Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae (D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109kDa (98/109kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.     

Characterization of house dust mite and scabies mite allergens by use of canine serum antibodies

OBJECTIVE: To identify the major allergenic proteins from the 3 main species of dust mites to which dogs react (Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei) and evaluate the potential cross-reactivity of dust mite allergens with antigens from the ectoparasitic mite Sarcoptes scabiei var canis. SAMPLE POPULATION: Sera from 83 dogs with atopic dermatitis. PROCEDURE: Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting using serum from atopic dogs was used to identify IgE-binding proteins in extracts of the 4 mite species. RESULTS: Sera of atopic dogs contained IgE against 23, 17, 25, and 17 allergens from D. farinae, D. pteronyssinus, E. maynei, and S. scabiei, respectively. Unlike the situation for humans, the major allergens for dogs are mostly proteins that are larger than 90 kd molecular weight. Dermatophagoides farinae and E. maynei appear to be more allergenic for dogs than is D. pteronyssinus. Some dogs with serum IgE against dust mites also had IgE against antigens of S. scabiei var canis. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple dust mite allergens induce an IgE response in dogs. These allergens are mostly greater than 90 kd molecular weight.     

Positive reactions to common allergens in 42 atopic dogs in Japan

Clinically important allergens for the diagnosis and treatment of atopic dermatitis vary geographically. In order to identify the most prevalent allergens in atopic dogs in Japan, 42 dogs with a clinical diagnosis of atopy were tested using both in vivo (intradermal skin test (IDST)) and in vitro (antigen-specific IgE assay) allergy tests. Allergens used for IDST included 26 allergen extracts from eight allergen groups: trees, weeds, grasses, house dust mites (HDM), molds, foods, epithelia, and arthropods. Immunodot assay was used to measure antigen-specific IgE against 24 allergens from these eight groups and against fish such as cod and sole. In the 42 dogs, the most common positive allergen reaction was to HDM on both IDST (29/42 dogs or 69%) and in vitro testing (23/42 or 54.8%). The second most frequent positive allergen reaction was to Japanese cedar pollen (21/42 or 50.0% for IDST and 7/42 or 16.7% for in vitro testing). In both tests, less than 20% of dogs had positive reactions to molds or foods. Positive reactions to cat epithelia were frequently found on IDST, but rarely found on in vitro testing. Agreement between the two tests was found in 26 instances: HDM (21 dogs), Japanese cedar pollen (five dogs) and wheat (one dog). In this study, the two most common allergens involved in atopic dermatitis in dogs in Japan were HDM and Japanese cedar pollen.     

Quantitation of house dust mite allergens (Der f 1 and group 2) on the skin and hair of dogs

OBJECTIVE: To determine the concentration of house dust mite (HDM) allergens, Der f 1 and group 2, on the skin and hair of dogs and whether associations exist between the presence of Der f 1 and group 2 allergens on the skin and hair of dogs and household and dog characteristics. ANIMALS: 63 pet dogs from 50 homes. PROCEDURE: Dogs were weighed and body surface area in square meters was determined. Skin and hair samples were obtained by vacuuming dogs. Collected dust was analyzed by use of standard ELISA techniques. RESULTS: HDM allergen was detected in 21 of 59 skin and hair samples. Presence of group 2 allergen on skin and hair of dogs was significantly associated with long hair, compared with short or medium length hair. Median house dust sample concentrations of Der f 1 and group 2 allergens were high in homes with dogs that had skin and hair samples that were positive for Der f 1 and group 2 allergens. Dogs with skin and hair samples that were positive for Der f 1 and group 2 allergens resided in homes with a high number of house dust samples that were positive for Der f 1, group 2, or both allergens and in homes with a mean house dust sample allergen concentration of > or =2 microg/g of dust. CONCLUSIONS AND CLINICAL RELEVANCE: Associations exist between environmental HDM allergen concentrations and HDM allergens on the skin and hair samples of dogs. Environmental allergen load is a major factor in accumulation of allergens on the skin and hair of dogs.     

