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A cDNA clone coding for a mite allergen of mol wt approximately 14,000 has been isolated and its DNA sequence determined. The native component from mite extracts encoded by this DNA was identified by immunoprobing blots of mite body extract with human IgE eluted from the electroblotted cloned fusion polypeptides derived from the expressed cDNA clone. The clone encodes a polypeptide with a deduced mol wt of 17,460. The deduced amino acid sequence was not homologous to any known protein sequences and it contains no cysteine or tryptophan. On blots, 21 of 38 sera from mite-allergic subjects recognized the cloned material, and this recognition strongly correlated with IgE-binding to the native component on protein blots of mite extract.

Samples of dust were collected from three articles of household bedding in twenty-two Sydney homes during a six-week period beginning in mid December, 1973. Mites, sometimes in large numbers, were found in all homes. The mean number of mites recovered from nursery sheepskins (all woollen) was thirteen times as high as the mean number from other forms of adult or infant bedding sampled. The high mean number in sheepskins is the result of massively high populations in seven of the eighteen skins sampled. Hospital sheepskins were almost almost uniformly mite free. Far fewer mites were collected from cot mattresses which were either vinyl topped or covered with a plastic coverslip than were collected from a cot mattress with a cotton top alone.

Monoclonal antibodies that recognize allergens are potentially extremely useful reagents for use in a variety of applications, including highly specific and sensitive assays for the standardization of allergen extracts and the measurement of environmental allergens, epitope mapping of allergens, comparison of IgE-binding determinants, and the immunolocalization of allergens in thin sections. Although monoclonal antibodies have many properties that enable them to be shared internationally, it is difficult to know from the literature what monoclonal antibodies are available and, in some cases, what are the individual characteristics of the antibodies. We are organizing an on-line data base of information about allergen-reactive monoclonal antibodies, which can be accessed by computer with international telecommunications networks. Each record of a monoclonal antibody includes details, if they are known, of allergen source and name, antibody production, complimentary antigenic determinant, names and addresses of the owners, availability of the antibody, and other important information. Investigators who have produced monoclonal antibodies that react with allergens are invited to submit details of the antibodies for inclusion in the data base.

Eight Dermatophagoides pteronyssinus extracts (three culture extracts, four mite body extracts, and the World Health Organization International Standard [IS]) were investigated by side-by-side sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by electrotransfer to nitrocellulose, and by probing with a pooled serum from mite-allergic subjects. Representative body and mite culture extracts were compared by probing with individual sera, and both types of extract were also compared by RAST-inhibition studies. Extracts from the same source differed in the molecular weight (MW) of some of their IgE-binding components. In general, most IgE-binding components in culture extracts and the IS were in the 14 to 35 kd MW region, whereas extracts from mite bodies and one culture extract contained more IgE-binding components of higher MW (35 to 110 kd). Comparison of representative mite body and culture extracts by use of 22 separate sera resolved 26 and 19 IgE-binding components in the two extracts, respectively. Patterns of RAST inhibition produced by both types of extracts when they were used either as the inhibitor or as the allergosorbent demonstrated qualitative differences between the two types of extracts. These results demonstrate that mite extracts may differ considerably in their allergenic composition and emphasize the need for standardization of mite allergenic extracts and the reexamination of the suitability of the D. pteronyssinus IS.

The resolution and detection of individual components in complex extracts by protein blotting have been investigated. By probing nitrocellulose transfers with monospecific and multispecific antisera, it was demonstrated that dissociating conditions were required for the maximum resolution of antigens by polyacrylamide gel electrophoresis, a conclusion reinforced by results from 2-D electrophoresis. The dissociating and reducing treatments employed, however, were both shown to be responsible for some loss of total antigenicity and included the complete loss of at least one important antigen. Assays with nitrocelluloses of different pore sizes demonstrated that both higher protein-binding capacities and higher backgrounds were associated with the use of the smallest pore size, while the sensitivity of the assay was greatest when a non-ionic detergent, and not proteins, were used for blocking. Nitrocellulose-bound proteins may be stained with amido black, India ink, toluidine blue, Ponceau S or a gold sol, but these agents do not always give identical staining patterns. While detection of components with immuno-enzyme staining methods had some advantages, problems with non-specific binding were encountered. These did not occur with affinity purified radiolabelled second antibodies, which in combination with scanning of autoradiographs allowed a quantitative approach to be adopted.

