Radioallergosorbent Technique Research Articles

In vitro analysis of mediators of contact urticaria caused by cefotiam hydrochloride

ABSTRACT Contact urticaria syndrome is a rare but potentially serious problem for hospital staff that handle antibiotics and other agents. While the cause is immunologic, little is known about the mediators involved. To investigate these mediators, three nurses with contact urticaria syndrome caused by exposure to cefotiam hydrochloride (CTM), and five normal controls were evaluated. IgE antibodies specific for CTM were detected by the radioallergosorbent technique (RAST). In response to CTM, cytokines were released from peripheral blood mononuclear cells (PBMC), and sulfidoleukotrienes were released from peripheral blood leukocytes. RAST counts in the three nurses with this syndrome exceeded those in the normal controls. The stimulation index of the leukotrienes released from the peripheral blood leukocytes in response to CTM also exceeded those in the normal controls. Higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) were also detected in the supernatant of the patients' PBMC. The results suggest that contact urticaria syndrome caused by exposure to CTM was induced by an immunologic mechanism via IgE antibodies, and that GM-CSF and leukotrienes may be involved in this reaction.

IgE antibody‐mediated shock reaction caused by topical application of chlorhexidine

A case of an anaphylactic shock following topical application of chlorhexidine preparation is reported. Specific skin-sensitizing antibodies against chlorhexidine were demonstrated in the serum from the patient by a passive transfer test. IgE antibodies against chlorhexidine were also detected by radioallergosorbent technique (RAST). Paper discs conjugated with chlorhexidine-HSA (human serum albumin) significantly bound the IgE antibodies. Furthermore, all of the sera from seven other patients with shock reactions following the topical application of chlorhexidine preparation also showed high RAST counts. Both chlorhexidine gluconate and chlorguanide which represents approximately half a molecule of chlorhexidine inhibited the reaction in a dose-dependent fashion. It is suggested that the shock reactions following topical application of chlorhexidine are mediated by IgE antibodies against chlorhexidine and that chlorhexidine and chlorguanide share the same antigenic determinant.

Evaluation Of Specific Ige Antibody In Clonorchis Sinensis Infection.

Serum IgE level and Clonorchis specific IgE in individuals with Clonorchis sinensis were determined by radioimmunosorbent(RIST) and radioallergosorbent technique(RAST) respectively. Highly significant elevations of serum IgE (P<0.001) and specific IgE antibodies (P<0.01) were observed in area from individuals with clonorchiasis. The mean values of serum IgE in individuals with clonorchiasis and healthy individuals were 2,372 IU/ml and 364 IU/ml respectively and specific IgE antibodies of both groups were 52.0 and 4.4%. A close correlation(r=0.9451) between serum IgE level and specific IgE antibodies were observed and correlation (r=0.6056) between serum IgE and EPG and between specific IgE and EPG(r=0.5693) were also observed.

Total and specific IgE levels in patients with atopic dermatitis. The correlation between prick testing, clinical history of allergy, and in vitro quantification of IgE during clinical exacerbation and remission.

Summary Ten young adults with chronic atopic dermatitis have been followed for up to 18 months through periods of clinical exacerbation and remission. Sequential estimation of total levels of circulating IgE and specific IgE by the radioallergosorbent technique (RAST) showed no significant correlation between IgE levels and severity of skin involvement. However, a clear correlation between clinical history, prick tests and RAST tests was obtained in several cases. Serum IgE levels in seven patients with ‘pure’ atopic dermatitis were elevated to levels similar to those found in three patients with additional respiratory symptoms.

IgE and IgE autoantibodies in patients with autoimmune thyroid disorders and their relatives.

We have investigated the possibility that immunoglobulin E (IgE) antibodies are involved in the pathogenesis of autoimmune thyroid diseases. For this purpose, the radioallergosorbent technique was used to search for IgE autoantibodies to various thyroidal antigens and to aggregated IgG in patients with Hashimoto's thyroiditis, Graves' disease, and their immediate relatives. Thyroidal antigens used were thyroglobulin, thyroid extract and thyroid homogenate. No definite evidence, however, was found for the presence of circulating IgE autoantibodies to these antigens.

