Identification of collagen as a new fish allergen

This study was intended to identify a high molecular weight allergen that had been detected in fish. Analyses by ELISA of five protein fractions prepared from bigeye tuna muscle showed that the high molecular weight allergen was contained in the myostromal protein fraction. Based on the results of SDS-PAGE, immunoblotting and amino acid analysis of the myostromal protein fraction, the high molecular weight allergen was judged to be collagen. Five of the eight patient sera used were found to react to the bigeye tuna collagen. In competitive ELISA inhibition experiments, the bigeye tuna collagen almost completely inhibited the IgE reactivity to the heated extracts from five species of fish, suggesting that collagen is commonly allergic regardless of fish species. However, no antigenic cross-reactivity was observed between collagens from fish and other animals.     

A comparative immunochemical analysis of allergoids and allergens]

In comparison with allergens having protein fragments with a molecular weight not exceeding 110 kD, allergoids have been found to consist of larger fragments with a molecular weight of 10-150 kD. Allergoids have less charged components than initial allergens and less antigenic components. Allergoids retain their capacity for stimulating the production of antibodies, specific to all antigenic components.     

Role of pepsin in modifying the allergenicity of bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) fish

The effect of pepsin digestion on the allergenicity of raw and thermally processed (boiled and fried) fish muscle extracts of two widely consumed fishes bhetki (Lates calcarifer) and mackerel (Rastrelliger kanagurta) was studied. Sere were collected from 110 patients who were hypersensitive to fish, as evidenced by their clinical history, symptoms and positive skin-prick test results. The various extracts after digestion with pepsin at different times of incubation were tested for specific IgE-binding activity by ELISA and immunoblotting with patients' sera. All the extracts of both the fishes retained their allergenicity as evidenced by ELISA and immunoblotting. In bhetki, maximum allergenicity was found in the pepsin-digested fried extract, whereas similar treatment decreased the allergenicity in fried mackerel. Results showed that raw as well as thermally processed allergens of both the fishes maintained strong allergenicity, even after digestion with pepsin for different time periods. The study revealed that the fish proteins played an important role in manifestation of allergy, due to their stable structure, which was retained even after pepsin and heat treatment.     

Phthalocyanine-dyed fibers adsorb allergenic proteins

Summary. Phthalocyanine (Pc)-dyed fiber is reported to reduce atopic symptoms in some patients when they use underwear made of the fiber. We investigated the adsorption of allergens on Pc-fiber. Pc-fiber trapped house dust/pollen/food allergens with varied molecular weight and pI. The adsorbed allergens were released in the presence of mild detergent. Pc-fiber did not change the molecular weight or disulfide bonding of the allergens. These observations imply that Pc-fiber is applicable as an “allergen trap” for a wide variety of products.     

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS–PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.     

Microarrayed allergens for IgE profiling

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.     

Hypoallergenic derivatives of major grass pollen allergens for allergy vaccination

Grass pollen-induced hay-fever and allergic asthma represent a major health problem in industrialized countries. Whereas the symptoms of these allergic conditions can be controlled by pharmacotherapy, specific immunotherapy vaccination is the only causative approach towards the treatment of these type 1 allergies. Specific immunotherapy is based on administration of increasing amounts of the disease-causing allergens in the form of allergen-containing extracts. However, the extracts used for immunotherapy consist of allergenic and non-allergenic components and may induce severe anaphylactic side-effects upon therapeutic administration. With recent developments in molecular biology of pollen allergens it has become feasible to produce modified hypoallergenic derivatives of recombinant allergens with abrogated or greatly reduced likelihood of anaphylactic side-effects as compared to extract-based treatments. We have demonstrated this concept through reducing the anaphylactic potential of major rye grass pollen allergens by introducing a few point mutations which leave the overall structural fold of the molecule unaltered. These modified forms are expected to make allergen-specific immunotherapy more widely used in the future.     

