be recognized in 13.7% of the patients and in 7% of the controls. Antibodies against EBV nuclear antigen were not detected in the IgM-positive sera.     

IgM antibodies to Epstein-Barr virus in infectious mononucleosis

Summary IgM antibodies to Epstein-Barr virus (EBV) could be demonstrated by immunofluorescence in the sera of all patients with clinical infectious mononucleosis (IM) and a heterophile antibody titer of 40. The EBV-IgM antibodies were found only over a period of 2 to 3 months after onset of symptoms. Demonstration of IgM antibodies in the sera of patients therefore indicates recent infection and may be useful for diagnosis. The fact that EBV-IgM antibodies can only be demonstrated during a relatively short period supports the assumption that EBV is indeed the etiologic agent of IM.     

High incidence of elevated antibody titers to Epstein-Barr virus in patients with uveitis

Summary. We assayed Epstein-Barr virus (EBV) antibody titers in patients’ sera using indirect immunofluorescence and tested for the presence of antibody to EBV immediate-early BZLF1 protein ZEBRA by Western blotting to explore the association of EBV infection with uveitis. IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to early antigen (EA), and antibodies to EBV nuclear antigen were detected at higher titers in sera of patients with uveitis than in the sera of healthy controls. Neither IgM antibody to VCA nor EA was detected in the patients’ sera. Anti-ZEBRA-IgG antibodies were detected in most patients’ sera, but not in those of healthy controls. These results suggest that uveitis might be a disease accompanied by EBV reactivation.     

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.     

Antibody response to Epstein-Barr virus in infectious mononucleosis.

Altogether 171 serum specimens from 58 patients with heterophil antibody-positive infectious monomucleosis were studied for antibody response to Epstein-Barr virus (EBV). The sera were tested for fluorescent immunoglobulin G (IgG) and IgM gel-precipitating (GP) and complement-fixing (CF) antibodies to EBV. All 58 patients had IgG and IgM antibodies to EBV. Both IgG and IgM antibodies developed rapidly; the IgM antibodies disappeared within 8 to 10 weeks, whereas the IgG antibodies remained at an almost constant level. The development of IgG antibodies was so rapid that a fourfold or greater rise in titers was noted only in 22% of the patients. Both GP and CF antibodies to EBV (crude P3HR-1 Burkitt cell antigen) developed slowly; the mean titers kept rising for more than 12 weeks. The micro GP technique seemed to be more sensitive than the CF method, because 86% of the patients with infectious mononucleosis had GP antibodies compared with 72% having CF antibodies. In patients with infectious mononucleosis, a seroconversion or significant rise in GP antibodies was noted in 57%, whereas only 19% had a similar change in CF antibodies. The most promising of these antibody assays in the diagnosis of recent infections was the EBV-specific IgM antibody technique, which enables one to make the diagnosis on the basis of only one serum specimen. In cases where the acute-phase serum specimen is missing, the diagnosis can be made later by using the GP and CF techniques.     

Anti-human cytomegalovirus nuclear antigen antibodies of different immunoglobulin classes

IgG, IgM and IgA immunoglobulin classes of antibodies to human cytomegalovirus nuclear antigens (CMNA) were studied by the acid-fixed nuclear binding technique (AFNB) and combined anti-complement immunofluorescence (combined ACIF). In acute cases of infectious mononucleosis (IM) of human cytomegalovirus (HCMV) origin and in the so-called double virus infections (HCMV + Epstein-Barr virus), anti-CMNA IgM antibodies were detected. They were absent from both anti-HCMV positive sera of healthy donors and sera of patients suffering of IM caused by EBV used as controls. The presence of anti-CMNA IgM may thus serve as an additional evidence of acute HCMV infection. Non-complement-fixing IgA classes of the anti-CMNA antibodies were not found in some of the sera gathered during the acute phase of IM of EBV origin: in one fourth of the HCMV seropositive donors and in a number of late serum samples. But non-complement-fixing and complement-fixing anti-CMNA components of the IgG class were detected.     

