Identification of sole parvalbumin as a major allergen: study of cross‐reactivity between parvalbumins in a Spanish fish‐allergic population

Background Fish allergy is becoming an important health problem in Spain, a country with the third highest level of fish consumption after Japan and Portugal. The most common fish allergens are parvalbumins. In our area, the most widely consumed fish species are lean, such as whiff (Lepidorhombus whiffiagonis) and sole (Solea solea). Adverse reactions to fish are usually related to these species, a fact that is largely unknown to allergists in other countries. Objective The aim of this study was to identify and purify the major allergen implicated in allergic response to sole and evaluate the IgE cross‐reactivity of purified parvalbumins from whiff and sole, which are phylogenetically close, and more distant species (i.e. cod and salmon). Methods Eighteen Spanish fish‐allergic patients with a positive history of type I allergy to fish were recruited from the clinic. Total protein extracts and purified parvalbumins from whiff and sole were tested for their IgE‐binding properties by combining two‐dimensional Western blotting and mass spectrometry. The extent of cross‐reactivity between these parvalbumins along with cod and salmon parvalbumins was investigated by IgE ELISA inhibition assay. Results An IgE‐binding spot of approximately 14 kDa was identified as parvalbumin and confirmed as a major allergen in sole extract, which is recognized by almost 70% of the patients. Whiff parvalbumin was recognized by 83.4% of the patients. High cross‐reactivity was determined for all purified parvalbumins by IgE inhibition assay. Conclusions and Clinical Relevance Sole and whiff parvalbumin were confirmed as major allergens. The parvalbumins of sole, whiff, cod and salmon were highly cross‐reactive, thus suggesting a high amino acid sequence identity between them. Cite this as: M. Perez‐Gordo, J. Cuesta‐ Herranz, A. S. Maroto, B. Cases, M. D. Ibáñez, F. Vivanco and C. Pastor‐Vargas, Clinical & Experimental Allergy, 2011 (41) 750–758.     

Characterisation of purified parvalbumin from five fish species and nucleotide sequencing of this major allergen from Pacific pilchard, Sardinops sagax

IgE-mediated allergic reaction to seafood is a common cause of food allergy including anaphylactic reactions. Parvalbumin, the major fish allergen, has been shown to display IgE cross-reactivity among fish species consumed predominantly in Europe and the Far East. However, cross-reactivity studies of parvalbumin from fish species widely consumed in the Southern hemisphere are limited as is data relating to immunological and molecular characterisation. In this study, antigenic cross-reactivity and the presence of oligomers and isomers of parvalbumin from five highly consumed fish species in Southern Africa were assessed by immunoblotting using purified parvalbumin and crude fish extracts. Pilchard (Sardinops sagax) parvalbumin was found to display the strongest IgE reactivity among 10 fish-allergic consumers. The cDNA sequence of the β-form of pilchard parvalbumin was determined and designated Sar sa 1.0101 (accession number FM177701 EMBL/GenBank/DDBJ databases). Oligomeric forms of parvalbumin were observed in all fish species using a monoclonal anti-parvalbumin antibody and subject's sera. Isoforms varied between approximately 10–13 kDa. A highly cross-reactive allergenic isoform of parvalbumin was identified and sequenced, providing a successful primary step towards the generation of a recombinant form that could be used for diagnostic and potential therapeutic use in allergic individuals.     

