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.     

T cell responses to the purified major allergens from the house dust mite Dermatophagoides pteronyssinus.

Proliferation assays were used to determine peripheral blood T cell responses to affinity-purified Der p I, Der p II, and Der f II allergens. Patients studied were sensitive to the house dust mite (HDM) either alone or in combination with other allergens. Control subjects were both nonatopic and atopic non-HDM sensitive. In general, only HDM-sensitive patients responded to the mite allergens. Mean stimulation indices (SI) were 10.2 +/- 2.8 (SEM) for Der p I and 10.0 +/- 2.2 for Der p II in HDM-sensitive individuals, and 2.0 +/- 0.2 and 2.8 +/- 0.6 for non-HDM sensitive control subjects (p less than 0.001). Patients sensitive to HDM and other allergens had higher responses than subjects sensitive to HDM alone, and the degree of proliferation to Der p I and Der p II was well correlated in individual patients (r = 0.71; p less than 0.001). Total IgE levels were elevated in the allergic patients, and values were highly correlated with the SI for both Der p I and Der p II (p less than 0.001). Responses to Der p II and Der f II were equivalent in 82% of individuals; however, in 18%, there was a marked discordance with strong responses to Der p II only. SIs were compared between different clinical groups of patients, and patients with asthma had significantly higher values to both Der p I and Der p II than patients with rhinitis alone (17.0 +/- 5.4 versus 5.7 +/- 1.0 for Der p I, p less than 0.05; 14.3 +/- 6.8 versus 5.0 +/- 1.0 for Der p II, p less than 0.05).     

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.     

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.     

In vitro translation of messenger RNA from the house dust mite Dermatophagoides pteronyssinus

RNA was extracted from the house dust mite Dermatophagoides pteronyssinus and a polyadenylated [poly(A)+] and non-polyadenylated [poly(A)-] mRNA isolated by oligothymidylic acid-cellulose chromatography. Both preparations were translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly(A)+ and poly(A)- mRNA was 21- and 1.3-fold, respectively, when compared with control translations performed without added mRNA. Sodium dodecylsulphate-polyacrylamide gel electrophoretic analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14,000- greater than 67,000 daltons were synthesized. Immunoprecipitation studies performed using a sheep anti-whole mite antiserum showed that several of the synthesized proteins corresponded with mite antigens. These findings were confirmed by coprecipitation studies using crossed immunoelectrophoresis (CIE). A comparison of the CIE data with those obtained with crossed radio-immunoelectrophoresis showed that 6 of the 9 allergens previously described in our laboratory were synthesized, namely Der p I, Dpt 1, 3, 4, 8 and 17. The allergenicity of the lysates was also confirmed by immunoprecipitation studies using a specific anti-human IgE reagent.     

Identification of major allergens from the house dust mites, Dermatophagoides farinae and Dermatophagoides pteronyssinus, by electroblotting

The allergens were separated from the extracts of house dust mites by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by autoradiography. Over 30 protein bands of the whole body extract of Dermatophagoides farinae were apparent on 10-20% gradient SDS-PAGE, and 13 bands with MW between 93KD and 12KD bound with specific IgE antibodies in patients' sera sensitive to house dust mites. The major allergenic component of the whole body extract of D. farinae was the protein of MW 14-15KD, which was detected in 95.7% of 47 patients' sera sensitive to house dust mites. The extract of Dermatophagoides pteronyssinus supplied by Bencard Company, England was thought to contain feces enriched material as noted in a few broad protein bands on SDS-PAGE. Seven allergenic components were shown by autoradiography. The protein band of MW 14-15KD was one of the most frequently revealed allergens on autoradiography, which has appeared in 32.5% of 40 patients' sera sensitive to house dust mites. The electrobotting technique used in the present study was fast, convenient and highly useful for both the identification of allergen components and the screening of specific IgE antibody. The individual variations of IgE immune responses to the allergenic components of the two house dust mites were discussed.     

