RAST Inhibition Research Articles

Book Reviews

Book reviewed in this article: Dieges, P. H. & de Monchy, J. G. R. (eds.): Skin test and RAST inhibition procedures in the standardization of allergenic extracts.Veldman, J. E. (ed.): Immunology, histophysiology, tumour immunology in otorhinolaryngology.

Investigation of the allergenic activity of isolated fractions of a standardized partly purified whole mite body (Dermatophagoides pteronyssinus) extract.

Twelve fractions of a molecular weight range of 1.35-670.00 kilodaltons (kD) were isolated from a biologically standardized partly purified whole mite body extract (Dermatophagoides pteronyssinus) by preparative size exclusion high-performance liquid chromatography. The allergenic activity and the antigen and allergen patterns of the isolated fractions were investigated by RAST, RAST inhibition and crossed (radio)immunoelectrophoresis (CIE/CRIE). By CRIE, each of the fractions showed allergen patterns, mostly of different compositions. Each fraction showed allergenic activity of different degrees by RAST inhibition. The highest allergenic activity could be measured by RAST inhibition with fractions which contained predominantly the major allergens Der pI and PY as detected by CRIE. Also proteins of higher molecular weights (greater than 158 kD) showed IgE-binding capacities. Nearly all antigens detected by CIE could be identified as allergens using CRIE.

Immunochemical estimations of allergenic activities from outdoor aero-allergens, collected by a high-volume air sampler.

To quantify airborne allergens in amorphus and morphological particles, a survey with collection of aero-allergens on glass fibre filters by means of a high-volume air-sampler (HIVOL) was conducted. In preliminary laboratory experiments we compared various filter elution techniques, and the pulverizing elution technique was found to be optimal with regard to yield and convenience. When a surfactant, Tween 20 (0.5% v/v), was added to the elution buffer, a recovery of 80% could be obtained. Allergens in eluates were analysed by means of an IgG-subclass RAST inhibition assay. This immunochemical method for quantification of airborne allergens was validated, as a high recovery of timothy grass pollen allergens was eluted from air filters, and eluates were shown specific by RAST inhibition. The amount of immunochemically measured airborne timothy and birch allergens collected by means of the HIVOL sampler was highly correlated with pollen counts obtained with a Burkard sampler (pollen trap) situated in the same place.

Immunologic specificity of isocyanate-induced IgE antibodies in serum from 10 sensitized workers

A procedure for the preparation of RAST disks used to assay isocyanate-specific IgE antibodies was developed. The outcome of the RAST was found to be strongly dependent on how the isocyanate test antigen was synthesized. Specific IgE from selected workers with isocyanate asthma reacted optimally to conjugates with ⩽10 isocyanate molecules bound per molecule of human serum albumin. Further haptenization resulted in decreased specific binding and increased nonspecific binding because of high levels of total IgE. The hapten and carrier specificity of isocyanate-induced IgE antibodies were studied by direct RAST and RAST inhibition. The existence of new antigenic determinants related to both the isocyanate hapten and the carrier could be demonstrated. It was important to use a test antigen prepared from the same isocyanate as that to which the worker had been exposed, since the cross-reactivity between different isocyanate haptens was partial and varied from one patient to another, it was confirmed that isocyanate-specific IgE antibodies can be demonstrated only in a subgroup of workers with isocyanate-related bronchial asthma.

The international collaborative study on the first international standard of birch ( Betula verrucosa)-pollen extract

A selected candidate international standard preparation of birch ( Betula verrucosa)-pollen extract was studied together with other birch-pollen extracts in a multinational study involving 20 laboratories in 11 countries. The biologic activity of the extract had previously been demonstrated in quantitative skin prick testing. The study methods comprised RAST inhibition, histamine release, quantitative immunoelectrophoresis, isoelectric focusing, and other methods. The results from RAST inhibition were calculated as parallel-line assays with statistical tests for linearity and parallelism. Analysis of variance was applied to test the significance of differences between potency estimates. In all assay methods, the candidate standard could be used to assign relative potencies to other birch-pollen extracts. The candidate standard was adequately stable during 36 months of storage at or below 5 °C. On the basis of this study, the World Health Organization has established the preparation as the International Standard for birch-pollen extract with assigned units of 100,000 IU per ampule.