IgE antibodies of fish allergic patients cross-react with frog parvalbumin

BACKGROUND: The major allergens in fish are parvalbumins. Important immunoglobulin (Ig)E cross-recognition of parvalbumins from different fish species has been shown. Recently frog parvalbumin alpha has been found to be responsible for a case of IgE-mediated anaphylaxis triggered by the ingestion of frog meat. The aim of this study was to investigate whether IgE antibodies of fish allergic persons cross-react with frog parvalbumin and to appreciate its clinical relevance. METHODS: The sera of 15 fish allergic patients and one fish and frog allergic patient were tested by IgE-immunoblotting against frog muscle extract. Sera were tested against recombinant parvalbumin alpha and beta from Rana esculenta. Skin prick tests were performed in selected patients with recombinant frog parvalbumin. Ca(2+) depletion experiments and inhibition studies with purified cod and frog recombinant parvalbumin were done to characterize the cross-reactive pattern. RESULTS: Fourteen of the sera tested had IgE antibodies recognizing low molecular weight components in frog muscle extract. Calcium depletion experiments or inhibition of patient sera with purified cod parvalbumin led to a significant or complete decrease in IgE binding. When tested against recombinant parvalbumins, three of 13 sera reacted with alpha parvalbumin and 11 of 12 reacted with beta parvalbumin from R. esculenta. Skin prick tests performed with recombinant frog parvalbumin were positive in fish allergic patients. Inhibition studies showed that a fish and frog allergic patient was primarily sensitized to fish parvalbumin. CONCLUSION: Cod parvalbumin, a major cross-reactive allergen among different fish species, shares IgE binding epitopes with frog parvalbumin. This in vitro cross-reactivity seems to be also clinically relevant. Parvalbumins probably represent a new family of cross-reactive allergens.     

Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen

BACKGROUND: The identity of allergenic almond proteins is incomplete. OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen. METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin. RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins. CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.     

Antacid medication inhibits digestion of dietary proteins and causes food allergy: a fish allergy model in BALB/c mice

BACKGROUND: Digestible proteins were supposed to be irrelevant for oral sensitization and induction of food allergy. Approximately 10% of the adult population uses antacids for the treatment of dyspeptic disorders, drugs that hinder peptic digestion. In these patients, proteins that are normally degradable might act as food allergens. OBJECTIVE: We aimed to study the influence of antacid intake on the allergenicity of dietary proteins, taking sturgeon caviar and parvalbumin, the major fish allergen, as examples. METHODS: Caviar proteins and recombinant parvalbumin from carp, rCyp c 1, were applied for intragastric feedings with or without the antacids sucralfate, ranitidine or omeprazole, using a Balb/c mouse model. RESULTS: Both caviar proteins and parvalbumin were rapidly degraded in an in vitro digestion assay at pH 2.0, but not at pH 5.0, imitating the effect of antacids. The groups fed with caviar in combination with ranitidine hydrochloride intramuscularly or sucralfate orally had significant levels of caviar-specific IgE antibodies (P <.01), T-cell reactivity, and elevated counts of gastrointestinal eosinophils and mast cells. Food allergy in these groups was further evidenced by oral provocation tests and positive immediate-type skin reactivity. In contrast, feedings with caviar alone led to antigen-specific T-cell tolerance. None of the groups showed immune reactivity against the daily mouse diet. As a proof of the principle, feeding mice with parvalbumin in combination with ranitidine or omeprazole intramuscularly induced allergen-specific IgE antibodies (P <.05). CONCLUSIONS: When antacid medication impairs the gastric digestion, IgE synthesis toward novel dietary proteins is promoted, leading to food allergy.     

Differential responses to natural and recombinant allergens in a murine model of fish allergy

Aerosolized fish proteins are an important cause of allergic airway reactions in both the domestic and the occupational environment. The aim of this study was to investigate inhalant fish-induced allergy in a mouse model and compare immune responses generated by raw and heat-treated fish extracts as well as natural and recombinant forms of the major fish allergen parvalbumin. Mice were sensitized with raw or cooked pilchard extract and challenged intranasally with cooked pilchard extract, purified natural pilchard parvalbumin or recombinant carp parvalbumin (rCyp c1.01). Cooked pilchard extract predominantly sensitized mice to parvalbumin and induced specific IgG1 and IgE antibodies against both pilchard parvalbumin and rCyp c1.01, whereas additional allergens were recognized by mice sensitized with raw extract, including a 36 kDa allergen that was also recognized by fish processing workers and was identified as glyceraldehyde-3-phosphate dehydrogenase. Mice challenged with cooked extract and purified pilchard parvalbumin had increased Th2 cytokine production in mediastinal lymph node cells and splenocytes, whereas mice challenged with rCyp c1.01 did not. This study identifies a new IgE-binding protein that may be important in occupational allergy to fish and demonstrates the feasibility of testing recombinant allergens for immunotherapeutic potential in vivo.     

