Specific IgE density assay: A new reverse enzyme allergosorbent test-based procedure for the quantitative detection of allergen-specific IgE

A new method is described for the quantitative detection of IgE antibodies, based on IgE capture with a specific antibody, reaction with liquid-biotinylated allergens and biotinylated anti-IgE and immunoenzymatic development of the reaction (reverse enzyme allergosorbent test). Using a reference system based on the World Health Organization IgE International standard, this method determines total IgE in the range 2-100kU/L and specific IgE in the range 0.2-100 kU/L, from which the specific/total ratio, called 'specific IgE density', can be calculated. This procedure has been applied to the study of specific IgE in 23 sera from patients polysensitized to pollen and mite allergens: 11 with asthma and 12 with rhinitis. The sensitivity and reproducibility of the method were evaluted. Sera from asthmatic patients showed higher cumulative levels of specific IgE (mean density 57.7%) than sera from rhinitic patients (mean density 32.6%). The clinical significance of specific IgE density in patients with multiple sensitizations is discussed.     

A new ImmunoCAP assay for detection of Echinococcus multilocularis-specific IgE

In alveolar echinococcosis (AE), caused by Echinococcus multilocularis (E.m.), increased levels of total and parasite-specific IgE are frequently found. These may not only have diagnostic but are also supposed to have prognostic value in the follow-up of AE patients. However, there is no commercial test available for quantification of E. m.-specific IgE (sIgE). The only commercial test available is based on E. granulosus (g.) hydatid antigen, which is not optimal for detection of E. m.-specific IgE. Therefore, a new ImmunoCAP with covalently bound crude antigen of E. m. was developed in cooperation with Pharmacia Research Forum for the analysis of E. m. sIgE. The E. m. ImmunoCAP was evaluated in 53 AE patients with different clinical disease progression and 20 healthy controls. Our data showed a higher sensitivity for sIgE determination with E. m. ImmunoCAP compared to the E. g. ImmunoCAP (73.6% vs 61.5%) and a positive correlation between total IgE and sIgE. Furthermore, there was a significant correlation between sIgE in both tests. In conclusion, the new E. m.-specific ImmunoCAP test proved to be a valuable tool for determination of sIgE. It may provide the basis for the development of further E. multilocularis-specific IgE immunotests which are essential for evaluation of sIgE during clinical course of AE.     

In vitro methods for specific IgE detection in cow's milk allergy

BackgroundA new method for determining serum specific IgE (IMMULITE^® 2000 3gAllergy) has recently become available.ObjectiveTo evaluate the clinical performance of IMMULITE 2000 in the diagnosis of cow's milk allergy compared with that of UniCAP^®. Additionally, we verified the behavior of both methods at two diagnostic decision points proposed by other authors.MethodsThe study population consisted of 31 children with cow's milk allergy (group A) and a control group of 19 atopic children without food allergy (group B). A blood sample from each child was tested using both methods and the results were compared.ResultsIn group A, the values for cow's milk IgE ranged from 0.35 kU/L (the lowest common detection limit) to above 100 kU/L. In group B, the values were less than 1.1 kU/L for IMMULITE 2000 and less than 1.6 kU/L for UniCAP. An agreement of 90 % in IgE classes was obtained. Both methods demonstrated exactly the same diagnostic performance (sensitivity: 100 %; specificity: 78.9 %; negative predictive value: 100 %; positive predictive value: 84.6%; efficiency: 90.2 %). The evaluation of the two methods at the two different decision points proposed in the literature showed a better positive predictive value with UniCAP, but we obtained equivalent performance with IMMULITE 2000 by choosing higher cutoff values.ConclusionsWe conclude that IMMULITE 2000 is as effective as UniCAP in the diagnosis of cow's milk allergy. Both methods can be used to obtain site-specific decision points that are population, age and disease dependent.     

Value of quantitative IgE measurement

The results of assay of IgE can only be expressed by a very imprecise system of classes. Quantification of the measurement of IgE by the method of the CAP-system of prognosis of allergic patients. Expression of IgE by a measurement of intensity of fluorescence allows the establishment of a decent curve for each allergen. It makes a method of predictive evaluation of risk of the allergy pathology for each allergen. It significantly improves the sensitivity, since it allows detection of IgE at levels less than 0.35 KUA/l. Thus it gives a considerable improvement for the clinician at the level of diagnosis avoiding the "gold standard" provocation test in allergy diagnosis as well at the level of prognosis.     

A new case of IgE myeloma

A new case of IgE myeloma is described. A 77-year-old woman presented with bone pain and fatigue. Serum protein analysis revealed a paraprotein of the IgE kappa type; bone marrow aspirate and immunofluorescence confirmed the diagnosis; ultrastructural examination showed immature plasma cells. Treatment with prednisone, melphalan, cyclophosphamide and interferon alfa did not produce any improvement and the patient died 5 months after diagnosis. The patient's clinical and laboratory data are compared with those of IgE myeloma cases reported in the literature.     

