Crystal Structures of Mite Allergens Der f 1 and Der p 1 Reveal Differences in Surface-Exposed Residues that May Influence Antibody Binding

The group 1 mite allergens Der f 1 and Der p 1 are potent allergens excreted by Dermatophagoides farinae and Dermatophagoides pteronyssinus, respectively. The human immunoglobulin E antibody responses to the group 1 allergens show more cross-reactivity than the murine immunoglobulin G antibody responses, which are largely species specific. Here, we report the crystal structure of the mature form of Der f 1, which was isolated from its natural source, and a new high-resolution structure of mature recombinant Der p 1. Unlike Der p 1, Der f 1 is monomeric both in the crystalline state and in solution. Moreover, no metal binding is observed in the structure of Der f 1 despite the fact that all amino acids involved in Ca²⁺ binding in Der p 1 are completely conserved in Der f 1. Although Der p 1 and Der f 1 share an extensive sequence identity, comparison of the crystal structures of both allergens revealed structural features that could explain the differences in murine IgG and human IgE antibody responses to these allergens. There are structural differences between Der f 1 and Der p 1 that are unevenly distributed on the allergens' surfaces. This uneven spatial arrangement of conserved versus altered residues could explain both the specificity and cross-reactivity of antibodies against Der f 1 and Der p 1.     

Crystal Structure of Der f 7, a Dust Mite Allergen from Dermatophagoides farinae

Background

Der f 7 is the group 7 allergen from the dust mite Dermatophagoides farinae, homologous to the major allergen Der p 7 from D. pteronyssinus. Monoclonal antibody that bind to residues Leu48 and Phe50 was found to inhibit IgE binding to residue Asp159, which is important for the cross-reactivity between Der f 7 and Der p 7.

Methodology/Principal Findings

Here, we report the crystal structure of Der f 7 that shows an elongated and curved molecule consisting of two anti-parallel β-sheets – one 4-stranded and the other 5-stranded – that wrap around a long C-terminal helix. The overall fold of Der f 7 is similar to Der p 7 but key difference was found in the β1–β2 loop region. In Der f 7, Leu48 and Phe50 are in close proximity to Asp159, explaining why monoclonal antibody binding to Leu48 and Phe50 can inhibit IgE binding to Asp159. Both Der f 7 and Der p 7 bind weakly to polymyxin B via a similar binding site that is formed by the N-terminal helix, the 4-stranded β-sheet and the C-terminal helix. The thermal stability of Der f 7 is significantly lower than that of Der p 7, and the stabilities of both allergens are highly depend on pH.

Conclusion/Significance

Der f 7 is homologous to Der p 7 in terms of the amino acid sequence and overall 3D structure but with significant differences in the region proximal to the IgE epitope and in thermal stability. The crystal structure of Der f 7 provides a basis for studying the function and allergenicity of this group of allergens.     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

Molecular cloning, expression, sequence analyses of dust mite allergen Der f 6 and its IgE-binding reactivity with mite allergic asthma patients in southeast China

We report the cloning and molecular characterization of a full-length cDNA encoding house dust mite allergen, Der f 6 from D. farinae isolated in China. The full-length Der f 6 cDNA was obtained with 840 nucleotides long. Nucleotide sequencing analyses showed a total of 36 mutations in five Der f 6 cDNA clones, corresponding to 23 incompatible amino acid residues. Recombinant Der f 6 (rDer f 6) protein was successfully expressed in and purified from E. coli BL21. Among 20 asthmatic patients, 45% was positive to rDer f 6 by ELISA. Bioinformatics analyses revealed that the mature Der f 6 was a hydrophobic and extracellular protein with chymotrypsin-like serine protease activity, its secondary structure was composed of alpha helix (7.69%), extended strand (34.62%), random coils (57.69%), and the similarity of Der f 6 to Blo t 6, Sui m 6, Der f 3 and Der f 9 was 64, 65, 35, and 38%, respectively.     