Antiviral antibodies in rheumatoid synovial fluid and cryoprecipitates

Distribution of viral antibodies was examined by the indirect fluorescent antibody technique in specimens from rheumatoid arthritis and from systemic lupus erythematosus patients. Titres in sera from rheumatoid arthritis patients were usually equal to, or greater than, those in joint fluids. Viral antibodies were found in whole cryoprecipitates from joint fluids and in the IgG fractions of the cryoprecipitates. Anti-herpes simplex antibodies were most frequently found in cryoprecipitates and were at highest titre in sera and joint fluids. Anti-respiratory syncytial virus antibodies were next in frequency and in titre. No evidence was presented to support the concept of localized viral antibody synthesis in joints of rheumatoid arthritis patients. Serum antibody titres in systemic lupus erythematosus patients were similar to those in rheumatoid arthritis patients; however, viral antibodies were not found in the serum cryoprecipitates of systemic lupus erythematosus patients.     

Persistent Epstein-Barr virus infection in patients with type II essential mixed cryoglobulinemia

The pattern of response to Epstein-Barr virus (EBV) has been investigated in 17 patients with essential mixed cryoglobulinemia (EMC) and in 17 control subjects. All subjects in both groups had IgG to the viral capsid antigen at comparable titers. In the absence of signs of recent infection, 13 patients had IgM to viral capsid antigen and 5 had also IgA, while all the controls were negative. EBV genome was present in bone marrow lymphocytes obtained from 4 patients with type II EMC, but not in those of one patient with type III disease; the latter patient's lymphocytes also failed to produce detectable levels of rheumatoid factor in culture, while the other four patients' lymphocytes released high amounts in culture supernatants. These data support the evidence of an association between type II EMC and persistent EBV infection.     

Antibody response to Epstein-Barr virus antigens in patients with chronic viral infection

We tested antibody titres against Epstein-Barr virus (EBV) antigens in patients suffering from chronic viral disease and compared them with those determined in sex- and age-matched healthy controls. Patient sera showed signs of active EBV infection [antibodies against early antigen (EA) and/or viral capsid antigen (VCA) in the IgM or IgA classes] significantly more frequently than the control group. Correspondingly, geometric mean titres (GMT) of antibodies against all viral antigens were elevated in the patients. The strongest association with EBV was observed in patients whose clinical symptoms closely resembled infectious mononucleosis: 92% of the subjects in this subgroup possessed anti-EA and 41 and 25% had IgM and IgA anti-VCA antibody, respectively. In patients with signs of lymphoproliferation only and in those suffering from frequent respiratory infections the association with EBV was less marked but still significant. Patients with transient defects in humoral and cellular immunity mounted higher titres against VCA in the IgG class than those without immune defects.     

Measurement of heterophil antibody and antibodies to EB viral capsid antigen IgG and IgM in suspected cases of infectious mononucleosis.

The EBV IgG titres in acute and convalescent specimens from 97 cases of infectious mononucleosis were compared with titres from acute and convalescent sera from 96 students with illnesses resembling infectious mononucleosis but without heterophil antibody, EB IgM or EB IgG seroconversion; and also with titres from 91 healthy students known to have had EB IgG antibody for at least six months. These titres were related to the titre of the Research Standard A.66/235 for infectious mononucleosis serum prepared by the National Institute for Biological Standards and Control. Serial sera were tested for heterophil antibody and EBVCA specific IgG and IgM from 61 university students with infectious mononucleosis. The period of persistence of heterophil antibody and EBV IgM after illness was outlined from the results of the tests. Single sera from 406 patients in hospital or general practice sent to the diagnostic laboratory for heterophil antibody tests were also tested for EBV antibodies without prior knowledge of the heterophil antibody result. The close agreement between heterophil antibody and EBV IgM results is shown. False positive EB IgM results were correlated with the presence of rheumatoid factor.     

A mu-capture immunoassay for detection of human herpes virus-6 (HHV-6) IgM antibodies in human serum.