Potentiation of IgE production to common environmental allergens by storage house dust mite Dermatophagoides farinae

Total scrum IgE and IgE antibodies against ten common antigens, including Dermatophagoides farinae (D.f.), moth, ragweed, orchard grass, cryptomeria, alternaria, aspergillus, dog dander, cat dander and tetanus toxoid, were determined using the PRIST and the Sepharose-RAST, respectively, in 100 clinically non-allergic Japanese subjects and interrelations of IgE responses to these antigens were investigated. We obtained following results. (1) The number of positive RAST antigens, to which the subjects responded, increased in parallel to their total serum IgE levels. (2) Among ten antigens, D.f. and moth antigens contributed a great deal to the elevation of total serum IgE level as compared with other antigens. (3) When the relationship between IgE response to D.f. and similar responses to the other nine antigens was investigated, positive RAST responses to various antigens occurred selectively in the subjects with positive RAST for D.f., and the number of positive RAST antigens to which the subjects responded increased depending upon their D.f. specific RAST levels. (4) Such an association was not found between IgE response to moth and the other nine antigens. These findings suggest that non-specific activation of IgE-producing B cells occurs as a result of continuing stimulation by D.f.     

Human immune response to allergens of house dust mite, Dermatophagoides pteronyssinus. V. Auto-anti-idiotypic antibody characterization and cross-reactivity

From the serum of 10 allergic subjects we have prepared IgG antibodies recognizing idiotopes carried by specific antibodies to Dermatophagoides pteronyssinus (Dpt) allergens, and studied cross-reactivity of anti-Dpt IgG bystander and antigen-binding site-associated idiotopes by latex agglutination assays. Idiotopes of specific anti-Dpt IgE were evaluated by radioimmunoassays. Depending on the assay, a binding or inhibition of more than 50%, as compared to the reactivity of specific antibodies with the corresponding anti-idiotypic (anti-Id) IgG, was considered significant. Cross-reactivity of antigen-binding site-associated idiotopes attained a mean proportion of 6/10 for IgG and 9.6/10 for IgE. By contrast, bystander idiotopes cross-reacted only occasionally with a mean proportion of 2/10 for both IgG and IgE antibodies. Anti-Id antibodies from two subjects have been isolated by adsorption on insolubilized anti-Dpt antibodies of the corresponding patient. Using this purified material we have confirmed that (a) the majority of anti-Id antibodies carry an "internal image" of the initial antigen and compete in a dose-dependent manner with Dpt allergens for the binding to the anti-Dpt antibodies and (b) paratope-associated idiotopes of anti-Dpt antibodies are shared by unrelated individuals.     

House dust mite, Dermatophagoides pteronyssinus, and its allergens: effects of washing

The study was undertaken to evaluate the effect washing at various temperatures would have on the house dust mite, Dermatophagoides pteronyssinus, and its allergens. Young and old mite cultures underwent a simulated washing programme. To record the number of live mites prior to and after washing a new counting method was applied. The findings demonstrated that although washing reduced the number of live mites, a washing programme at minimum temperature 58 degrees C was required in order to achieve complete extermination of the house dust mites. A distinct protein loss was observed when washing at higher temperature, thus further reducing the total allergen pool without loss of allergen activity as determined by direct RAST.     

Enzymatic analyses of house dust mite extracts from Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) during different phases of culture growth.

The majority of clinically important allergens of Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 present enzymatic activity. The allergenic enzymes described include cysteine proteases in group 1 allergens, trypsins in group 3, amylases in group 4, and chymotrypsins in group 6. Apart from these, other possibly allergenic enzymes also have been identified. Therefore, enzymatic profiles were studied during the 3 growth periods of the mite population--latency phase, exponential growth phase, and death phase. The activity of 19 different enzymes was analyzed by means of the Api Zym system, a method that has been used to study both mite extracts and other allergenic materials. Our study has demonstrated that the extracts contain a large variety of enzymes. It has been observed that enzymatic activity is caused exclusively by mites because the control carried out on the culture medium was negative for all the enzymes studied. Generally, the levels of diverse enzymatic activity increased with the growth of the culture, and decreased later, in both species. However, proteases are the exception; they maintain a high level of activity during the death phase of the cultured mites. The ratio between trypsin and chymotrypsin activity can be used as an excellent tool for quality control parameters during obtention of allergenic mite extracts.     