Population growth and allergen accumulation of Dermatophagoides farinae cultured at 20 and 25 °C

House dust mites are cultured to obtain mite allergen material to produce allergen extracts (vaccines) for diagnostic tests, immunotherapy, and research purposes. Research laboratories and manufacturers have their own culturing protocols to grow these mites and these may vary between manufacturers and between research laboratories. The temperature at which mites are cultured may influence the allergen composition, allergen ratio of Der 1: Der 2 and endotoxin levels in the extracts produced from these cultured mites. In order to produce standardized and uniform extracts, across the industry and in various research laboratories, the influence of culture conditions must be understood. Here we determined how temperature affects mite population growth rates, dynamics of allergen production, Der f 1: Der f 2 ratio and endotoxin levels in extracts made from Dermatophagoides farinae mites cultured at 20 and 25 °C. We found that Der f 1 and Der f 2 accumulated exponentially in the cultures with Der f 1 accumulating faster than Der f 2. When the live mite populations peaked, the ratios for Der f 1: Der f 2 were 4.1 and 4.7 for cultures reared at 20 and 25 °C, respectively. Most of the Der f 1 and Der f 2 allergen in whole cultures is not in mite bodies and is lost when the mite material is washed. Thus, if the ratio of Der f 1 and Der f 2 is an important consideration for commercial and research extracts, then the temperature at which the mites are cultured and the collection procedure are important considerations.     

Sequence polymorphisms of Der f 1, Der p 1, Der f 2 and Der p 2 from Korean house dust mite isolates

Amino acid sequence variations have possible influences on the allergenicity of allergens and may be important factors in allergen standardization. This study was undertaken to investigate the sequence polymorphisms of group 1 and 2 allergens from Korean isolates of the house dust mites Dermatophagoides farinae and D. pteronyssinus. cDNA sequences encoding group 1 and 2 allergens were amplified by RT-PCR and compared the deduced amino acid sequences. Der f 1.0101, which appeared in 64.0 % of the 50 sequences analyzed, was found to be predominant. Among the Der p 1 sequences, Der p 1.0102 and 1.0105 were predominant (58 %). Among the Der f 2 sequences, Der f 2.0102 (40.7 %) and a new variant with Gly at position 42 (27.8 %) were predominant. The deduced amino acid sequences of 60 Der p 2 clones were examined, and 28 variants with 1-5 amino acid substitutions were found. Interestingly, all of the Der p 2 sequences had Thr instead of Lys at position 49. Two variants (Leu40, Thr49, and Asn114 (26.6 %); Val40, Thr49, and Asn114 (20.0 %)) were found to be the most predominant forms of Der p 2. Der p 1 has a high rate of sporadic substitutions and the group 2 allergens show a more regular pattern with orderly associations of amino acid substitutions. Der f 1 and Der p 2 from Korean mite isolates have unique amino acid sequence polymorphisms. These findings provide important data for house dust mite allergen standardization.     

A comparative study of allergenic and potentially allergenic enzymes fromDermatophagoides pteronyssinus,D. farinae andEuroglyphus maynei

The presence of the enzymatically active allergens equivalent toDer p I (cysteine protease),Der p III (serine protease) and amylase in extracts ofDermatophagoides pteronyssinus, D. farinae andEuroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived fromD. pteronyssinus andD. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared fromD. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.     