BACKGROUND: Human herpes virus-6 (HHV-6) was first isolated in 1986. It has been shown to cause exanthema subitum and has been associated with various other diseases. HHV-6 infection is widespread, and more than 90% of the population have antibodies against HHV-6 at the age of 2 years. Once acquired, the virus remains latent in the body. This makes it difficult to draw any conclusions about a causal relationship between the demonstration of HHV-6 and a specific disease. OBJECTIVES: This work was to develop a mu-capture HHV-6 IgM enzyme linked immuno sorbent assay (ELISA) for use in routine diagnosis and for wide scale patient population analysis. STUDY DESIGN: A mu-capture HHV-6 IgM ELISA was established. A total of 682 sera consisting of 585 sera from Danish blood donors and 97 sera from patients with autoimmune antibodies were analysed in the HHV-6 IGM ELISA. One hundred and ninety-two sera had earlier been analysed for total HHV-6 antibody content in a competitive ELISA, 94 sera were analysed for cytomegalovirus (CMV) IgM and 57 sera for Epstein Barr virus (EBV) antibodies, using different ELISA assays. The results for 12 primary infections with HHV-6 are also reported. RESULTS: A HHV-6 IgM optical density (OD)-ratio was calculated according to a constant positive control. An empirical cut off of 0.5 HHV-6 IgM OD-ratio was chosen (with regard to the 10 HHV-6 seroconverters), which resulted in a specificity of 97.5% of the HHV-6 IgM ELISA. Two of the three donor sera with HHV-6 IgM OD-ratios more than 1.05 had total HHV-6 antibody titers significantly above the group with IgM OD-ratios below 0.7 consisting with HHV-6 reactivation. There was no cross reactions to EBV or CMV IgM positive sera. CONCLUSION: The HHV-6 IgM ELISA seems valid to diagnose primary HHV-6 infection in particular in combination with the HHV-6 total antibody assay.     

The response to Epstein-Barr virus infection in Sj?gren's syndrome

To assess the response to Epstein-Barr virus (EBV) infection in patients with primary Sj?gren's syndrome (SS), the frequency of detection of EBV DNA was studied in salivary gland biopsies and the antibody and idiotypic response to the virus was compared with healthy controls and infectious mononucleosis (IM). Viral DNA, detected by in-situ hybridization, was found in biopsies from two out of 12 patients with SS and six out of 10 controls. IgG, IgA and IgM antibodies to the virus, measured by ELISA using synthetic peptides (early antigen and EBNA-1) and a cloned fusion protein (EBNA-1), were normal in sera from 20 patients with SS, whereas infectious mononucleosis patients showed an increase in IgM antibodies to EBNA-1 and IgG antibodies to early antigen. One similarity between infectious mononucleosis and Sj?gren's syndrome was a significant increase in the germline heavy chain idiotype G6 in both diseases, suggesting activation of similar B-cell subsets. It is possible that this is due to EBV, though the low frequency of EBV DNA in biopsies and the normal levels of EBV antibodies in SS does not lend any evidence that the virus itself is the causative agent.     

Improved Epstein-Barr virus immunoglobulin M antibody test.

The successful demonstration of Epstein-Barr virus immunoglobulin M (EBV IgM) antibody in human sera has been accomplished to date by at least four groups of workers. Many, however, including ourselves, have had difficulty in getting reproducible results with the techniques described. The three-coat technique described by H. Schmitz and M. Scherer (1972) on both fractionated and unfractioned sera was adopted with minor modifications. The Hyland antihuman IgM antiserum used in the second coat was made specific by absorption on Cohn fraction II. This step in the procedure was found to be the single most important factor in arriving at reproducible results in the IgM test. The EBV IgM antibodies from our results to date with this method in 14 cases of heterophil-positive cases of mononucleosis appear short lived, lasting 2 months or less. These antibodies were found in only 2 of 18 selected non-mononucleosis cases, in both associated with EBV-viral capsid antigen antibody rise or seroconversion. The successful elimination of nonspecific fluorescence by a simple, inexpensive procedure and the possibility of testing unabsorbed, unfractionated sera directly will facilitate the use oe the EBV IgM antibody test in the future.     

