Antigenic and allergenic properties of the storage mite Tyrophagus putrescentiae

The antigens and allergens in the storage mite Tyrophagus putrescentiae, commonly found in grain and hay and sometimes in house dust, were characterized by crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis. Three mite extracts, T. putrescentiae (TP) mite bodies, feces, and a combination of mites, feces, and culture medium (TP + CM) were studied. TP and TP + CM (primarily feces) extracts exhibited 20 and 18 antigens, respectively. By use of serum from two skin test-positive patients, autoradiograms demonstrated TP contained two allergens and TP + CM contained five allergens, three of which probably originated from feces. CM (whole wheat flour) did not stimulate antibody production in rabbits. TP feces shared 10 antigenic determinants with TP and 14 with TP + CM. Two antigens common to TP feces and TP were also shared allergens.     

Antigens and allergens from the common house dust mite Dermatophagoides pteronyssinus. Part II. Identification of the major IgE-binding antigens by crossed radioimmunoelectrophoresis

Crossed immunoelectrophoresis demonstrated 51 antigens in a water-soluble extract of the house dust mite Dermatophagoides pteronyssinus. Autoradiography demonstrated that 11 of the antigens bound IgE antibodies in the sera of house dust mite-allergic patients. IgE antibodies in 23 different sera reacted with from one to eight antigens. On the basis of Lowenstein's (1978) definition of a "major allergen," two of the antigens, numbers one and 36 would be described as "major allergens." Apart from antigen number 36 that has already been demonstrated to be an important allergen in patients allergic to D. pteronyssinus, the clinical importance of the other 10 IgE-binding antigens has yet to be assessed. Reasons against use of the term "major allergen" and its replacement with the title "clinically important allergen" are advanced.     

Purification and partial characterization of two major allergens from the house dust mite Dermatophagoides pteronyssinus

Two major allergens of the house dust mite, Dermatophagoides pteronyssinus (Dp), were purified, and their molecular weight and isoelectric points (pIs) were determined. Dp 42 was purified from an acetone-precipitated mite-excrement extract by a combination of hydrophobic interaction chromatography on phenyl Sepharose and copper-chelate chromatography. The molecular weight was determined to be 18,000 and 25,000 to 30,000 by gel filtration (G-75) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively, and pI values of 4.6, 5.6, and 6.6 were obtained by sucrose gradient isoelectric focusing (IEF). These values correspond well with those described for the identical allergen, P1. The pI 6.6 variant was considerably enriched in the purified material. Dp 42 constituted 6.4% of the dry weight of a reference whole mite-culture extract. Dp X was obtained partially purified by gel filtration (G-75), ammonium sulphate precipitation, and hydrophobic interaction chromatography. The molecular weight was 18,000 to 20,000 by gel filtration and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Multiple pIs in the range 5 to 7 were found by sucrose gradient IEF and crossed IEF. The two purified allergens carried clearly distinct activities toward human IgE and appeared as potent allergens in crossed radioimmunoelectrophoresis, RAST, and RAST inhibition.     

Purification and Characterization of a Major Allergen from the House Dust Mite Dermatophagoides farinae

A major allergen from an extract of the house dust mite, Dermatophagoides farinae, was shown to be extremely heterogenic with respect to charge. A slightly basic component of this allergen with a pI of 8, was purified by isoelectric focusing in two steps. The purified component, denoted antigen 19/20 IIa, seemed to be representative for the allergenic activity of the major allergen. The amino acid analysis suggested that antigen 19/20 IIa had a molecular weight of 9400 and contained one residue of galactosamine. SDS polyacrylamide gel electrophoresis did indicate a somewhat higher molecular weight of 14,500. Antibodies against the purified component cross-reacted with a crude extract of the mite Dermatophagoides pteronyssinus.     

A comparative study of the allergens of cat urine, serum, saliva, and pelt

In direct RAST analyses of sera from 43 individuals with a history of cat allergy, 39.5% were positive to cat pelt, 37.5% to cat saliva, and 12% each to cat urine and serum. The cat pelt and saliva extracts contained allergen 1, but cat serum and cat urine collected by bladder puncture had no detectable levels of this allergen. A crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis analysis failed to reveal any allergen in urine or serum that was not also present in the saliva or pelt preparations, although urine had two allergens not present in serum. When serum from a patient who was direct RAST positive to cat pelt, serum, saliva, and urine was tested by crossed radioimmunoelectrophoresis, it was determined that a total of six allergens were detectable in cat pelt, three in cat urine, and six in cat serum. Since cat serum contains no detectable cat allergen 1, it may be concluded that at least seven allergens derived from the cat are capable of binding to IgE antibody in humans.     

