Immunogenic peptides: B & T Cell Epitopes of Per a 10 Allergen of Periplaneta americana

Mapping of B and T cell epitopes of an allergen can be utilised in the development of alternative therapeutic modalities and diagnostics. The present study was aimed to identify B and T cell epitopes of Per a 10, a major cockroach allergen, by computational tools and subsequent validation by in vitro experiments. Per a 10 three-dimensional structure was homology modelled using structure of anionic trypsin from pacific chum salmon as a template. Seven B cell epitopes (B-P1 to B-P7) were predicted by sequence and structure based methods. Three T cell epitopes (T-P8 to T-P10) were predicted by binding score and inhibitory concentration dependent prediction tools. Predicted epitopes were synthesized and biological activity was assessed by ELISA, ELISA inhibition and PBMC proliferation assays. B cell peptides B-P5, B-P6 and B-P7 showed significantly high IgE binding with pooled and individual cockroach hypersensitive patients’ sera while the T cell peptides did not show IgE binding. ELISA inhibition was performed to determine the potency of the predicted peptides. Fifty nanogram of peptide B-P7 was required for 50% IgE binding inhibition of surface bound Per a 10 whereas seventy five nanogram and ninety nanogram of B-P5 and B-P6 were required for the same respectively. Upon stimulation with T-P8 and T-P10 peptides, PBMCs from cockroach allergic patients’ (n = 15) showed significant lymphocyte proliferation and induced IL-4 and IL-5 cytokine release in the culture supernatant demonstrating Th2 dominant cell mediated response of predicted T cell peptides. In conclusion, Per a 10 3-D structure obtained by homology modelling was used to identify B and T cell epitopes, followed by in vitro validation. The identified peptides can be potentially used in designing diagnostics and therapies for cockroach allergy.     

IgE-binding epitopes of the American cockroach Per a 1 allergen

Cockroach is one of the major indoor allergens for IgE-mediated allergic respiratory illnesses throughout the world. The American cockroach (Periplaneta americana) Per a 1 allergen is antigenically cross-reactive with the German cockroach (Blattella germanica) Bla g 1 allergen. The aim of this study was to identify linear B cell epitopes of Per a 1 that are recognized by human IgE. Per a 1 deletion mutants were generated from the recombinant Per a 1.0104 allergen (274 amino acid residues), and antigenicities were assessed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and binding inhibition. Human atopic sera were not able to recognize deletion mutants consisting of amino acids 1-77, 86-205, and 200-266. However, human sera did recognize the N-terminal mutant containing amino acids 1-87 and the C-terminal mutant containing amino acids 200-274, demonstrating positive IgE binding that was heterogeneously distributed among the different sera tested. Amino acid residues 78-85 and 267-274, containing internal repeats, were shown to be required for IgE binding to the Per a 1.0104 protein. Two peptides corresponding to these IgE-binding amino acid sequences were synthesized. Peptide 78-85 showed a positive IgE interaction with 80% of the sera, while peptide 267-274 was capable of IgE binding to all of the sera tested. Moreover, preincubation of atopic sera with IgE-positive recombinants and peptides resulted in marked inhibition of the IgE binding to purified Per a 1.0104 allergen. Amino acid sequences 78LIRALFGL85 and 267IRSWFGLP1274 of the American cockroach Per a 1.0104 allergen were involved in IgE binding. These findings will advance the understanding of the specific reactivity of the epitopes of cockroach allergens, thereby contributing to the development of specific immunotherapies for clinical use.     

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.     

Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens

BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied. OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects. METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques. RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon. CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.     

IgE-Binding Epitope Mapping and Tissue Localization of the Major American Cockroach Allergen Per a 2

Purpose

Cockroaches are the second leading allergen in Taiwan. Sensitization to Per a 2, the major American cockroach allergen, correlates with clinical severity among patients with airway allergy, but there is limited information on IgE epitopes and tissue localization of Per a 2. This study aimed to identify Per a 2 linear IgE-binding epitopes and its distribution in the body of a cockroach.