Modelling and Bioinformatics Analysis of the Dimeric Structure of House Dust Mite Allergens from Families 5 and 21: Der f 5 Could Dimerize as Der p 5

Abstract

Allergy represents an increasing thread to public health in both developed and emerging countries and the dust mites Dermatophagoides pteronyssinus (Der p), Blomia tropicalis (Blo t), Dermatophagoides farinae (Der f), Lepidoglyphus destructor (Lep d) and Suidasia medanensis (Sui m) strongly contribute to this problem. Their allergens are classified in several families among which families 5 and 21 which are the subject of this work. Indeed, their biological function as well as the mechanism or epitopes by which they are contributing to the allergic response remain unknown and their tridimensional structures have not been resolved experimentally except for Blo t 5 and Der p 5. Blo t 5 is a monomeric three helical bundle, whereas Der p 5 shows a three helical bundle with a kinked N-terminal helix that assembles in an entangled dimeric structure with a large hydrophobic cavity. This cavity could be involved in the binding of hydrophobic ligands, which in turn could be responsible for the shift of the immune response from tolerance to allergic inflammation. We used molecular modelling approaches to bring out if other house dust mite allergens of families 5 and 21 (Der f 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) could dimerize and form a large cavity in the same way as Der p 5. Monomeric models were first performed with MODELLER using the experimental structures of Der p 5 and Blo t 5 as templates. The ClusPro server processed the selected monomers in order to assess their capacity to form dimeric structures with a positive result for Der p 5 and Der f 5 only. The other allergens (Blo t 5, Sui m 5, Lep d 5, Der p 21 and Der f 21) did not present such a propensity. Moreover, we identified mutations that should destabilize and/or prevent the formation of the Der p 5 dimeric structure. The production of these mutated proteins could help us to understand the role of the dimerization process in the allergic response induced by Der p 5, and if Der p 5 and Der f 5 behave similarly.     

Cloning and expression of Der f 6, a serine protease allergen from the house dust mite, Dermatophagoides farinae.

House dust mite allergen is thought to be a major cause of asthma. Characterization of these allergen molecules is therefore an important step for the development of effective diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report molecular cloning and expression of the group 6 (chymotrypsin-like) allergen from the house dust mite, Dermatophagoides farinae. Sequencing analysis indicates that cloned cDNA, designated Der f 6, encodes a 279 amino acid polypeptide which conserves a primary structure characteristic for chymotrypsin-like serine proteases found in mammals. Recombinant Der f 6 expressed in Escherichia coli bound IgE in a pool made of 20 sera, and induced histamine release from patients' peripheral blood cells.     

Expression of house dust mite allergens Der f 1 and Der f 2 in leaves of Nicotiana benthamiana

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE.     

Population growth and allergen accumulation of <Emphasis Type="Italic">Dermatophagoides pteronyssinus</Emphasis> cultured at 20 and 25°C

The house dust mites, Dermatophagoides pteronyssinus and D. farinae are cultured commercially and in research laboratories and material is harvested from these cultures to make extracts that are used for diagnosis, immunotherapy and research. Temperature and other climatic conditions can influence population growth rates, dynamics of allergen production, and the associated endotoxin, enzyme and protein levels of the mite material harvested from these cultures. Here we determined how temperature affected these parameters. Dermatophagoides pteronyssinus was cultured at 20 and 25°C at 75% relative humidity, and at 2-week intervals the concentrations of mites, Der p 1 and Der p 2 allergens, endotoxin, and selected enzymes were determined. Mite density increased exponentially but growth rate and final population density were greater at 25°C compared to 20°C. The combined allergen (Der p 1 + Der p 2) concentrations accumulated in the cultures at about the same rate at both temperatures. However, individual Der p 1 and Der p 2 accumulation rates varied independently at the two temperatures. Der p 1 accumulated faster at 20°C whereas Der p 2 accumulated faster at 25°C. The amount of Der p 1 in whole cultures was greater than the amount of Der p 2. The concentration of allergen for washed mites harvested from the cultures was much less than for the whole cultures. Our study demonstrated that temperature is an important factor in population growth and the dynamics of allergen production in cultured mites.     