A role for measurement of nasal IgE antibodies in diagnosis of Alternaria-induced rhinitis in children

BackgroundRhinitis is a very common disease, frequently caused by sensitisation to inhalant allergens. Negative results from skin prick tests (SPT) and in vitro IgE tests generally lead to a diagnosis of non-allergic rhinitis. However, it is possible, as indicated by studies addressed with dust mites or pollens that the production of specific IgE occurs exclusively at nasal level.MethodsWe measured specific nasal IgE in children suffering from rhinitis in the periods when Alternaria spores were present in the air.All subjects underwent SPT with a standard panel of aeroallergens (Stallergenes, Milan, Italy) and, in the same session, to nasal IgE test (NT). Nasal provocation test (NPT) with Alternaria was used as reference.ResultsFifty-six subjects were included in the study. Of them, 20 (37.5%) were positive to SPT and 45 (80.3%) were positive to NT. In particular, 11 subjects (19.6%) had a positive SPT and a negative NT; 36 (64.3%) had a negative SPT and a positive NT; and 9 (16.1%) were positive to both tests. Positivity of NT and NPT was observed in 36 patients (69.6%), while positivity of SPT and NPT was observed in 15 patients (26.8%). This difference was highly significant (p < 0.0001).ConclusionsThese findings suggest that sensitisation to Alternaria is frequently expressed by exclusive production of specific IgE in the nasal mucosa. Thus, measuring nasal IgE in children with rhinitis and negative SPT during the period of presence of Alternaria spores seems helpful to avoid a mistaken diagnosis of non-allergic rhinitis.     

Immunological detection of in vitro formed phosphatidylethanol--an alcohol biomarker--with monoclonal antibodies

Background:  Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography–mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development.
Methods:  C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis.
Results:  We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth.
Conclusions:  The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes.     

A staphylococcal slide test for detection of antineutrophil antibodies

We describe a simple test for direct or indrect detection of antineutrophil antibodies. Sensitized leukocytes adherent to glass slides and fixed with paraformaldehyde can be stored in buffer for at least 3 wk. Killed Cowan I staphylococci, containing protein A, bind to sensitized but not control cells, and binding is ascertainable by light microscopy. Indirect tests were positive for 39/41 patients suspected of having immune neutropenia and found to have antineutrophil antibodies by an indirect radiochemical opsonic method. Fifty-four control sera from healthy persons, patients with bone marrow failure, or with immune complex diseases without neutropenia, gave negative indirect tests. Direct tests for cell-bound antibody could be done even during severe neutropenia by reacting fixed autologous cells with staphylococci in the absence of added serum. In some patients only the direct test was positive.     

Characterization of allergens of the cat flea, Ctenocephalides felis: detection and frequency of IgE antibodies in canine sera

Flea allergens, fractionated by polyacrylamide gel electro-phoresis and transferred to nitrocellulose, were identified using 20 flea-allergic dog sera in an enhanced chemilumi-nescent assay for canine IgE antibodies. At least 15 different flea components in the molecular weight range of 14–150 K bound IgE and every serum demonstrated a different pattern of binding. Three of the components with apparent molecular weights of 25, 40 and 58 K were each bound by at least 40% of the sera. No reactivity was seen when normal dog sera were used. These results demonstrate a greater number of flea allergens and a far greater diversity of the IgE antibody response to flea allergens than has previously been described, and suggest that immediate hypersensitivity may be an important mechanism in the pathogenesis of canine flea allergy.     

Serum IgE anti-cartilage collagen antibodies in rheumatoid patients

Summary The presence of circulating IgG, IgA and IgM antibodies to native cartilage collagens in some patients with rheumatoid arthritis (RA) suggests that an autoimmune response to cartilage collagens may be involved in the pathogenesis of RA. However, the relevance of such antibodies to the pathological process remains unclear, and it is likely that many humoral and cellular derived factors combine to trigger events leading to the chronicity of the rheumatoid lesion. Since histological and biochemcal studies have suggested the involvement of mast cells in the rheumatoid joint, we have studied the frequency of IgE antibodies directed against the cartilage collagens type II, IX and XI in patients with active rheumatoid disease. Of the 91 patients' sera tested, 32 had significant levels of IgE anti-cartilage collagen antibodies when compared with non-arthritic controls. Total serum IgE levels did not correlate with the presence of IgE anti-collagen antibodies, nor were any patients positive for IgE antibodies to fibronectin, a widely distributed extracellular matrix component. These results are consistent with an allergic type I hypersensitivity reaction to cartilage antigens in RA involving mast cell and basophil degranulation.     

125 I-anti-immunoglobulin test: a new tool for the detection of drug-allergic platelet antibodies.

It is shown that the 125I-anti-immunoglobulin test with platelets is as sensitive and reproducible for the detection of quinidine-associated antibodies as platelet complement fixation. Because of its independence from complement activation, this test might prove most valuable for the demonstration of non-complement-fixing, drug-associated antibodies.     

A new quantitative measurement for surgical needle ductility

Because surgical needle ductility is such an important factor in the selection of surgical needles, a new curved needle ductility test instrument was devised to measure the ductility of needles from three commercial manufacturers. The superior ductility of needles made by one manufacturer was related to the specific alloy, stainless steel ASTM 45500, used in their production. The ASTM 45500 stainless steel has a significantly greater tensile and yield strength than the other stainless steel alloys, accounting for its superior ductility.     

A comparison of assays used for the detection of antibodies to DNA

Summary To determine whether the osteopenia of rheumatoid arthritis (RA) is due to reduction of trabecular bone mass (TBV) and/or cortical width (CW), we evaluated these parameters by bone histomorphometry; we also measured the calciotropic hormones parathormone(PTH) and calcitonin (CT), vitamin D [25(OH)D] and the biological markers of bone remodeling in a group of patients with RA. Study subjects were divided into Group C — premenopausal patients, and Group A — menopausal patients and men of the same ages. These groups were compared to two agematched control groups, B and D. In both A vs. B and C vs. D, TBV and CW were significantly lower in patients. There were no differences in PTH or CT, but 25(OH)D was significantly reduced, and BGP, OHP/Cr and AP were raised in patients. Patients also exhibited TBV loss in more than 55% and CW loss in more than 98%. These changes suggest that the decline in bone mass, mainly cortical, but also trabecular, is due to increased bone turnover and enhanced resorption and seem to reflect intrinsic alterations of RA.