Humoral immune response in Epstein-Barr virus infections. I. Elevated serum concentration of the IgG1 subclass in infectious mononucleosis and nasopharyngeal carcinoma

Using radial immunodiffusion we measured IgG subclass concentrations and studied their distribution in serum samples from patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC), two Epstein-Barr virus (EBV)-associated diseases, in comparison with two control groups [completely anti-EBV negative persons and subjects carrying antibodies to the viral capsid antigen (VCA)]. Antibody titres to VCA and to the early antigen (EA) were determined by indirect immunofluorescence and revealed characteristic patterns for the respective diagnostic groups. Nephelometric assays served for quantitating total protein, albumin, total IgG, IgA and IgM in all the sera. In the IM and NPC groups the concentration of IgG1 was significantly elevated by more than 50% whereas the other three subclasses remained unchanged as compared with the controls. Correspondingly, we found a significant increase of total IgG in IM and NPC. In IM, the only disease where VCA-specific IgM antibodies have been reported to occur, IgM levels were markedly elevated. Our data suggest that the IgG1 subclass plays an important role in the humoral immune response to EBV-determined antigens and that it is possibly involved in the control of virus infection.     

Isotype and immunoglobulin subclass distribution of eye muscle membrane reactive antibodies in the serum of patients with thyroid-associated ophthalmopathy as detected in Western blotting.

We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity Purification of IgG Subclasses and the Distribution of Thyroid Auto-Antibody Reactivity in Hashimoto''s Thyroiditis

To delineate accurately the IgG subclass distribution of thyroid auto-antibodies, sera from nine patients with Hashimoto's thyroiditis were fractionated into IgG subclasses by complete depletion of the other IgG subclasses on affinity columns. All IgG subclass fractions contained thyroglobulin and microsomal (or thyroid peroxidase) antibody activity, although when compared to the total serum concentrations of IgG subclasses, IgG4 antibodies were overrepresented. However, in contrast to recent studies, this particular subclass never predominated--IgG4 antibody levels being exceeded by those of the IgG1 and IgG2 subclasses; it seems likely that these differences relate to varying sensitivity for different subclasses in previously used assay methods. This pattern of subclass activity differed from that of tetanus toxoid antibodies, which were found in six subjects. There was no light chain restriction within any subclass, showing that the overproduction of IgG4 thyroid antibodies is not of monoclonal origin. The functional affinity of subclasses for both thyroid antigens varied between patients, but IgG2 subclass fractions showed the highest functional affinity in the majority of samples. We also found that IgG2 subclass thyroid antibodies were ineffective in eliciting antibody-dependent cell-mediated cytotoxicity, as distinct from the other three subclasses. Our results show that thyroid antibodies are less restricted in their IgG subclass distribution and patients are less heterogeneous than previously described. Moreover, IgG2 thyroid antibodies are quantitatively important and differ in relative functional affinity and effector function from IgG1 and IgG4 thyroid antibodies.     

IgG subclasses in children with nephrotic syndrome

To determine whether the hypogammaglobulinemia of childhood nephrotic syndrome is characterized by symmetric depression of the IgG subclasses, the authors compared the IgG subclass concentrations in nephrotic patients in relapse versus remission. The authors used a highly sensitive monoclonal antibody-based enzyme immunoassay that allows quantitation with comparable precision of all four subclasses. They analyzed 28 sera obtained from 22 nephrotic patients during relapse (n = 16) and/or remission (n = 12). The mean ages of the two groups were similar. IgG1 and IgG2 were significantly decreased during relapse compared with remission, whereas IgG3 and IgG4 were not significantly different. This pattern of asymmetric depression of IgG subclasses supports a cause other than urinary losses in the pathogenesis of this abnormality.     

Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors.

The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine-alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex.     

Autoantibodies in three populations of Burkitt''s lymphoma patients.

Autoantibodies against actin, intermediate filaments, single stranded DNA and histones were found in the sera of Burkitt's lymphoma (BL) patients and normal controls from tropical Africa, where there is a high incidence of BL. Both groups had significantly higher levels of these autoantibodies than Caucasian normal controls or patients with other malignant diseases, as well as Epstein-Barr (EB) virus positive and EB virus negative BL patients from regions of North Africa and France where the incidence is lower than in tropical Africa. No linear correlation could be seen between autoantibody levels and anti-EB virus/VCA or EBNA titres in patients and controls from tropical Africa. The results suggest that a factor independent of EB virus causes an immunological imbalance and autoantibody production in the population from tropical Africa. This factor may be one of several which results in a high incidence of BL in tropical Africa.     

