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.     

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.     

Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum

Cladosporium herbarum was supplied from two sources and extracted identically. Antisera against the extracts were produced in rabbits and two reference patterns were established using crossed immunoelectrophoresis (CIE). Both patterns showed more than 60 precipitates but less than 50% of the detectable antigens appeared to be identical in the two extracts. The allergens were identified by means of crossed radioimmunoelectrophoresis (CRIE) using sera from 35 individuals with proven or suspected allergy to C. herbarum. Four important and 10-20 less important allergens were demonstrated. Among the allergens present, there were none reacting with all patient sera. Only 1 out of 3 rabbits immunized with a suspension of broken cells of C. herbarum showed precipitating antibodies to the statistically most important allergen, while 9 rabbits immunized with aqueous extracts of the mold did not. The composition of the two extracts with respect to allergens differed. Allergens present in one extract were not always detectable in the other. The experiments also showed how CIE/CRIE with various combinations of antigens and antisera may be combined with CRIE inhibition, radioallergosorbent test (RAST) and RAST inhibition for comparing complex allergen extracts at the molecular level.     

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.     

Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-I and Ag 1)

Partially purified preparations of Alt-I, the main allergenic fraction of Alternaria alternata isolated by Yunginger, and of Ag 1, shown in crossed radioimmunoelectrophoresis (CRIE) to be the dominating major allergen of A. alternata (Løwenstein, Nyholm), were compared by tandem crossed immunoelectrophoresis (CIE), RAST inhibition, and the CRIE-related technique, single radial radioimmunodiffusion (SRRID). The two allergen preparations showed reaction of identity in tandem-CIE and indistinguishable specific IgE binding in CRIE and SRRID, regardless of antibodies and serum pools used. In RAST inhibition, the relative potencies of the allergen preparations and of the crude extracts correlated well with their Alt-I/Ag 1 content as estimated by rocket immunoelectrophoresis. Moreover, all inhibition curves were parallel, confirming identical IgE binding by Alt-I and Ag 1 with the serum pools used. A second preparation of Alt-I, isolated from another strain of Alternaria, showed reaction of partial identity with Ag 1 in tandem-CIE, indicating that different variants of Alt-I (Ag 1) may exist in different strains of A. alternata.     

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 the biochemical structure of the major cat allergen Felis domesticus I.

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.     

Comparative studies on tree-pollen allergens. VI. The effect of thermal denaturation on the reactivity of birch pollen (Betula verrucosa) allergens

The structural changes as due to thermal denaturation of antigens and allergens of birch pollen produced by gel filtration chromatography were studied by circular dichroism, RAST inhibition and immunoelectrophoretic techniques. Results of circular dichroism showed that both BV3 and BV4 contained 20-25% alfa-helix, while BV45 consists of beta-pleated sheet and random coil. Fractions BV3 and BV4 lost about 50% of their native tertiary structures when heated at 100 degrees C for 3 h. The number of antigenic lines found in the CIE plates for the heat-treated fractions was reduced as well. The CRIE preparations showed unchanged radiostaining for the heated BV3 fraction (correlated to the untreated BV3 fraction). For the BV4 fraction a diminished radiostaining was observed. A reduction of 8-fold and 4-fold of the IgE binding capacity was observed in RAST inhibition for fractions BV3 and BV4, respectively after heating. The purified fraction BV45 showed 50% decrease in CD absorption after heating to 100 degrees C for 1 h. The CIE pattern of the heated fraction gave a single precipitate line and the area under the precipitate line was reduced as compared to the unheated fraction. The CRIE plate showed reduced intensity of radiostaining. Skin prick tests and PK tests confirmed the reduced allergenicity of the heated fractions. These findings confirm the relative thermostability of the allergens in birch pollen. However, heating infers irreversible changes on the tertiary structure of the molecules, and these structural changes can be correlated to a quantitative reduction of allergenic reactivity.     

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.     

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 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.     