Serum immunoglobulin E against storage mite allergens in dogs with atopic dermatitis

OBJECTIVE: To determine the prevalence of serum IgE against the storage mites Acarus siro, Blomia tropicalis, and Tyrophagus putrescentiae in a population of dogs with atopic dermatitis. SAMPLE POPULATION: Sera from 84 dogs with atopic dermatitis residing in various regions of the United States and Europe. PROCEDURE: Immunoblotting of sera from atopic dogs was used to identify proteins in mite extracts that bound IgE. RESULTS: 94% of the dogs had serum IgE against proteins in extracts of 1 or more of the storage mite species. Ninety-five, 92, and 89% of the storage mite-sensitive dogs had serum IgE against proteins in extracts of A siro, B tropicalis, and T putrescentiae, respectively. Eighty-two percent had serum IgE against at least 1 protein in all 3 species. Most of the major allergens had molecular weights > 80 kd. A greater percentage of the dog sera had IgE against storage mite proteins, compared with proteins of the house dust mites Dermatophagoides farinae and D pteronyssinus. CONCLUSION AND CLINICAL RELEVANCE: Many dogs with atopic dermatitis have serum IgE against many allergens of storage mites. Most of these allergens, like allergens of dust mites, had molecular weights > 80 kd. Storage mite sensitivity in dogs may be as important, if not more important, than dust mite sensitivity.     

Prevalence of the group 1 Dermatophagoides allergens Der p 1 and Der f 1 in homes with no dogs, healthy dogs and Dermatophagoides-sensitized atopic dogs in Liverpool

Dermatophagoides farinae is a frequent allergen in canine atopic dermatitis despite its reported scarcity in the UK, and the aim of this study was to determine whether dogs were uniquely exposed to this species. Der f 1 and Der p 1 in dust collected from living room carpets, bedroom carpets and dog beds of 13 houses with no dogs, 13 with healthy dogs, and 16 with Dermatophagoides-sensitized atopic dogs were quantified by ELISA. Der p 1 levels (microg g(-1) house dust) were significantly higher than Der f 1 in living rooms (Der p 1 median = 1.9, 95% CI = 2.05-6.32, n = 42; Der f 1 median = 0.07, 95% CI = 0.01-0.06, n = 42), bedrooms (Der p 1 median = 4.35, SD = 5.52; Der f 1 median = 0.01, 95% CI = 0.001-0.1, n = 42) and dog beds (Der p 1 median = 1.04, 95% CI = 1.4-8.1, n = 29; Der f 1 median = 0.008, 95% CI = 0.01-0.04, n = 29) (P < 0.0001). Living rooms in houses without dogs had significantly greater Der p 1 levels (median = 7.0, 95% CI = 3.53-15.8, n = 13) than houses with healthy (median = 1.19, 95% CI = 0.44-3.49, n = 13) or atopic dogs (median = 0.78, 95% CI = 0.63-2.42, n = 16) (P = 0.0004). Environmental flea control in living rooms and washing dog beds was associated with significantly reduced Der p 1 levels. This confirms that D. pteronyssinus is common but D. farinae is rare in the sampling area. Apparent sensitization to D. farinae is probably due to cross-reaction. A combination of environmental measures could reduce allergen exposure.     

Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.     

Use of an amplified ELISA technique for detection of a house dust mite allergen (Der f 1) in skin and coat dust samples from dogs

OBJECTIVE: To use an amplified ELISA technique to document the presence and quantify the concentration of the house dust mite allergen, Der f 1, in skin and coat dust samples collected from dogs. ANIMALS: 29 pet dogs of various breeds. PROCEDURE: Dogs were weighed, and body surface area in square meters was determined. Skin and coat dust samples were obtained by vacuuming dogs. Collected dust was analyzed by use of standard and amplified ELISA techniques. RESULTS: By use of the standard ELISA technique, Der f 1 was detected in skin and coat dust samples from 6 of 29 (21%) dogs. Mean concentration of Der f 1 in the 6 samples with positive assay results was 16.16 ng/mL (range, 5.61 to 31.24 ng/mL). Samples with negative assay results were retested for dust mite allergen by use of an amplified ELISA technique; an additional 14 dogs had positive assay results. Mean concentration of allergen was 0.36 ng/mL (range, 0.19 to 2.20 ng/mL). Combining both techniques, 20 of 29 (69%) dogs had positive assay results for Der f 1. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study indicate that house dust mite allergens are present on the skin and in the coat of dogs, and this source of allergen may act as a reservoir for allergen exposure in hypersensitive dogs. Use of an amplified ELISA technique to determine environmental concentrations of house dust mite allergens in homes and on dogs will help to identify the relationship between immunologic findings and environmental exposures in dogs with atopic dermatitis.     