IgG subclass distribution in organ specific autoantibodies. The relationship to complement fixing ability

Indirect immunofluorescence techniques employing sheep monospecific antisera to human IgG subclasses on unfixed cryostat sections have revealed the IgG subclass distribution in autoantibodies to pancreatic islets (ICA), thyroid epithelial (TMA), gastric parietal (PCA) and adrenal fasciculata (AdA) cells. Whereas antibodies were detected in all four subclasses in 21 of 27 TMA positive sera (mean IgG titre 29 +/- 5 . 2), 13 of 15 PCA (mean IgG titre 41 +/- 3 . 8) and eight of 14 AdA sera (mean IgG titre 10 +/- 3 . 1), only four of 35 ICA positive sera (mean IgG titre 45 +/- 3 . 6) reacted in all four subclasses. Approximately 50% of ICA positive sera showed a restricted polyclonal response to the 'common' pancreatic antigen and 12% of these sera reacted only with IgG2 subclass. The restriction rarely applied to co-existent thyrogastric antibodies in these sera and was independent of the ability of ICA to fix complement. Lesser subclass restrictions were observed in antibody responses to the 'common' antigen of the adrenal cortex.     

Anti-mitochondrial antibody IgG subclass distribution and affinity in primary biliary cirrhosis.

We have studied the total IgG subclass and anti-mitochondrial antibody (AMA) specific IgG subclass distribution in primary biliary cirrhosis (PBC) sera. In order to solve the problems caused by the differing affinities of subclass specific monoclonals and the competitive inhibition of antibodies in a whole serum assay, six sera were separated into subclass-specific fractions by affinity-depletion chromatography. AMA subclass distribution of 20 further sera from patients with PBC was assessed using conventional methods and the results were calibrated against one of the fractionated sera. Light chain distribution and AMA functional affinity were also assessed for the fractionated subclasses. Total amounts of IgG3 were significantly increased compared with normal controls. AMA were found in all IgG subclasses and not restricted predominantly to IgG3 as previously described. The functional affinity of IgG3 AMA is generally lower as compared with that of other subclasses. No light chain restriction was found.     

Quantification of IgG subclasses in sera of normal adults and healthy children between 4 and 12 years of age.

The concentration of the four subclasses of IgG was determined in sera of normal adults and healthy children between 4 and 12 years of age, using the radial immunodiffusion technique. A relation between the concentration of IgG subclasses and Gm type was studied in adults. No influence of Gm type on IgG1 concentration could be shown, except that the group of Gm(fb) individuals had a higher level than the others. The mean concentration of IgG2 was higher in sera positive for Gm(n) than in those lacking this genetic marker. High IgG3 concentrations corresponded to the presence of Gm(b). No clearcut evidence was obtained for a relation between IgG4 concentration and Gm factors, although in general Gm(n) positive individuals had higher and Gm (zag) positive individuals lower concentrations of this subclass in their serum. Quantification of IgG subclasses in sera from healthy children of different ages revealed that the amount of IgG2 rises slowly with age, having not yet reached the adult level at the age of 12 years. This also holds for IgG4, although in a lesser degree. No significant differences from the adult level were found for the concentrations of IgG1 and IgG3.     

IgG subclasses in human chronic schistosomiasis: over-production of schistosome-specific and non-specific IgG4

IgG subclasses were determined quantitatively in sera from 63 Egyptian men who were infected with Schistosoma mansoni. Total and antigen-specific IgG was measured pre- and post-treatment. Total IgG subclass antibodies were determined by immunoradiometric assay (IRMA) using monoclonal antibodies (MoAbs). The anti-worm and anti-egg specific S. mansoni IgG subclass antibodies were quantitatively measured by ELISA using specific MoAbs and standards obtained by affinity chromatography. Our data show that total IgG of the patients was elevated in the range of two to three times above normal. The magnitude of increase differed markedly among the four subclasses of IgG. The IgG1, IgG2 and IgG3 concentrations were approximately two to four times higher than normal, whereas the IgG4 concentrations was 20 times normal (9000 mg/l). IgG1 and IgG4 tended to dominate the IgG subclass distribution of anti-soluble worm antigen preparation (SWAP) antibodies followed by IgG2 and IgG3. On the other hand, IgG1 and IgG2 dominated the IgG subclass distribution of anti-soluble egg antigen (SEA) antibodies. As with IgG1, IgG2 and IgG3, most IgG4 was non-specific. The role of IgG subclasses in the pathogenesis of schistosomiasis is not clear. However, the high concentration of IgG4 might act as IgE blocking antibody, possibly as anti-idiotypes that may play a role in down-regulation of the immune system when it is challenged with an excess of antigen.     

