Management of primary antibody deficiency by consultant immunologists in the United Kingdom: a paradigm for other rare diseases.
Variation in clinical practice and its effect on outcome is little known for rare diseases such as primary antibody deficiency. As part of a national audit a survey of all 30 consultant immunologists in the United Kingdom dealing with primary antibody deficiency syndromes in adults and children was carried out in 1993 to ascertain their practices in diagnosis and management. Consensus guidelines were published after the survey was completed. Comparison of the survey results of clinical practice at the time the guidelines were published with the standards identified highlighted that the practice of a minority of specialists was at variance with their peers and with the consensus document, particularly in the use of intramuscular immunoglobulin, the dose and frequency of intravenous immunoglobulin, and target trough immunoglobulin G concentration, which has implications for the quality of patient care. However, much closer agreement existed in the key areas of management, such as diagnosis and selection of intravenous immunoglobulin. The approach and the problems identified are relevant to the management of other rare diseases, in which diagnosis and management is complex and there are few specialists with the necessary knowledge to undertake such care. This survey, the first attempted audit of practice, shows that within a motivated group of specialists highly significant differences in practice may exist and the authors emphasise the importance of setting clear guidelines against which care can be assessed. (+info)
Detection of allergen-induced basophil activation by expression of CD63 antigen using a tricolour flow cytometric method.
In the field of allergy diagnosis, most in vitro functional tests are focused on basophils. Nevertheless, the very small number of circulating basophils limits these experiments and their clinical benefit remains controversial. As flow cytometry is a valuable tool for identifying cell populations, even at low concentrations, we developed a tricolour flow cytometric method for the study of allergen-induced basophil activation. Identification of cells was based both on CD45 expression and on the presence of IgE on the cell surface, since basophils express high-affinity receptors for IgE (Fc epsilon RI). Cell activation upon allergen challenge was assessed by the expression of CD63 antigen on the plasma membrane. Basophil isolation and activation (with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine) were validated in 32 non-allergic patients. In 12 allergic patients, basophil stimulation by a relevant allergen was in most cases positive (10/12). Furthermore a concentration-dependent hook effect was observed. Of the allergic and non-allergic patients, none showed non-specific activation with an irrelevant allergen (specificity 100%). Overall, our preliminary results, even in a small population, suggest that this is a reliable and valuable method for the diagnosis of allergies complementing specific allergen IgE and skin test results. Obviously, additional clinical studies are needed to validate these first results. (+info)
New immunological assays for the diagnosis of Helicobacter pylori infection.
There are several types of immunological tests available for the diagnosis and management of Helicobacter pylori infection. Most commercially available serological kits use the enzyme linked immunosorbent assay (ELISA) test format. Originally the kits used crude antigen preparations although many of the newer kits use a more purified antigen preparation, with often increased specificity but lower sensitivity. Near patient test kits are based either on latex agglutination or immunochromatography. Generally they have low sensitivities compared with laboratory tests. Western blotting, ELISA, and recombinant immunoblot assays (RIBA) have also been developed into commercially available kits and can be used to indicate the presence of specific virulence markers. An antigen detection kit has been developed for the detection of Helicobacter pylori in faeces. Immunological reagents have also been combined with other diagnostic modalities to develop immunohistochemical stains and DNA immunoassays. Helicobacter pylori is now recognised as the cause of gastritis and most cases of peptic ulcer disease (PUD); its long term carriage increases the risk of gastric adenocarcinoma sixfold and it is designated as a class I carcinogen. H pylori has also been implicated as a cause of gastric mucosa associated lymphoid tissue lymphomas. Its relation to non-ulcer dyspepsia remains controversial. Additionally, long term carriage of the organism may be associated with short stature in young girls and, in the general population, as a possible risk factor for the development of vasospastic disorders and possibly skin immunopathology such as urticaria. With the recognition of H pylori as an important human pathogen, it has become one of the growing number of organisms to have its complete genome sequence mapped. Serology is an important method of determining colonisation status and can be used for diagnosis, as a screening procedure, or to follow the efficacy of eradication regimens. Most serological assays are in the ELISA format although some are based on the latex agglutination reaction. These latter are used principally as near patient assays. Most assays detect IgG in serum although some detect serum IgA. More recently developed assays detect IgA in saliva and the production of affinity purified antibodies has led to the development of an antigen detection assay for faecal specimens. Serological reagents have also been used in immunocytochemistry and to speed up the detection of amplified products of the polymerase chain reaction (PCR)-DNA immunoassays. (+info)
A new method with general diagnostic utility for the calculation of immunoglobulin G avidity.
The reference method for immunoglobulin G (IgG) avidity determination includes reagent-consuming serum titration. Aiming at better IgG avidity diagnostics, we applied a logistic model for the reproduction of antibody titration curves. This method was tested with well-characterized serum panels for cytomegalovirus, Epstein-Barr virus, rubella virus, parvovirus B19, and Toxoplasma gondii. This approach for IgG avidity calculation is generally applicable and attains the diagnostic performance of the reference method while being less laborious and twice as cost-effective. (+info)
Detection of Francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a PCR.
The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida, Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp., Yersinia spp., Escherichia coli, and Burkholderia spp., was observed. The detection limit of the assay was 10(3) to 10(4) bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR. (+info)
Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus.
Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical. (+info)
Tissue-specific expression of c-series gangliosides in the extraneural system.
c-Series gangliosides in extraneural tissues from young and adult rats were examined using thin-layer chromatographic (TLC) immunostaining with a specific monoclonal antibody A2B5. The composition of c-series gangliosides significantly differed among tissues. In adult rats, while liver tissue contained GT1c, GQ1c, and GP1c, renal tissue had GT3 as the major c-series ganglioside with GT2 in a lesser amount. Pancreatic tissue expressed c-series gangliosides that consisted of GT3, GT2, GQ1c, and GP1c. In other tissues including adrenal, thyroid, and eye lens, GT3 constituted the main c-series ganglioside species. While total ganglioside contents of extraneural tissues were much lower than that of brain tissue, the proportions of c-series gangliosides to total gangliosides were higher in many extraneural tissues. Interestingly, eye lens had the highest GT3 content among rat tissues examined. The compositions and concentrations of c-series gangliosides in liver and kidney significantly differed between 5-day-old and 7-week-old rats, suggesting the development-dependent expression of c-series gangliosides in these tissues. These results suggest that the expression of c-series gangliosides in extraneural tissues is regulated in a tissue-specific manner. (+info)
Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides.
The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guerin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96, P < 0.0001 for ESAT-6; r = 0.98, P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6; r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost. (+info)