Characterization of allergenic components from house dust mite Dermatophagoides siboney. Purification of Der s 1 and Der s 2 allergens

Background Allergic reactions to house dust miles of the genus Dermatophagoides play an important role in the pathogenesis of asthma and other atopic diseases. Dermatophagoides siboney has been described as a species from Cuba. Together with D. pteronyssinus and Blomia tropicalis , it is frequently found in house dust from homes of asthmatics.
Objective The aim of this study was to investigate the allergenic composition from the house dust mite D. siboney .
Methods The charactcrization of D. siboney extract was performed by SDS-gPAGE and immunoblotting. Purification of individual components was performed by affinity ehromatography.
Results At least 16 components between 13 and 98 k Da stained by Coomassie Blue were found. Using a panel of 35 sera from a topic mile sensitive patients 13 components reacted to different extent with patient IgE. Two components, 25 and 14 kDa, bound to specific IgE strongly and frequently, i.e. 80 and 91% of the patients, respectively. Affinity ehromatography using crossreacting monoclonal antibodies to group 1 and 2 allergens resulted in purified preparations of 25 and 14 kDa proteins, which showed IgE-binding with the majority of the human sera when tested by immune-dot.
Conclusion Based on the IgE binding profile of D. siboney and on the capacity to react with crossreacting monoclonal antibodies for groups I and 2, it is proposed to name these two allergens, 25 and 14 k Da, Der s 1 and Der s 2, respectively.     

Altered antigenicity of M-177, a 177-kDa allergen from the house dust mite Dermatophagoides farinae, in stored extract

BACKGROUND: A high molecular weight allergen, M-177 (177 kDa) was isolated from Dermatophagoides farinae using a specific antibody raised to an allergenic clone Mag 3, which was obtained by immunoscreening a mite cDNA library. The potent IgE reactivity of M-177 is comparable with that of Der f 2. OBJECTIVE: The aim of this study was to analyse the molecular characteristics and the allergenic activity of M-177 in stored mite extracts. METHODS: Antigens were analysed by immunoblotting and enzyme-linked immunosorbent assay (ELISA; inhibition). Allergenic activity was estimated from IgE reactivity and the results of a histamine release assay. RESULTS: The intact M-177 molecule was present in high concentrations in fresh extract obtained from purified mite bodies, but was only detected in small amounts in stored extracts. Instead of the intact molecule, anti-Mag 3 antibody detected various cross-reactive antigens in the stored preparations. Studies of a stored liquid extract showed that these cross-reactive antigens were produced by the degradation of M-177, and that this change was suppressed by the addition of protease inhibitors. Interestingly, the allergenic activity of the fragmented M-177 (sM-177) isolated from the stored extracts was greater than that of the intact antigen. Specific IgE reacted with sM-177 in 84.2% of 38 sera samples from patients allergic to mites, while 65.8% were positive for M-177-specific IgE. Similarly, the histamine release test showed that sM-177 had greater allergenic activity in vitro. ELISA inhibition indicated that the increased allergenic activity resulted from alteration of the antigenicity with the degradation of M-177. CONCLUSIONS: M-177 is a protease-sensitive allergen. The breakdown products of M-177 provoked higher allergenic activity than the intact allergen.     

Allergenic and antigenic relationship between three species of storage mite and the house dust mite, Dermatophagoides pteronyssinus

We have explored the antigenic and allergenic relationship between the house dust mite Dermatophagoides pteronyssinus and three species of storage mite, Glycyphagus destructor, Acarus siro, and Tyrophagus longior. Crossed immunoelectrophoresis demonstrated that all the mite extracts contained multiple antigens but that there was only limited cross-reactivity between the different species. Six sera were obtained from workers exposed to storage mites and with occupationally related lower respiratory tract symptoms. All workers had specific IgE to D. pteronyssinus and to one or more of the storage mites. The pattern of reactivity varied between the different sera, two responded primarily to D. pteronyssinus and A. siro and four sera to D. pteronyssinus and G. destructor. Only weak responses were observed to T. longior. RAST-inhibition and affinity-absorption experiments demonstrated that D. pteronyssinus had at least three groups of distinct allergenic determinants, determinants specific to D. pteronyssinus, determinants shared with A. siro, and determinants shared with G. destructor. Similarly, both A. siro and G. destructor have specific allergenic determinants and determinants shared with D. pteronyssinus. The findings demonstrate the complexity of the immunologic responses to the different mite species.     

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.     

Cloning,bioinformatics analysis,and expression of the dust mite allergen Der f 5 of Dermatophagoides farinae

Crude extracts of house dust mites are used clinically for diagnosis and immunotherapy of allergic diseases, including bronchial asthma, perennial rhinitis, and atopic dermatitis. However, crude extracts are complexes with non-allergenic antigens and lack effective concentrations of important allergens, resulting in several side effects. Dermatophagoides farinae (Hughes; Acari: Pyroglyphidae) is one of the predominant sources of dust mite allergens, which has more than 30 groups of allergen. The cDNA coding for the group 5 allergen of D. farinae from China was cloned, sequenced and expressed. According to alignment using the VECTOR NTI 9.0 software, there were eight mismatched nucleotides in five cDNA clones resulting in seven incompatible amino acid residues, suggesting that the Der f 5 allergen might have sequence polymorphism. Bioinformatics analysis revealed that the matured Der f 5 allergen has a molecular mass of 13604.03 Da, a theoretical pI of 5.43 and is probably hydrophobic and cytoplasmic. Similarities in amino acid sequences between Der f 5 and allergens of other domestic mite species, viz. Der p 5, Blo t 5, Sui m 5, and Lep d 5, were 79, 48, 53, and 37%, respectively. Phylogenetic analysis indicated that Der f 5 and Der p 5 clustered together. Blo t 5 and Ale o 5 also clustered together, although Blomia tropicalis and Aleuroglyphus ovatus belong to different mite families, viz. Echimyopodidae and Acaridae, respectively.     