Celery allergens in patients with positive double-blind placebo-controlled food challenge

BACKGROUND: Recently, for the first time, allergy to celery was confirmed by double-blind placebo-controlled food challenge (DBPCFC). Api g 1, Api g 4, cross-reactive carbohydrate determinants (CCD), and a 60 kDa allergen have been described as celery allergens. OBJECTIVE: To get insights in IgE responses of patients with a positive DBPCFC to celery tuber (celeriac) compared with patients with a negative challenge test. METHODS: Specific IgE to native and heated celery tuber and to recombinant Api g 1, the major celery allergen, were determined by enzyme allergosorbent test and immunoblotting. IgE binding to Api g 1, Api g 4, and CCD was confirmed by inhibition experiments that used recombinant Api g 1, recombinant Api g 4, pure N-glycans, and extracts of celeriac, lychee fruit, and pollens of birch, mugwort, and timothy grass as inhibitors. RESULTS: Immunoblotting with sera from 22 patients with a positive DBPCFC to celeriac confirmed the presence of known allergenic structures: The major allergen Api g 1 (16 kDa) was recognized by IgE from 13 of 22 patients (59%). Another major allergen was CCD, determined by IgE reactivity in 12 of 22 patients (55%). Celery profilin, Api g 4, was recognized by IgE from 5 of 22 patients (23%). CONCLUSION: Our DBPCFC-positive patients exclusively presented IgE to known celery allergens, although the prevalences were slightly different than were previously reported. No obvious differences were found in patients with positive IgE antibody but negative challenge test. IgE binding to all 3 structures in celeriac extract was inhibited by birch pollen extract, whereas mugwort pollen extract could only inhibit IgE reactivity to Api g 4 and CCD. Inhibition experiments with a purified carbohydrate moiety clearly showed that the IgE epitope mannose-xylose-fucose-glycan (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[ Fucalpha1-3]GlcNAc) or a closely related structure is present in celeriac extract and is important in patients with clinical allergy to celery.     

Transaldolases are novel and immunoglobulin E cross‐reacting fungal allergens

Background Mould‐induced atopic respiratory diseases are a worldwide problem. Characterization of fungal allergens is of major clinical importance. Objective We identified a novel transaldolase family allergen of Cladosporium and Penicillium species. Methods Fungal allergens were identified by immunoblotting, peptide mass mapping and partial sequencing, cDNA cloning and IgE epitope mapping. Results A 36.5 kDa IgE‐binding component in a partially purified C. cladosporioides preparation was identified. Mass spectrometric analyses suggest that this novel IgE‐reacting allergen is a transaldolase. A corresponding full‐length 1246 bp cDNA encoding a polypeptide of 325 residues was isolated. The newly identified transaldolase allergen has been designated as Cla c 14.0101. The cDNA encoding the Pencillium chrysogenum transaldolase was isolated by RT‐PCR according to the cDNA sequence encoding a P. chrysogenum Wisconsin 54‐1255 hypothetical protein. The purified rCla c 14.0101 protein reacted with IgE antibodies in 10 (38%) of 26 Cladosporium cladosporioides‐sensitized asthmatic patients. Nine of the 10 rCla c 14.0101‐positive sera have IgE binding against the recombinant Penicillium transaldolase (rPen ch 35.0101). Among the eight fungal transaldolase‐positive sera tested, three showed IgE binding against the recombinant human transaldolase. To determine cross‐reactivity between the Cladosporium and Penicillium fungi, IgE cross‐reactivity was detected between these two fungal transaldolase allergens by inhibition assays. Both the N‐ and the C‐terminal fragments of Cla c 14.0101 were recognized by IgE antibodies. The C‐terminal IgE‐reacting determinant was narrowed down to a region encompassing Thr257 to Ser278 of Cla c 14.0101. It was mapped onto a loop‐like structure of a 3D model constructed for Cla c 14.0101. Conclusion and Clinical Relevance We identified transaldolase as a novel and IgE cross‐reactive allergen family of C. cladosporioides and P. chrysogenum. In addition, an IgE‐reacting fragment (Thr257 to Ser278) was pinpointed to a loop‐like structure on Cla c 14.0101. Results obtained provide important information in clinical mould allergy. Cite this as: H. Chou, M. F. Tam, C.‐H. Chiang, C.‐T. Chou, H.‐Y. Tai and H.‐D. Shen, Clinical & Experimental Allergy, 2011 (41) 739–749.     