A study of allergens in celery with cross-sensitivity to mugwort and birch pollens

Sixty-one sera with positive RAST to mugwort pollen ( Artemisiae vulgaris ) were submitted to RASTs for birch pollen ( Betula verrucosa) and celery ( Apium graveolens ). In 36 cases RAST results were positive for celery. In addition, 23 sera presented specific IgE to birch pollen. The binding of specific IgE to individual allergens in celery, mugwort pollen and birch pollen was studied by the immunoblotting technique. This involved electrophoretic separation of allergenic extracts, electrotransfer of proteins onto nitrocellulose sheets and sensitive immunoenzymatic detection. Eighteen sera had specific IgE binding to two celery components of molecular weight around 15 kD. All these sera also detected a 15 kD allergen in mugwort and two allergens in birch of 14 kD and 16 kD molecular weight. The sera that did not detect the 15 kD bands in celery failed to react with both the 15 kD mugwort component and the 14 and 16 kD birch components. Specific cross-inhibitions of the detection of these allergens on immunoblots were obtained by pre-incubation of the sera with crude extract of the three species. These results strongly suggest that such allergens display some structural identity and that they could be at the origin of some cases of crossed hypersensitivity to celery, mugwort pollen and birch pollen.     

Detection of Chlorella-specific IgE in mould-sensitized children

The content of IgE, specific to the unicellular green alga Chlorella sp., was analysed in sera from 46 atopic children sensitized to moulds, using radioallergosorbent test (RAST), immunoblotting and crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis (CIE/CRIE). Chlorella-specific IgE was found in 23/46 sera by RAST, in 28/41 sera by immunoblotting and in 6/30 sera by CIE/CRIE. The Chlorella components most frequently binding IgE as analysed by gradient gel electrophoresis and immunoblotting were of molecular weights of approximately 13, 17, 19, 26 and 49 kD. Twenty-nine precipitating antigens, including seven IgE-binding precipitates were detected by CIE/CRIE. The study shows that low concentrations of specific IgE are formed to the green alga Chlorella in sera from atopic individuals sensitized to moulds.     

Characterization and partial purification of the cockroach antigen in relation to housedust and housedust mite (D.f.) antigens

Allergens in crude cockroach antigens (CRa) have not been well defined. In order to characterize the CRa, the authors partially purified CRa by gel filtration (G-75) and analyzed it by polyacrylamide electrophoresis (PAGE) and allergy skin testing (AST). Hyperimmune serum was produced against CRa in rabbit (R anti-CRa) and used for immunoelectrophoresis (IEP), double immunodiffusion (DID), crossed immunoelectrophoresis (CIE), and crossed radio immunoelectrophoresis (CRIE) to further characterize the CRa. The crude CRa contained 9 and 15 precipitating arcs by IEP and CIE, respectively. Indirect CRIE revealed that CRa contained nine to 15 different allergens. There were no lines of identity between the CRa and housedust mite (D.f.) or between the CRa and commercial housedust by DID or IEP. Autologous housedust of cockroach asthmatic subjects showed lines of identity with the CRa. Fractionation of CRa on a Sephadex G-75 column chromatography showed two distinct peaks (B1 and B2) separated by a broad valley. The high molecular fraction B1 was most allergenic (100%) and contained three antigens of high molecular weight. These three antigens were identifiable by the techniques of IE, CIE, and DID. B2, the low molecular fraction (molecular weight less than 13,000 dalton) contained minor allergen(s). B2 elicited positive AST in 55% of cockroach-sensitive individuals, but it was not detected by IEP, CIE, nor by DID.     

Shared allergenic and antigenic determinants in Alternaria and Stemphylium 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.     

Identification of crustacea allergens by crossed radioimmunoelectrophoresis

Crossed immunoelectrophoresis (CIE) detected 18 precipitating antigens in extracts of shrimp. Of these antigens, crossed-line immunoelectrophoresis (CLIE) of shrimp extract demonstrated that 5 cross-reacted with crayfish, 3 with lobster and 1 with crab extract. Allergens present in the shrimp CIE plates were identified by crossed radioimmunoelectrophoresis (CRIE) using sera from 6 study subjects who were skin-test and RAST positive to shrimp extract. Of the 7 allergens detected, 3 (precipitins 1, 3 and 6) reacted with most of the 6 sera tested from shrimp-sensitive subjects. Precipitins 1 and 6 appear to be common crustacea allergens (present in shrimp, crayfish, lobster and crab) whereas precipitin 3 may be a specific allergen since it is present only in shrimp.     

