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.     

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.     

A collaborative study on the first international standard of Dermatophagoides pteronyssinus (house dust mite) extract

A collaborative study was carried out to assess the suitability of a preparation to serve as the International Standard for Dermatophagoides pteronyssinus (house dust mite) extract. The proposed international standard of D. pteronyssinus, two additional freeze-dried extracts, and a commercially available skin testing solution were tested in the study. Nineteen laboratories in 11 different countries participated. The assay methods used included RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, isoelectric focusing, quantitative skin testing, and various other methods for assessing total allergenic activity. In addition, six laboratories measured the quantity of antigen P1, and three laboratories measured antigen DpX in each of the preparations. On the basis of the results from this study, the World Health Organization established the preparation as the International Standard for D. pteronyssinus extract with an assigned unitage of 100,000 IU per ampule. The units refer both to the total allergenic activity of the ampule and to that of the individual allergens, such as P1 and DpX.     

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 degrees 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.     

Production of an international reference standard alternaria extract. I. Testing of candidate extracts

As part of a program to establish international standards of selected allergens, 6 coded extracts of Alternaria were assessed in 6 laboratories by immunochemical, biochemical and physicochemical procedures. Direct RAST, RAST inhibition, quantitative skin tests and leukocyte histamine release were used to assign relative orders of potency to the 6 extracts. The composition and major allergen content was tested by thin-layer isoelectric focusing and quantitative immunoelectrophoresis (crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis). Three laboratories determined the quantity of purified allergens in each of the preparations. In addition, source materials were sent to an expert Alternaria taxonomist for independent identification. The results showed considerable variation with respect to total allergenic potency and content of individual allergens. Source materials could not be confirmed as Alternaria in some instances. Based on fulfillment of written specifications and assay results, extract No. 6 was recommended by the Alternaria Working Group as the candidate international standard to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies.     

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.     

Production and testing of an international reference standard of short ragweed pollen extract

A lyophilized candidate International Reference Standard of short ragweed pollen extract was prepared by use of defined source material. In preliminary experiments, this extract was demonstrated by RAST inhibition and crossed radioimmunoelectrophoresis assays to contain several well-characterized ragweed allergens and to contain multiple antigenic bands by crossed immunoelectrophoresis analysis. In a subsequent multinational collaborative study involving 12 laboratories in five countries, the candidate extract was compared with existing national reference or commercial ragweed extracts by a variety of immunochemical, biochemical, and physicochemical procedures. The candidate extract could be used to assign relative orders of potency to the comparison-test extracts. In separate studies, the candidate extract was demonstrated to be stable when it was stored at either -20 degrees C or +5 degrees C for at least 2 yr. The candidate extract has been accepted as an International Reference Standard with an assigned arbitrary potency of 100,000 units per ampule .     

Comparative studies on tree pollen allergens. XVII. Immunochemical analysis of the international standardization extracts of birch (Betula verrucosa) pollen as compared with a local partially purified extract

Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.     

Comparison of allergenic potentials of timothy (Phleum pratense) pollens from different pollen seasons collected in the Belgrade area

We investigated extracts of timothy grass pollen from four seasons (1989, 1990, 1991, and 1994) by protein content, SDS-PAGE, immunoblot, RAST, RAST inhibition, and crossed immunoelectrophoresis. Extract of the pollen from 1991 showed the lowest yield in quantitative assays. SDS-PAGE, crossed immunoelectrophoresis, RAST, and RAST inhibition expressed approximately comparable patterns for all extracts except that from 1991. Obviously, the quality of grass pollens, as shown for some ragweed ( Ambrosia elatior ) pollens depend on year of collection. Our findings are partially in agreement with some earlier examinations of the quality of timothy pollen from different pollen seasons.     

A comparative study of the allergens of cat urine, serum, saliva, and pelt

In direct RAST analyses of sera from 43 individuals with a history of cat allergy, 39.5% were positive to cat pelt, 37.5% to cat saliva, and 12% each to cat urine and serum. The cat pelt and saliva extracts contained allergen 1, but cat serum and cat urine collected by bladder puncture had no detectable levels of this allergen. A crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis analysis failed to reveal any allergen in urine or serum that was not also present in the saliva or pelt preparations, although urine had two allergens not present in serum. When serum from a patient who was direct RAST positive to cat pelt, serum, saliva, and urine was tested by crossed radioimmunoelectrophoresis, it was determined that a total of six allergens were detectable in cat pelt, three in cat urine, and six in cat serum. Since cat serum contains no detectable cat allergen 1, it may be concluded that at least seven allergens derived from the cat are capable of binding to IgE antibody in humans.     