Fish collagen is an important panallergen in the Japanese population

Collagen was identified as a fish allergen in early 2000s. Although its allergenic potential has been suggested to be low, risks associated with collagen as a fish allergen have not been evaluated to a greater extent. In this study, we aimed to clarify the importance of collagen as a fish allergen. Our results showed that 50% of Japanese patients with fish allergy had immunoglobulin E (IgE) against mackerel collagen, whereas 44% had IgE against mackerel parvalbumin. IgE inhibition assay revealed high cross‐reactivity of mackerel collagen to 22 fish species (inhibition rates: 87–98%). Furthermore, a recently developed allergy test demonstrated that collagen triggered IgE cross‐linking on mast cells. These data indicate that fish collagen is an important and very common panallergen in fish consumed in Japan. The high rate of individuals' collagen allergy may be attributable to the traditional Japanese custom of raw fish consumption.     

Differences between specificities of IgE and IgG4 antibodies: studies using recombinant chain 1 and chain 2 of the major cat allergen Felis domesticus (Fel d) I

IgE- and IgG4 antibodies were compared for reactivity with recombinant chain 1 and chain 2 of the cat allergen Felis domesticus (Fei d) I. Recombinanl chain 1 and chain 2 were coupled to sepharose and tested in IgE- and IgG4 radioallergosorbent test (RAST) experiments. Substantial IgE- and IgG4 binding was found. The fraction of Pel d I-specific antibody that bound to the recombinant chains was calculated. For chain 1, the mean value of this fraction was 0.30 for IgE and 0.23 for lgG4 (P= 0.05). For chain 2, the mean value of this fraction was 0.19 for IgE and 0.13 for IgG4 (P= 0.02). These results indicate that differences in fine specificity exist between IgE and IgG4 antibodies. Moreover, these findings support our results with chemically prepared peptides derived from these two chains and suggest that the B cells producing IgE antibodies are more likely to recognize a less ‘native’ form of Pel d I, compared with IgG4.     

Assessment of recombinant dog allergens Can f 1 and Can f 2 for the diagnosis of dog allergy

BACKGROUND: The use of recombinant allergens for the diagnosis and immunotherapy of allergy may offer several advantages over allergen extracts. OBJECTIVE: To produce recombinant dog allergens Can f 1 and Can f 2 in Pichia pastoris yeast and to assess their suitability for the diagnosis of dog allergy. METHODS: Clinically diagnosed dog-allergic patients' and healthy non-atopic dog owners' reactivities against recombinant Can f 1 and Can f 2 and commercial dog epithelial extract were studied by a panel of methods including skin prick test (SPT), ELISA and IgE immunoblotting. RESULTS: Recombinant Can f 1 and Can f 2 were found immunologically functional: they bound dog-allergic patients' IgE in immunoblotting and inhibited specifically the binding of IgE to their natural counterparts in the dog allergen extract. Moreover, patients' IgE reactivity in immunoblotting to natural Can f 1 and their SPT with the recombinant allergen were perfectly concordant (phi coefficient 1.0, P<0.001). The concordance was slightly lower with recombinant Can f 2 (phi coefficient 0.92, P<0.001). A lower number of dog-allergic patients, 52%, reacted against Can f 1 than previously reported. About one-third of the patients reacted to Can f 2. In immunoblotting, the highest prevalence of reactivity, 60%, was directed to an 18 kDa component. Aminoterminal sequencing showed this to be a previously unidentified allergenic protein. CONCLUSIONS: The recombinant allergens can be used reliably to identify Can f 1 and Can f 2-sensitized individuals. However, the two allergens are insufficient as reagents for diagnosing dog allergy.     

