Allergenic Activity Research Articles

Protease-Activated Receptor-2 Activation Contributes to House Dust Mite-Induced IgE Responses in Mice

Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif) ligand 17 (CCL17) and thymic stromal lymphopoietin (TSLP), were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT) and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM-driven allergic airway disease.

The role of N‐glycosylation in kiwi allergy

The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen. In this sense, N-glycosylation is an exclusive characteristic of plant allergens not present in mammals and it could be implied in allergenic sensitization. With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2. The natural allergen, Act d 2, was deglycosylated by trifluoromethanesulfonic acid treatment; the N-glycan fraction was obtained by extended treatment with proteinase K. N-glycan- and protein- fractions were recognized by specific IgE of kiwi-allergic patients. By contrast, the sugar moiety showed a reduced capacity to activate basophils and T cells, but not dendritic cells derived from patients' monocytes. Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety. Thus, the sugar moiety plays a significant role in sensitization, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions.

A molecular model system based on human monoclonal allergen-specific IgE antibodies to study basophil activation

Methods Monoclonal human IgE antibodies specific for the major birch pollen allergen Bet v 1 and for the major grass pollen allergen Phl p 5 and the corresponding allergens as well as a hybrid protein consisting of the Bet v 1 and Phl p 5 allergens were expressed. Using circular dichroism spectroscopy we compared the fold of the hybrid protein with that of the mixture of the two allergens. Rat basophil leukemia cells transfected with the human FceRI receptor were then loaded with the monoclonal IgE antibodies or patients serum containing polyclonal allergen-specific IgE and then exposed to the allergens and the hybrid protein to study basophil activation. Results We found that basophil activation was obtained with a mixture of two monoclonal IgE antibodies and the hybrid protein which increased in magnitude with rising concentrations of allergen-specific IgE antibodies. Thus increases in allergen-specific IgE lead to the release of larger amounts of mediators. When polyclonal IgE from allergic patients was used, basophil activation was obtained already with lower concentrations of the hybrid protein. An augmentation of the number of IgE epitopes thus increased the allergenic activity and thus the “potency” of the allergen. Preliminary titration experiments of the two monoclonal IgE antibodies also suggested that equimolar concentrations of the IgE antibodies with different specificity were most efficient in causing basophil activation, most likely due to the formation of large immune complexes. Conclusion Results obtained in our molecular model for human basophil activation show that the levels of allergen-specific IgE dictate the extent of basophil degranulation and that the number of IgE epitopes determines the potency of an allergen to induce degranulation. The model should be useful to explore the factors determining allergen-induced basophil activation and to study therapeutic strategies controlling this event. This study was supported by grant P23318-B11 and in part by grants P23350-B11 and F4605 of the Austrian Science Fund (FWF).

Criteria used to categorise proteins as allergens for inclusion in allergenonline.org: a curated database for risk assessment

Background New proteins introduced in foods through genetic engineering or processing are often evaluated for potential risks of eliciting food allergy (Codex, 2003). The primary risk is the transfer of an allergen or a protein nearly identical to an allergen and capable of causing IgE mediated cross-reactions into a new food source. The AllergenOnline.org database was developed to provide a curated set of allergenic sequences for bioinformatics comparisons to identify proteins that should be tested further (e.g. serum IgE binding). Curation is necessary as the scientific literature and general sequence databases include thousands of proteins labeled as allergens that lack proof of allergenic activity.