B-Lymphotropic papovavirus and possibility of infections in humans

A novel member of the papovavirus group has been isolated from EBV-transformed African green monkey (AGM) B-lymphoblasts. The virus is characterized by its B-lymphotropic host range and has tentatively been named “lymphotropic papovavirus” (LPV). Seroepidemiological studies revealed that besides sera from African green monkeys a substantial proportion of human sera reacted with antigens of this virus. The specificity of this reaction observed in indirect immunofluorescence tests was underlined by immunoprecipitation (IP) and neutralization studies. By IP we demonstrated that AGM as well as human sera reacting in immunofluorescence with LPV antigens precipitated polypeptides of about 40 K. Those polypeptides were detected only in LPV-infected cultures. Sera of high reactivity in immunofluorescence tests also neutralized viral infectivity. These data suggest that an agent antigenetically closely related to AGM-LPV also infects part of the human population. The age distribution of the human antibody response revealed that a low percentage of sera reacted in age groups below 30 years. Thereafter, however, about 30% of all sera exhibited antibodies to LPV antigens. There was a slight increase in seroreactivity in sera from patients tested for infectious hepatitis when compared to non-selected human sera.     

Subclass distribution and IgE responses after treatment in human schistosomiasis

The IgG and IgA subclass distribution of specific antibodies as well as the distribution of total and specific IgE in 15 patients with schistosomiasis was determined in consecutive samples before and after initiation of treatment. An adult worm antigen preparation and a soluble egg antigen preparation were used as antigens in the ELISA assays. After initiation of treatment a rise was noted in certain subclasses and a correlation was found for specific IgG1 and IgG4 serum levels in the egg-excreting patients against adult worm antigen and for specific IgG4 and IgE levels in sera from the eight patients with a chronic disease. They also had a rise of the specific IgA1 titre and six of them also of specific IgA2. Members of eosinophilic granulocytes reached a peak after 2 weeks in seven of the eight patients. The increase of eosinophils was an early event as opposed to the incidence of peak of the determined specific isotypes. The associated rise in IgG1, IgG4 and IgE antibody concentrations and eosinophils may suggest a causal relation possibly induced by common interleukins.     

Precipitating and non-precipitating complement consuming IgG subclass antibodies in pigeon breeders' disease

The sera of patients with pigeon breeder's disease usually contain precipitating serum factors as well as human complement consuming factors as shown by incubation of the serum with pigeon dropping antigens. Although a single serum factor, possibly an IgG antibody, might account for both phenomena, affinity chromatography experiments revealed that the sera of patients with pigeon breeders' disease contain non-recipitating, human complement consuming, serum factors besides precipitating serum factors which are also capable of complement consumption. The non-precipitating serum factors most likely belong to the IgG3 immunoglobulin subclass, whereas the precipitating antibodies belong to the subclasses IgG1 and IgG2.     

Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia

Nasopharyngeal carcinoma (NPC) in Tunisia is characterized by its bimodal age distribution involving juvenile patients of 10-24 years and adult patients of 40-60 years. Three serological techniques were compared for primary diagnosis (N = 117) and post-treatment monitoring (N = 21) of NPC patients separated in two age groups. Immunofluorescence assay (IFA) was used as the "gold standard" for detection of IgG and IgA antibodies reactive with Epstein-Barr virus (EBV) early (EA) and viral capsid (VCA) antigens. Results were compared with ELISA measuring IgG and IgA antibody reactivity to defined EBNA1, EA, and VCA antigens. Immunoblot was used to reveal the molecular diversity underlying the anti-EBV IgG and IgA antibody responses. The results indicate that young NPC patients have significantly more restricted anti-EBV IgG and IgA antibody responses with aberrant IgG VCA/EA levels in 78% compared to 91.7% in elder patients. IgA VCA/EA was detected in 50% of young patients versus 89.4% for the elder group (P < 0.001). Immunoblot revealed a reduced overall diversity of EBV antigen recognition for both IgG and IgA in young patients. A good concordance was observed between ELISA and IFA for primary NPC diagnosis with 81-91% overall agreement. Even better agreement (95-100%) was found for antibody changes during follow-up monitoring, showing declining reactivity in patients in remission and increasing reactivity in patients with persistent disease or relapse. ELISA for IgA anti-VCA-p18 and immunoblot proved most sensitive for predicting tumor relapse. VCA-p18 IgA ELISA seems suitable for routine diagnosis and early detection of NPC complication.     

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.     

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。     

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.     

