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.     

A reference allergen preparation of the house dust mite D. pteronyssinus, produced from whole mite culture--a part of the DAS 76 study. Comparison with allergen preparations from other raw materials

A D. pteronyssinus whole culture allergen preparation contained 49 antigens as revealed by crossed immunoelectrophoresis (CIE), using polyspecific rabbit antibodies. Crossed radio-immunoelectrophoresis ( CRIE ) with sera from 30 patients revealed nine allergens, antigens 42, X, Y and 23 (in rank order) showing the most frequent and intense IgE-uptake. Nine antigens originated from the culture medium (human dander + yeast), but none of these gave rise to specific IgE-uptake. Extremely few and weak reactions were observed in radioallergosorbent (RAST) with 129 sera, using media extracts on the discs. Purified mite body extract (PMB) contained less ag 42 and more ag Y and ag 23 than whole mite culture extract ( WMC ), whereas an acetone-extracted mite excreta preparation (AML) contained 5 times more ag 42, but was devoid of ag Y and ag 23. Ag X was present in all preparations. The RAST-inhibitory potency of PMB was best correlated with the content of ag X. Preparations with properties similar to WMC and PMB were judged as suitable for clinical application.     

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.     

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.     

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.     

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.     

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.     

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.     

Reproducibility of skin prick testing with allergen extracts from different manufacturers

Allergen extracts for skin prick testing (SPT) are available from several manufacturers. Although these solutions are specified according to well-defined internal company standards, there is no generally accepted overall standardization. To assess the comparability of skin prick test solutions of various manufacturers, we compared extracts of nine different allergens from four companies by SPT in 29 children sensitive to one or more of the allergens, in a double blind fashion. RAST (Pharmacia) and EAST (Kallestad) were determined in simultaneous blood samples. Allergen extracts were also examined by rocket immunoelectrophoresis for their content of major allergens. Skin reactions, assessed by mean diameter of wheals, to identical allergen extracts varied significantly between the four vendors (P less than 0.05-0.001). Correlation between RAST and EAST was good for all allergens except birch pollen and mugwort. Content of the major allergen in corresponding extracts varied significantly between the different companies (less than 1%-2000%). These data underline the need for international reference extracts as intracompany standardization of test solutions alone is not enough to yield general reproducibility of skin prick test results.     

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.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

Standardization of allergen extracts by inhibition of RAST, skin test, and chemical composition

Five allergen extracts of Dermatophagoides pteronyssinus, Lolium perenne, Alternaria tenuis, Aspergillus fumigatus and Cladosporium herbarum, obtained from four different manufacturers, were examined by inhibition of RAST, content of protein and carbohydrate, contents of phosphorylcholine (Pc) and tridacnin reactive components, and by skin test. Inhibition of RAST was used as a primary method for establishing allergenic potency and demonstrated wide variations for each preparation supplied by the different manufacturers. The extracts also varied widely in protein and carbohydrate content and in the ratio of these parameters, indicating internal heterogeneity. Pc content was significantly related to RAST potency for extracts of A. fumigatus and A. tenuis, suggesting that Pc content may be used as a primary standarization procedure for these extracts. Skin test reactions undertaken at a single concentration did not show any significant variation in weal size between preparations of a given allergen extract. However, of particular importance to practising clinicians is the finding that varying numbers of patients showed negative skin reactions to one preparation of a particular allergen yet were positive to the corresponding preparations supplied by the other companies.     

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.     

Antigenic/Allergenic Composition of Poodle/Alsatian Dandruff Extract

Poodle and Alsatian dog dandruff extracts were characterized by crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) using sera from 24 individuals clinically sensitive to dogs. By using a system with intermediate gel in immunoelectrophoresis, the content of dander-specific and serum-specific allergens was established. 29 antigens (18 dander-specific and 11 serum-specific) were identified in the mixed breed Poodle/Alsation dandruff extract. Of these, 24 antigens were radiostained in CRIE. 16 allergens were dander specific and the remaining eight were serum specific. Positive dog dander RAST (e5 and Poodle/Alsatian dandruff extract) results were observed in the tested dog hypersensitive subjects. Our results suggest that the mixture of Poodle/Alsatian dandruff extract may be a suitable preparation for the diagnosis and treatment of dog allergy.     