Methods

The cDNA of Per a 2 was used as a template and combined with oligonucleotide primers specific to the target areas with appropriate restriction enzyme sites. Eleven overlapping fragments of Per a 2 covering the whole allergen molecule, except 20 residues of signal peptide, were generated by PCR. Mature Per a 2 and overlapping deletion mutants were affinity-purified and assayed for IgE reactivity by immunoblotting. Three synthetic peptides comprising the B cell epitopes were evaluated by direct binding ELISA. Rabbit anti-Per a 2 antibody was used for immunohistochemistry.

Results

Human linear IgE-binding epitopes of Per a 2 were located at the amino acid sequences 57-86, 200-211, and 299-309. There was positive IgE binding to 10 tested Per a 2-allergic sera in 3 synthetic peptides, but none in the controls. Immunostaining revealed that Per a 2 was localized partly in the mouth and midgut of the cockroach, with the most intense staining observed in the hindgut, suggesting that the Per a 2 allergen might be excreted through the feces.

Conclusions

Information on the IgE-binding epitope of Per a 2 may be used for designing more specific diagnostic and therapeutic approaches to cockroach allergy.     

Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts

BackgroundCurrent practice guidelines state that protease-rich fungal and insect extracts can be combined when preparing immunotherapy vaccines, but data supporting the stability of allergens in these mixtures have not been reported.ObjectiveTo determine the stabilities and compatibilities of Alternaria alternata and German cockroach allergens in mixtures with other high-protease fungal and insect (cockroach, imported fire ant) extracts at final extract concentrations consistent with injection dose targets for maintenance immunotherapy.MethodsMixtures containing Alternaria, German cockroach, and other fungal and insect extracts frequently included in immunotherapy vaccines were analyzed by a combination of quantitative analyses (enzyme-linked immunosorbent assays for multiallergen immunoglobulin E [IgE]-binding potency, major Alternaria allergen Alt a 1, and major German cockroach allergens Bla g 1 and Bla g 2) and qualitative methods (immunoblotting). Mixtures and analogous single-extract controls containing 10 to 50% glycerin were evaluated after storage for up to 12 months at 2°C to 8°C.ResultsMixtures of extracts within the same phylogenetic groups (fungal-fungal, insect-insect) retained favorable Alternaria and German cockroach allergen levels and activities under most conditions examined. For several cross-taxonomic (fungal–insect) extract combinations at 10 to 25% glycerin concentrations, different immunochemical test methods measuring single (major) or multiple allergens yielded threefold to 10-fold variations in allergen recoveries.ConclusionAllergen compatibilities can be compromised in some fungal–insect extract mixtures, contrary to current immunotherapy practice parameter recommendations. Separation of these products into different treatment vials may be required to produce stable mixtures for subcutaneous immunotherapy. Data from assay methodologies with distinct binding specificities provide a critical assessment of allergen activities in high-protease extract mixtures.     

Molecular basis of arthropod cross-reactivity: IgE-binding cross-reactive epitopes of shrimp,house dust mite and cockroach tropomyosins

