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.     

Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA

The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.     

Isolation and partial characterization of a major peanut allergen

We isolated and partially characterized a major peanut allergen (Peanut-I) using the radioallergosorbent test (RAST) to monitor the allergenicity of various fractions. Raw peanuts were pulverized, defatted, and extracted; the resulting crude extract (CPE) was fractionated by diethylaminoethyl (DEAE) cellulose anion-exchange chromatography using a linear salt gradient. Peak allergenic fractions were further purified by a second DEAE cellulose chromatography step, followed by a preparative polyacrylamide gel electrophoresis (PAGE) step. The final product, Peanut-I, contained 11% nitrogen and 8.7% carbohydrate and was homogeneous by both analytical PAGE and immunoelectrophoresis against rabbit anti-CPE. Peanut-I was heterogeneous by thin-layer electrofocusing and by PAGE in 1% sodium dodecyl sulfate. Biologic activity of Peanut-I was demonstrated by positive skin tests and leukocyte histamine release assays in patients with peanut allergy. By RAST inhibition assay Peanut-I did not account for all of the allergenic activity of CPE.     

Monoclonal antibodies against selected peanut allergens: Production and use as affinity agents

An extract of dry‐roasted commercial peanut mix (CPE) was examined for allergenic activity in peanut‐sensitive individuals, using skin tests and radioallergosorbent (RAST) assays. Proteins in the extract were characterized by sodium dodecyl sulfate polyacry‐lamide gel electrophoresis (SDS‐PAGE) and immunoblotting. The proteins were electro‐eluted in three fractions in the ranges 15–25, 26–58 and 65 kDa. The 15–25 kDa molecular weight fraction produced the most reactive skin tests in peanut‐sensitive subjects and was chosen for monoclonal antibody production. Six hybridoma cell lines secreting peanut‐specific antibodies of the IgM isotype (kappa light chain) were produced. Immunopurified CPE proteins were then subjected to SDS‐PAGE, resulting in five major bands with approximate molecular weights of 14, 25, 38, 40 and 44 kDa. Immunoblotting of these separated proteins revealed: (1) three bands with approximate molecular weights of 38, 44 and 65 kDa, which bound IgE from peanut‐sensitive patients; and (2) that the monoclonal antibodies recognized epitopes in bands at approximate molecular weights of 12, 14, 23 and 25 kDa. RAST inhibition assays showed that the affinity‐purified proteins were able to inhibit the binding of serum IgE from peanut‐allergic individuals to solid‐phase CPE.     

Allergenic cross-reactivity between Callistemon citrinis and Melaleuca quinquenervia pollens

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH4B) and melaleuca (NH4M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH4B and NH4M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH4B1-NH4B4 and NH4M1-NH4M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH4B1 and NH4M1, respectively, contained allergenic components. These allergenic components, NH4B1 and NH4M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.     

Bee moth (Galleria mellonella) allergic reactions are caused by several thermolabile antigens

BACKGROUND: Exposure and contact with bee moth (Galleria mellonella) larvae (Gm) can cause an allergic reaction both in anglers and breeders. We described the case of an amateur fisherman who experienced an allergic reaction using Gm but not using heat-treated Gm (h-Gm) (mummies). The aim of this study was to demonstrate by immunoblotting and radioallergosorbent test (RAST)-inhibition experiments the loss of allergenic epitopes in h-Gm extracts. METHODS: Galleria mellonella larvae and h-Gm were homogenized and extracted at 10% (w/v) in 0.5 M phosphate-buffered saline, pH 7.4 containing 0.5% NaN(3) for 16 h at 4 degrees C. Gm and h-Gm extracts were electrophoresed in a 10% polyacrylamide precast Nupage Bis-Tris gel at 180 mA for 1 h and the resolved proteins stained with 0.1% Coomassie brilliant blue and the molecular weight calculated. For the immunoblotting detection of allergenic components the resolved extracts were transferred onto a nitrocellulose membrane and incubated with the patient's serum. Bound specific-IgE was detected by peroxidase-conjugated anti-human IgE. RAST inhibition experiments were performed according to the Ceska method. RESULTS: The protein profile of Gm and h-Gm extracts resulted markedly different in number, intensity and the position of bands, indicating that heat-treatment modifies the chemical-physical characteristics of the protein contents. The Gm extract showed a strong-coloured band at 73 kDa and more than 20 components ranging from 12 to 133 kDa; h-Gm showed two main band at 77 and 38 kDa and about 15 faint bands between 20 and 133 kDa apparently without any correspondence to the bands present in the Gm extract. Immunoblotting with the patient's serum demonstrated several bands of reactivity with the Gm extract ranging from 20 to 100 kDa and no recognizable bands, but only a diffuse smear with h-Gm. When used in a RAST inhibition experiment the h-Gm extract demonstrated an inability to compete with the Gm one for the binding to patient's IgE serum. CONCLUSIONS: The h-Gm seems to lose the allergenic epitopes and has two advantages for anglers: to avoid new possible sensitizations as well as allergic symptoms in sensitized people, without interfering with their skills and satisfaction in their fishing performance.     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

