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1.
ELISA quantitation of IgG subclass antibodies to dietary antigens   总被引:3,自引:0,他引:3  
The IgG subclasses of human antibodies against 2 dietary antigens, ovalbumin (OA) and beta-lactoglobulin (BLG), were studied by ELISA using monoclonal anti-human IgG subclass antibodies. Under the assay conditions used, the anti-IgG subclass antibodies were subclass specific. Quantitative estimates of the subclass antibodies were obtained by reference to a 'capture' assay using F(ab')2 anti-light chain antibody as ligand and IgG myelomas as standards. The validity of these estimates was supported by antibody quantitation using the Farr assay. In healthy adults with serum anti-OA or anti-BLG antibodies, anti-OA antibodies were found mainly in the IgG1 (9/11) and IgG4 (6/11) subclasses, whereas 5 sera showed high levels of IgG2 antibodies. In contrast, the IgG subclass distribution of anti-BLG antibodies was predominantly IgG4 (10/10).  相似文献   

2.
IgG antibody response to the inhalant allergen Parietaria judaica (Pj) and IgG subclass distribution were studied in 82 normal subjects, divided into three groups according to age (0–1, 1–20, and 20–60 years) and in 32 allergic subjects aged 20–60 years. Both normal and allergic subjects showed an IgG response, and all had IgG1 antibodies specific for PjE. Serum IgG2, IgG3, and IgG4 against PjE were detectable in 36%, 46%, and 22% of normal subjects, and in 58%, 31%, and 65% of allergic subjects, respectively. A significant difference in class distribution between allergic and age-matched normal subjects was found only for IgG4 antibodies against PjE (65% and 17%; P<0.01). The ELISA results were also analyzed quantitatively, taking into account the relative proportion of specific antibodies. Thus, in normal subjects IgG1 antibodies showed a decreasing trend as the age rose, while no differences according to the age of the subject were found for IgG2 and IgG4. When data from allergic subjects (20–60 years) and the age-matched normal group were compared, they were different for the relative percentage of IgG2 only, showing for this a significantly lower value (P< 0.001). The present data indicate that normal and allergic subjects show differences in the IgG isotype distribution depending on their sensitivity and duration of allergen exposure.  相似文献   

3.
U. TIIKKAINEN  M. KLOCKARS 《Allergy》1989,44(6):419-426
We describe the assay conditions for an enzyme-linked immunoassay for the determination of IgG and IgG subclass antibodies in serum to water-soluble wheat flour antigens. The optimal antigen coating concentration was 5 micrograms/ml for total IgG, IgG1, IgG4 and 100 micrograms/ml for IgG2. Serial dilutions of test sera were used and commercially available monoclonal mouse anti-human IgG isotype antibodies (as ascites fluid) were diluted 1:500-1:1000. Specific wheat flour antibodies belonging to the IgG1, IgG2 and IgG4 subclasses were detected. Despite the lack of standardized isotype-specific second mouse monoclonal antibodies, the subclass antibody levels between flour-exposed bakers and controls could be compared. We observed significantly higher IgG1, IgG2, and IgG4 subclass antibodies among 23 bakers than among 12 non-exposed controls, but no IgG3 antibodies were detected. The differences in biological activities of the IgG subclass antibodies may explain the clinical and pathophysiological features for fluor-induced occupational allergic diseases among bakers.  相似文献   

4.
U. Tiikkainen  M. Klockers 《Allergy》1990,45(7):497-504
We measured the IgG subclass antibody levels to wheat flour in 42 bakers and 20 controls with an enzyme immunoassay. The levels of total IgG, IgG1 IgG2 and IgG4 antibodies were significantly higher in the bakers than in the unexposed controls. The presence of anti-wheat flour IgG subclass antibodies in the bakers was correlated with various clinical variables including IgE levels, duration of asthmatic or rhinitis symptoms, skin prick test response, peripheral blood eosinophil levels, bronchial histamine reactivity and responses to nasal challenge with wheat flour. The IgG subclass antibody levels of the total cohort of bakers did not correlate with any of the measured clinical variables. However, among men specific IgG4 and IgG1 antibody levels correlated negatively with total IgE levels and duration of rhinitis, respectively. We conclude that IgG and IgG subclass levels to wheat flour in bakers reflect exposure, but that it is not related to any specific clinical situation. The exact pathogenic role of these antibodies in the development of occupational asthma and rhinitis is thus not clear.  相似文献   