Protein fractions of the ova, embryos and skeletal muscles of the loach affecting the stability and activity of lactate dehydrogenase

When lactate dehydrogenase obtained from Misgurnus muscles and purified to the homogeneous state is incubated for 16 hat 38 degrees C, its activity lowers down to 10% of the initial value. Extracts of egg cells, embryos or skeletal muscles of the mentioned fish species added to the enzyme solution decrease considerably its inactivation. Proteins stabilizing the activity of lactate dehydrogenase are revealed in the supernatant liquid obtained after salting out of the above extracts with 60% sulphate ammonium saturation. These proteins are in fractions with the molecular weight below 45 kDa. Among proteins with the molecular weight 10 kDa there are polypeptides which exert an activation effect on lactate dehydrogenase. This effect is intensified with the presence of insulin.     

Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts

Background

German cockroach (GCr) allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.

Objective

Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.

Methods

Single chain fragment variable (scFv) antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human) coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.

Results

Chicken scFv’s generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv’s recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV) of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.

Conclusions

An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.     

Evidence that the castor bean allergens are the albumin storage proteins in the protein bodies of castor bean

The well characterized castor bean (Ricinus communis L.) allergens were identified as the low molecular weight albumin storage proteins in the matrix of the protein bodies in the endosperm. The methods of identification involved molecular weight estimation, amino acid composition, stability at 100° C, solubility in various solvents, gel electrophoresis, and immunological techniques. The finding explains the wide distribution of allergens in various seeds.     

Recombinant carp parvalbumin, the major cross-reactive fish allergen: a tool for diagnosis and therapy of fish allergy

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage lambda gt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the beta-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly alpha-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.     

Fractionation and immunochemical characterization of Prosopis juliflora pollen allergen

Prosopis juliflora pollen grain crude extract gave six different molecular weight fractions varied from 81,000 to 13,000 dalton on Sephadex G-100 gel filtration. The purity of fractions of Prosopis juliflora pollen extract were checked by polyacrylamide gel electrophoresis. The fraction had an molecular weight 20,000 dalton showed four absorption maxima whereas other fractions had single absorption maxima. Allergenic activity and nature of allergens were evaluated by in vitro Radioallergosorbent test and in vivo Passive Cutaneous Anaphylaxis test. All these tests indicated that most allergenic fractions were in the 20,000 molecular weight.     

Dermatophagoides farinae Allergens Diversity Identification by Proteomics

The most important indoor allergens for humans are house dust mites (HDM). Fourteen Dermatophagoides farinae allergens (Der f 1–3, 6, 7, 10, 11, 13–18, and 22) are reported although more than 30 allergens have been estimated in D. farinae. Seventeen allergens belonging to 12 different groups were identified by a procedure of proteomics combined with two-dimensional immunoblotting from D. farina extracts. Their sequences were determined by Edman degradation, mass spectrometry analysis, and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests, immunoblots, basophil activation test, and skin prick tests. Eight of them are the first report as D. farinae allergens. The procedure of using a proteomic approach combined with a purely discovery approach using sera of patients with broad IgE reactivity profiles to mite allergens was an effective method to investigate a more complete repertoire of D. farinae allergens. The identification of eight new D. farinae allergens will be helpful for HDM allergy diagnosis and therapy, especially for patients without response for HDM major allergens. In addition, the current work significantly extendedthe repertoire of D. farinae allergens.The house dust mites (HDM)¹ are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases (1). Extensive studies have been conducted to understand the biological, chemical, and structural properties of dust mite allergens. Most of the best characterized allergens are from dust mites Dermatophagoides pteronyssinus and D. farinae (Acari: Pyroglyphidae). Twenty-three groups of dust mite allergens are listed in the (IUIS) nomenclature data set, and 21 of them have been identified from Dermatophagoides spp (http://www.allergen.org/). There is an extreme diversity of dust mite allergens. Western blotting studies with human sera containing high levels of anti-mite IgE showed more than 32 bands with molecular weights ranging from 11 to greater than 100 kDa (2). Two groups of mite allergens (group 1 and 2) have been extensively studied. They are a 25-kDa cysteine protease and a 14-kDa epididymal protein, respectively. More than 80% of humans with house dust mite allergy mount an IgE response to the group 1 and more than 90% to the group 2 (3–6).The group 1 and 2 molecules are major allergens in HDMs but about 20% of patients do not have IgE antibody to the two group allergens (3). It has been found that there are also many other HDM allergens containing high IgE binding activity although these are present in low and variable concentrations in mite extracts (minor allergens), usually at less than 1% of the group 1 and 2 allergens (3). Allergens present in low amount in mite extracts, which can induce high titers of IgE, suggest that they are potent at low concentration. Another possibility is that the amount of allergen required to induce allergic responses in the airways is more than that required to induce IgE. It has been estimated that there are at least 30 allergens in the extracts of D. farinae by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) combined with autoradiography analysis (7). Two-dimensional (2-D) immunoblotting has been applied to study mapping of D. farinae mite allergens (7). Seven allergens including Der f 1, Der f 2, Der f 3, Der f 4, Der f 5, and 2 high molecular mass allergens, which share significant homologies with allergen Mag 3 from D. farinae and with a chitinase from prawn Penaeus japonicus, have been identified from the 2-D immunoblotting analysis (7). Up to now, 14 allergens from D. farinae have been named. Most of them are in the molecular weight range of 14 to 60 kDa. Given the extreme diversity of mite allergens, many investigations with novel allergen identification are still in progress or are yet to be undertaken. It is well known that many mite allergens are not identified on the basis of two possible reasons: (1) it is difficult to purify and characterize minor allergens because they present in low concentration in mite extracts; (2) some minor allergens are neglected because of their minor amount or abilities to only induced allergy to a minor population. It is necessary to develop efficient procedure with high accuracy and resolution to purify and characterize allergens from mite extracts. In this work, 17 allergens or their isoforms have been identified from the mite extracts of D. farinae by a procedure of proteomics combined with two-dimensional immunoblotting. Eight of them are the first to be reported as mite allergens.     