Isolation and partial characterization of three major allergens of horse hair and dandruff.

Three major allergens of horse hair and dandruff have been isolated. The fractionation procedures involved various combinations, described in detail, of ethanol precipitation below --5degreesC, cation- and anion-exchange chromatography, and gel filtration. UV absorption, quantitative immunoelectrophoresis and RAST inhibition were used to monitor the separations. Protein impurities constituted less than 5% in all cases. The molecular weights of the isolated proteins were 1.9 X 10(4), 5.1 X 10(4) and 3.1 X 10(4) daltons, respectively. The pIs were determined as 4.1, 3.8 and 3.9, respectively. The amino acid analysis of the isolated allergens revealed large variations in their amino acid composition which might explain different reactivities in RAST experiments. The allergenic activities of the isolated antigens were determined by RAST inhibition and prick tests.     

Demonstration of distinct allergens by means of immunological methods. Comparison of crossed radioimmunoelectrophoresis, radioallergosorbent test and in vivo passive transfer test

An immunosorbent column was prepared containing a purified major allergen fraction from codfish (DS 22) covalently coupled to agarose. Sera from patients allergic to codfish were run through the column at pH 7.2. After extensive washing, the IgE retarded in the column was eluted with a buffer at pH2.5. The original sera and fractions from the chromatography experiments were examined by means of crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), radioallergosorbent test (RAST) and in vivo passive transfer (PK) tests using the DS 22 from codfish and a crude codfish extract. The experiments demonstrated that the crude extract contained a minor codfish allergen (antigen-17-cod) which was distinct from DS 22. RAST was the most convenient technique for the identification of fractions containing allergenic activity. The PK tests served to prove the biological activity in vivo. CIE/CRIE were superior to RAST and PK tests regarding their ability to identify distinct allergens. Full agreement was found between results using different techniques including the immunosorbent experiments. Some of the radiostaining in CRIE, however, was misleading due to coprecipitation of DS 22 in several precipitates in the CIE preparations of the crude codfish extract.     

Studies of dialysates from Dermatophagoides farinae: partial purification and haptenic properties of dialysates from Dermatophagoides farinae.

Dialysates from Dermatophagoides farinae were partially purified. Fractionation on HPLC and anion exchange chromatography revealed that the dialysates consisted of 5 major fractions of glycoprotein whose apparent molecular sizes were 5.1, 4.1, 3.2, and less than 1.35 kD on HPLC. The apparent molecular size of two fractions was 5.3 and 2.9 kD on SDS-PAGE. They were basic glycoproteins which had a pI ranging from 7.46 to 8.71 on PAG-IEF. These fractions were allergenic in the RAST and ELISA inhibition tests but not in the skin prick test (SPT). Our results suggest that the dialysates from D. farinae have haptenic properties. The dialysates from D. farinae (low molecular weight) and its 5 fractions bound noncovalently to human serum albumin (HSA) at the free tyrosine residues of HSA. They proved to bind noncovalently to serum proteins and collagens. Once they bound to proteins, the conjugates became allergenic not only in the RAST and ELISA inhibition test but also in the SPT. Our results provide evidence that the dialysates from D. farinae have haptenic properties.     