The group III allergen from the house dust mite Dermatophagoides pteronyssinus is a trypsin-like enzyme.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.     

The human immune response against major allergens from house dust mite, Dermatophagoides pteronyssinus. II. Idiotypic cross-reactions of allergen-specific antibodies

Antisera were prepared in rabbits against the idiotypic (Id) determinants of antiallergen antibodies. These antibodies were isolated from the plasma of 10 unrelated patients by immunoabsorption. Three major allergens isolated from the house dust mite, Dermatophagoides pteronyssinus (DPT), were used. The anti-Id antisera were rendered specific by successive absorption on insolubilized allergens, IgG and IgM from a pool of human sera and Ig from the donor after removal of anti-DPT antibodies by absorption. Anti-Id antibodies raised essentially against IgG antibodies were found to inhibit the agglutinating activity of both IgG and IgM anti-DPT antibodies toward allergen-coated latex. These anti-Id antibodies were also able to inhibit partly the binding of radiolabeled allergen to IgE anti-DPT antibodies. Matching the anti-Id antisera with the various individual anti-DPT antibodies revealed an Id cross-reactivity of about 71% for IgG, 61% for IgM and 62% for IgE. A second and predominant type of anti-Id antibodies recognized bystander idiotopes as the allergen did not inhibit Id-anti-Id reaction. These idiotopes were apparently more specific to individuals (private) as the anti-DPT antibodies of a given patient did not agglutinate latex particles coated with anti-Id antibodies prepared against Id of another patient.     

Localization of antigens and allergens in thin sections of the house dust mite, Dermatophagoides pteronyssinus (Acari: Pyroglyphidae)

Cryostat sections of the house dust mite, Dermatophagoides pteronyssinus, were probed in fluorescent microscopy studies with rabbit polyclonal and mouse monoclonal antibodies specific for mite allergens including the major allergen, Der p I. Sections also were probed for allergens with sera from human mite-allergic subjects containing IgE antibodies to mite allergens and with lectins. Antibody binding was mainly to the gut lining and gut contents of the mite, although some specific labeling also was associated with the head region and cuticle. This is the first detailed localization of mite allergens in situ. The morphology of the mite was investigated using plastic embedded thin sections and was found to be similar to that previously described for D. farinae.     

Cross-reactivity between the house dust mite Dermatophagoides pteronyssinus and the mange mites Psoroptes cuniculi and P. ovis. I. Demonstration of antibodies to the house dust mite allergen Dpt 12 in sera from P. cuniculi-infested rabbits

The cross-reactivity of the mange mites Psoroptes cuniculi (PC) and Psoroptes ovis (PO) antigens with the house dust mite Dermatophagoides pteronyssinus (DP) antigens has been studied. Cross-reactivity between mange mite and house dust mite antigens was demonstrated by both ELISA and immunoelectrophoresis by use of a sheep anti-DP antiserum. Both PC and PO were demonstrated to contain eight cross-reacting antigens. Sera from rabbits infested with PC were demonstrated to produce antibodies to the homologous immunogen, to PO antigens, and to DP antigens. Of the seven sera from infested rabbits tested, four were demonstrated to produce a strong antibody response to a major DP antigen Dpt 12, and two were demonstrated to produce a weak response that was judged empirically by double-diffusion analysis. Two sera were judged to react with the DP lipoprotein, Dpt 4. Sera from control rabbits did not demonstrate reactivity with any extract tested. Despite the detection of anti-Dpt 12 antibodies, however, an antigen corresponding to Dpt 12 was not detected in either PC or PO extracts. The findings that mange mite-infested rabbits produce antibodies that recognize DP antigens probably explain previous observations in which it was demonstrated that commercially obtained rabbit anti-immunoglobulin antisera contain anti-DP antibodies, a finding that suggests caution in the use of such reagents in studies designed to measure antibody responses to DP allergens.     