Limonene hydroperoxide analogues show specific patch test reactions

The fragrance terpene R-limonene is a very weak sensitizer, but forms allergenic oxidation products upon contact with air. The primary oxidation products of oxidized limonene, the hydroperoxides, have an important impact on the sensitizing potency of the oxidation mixture. One analogue, limonene-1-hydroperoxide, was experimentally shown to be a significantly more potent sensitizer than limonene-2-hydroperoxide in the local lymph node assay with non-pooled lymph nodes. To investigate the pattern of reactivity among consecutive dermatitis patients to two structurally closely related limonene hydroperoxides, limonene-1-hydroperoxide and limonene-2-hydroperoxide. Limonene-1-hydroperoxide, limonene-2-hydroperoxide, at 0.5% in petrolatum, and oxidized limonene 3.0% pet. were tested in 763 consecutive dermatitis patients. Of the tested materials, limonene-1-hydroperoxide gave most reactions, with 2.4% of the patients showing positive patch test reactions. Limonene-2-hydroperoxide and oxidized R-limonene gave 1.7% and 1.2% positive patch test reactions, respectively. Concomitant positive patch test reactions to other fragrance markers in the baseline series were frequently noted. The results are in accordance with the experimental studies, as limonene-1-hydroperoxide gave more positive patch test reactions in the tested patients than limonene-2-hydroperoxide. Furthermore, the results support the specificity of the allergenic activity of the limonene hydroperoxide analogues and the importance of oxidized limonene as a cause of contact allergy.

Allergy To Ferret

S U N D A Y 354 Characterization Of The Allergenic Activity Of Tropomyosin From Aedes Aegypti Mr. Jose F. Cantillo, Prof. Leonardo Puerta, Dr. Enrique FernandezCaldas, Dr. Sylvie Lafosse-Marin; Complutense University of Madrid, Spain; Inmunotek S.L., Alcal a de Henares, Spain, Institute for Immunological Research/University of Cartagena, Cartagena, Colombia, Inmunotek, S.L, Spain, Cabinet de Inmunoallergology, Fort de France, Martinique. RATIONALE: Mosquito bites are an important cause of skin lesions in allergic and non-allergic individuals worldwide. The inhalation of mosquito allergens may also be a cause of allergic respiratory diseases. It has been suggested that there may be cross-reactivity among mosquitoes and other arthropods such as mites, Chironomidae and shrimps. We hypothesized that the panallergen tropomyosin may be involved in this process. METHODS: Tropomyosin was purified from a whole body A. aegypti extract by size exclusion and anionic exchange chromatography. Specific IgE binding to the purified tropomyosin was evaluated by immunoblot and ELISA in allergic patients with positive ELISA to A. aegypti allergenic extract and control individuals. Cross-reactivitywas evaluated by immunoblotting and ELISA inhibitions using tropomyosin derived from the giant tiger prawn Penaeus monodon (nPen m 1) and from the whiteleg shrimp Litopenaeus vannamei (rLit v 1). RESULTS: A purified fraction from the allergenic A. aegypti extract showed four bands of several molecular weights with reactivity to antiPen m 1 rabbit serum, which showed also IgE reactivity to positive sera to mosquito extract. The IgE reactivity to these bands disappears when the sera where adsorbed with shrimps tropomyosin (nPen m 1 and rLit v 1). 25% of the mosquito allergic individuals had detectable specific IgE levels to this fraction. Inhibition of IgE reactivity higher than 90 % was demonstrated in a sera pool adsorded with nPen m 1 and rLit v 1. CONCLUSIONS: We have demonstrated the presence of tropomyosin in A. aegypti extract, which cross-react with shrimp tropomyosins.

Role Of Basophil Activation Test For Identifying Subjects With Local Allergic Rhinitis

A previous pilot study showed the presence of allergen-specific IgE in the surface of peripheral basophils in 50% of subjects with confirmed local allergic rhinitis (LAR). However, this phenomenon was investigated in a small number of patients. The aim of this study is confirming the presence of specific allergen activation of basophils, by means of basophil activation test (BAT) in a large population of LAR subjects sensitized to D. pteronyssinus (DP).

Mammalian airborne allergens.

