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A radioimmunoassay for evaluation of the IgE and IgG antibody responses in the rat.

An assay, the paper radioallergosorbent test (PRAST), for the measurement of specific serum IgE antibody in the rat is described in detail. This assay has been used, in conjunction with a modified PRAST for the determination of relative specific serum IgG antibody and the PRIST assay for total serum IgE [13], to measure specific IgE and IgG and total IgE immune responses in normal parasite infected rats immunized using various protocols. The results indicate that there is a relationship between the basic IgE level and the immune response, i.e. a rat strain with a low constitutive IgE level demonstrates a weak response whereas a high level strain reacts strongly. When PRAST and passive cutaneous anaphylaxis (PCA) were compared, using standardized IgE antibody containing sera, the results were in good agreement. However, PRAST is the preferable assay as it shows less intrinsic variation, is more sensitive than PCA, and is not influenced by high serum IgE levels in the recipient animal.

Anaphylaxis↗

Ethylene oxide sensitivity in hemodialysis patients.

Sera from 138 patients who had experienced hypersensitivity-type reactions while on hemodialysis (reactors) were examined retrospectively by the radioallergosorbent test (RAST) for specific IgE antibody to ethylene oxide (ETO). Seventy-eight hemodialysis patients without a history of reaction were also evaluated as controls. Elevated serum RAST values (greater than 2.0) were more common in reactors (63%) than in controls (11%, p less than 0.001). In a second study, RAST assays were performed using human serum albumin conjugated to ETO (HSA-ETO) as antigen and also using a concentrate of fluid used to rinse ETO-sterilized dialyzers ("dialyzer extract") as antigen. The RAST ratios obtained with HSA-ETO were similar to those obtained using the dialyzer extract (rank order correlation coefficient = 0.829, p less than 0.001). In a third study, RAST inhibition was demonstrated both by HSA-ETO and dialyzer extract. Our results, extending previously published reports, suggest that hypersensitivity to ETO might play an important role in hemodialysis-associated hypersensitivity-type reactions.

Antigens↗

Total serum IgE and IgE antibodies specific to house dust mite found in two aged-matched cohorts of children with and without otitis media with effusion.

OBJECTIVES: To determine whether otitis media with effusion (OME) is associated with elevated serum immunoglobulin E (IgE) and IgE specific for house dust mite. DESIGN: Forty-seven children who had evidence of bilateral OME, both otoscopically and on tympanometry, on two separate occasions, 3 months apart were admitted for ventilation tubes. Forty-eight children admitted for minor eye surgery who had otoscopically normal ears and no history of middle ear problems were used as controls. Bloods samples were taken under anaesthesia. Total IgE and IgE radioallergosorbent test (RAST) to house dust mite was measured by the Pharmacia Unicap 100 system. The results from the two groups were compared. SETTING: Birmingham Children's Hospital. PARTICIPANTS: Children between the ages of 3 and 10. Children with Down's syndrome, cleft lip and palate, ciliary abnormalities, known immunodeficiencies and cardiac abnormalities were excluded. MAIN OUTCOME MEASURES: Total IgE and RAST to house dust mite. A RAST of >0.35 was taken to be positive. RESULTS: There was no statistical difference between the control and study groups for the total IgE. Six children from both study and control groups had a raised house dust mite RAST. There was no difference in the levels between either group. CONCLUSIONS: Our findings indicate that there is no direct relationship between OME and biochemical evidence of allergy, specifically to house dust mite.

Allergens↗

Identification of sunflower seed IgE-binding proteins.

