21 resultados para antibody specificity

em DI-fusion - The institutional repository of Université Libre de Bruxelles


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BACKGROUND: Persistent polyclonal B cell lymphocytosis (PPBL) is a rare condition characterized by increased IgM and large excess of B cells with an IgD(+) CD27(+) phenotype. In normal individuals, these cells play a central role in the defense against pneumococcal infection. So far, few studies have characterized humoral immune responses in PPBL patients. We therefore measured IgG directed against S. pneumoniae antigens in a 51 yr-old woman with PPBL before and after vaccination with a pneumococcal 23-valent polysaccharide vaccine. METHODS: Antibodies against pneumococcal antigens were measured first with an overall immunoassay using microplates coated with the 23-valent pneumococcal vaccine. A serotype-specific test was also performed according to the WHO consensus protocol. RESULTS: Despite a large number of IgD(+) CD27(+) cells, our patient had low baseline titers of IgG directed against pneumococcal antigens and did not significantly respond to a 23-valent polysaccharide vaccine against S. pneumoniae. On the contrary, she had good titers of IgG directed against tetanus toxoid. CONCLUSION: IgM(+) IgD(+) CD27(+) cells which accumulate in this patient with typical PPBL patient failed to perform IgG isotype switch after a polysaccharide vaccine. The potential mechanisms and relationships with the main features of PPBL are discussed. Further studies on a larger number of similar patients are needed.

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Overexpression and amplification of the HER-2 oncogene in patients with breast cancer has correlated with early onset of metastasis, resistance to hormonal therapy and some forms of chemotherapy, and shortened survival. Therefore, evaluation of this putative prognostic or predictive factor seems critical. Because different antibodies are used for the detection of the 185-kd HER-2 oncoprotein, we studied the sensitivity of 3 frequently used antibodies. Immunohistochemistry results were correlated with gene amplification level as assessed by fluorescence in situ hybridization. Protein overexpression was found in 17.2% and 12.5% of cases using antibodies against the external (TAB250) and internal (CB11) domains of the protein, respectively, and in 38.0% of cases using a rabbit polyclonal antibody. Fluorescence in situ hybridization was successful in all 160 tumors, and amplification was found in 37 tumors (23.1%). The monoclonal antibody TAB250 had the lowest misclassification rate, 9.6% (sensitivity, 67%; specificity, 97.5%).

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Journal Article

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Journal Article

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Peptide microarrays are useful tools for characterizing the humoral response against methylated antigens. They are usually prepared by printing unmodified and methylated peptides on substrates such as functionalized microscope glass slides. The preferential capture of antibodies by methylated peptides suggests the specific recognition of methylated epitopes. However, unmodified peptide epitopes can be masked due to their interaction with the substrate. The accessibility of unmodified peptides and thus the specificity of the recognition of methylated peptide epitopes can be probed using the in situ methylation procedure described here. Alternately, the in situ methylation of peptide microarrays allows probing the presence of antibodies directed toward methylated epitopes starting from easy-to-make and cost-effective unmodified peptide libraries. In situ methylation was performed using formaldehyde in the presence of sodium cyanoborohydride and nickel chloride. This chemical procedure converts lysine residues into mono- or dimethyl lysines.

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BACKGROUND: Tissue transglutaminase (t-TG) is the main autoantigen recognized by the endomysium antibodies (EMA) observed in patients with celiac disease (CD). The aim of the study was to assess an ELISA method for t-TG antibodies (t-TGA) with respect to EMA IF assay in pediatric and adult patients. METHODS: t-TGA were analyzed by ELISA in 220 sera samples: 82 patients with biopsy-proven untreated CD (23 adults and 59 children), 14 CD children on gluten-free diet, 18 asymptomatic relatives of CD patients, and 106 age-matched control patients with gluten-unrelated gastrointestinal diseases (58 adults and 48 children). Serum IgA EMA were tested on umbilical cord sections in all patients. RESULTS: The great majority (92.7%) of untreated CD patients (both adults and children) were t-TGA positive (values ranging from 20.1 to > 300 AU). None of the child control patients and only two out of 58 (3.4%) of the adults with unrelated gastrointestinal diseases had serum t-TGA positivity; two out of 18 first-degree relatives with biopsy-proved silent CD were t-TGA (as well as EMA) positive. Finally, two out of 14 CD children, assuming a gluten-free diet, had serum t-TGA (as well as EMA). A highly significant correlation (P < 0.001) was observed between t-TGA concentrations and EMA. t-TGA showed a sensitivity of 87% and 95%, a specificity of 97% and 100% for adults and children, respectively. CONCLUSION: The method is highly sensitive and specific in the diagnosis of CD and is promising as a tool for routine diagnostic use and population screening, especially in children.