Sera from 30 workers in an entomological research institute and from five Sydney asthmatics, all with serum IgE antibodies to sheep blowfly (Lucilia cuprina) antigens, were studied with the aim of identifying individual IgE-binding proteins in extracts of L. cuprina adult flies, larvae and eggs, and in extracts of related species of flies from the order Diptera. Using protein blotting, 21, 18 and nine different IgE-binding components were identified in extracts of L. cuprina adult flies, larvae and eggs, respectively. A component(s) of MW 67 kD found in all three developmental stages, showed the highest frequency of IgE-binding; reacting, for example, with 70% of the sera tested with the adult fly extract. Some components were detected in only one of the three developmental stages. Investigations of possible allergenic cross-reactivity between L. cuprina and extracts from six other related species of flies revealed IgE-binding bands in each of the extracts as well as in an extract of Agrotis infusa, a cutworm not belonging to the order Diptera. One strongly reacting component of MW 20 kD was detected in extracts of five different fly species as well as in the A. infusa extract. The results suggested that allergenic cross-reactivity between some fly species exists, and may extend to taxonomically unrelated insect species.

The effect of blocking nitrocellulose membranes with the nonionic detergent Tween 20 on the detection, by protein blotting, of IgE-binding to house dust mite Dermatophagoides pteronyssinus allergens has been investigated. Tween blocking led to enhanced immunodetection of allergens despite removal of proteins from the membrane when compared to protein blocking agents which did not displace transferred components. The enhancement varied with the different mite components and, for one in particular, antigen Der p II, an increase of more than 100-fold in IgE antibody binding occurred despite a concurrent loss of more than 90% of Der p II from the membrane. Both the enhancement of binding and loss of components from the membrane were dependent upon the time course of blocking and the concentration of Tween used.

The reproducibility of the radioallergosorbent (RAST) inhibition and direct binding assys with mite allergen were investigated in the presence of heterogeneous extracts and non-mite-sensitive atopic sera. Both contain components similar to potential contaminants which would occur in the assay of mite allergen and dust allergen and dust allergen extracts. The standardized inhibition and direct binding assays employed had a day to day (n = 4) coefficient of variation [(s.d. x 100)/mean] of 15% and 24% respectively. The inhibition assay for mite allergen was reproducible in the presence of protein concentrations of added plant, fungal, arthropod and animal extracts in excess of the protein concentrations that occur under the operational mite assay conditions. The mite inhibition assay was also reproducible in the presence of non-mite allergen extracts, with and without additional sera containing IgE specific for the non-mite allergens. The binding of a additional sera containing IgE specific for the non-mite allergens. The binding of a constant quantity of mite allergen to the activated solid phase in the direct binding assay was reproducible in the presence of added bovine serum albumin, and of a fungal or arthropod extract, representing the heterogeneous components of an allergen extract at the concentrations of total protein known to occur in the direct binding assay of mite extracts.

The association between house dust allergy and asthma has long been recognized, and it has been demonstrated that a major allergen in house dust is related to the presence of mites of the genus Dermatophagoides. Using extracts of mite culture for skin testing, as many as 10% of the population and up to 90% of allergic asthmatics give positive immediate reactions. Although mites may occasionally become airborne during bed-making, it has also been demonstrated that they 'secrete or excrete' some allergen. Recently, we have shown that up to three-quarters of the serum IgE antibodies to mites are directed against a major allergen-antigen P1 (molecular weight 24,000). Using a radioimmunoassay it is possible to measure the concentration of this glycoprotein in both dust samples and mite cultures. These measurements, which are reported here, show that more than 95% of the allergen accumulating in mite cultures is associated with faecal particles.

Mite allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 45 mite atopic sera and an enzyme immunostaining assay for IgE. More than 20 different mite components bound IgE and almost every serum showed a different pattern of binding. 7 of the 20 components were bound by half of the sera and 70% were bound by at least 20% of the sera. These results demonstrate a greater number of house dust mite allergens and a far greater diversity of the IgE antibody response to mite allergens than has previously been described.

To study the long-term effects of avoiding domestic allergens, nine asthmatic patients who were allergic to dust mites lived in hospital rooms for two months or more. In all patients symptoms and early morning peak flows improved. In seven patients anti-asthma treatment could be reduced and it was possible to carry out repeated bronchial provocation with histamine. Five of these patients showed a progressive eightfold or greater increase in the concentration of histamine necessary to provoke a 30% fall in forced expiratory volume in one second (PD30). The increase in PD30 in the seven patients during their period of living in hospital was highly significant. Avoidance of important allergens seems not only to result in clinical remissions but in many cases also reduce bronchial hyperreactivity.

Employment of reducing conditions during sample preparation alters the mobility of the house dust mite (Dermatophagoides pteronyssinus) allergen Der p I as determined by sodium dodecylsulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) from an apparent MW of approximately 25 kD (unreduced) to an apparent MW of approximately 30 kD. Probing of nitrocellulose transfers with sera from subjects allergic to D. pteronyssinus showed that reduction of Der p I was accompanied by a substantial loss of IgE-antibody-binding capacity by this allergen. An important consequence of the effect of reduction on Der p I is that the electrophoretic mobility of this protein becomes very similar to a closely spaced pair of protein-staining bands, probably Der p III, of MWs 30-31 kD. These bands bind IgE antibodies strongly and with high frequency and exhibit the same electrophoretic mobilities under both reducing and non-reducing conditions. Thus, for the clear resolution of allergen Der p I from other IgE-binding components in the same MW region, including Der p III, house dust mite samples for analysis by SDS-PAGE and blotting should not be reduced.