Standardization of Dermatophagoides pteronyssinus: assessment of potency and allergen content in ten coded extracts

As part of the studies to establish an international reference preparation of Dermatophagoides pteronyssinus allergens, ten coded extracts of this mite were assessed in four laboratories. The extracts were compared for total potency using direct RAST, RAST inhibition and quantitative skin tests, and also for composition and major allergen content using crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, rocket immunoelectrophoresis and radioimmunoassay. In addition, the source materials were examined by light microscopy, and the extracts were examined for the presence of proteins/allergens derived from the culture media. To help with standardization, a new reference pool of sera from patients allergic to D. pteronyssinus was also established (National Institute of Biological Standards and Control, NIBSC, 82/528). The results showed that techniques are available for measurements of potency and allergen content. In several of the extracts, culture medium derived allergens and antigens were demonstrated. It also became clear that extracts varied not only in their total potency but also in the distribution of the identifiable allergens. In particular, extracts derived from isolated mites contained more AgX and/or Ag 23 relative to their content of antigen P1 (= Ag42). These studies lead to the choice of an extract for an international reference preparation (NIBSC 82/518) and helped to establish the methods used subsequently in the international collaborative study.     

Cross antigenic and allergenic properties of the house dust mite Dermatophagoides farinae and the storage mite Tyrophagus putrescentiae

The crossed antigenicity and allergenicity of the storage mite Tyrophagus putrescentiae (TP) and the house dust mite Dermatophagoides farinae (DF) were characterized by means of crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. DF extracts exhibited 32 antigens and as many as eight were demonstrated to be allergens. DF feces exhibited 20 antigens and six of these were allergens. Twenty antigens and two allergens were demonstrated for TP. Two antigenic and allergenic determinants were shared by DF and TP, and two determinants were also shared by DF feces and TP feces. TP feces and DF shared two antigenic and allergenic determinants. Our results demonstrated that the two mites and their feces extracts contain multiple antigens and allergens.     

Standardization of rye-grass pollen (Lolium perenne) extract. An immunochemical and physicochemical assessment of six candidate international reference preparations

Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.     

Quality of timothy pollen (Phleum pratense) from different pollen seasons and different suppliers

We investigated seven batches of timothy (Phleum pratense) pollen from four different pollen seasons and three different manufacturers in the USA and Europe by several biochemical and immunochemical methods. By measuring the contents of hexoses, proteins and the total allergenic activity using RAST inhibition we could not find any significant differences in quality between pollen of different seasons, different geographical origin and different manufacturers. There were only slight differences in the staining of some protein bands and in peak height of some antigens in isoelectric focusing (IEF), SDS-PAGE and crossed immunoelectrophoresis. These differences in IEF and SDS-PAGE patterns seem to be specific for the pollen samples from the USA and Europe, respectively. We also observed minor differences in IEF immunoprint patterns, mainly for some basic proteins. The allergen patterns in crossed radioimmunoelectrophoresis were very similar, with only minor differences in concentration of the basic allergens. It appears that timothy pollen from different years can be produced in different geographical areas by different manufacturers with fairly constant allergenic activity.     

Fusarium solani: Evidence for common antigenic/allergenic determinants with other Fungi Imperfecti

Aqueous extracts of Fusarium solani and other members of the Fungi Imperfecti were evaluated for the presence of common antigenic/allergenic determinants using skin-prick testing, radio-allergo-sorbent test (RAST) inhibition, and immunoelectrophoretic methods. Prevalence of skin reactivity in forty-four atopic individuals, tested with commercially available fungal extracts, ranged from 27.3% for Alternaria tenuis to 6.8% for Penicillium notatum. No specific patterns of reactivity emerged from statistical analyses of skin test data. In contrast, RAST inhibition demonstrated common allergenic determinants. P. notatum and Aspergillus glaucus inhibited F. solani RAST by 79% and 84%, respectively. This was supported by crossed line immunoelectrophoresis; both P. notatum and A. glaucus had antigenic determinants in common with F. solani. Collectively, these studies suggest that F. solani, P. notatum , and A. glaucus have several common antigenic/allergenic determinants.     

Studies on Alternaria allergens: II. Measurement of the relative potency of commercial Alternaria extracts by the direct RAST and by RAST inhibition

The relative potency of 12 commercial Alternaria extracts was analyzed by end-point skin test titrations and compared to in vitro measurements of potency, including (1) the radioallergosorbent test (RAST) inhibition procedure; (2) the direct RAST procedure; and (3) the protein nitrogen unit (PNU) content. Potencies determined by skin testing 10 sensitive patients were strongly correlated among the various patients. Measurements of potency by both RAST inhibition and direct RAST assay were strongly correlated to potency as measured by skin testing. In contrast, neither the weight:volume nor the PNU content bore any relationship to allergenic potency as measured by skin testing or by either of the RAST procedures. Extracts differed by as much as 3,000-fold in allergen content by skin testing. Moreover, the extracts appeared to contain different allergenic determinants when tested by RAST inhibition. RAST inhibition offered several technical advantages over the direct RAST procedure, in that only one solid-phase RAST reagent was required, slopes of dose-response curves could be more easily compared, and a greater discrimination in allergenic potencies among extracts could be made. The RAST appears to offer an excellent method for measuring the potency of allergy extracts, pending the isolation and characterization of actual allergens.     