実験動物アレルギーの臨床的，免疫学的研究 とくにモルモット上気道アレルギーを中心に

With the increasing numbers of individuals who take care animals, there has been a significant increase in the incidence of allergy to laboratory animals, and this has become an important social problem.The present study was carried out to clarify the mechanism of LAA, to identify the major allergens in guinea pig urine and saliva, and to find a way to prevent allergic symptoms.The patients were apt to have positive skin tests, high levels of serum specific IgE and immediate positive reactions to intranasal allergen challenge tests. LAA therefore seems to be a type 1 allergy. The molecular weights of major allergens determined by polyacrylamide gel electrophoresis and gel chromatography were 14, 000 to 18, 000 daltons in guinea pig urine and 15, 000 to 52, 000 daltons in guinea pig saliva. The results of RAST inhibition assays showed cross-allergenicity among guinea pig urine and saliva and serum and pelt and rat urine.A new type of air conditioning with better air flow (laminary air flow in one direction) than in the conventional turbulent flow system appeared to be effective for one of the ways to prevent LAA.

Characterization of allergens from spores of the oyster mushroom, Pleurotus ostreatus

Crude extracts of Pleurotus ostreatus spores obtained from a single local source were fractionated by gel filtration to resolve the allergenic components. The fraction pool corresponding to 10.5 to 25 kd molecular weight contained allergenic activity as demonstrated by both RAST and skin testing. Similar results were obtained with extracts from spores that originated in four other areas and with extracts prepared from P. sajor-caju spores obtained from commercially produced caps. The RAST-active fraction was further separated by hydrophobic interaction chromatography (HIC). HIC fraction pools were assayed for allergens) by RAST inhibition and immunoblotting of isoelectric focused polyacrylamide gels. RAST-inhibition data indicated that the allergen(s) was reversibly bound to the HIC column, eluting with 2, 1, and 0.15 mol/L of buffered salt solutions. After eiectrofocusing, these fractions yielded 15, 12, and 11 Coomassie brilliant blue-staining bands, respectively. IgE binding occurred with 7, 8, and 6 of these bands, as revealed by radiostaining of the immunoblots. These procedures help identify P. ostreatus spore allergens and allow a greater degree of standardization in the preparation of allergen extracts from basidiospores for use in diagnosis and therapy of fungal allergy.

Occupational sensitivity to Atphitabius diaperinus (Panzer) (lesser mealworm)

Alphitobius diaperinus is an important beetle in the grain and poultry industries. We evaluated three individuals with work-related symptoms of asthma, rhinitis, conjunctivitis, urticaria, and angioedema on exposure to the insect. Prick skin tests with extracts prepared from the larval, pupal, and adult life stages were positive in all three patients. Specific IgE antibodies to these extracts were demonstrated by RAST or radioimmunoassay. RAST and radioimmunoassay inhibition confirmed the specificity of IgE binding and further demonstrated immunologic cross-reactivity between the three life stages. Peripheral blood leukocytes from two of the individuals demonstrated significant histamine release when they were compared with cells from nonexposed atopie and normal control subjects. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. More than 30 protein bands were detected in each of the extracts; however, the patterns of separation were different for each life stage. After immunoblotting and autoradiography, IgE-binding proteins were recognized by sera from all three individuals in the larval extract at 90 kilodaltons (kd), in the pupal extract at 90, 64, and 38 kd, and in the adult extract at 84 kd. Additionally, several other proteins were identified as being allergenic in some of the patients. We conclude that these three patients developed IgE-mediated sensitivity to A. diaperinus antigens as the result of occupational exposure. To our knowledge, this is the first description of sensitivity to this grain beetle.