Twenty eight per cent (15) of 53 workers engaged in a sheep blowfly breeding programme designed to control genetically the pest Lucilia cuprina experienced allergic manifestations resulting from contact with this insect. The most common symptoms were rhinitis, affected eyes, rashes, and lower respiratory symptoms, usually, but not always, immediate in type. A personal history of non-insect related asthma, allergic rhinitis, or eczema, or a combination of these was more common in the fly allergic group but some workers experienced allergic symptoms only when exposed to the adult sheep blowfly. Raised levels of serum IgE antibodies specific for adult and larval allergens were found in approximately 70% of symptomatic workers, whereas only 30% and 7% of two groups of asymptomatic workers were found to have these antibodies. It is concluded that the sheep blowfly is an important source of airborne allergens and can prove a considerable occupational health hazard. Measures designed to reduce worker contact with blowflies and their emanations considerably reduced the incidence of allergic symptoms in the exposed workers.

Using an inhibition radioimmunoassay for the major allergen from Dermatophagoides pteronyssinus (antigen P1), we studied the distribution of this dust allergen in the houses of patients with asthma. Both bed and floor dust samples contained a wide range of antigen P1, 100 to 100,000 ng/g of fine dust, and this concentration correlated well with the number of mite bodies (r = 0.81, p less than 0.001). We were unable to detect antigen P1 in the air of undisturbed rooms. However, during domestic activity, between 1 and 30 ng were collected on a filter than sampled air for 45 min at 17 L/min. Using a cascade impactor it was shown that greater than 80% of the airborne antigen P1 was associated with particles greater than 10 mu in diameter. Some of the particles containing allergen could be identified because they formed precipitin rings when impacted onto agarose containing rabbit antimite antiserum. These particles had the physical appearance of mite feces, which are the major source of antigen P1 in mite cultures. The results suggested that natural exposure to this dust allergen allows occasional fecal particles to enter the lungs and that these particles contain very concentrated allergen.

Immediate, type 1 allergic reactions occur following interaction between allergen, immunoglobulin E (IgE), and the mast cell or basophil. Allergen-induced activation of these cells leads to the release of a number of mediators that are responsible for the symptoms observed during an allergic reaction. Recent progress in elucidating the structures, and in developing syntheses for platelet aggregating factor and the leukotrienes, promises to increase our understanding of the immunopharmacology of allergic and inflammatory reactions. The need for the standardization of allergen extracts is emphasized, and current activity in this area is highlighted.

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.

Five different brands of nitrocellulose (NC), each of pore size 0.45 micron and without adsorbed antigen, bound different amounts of two labelled antisera and labelled protein A. Experiments with some non-ionic surface active agents and proteins showed that milk powder and bovine serum albumin were the most effective agents for blocking non-specific binding of labelled protein to NC. With some of the NCs, Nonidet P-40 (NP-40) and Tween 20 were almost as effective as milk powder. The protein-binding capacity of unblocked NC and the level of protein binding after blocking were found to be inversely proportional to the pore size of the NC. A comparison of blocking agents in an immunoassay with pollen proteins adsorbed to NC discs revealed that the highest specific uptakes of antiserum occurred with NP-40 and Tween and not with any of the protein blocking agents such as milk powder. Hence, for the detection of proteins using NC-based assays (but not necessarily following electroblotting), the best choices would appear to be: NC of pore size 0.45 micron; a brand of NC that provides a suitable balance between protein binding capacity and non-specific uptake of protein after blocking; a non-ionic detergent such as NP-40 or Tween 20.

An allergic reaction, provoked by exposure to the blowfly Lucilia cuprina and shown to be IgE-mediated, occurred in a subject employed in an entomological research laboratory. The subject's serum, and sera from three other asthmatic patients with IgE antibodies to blowfly extracts, also reacted with extracts from the screw-worm fly (Chrysomya bezziana). Results suggested that antigens from the two species share immunological cross-reactivity. Cross-reactions also exist between the different developmental stages of both species. Allergic reactions to inhaled insect allergens may not be uncommon in the Australian community.

Components of an extract of Dermatophagoides pteronyssinus were electrophoretically separated, transferred to nitrocellulose membranes, and then probed with a variety of antibodies and lectins. Using mite-allergic human sera as the first antibody source, IgE-binding components were identified. Binding patterns by monoclonal and heterologous sera were also obtained. The method is suitable for the qualitative, and ultimately the quantitative, characterization of allergen extracts. The electroblotting procedure employing specific probes, therefore, promises to be a valuable aid for allergen standardization.