BACKGROUND: Shrimp may cross-react with other crustaceans and mollusks and nonedible arthropods such as insects (cockroach and chironomids), arachnids (house dust mites) and even nematodes. Since the muscle protein tropomyosin has been implicated as a possible cross-reacting allergen, this study characterized the IgE-binding epitopes in shrimp tropomyosin, Pen a 1, that cross-react with other allergenic invertebrate tropomyosins in house dust mites (Der p 10, Der f 10) and cockroaches (Per a 7). Pen a 1-reactive sera from shrimp-allergic subjects were used to evaluate the effect on IgE binding of different amino acid substitutions in Pen a 1 epitopes based on homologous sequences in Per a 7 and Der p 10/Der f 10. METHODS: Peptides were synthesized spanning the length of Pen a 1 IgE-binding epitopes and amino acid substitutions were performed based on homologous amino acid sequences from Per a 7 and Der p 10/Der f 10. RESULTS: 7/8 individually recognized Pen a 1 epitopes (2, 3a, 3b, 4, 5a, 5b and 5c) had an identical amino acid sequence with lobster allergen, Hom a 1, 4/8 (3a, 3b, 4 and 5a) with Der p 10 and Der f 10, and 5/8 (2, 3a, 3b, 4 and 5a) with Per a 7. In addition, even homologous regions of other arthropod tropomyosins that differ in one or more amino acids from the sequences of Pen a 1 epitopes are still recognized by shrimp-allergic IgE antibodies. In total, shrimp-allergic sera recognize 6/8 peptides homologous to Pen a 1 epitopes in Per a 7, 7/8 in Der p 10/Der f 10, and 7/8 epitopes in Hom a 1. CONCLUSIONS: The IgE recognition by shrimp-allergic individuals of identified and/or similar amino acid sequences homologous to Pen a 1 epitopes in mite, cockroach and lobster tropomyosins are the basis of the in vitro cross-reactivity among invertebrate species. Based on amino acid sequence similarity and epitope reactivity, lobster tropomyosin has the strongest and cockroach the least cross-reactivity with shrimp. The clinical relevance of these cross-reactivities in developing allergic reactions to different arthropods needs to be determined.     

Allergenic Characterization of a Novel Allergen,Homologous to Chymotrypsin,from German Cockroach

Purpose

Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease.

Methods

A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA.

Results

The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract.

Conclusions

A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.     

Bla g 3: a novel allergen of German cockroach identified using cockroach-specific avian single-chain variable fragment antibody

BackgroundThe IgE response to cockroach allergens is thought to be associated with asthma. German cockroach (GCr) allergen extract is a complex mixture of allergens, and the identification and characterization of immunodominant allergens is important for the effective diagnosis and treatment of GCr-induced asthma.ObjectiveTo characterize a novel GCr allergen homologous to the American cockroach allergen Per a 3.MethodsGCr-specific avian monoclonal antibodies were used for direct immunoprecipitation of specific targets from whole-body GCr extract. Precipitated protein was identified by mass spectrometry and sequence analysis. Putative recombinant protein also was expressed, purified, and used for determination of allergenicity, determined by IgE enzyme-linked immunosorbent assay with serum from 61 GCr-allergic patients. The identified target also was analyzed for heat stability using a bead-based assay.ResultsThe immunoprecipitated target of monoclonal antibody 2A1 was identified as a novel allergen of GCr homologous to American cockroach allergen Per a 3. This homolog, designated Bla g 3, has an apparent mass of 78 kDa, can be measured in GCr extract using antibody 2A1, and is a heat-stable protein. Screening of 61 serum samples from GCr-allergic patients showed a 22% prevalence of Bla g 3-specific IgE.ConclusionBla g 3 is a GCr allergen with structural homology to American cockroach allergen Per a 3.     

Analysis of cross-reactive allergens from American and German cockroaches by human IgE

Serum from atopics hypersensitive to the American cockroach were examined for their specific IgE to American and German cockroaches by the fluoroallergosorbent test (FAST). Of 44 sera tested, 86.4% (38/44) contained IgE to American and German cockroaches, and 13.6% (6/44) were found to be positive for American cockroach alone by FAST. Nine individual sera containing IgE antibodies to both cockroaches were used to analyze the cross-reacting allergens in the crude American and German cockroach extracts by FAST inhibition and immunoblotting. FAST-inhibition studies showed various degrees but similar inhibition of binding of human IgE to solid-phase American cockroach extract. Proteins from both cockroach extracts were separated on SDS-PAGE followed by immunoblotting, and the results showed considerable heterogeneity in the IgE-binding patterns with each of the cockroach extracts for the same nine individual sera. Components with apparent molecular weights of 60, 52, 49, 38, and 12 kDa from both the American and German cockroaches were able to bind IgE antibody. These results suggest the presence of cross-reactive allergens in the American and the German cockroaches.     

Similar localization of conformational IgE epitopes on the house dust mite allergens Der p 5 and Der p 21 despite limited IgE cross‐reactivity

Background

Due to high IgE recognition frequency and high allergenic activity, Der p 5 and Der p 21 are clinically important house dust mite (HDM) allergens. The objective of this study was to characterize the immunodominant IgE epitopes of Der p 5 and Der p 21 responsible for their high allergenic activity.