BACKGROUND: Sunflower seed can cause severe anaphylactic reactions in some susceptible individuals. It is conceivable that the 2S sunflower seed protein is an allergen based on its high degree of homology (34%) with the allergenic mature 2S albumin protein of the Brazil nut. The first step in determining the allergenicity of sunflower seed proteins is to identify IgE-binding proteins. METHODS: Sera from sunflower seed-sensitive individuals were evaluated by radioallergosorbent test (RAST), isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with sunflower seed proteins. RESULTS: Positive RAST scores (>2) were observed in 3 individuals and immunoblotting demonstrated IgE-binding to 2-7 distinct proteins ranging in size from 10 to 50 kD. Two out of 3 sera recognized two proteins between 16 and 17 kD. The lower molecular weight protein (16 kD) approximates to the prepo region of the precursor methionine-rich 2S albumin protein found in sunflower seed (SFA-8/SSA). IEF followed by immunoblotting demonstrated several IgE-binding proteins, including two proteins with isoelectric points of 5.97 and 5.3, respectively, which are consistent with the mature and immature forms of the SFA-8/SSA region. CONCLUSIONS: Sunflower seed contains several IgE-binding proteins, including regions of the high-methionine 2S albumin SFA-8/SSA.

2S Albumins, Plant↗

[Environmental and immunological survey of sensitization with Japanese cedar (Cryotomeria japonica) pollen in children. Part 2. The clinical significance of Japanese cedar (Cryptomeria japonica) pollen specific IgE antibody and IgG4 antibody].

The clinical manifestations and Japanese cedar (sugi in Japanese) pollen specific antibodies were studied in 340 children who had not received specific hyposensitization. Specific IgE antibodies were measured by radioallergosorbent test (RAST) and IgG4 antibody by enzyme linked immunosorbent assay (ELISA). There were 52 children (15%) with RAST scores of over 2, and 44 children (13%) with ELISA of over 101 mu/ml. The specific IgG4 level was significantly higher in the RAST positive group than in the negative group. The incidence of pollinosis among children with RAST negative and IgG4 below 100 mu/ml was 13.3%, while combinations of RAST negative and higher ELISA, RAST positive and low ELISA, and RAST positive and higher ELISA were 50.0+-62.5%. There were 5 children with RAST scores of 4, and 6 children with ELISA of over 201 mu/ml. Symptoms of pollinosis manifested in all of them. Since the children with low IgE and high IgG4 antibodies had comparatively more symptoms, the IgG4 antibodies might be reaginic. It was concluded that measurement of sugi specific IgG4 antibodies as well as IgE antibodies could provide useful data on pollinosis in children.

Adolescent↗

Pattern of sensitization to common environmental allergens amongst atopic Singapore children in the first 3 years of life.

This study was aimed to investigate the sensitization pattern to a range of common allergens in young Singaporean children. A cross-sectional study involving 75 children aged below 3 years was carried out. They presented between December 1995 and April 2000 with symptoms of asthma, rhinitis, eczema, or food allergy. Their levels of allergen-specific serum IgE to a panel of foods (egg white, milk, soy protein, shrimp, wheat and peanut), pet dander, dust mites and cockroaches were measured with Pharmacia CAP System radioallergosorbent test kits. Serum IgE levels greater than 0.35 kU/l represented a positive result. Four children could not be tested with the complete panel because of insufficient serum. The prevalence of sensitization was highest for cow's milk (45.9%) followed by egg white (38.7%), dust mites Dermatophagoides pteronyssinus (31.4%) and Blomia tropicalis (25.5%). Sensitization to ingested allergens was significantly more prevalent in children aged 1 year or younger than in the older children (70.4% of those below 1 year, and 50% of those aged 1-3 years; p < 0.02). Sensitization to inhaled allergens, such as dust mites, was more likely to manifest as respiratory symptoms (allergic rhinitis and asthma), while ingested allergens were associated with gastrointestinal symptoms and eczema (p < 0.001). It was concluded that infants and young children are at high risk of sensitization to common environmental substances. Allergen avoidance is therefore important even in the very young. The prevalence of sensitization to food allergens is higher compared to inhalant allergens in young children.

Allergens↗

Studies on Dermatophagoides pteronyssinus allergens: measurement of the relative potencies of D. pteronyssinus purified extracts by in vitro and in vivo methods.