5.
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.  相似文献   

6.
The distribution and affinity of IgG subclasses against various aeroallergens were assessed by inhibition of specific antibody binding. Two parameters from the dose-response curves were taken as indicative of antibody affinity: the point of 50% inhibition and the value of the slopes on double-log plots. It was found that IgG4 antibody specific for aeroallergens (i.e., from pollens of several species of Gramineae, Olea europaea , and Parietaria judaica and from house dust) usually exhibits high affinity, except for Dermatophagoides pteronyssinus . High binding affinity was also displayed by IgGl subclass antibodies against the allergens of O. europaea and P. judaica . Distinct IgG subclass affinity profiles were observed for the allergens of grass pollen (i.e., Holcus lanatus ) and dust mites (i.e., D. pteronyssinus ). These results demonstrate that IgG subclass distribution, as well as antibody affinity, depends on the nature of the sensitizing allergen.  相似文献   

7.
Immunoenzymatic assays were developed for the measurement of antibodies against mycobacterial lipoarabinomannan (LAM), a cell-free proteic extract (CFX) of Mycobacterium leprae, and the 38-kD protein antigen of M. tuberculosis. Sera from 108 leprosy patients, belonging to all clinical–immunological forms of the spectrum, and 81 patients with localized or disseminated tuberculosis (TB) were tested for antibodies of the four IgG subclasses. Standard calibration curves were used to allow comparisons between results of different isotypes and specificities. Mean concentrations of total IgG antibodies were higher in the overall leprosy population than in TB patients. In leprosy, levels of anti-CFX increased from tuberculoid toward lepromatous forms, with a clear switch from IgG1 to IgG2 subclass predominance. A similar IgG1 to IgG2 conversion was observed in anti-LAM antibodies, although total levels of anti-LAM were similar in patients with tuberculoid and lepromatous forms. In TB, antibodies against polysaccharide and protein antigens were both predominantly of IgG1 subclass, whatever the patient's clinical status, although lower in disseminated forms, probably due to concomitant HIV infection. A hypergammaglobulinaemia was also found in most leprosy and TB patients. In TB this was due to increased IgG1 and IgG3, especially in HIV co-infected patients. Based on the current knowledge of the influence of T cell-secreted cytokines on human immunoglobulin isotype expression, these results do not fit with a putative role of Th1 (such as found in TB and tuberculoid leprosy (TT)) and Th2 (such as found in leprosy lepromatous (LL) leprosy) environment in the isotypy of antibody responses in mycobacterial infections. Nor do variations of isotypy according to pathological conditions seem to be related to the biochemical nature of antigens, since antibodies to LAM and protein antigens had comparable evolutions of their subclass distribution. Other factors are to be investigated in order to understand better the significance and possible roles of antibodies in mycobacterial diseases.  相似文献   

8.
ELISA is increasingly used to measure antibodies in new circumstances. Recently, it has been applied to the measurement of IgG subclass antibodies against common antigens in early childhood. These studies have raised concerns about the specificity of some of these assays. This paper details the results of experiments which have assessed the specificity of IgG 1 binding to allergens of dust mite ( Dermatophagoides pteronyssinus ) and ryegrass ( Lolium perenne ) pollen by inhibition ELISA in the sera of 2-year-old children of atopic parents. Six sera which showed binding of IgG 1 to D. pteronyssinus and six to L. perenne were used. All had IgG1 antibody against ovalbumin. In the children's sera, binding to D. pteronyssinus was substantially inhibited by preincubation with the homologous antigen, but not with ovalbumin, thereby confirming the specificity of the assay. However, suppression of IgGl binding to L. perenne with the homologous antigen was comparatively small, and ovalbumin could cause an equivalent inhibition, indicating poor specificity. Furthermore, the level of IgG1 binding to L. perenne was closely correlated to the level of IgG1 binding to ovalbumin ( r = 0.98; P < 0.001). When the assay was reversed, IgG1 binding to ovalbumin was only slightly inhibited by L. perenne , indicating that most antibody binding to ovalbumin was specific. Thus, binding IgG1 in both adult and child sera to D. pteronyssinus appeared to be specific, while child, but not adult, IgG1 binding to L. perenne showed poor specificity. This disparity may be due to differences in the affinities of the respective antibodies, and it illustrates the importance of determining assay specificity when making measurements in early childhood.  相似文献   