Evaluation of commercial imported fire ant extracts by crossed immunoelectrophoresis and radioallergosorbent test

Imported fire ant whole-body extracts (IFAWBE) from three major U.S. commercial suppliers and a reference IFAWBE and imported fire ant venom (IFAV) preparation were compared by crossed immunoelectrophoresis and RAST-inhibition testing. Results of crossed immunoelectrophoretic studies showed major differences in antigen content between commercial preparations. IFAWBE and IFAV RAST-inhibition testing showed that no commercial extract had equivalent inhibitory activity to the reference IFAWBE. Reference IFAWBE reached 50% inhibition of IFAWBE and IFAV RAST at 52 and 492 μg/ml, respectively. IFAV gave 50% inhibition of IFAWBE and IFAV RAST at 5.9 and 58 μg/ml, respectively. Of the commercial extracts, only those from one supplier were able to reach 50% inhibition of the reference IFAWBE RAST, and no commercial extract from any supplier was able to reach 50% inhibition of IFAV RAST. Analysis of inhibition slopes and intercepts confirmed that there were both qualitative and quantitative differences among the commercial extracts and the reference IFAWBE and IFAV. Our findings suggest possible reasons why some individuals fail to respond to testing or immunotherapy with IFAWBE and show the urgent need for standardization of allergenic extracts for diagnosis and treatment of imported fire ant sensitivity.

Allergens in Hymenoptera venom : XXI. Cross-reactivity and multiple reactivity between fire ant venom and bee and wasp venoms

The relationships between fire ant venom and bee and wasp venoms were explored by studying sera from five groups of subjects. Group 1 included adults not allergic to any venoms and who were not exposed to fire ants. Group 2 included adults with fire ant exposure who were not allergic to venoms. Group 3 included patients with recent systemic reactions to fire ant venom. Group 4 included patients allergic to bee and vespid venoms with no fire ant exposure. Last, group 5 included patients allergic to bee and vespid venoms with fire ant exposure. None of the serum samples from group 1 was RAST reactive to fire ant venom, but 24% of those from group 2 were fire ant positive, as were 100% of those from group 3, 51% of those from group 4, and 87% of those from group 5. The RAST-positive patients in groups 2 and 5 were also skin test positive. RAST inhibition studies demonstrated cross-reactivity in some cases and multiple reactivity in others. The serum samples were further investigated via nondenaturing electrophoretic immunoblot studies and RAST with highly purified allergens. Serum samples from group 4 reacted to a single band on immunoblots and with only one of the four purified allergens from fire ant venom ( Solenopsis invicta I, or Sol i I). Serum samples from groups 2, 3, and 5 showed various patterns of allergen reactivity. All serum samples from patients allergic to fire ant venom who also reacted to bee and/or vespid venoms by RAST contained IgE antibodies binding to Sol i I. A few serum samples that did not bind to bee or vespids did not have IgE antibodies against Sol i I. Sol i I, the 37,000 dalton molecular weight allergen, is the major cross-reactive allergen of imported fire ant venom.

Experiences of specific IgE in asthma due to diisocyanates.

The specific IgE antibodies were studied with the Phadebas RAST technique in 35 patients with asthma due to diisocyanates. Two had been sensitized to toluene diisocyanate (TDI), 17 to methylene diisocyanate (MDI) and 16 to hexamethylene diisocyanate (HDI). In each case the diagnosis was confirmed with a bronchial provocation test (BPT). The asthmatic reaction was immediate in 17 cases, of which three had also a late reaction (dual). Eighteen patients reacted only with a late reaction. Seven (20%) had specific IgE to diisocyanates. All RAST-positive patients had an immediate asthmatic reaction. None of the late reactors and referents had positive RASTs. RAST inhibition tests with 94-100% inhibition confirmed the specificity of the method. There was cross-reactivity between different diisocyanates, however. The patients with positive RAST to diisocyanates had a higher level of total IgE than the RAST negative group and the referents.