Dermatophagoides farinae Allergens Diversity Identification by Proteomics

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1–3, 6, 7, 10, 11, 13–18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.The house dust mites (HDM)¹ are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases (1). Extensive studies have been conducted to understand the biological, chemical, and structural properties of dust mite allergens. Most of the best characterized allergens are from dust mites Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae). Twenty-three groups of dust mite allergens are listed in the (IUIS) nomenclature data set, and 21 of them have been identified from Dermatophagoides spp (http://www.allergen.org/). There is an extreme diversity of dust mite allergens. Western blotting studies with human sera containing high levels of anti-mite IgE showed more than 32 bands with molecular weights ranging from 11 to greater than 100 kDa (2). Two groups of mite allergens (group 1 and 2) have been extensively studied. They are a 25-kDa cysteine protease and a 14-kDa epididymal protein, respectively. More than 80% of humans with house dust mite allergy mount an IgE response to the group 1 and more than 90% to the group 2 (3–6).The group 1 and 2 molecules are major allergens in HDMs but about 20% of patients do not have IgE antibody to the two group allergens (3). It has been found that there are also many other HDM allergens containing high IgE binding activity although these are present in low and variable concentrations in mite extracts (minor allergens), usually at less than 1% of the group 1 and 2 allergens (3). Allergens present in low amount in mite extracts, which can induce high titers of IgE, suggest that they are potent at low concentration. Another possibility is that the amount of allergen required to induce allergic responses in the airways is more than that required to induce IgE. It has been estimated that there are at least 30 allergens in the extracts of D. farinae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) combined with autoradiography analysis (7). Two-dimensional (2-D) immunoblotting has been applied to study mapping of D. farinae mite allergens (7). Seven allergens including Der f 1, Der f 2, Der f 3, Der f 4, Der f 5, and 2 high molecular mass allergens, which share significant homologies with allergen Mag 3 from D. farinae and with a chitinase from prawn Penaeus japonicus, have been identified from the 2-D immunoblotting analysis (7). Up to now, 14 allergens from D. farinae have been named. Most of them are in the molecular weight range of 14 to 60 kDa. Given the extreme diversity of mite allergens, many investigations with novel allergen identification are still in progress or are yet to be undertaken. It is well known that many mite allergens are not identified on the basis of two possible reasons: (1) it is difficult to purify and characterize minor allergens because they present in low concentration in mite extracts; (2) some minor allergens are neglected because of their minor amount or abilities to only induced allergy to a minor population. It is necessary to develop efficient procedure with high accuracy and resolution to purify and characterize allergens from mite extracts. In this work, 17 allergens or their isoforms have been identified from the mite extracts of D. farinae by a procedure of proteomics combined with two-dimensional immunoblotting. Eight of them are the first to be reported as mite allergens.     

Quantitative determination of Der p I allergen levels in allergenic extracts and house-dust samples

Summary House-dust mites are responsible for serious respiratory diseases in humans. An ELISA assay to detectDermatophagoides pteronyssinus (Dp) mites has been set up. This assay, based on quantitative determination of the majorDp allergen, called Der p I, has been applied for the analysis of growing mite cultures and house-dust samples.Der p I allergen levels in mite cultures are well correlated with the number of mites present as well as with the biological activity of the corresponding extracts. Different methods for detectingDp mites in house-dust samples were compared. The ELISA method shows good sensitivity and specificity, and is particularly suitable for routine analyses.     

Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides

The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans.     

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

Sensitivity to house dust mite allergens and prevalence of allergy-causing house dust mite species in Pothwar,Pakistan

This study is the first report on the epidemiological status of house dust mite (HDM) allergy in Pothwar, Pakistan. Allergy data of 2087 symptomatic patients were obtained, of whom 1706 (81.7%) patients were skin-prick-test positive for HDM allergens. This percentage was significantly higher than for pollen and food allergens. In the results of this study Dermatophagoides farinae (61%) and D. pteronyssinus (29%) were the predominant species in the study area. Besides these pyroglyphids, predatory Cheyletus sp. (10%) and an oribatid mite sp. (1%) were also observed. Random and patients’ houses showed 87.4 and 87.1% positive mite infestation, respectively. Mean (±?SEM) D. farinae counts per g of dust in random samples was 235.4 ± 7.93 compared to 274.7 ± 10.78 from patients’ homes. Mean D. pteronyssinus counts from random houses compared to patients’ houses were 115.0 ± 4.57 and 124.6 ± 5.76, respectively. Mite counts depicted seasonal variation, with peaks during monsoon season. ELISA results of dust samples demonstrated that of the dust samples with?>?10 µg/g of dust, the threshold value described as a risk factor for developing asthma, 57.6% had Der f1 and 20% Der p1 allergen load. Mean Der f1 burden was significantly higher than Der p1, with maximum levels during monsoon and autumn seasons. This research established a better awareness about the epidemiological status of HDM allergy and prevalence of allergy causing HDM species in Pakistan.     