Measurement of IgE Antibody by an Antigen-Binding Assay: Correlation with PK Activity and IgG and IgA Antibodies to Allergens

Abstract A quantitative radioimmunoassay for IgE antibodies (ab) is described. The assay measures IgE antigen-binding activity (BA) by using purified allergens radiolabeled with 125I, IgE myeloma serum as a source of carrier protein, and monospecific goat anti-IgE (anti-IgE) to precipitate the IgE. IgE BA for the Group I protein of rye grass pollen (Rye I) was measured in the serum of patients with grass pollen hay fever who had never received immunotherapy. Values for IgE BA were found to correlate well with radioallergosorbent technique (RAST) results (rs = 0.89, p&amp;lt;0.001). By assaying IgE-rich fractions, pure IgG, and whole serum, we found a good correlation between IgE BA, Prausnitz-Küstner (PK) activity, and total IgE. A positive PK test could be elicited with 0.05 ml of dilutions containing as little as 1 or 2 units IgE BA for Rye I/ml. PK testing was more sensitive than either RAST or the antigen-binding assay. Estimates based on the quantity of antigen bound in antigen excess suggested that 1 unit IgE BA represented 0.1 ng IgE ab. With higher concentrations of antigen (up to 1 µg/ml), however, estimates suggested that 1 unit IgE BA represented as much as 0.4 ng IgE ab. These higher estimates appeared to correlate reasonably well with estimates based on RAST elution or RAST absorption. The assay for IgE BA was directly comparable with assays for IgG BA and IgA BA. Results of these assays on sera from the patients showed that IgG BA for Rye I was detectable in 25 of 25 IgE BA in 24 of 25, and IgA BA in 21 of 25. Quantitatively we found a characteristic pattern IgG BA &amp;gt; IgE BA &amp;gt; IgA BA (geometric mean values 862, 214, and 60.2 units/ml, respectively). In keeping with this there was a highly significant quantitative correlation between IgG BA and IgE BA (r = 0.72, p&amp;lt;0.001). None of the sera from nonallergic persons contained detectable IgA or IgE BA for Rye I; however, 20% of these sera (10 of 48) contained IgG BA.

Allergens of mammalian origin: IV. Evidence for common allergens in cat and dog serum

IgE antibodies present in serum from 3 patients clinically sensitive to cat and dog were shown to combine with whole cat and dog sera linked to cellulose particles. Employing a modified radioallergosorbent technique (RAST), it was shown that cat and dog sera inhibited the binding of IgE antibodies to insolubilized cat serum and to insolubilized dog serum. These findings suggest that cat and dog sera have common allergens. Other mammalian sera were also tested and found to have little inhibitory activity. Cat and dog sera did not inhibit the binding of IgE antiragweed antibodies to an insolubilized ragweed fraction. Following separation of cat serum by gel filtration on Sephadex G-200 or electrophoresis in acrylamide gel, several fractions were found to inhibit the binding of IgE antibodies to insolubilized cat and dog sera. Whether the heterogeneity observed reflects the presence of single allergens with diverse size and charge properties or multiple allergens remains to be determined.

In vitro cross-allergenicity of major aeroallergenic pollens by the radioallergosorbent technique

Cross-reactivity between major classes of aeroallergenic pollens has been estimated by comparing the inhibitory effects of selected allergens upon the reaginic activity of other native and non-native varieties, as measured by the radioallergosorbent technique (RAST). Common allergenic determinants were demonstrated in indigenous and nonindigenous ragweed species. Although patterns of inhibitory capacity were less uniform among grass pollens, endemic species tended to have more common allergenic properties than nonendemic species. Tree pollens exhibited the greatest degree of heterogeneity, confirming the previous view that these pollens tend to be less cross-reactive. Concomitant skin test threshold studies utilising the same groups of pollens were in general agreement with in vitro results. Because threshold skin testing was affected by a number of uncontrollable variables, however, the current modification of RAST in estimating cross-allergenicity was more accurate and reliable than data based upon cutaneous tests. It was concluded that reagin neutralization by the RAST method is the best currently available method of assessing cross-allergenic properties of pollens.