9.
In this study we describe an ELISA using monoclonal antibodies to IgG 1, 2, 3, 4, IgA1 and IgA2 for determining the subclass distribution of human-specific antibodies. No cross-reactivity of the subclass-specific reagents under the conditions used was observed. The sensitivity was 0.5 ng/ml for IgG1, 3, 4; 1.5 ng/ml for IgG2 and 50 ng/ml for IgA1 and IgA2. The reproducibility as described by the coefficient of variation calculated on repeated runs was 8-26% if the data were obtained by relating the absorbance values to a positive serum run in the assay, 17-58% when relating the OD figures to those of a standard myeloma plate. The method may be considered semiquantitative with high sensitivity and specificity, easy to handle and with small day-to-day variation. The assay has been applied to a number of antigens of protein and polysaccharide nature.  相似文献   

10.
We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93·4% sensitivity, 93·7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG I> IgG2> IgG4.  相似文献   

11.
A characteristic feature of Wegener's granulomatosis is the presence of antineutrophil cytoplasm antibodies (ANCA) to proteinase 3 (PR3). In vitro, ANCA activate neutrophils by co-ligating PR3 and FcgammaRIIa/IIIb receptors. ANCA are predominantly of the IgG isotype, and IgG1, IgG3 and IgG4 subclasses are particularly represented. To address the pathogenic role of individual ANCA-IgG subclass antibodies, patients' sera were screened using indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and subclass PR3-ELISA to identify patients with high titres of PR3-ANCA within the IgG1, IgG3 or IgG4 subclasses. Unfractionated ANCA-IgG and subclass fractions were isolated by affinity chromatography and compared for their capacities to stimulate superoxide production by primed human neutrophils. Donor neutrophils were analysed for constitutive and induced FcgammaRI expression by flow cytometry. The IgG1, IgG3 and IgG4 subclass fractions, isolated from three different ANCA sera, each stimulated superoxide production from neutrophils derived from multiple donors. Subsequently, IgG4 subclass fractions isolated from a further four ANCA positive sera demonstrated varying abilities to stimulate release of superoxide; unrelated to PR3-ANCA titre, neutrophil donor, or neutrophil FcgammaRI expression. The stimulation of superoxide release by IgG1- and IgG3-ANCA subclass fractions is consistent with the proposed mechanism of co-ligation of PR3 antigen and FcgammaRIIa/IIIb receptors. However, the demonstration of similar activity for the IgG4-ANCA subclass fractions isolated from some sera was unexpected. This activity was independent of neutrophil donor and expression of FcgammaRI, suggesting it was capable of activating neutrophils via constitutively expressed FcgammaRIIa/IIIb or co-ligation of other, unidentified, cell surface molecules.  相似文献   