Absence of continuous epitopes in the house dust mite major allergens Der p I from Dermatophagoides pteronyssinus and Der f I from Dermatophagoides farinae

Background The house dust mite has been shown to be an important source of domestic allergens associated with immediate hypersensitivities. The Group I mite allergens Der p I from Dermatophagoides pteronyssinus and Der f I from D. farinae display extensive amino acid sequence homology and have similarities with cysteine protease enzymes.
Objective The availability of the complete amino acid sequences for these allergens allowed us to search for the allergic detertninants within these molecules. The aim of the present investigation was to identify any continuous IgE-binding epitopes within these amino acid sequences. We also sought to test the validity of previously reported Der p I peptide epitope sequences.
Methods In order to identity any continuous IgE epitopes, the amino acid sequences of Der p I and Der f I were synthesized as decapeptides overlapping in sequence and coupled to plastic pins. The specific IgE-binding capacity of these peptides was assayed using an enzyme-linked biotin-streptavidin procedure and sera from patients known to be sensitive to these allergens. Previously reported Der p I peptide epitopes were synthesized as free peptides and tested for their ability to inhibit specific IgE binding to allergen extract discs.
Results None of the pin-coupled Der p I or Der f I peptides was found by the continuous epitope mapping procedure to bind significantly to specific IgE in the sera of hypersensitive patients. The previously reported Der p I peptide epitopes did not inhibit specific IgE binding to mite extract discs.
Conclusion The specific IgE binding epitopes of the house dust mite allergens Der p I and Der f I are discontinuous in nature.     

HLA-DRB1*07 may have a susceptibility and DRB1*04 a protective effect upon the development of a sensitization to house dust mite Dermatophagoides pteronyssinus

BACKGROUND: IgE responses to house dust mite-derived allergens seem to be the most important in the development of atopic asthma and rhinitis, but it has been difficult to demonstrate genetic control of the IgE response to the allergens. OBJECTIVE: This study was undertaken to investigate the association between sensitization to house dust mite, D. pteronyssinus (DP), and genotypes of HLA-DRB1 alleles. METHODS: DNAs were extracted from two groups of unrelated Koreans: (1) 178 with sensitization to DP; and (2) 99 age-matched non-atopic controls. Genotypes of the HLA-DRB1 alleles were determined using polymerase chain reaction (PCR)-based methods. RESULTS: The frequency of HLA-DRB1*07 was significantly increased in the DP-sensitive subjects compared with the controls (15.7% vs 4.0%, P = 0.009). Conversely, the frequencies of HLA-DRB1*04 and *14 were decreased in the DP-sensitive subjects compared with the controls (27.5% vs 45.5%, P = 0.002; 13.5% vs 24.2%, P = 0.02). Of the DRB1*04 alleles, DRB1*0403 was significantly decreased in the DP-sensitive subjects compared with the controls (3.9% vs 13.1%, P = 0.005). No significant differences were found in the distributions of the other HLA-DRB1 alleles between the two groups. CONCLUSION: HLA-DRB1*07 may have a susceptibility, and DRB1*04 and DRB1*14 may have a protective effect, upon the development of a sensitization to DP.     

Prevalence of sensitization to the storage mites Acarus siro, Tyrophagus putrescentiae, and Lepidoglyphus destructor in allergic patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus

The prevalence of sensitization to the storage mites Acarus siro (AS), Tyrophagus putrescentiae (TP), and Lepidoglyphus destructor (LD) was studied in 250 sera of patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus (DP) by measuring IgE binding to extracts of the storage mites. Additionally, allergenic cross-reactivity between DP and the storage mite species was studied by RAST inhibition with five individual sera (and a pool of these sera) with moderate IgE levels to all three storage mites and to DP. Increased serum IgE to storage mites was found in 46% of the 200 patients sensitized to DP. Increased prevalence rates of IgE titers to storage mites were associated with higher IgE levels to DP In 50 sera without sensitization to DP, only five sera showed increased IgE to one of the storage mites. Extracts of TP almost completely inhibited the IgE binding to AS, and vice versa. DP inhibited IgE binding to all storage mites up to 60%, whereas IgE binding to DP was only minimally inhibited by extracts of storage mites. In conclusion, cosensitization to storage mites is a frequent finding in patients sensitized to DP. Although this is largely the result of cross-reactivity between different mite species, it may nevertheless be of clinical significance in patients exposed to storage mites.     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.