IgE-reactivity to major Japanese cedar (Cryptomeria japonica) pollen allergens (Cry j 1 and Cry j 2) by ELISA in dogs with atopic dermatitis

The present study investigated IgE-reactivity to two major Japanese cedar (Cryptomeria japonica, C. japonica) pollen allergens (Cry j 1 and Cry j 2) in dogs with atopic dermatitis by use of a fluorometric ELISA. The serum samples from 27 dogs that showed IgE-sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to the two major allergens. All 27 dogs had anti-Cry j 1 IgE, and 10 (37%) had anti-Cry j 2 IgE. Inhibition of binding of dog specific IgE to crude C. japonica pollen allergen was carried out by addition of Cry j 1. When serum samples containing anti-Cry j 1 IgE but no anti-Cry j 2 IgE were incubated with Cry j 1, specific IgE binding to crude C. japonica pollen allergen was almost abolished. These findings suggest that Cry j 1 is a major allergen in dogs.     

Quantitation of house dust mites and house dust mite allergens in the microenvironment of dogs

OBJECTIVE: To quantitate the density of Dermatophagoides farinae and D pteronyssinus and concentrations of house dust mite (HDM) allergens (Der f 1, Der p 1, and Group 2 allergens) in the indoor microenvironment of dogs. Sample Population-50 homes in Columbus, Ohio. PROCEDURES: n each home, samples of dust were collected from 3 locations in which dogs spent most time. Whenever possible, the species of mites collected was identified. Mite density (mites/g of dust) was assessed, and allergen concentrations were assayed by standardized ELISAs. Relative humidity and temperature in each home were monitored during a 5-day period. Characteristics of homes and sample sources were evaluated. RESULTS: Dust samples from all 50 homes contained > or = 1 HDM allergen; Der f 1 and Der p 1 were detected in 100 and 74% of homes, respectively. Fifteen homes had HDMs; compared with D pteronyssinus, D farinae was found more commonly (14/15 homes) and at a higher density. Basements, homes without central air-conditioning, and dog beds that were > or = 1 year old had high HDM allergen concentrations. Homes with > or = 2 microg of Der f 1 or Group 2 allergens/g of dust or > or = 100 mites/g of dust were significantly more likely to have a maximum relative humidity > or = 75%. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate the presence of HDMs and HDM allergens in the specific microenvironment of dogs in homes. Factors associated with high levels of exposure were identified, which may be associated with increased risk for sensitization and development of atopic diseases.     

Quantitation of house dust mites and house dust mite allergens in the microenvironment of dogs

OBJECTIVE: To quantitate the density of Dermatophagoides farinae and D pteronyssinus and concentrations of house dust mite (HDM) allergens (Der f 1, Der p 1, and Group 2 allergens) in the indoor microenvironment of dogs. SAMPLE POPULATION: 50 homes in Columbus, Ohio. PROCEDURES: In each home, samples of dust were collected from 3 locations in which dogs spent most time. Whenever possible, the species of mites collected was identified. Mite density (mites/g of dust) was assessed, and allergen concentrations were assayed by standardized ELISAs. Relative humidity and temperature in each home were monitored during a 5-day period. Characteristics of homes and sample sources were evaluated. RESULTS: Dust samples from all 50 homes contained > or = 1 HDM allergen; Der f 1 and Der p 1 were detected in 100 and 74% of homes, respectively. Fifteen homes had HDMs; compared with D pteronyssinus, D farinae was found more commonly (14/15 homes) and at a higher density. Basements, homes without central air-conditioning, and dog beds that were > or = 1 year old had high HDM allergen concentrations. Homes with > or = 2 microg of Der f 1 or Group 2 allergens/g of dust or > or = 100 mites/g of dust were significantly more likely to have a maximum relative humidity > or = 75%. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated the presence of HDMs and HDM allergens in the specific microenvironment of dogs in homes. Factors associated with high levels of exposure were identified, which may be associated with increased risk for sensitization and development of atopic diseases.     