Anaphylaxis to amoxycillin but good tolerance for benzyl penicillin. In vivo and in vitro studies of specific IgE antibodies.

Three patients are reported on who suffered anaphylactic reactions after amoxycillin (AX) treatment and challenge but tolerated benzylpenicillin (BP) parenterally and orally. Two of the three patients had positive skin tests and RAST to AX reagents but negative responses to benzyl penicilloyl (BPO) specific skin tests and RAST and the minor determinant mixture (MDM) skin test reagent. The third case was negative to all skin tests and RAST. RAST and RAST inhibition on the two positive sera suggest that the response is related to the acyl side chain of AX.

The collaborative study of the international standard of dog, Canis domesticus, hair/dander extract

A collaborative study was carried out to assess the suitability of a chosen preparation to serve as the international standard (IS) for dog ( Canis domesticus) hair/dander extract. Sets of five coded preparations of dog extract, including the proposed IS, were delivered to each participating laboratory in glass-sealed ampules. Fifteen laboratories in nine different countries examined the preparations by RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, histamine release, and by other methods. In spite of the use of different versions of methods and different reagents, quite consistent results were obtained, and the proposed IS was found to have a satisfactory activity in all assays and to be useful as a calibrator when allergenic potency of similar dog allergenic extracts was estimated. Four laboratories estimated the specific content of allergens Ag 3, Ag 13, and albumin and found the proposed IS to be a suitable standard for these assays. On the basis of the results from this study, the World Health Organization established the preparation as the international standard for dog hair/dander extract with an assigned unitage of 100,000 IU per ampule. The units refer to both the total allergenic activity of the ampule and to that of individual allergens.

Immunogenicity and Allergenicity of Whey Protein Hydrolysates

ABSTRACTAllergic reactions to cow's milk are common in children. The sera of allergic subjects have been found to contain specific IgE antibodies against several whey proteins such as α‐lactalbumin (α‐la) and β‐lactoglobulin (β‐lg). This work aims to study the effect of hydrolysis on the immunogenicity and the allergenicity of α‐la and β‐lg. Whey proteins were submitted to hydrolysis with immobilized α‐chymotrypsin. High performance liquid chromatography analysis showed a decrease of native whey proteins and the appearance of low molecular weight peptides. The immunogenicity of these hydrolysates was not modified, but their allergenicity, as assessed by RAST inhibition, was decreased. The results may have important clinical implications.

RAST-inhibition studies of the imported fire ant Solenopsis invicta with whole body extracts and venom preparations

Whole body extracts of imported fire ants (IFAWBE) are the only reagents currently available for diagnosis and immunotherapy of patients with anaphylaxis to these Hymenoptera. To characterize better IFAWBE of the species Solenopsis invicta, we evaluated the sera of 29 patients with systemic or large local reactions to imported fire ant (IFA) stings. Forty-eight percent ( 14 29 ) of these sting-sensitive patients were IFAWBE RAST positive (⩾6% binding of total radioactivity added). With a pool of sera with an initial IFAWBE-RAST value of 16.2% binding, we evaluated RAST inhibition by IFA venom (IFAV), IFAWBE, and the venom component, transpiperidine. Maximum RAST inhibition obtained was 84% with 300 μg/ml of IFAV, 95% with 5 mg/ml of protein IFAWBE, and insignificant with undiluted transpiperidine. We conclude that IFAWBE contains large quantities of immunoreactive venom components other than transpiperidine and that the allergenicity of IFAWBE and venom resides in the small amount of protein present in IFAV.