Historically, horse dandruff was a favorite allergen source material. Today, however, allergic symptoms due to airborne mammalian allergens are mostly a result of indoor exposure, be it at home, at work or even at school. The relevance of mammalian allergens in relation to the allergenic activity of house dust extract is briefly discussed in the historical context of two other proposed sources of house dust allergenic activity: mites and Maillard-type lysine-sugar conjugates. Mammalian proteins involved in allergic reactions to airborne dust are largely found in only 2 protein families: lipocalins and secretoglobins (Fel d 1-like proteins), with a relatively minor contribution of serum albumins, cystatins and latherins. Both the lipocalin and the secretoglobin family are very complex. In some instances this results in a blurred separation between important and less important allergenic family members. The past 50 years have provided us with much detailed information on the genomic organization and protein structure of many of these allergens. However, the complex family relations, combined with the wide range of post-translational enzymatic and non-enzymatic modifications, make a proper qualitative and quantitative description of the important mammalian indoor airborne allergens still a significant proteomic challenge.

Multiple House Dust Mite Allergen- Sensitization Profiles in Children with Allergic Asthma

Background: Allergic asthma is a common chronic inflammatory disorder which affects populations, with increasing prevalence among children. The risk of developing asthma is uncertain but depends on the interaction of environmental and genetic factors. The role of house dust mite (HDM) allergen exposure in the development of sensitization and asthma remains unclear. Objective: This study determined the sensitization profiles of common HDM species in a population of asthmatic children, which is essential in the development of highly specific and accurate diagnostic and therapeutic strategies for asthma in children. Methods: The immunoglobulin E-binding activity of allergens from the HDM species Blomia tropicalis (Bt), Dermatophagoides farina (Df), and Dermatopahagoides pteronyssinus (Dp) were determined in 250 age- and sexmatched paediatric allergic asthma and non-atopic Filipino subjects using Enzyme-linked immunosorbent assay and western blot analysis. Results: Majority of the allergic asthma patients tested were sensitized with multiple allergens from different HDM species where 33% exhibited sensitizations to any two HDM species and 26% were sensitized with allergens from three HDMs. HDM allergens of different molecular weights bind to IgE in allergic asthma patients tested. In addition a significant correlation was observed between total IgE and HDM-specific IgE level among allergic asthma patients (Bt p-value=0.038; Df p-value=0.045; Dp p-value=0.003), suggesting a significant contribution of dust mite allergens in the up-regulation of total serum IgE levels of allergic asthma patients. Conclusion: The results obtained in this study suggest that the HDM species Bt, Dp, and Df are important sources of allergens that trigger multiple sensitization in children with allergic asthma in the Filipino population. The incorporation of Bt, Dp, and Df allergens in the panel of diagnostic allergens for HDM allergy and allergic asthma is highly recommended.

Allergic sensitization: food- and protein-related factors.

Presented here are emerging capabilities to precisely measure endogenous allergens in soybean and maize, consideration of food matrices on allergens, and proteolytic activity of allergens. Also examined are observations of global allergy surveys and the prevalence of food allergy across different locales. Allergenic potential is considered in the context of how allergens can be characterized for their biochemical features and the potential for proteins to initiate a specific immune response. Some of the limitations in performing allergen characterization studies are examined. A combination of physical traits of proteins, the molecular interaction between cells and proteins in the human body, and the uniqueness of human culture play a role in understanding and eventually predicting protein allergy potential. The impact of measuring food allergens on determining safety for novel food crops and existing allergenic foods was highlighted with the conclusion that measuring content without the context of clinically relevant thresholds adds little value to safety. These data and findings were presented at a 2012 international symposium in Prague organized by the Protein Allergenicity Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute.