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BACKGROUND: Serologic methods have been used widely to test for celiac disease and have gained importance in diagnostic definition and in new epidemiologic findings. However, there is no standardization, and there are no reference protocols and materials. METHODS: The European working group on Serological Screening for Celiac Disease has defined robust noncommercial test protocols for immunoglobulin (Ig)G and IgA gliadin antibodies and for IgA autoantibodies against endomysium and tissue transglutaminase. Standard curves were linear in the decisive range, and intra-assay variation coefficients were less than 5% to 10%. Calibration was performed with a group reference serum. Joint cutoff limits were used. Seven laboratories took part in the final collaborative study on 252 randomized sera classified by histology (103 pediatric and adult patients with active celiac disease, 89 disease control subjects, and 60 blood donors). RESULTS: IgA autoantibodies against endomysium and tissue transglutaminase rendered superior sensitivity (90% and 93%, respectively) and specificity (99% and 95%, respectively) over IgA and IgG gliadin antibodies. Tissue transglutaminase antibody testing showed superior receiver operating characteristic performance compared with gliadin antibodies. The K values for interlaboratory reproducibility showed superiority for IgA endomysium (0.93) in comparison with tissue transglutaminase antibodies (0.83) and gliadin antibodies (0.82 for IgG, 0.62 for IgA). CONCLUSIONS: Basic criteria of standardization and quality assessment must be fulfilled by any given test protocol proposed for serologic investigation of celiac disease. The working group has produced robust test protocols and reference materials available for standardization to further improve reliability of serologic testing for celiac disease.

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OBJECTIVE: Strict lifelong compliance to a gluten-free diet (GFD) minimizes the long-term risk of mortality, especially from lymphoma, in adult celiac disease (CD). Although serum IgA antitransglutaminase (IgA-tTG-ab), like antiendomysium (IgA-EMA) antibodies, are sensitive and specific screening tests for untreated CD, their reliability as predictors of strict compliance to and dietary transgressions from a GFD is not precisely known. We aimed to address this question in consecutively treated adult celiacs. METHODS: In a cross-sectional study, 95 non-IgA deficient adult (median age: 41 yr) celiacs on a GFD for at least 1 yr (median: 6 yr) were subjected to 1) a dietician-administered inquiry to pinpoint and quantify the number and levels of transgressions (classified as moderate or large, using as a cutoff value the median gluten amount ingested in the overall noncompliant patients of the series) over the previous 2 months, 2) a search for IgA-tTG-ab and -EMA, and 3) perendoscopic duodenal biopsies. The ability of both antibodies to discriminate celiacs with and without detected transgressions was described using receiver operating characteristic curves and quantified as to sensitivity and specificity, according to the level of transgressions. RESULTS: Forty (42%) patients strictly adhered to a GFD, 55 (58%) had committed transgressions, classified as moderate (< or = 18 g of gluten/2 months; median number 6) in 27 and large (>18 g; median number 69) in 28. IgA-tTG-ab and -EMA specificity (proportion of correct recognition of strictly compliant celiacs) was 0.97 and 0.98, respectively, and sensitivity (proportion of correct recognition of overall, moderate, and large levels of transgressions) was 0.52, 0.31, and 0.77, and 0.62, 0.37, and 0.86, respectively. IgA-tTG-ab and -EMA titers were correlated (p < 0.001) to transgression levels (r = 0.560 and R = 0.631, respectively) and one to another (p < 0.001) in the whole patient population (r = 0.834, N = 84) as in the noncompliant (r = 0.915, N = 48) group. Specificity and sensitivity of IgA-tTG-ab and IgA-EMA for recognition of total villous atrophy in patients under a GFD were 0.90 and 0.91, and 0.60 and 0.73, respectively. CONCLUSIONS: In adult CD patients on a GFD, IgA-tTG-ab are poor predictors of dietary transgressions. Their negativity is a falsely secure marker of strict diet compliance.