Molecular cloning and immunological characterization of the house dust mite allergen Der f 7

Background The allergen Der p 7 from Dermatophagoides pteronyssinus has been defined by molecular cloning and shown to be an important specificity in 50% of miteallergic patients. Objective This study compares the cDNA sequence and serological reactivity of Der f 7 from D. farinae with Der p 7. Method cDNA encoding Der f 7 was amplified by polymerase chain reaction. sequenced and expressed as a fusion with glutathione-S-transferase for IgE and monoclonal antibody binding studies. Results Der f 7 cDNA encoded a 213 polypeptide containing a predicted 17 amino acid leader sequence, no cysteines and a single N-glycosylation site similar to Der p 7. The predicted 196 residue mature polypeptide had 86% identity lo Der p 7 and a calculated molecular weight of 22 34SDa. No homologues were found in searches of the data banks. The Der f 7 fusion protein showed a single band of 46 k Da by sodium dodccyl snlfute-polyaerylamide gel electrophoresis (SDS-PAGF) and reacted with IgE antibodies in 19/41 (46%) of sera from asthmatic children. The degree of binding was usually 30% of that to Der p 7 consistent with the exposure of the patients to D. Pteronyssinus. Monoclonal antibodies (WH9 and WH22) against Der p 7 reacted with Der f 7 but inhibition studies showed a 10-fold difference in reactivity. Conclusion Der f 7 has a predicted 213 residue polypeptide with 86% homology and serological cross reactivity to Der p 7.     

Calcium-binding proteins and their role in allergic diseases

Calcium-binding proteins (CBPs) are ubiquitous pollen allergens and important food allergens in fish and amphibians. Calcium-binding allergens containing two EF-hands (polcalcins) have been detected and characterized in pollen from trees, grasses, and weeds. Timothy grass Phl p 7 is the most cross-reactive allergen among polcalcins. Although there is cross-reactivity described within the subfamilies of calcium-binding allergens, there are no strong indications for IgE cross-reactivity between CBPs from plants, fish, and humans. Therefore, Phl p 7 could be used as marker to identify multiple pollen-sensitized patients, whereas cod Gad c 1 or carp Cyp c 1 could be selected for the diagnosis of fish allergy. Hom s 4, a calcium-binding autoantigen, might be an interesting candidate to monitor chronic skin inflammation in atopic and nonatopic individuals. Diagnostic tests containing these molecules could allow the identification of most patients sensitized to calcium-binding allergens/antigens. In general, IgE recognition of calcium-binding allergens is influenced by binding or release of calcium ions. This knowledge could be used to engineer hypoallergenic CBPs for specific immunotherapy.     

Molecular cloning and immunological characterisation of potential allergens from the mould Fusarium culmorum

BACKGROUND: High quality and stability are essential requirements of commercial allergen preparations. Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum.OBJECTIVE: The present study was performed to identify, isolate and characterise allergens of F. culmorum as a basis for a stable allergenic reference material. In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated.METHODS: Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F. culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST). Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection. A complementary (c)DNA expression library of F. culmorum was prepared and screened for IgE-binding clones using sera from F. culmorum-sensitive individuals. Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E. coli. The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST.RESULTS: Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F. culmorum-specific IgE antibodies. Compared to the in-house extract, nearly all IgE-reactivties in the range of 15-30kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F. culmorum-specific IgE by EAST. IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts. Three IgE binding clones were isolated from the cDNA-library. One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds. The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2). The third clone (Fus c 3) was not related to known proteins. With sera from 26 individuals sensitised to F. culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively.CONCLUSIONS: F. culmorum may represent an underestimated source of aeroallergens. In contrast to highly labile and poorly standardised F. culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material. A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F. culmorum-sensitised subjects. IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.     