To standardize the Dermatophagoides pteronyssinus extracts used in clinical allergy practice, the relative potencies of several purified extracts were estimated by radioallergosorbent test (RAST) inhibition, histamine release, and intracutaneous titration testing, and the results were compared. The potencies of nine independently prepared D. pteronyssinus extracts were assayed with respect to an extract adopted as a reference. In vivo the cutaneous wheal surface after intradermal injections was measured on a population of D. pteronyssinus-sensitive subjects. In vitro RAST inhibition was performed with the reference extract as allergosorbent and with a pool of human IgE-rich sera from patients sensitized to D. pteronyssinus but untreated. For histamine release human basophils from patients allergic to D. pteronyssinus were used. In the three method dose-response curves were plotted for the reference extract and the other extracts. A comparison of the measurement of in vitro and in vivo potencies is reported. Simialr results were obtained with the three methods: all the extracts but one were as potent as the reference preparation. However, for routine purposes RAST inhibition appears to be the most convenient and reliable procedure.

Allergens↗

Analysis of the allergenic composition of rat dust.

BACKGROUND: Allergy to rats is an important occupational health problem. The allergens of rat urine have been well defined but those in rat room dust, a potentially important source of inhalant exposure, have not. OBJECTIVE: To describe the allergens present in rat room dust and to identify a suitable marker protein which may be used to quantify airborne rat allergen. METHODS: Dust collected from the air-conditioning system (bulk dust, 'bd') and with an air sampler (airborne dust, 'ad') were analysed by radioallergosorbent test (RAST) inhibition, immunoblotting and immunoblot inhibition techniques and comparisons made with hair and urine extracts prepared from adult male Wistar rats. RESULTS: Extensive crossreactivity was found between the extracts by RAST inhibition under different experimental conditions. Dust was more potent as an inhibitor than other extracts. The immunoblotting patterns of both dusts were similar although 'ad' contained an allergen at 29 kDa not found in 'bd'. Forty-two sera from rat allergic subjects were used to identify 18 allergens in 'bd'. Three 'major' allergens were found; 100% of subjects had immunoglobulin (Ig)E to a 44 kDa allergen and 74% and 88% of subjects had IgE with bound to the 20.5 and 17 kDa allergens respectively. Immunoblot inhibition experiments identified the 17 kDa dust allergen as alpha 2u-globulin (Rat nI). CONCLUSIONS: Rat dust is a complex allergenic source. The 17 kDa dust allergen has immunological identity with Rat nI and is a suitable marker protein for the quantitation of airborne rat allergen.

Air Pollutants↗

Studies of the specificities of IgE antibodies found in sera from subjects with allergic reactions to penicillins.

Penicillins are immunogenic when administered to humans and in some instances they can also be allergenic, inducing specific IgE antibodies. Whilst the major haptenic group, the penicilloyl, is well characterised, less is known about the relative importance of the different parts of the structure for antibody binding and how this can influence the specificity of patients response. In order to investigate this further, sera from subjects who had suffered an IgE-mediated reaction to penicillins were studied using the radioallergosorbent test (RAST) and RAST inhibition. The assays employed reagents related to the penicillins causing the reaction. Using 173 sera, positive RAST results were only found with reagents based on benzyl penicillin (BP) and amoxicillin (AX). Fifty-three positive sera were selected for further studies and categorized into three groups: (A) sera only RAST positive to AX, (B) sera only positive to BP and (C) sera positive to both penicillins. RAST inhibition studies were then carried out using monomeric penicilloyl conjugates and compounds representing parts of the penicilloyl structures of BP and AX. For all three groups, monomeric penicilloyl conjugates were the most efficient inhibitors but there were differences for the other compounds. Group A sera were also inhibited by the side chain amoxicillin, whereas group B sera were poorly inhibited by all other inhibitors. Group C sera showed two patterns of inhibition, both consistent with their more cross-reactive profile.

Amoxicillin↗

The role of cereal and fungal amylases in cereal flour hypersensitivity.