Reactivity of ragweed allergens with IgE antibodies. Analyses by leukocyte histamine release and the radioallergosorbent test and determination of cross-reactivity.

Five distinct proteins with allergenic activity have been isolated from short ragweed pollen. We initially tested three of these, AgE, AgK, and Ra3, for reactivity with IgE antibodies by leukocyte histamine release and by the radioallergosorbent test (RAST). We found highly significant correlations between the reactivities of these allergens by leukocyte histamine release and by the RAST, consistent with the view that both procedures detected comparable allergenic activity. We next tested the allergenic cross-reactivity of all five ragweed allergens. AgE, AgK, Ra3, Ra4, and Ra5, by RAST inhibition. With solid-phase AgE the only nonhomologous inhibitor was AgK, which cross-reacted weakly and required a 140-fold mass excess of AgK compared to AgE. With solid-phase AgK both AgK and AgE produced significant inhibition; AgE was slightly more potent than the homologous AgK, Ra3 and Ra5 were allergenically unique, because only the homologous allergen produced 50% inhibition. Ra4 was weakly inhibited by AgE, Ra3, and Ra5 when these allergens were added in 300- to 5---fold mass excesses; this weak inhibition may represent either cross-reaction or cross-contamination. We found that RAST inhibition could be used as an assay for the individual ragweed allergens and we demonstrated the presence of all of the allergens in a whole ragweed extract. The sensitivity of the RAST inhibition assay ranged from 10 ng to 100 ng for 50% inhibition. Finally, the solid-phase ragweed allergens were used to determine the frequency of elevated IgE antibody levels in 65 patients with ragweed hay fever. Virtually all of the patients reacted with AgE (97%), while 88% reacted with AgK, 51% reacted with Ra3, 28% reacted with Ra4, and 17% reacted with Ra5. These results highlight the usefulness of the RAST as a specific and sensitive tool for immunochemical studies of allergens.     

Identification of dander-specific and serum-specific allergens in cat dandruff extract

Cat dandruff extract was characterized by crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) using sera from 27 cat-sensitive individuals. By applying a CIE/CRIE system with intermediate gel, the content of dander-specific and serum-specific allergens was established. Thirteen serum-related and eight dander-related antigens were identified in cat dandruff. Of these, eight antigens were visibly radiostained in CRIE. Five allergens were serum-specific and of these allergens cat albumin was the most important (intermediate allergen). Three allergens were dander-specific and of these three only cat allergen I fulfilled the criteria for a major allergen.     

Characterization of Chenopodiales (Amaranthus retroflexus, Chenopodium album, Kochia scoparia, Salsola pestifer) pollen allergens

Pollen extracts of the four taxonomically related weeds, Amaranthus retroflexus (Ama r), Chenopodium album (Che a), Kochia scoparia (Koc s) , and Salsola pestifer (S. kali) (Sal p) , were characterized by various methods including crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), and SDS-PAGE immunoblotting. The allergen profiles were determined by CRIE and SDS-PAGE IgE immunoblotting. CRIE detected from one to four important allergens, while SDS showed up to four bands that bound IgE from a number of patient sera. CRIE and SDS-PAGE immunoblotting did not recognize the same number of important allergens in the individual weeds, and the number of allergens detected by the two methods differed considerably, suggesting that IgE-binding epitopes may be denatured during SDS-PAGE. However, it was possible to correlate the identity of some of the important allergens detected by CRIE and SDS-PAGE immunoblotting in all four weeds.     

Expression of birch pollen-specific IgE-binding activity in seeds and other plant parts of birch trees (Betula verrucosa Ehrh.).

Dot immunoblotting of crude extracts of various aerial parts of birch trees, using patient serum rich in birch pollen IgE, showed IgE-binding activity in leaves, buds, twigs, seeds, bark, and old male catkins. Seed extracts analysed by SDS-PAGE, electroblotting to nitrocellulose and immune detection using isotope-conjugated anti-IgE verified the presence in seeds of an IgE-binding protein of an approximate molecular weight of 12 kD, distinct from the major allergen (molecular weight 17 kD) of Betula verrucosa pollen. The allergen of birch seeds was readily leachable from the seeds. Many of the birch plant part extracts were active in RAST inhibition using birch pollen RAST discs, but showed low potency relative to the allergenicity of birch pollen allergens.