Multiplicity of allergens in peanuts

Crude peanut protein fractions from raw and roasted peanuts were examined in the RAST with 10 sera from patients showing clinical peanut sensitivity. The radioactive uptake results, which were generally high, did not reveal any distinguishable pattern. Two commercially available peanut proteins, peanut lectin and phospholipase D, gave poor RAST responses. Three purified peanut proteins, α-arachin, conarachin I, and concanavalin A-reactive glycoprotein, all gave significant RAST results that were generally lower than those obtained with the crude extracts. The extent of RAST inhibition obtained with these materials was inversely related to their abundance in the total peanut protein. Crossed immunoelectrophoresis with extracts from raw and roasted peanut indicated the presence of 22 and 10 anodically migrating antigens, respectively. Sixteen IgE binding antigens were revealed for raw peanut and seven for roasted peanut after incubation with a mixed serum from the 10 patients in crossed radioimmunoelectrophoresis (CRIE) using ¹²⁵I-labeled anti-IgE. CRIE plates treated with individual serum samples showed that all the patients had specific IgE for the major antigen peak, which has been tentatively identified as α-arachin. This major storage protein of peanut, which is known to be particularly heat resistant, may be of greater clinical significance than its apparently low RAST activity would seem to indicate.     

Guanine content and Dermatophagoides pteronyssinus allergens in house dust samples

The study of the relationship between the guanine content and Dermatophagoides pteronyssinus (Dp) allergens in house dust samples is reported. Mattress and carpet dust of bedrooms from 22 different homes constituted the house dust samples. The guanine content was determined by quantitative measurements and the mite allergenicity by two immunochemical assays with a partially purified extract of Dp as internal reference: RAST inhibition and crossed and rocket line immunoelectrophoresis. A large scale range of guanine content was obtained among the 22 house dust samples studied (0.01 to 1.78 mg/0.1 gm of dust). Data of RAST inhibition, analyzed according to parallel line bioassay, demonstrated no significant difference between the slopes of the reference and the house dust sample lines, but a 100-fold variation in the relative Dp potencies was observed. By crossed and rocket line immunoelectrophoresis technique, the presence and the amounts of major Dp allergens (Der p I and Dp 4) were established in most house dust extracts. A significant correlation was found between the guanine content of the house dust samples and their relative Dp potencies (r = 0.86) on the one hand, and with their relative content of Der p I and Dp 4, two major Dp allergens (r = 0.75 and r = 0.74, respectively) on the other hand; in each case, a quantitative relationship was established. These results suggest that the guanine determination could assess mite allergens in house dust and may be a useful tool in large-scale investigations of house dust.     

Crawfish and lobster allergens: identification and structural similarities with other crustacea

Antigenic and allergenic components in crawfish and lobster extracts were studied using crossed immunoelectrophoretic techniques. Crossed immunoelectrophoresis with rabbit antisera revealed 23 antigens in crawfish and 17 antigens in lobster extracts. Both extracts exhibited structural similarities in antigens mutually and with other crustacea in cross-line immunoelectrophoresis. Crossed radioimmunoelectrophoresis (CRIE) demonstrated 6 crawfish and 4 lobster allergens when individual or pooled sera from radioallergosorbent test (RAST)-positive crustacea-sensitive subjects were used. Since radiostaining was also observed with sera from RAST-negative nonsensitive subjects, specificity of IgE binding was tested using CRIE-inhibition. Preincubation of RAST-positive sera with crawfish or lobster extract decreased radiostaining in CRIE, while no changes occurred when using control sera. These results confirmed the presence of IgE-mediated mechanisms in seafood allergy and demonstrated a number of shared antigenic determinants among crustacea allergens.     

Dust from carpeted and smooth floors

Dust samples were collected twice from smooth and carpeted floors in 10 Norwegian schools. The content of antigens and allergens of alder (Alnus incana), birch (Betula verrucosa), timothy (Phleum pratense), cat and dog dander, house dust mite (Dermatophagoides farinae), mould (Cladosporium herbarum), hen egg white and codfish (DIII) were investigated by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE), radio allergosorbent test (RAST) inhibition and quantitative precipitation inhibition analysis by laser nephelometry. Antigens and allergens of cat and dog dander and hen egg white were most prevalent in the dust samples investigated. With the exception of hen egg white and codfish allergens, no statistically significant differences in mean allergen content were shown in identical quantities of freeze-dried dust extracts from carpeted and smooth floors. RAST-inhibition analyses of identical amounts of dust from either floors showed higher content of allergens of cat, dog, hen egg white, codfish, mould and timothy pollen in classrooms with carpets.