To investigate the role of cereal alpha and beta-amylase in bakers' asthma, we have compared the IgE response of 30 wheat-flour-allergic individuals to barley alpha and beta-amylases with that of fungal alpha-amylase using radioallergosorbent test (RAST), RAST inhibition assays and Western blotting. RAST analysis showed 29 of the 30 subjects with inhalant induced cereal allergy had positive IgE to cereal amylases, but only 16 were positive to fungal alpha-amylase. Regression analysis showed an association between specific IgE to wheat-flour and to barley alpha-amylase (r = 0.70) and barley beta-amylase (r = 0.92) but a poor association with fungal alpha-amylase (r = 0.34). RAST inhibition showed minimal crossreactivity between barley alpha or beta-amylase and barley and fungal alpha-amylase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that non-reduced barley alpha-amylase had a molecular weight of 54 kDa and barley beta-amylase a molecular weight of 64 kDa. Reduced fungal alpha-amylase had a molecular weight of 54 kDa. Cereal alpha and beta-amylase appear to be important allergens in patients with allergy to flour.

Air Pollutants, Occupational↗

Plantago lanceolata (English plantain) pollinosis in Japan.

OBJECTIVE: The objective was to ascertain the prevalence of Plantago lanceolata (English plantain) pollinosis in Japan. METHODS: A total of 160 patients with allergic rhinitis were examined at the Hokkaido University Hospital Otolaryngology Clinic between January 2002 and December 2003. We investigated the frequency of P. lanceolata antigen-specific immunoglobulin E (IgE) antibody-positive serum using the radioallergosorbent test (RAST), the P. lanceolata pollen counts in the Sapporo area and the clinical symptoms of patients with allergic rhinitis caused by this pollen. RESULTS: P. lanceolata pollen was dispersed throughout the Sapporo area from mid-May to early September, peaking in the latter half of May. The airborne pollen count showed little variation between years. The P. lanceolata RAST-positive rate was 12.8%, which was close to those for ragweed and sagebrush. All subjects showed overlapping antigen sensitization, that is, none displayed sensitization (by RAST) exclusively to P. lanceolata. The onset of subjective symptoms peaked in June and the symptoms worsened from April to October. CONCLUSION: P. lanceolata was dispersed over a long period, from mid-May to early September. The P. lanceolata RAST-positive rate (12.8%) was similar to those for sagebrush and ragweed, which are dispersed during the fall. These results suggest that P. lanceolata is an important causative agent of pollinosis in Japan.

Adolescent↗

Nitrocellulose-RAST analysis of allergenic cross-reactivity of Candida albicans and Saccharomyces cerevisiae mannans.

Two chemically purified mannan preparations and one affinity purified mannan preparation of Candida albicans and Saccharomyces cerevisiae were analyzed for allergenic cross-reactivity. Simultaneous IgE binding in radioallergosorbent test (RAST) was studied with all preparations and the chemically purified mannans were also used for RAST inhibitions. The yeast mannans were purified with the Peat method, using repeated ethanol precipitations and Fehling's precipitation in alkaline conditions, and the Cetavlon method, using hexadecyltrimethylammonium bromide and ethanol precipitations. Affinity purification of the mannans was performed with concanavalin A lectin bound to a Sepharose column. Mannan from Pityrosporum ovale was purified with the Cetavlon method for analysis of simultaneous RAST reactivity. Simultaneous IgE binding to all the studied yeast mannans was generally seen in all the studied sera of patients suffering from atopic dermatitis. The strongest responses were seen against C. albicans mannan, especially the Cetavlon mannan. In the RAST inhibition studies IgE binding to S. cerevisiae mannan was completely inhibited by C. albicans mannan preparations, whereas reciprocal inhibition was not complete. These results indicate that in atopic dermatitis simultaneous IgE response to yeast polysaccharides occurs and that the major sensitizer is C. albicans and IgE antibodies against S. cerevisiae mannan are cross-reacting.

Candida albicans↗

Use of a screening RAST in a large neuro-otologic practice.