IgE reactivity and cross-reactivity to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens in dogs with atopic dermatitis

The natural occurrence of Japanese cedar (Cryptomeria japonica) pollinosis has been reported in dogs with atopic dermatitis. However, the reactivity to Japanese cypress (Chamaecyparis obtusa) pollen allergens in these dogs has not been reported. The present study was designed to investigate the reactivity to Japanese cypress pollen allergens in dogs sensitized to Japanese cedar pollen allergens. In 19 dogs with specific IgE to C. japonica pollen allergen, we measured the specific IgE to C. obtusa pollen allergen and examined the reactivity to the allergen by intradermal test. Of the 19 dogs, 18 had specific IgE to crude and purified major allergens (Cha o 1) of C. obtusa pollen. Most of the dogs showed a positive reaction to C. obtusa pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in C. japonica pollen) was observed by the ELISA inhibition method. Dogs sensitized to Japanese cedar pollen allergens demonstrate reactivity to Japanese cypress pollen allergens.     

Group 1 and 2 Dermatophagoides house dust mite allergens in the microenvironment of cats

House dust mite allergens (HDMAs) are some of the most common allergens associated with allergic diseases in humans and dogs. The purpose of this study was to determine whether HDMAs could be detected in cat‐associated household microenvironments. From 50 cat‐only households with 95 cats, dust samples were collected by vacuuming for 2 min m^?2 from three areas where cats slept or rested regularly from September to October 2006. Relative humidity and temperature were measured in each household using a data logger. Each owner completed a questionnaire on potential factors that might influence the prevalence of house dust mites (HDMs). Dust samples were analysed utilizing an ELISA for Der p 1, Der f 1 and HDM group 2 allergens. In 38 of 50 households there was greater than 2 μg g^?1 of dust for at least one HDMA. Using stepwise logistic regression, factors associated with increased HDMA levels included: free‐standing houses, number of humans in household, longhaired cats and age of the cat. Factors associated with decreased HDMA concentrations included: forced air heating and central air conditioning, less than 50% carpeting of the home, use of flea control, cats suffering from dermatological disease and the average temperature of the household. Many sleeping/resting areas utilized by cats contain sufficiently high levels of HDMAs to be potential sources of sensitization. This finding should lead to further determination of the role of HDMs in cats suffering from putative allergic conditions such as atopic dermatitis or asthma.     

Prevalence of house dust mites and dermatophagoides group 1 antigens collected from bedding, skin and hair coat of dogs in south-west England

The house dust mites Dermatophagoides farinae (Df) and D. pteronyssinus (Dpt) are commonly implicated as allergens causing canine atopic dermatitis in the UK. However, there are few studies that characterize the exposure of UK pet dogs to these mites. The objectives of this study were to determine the prevalence of the mite species on the skin, hair coat and bedding of a population of pet dogs. Dust samples (n = 68) were collected from both dogs and their beds using a standardized vacuuming technique and stored at -20 degrees C. Mites were identified using accepted morphological criteria. House dust mite allergen concentrations were assayed using standardized ELISA for Dpt and Df group 1 allergens (Der p 1 and Der f 1). Mites were identified in 15/68 samples (22%) and Dpt was the most common. Df mites were not present. Der p 1 allergens were detected in 60% of samples, and Der f 1 in 6% of samples. There were no significant differences between the number of Der p 1 positive samples from dogs and the number of those from their bedding, or between the average Der p 1 concentrations from dogs and the number of those from their bedding. Contrary to studies elsewhere in Europe and the USA, these findings support studies of human asthma patients in the UK, where exposure to Df is rare, but to Dpt is common. As the prevalence of positive intradermal and serological reactions to Df in atopic dogs is high, further investigations are warranted to clarify true Df hypersensitivity or potential immunological cross-reactivity between mite allergens.     

House dust and forage mite allergens and their role in human and canine atopic dermatitis

This article reviews the literature regarding the role of house dust and forage mite allergens in canine atopic dermatitis. The presence of immunoglobulin E (IgE) to these mites, especially to Dermatophagoides farinae, is common in both normal and atopic dogs. Exposure of dogs to the different mites is described both in the direct environment and in the coat of animals for house dust mites and in the food for forage mites. Allergens causing allergic disease in dogs seem to be different from those in humans. Dogs seem to react to high molecular weight allergens, compared to the low molecular weight group 1 and group 2 proteases that are commonly implicated in humans with atopic diseases. Despite numerous published studies dealing with this subject, a number of questions still need to be addressed to better understand the exact role of these mites in the pathogenesis of canine atopic dermatitis and to improve the quality of the allergens used in practice.     