深圳地区粉尘螨Ⅱ类变应原基因的多态性分析及其表达蛋白的变应原性鉴定

粉尘螨Ⅱ类变应原(Der fⅡ)是粉尘螨Dermatophagoides farinae主要变应原之一,可引发Ⅰ型变态反应。从深圳地区挑取活粉尘螨,经形态鉴定后纯培养,提取其总RNA,RT-PCR扩增出Der fⅡ 基因,克隆到pMD18-T载体后测序。通过计算机软件分析该基因的多态性。将该目的基因克隆到pET-His表达载体上得到重组质粒pET-Der fⅡ。工程菌经IPTG诱导培养,表达Der fⅡ目的蛋白,该蛋白主要以包涵体形式存在。重组Der fⅡ蛋白通过6His-tag蛋白纯化系统进行分离、纯化,并进行Western blot检测该重组蛋白与对粉尘螨过敏患者血清中IgE的反应性。结果表明,克隆的5株深圳地区的Der fⅡ 基因与GenBank公布的Der fⅡ(AB195580.1)核苷酸序列同源性为96.8%～99.3%,推导的氨基酸序列同源性为93.8%～98.6%。表达纯化的5种重组Der fⅡ蛋白经Western blot检测,表明都具有良好的变应原性。     

Domestic Mite Antigens in Floor and Airborne Dust at Workplaces in Comparison to Living Areas: A New Immunoassay to Assess Personal Airborne Allergen Exposure

Objectives

Allergens produced by domestic mites (DM) are among the most common allergic sensitizers and risk factors for asthma. To compare exposure levels between workplaces and living areas a new assay able to measure airborne DM antigen concentrations was developed.

Methods

At workplaces and in living areas, 213 floor dust samples and 92 personal inhalable dust samples were collected. For sensitive quantification of DM antigens, a new enzyme immunoassay (EIA) based on polyclonal antibodies to Dermatophagoides farinae extract was developed. Reactivity of five house dust mite and four storage mite species was tested. All dust samples were tested with the new EIA and with the Der f 1 and Der p 1-EIAs (Indoor Biotechnologies, UK) which detect major allergens from D. farinae and D. pteronyssinus by monoclonal antibodies. Samples below the detection limit in the DM-EIA were retested in an assay variant with a fluorogenic substrate (DM-FEIA).

Results

The newly developed DM-EIA detects antigens from all nine tested domestic mite species. It has a lower detection limit of 200 pg/ml of D.farinae protein, compared to 50 pg/ml for the DM-FEIA. DM antigens were detected by DM-EIA/FEIA in all floor dust and 80 (87%) of airborne samples. Der f 1 was found in 133 (62%) floor dust and in only 6 airborne samples, Der p 1 was found in 70 (33%) of floor samples and in one airborne sample. Der f 1 and DM concentrations were highly correlated. DM-antigens were significantly higher in inhalable airborne samples from textile recycling, bed feather filling, feed production, grain storage and cattle stables in comparison to living areas.

Conclusions

A new sensitive EIA directed at DM antigens was developed. DM antigen quantities were well correlated to Der f 1 values and were measurable in the majority (87%) of airborne dust samples. Some workplaces had significantly higher DM antigen concentrations than living areas.