Antigens and Allergens in Birch Pollen Extract

More than 70% of the total allergenic activity of a birch pollen (BP) extract was detected within the first 30 min of extraction. Fractionation of the BP extract by gel filtration and analysis of the eluted antigens by a fused rocket immunoelectrophoresis revealed at least three antigens with molecular weights of about 29 000, and 17 000-10 000, corresponding to antigens Nos. 7-8 and No. 2, respectively, in crossed-immunoelectrophoresis (CIE) and in crossed-radioimmuno-electrophoresis (CRIE). Gel isoelectrofocusing of the pooled allergenic fractions revealed two major protein bands with pI's around 5.6 and 5.7, probably corresponding to antigens Nos.7-8 and No. 2, respectively. Antigens Nos. 7-8 were thermoresistant, while antigen No. 2 was thermolabile. The allergenic activity was determined by prick skin testing and by the RAST inhibition method. More than 90% of the allergenic activity in the fractions was located in the protein peak C (mol. wt. 10 000-17 000) containing antigens 7-8. About 30% of the total allergenic activity of the extract (1:10 w/v) was recovered in the peak C fractions, and only less than 0.5% outside these fractions. Higher allergenic activity was obtained for the peak B fractions (mol. wt. 29 000) by skin prick testing than by the RAST. Peak B contained allergens (antigen 2) distinct from those of peak C by the CRIE and by the RAST. The allergenic material in the low molecular weight fractions of peak D (mol. wt. 2000-5000) was allergenically similar to that of peak C in the RAST. Only weak and even negative skin reactions were observed with the peak D fractions in allergic subjects.     

Cytochrome c allergens isolated from the pollens of the dicotyledons English plantain (Plantago lanceolata) and Paterson's curse (Echium plantagineum)

Two cytochrome c allergens were isolated from extracts of the pollens of the dicotyledons English plantain (Plantago lanceolata) and Paterson's Curse (Echium plantagineum) by ion exchange chromatography, gel filtration and preparative isoelectric focusing. They were characterized by their absorption spectra, mol. wt, pI and amino acid composition. The cytochromes c bound specific IgE in the sera of hypersensitive patients by RAST. Preliminary evidence for allergenic cross-reactivity between them was obtained by RAST inhibition.     

Studies of the allergens of Olea europea pollen

Allergens of Olea eurupea have been prepared by isoelectrofocusing and tested by RAST, RAST inhibition and skin tests. The major allergen was found to be an acidic protein of pI 6, consisting of two polypeptide chains of molecular weight 15000 and 17000 daltons, possibly non-covalently associated into a dimer. Most other allergens were acidic proteins, but basic proteins were also observed with allergenic activity. The RAST inhibition studies show a considerable degree of allergic cross-reactivity between the isoelectrofocusing fractions.     

Basidiospore allergens: analysis of Coprinus quadrifidus spore, cap, and stalk extracts

Atopic individuals with symptoms of respiratory allergy have been shown to have IgE-mediated reactions to spores from the basidiomycete fungi. Because our earlier studies suggested that parts of the fungus other than spores may contain allergens, the current study was performed. Extracts of Coprinus quadrifidus spores, caps, and stalks were prepared and fractionated by gel filtration column chromatography on Sephadex G-75. Analysis of column fractions of each separation by ultraviolet absorption demonstrated at least three peaks of absorbency in spore, cap, and stalk extracts. Pooled column fractions were analysed by direct radio-allergosorbent test (RAST) using pooled sera from C. quadrifidus skin-test positive subjects. Enhanced allergenic activity was present in the same portion of the column eluate for cap, spore, and stalk fractionations, corresponding to a molecular weight of approximately 10.5–25 kD. Pools with allergenic activity were used to test volunteers by skin prick and RAST. Skin test and RAST activities were similar for each of the three Coprinus extracts, with stalk being the most potent. Evidence of common allergenic epitopes was demonstrated by inhibition of spore RAST by spore, cap, and stalk extracts. These results suggest that C. quadrifidus cap and stalk extracts contain allergens similar to those in spores extract and may provide useful sources of allergen for further study.     

Studies on allergens of Dermatophagoides pteronyssinus by direct and indirect RAST.

Dermatophagoides pteronyssinus (DP) extract was fractionated by Sephadex G-75 and liquid isoelectric focusing (IEF) and the allergenic activity of different fractions was monitored by direct and indirect RAST. The fractionation on Sephadex G-75 showed that the allergenic activity of DP extract was related to wide molecular weight spectrum components, even though the maximum amount was recovered in effluent that contained protein with a molecular weight ranging between 25,000 and 12,500 daltons. By fractionation of the mite extract on IEF, three main peaks of allergenic activity (pI less than 3.0; pI = 4.3 +/- 0.25; pI = 6.4 +/- 0.25) were found. Cross-inhibition experiments showed a high degree of cross-reactivity between allergenic material eluted in very distant regions of molecular weight or isoelectric point. The allergenic activity of unfractionated mite extract and of its IEF fractions was destroyed by pronase - but not by neuraminidase - treatment. These results suggest that DP extract probably contains one main allergen existing in multiple molecular forms rather than several distinct allergens and that a protein moiety of the allergen is necessary for the combination with IgE.     