Stratum corneum removal facilitates experimental sensitization to mite allergens in atopic dogs

In humans with atopic dermatitis and in mouse models of IgE-mediated allergic diseases, evidence is mounting that the stratum corneum (SC) provides an important barrier against environmental allergens. At this time, it is not known whether the SC has a similar role in dogs, especially in those with atopic dermatitis. The objectives of this pilot study were to determine whether SC removal led to earlier and stronger sensitization of atopic dogs to Dermatophagoides farinae (Df) house dust mites. Five Maltese-beagle atopic (MBA) dogs were sensitized epicutaneously after the SC was removed with ten tape strips (TS group), while sensitization was done without tape strips in five other MBA dogs (nontape stripping; NTS group). During this 16 week study, sensitization was assessed with allergen-specific IgE serology, intradermal testing with Df allergens and determination of stimulation indices of blood mononuclear cells cultured with Df and stained for CD4 and the activation markers CD25 or CD30. Compared with dogs from the NTS group, those of the TS group exhibited earlier rises in Df-specific IgE serum levels, usually had higher allergen-specific IgE titres, showed higher intradermal test reactivity and had earlier increases and higher percentages of CD25- or CD30-positive activated allergen-specific peripheral CD4-positive T lymphocytes. These observations implicate a role of the SC as a barrier limiting sensitization to exogenous allergens in this experimental atopic dog model.     

Comparison of response to immunotherapy by intradermal skin test and antigen-specific IgE in canine atopy

The intradermal skin test (IDST) and serologic allergy test (SAT) has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy. To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens. In addition, we compared agreement rate, sensitivity and specificity between them (using IDST as the standard). The allergen most commonly positive in both tests was house dust mites (IDST: 69.5%, SAT: 48.4%). Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens. Agreement rates between the two tests ranged from 67.4% to 96.8%; the overall mean agreement rate were 81%. SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%. The specificities were very high for all allergens, on the order of 94.9-100% (median=98.7%). Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST (15 dogs) and SAT (12 dogs) results. Overall, 60% (9/15) of the IDST group and 66.8% (8/12) of the SAT group experienced a 50% to 100% reduction in their symptomatology. No significant differences were found in response to immunotherapy during the follow-up period between allergen selection methods. These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.     

Comparison between an intradermal skin test and allergen-specific IgE-ELISA for canine atopic dermatitis

The aim of this study was to compare the results of an intradermal skin test (IDST) with those of an allergen-specific IgE-ELISA in 210 dogs with atopic dermatitis. All the dogs had a clinical diagnosis of atopic dermatitis and underwent an IDST. The sera of all dogs were analysed for allergen-specific IgE by ELISA using the monoclonal antibody D9 against dog IgE. IDST was used as the standard assay. In both methods, the following antigens provided a positive test result: Dermatophagoides farinae, Acarus siro, Tyrophagus putrescentiae, ragweed, mugwort and Lepidoglyphus destructor. ELISA had an overall sensitivity of 82.4% and an overall specificity of 93.8%. The overall accuracy of the ELISA was 91.3%. The evaluated monoclonal D9 ELISA was found to be a reliable tool for the diagnosis of those allergens that cause clinical atopy, and can be recommended for use in dogs when immunotherapy is a therapeutic option.     

Characterisation of major and minor Dermatophagoides allergens in canine atopic dermatitis

Atopic dermatitis is a well-recognised chronic inflammatory skin disease of humans and dogs. Most atopic dogs are sensitised to Dermatophagoides mites. The aim of this study was to characterise allergens in different Dermatophagoides species using polyclonal and monoclonal antibodies to canine IgE. Western blots were prepared from crude extracts of D farinae, D pteronyssinus and D microceras, and purified group 1 and 2 allergens under reducing and non-reducing conditions. They were probed with sera from atopic (n = 33) and healthy (n = 27) dogs. There was no significant difference in the sensitivity or specificity between the polyclonal and monoclonal sera in detecting Dermatophagoides -specific IgE. Major allergens common to both D farinae and D pteronyssinus were detected at 97-98 kDa, 103-104 kDa and 134-139 kDa on both reducing and non-reducing blots. Major allergens at 84-85 kDa, 65-69 kDa and 44-45 kDa were only recognised on reducing blots, suggesting that these are fragments of the larger allergens. Only a few sera recognised group 1 or 2 allergens on blots of crude extracts or purified allergens. These results confirm that, in atopic dogs, high molecular weight allergens are the most important Dermatophagoides allergens, rather than the low molecular weight group 1 and 2 proteins.