Obtaining and Experimental Study of Candida Allergens

Due to the need of diagnostics of the widespread allergic diseases connected with infection ofa person by fungi, the question of creation of highly sensitive diagnostic panels of fungal allergens including molds and yeasts is important. Work purpose: creation of experimental technology of preparation of allergens from culture of C. albicans and studying of their physical, chemical and immunobiological properties. For receiving allergens we used the inactivated biomass of clinical strains of Candida albicans. Cultivation was carried out in the original mineral (without protein) CC1 and ML media in liquid and agarized variants with addition of sugar in various concentrations. In samples of allergens we defined amount of protein, carbohydrates, nucleic acids, specific activity in vitro (reaction of degranulation of rat mast cells). It was shown that the content of protein nitrogen varied from 72 to 18900 PNU, carbohydrates from 0,001 to 0,079 mg/ ml depending on physiological properties of a population of C. albicans and cultivation conditions. For determination of specific activity (reaction of rat mast cells degranulation) of samples used sera from patients with a sensitization to fungi. Allergenic activity of preparations in reaction of degranulation of rat mast cells varied from 3% (spontaneous) to 52%. The highest specific activity was shown for samples of the allergens obtained from C. albicans, grown in the ML medium. The results obtained using of 18 samples of preparations, can be a basis for a choice of an optimum technological mode of allocation of allergens from C. albicans.

Potential Utility of High-Pressure Processing to Address the Risk of Food Allergen Concerns.

High-pressure processing (HPP) technology is a novel, nonthermal processing technology for food. This special processing method can inactivate microorganisms and enzymes in food at room temperature using ultra-high pressures of above 100 MPa, while the original flavor and nutritional value of the food are maintained, with an extended refrigerated shelf-life of the food in distribution. In recent years, because of the rising prevalence of food allergies, many researchers have actively sought processing methods that reduce the allergenicity of food allergens. This study describes the effects of the current HPP technology on allergen activity. Our main goal was to provide an overview of the current research achievements of the application of HPP to eliminate the allergenicity of various foods, including legumes, grains, seafood, meat, dairy products, fruits, and vegetables. In addition, the processing parameters, principles, and mechanisms of HPP for allergen destruction are discussed, such as the induction of protein denaturation, the change in protein conformation, allergen removal using the high-pressure extraction technology, and the promotion of enzymatic hydrolysis to alter the sensitization of the allergens. In the future, the application of HPP technology as a pretreatment step for raw food materials may contribute to the development of food products with low or no allergenic ingredients, which then can effectively reduce the concern for consumers with allergies, reduce the risk of mistaken ingestion, and reduce the overall incidence of allergic reactions from food.

Allergen-specific immunoglobulin, histamine and T-cell responses induced by soybean glycinin and β-conglycinin in BALB/c mice of oral sensitisation

Glycinin and β-conglycinin are major soybean allergens involved in food hypersensitivity. However, the mechanism of immune responses induced by glycinin and β-conglycinin has not been fully understood. Balb/c mice were oral sensitised with different doses (0.1, 1.0 and 10 mg/day) of soybean glycinin and β-conglycinin for five weeks. Allergen-specific immunoglobulin (Ig), serum histamine and T-cell responses were tested to assess the allergenic activity of glycinin and β-conglycinin. Mice sensitised with 0.1 or 1.0 mg/day allergens induced high levels of specific IgE, IgG1 and serum histamine compared with mice treated with saline. Furthermore, specific T-cell proliferation and significant up-regulation of interleukin (IL)-4, IL-5 and interferon (IFN)-γ were observed in splenocytes from mice gavaged with 0.1 or 1.0 mg/day soybean proteins. Low doses of glycinin or β-conglycinin can induce allergic reactions in BALB/c mice, which might be associated with increased IgE and cytokine production.