Immunochemical characterization of cocos nucifera pollen

The Cocos nucifera pollen, as one of the sources of allergen responsible for immediate hypersensitivity reaction, was confirmed by skin prick test, bronchial provocation test, and RAST. The whole pollen extract (WPE) of C. nucifera was fractionated by combination of gel filtration and ion-exchange columns with fast protein liquid chromatography (Pharmacia, Uppsala, Sweden). Three protein peaks designated Cocos II, Cocos VI, and Cocos VII exhibited allergenic properties, as tested by skin prick test, direct IgE ELISA, bronchial provocation test, and immunoblot analysis. In RAST inhibition, Cocos IIa (a high-molecular-weight protein) obtained by fractionation of Cocos II on Mono Q column (fast protein liquid chromatography) (Pharmacia) was found to be the most potent allergen in Cocos WPE, followed by Cocos VI and Cocos VII, which are low-molecular-weight proteins. The reference patterns of Cocos WPE on crossed immunoelectrophoresis and thin-layer isoelectric focusing were established for future standardization of Cocos WPE to be used in the diagnosis and immunotherapy of allergic patients.     

Comparative studies on tree pollen allergens. VIII. Immunological properties of the alder (Alnus incana) pollen extract

The immunological properties of the aqueous crude alder pollen extract (AI crude) and gel filtration fractions AI 3, AI 4 and AI 34 (pool of fractions AI 3 and AI 4) were examined by immuno- and radioimmuno-electrophoretic techniques, RAST titration, RAST inhibition and skin prick tests (SPT). In CIE, the AI crude extract and AI 34 displayed reference precipitate patterns consisting of 27 and 24 visible Coomassie brilliant blue stained lines, respectively. The CRIE allergogram performed by incubation with 18 individual reaginic sera detected three IgE-binding antigens characterized by different IgE-binding properties. Antigen No. 7 (Ag 7) was demonstrated to be the major IgE antibody-binding antigen of alder pollen, while Ag 1 and Ag 11 were classified as intermediate allergens. The allergens of alder pollen were located in fractions AI 3 and AI 4 of the gel filtration chromatogram. Ag 7 was present in both fractions as demonstrated by FRIE with autoradiography (FRIEWA) on the gel filtration fractions and tandem-CRIE of AI 3 and AI 4. The CRIE allergogram, RAST, RAST inhibition and SPT demonstrated fraction AI 34 to be allergenically representative of the AI crude extract both qualitatively and quantitatively. Thus, fraction AI 34 was considered an optimal purified allergen extract of alder pollen, a suitable material for further biochemical characterization and trials on purification of the allergenic reactive antigens.     

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 allergen(s) 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 electrofocusing, 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.     

Partial characterization of allergens associated with hypersensitivity to the 'green nimitti' midge (Cladotanytarsus lewisi, Diptera: Chironomidae)

Allergens in extracts of the ‘green nimitti’ midge, Cladotanytarsus lewisi Freeman (Diptera: Chironomidae), a cause of widespread hypersensitivity in the Sudan, were isolated and partially characterized by assays which depend on the binding of ¹²⁵I-anti-IgE to allergen-IgE complexes. These methods included RAST inhibition, crossed radioimmunoelectrophoresis (CRIE) and rocket radioimmunoelectrophoresis (RRIE). Following Sephadex G100 chromatography the ‘major peak’ of allergcnicity as determined by RAST inhibition. RRIE and SDS-PAGE was associated with molecules of approximately 17 000 daltons. The peak eluting at Vo contained material of molecular weight 66000 daltons which also bound ¹²⁵-I-anti-IgE. but had only 61% of the activity of the ‘major peak’ by RAST inhibition. By isoelectric focusing and RRIE of fractions obtained by chromatofocusing with polybuffer exchanger 94, the ‘major peak’ was associated with multiple bands with a pI range of 3.5-5.5. These results indicate that the major allergens from C. lewisi are a group of closely related acidic peptides.     

Immunochemical and physicochemical characterization of commercial Altemaria extracts: a model for standardization of mold allergen extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.