Dextran-induced Anaphylactoid Reactions in Man

Dextran reactive antibodies (DRA) were studied in 123 patients having experienced dextran-induced anaphylactoid reactions (DIAR) during 1970-1975. No evidence for reaginic DRA was obtained by radioallergosorbent technique and passive cutaneous anaphylaxis in Cynomolgus monkeys; total IgE levels were within normal range. It is concluded that DIAR are not mediated by dextran-specific reagins. Further, no reaginic antibodies against potential contaminants from the dextran manufacturing process were demonstrable. In two population samples of normal human sera from Sweden and Germany hemagglutinating DRA (IgG, IgA, and IgM classes) were found in 63 and 74%, high titres (16-256) comprising 14 and 25%. In dextran reactors a direct positive correlation between titres of hemagglutinating DRA and increasing severity of DIAR was observed. The accumulation of high DRA titres in severe reactions may be taken as circumstantial evidence for the causal role of hemagglutinating DRA in these cases. However, if high titres of DRA alone were responsible for triggering DIAR, the expected frequency would be more than thousand times higher than the reported global incidence of DIAR. To explain this discrepancy, involvement of certain Ig classes or subgroups, possibly in combination with other predisposing factors is suggested. In mild reactions DRA appear to play a negligible role. Positive dextran wheal and flare reactions, often correlated with high titres of hemagglutinating DRA, were seen in 32% of dextran reactors, indicating that skin tests are of limited predictive value. No significant difference between sexes, age groups, or pre- and intraoperatively started dextran infusions was observed; association with certain diseases was not apparent.

Studies on allergens of horse epithelium with two variants of RAST.

Application of the radioallergosorbent technique (RAST) as a method for determining allergenic activity in horse epithelia extracts was investigated. Two variants of RAST are described: a direct and an indirect RAST allergen assay. Different batches of commercial horse epithelia extracts showed large variations with regard to allergenic activity. Gel chromatographic separation of the extracts showed that they contained a wide molecular weight spectrum of allergenic components in the range 10,000–75,000 g/mol with large variations between different extracts as to the composition of allergenic components. Large variations were also found between different patients with regard to the pattern of components in the extracts to which the patients were allergic. Irrespective of whether a skin test or RAST is used as a test method, the measure of the allergenic activity of an extract is dependent on the reagin pattern of the patients selected for the test. The extracts contain several components without allergenic activity. Results from fractionation of allergen extracts by ion-exchange chromatography indicate that this method might be suitable for isolating allergenic material in horse epithelia. It is suggested that RAST should be used for monitoring preparation and quality control of allergenic extracts.

Tryptic cleavage of a homogeneous cod fish Allergen and isolation of two active polypeptide fragments

A homogeneous allergenic protein from cod designated Allergen M was subjected to Edman degradation and found to be blocked at its amino-terminal. By the hydrazine dansyl chloride method the amino-terminal was found to be N-acetylated. Peptide maps of tryptic digested Allergen M yielded 15 ninhydrin-positive spots. The ϵ-amino groups of lysine were masked by ethyl trifluoro thioacetate and maleic anhydride respectively. Hydrolysis by trypsin at the single arginie site of TFA-Allergen M or maleyl-Allergen M, resulted in two polypeptide fragments, which were separated on Sephadex G-50 and designated TM 1 and TM 2. Both fragments were equally active allergenically as assessed by direct skin tests, passive transfer tests and by the radioallergosorbent technique (RAST). Amino acid composition of Allergen M and the fragments TM 1 and TM 2 were determined. Molecular weight estimations by sodium dodecyl sulphate/7·5 per cent polyacrylamide gel electrophoresis revealed 15,000 for Allergen M, 8500 for TM 1 and 6500 for TM 2. Fragment TM 1 was found to be the amino terminal one, with the arginine at its carboxyl-terminal. Allergen M as well as fragment TM 1 contained one residue of glucose. Fragment TM 2 had a freely available alanine as the NH 2-terminal. This fragment was free of carbohydrate as well as sulphur containing amino acids.

&lt;i&gt;In vitro&lt;/i&gt; Diagnosis of Atopic Allergy

In 20 children with symptoms of asthma or allergic rhinoconjunctivitis the serum concentrations of IgE antibodies (reagins) and of total IgE were followed during and after rush desensitization. The reaginic activity in serum was determined by the radioallergosorbent technique (RAST). In the majority of children an increase was found 2–4 weeks after the start of the treatment both of the IgE antibodies and of the total concentration of IgE. In some cases this increase was very pronounced. Usually the increase of IgE antibodies was more pronounced than that of total IgE. The reaginic increase was often but not always observed during the first days of treatment. The quantitative changes were most varying, both in the group and in the same individual concerning IgE antibodies to different allergens.