Evidence in the literature emphasizes the role of the immune system in disorders of the inner ear and eustachian tube. We initially investigated the presence of inhalant allergy in selected patients seen for otologic problems by means of a screening radioallergosorbent test (RAST), using either a microscreen or a limited antigen panel. This study analyzed the results of tests performed over a 2-year period on 186 patients seen by one of us (WLM) for treatment of vertigo (66%), tinnitus (63%), hearing loss (49%), aural fullness (48%), Meniere's quadrad (27%), balance disturbance other than true vertigo (21%), and eustachian tube dysfunction (4%). We found an incidence of immunoglobulin E-mediated hypersensitivity of nearly 40% in a patient population selected solely for neuro-otologic symptoms and not for sinonasal symptoms. This figure is more than double that quoted for the general population. We also found a surprisingly high incidence of mold antigen atopy in this selected population. Allergy can contribute to a number of otologic symptoms, including eustachian tube dysfunction, vertigo, tinnitus, hearing loss, aural fullness, and nonspecific balance disturbance. Allergy also has been emphasized as an etiologic factor in a portion of patients diagnosed with Meniere's syndrome. A screening RAST, combined with clinical evaluation, appears to be an excellent tool for evaluating these patients for inhalant allergy as part of a comprehensive workup.

Adolescent↗

Elevated nonspecific plasma proteins in allergic patients.

Several allergen-specific plasma proteins, such as IgE and IgG subclasses, are commonly used for the evaluation of grade of allergy. In the present investigation, we compared the concentration of various nonspecific plasma proteins, mostly known as inflammation markers, in an allergic and a healthy population. Plasma from 130 children with single inhalation allergies to grass pollen, birch pollen, or house dust mites as well as from 42 healthy children was obtained during the symptom-free period. Patients showed symptoms including allergic rhinitis, dermatitis, and asthma with one single radioallergosorbent test (RAST) class 3 or higher. Plasma concentrations of soluble intercellular adhesion molecule-1(sICAM-1), soluble interleukin-2 receptor(sIL-2R), sE-selectin, and soluble vascular cell adhesion molecule-1 (1sVCAM-1) were analyzed by enzyme linked immunosorbent assay (ELISA) technique. Concentrations of sICAM-1 and sE-selectin were significantly increased in all patients compared to controls. In the single allergen groups, sICAM-1 elevation was significant in the grass and mite groups, but not in the birch group; while sE-selection increase was significant in the birch and mite groups, but not in the grass group. The elevation of sIL-2R in the allergic patients was obvious in each single allergen group, but not significant. No difference was observed in sVCAM-1 expression. In two groups of patients with mean age of 9.5 years versus 17.5 years, the analyzed parameters were not age dependent. The increased proteins may be useful as additional markers for efficacy and follow-up investigations of allergy therapies.

Adolescent↗

Exposure to pets and allergies in children.

Currently, there is ongoing discussion regarding potential protective effects of exposure to pets during early childhood on the development of atopic disorders in children later in life. We used data from three consecutive cross-sectional surveys to study the relationship between contact with dogs, cats and other pets, and allergic diseases in schoolchildren 5-14 years of age. In three study areas of the former East Germany, 7,611 questionnaires were received from 5,360 different children who were examined between 1992 and 1999 as school entrants, or third- or sixth-graders. Allergic sensitization to common aeroallergens (birch, grass, mite, cat) was assessed by specific serum immunoglobulin E (IgE) concentrations [using radioallergosorbent testing (RAST)] for 85% of the children. After adjustment for possible confounders, inverse associations were found between contact with dogs in the first year of life and lifetime prevalences of asthma [odds ratio (OR) = 0.68; 95% confidence interval (CI): 0.43-1.08], hay fever (OR = 0.61; 95% CI: 0.39-0.95), eczema (OR = 0.76; 95% CI: 0.61-0.94), itchy rash (OR = 0.76; 95% CI: 0.61-0.94), and pollen sensitization (RAST >/= 4: OR = 0.56; 95% CI: 0.38-0.82). These effects were more pronounced for children with atopic parents. Similar associations were observed for current contact with dogs. We identified no clear relationships for the other pets (cats, rodents, birds), with the exception that children currently exposed to cats were more likely to be sensitized against cats. In conclusion, this study supports the hypothesis of a potential protective mechanism related to dog exposure in early life, especially for children of atopic parents. However, this association was found only for dogs and not for cats.