12.
Prospectively, serum levels of IgE, specific IgE antibodies (AB) to whole cow milk protein (CMP), bovine se-albumin, bovine immunoglobulin, bovine lactoferrin, bovine lactalbumin and beta-lactoglobulin (BLG), IgG and IgG subclass antibodies to ovalbumin (OA) and BLG, and IgG4 RAST to CMP (bovine whey) were measured in 39 infants with cow milk protein allergy (CMPA) at birth (cord blood), at time of diagnosis and before and after milk challenge at the age of 12 months. Immunological measurements were also undertaken in 33 control infants without CMPA at birth, at 6 months and at 18 months. At no time, were differences found between the levels of IgG and IgG subclass AB to OA and BLG in control versus infants with CMPA. In the 39 infants with CMPA no correlation was found between the levels of IgE, IgG and IgG subclass AB in cord blood and subsequent levels of these values, irrespective of the type of CMPA (IgE-mediated (CMA) or non-IgE-mediated (CMI)), and irrespective of whether remission had occurred. In cord blood 25/33 (76%) of the infants with CMPA had specific IgE-AB to one or more of the bovine milk proteins indicating a prenatal intrauterine sensitization to cow milk protein. At 6 months the frequency of specific IgE-AB to bovine milk proteins was significantly (p less than 0.05) higher in infants with CMA versus CMI, and at 12 months total serum-IgE and the increase of these specific IGE-AB and RAST to CMP were significantly higher (p less than 0.05) in infants with persistent CMA. From 6 to 12 months withholding milk resulted in a significant fall in specific IgE-AB to CMP, and IgG, IgG1 and IgG4 anti-BLG followed by an increase after milk challenge. Decreasing levels of IgG anti-OA from birth to 6 months reflect passive maternal transfer of IgG through the placenta, and increasing levels of IgG anti-BLG, already from birth to 6 months, may represent an early exposure to CMP in all infants. Significantly higher levels (p less than 0.05) of IgG anti-OA AB, IgG1 and IgG4 anti-BLG AB were found in infants with persistent CMA, indicating a close relation between the synthesis of IgE and IgG and between IgE and IgG subclasses (IgG1 and IgG4) in symptomatic cow milk-allergic individuals.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

13.
Mouse monoclonal antibodies (MAb) specific for each of the four human IgG subclasses and immunofluorescence flow cytometry were used to evaluate the subclass of the IgG antibody response to sperm in serum samples from 13 men and 6 women with a high titer (greater than 1:15,625) of IgG antisperm antibodies (ASA] determined by an indirect immunobead test. Five sera without ASA were also studied as a control. All 19 (100%) of the ASA-positive sera contained immunoglobulin (Ig)G ASA of the IgG1 and IgG3 subclasses. A 1:1 correlation was observed between the presence of IgG1 and IgG3 ASA. IgG2 was essentially undetectable, while IgG4 reactivity, although less intense than IgG1 and IgG3, was more prominent in the sera from the five vasectomized men. The ability of the IgG1 and IgG3 ASA-positive sera to deposit complement (C) on sperm was demonstrated by the concomitant binding to antibody-laden sperm of polyclonal antibodies to the membrane attack complex (C5b-9) of C. Both C-fixing and non-C-fixing ASA-positive sera were found to possess IgG1 and IgG3 antisperm antibodies. The predominance of IgG1 and IgG3 subclasses suggested a T-cell dependent immune response to sperm antigens.  相似文献   

14.
A standard enzyme-linked immunosorbent assay method for anti-Aspergillus fumigatus IgG in human serum was modified to produce a quantitative assay. The resulting assay was reproducible and capable of separating individual precipitin line groups and provided a means of monitoring the variation in antibody levels over long periods in patients with pulmonary aspergillosis.  相似文献   

15.
Twenty-one IgG subclass-deficient adult patients with repeated infections of the respiratory tract, were immunized with Haemophilus influenzae type b capsular polysaccharide (HibCP) covalently bound to tetanus toxoid (TT). Specific immunoglobulin and IgG subclasses to HibCP and TT were quantified; the biological activities of HibCP antibodies were also investigated. Most patients showed an antibody response similar to that observed in healthy adults, and the bactericidal activity related to the post-immunization levels of HibCP antibodies. No relation was found between immunoglobulin isotype deficiency, the clinical symptoms and the IgG subclass responsiveness, and no relation was observed between HibCP and TT antibody responses. Our data indicate that some, but not all, patients with recurrent infections and IgG subclass deficiency have an abnormal serum antibody response to polysaccharide and protein epitopes of Hib-TT conjugate vaccine. Analysis of the antibody response after vaccination with HibCP-TT conjugate vaccine did not seem to predict the clinical course of such patients.  相似文献   