Methods

A panel of 12 overlapping peptides spanning the Der p 5 and Der p 21 sequence were synthesized to search for sequential IgE epitopes by direct testing for allergic patients' IgE reactivity. Peptide‐specific antibodies raised in rabbits were used in inhibition studies for localizing conformational IgE epitopes which were visualized on the surfaces of the allergen structures by molecular modelling. IgE cross‐reactivity between the allergens was investigated by IgE inhibition studies.

Results

Immunodominant IgE epitopes defined by allergic patients' IgE on Der p 5 and Der p 21 were primarily of the conformational, discontinuous type including N‐ and C‐terminal portions of the protein. They could be located on each allergen on one area with similar localization, but despite similar structure of the allergens, no relevant IgE cross‐reactivity could be detected.

Conclusion

Our study shows that Der p 5 and Der p 21 contain a major conformational IgE epitope‐containing area located on similar portions of their structure, but they lack relevant IgE cross‐reactivity. These data are important for the development of modern allergy vaccines based on defined molecules for allergen‐specific immunotherapy of HDM allergy.     

Comparison of antigenic and allergenic composition of two partially puriflied extracts from Dermatophagoïdes farinae and Dermatophagoïdes pteronyssinus mite cultures

Dermatophagoïdes farinae (Df 80d) and Dermatophagoïdes pteronyssinus (Dp 80d) extracts were analyzed,for their antigenic and allergenic composition by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). By CIE, II antigens could be numbered in Df 80d (Df I to Df II) and seven antigens in Dp 80d (Dp I to Dp 7). This technique allowed us also to define antigens with common as well as specific parts for the two mite species. Among the antigens of D. farinae and D. pteronyssinus, only the antigen corresponding to DO and Dp 5 seems to bear common epitopes to the two mite species, whereas Df 6 and Dp 4 appear to, bear, respectively, specific epitopes 4f each species. Moreover, Df lI appears to bear specific epitopes of D. farinae, although it shows a partial identity with Dp 7. By CRIE, on 20 mite-sensitive patients' sera, we identified, for each mite extract, the allergens responsive to human specific IgE. The allergograms show that the majority of mite-sensitive patients react with Df 11 and Df 6 and with Dp 7 and Dp 4. Tines, these antigens can be considered as major allergens. The minor allergens were also identified. None of these antigens was recognized by the control sera. Moreover, we observed that, for one antigen (antigen 5) there exist antigenic determinants common to the two species of mites toward the rabbit serum and specific allergenic determinants to the human IgE response. A significant correlation was found between the specific IgE binding in CRIE and in RAST (Spearman coefficient: “rs” = 0.61 p < 0.0l ,for Df; “rs” = 0.78 p < 0.01 for Dp).     

Mimotopes identify conformational B-cell epitopes on the two major house dust mite allergens Der p 1 and Der p 2

House dust mite allergy occurs in 10-20% of the population. Improvement of the present immunotherapy requires detailed knowledge about the structure of the allergens. Mimotopes selected from phage peptide libraries imitate the conformational epitopes of a natural allergen. The aim of our study was to generate epitope mimics for the two major allergens of house dust mite. When the monoclonal anti-Der p 1 and anti-Der p 2 antibodies were used for biopannings, mimotopes were selected which bound also specific IgE from human allergic patients' sera. The conformational matching of these mimotopes on the 3D structure of the natural allergens determined discontinuous epitopes in both cases, representing conformational B-cell epitopes relevant for binding of human IgE. Therefore, these mimotopes are potential candidates for the directed induction of blocking antibodies and epitope-specific immunotherapy of mite allergy.     