Allergy to mice. I. Identification of two major mouse allergens (Ag 1 and Ag 3) and investigation of their possible origin

An extract of dust from the outlet filters of a mouse isolator was used as a basis for determining the source of inhalant allergens for subjects sensitive to this species. The antigenic components, identified by crossed immunoelectrophoresis (XIE), were compared to those found in extracts of other mouse-derived source materials, i.e. urine, fur, dander and saliva. Of the eight dust components, one (Ag 1) was identified as antigenically identical to the major urinary pre-albumin whilst the others were detected in fur, and to a lesser extent dander and saliva. None of the dust antigens was detected as a component of food or bedding.
Crossed radio-immunoelectrophoresis (XRIE), performed using sera from a group of fifteen mouse-allergic subjects (positive by RAST to mouse extracts), identified seven of the dust antigens as IgE-binding components. Antigens 1 and 3 were reactive with all the sera tested and have, therefore, been termed the'major'allergens. Varied responses were obtained to the other'minor'antigens.
Ag 1 (urinary pre-albumin) and Ag 3 were detected in all samples of mouse dust studied. RAST and RAST inhibition also indicated the presence of urinary prealbumin. These findings suggest that the major mouse inhalant allergens may be derived predominantly from urine and secretions originating in the skin and present on the fur.     

Identification and Clinical Significance of Allergenic Molecules of Cat Origin

Freeze-dried extracts from cat dander and the corresponding rabbit antibodies were used for establishing the CIE reference pattern for cat dander extracts. Anti-Cat Ag 1 and anti-cat albumin were used for identification of the corresponding antigens. CRIE on sera from selected groups of American and Danish cat-allergic patients demonstrated antigen-specific IgE binding to 10 of 15 cat dander antigens (Cat Ag 1 being the major allergen). Only minor differences were found between the two groups. Four of these allergens were serum proteins. Variable amounts of many of the 10 allergens were measured by QIE in saliva, serum, urine and three cat pelt extracts. However, extremely wide ranges for content of the serum allergens and the non-serum allergens were found. This was exemplified by an albumin/cat Ag 1 ratio between 1 and 400, smallest in cat dander. Immunoabsorption using anti-cat dander, anti-cat albumin and anti-Cat Ag 1 indicated that the anti-cat dander, anti-cat albumin, and the anti-Cat Ag 1 absorbed approximately 90%, 25%, and 56%, respectively, of the dander RAST activity, and 87%, 11%, and 45%, respectively, of the saliva RAST activity, confirming the major importance of Ag 1. It is concluded that cat allergenic extracts should contain only modest amounts of serum albumin and other serum-derived antigens and that any relevant standardization must include quantification of at least Cat Ag 1 and cat albumin.     

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 .     

Dust from carpeted and smooth floors. I. Comparative measurements of antigenic and allergenic proteins in dust vacuumed from carpeted and non-carpeted classrooms in Norwegian schools

Dust samples from fitted-carpets and linoleum floors in 12 schools in Norway were collected by vacuum cleaning. The presence of antigens and allergens of alder (Alnus incana), birch (Betula verrucosa), timothy (Phleum pratense), mould (Cladosporium herbarum), house dust mite (Dermatophagoides farinae), cat and dog dander, codfish, hen egg white and human dander were investigated by crossed immunoelectrophoresis (CIE), crossed radio-immunoelectrophoresis (CRIE) and radio-allergosorbent test (RAST) inhibition. No qualitative differences in allergen contents of dust from both types of floor tested were noted. Similarly, no relationship could be demonstrated between floor-type and allergen concentration under identical experimental conditions. Antigens and allergens of both cat and dog were frequently demonstrated in dust extracts. All extracts included human dander and mould allergens. In addition, most dust samples from both carpeted and smooth floors contained hen egg white and codfish allergens. Furthermore, the study demonstrated that dust from smooth floors and fitted-carpets was relatively free of mite and pollen from alder, birch and timothy.     

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity

A solid-phase, monoclonal antibody-based ELISA was set up to quantitale group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1–100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleumpratense extracts, and a very good quantitative correlation was found ( r =0.98; P <0.0001). A highly significant correlation ( r >0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.