Dissection of the IgE and T-cell recognition of the major group 5 grass pollen allergen Phl p 5

The major timothy grass pollen allergen Phl p 5 belongs to the most potent allergens involved in hay fever and asthma. This study characterized immune-dominant IgE- and T-cell-recognition sites of Phl p 5. Seven peptides, P1 to P7 with a length of 31 to 38 amino acids that spanned the Phl p 5 sequence, were synthesized, characterized by circular dichroism spectroscopy, and tested for IgE reactivity, basophil activation, and T-cell reactivity. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Phl p 5 or cross-reactive allergens from different grass species. Peptide-specific antibodies were tested for the capability to inhibit IgE reactivity to Phl p 5 and allergen-induced basophil activation of patients with allergy. The peptides exhibited no secondary structure and showed no IgE reactivity or relevant allergenic activity, indicating that Phl p 5 IgE epitopes are conformational. Except for P3, peptide-specific IgG antibodies blocked IgE binding to Phl p 5 of patients with allergy and cross-reacted with temperate grasses. IgE inhibition experiments and molecular modeling identified several clustered conformational IgE epitopes on the N- as well as C-terminal domain of Phl p 5. P4, which stimulated the strongest T-cell and cytokine responses in patients, was not part of the major IgE-reactive regions. Our study shows an interesting dissociation of the major IgE- and T-cell-reactive domains in Phl p 5 which provides a basis for the development of novel forms of immunotherapy that selectively target IgE or T-cell responses.

Cloning, expression in E. coli and immunological characterization of Par j 3.0201, a Parietaria pollen profilin variant

Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.

New method of screening allergenic components from Shuanghuanglian injection: With RBL-2H3/CMC model online HPLC/MS system

Shuanghuanglian (SHL) injection is a traditional Chinese medicine (TCM) injection widely used in China to treat influenza, tonsillitis, bronchitis, and faucitis. Adverse drug reactions (ADR) of SHL injection, majorly manifested as allergic reactions, were among the leading causes of death from TCM injection. In this study, an RBL-2H3/CMC online LC/MS system was established to screen and identify allergenic components in SHL injection, by which Baicalin was identified as a potential allergenic component. Allergenic activities of baicalin were investigated in RBL-2H3 cell degranulation and β-hexosaminidase release tests in vitro. Our results showed that baicalin dose-dependently induced RBL-2H3 cell degranulation and β-hexosaminidase release in the range of 0–50μg/ml. The RBL-2H3/CMC online LC/MS system developed in this study may potentially be used to screen allergenic components in other TCM injections.

Allergen immobilisation and signal amplification by quantum dots for use in a biosensor assay of IgE in serum

The production of biosensors for point of care diagnostics usually requires the immobilisation and storage of protein (for example, antigen or antibody) on a sensor surface, in a manner that retains a high degree of activity and low levels of non-specific binding. These characteristics have been assessed for polymer immobilised antigens (allergens) using an IgG binding assay and demonstrated further by assay with serum containing reactive IgEs.The activity of allergens immobilised on sensor chips using copoly(DMA–NAS–MAPS) and a spotting technique, as well as the specificity of their binding interactions with cognate immunoglobulins was assessed using Dual Polarisation Interferometry (DPI). The data obtained indicate that the allergens studied remain stable over long periods of time (at least 114 days). This performance compared favourably with other immobilisation methods. Allergen coated chips were tested in an anti-casein IgE assay using human serum from allergic and non-allergic donors. Detection of both total Ig and specific IgE was demonstrated using a secondary anti-IgE antibody. Furthermore, optical signal enhancement with streptavidin conjugated quantum dots was shown to yield responses for samples below 0.84ng/mL (0.35KU/L) of IgE, which overlap with the industrial quasi-standard ImmunoCAP® and is the clinically relevant threshold used to classify serum samples from allergic individuals.

Allergenic relevance of nonspecific lipid transfer proteins 2: Identification and characterization of Api g 6 from celery tuber as representative of a novel IgE-binding protein family

