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.     

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.     

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 .     

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.     

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.     

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.     

Allergy to guinea pigs: II Identification of specific allergens in guinea pig dust by crossed radio-immunoelectrophoresis and investigation of the possible origin

An extract of dust from the air-vent filters of a room housing guinea pigs was analysed by quantitative immunoelectrophoretic procedures and compared with extracts of various materials derived from guinea pigs. Crossed radio-immunoelectrophoresis (CRIE) of the dust, performed with sera from twenty asthmatic patients who were positive by skin testing and RAST to guinea pig extracts, identified fourteen IgE-binding constituents. Although responses varied, most sera reacted with lour particular allergens, antigens 2. 3, 10 and SI. The numbers of allergens recognized by individual patients correlated with the RAST score, but not with total scrum IgE. All seventeen dust constituents detected by crossed immunoelectrophoresis (and all four major allergens), were also present in extracts of guinea pig dander, fur. saliva and urine; several of these components were absent in an epithelial extract, and there were even less in preparations of shaved pelt, serum or faeces. None of the dust extract antigens were detected in materials used in animal husbandry, dust samples from rooms without guinea pigs, or a D. pteronyssinus extract. These findings suggest that inhalant allergens may be derived predominantly from material shed from the guinea pig coat after contamination with saliva, and possibly to a lesser extent, urine.     

Studies on Alternaria allergens: I. Establishment of the radioallergosorbent test for measurement of Alternaria allergens

The ability to covalently couple Alternaria allergens to macrocrystalline cellulose particles has permitted not only the measurement of IgE antibodies to Alternaria in patient serums but also the identification of allergenic fractions from crude Alternaria extracts. Crude aqueous Alternaria extracts from 3 commerical suppliers were coupled to cellulose but failed to bind more than 5% of total radioactive counts (TRC) when reacted with serums from highly sensitive patients. Fractionation of a commercial extract through Sephadex G-25 showed that almost all allergenic activity was located in a protein and carbohydrate-containing peak eluting at the column void volume. These fractions were pooled and coupled to cellulose to yield a RAST polymer which produced up to 20% TRC binding when tested with serums from over 100 Alternariasensitive patients, and only up to 1% TRC binding with 17 nonallergic serums. The study of commercial Alternaria extracts by chromatographic and RAST inhibition techniques showed that present extracts are neither qualitatively or quantitatively comparable.     

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.     

Preparation of allergen extracts from the green alga Chlorella. Studies of growth variation, batch variation, and partial purification

Allergen extracts were prepared from different raw materials of three strains of the unicellular green alga Chlorella (C. vulgaris, C. homosphaera and C. saccharophila). Growth variation, batch variation and composition of partially purified extracts were studied by analyzing total protein and carbohydrate content, protein and IgE-binding patterns and allergenic potency. Chlorella allergens were produced in the same proportions in early and late exponential growth phases but appeared partly degraded in the stationary phase. Uniform growth and extraction from Chlorella cells in the late exponential phase were demonstrated. Partially purified extracts from cells in late exponential phase contained 50% protein of the dry weight compared to 20% in crude extracts. The carbohydrate content was 20-30% of the dry weight of both crude and purified extracts. The allergenic potency of the purified extracts was 150-340% of the crude extracts, as assayed by RAST inhibition. The purified C. vulgaris extract showed a reduced IgE binding compared to crude extracts, while C. homosphaera and C. saccharophila extracts exhibited consistent IgE binding and are therefore suitable for in vivo and in vitro studies of allergenic properties of Chlorella.     

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.     

Allergy to guinea pigs: I Allergenic activities of extracts derived from the pelt, saliva, urine and other sources

Guinea pig-sensitive patients with asthma and rhinitis were skin test positive to extracts of several materials derived from guinea pigs. A radioallergosorbent test (RAST) was developed to measure serum IgE specific for the dander, urine, saliva and also for dust from the air-vent filters of a room housing guinea pigs. A strong correlation was found between positive skin test reactions, and raised serum IgE to these extracts. Furthermore, the relative allergenic potency of extracts was similar when determined by skin-prick testing and by inhibition of the RAST to guinea pig dust. Non-guinea pig-derived extracts such as the hay, sawdust and diet had negligible activity in skin testing and RAST inhibition; and preparations of Dermatophagoides pteronyssinus, house dust and rat dust did not inhibit the RAST for guinea pig room dust. The guinea pig dust, dander, fur, urine and saliva were the more potent extracts; while whole pelt, faces and serum were considerably less active. Extracts from different sexes were not appreciably different in potency. The results of skin testing. RAST and RAST inhibition suggest cross-allergenicity between the various extracts. Although material shed from the pelt may have been derived from saliva, or even urine, allergenic activities of urinary and salivary preparations were found to be less than those of the dander, fur or dust. This suggests that allergens have become concentrated on the pelt.     

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.     

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.     

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.     

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.     

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.     

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.     

In vivo tests with pollen extracts previously investigated by means of direct RAST titration allergen assay

The allergenic potency of different birch, Timothy and mugwort pollen extracts was determined by means of a direct RAST titration allergen assay. For birch and Timothy allergens, the results of skin and provocation tests did not confirm the results of the in vitro determinations of allergenicity. There was a poor correlation between the results of skin tests and the results of Phadebas RAST for determination of specific IgE to mugwort, whereas the correlation between skin tests and RAST for other allergens was excellent. It is concluded that direct RAST titration allergen assay is not adequate for all kinds of allergen preparations and that the Phadebas RAST for mugwort is less sensitive than the RAST for other allergens. The diagnostic efficacy of the different allergen preparations could not be evaluated.