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OBJECTIVE: To investigate the value of serum antitissue transglutaminase IgA antibodies (IgA-TTG) and IgA antiendomysial antibodies (IgA-EMA) in the diagnosis of coeliac disease in cohorts from different geographical areas in Europe. The setting allowed a further comparison between the antibody results and the conventional small-intestinal histology. METHODS: A total of 144 cases with coeliac disease [median age 19.5 years (range 0.9-81.4)], and 127 disease controls [median age 29.2 years (range 0.5-79.0)], were recruited, on the basis of biopsy, from 13 centres in nine countries. All biopsy specimens were re-evaluated and classified blindly a second time by two investigators. IgA-TTG were determined by ELISA with human recombinant antigen and IgA-EMA by an immunofluorescence test with human umbilical cord as antigen. RESULTS: The quality of the biopsy specimens was not acceptable in 29 (10.7%) of 271 cases and a reliable judgement could not be made, mainly due to poor orientation of the samples. The primary clinical diagnosis and the second classification of the biopsy specimens were divergent in nine cases, and one patient was initially enrolled in the wrong group. Thus, 126 coeliac patients and 106 controls, verified by biopsy, remained for final analysis. The sensitivity of IgA-TTG was 94% and IgA-EMA 89%, the specificity was 99% and 98%, respectively. CONCLUSIONS: Serum IgA-TTG measurement is effective and at least as good as IgA-EMA in the identification of coeliac disease. Due to a high percentage of poor histological specimens, the diagnosis of coeliac disease should not depend only on biopsy, but in addition the clinical picture and serology should be considered.

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Anti-neutrophil cytoplasmic antibodies (ANCA) are diagnostic markers for systemic vasculitis. They are classically I detected by an indirect immunofluorescence test using normal donor neutrophils as substrate. This assay lacks antigenic specificity and is not quantitative. The 'EC/BCR Project for ANCA Assay Standardization' is an international collaboration study with the aim to develop and standardize solid phase assays for ANCA detection. In this part of the study the isolation and characterization of proteinase-3 and myeloperoxidase, the two main target molecules for ANCA, and the development and standardization of ELISAs with these antigens are described. Six laboratories successfully isolated purified proteinase-3 preparations that could be used. Three of these preparations, together with one myeloperoxidase preparation, were subsequently used for ANCA testing by ELISA. The ELISA technique was standardized in two rounds of testing in the 14 participating laboratories. The coefficient of variation of these new assays decreased from values of approx. 50% in the first round to approx. 20% in the second round. We conclude that purified proteinase-3 and myeloperoxidase can be used in standardized ELISAs for ANCA detection. Whether such procedures offer advantages over the IIF test will be determined in a prospective clinical study.

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To evaluate the immunogenicity and safety of a 23-valent pneumococcal vaccine in human immunodeficiency virus (HIV)-seropositive patients, 80 men and 18 women received 1 dose of the vaccine (Pneumo 23; Pasteur Mérieux MSD, Brussels). The total IgG antibody response against all 23 Streptococcus pneumoniae capsular antigens was measured. Antibody levels were expressed in arbitrary units per microliter, referring to a standard curve. Geometric mean titers of the total IgG capsular antibodies on the day of vaccination and 30-45 days later were compared. The ratios of titers after and before vaccination in patients with > 500, 200-500, and < 200 CD4 lymphocytes/microL were 10, 10, and 12.6, respectively. Nonresponse (ratio < 4) occurred in 17% of patients and was unrelated to CD4 cell count. The vaccine was well tolerated; no serious side effects occurred. In 83% of the patients with HIV infection, the total antipneumococcal IgG level was higher after vaccination.

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Individuals with genetic defects in CD40 ligand (CD40L) or B-cell antigen receptor coreceptor molecules CD19 and CD81 suffer from an antibody deficiency. Still, these patients carry low levels of memory B cells and serum antibodies.

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Two different types of pertussis vaccines are currently available to protect children against whooping cough, the first-generation whole-cell (Pw) vaccines and the more recent acellular (Pa) vaccines. Both types provide good protection, yet induce different types of immune responses in 6-month-old infants, with a strong Th1 response induced by Pw vaccines compared to a mixed Th1/Th2 response and a delay in non-specific IFN-gamma secretions after the administration of Pa vaccines. We show here that at 13 months of age, most Pw- or Pa-vaccinated children display Bordetella pertussis-specific T-cell responses, in addition to significant antibody levels, although a higher Th2/Th1 cytokine ratio remained in Pa recipients compared to Pw recipients. In contrast, the proportion of children with tetanus toxin-specific T-cell responses was lower in Pa than in Pw vaccine recipients, although most children had protective anti-tetanus toxin IgG levels. In addition, the global Th2 bias observed in 6-month-old infants vaccinated with a Pa vaccine was normalized at 13 months.