Allergenicity and cross‐reactivity of cat and dog allergenic extracts

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophoretic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody based radioimmunoassay for the quantitation of the main cat allergen (Fel d I or cat-1)

A two-site solid-phase radioimmunoassay has been developed for the quantitation of the main cat allergen, Fel d I or cat allergen 1. The assay is based on two different monoclonal antibodies which recognize different epitopes on the Fel d I molecule; one antibody (C5/24) was immobilized on the solid phase and the other (C5/8) was labeled with 125I, being the allergen molecule sandwiched between them. The assay is specific for the Fel d I molecule and sensitive enough to detect as little as 0.25 ng/ml of allergen. The Fel d I RIA was compared with a radial immunodiffusion technique for the determination of allergen levels in several cat extracts and a good quantitative correlation was found. The same good correlation was found when the results obtained with the Fel d I RIA were compared with the determination of the total allergenic activity by RAST inhibition. The results indicate that the MAb RIA could be very useful in the standardization of allergenic extracts from feline origin.

Sensitivity to tomato and peanut allergens in children monosensitized to grass pollen.

Possible associations between allergy to grass pollen and positive skin tests to food allergens were studied in 102 children monosensitized (as to inhalant allergens) to grass pollen, and in 117 children monosensitized (as to inhalant allergens) to Dermatophagoides. Thirty-two foods were tested by an epicutaneous method. Positive skin tests to food allergens were more frequent in children with allergy to grass pollen (59.8%) than in children with allergy to Dermatophagoides (9.4%). A considerably high frequency of positive reactions to tomato (39.2%), peanut (22,5%), green pea (13.7%), and wheat (11.7%) was observed in children with allergy to grass pollen. Positive skin tests to peanut closely correlated with positive RAST results and nasal provocation tests, whereas in children with skin test positivity to tomato a close correlation with nasal provocation tests but a 45% correlation with a positive RAST result were observed. RAST inhibition experiments were carried out, and the results may suggest the presence of cross-reacting IgE to grass pollen, tomato, and peanut antigens. Clinical implications of these findings are discussed in the light of histories of food hypersensitivity, urticaria-angioedema, and atopic dermatitis in children with allergy to grass pollen.

Production of a proposed International Reference Standard Altenaria extract : II. Results of a collaborative trial

A lyophilized Alternaria extract prepared from a defined eight-strain source material was compared with three other Alternaria extracts to assess its potential suitability as an international standard (IS). Twenty-two laboratories in 12 different countries collaborated. Assay methods included RAST inhibition, quantitative immunoelectrophoresis, thin-layer isoelectric focusing, skin testing, leukocyte histamine release, and various other methods for thorough characterization of the extracts. In addition, three laboratories quantitated specific allergen in each extract. The proposed IS extract could be used to assign a relative potency to other test extracts. In separate studies, the proposed IS extract was demonstrated to be stable for at least 21 months when it is stored at −70 °C, −20 °C, and 4 °C.

Modification of House Dust Mite Allergens by Monomethoxypolyethylene Glycol

In animal models, allergen modification by coupling to monomethoxypolyethylene glycol (mPEG) molecules can reduce allergenicity of the extract and makes the allergen capable of suppressing boosted IgE response. To investigate in a human system the degree of attenuation implied by a mPEG modification of a house dust mite (Dermatophagoides pteronyssinus) extract, 55 adults with asthma caused by house dust mites were tested by skin prick test (SPT) and histamine release assay (HR). RAST inhibition was performed on sera from 6 additional patients. Modified extract containing 0.42 mmol mPEG/g protein was used for the analyses. In order to get the same response of the two extracts when assessed by HR and SPT, a median increase in concentration of 10-fold of the mPEG-modified extract compared to the unmodified extract was needed. Interindividual variation was limited. Sixty-four to 72% needed a dose increase within +/- half a decade from this value. In 42-49% of the patients, results from SPT and HR deviated less than half a decade. The relative potency of the modified extract as measured by RAST inhibition was reduced to 17-78% (mean 39%). Reduced allergenicity would by itself mean less side effects in immunotherapy. When planning such therapy it is important to know that mPEG modification reduces the allergenicity to a similar extent in a majority of patients.