Sensitization to major Dermatophagoides pteronyssinus allergens in house dust mite allergic patients from North Eastern Poland developing rhinitis or asthma

PurposeRecognition of individual allergens by IgE is crucial for triggering symptoms in allergic rhinitis (AR) or asthmatic (AA) patients. House dust mite (HDM) allergy is frequent around the world, the sensitization profile to individual HDM allergens varies in individual HDM-allergic patients (APs). The aim of this study was to evaluate the pattern of IgE sensitization to three major Dermatophagoides pteronyssinus (Dp) allergens among patients from North Eastern Poland suffering from HDM-AR and/or AA.Patients and methodsThe study was performed on 323 HDM-AR and/or AA patients and 106 controls (CG) including 30 healthy non-atopic subjects, 32 AR patients not sensitized to Dp and 44 non-atopic asthmatics. IgE levels to natural (n)Der p 1, nDer p 2, recombinant (r)Der p 2.0101 and rDer p 23 allergens were measured by ELISA.ResultsThe majority of HDM-APs were sensitized to nDer p 1 (72.1%), nDer p 2 (81.7%), rDer p 2.0101 (78.3%) and rDer p 23 (70.9%). The frequency of positive results to individual allergens depended on clinical manifestations and the level of IgE to the whole Dp extract. In HDM-AA patients, reactivity to nDer p 1 and rDer p 23 was detected more frequently than in HDM-AR patients. The whole Dp extract completely inhibited IgE binding to nDer p 1 and nDer p 2 but only partially to rDer p 23.ConclusionsHDM-APs from North-Eastern Poland display sensitization profile to major allergens which is similarly observed in western Europe. HDM-based diagnostic and therapeutic products should include all major allergens.     

Human immune response to allergens of house dust mite, Dermatophagoides pteronyssinus. IV. Occurrence of natural autologous anti-idiotypic antibodies

IgG isolated from the plasma of seven individuals hypersensitive to the common house dust mite Dermatophagoides pteronyssinus (DPT) was exhaustively adsorbed onto insolubilized DPT. The unbound fraction was found by radioimmunoassay to contain antibodies recognizing the variable region of both anti-DPT IgG and IgE antibodies. This recognition was idiotype (Id)-specific as it persisted after passage over insolubilized polyclonal IgG of unrelated specificity. Most of these anti-Id IgG carried the internal image of the initial antigen in that they competitively inhibited the binding of anti-DPT antibodies to DPT. Immunoadsorption of anti-Id IgG onto insolubilized anti-DPT IgG antibodies from the same individual completely eliminated their reaction with anti-DPT IgG but not with anti-DPT IgE, suggesting that idiotopes included in the antigen-binding site of specific IgG and IgE antibodies were not identical. Anti-Id IgG recognizing idiotopes located outside the antigen-binding site (bystander idiotopes) were also completely removed by passage over insolubilized anti-DPT IgG; in this case the reaction of the anti-Id IgG with both anti-DPT IgG and anti-DPT IgE was inhibited, indicating that, for a given individual, bystander idiotopes were shared between anti-DPT antibodies pertaining to these two isotypes.     

Investigation of house dust mite (Dermatophagoides pteronyssinus) allergen extracts prepared from purified mite bodies and whole mite culture

Allergen extracts prepared from purified mite bodies (PMB) and whole mite culture (WMC) were investigated by using in vivo and in vitro methods. PMB showed a more complex allergen pattern than WMC. In PMB, 6 out of 16 allergens could be detected as major allergens. In WMC, 5 out of 10 allergens could be detected as major allergens. Both extracts contained the same amount of the major allergen Der p I. By skin prick testing comparable results were obtained with both extracts. Investigation of the reproducibility showed that 15 production lots of PMB could be produced in a high conformity according to their allergenic potency and their protein and allergen composition.     

Ultrastructural localization of the allergen Der p I in the gut of the house dust mite Dermatophagoides pteronyssinus

The sites and concentrations of Der p I within the house dust mite were determined. Highest concentrations were found in the epithelium of the posterior end of the mite stomach, implying that this is the site of Der p I synthesis and secretion.