Structural property of soybean protein P34 and specific IgE response to recombinant P34 in patients with soybean allergy

Soybean allergy is one of the important food allergies because soybean is widely used in processed foods. P34 has been identified as the main allergen in soybeans. The main objective was to analyze the structural property of recombinant P34 and the P34 antigen-specific IgE response in soybean allergy using recombinant P34. Recombinant P34 was expressed by the BL21 (DE3) strain of Escherichia coli. Purified recombinant P34 showed oligomerization and binding to endotoxin. The binding of recombinant P34 to endotoxin was confirmed by LPS pull-down assay. High-density SDS treatment dissociated oligomeric recombinant P34 and removed endotoxin. Both native P34 and purified recombinant P34 showed almost identical structural properties as determined by circular dichroism analysis. We analyzed recombinant-P34-specific IgE antibodies by the ImmunoCAP System. In ImmunoCAP using recombinant P34, all sera from healthy controls were classified as negative. A correlation was found between the specific IgE antibodies to whole soybean and recombinant P34 (r=0.526, P<0.05). The sera from 3 of 9 (33%) patients with outgrown soybean allergy and 6 of 9 (66%) patients with soybean allergy were classified as positive. SDS-treated recombinant P34 retained its structure and biological activity. Recombinant P34 is a useful tool for the analysis of antigen-specific response in soybean allergy. It may be possible to develop a modified form of recombinant P34 for the diagnosis or treatment of soybean allergy using specific immunotherapy techniques.     

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.     

Characterization of IgE-binding epitopes on Candida albicans enolase

Candida albicans enolase is one of the important allergens in Candida allergy. We isolated and purified 46 kDa C. albicans enolase (CAE) from C. albicans and characterized epitopes for IgE antibody by lectin-blotting and enzymatic digestion followed by sodium dodecyl sulfale polyacrylamide gel electrophoresis (SDS-PAGE) and immunobiotting. Lectin blotting and deglycozilation indicated that this protein did not contain polysaccharide side chains. The purified CAE and recombinant fusion protein produced from CAE gene possessed common epitopes for IgE antibody. We estimated IgE binding epitopes on the basis of reported amino acid sequences from the analysis of cDNA encoding CAE. V8 protease digestion of CAE gave six polypeptide fragments (A-F). The N-termini of each fragment were confirmed by amino acid sequence and the C-termini were estimated by molecular weights of each fragment and the specific cutting site of V8 protease. Fragment C (25.0 kDa; F-171-I-399) reacted to 90% IgE antibodies examined, whereas fragments D (21.0 kDa; F-171-1-360), E (16.2kDa: F-171-D-317) and F (13.0kDa; A-47-E-170) showed no IgE binding. Our results suggest that epitopes for IgE antibodies exist near the C-terminal of the protein.     

Reactivity of the immunoglobulin E in bovine gelatin-sensitive children to gelatins from various animals

It has been reported that most children who showed anaphylaxis to measles, mumps and rubella vaccines containing bovine gelatin as a stabilizer have anti-bovine gelatin IgE. The present study was designed to investigate the reactivity of IgE in bovine gelatin-sensitive children to gelatins from various animals, and the antigenic cross-reactivity between the gelatins. Serum samples taken from 10 children who showed anaphylaxis to vaccines containing bovine gelatin were used in this study. The level of anti-bovine gelatin IgE in these serum samples ranged from 11.0 to 251 Ua/ml. The IgE in most of the children reacted to kangaroo and mouse gelatins, to which they had had little or no exposure as a food or a vaccine stabilizer. The IgE binding to kangaroo and mouse gelatins was completely inhibited by bovine gelatin, whereas reciprocal inhibition was not complete, indicating that antigenic cross-reactivity is present between the mammalian gelatins. Only one child had strong IgE reactivity to fish gelatins, and this reactivity was not inhibited by bovine gelatin, indicating that no antigenic cross-reactivity exists between bovine and fish gelatins. Most of the children who displayed sensitivity to bovine gelatin showed IgE reactivity to other mammalian gelatins. This reactivity may be due primarily to the antigenic cross-reactivity between mammalian gelatins.