Expression of the American cockroach Per a 1 allergen in mammalian cells

BACKGROUND: Cockroach allergens are one of the major etiologic risk factors for developing IgE-mediated allergic respiratory illness throughout the world. Per a 1 is a cross-reactive allergen of American and German cockroaches. This study aimed to investigate the expression of a recombinant American cockroach (Periplaneta americana) Per a 1, C42, allergen in mammalian COS-1 cells. METHODS: The COS-1 cells and Escherichia coli were used to express the P. americana C42 allergen. Recombinant proteins were purified with hydroxylapatite and DE52 chromatography. Biologic reactivities of recombinant proteins were examined by direct IgE binding and IgE inhibition studies with the enzyme-linked immunosorbent assay (ELISA). RESULTS: C42 was successfully expressed in the mammalian COS-1 cell as a 50-kDa secreted protein, and purified from the culture medium. The specific human IgE antibodies against recombinant C42 from either E. coli (C42-E. coli) or COS-1 (C42-COS-1) were compared by ELISA with 12 sera from Per a 1 and C42 skin-test-positive patients. All atopic sera contained specific IgE antibodies to C42 from either E. coli or COS-1. Moreover, recombinant C42-COS-1 bound higher levels of serum IgE than recombinant C42-E. coli among C42-sensitive atopic patients, and a statistically significant difference (P<0.01) was found between them. In addition, recombinant C42-COS-1 as an inhibitor revealed higher inhibition of IgE binding to natural Per a 1 than recombinant C42-E. coli. CONCLUSIONS: The biologically highly reactive recombinant C42 produced in the COS-1 cell provides an alternative expression system and will facilitate studies on the immune response of asthma patients to cockroach allergens.     

IgE and monoclonal antibody binding by the mite allergen Der p 7

Background The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergie patients. Objective This study was performed to compare the IgE bindinig activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies. Methods IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. Reslts A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f 7 was used for epitope analysis. All of these reaeted with eaeh allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, lour could inhibit the binding of IgE (WH9, WH22. WPS and HD19) while one (WH14) could not. Conclusions Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specilieities.     

Two new types of allergens from the cockroach,Periplaneta americana

Periplaneta americana cockroach is an important source of inhalant indoor allergen resource, and there are more than twenty IgE‐binding components identified in P. americana, but only nine allergens were characterized. Our knowledge about cockroach allergens remains poor. In this work, two novel allergen proteins Per a 11 (alpha‐amylase) and Per a 12 (chitinase) with molecular weight around 55 and 45 kDa, respectively, were purified and characterized from the midgut of cockroaches. Their primary sequences were determined by Edman degradation, mass spectrometry, and cDNA cloning. Sera from 39 and 30 of 47 (83.0% and 63.8%) patients reacted to Per a 11 and Per a 12 on immunoblots, respectively. The allergenicity of Per a 11 and Per a 12 was further confirmed by competitive ELISA, basophil activation test (BAT), and skin prick test (SPT). They appear to be of importance for the allergic reactions induced by cockroach and have a potential for component‐based diagnosis of allergy.     

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.     

Investigations of culture medium-free house dust mites. III. Antigens and allergens of body and fecal extract of Dermatophagoides farinae

Crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) were used to characterize antigens (Ags) and allergens derived from Dermatophagoides farinae (DF) culture media-free mite body and mite fecal matter extracts. CIE of DF body and DF feces extracts revealed the presence of 35 and 20 Ags, respectively. CRIE experiments demonstrated IgE binding by 14 and seven DF body and DF feces Ags, respectively, when CIE gels were incubated with reference sera from clinically mite-sensitive patients. Binding of specific IgE to the various Ags in the two extracts varied significantly both in frequency and in strength from patient to patient and within the same patient's serum. Sera from some patients demonstrated IgE binding predilection for specific DF body Ags, whereas other sera exhibited greater binding preference for DF feces Ags. Homologous, heterologous, and intermediate gel CIE and CRIE clearly demonstrated that DF bodies and DF feces share some common Ags or epitopes, but the two different extracts also were quantitatively different. Some Ags and allergens originate from mite body material and are not present in mite feces. These results indicate that only extracts containing high concentrations of both body and fecal allergens should be used in clinical testing and therapy.     

Effect of reagins and allergen extracts on radioallergosorbent assays for mite allergen

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