Adolescent↗

A clinical and immunological study of allergy to hen's egg white. IV. Specific IGE-antibodies to individual allergens in hen's egg white related to clinical and immunological parameters in egg-allergic patients.

Different subgroups of egg-allergic patients were established according to differences in allergic symptoms provoked by hen's egg white, total IgE-level, RAST (radioallergosorbent test) results to egg white, and age. In each group the pattern of specific IgE-antibodies to individual allergens in the egg white, determined by means of crossed radioimmunoelectrophoresis (CRIE), was compared with the pattern in a control group. The RAST value to egg white was found to exert the strongest influence on the CRIE results. A few statistically significant differences in CRIE results to certain allergens were also observed in other pairs of subgroups of egg-allergic patients. Possible explanations for this are discussed. It was concluded that CRIE studies of allergens in mixed protein solutions, using a series of sera from allergic patients, will most probably give the same results and the same classification of the allergens independent of the characteristics of the patients from which the sera were collected. However, it should be emphasized that the CRIE series must include a sufficient number of patients with high RAST values to the allergen in question. Otherwise some allergens may be missed and the classification of the allergens may be different.

Adolescent↗

Prevalence of atopy in an inner-city asthmatic population.

Seventy-three patients at The Mount Sinai Hospital Emergency Room were investigated to determine the prevalence of atopy in asthma in a predominantly black and Hispanic inner-city population. Serum IgE levels and radioallergosorbent tests (RASTs) to eight common inhalant allergens were measured in both the asthmatic group and a nonasthmatic emergency-room control group. The mean total IgE level for the asthma group was 263.8 IU/mL compared to 63.8 IU/mL in the control group (p = 0.032), and 60% of the asthmatics had IgE levels in the atopic range (> 100 IU/mL). Increases in IgE were associated with age under 50 years but did not reach statistical significance. Cockroach, dust mite, cat, and dog were the most common RASTs in the asthmatic group; there were no positive RASTs in the control group. There was a correlation (p = 0.04) between age (less than 50 years) and increased numbers of positive RASTs. These results are similar to those of other studies that have associated atopy with asthma in rural and suburban populations. These data demonstrate that atopy is common in the asthmatic patients seen in The Mount Sinai Hospital Emergency Room and strongly suggest that management of atopic factors should become routine in the care of adult asthmatic patients.

Adolescent↗

Comparison of the venom immunogenicity of various species of yellow jackets (genus Vespula).

Venoms from various yellow jacket species were examined by two-dimensional thin-layer chromatography (TDTLC), double-diffusion gel precipitation (DDGP) using rabbit antisera, and the radioallergosorbent test (RAST). Comparison of representative venoms by the TDTLC showed that the venoms of V. vulgaris and V. maculifrons have a larger number of Ninhydrin (triketohydrindene hydrate)-positive substances than the venom of V. squamosa. The results of the DDGP confirmed the differences; venoms of V. vulgaris, V. maculifrons, V. flavopilosa, and V. germanica have one or more major components with immunogenic identity. The venom of V. squamosa has a species-specific major component and some minor components immunologically identical to the other venoms examined. Sera from 21 patients with a history of anaphylaxis following yellow jacket stings were examined by the RAST. Using the venoms of V. maculifrons, V. vulgaris, V. flavopilosa, and V. germanica as coupling antigens, most sera reacted similarly. The sera did not react with V. squamosa. These results suggest that the major component in venom obtained from the four yellow jacket species has immunogenic identity. Venom of V. squamosa differs from the remaining venoms. As a practical corollary, with the exception of venom from V. squamosa, common sensitivity appears to exist among the yellow jacket venoms examined.

Animals↗