A modified immunofluorescence test for Epstein-Barr virus-specific IgM antibody

The fluorescent antibody (FA) test for Epstein-Barr virus (EBV)-specific IgM antibody was improved by the use of sodium butyrate to induce a higher level of EBV antigen expression in P3HR-1 slide preparations and by removal of rheumatoid factor (RF) and IgG antibodies from test sera by means of adsorption with suspensions of Sepharose-IgG and Streptococcus pyogenes strain AR1. This method was compared with the Paul-Bunnell test (PB) on 1106 sera submitted to a routine virus diagnostic laboratory for infectious mononucleosis serology and 96.4% of sera showed concordant results. Thus the EBV-IgM-FA method was suitable for routine diagnostic use. However, it proved helpful to test EBV-IgM positive sera by PB to assist in the detection of cross-reacting IgM antibodies sometimes present.     

Antibodies to Epstein-Barr virus-induced early antigens in blood donors

IgG class antibodies to the early antigen complex (EA; D + R components) of the Epstein-Barr virus (EBV) were found by indirect immunofluorescence in titres greater than or equal to 1:10 in 196 (49.4%) out of 397 blood donors and titres greater than or equal to 1:20 in 84 (21.2%) of these subjects. Anti-EA titres of greater than or equal to 1:2560 were detected in 6 sera, anti-EA(D) only in 17 donors (4.3%). Additional EBV-specific antibody tests were performed in sera with anti-EA-IgG titres of greater than or equal to 1:20 (n = 84), including IgM class antibodies to virus capsid antigen (anti-VCA-IgM). Specific IgM revealed active EBV infection in 12 blood donors. There was no correlation between the presence of anti-VCA-IgM and the magnitude of anti-EA-IgG titres. Therefore, it seems to be impossible to define the threshold titre for EA antibodies indicating active EBV infection. For this purpose probably a titre increase should be demonstrated in paired sera.     

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.     

Rheumatoid factor as a cause of positive reactions in tests for Epstein–Barr virus-specific IgM antibodies

Sera from twenty-eight patients with rheumatoid arthritis (RA) were titrated in indirect immunofluorescence tests for Epstein–Barr virus (EBV) specific antibodies. All had IgG antibodies to viral capsid antigen (VCA), 64% at titres [unk] 320, and 71% reacted also in tests for VCA-specific IgM antibodies at titres ranging from 20 to 640. The reactions observed in the IgM test were not due to VCA-specific IgM antibodies, however, but rather to rheumatoid factor (RF) usually an IgM antibody to the Fc regions of IgG. The titres recorded in the anti-VCA IgM test correlated significantly with the RF titres and both reactivities were abolished by adsorption onto IgG coated latex particles. In addition, they clearly depended upon the height of the IgG antibody titre to VCA, indicating that the more VCA-specific IgG molecules are present the more likely it is that RF will combine with them in sufficient quantity before or after their attachment to VCA-positive test cells so as to become detectable by the fluorescent antibodies to human IgM. Results comparable in every aspect were obtained with those sera from patients with Hodgkin's disease, nasopharyngeal or cervical carcinomas which reacted in the anti-VCA IgM test. Sera from patients with infectious mononucleosis may also contain RF, but in such cases its removal by adsorption onto IgG-coated latex particles did not generally reduce the VCA-specific IgM antibody titre. Removal of RF from any of the sera studied did not affect the titres of VCA-specific IgG and, where applicable, IgA or heterophil antibody titres. These results re-emphasize the pitfall created by RF noted previously in tests for virus-specific IgM antibodies.     

Specific concentration of antilymphocyte antibodies in the serum cryoprecipitates of patients with systemic lupus erythematosus.

Antibodies to surface determinants of human lymphocytes, recognized both by cytotoxicity of fluorescent antibody analysis, were shown to be specifically enriched over the serum levels in cryoprecipitates from patients with systemic lupus erythematosus (SLE). The antilymphocyte antibody was shown to be cold reactive and was exclusively IgM. It was distinct from IgM anti-IgG, which was also variably concentrated in the cryoprecipitates. The question whether the antilymphocyte antibodies appear in the cryoprecipitates as complexes because of interaction with surface membrane antigens, or simply because of cold reactive properties, remains to be determined. The possible clinical relevance of the cryoprecipitation of these antibodies in systemic lupus erythematosus is discussed.