Distribution of immunoglobulin G subclasses of anti-thyroid peroxidase antibody in sera from patients with Hashimoto's thyroiditis with different thyroid functional status

The mechanism of disease progression in Hashimoto's thyroiditis (HT) is still unclear. Anti-thyroid peroxidase antibody (TPOAb), a diagnostic hallmark of HT, is principally of the immunoglobulin G (IgG) isotype, and it appears to be a response to thyroid injury. The aim of our study was to evaluate the distribution of IgG subclasses of TPOAb in sera from patients with HT with different thyroid functional status. Sera from 168 patients with newly diagnosed HT were collected and divided into three groups according to thyroid function: patients with hypothyroidism (n = 66), subclinical hypothyroidism (n = 60) and euthyroidism (n = 42). Antigen-specific enzyme-linked immunosorbent assay was used to detect the distribution of TPOAb IgG subclasses. The prevalence of TPOAb IgG subclasses in all patients' sera with HT was IgG1 70.2%, IgG2 35.1%, IgG3 19.6% and IgG4 66.1% respectively. The prevalence of IgG2 in sera from patients with hypothyroidism (51.5%) was significantly higher than that of subclinical hypothyroidism (33.3%) (P < 0.05), and the latter was also significantly higher than that of euthyroidism (11.9%) (P < 0.05). The positive percentage of IgG2 subclass in sera from patients with hypothyroidism and subclinical hypothyroidism was significantly higher than that of euthyroidism (P < 0.05), the prevalence and positive percentage of IgG4 subclass in sera from patients with hypothyroidism and subclinical hypothyroidism was significantly higher than that of euthyroidism respectively (P < 0.05). The predominant TPOAb IgG subclasses in sera from patients with HT were IgG1 and IgG4. Patients with high levels of TPOAb IgG2, IgG4 subclasses might be at high risk of developing overt hypothyroidism.     

Detection of IgA antibodies to Epstein-Barr virus-associated antigens by ELISA

The detection of IgA antibodies to the Epstein-Barr virus (EBV)-associated viral capsid antigen (VCA) and early antigens (EA) is of diagnostic and prognostic importance for patients with nasopharyngeal carcinoma (NPC). An ELISA for the determination of serum IgG antibodies to these antigens has been developed which uses the double antibody method. 136 sera obtained from healthy donors and patients with non-EBV related tumors and lymphomas were tested by ELISA; only 3 sera, from patients with chronic lymphatic leukemia, hairy cell leukemia and Burkitt-like lymphoma, contained antibodies of IgA class to VCA and EA. Ninety-five sera from patients suspected of having NPC were tested. IgA anti-VCA was found in 28 sera (29.5%), 12 of which also contained IgA anti-EA. The assays described are suitable for diagnosis and follow-up of patients with EBV-associated nasopharyngeal carcinoma. Furthermore, isolated EA components may be tested for their reactivity with IgA antibodies, as was shown for the 60 kDa polypeptide associated with the EA complex.     

Utilizing self-prepared ELISA plates for a cross-population study of different anti-HBe IgG subclass profiles

Fourteen serum samples obtained from hepatitis B virus (HBV) chronic carriers and patients recovered from hepatitis B infection were used with four sodium dodecyl sulfate-treated enzyme-linked immunosorbent assay (ELISA) plates available commercially, and one self-prepared HBcAg analog for evaluation of anti-HBe subclass pattern absorbance. The self-prepared plates had the best performance and were thus used for samples obtained from 104 (60 male and 44 female) HBV chronic carriers and 439 (247 male and 192 female) recovered individuals. Tests for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also carried out in 21 of the subjects (>25 IU/ml). Statistical comparison of these patients with elevated ALT/AST levels with other ALT/AST-normal chronic carriers revealed no significant differences in the anti-HBe OD, although the mean optical density (OD) of patients with elevated ALT/AST levels was higher. The results suggest that the anti-HBe IgG subclass profiles in the chronic carriers did not change with inflammation of the liver, and were independent of sex and age. In contrast to previous anti-HBc findings, the distribution pattern of anti-HBe subclasses in HBV chronic carriers was IgG1 > IgG4 > IgG3 while in the recovered individuals it was IgG1 > IgG3 > IgG4, for both males and females. Subclasses IgG1 and IgG2 were the most and least prevalent isotypes, respectively, in both study groups. The results of the study suggest that induction of IgG1 and/or IgG3 antibodies is important for effective virus neutralization, while IgG2 antibodies are of limited importance. Significantly higher OD values for anti-HBe IgG4 were observed when comparing samples from the chronic carriers and recovered individuals, which may reflect the effects of persistence. Further, in contrast to previous anti-HBs results, the concentrations of total IgG and IgG1 were higher in the samples from chronic carriers relative to those from recovered individuals.