16.
Background: Different serological tests are used in serologic diagnosis of brucellosis. The most widely used of these are Standard Tube Agglutination and Coombs anti-brucella tests. Whereas ELISA Ig M and Ig G tests have been in use for a long time, immuncapture agglutination test has been recently introduced and used in serological diagnosis. The aim of this study was to compare diagnostic values of ELISA Ig M and Ig G and immuncapture agglutination tests with Coombs anti-brucella test.Methods: Sera from 200 patients with presumptive diagnosis of brucellosis were included into the study. Coombs anti-brucella test, ELISA Ig M and Ig G tests and Immuncapture test were investigated in these sera. Then, sensitivity, specificity, negative predictive and positive predictive values were calculated.Results: Sensitivity, specificity, negative predictive and positive predictive values were found to be 90,6 %, 76,3 %, 94,2 %, and 65,9 % respectively for the Immuncapture test, whereas they were found to be 73,7 %, 58,9 %, 84,2 %, and 42,8 % for Ig G and 72,2 %, 67,8 %, 85,2 %, and 48,7 % for Ig M. The Immuncapture test was found to be compatible with ELISA Ig M and Ig G tests but it was statistically incompatible with Coombs anti-brucella test.Conclusions: Immuncapture agglutination test yields similar results to those of Coombs anti-brucella test. This test is a useful test by virtue of the fact that it determines blocking antibodies in the diagnosis and follow-up of brucellosis.  相似文献   

17.
Enzyme-linked immunosorbent assays (ELISAs) were used to detect antibodies to the C6 peptide of the Borrelia burgdorferi VlsE protein and a selection of B. burgdorferi IgG antigens, separately and as a combination, in 355 serum specimens from blood donors and patients. Western immunoblotting was used as the reference method. The sensitivity of the combined analysis of IgG antigen and C6 peptide analysis was markedly superior to those of the separate analyses. When the C6 peptide and IgG results were concordant, the customary confirmatory Western immunoblotting assay could be omitted, thus reducing the time and cost of analysis.  相似文献   

18.
McAb ELISA间接夹心法检测HFRS病人血清IgM和IgG抗体的研究   总被引:2,自引:0,他引:2  
肾综合征出血热(HFRS)早期临床症状不典型,因此在病人血清中检出特异性抗体,特别是IgM抗体对辅助临床诊断有重要意义。此外,HFRS的流行病学监测和病原学研究等都需有敏感、特异、简便而客观的  相似文献   

19.
In 47 children with coeliac disease, IgG1 was the dominant IgG subclass (mean 61%) in antibodies to gliadin, a protein component of wheat; IgG2 (22%) and IgG3 (15%) were also found. Very little IgG4 (3%) was detected. Both G2m(n)allotype and the age of the patient had an independent effect on the subclass distribution. The effects of these two factors seem similar to those described earlier on responses to polysaccharides; both G2m(n) and age increase the expression of IgG2 antibodies and concomitantly the share of IgG1 is decreased. The average increase in the share of IgG2 brought about by n/n-genotype was 25.3% (95% confidence limits 12.2-38.4%; P less than 0.001) and that brought about by each additional year of age of the patient 1.1% (0.3-1.9%; P = 0.006). IgG antibodies to beta-lactoglobulin and ovalbumin consisted mainly of IgG1 and IgG4. The titres of the IgG subclasses were low and in many cases below detection level. No association with G2m(n) genotype and share of IgG1 or IgG4 could be demonstrated. Age was inversely correlated with the shares of IgG1 in antibodies to beta-lactoglobulin and ovalbumin.  相似文献   

20.
Routine determination of IgG and IgM levels is likely to be included in future international immunotoxicity assessment guidelines with the rat as the required animal species. Only a few historical data are available on these two parameters in the rat. We describe a quantitative ELISA sandwich method using commercially available purified rat immunoglobulin as standards and provide the normal range for IgG and IgM values in healthy untreated 10 ± 2 to 30 ± 3 weekold Sprague-Dawley rats. Our data indicate that both IgM and IgG levels increase gradually with age. In addition, we determined IgG values in rats hyperimmunised with protamine, lysozyme and casein.  相似文献   

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