Apium graveolens represents a relevant food allergen source linked with severe systemic reactions. We sought to identify an IgE-binding nonspecific lipid transfer protein (nsLTP) in celery tuber. A low molecular weight protein exclusively present in celery tuber was purified and designated Api g 6. The entire protein sequence was obtained by MS and classified as member of the nsLTP2 family. Api g 6 is monomeric in solution with a molecular mass of 6936 Da. The alpha-helical disulfide bond-stabilized structure confers tremendous thermal stability (Tm > 90°C) and high resistance to gastrointestinal digestion. Endolysosomal degradation demonstrated low susceptibility and the presence of a dominant peptide cluster at the C-terminus. Thirty-eight percent of A. graveolens allergic patients demonstrated IgE reactivity to purified natural Api g 6 in ELISA and heat treatment did only partially reduce its allergenic activity. No correlation in IgE binding and limited cross-reactivity was observed with Api g 2 and Art v 3, nsLTP1 from celery stalks and mugwort pollen. Api g 6, a novel nsLTP2 from celery tuber represents the first well-characterized allergen in this protein family. Despite similar structural and physicochemical features as nsLTP1, immunological properties of Api g 6 are distinct which warrants its inclusion in molecule-based diagnosis of A. graveolens allergy.

Recombinant house dust mite allergens

House dust mites (HDM) are a globally important source of allergen responsible for the sensitization of more than 50% of allergic patients. Specific immunotherapy with HDM extracts is effective but allergen extracts cannot be fully standardized and severe side-effects can occur during the protracted course of treatment. The introduction of molecular biological techniques into allergy research allowed the indentification of more than 20 groups of HDM allergens. Recombinant HDM allergens can be produced in defined concentrations and consistent quality and allow the development of vaccines for HDM allergy with reduced allergenic activity and retained immunogenicity. The immunotherapy trials in pollen allergic patients with recombinant pollen allergens/hypoallergenic allergen derivatives have shown that this treatment is effective and indicated that recombinant HDM vaccines might improve immunotherapy of HDM allergic patients. Here we report the steps for the development of vaccines for HDM allergy. After selection of the most prevalent HDM species, the panel of allergens to be included into a therapeutic vaccine for HDM allergy needs to be determined. HDM allergens with high IgE-binding frequency and clinical relevance will be modified into hypoallergenic variants and evaluated for their allergenic activity and immunogenicity. Derivatives with reduced allergenic activity but with retained immunogenicity would be good candidates for a HDM vaccine for safe and efficient immunotherapy.

Mutational analysis of immunodominant epitopes of caprine β-casein recognized by IgE antibodies from patients allergic to goat’s milk and tolerant to cow’s milk

Background Several cases of allergy to goat’s milk (GM) without allergy to cow’s milk (CM) have been reported. GM-allergy has also been reported in CM-allergic children successfully treated with oral immunotherapy. We previously demonstrated that IgE antibodies from GM-allergic/CM-tolerant patients recognize the caprine b-casein (bcap) without cross-reacting with the bovine b-casein (bbov) despite a high sequence identity (91%). We aimed in the present work to identify the critical amino acids in the non-crossreactive IgE-binding epitopes of bcap. Methods Using site-directed mutagenesis, recombinant bcap was modified by performing residue substitutions with the corresponding amino acids found in bbov. The IgEbinding capacity of the different modified bcap was then evaluated with sera from 9 GM-allergic/CM-tolerant patients and 9 CM-allergic patients. The specificity of murine monoclonal antibodies (mAb) raised against caprine caseins was also analyzed in order to further characterize non-cross-reactive epitopes. The allergenic activity of recombinant bcap was finally assessed by degranulation tests of RBL cells passively sensitized with human IgE antibodies. Results The substitutions A55T/T63P/L75P in the N-terminal part and P148H/S152P in the C-terminal part of bcap induced the greatest decrease of IgE-reactivity of GMallergic/CM-tolerant patients toward the caprine allergen. The threonine 63 was found to be particularly critical, as confirmed by the specificity of mAb SCB1D, whose ability to bind bcap was abolished by the substitution T63P. The recombinant bcap containing the five substitutions was unable to induce the degranulation of RBL cells passively sensitized with IgE from GM-allergic/CM-tolerant patients but was still fully allergenic when testing sera from CM-allergic patients. Conclusion