Identification of critical amino acids in the IgE epitopes of Ric c 1 and Ric c 3 and the application of glutamic acid as an IgE blocker

Background

The allergenicity of Ricinus communis L. (castor bean, Euphorbiaceae) is associated with components of its seeds and pollen. Castor bean allergy has been described not only in laboratory workers, but also in personnel working in oil processing mills, fertilizer retail, the upholstery industry and other industrial fields. In the present study, we describe the critical amino acids in the IgE-binding epitopes in Ric c 1 and Ric c 3, two major allergens of R. communis. In addition, we also investigate the cross-reactivity between castor bean and some air and food allergen extracts commonly used in allergy diagnosis.

Methodology/Principal Findings

The IgE reactivity of human sera from atopic patients was screened by immune-dot blot against castor bean allergens. Allergenic activity was evaluated in vitro using a rat mast cell activation assay and by ELISA. Cross-reactivity was observed between castor bean allergens and extracts from shrimp, fish, gluten, wheat, soybean, peanut, corn, house dust, tobacco and airborne fungal allergens. We observed that treatment of rat and human sera (from atopic patients) with glutamic acid reduced the IgE-epitope interaction.

Conclusions/Significance

The identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment.     

A novel grass pollen allergen mimotope identified by phage display peptide library inhibits allergen-human IgE antibody interaction

The aim of this study was to investigate the molecular basis of human IgE-allergen interaction by screening a phage-displayed peptide library with an allergen-specific human IgE-mimicking monoclonal antibody (mAb). A mAb that reacted with major grass pollen allergens was successfully identified and shown to inhibit human IgE-allergen interaction. Biopanning of a phage-displayed random peptide library with this mAb yielded a 12 amino acid long mimotope. A synthetic peptide based on this 12-mer mimotope inhibited mAb and human IgE binding to grass pollen extracts. Our results indicate that such synthetic peptide mimotopes of allergens have potential as novel therapeutic agents.     

Functional Interaction of Common Allergens and a C-type Lectin Receptor, Dendritic Cell-specific ICAM3-grabbing Non-integrin (DC-SIGN), on Human Dendritic Cells

Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the α1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le^x). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-α expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-α expression in MDDCs via, in part, Raf-1 signaling pathways.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

Purification and characterization of purine nucleoside phosphorylase from Proteus vulgaris.

Purine nucleoside phosphorylase was isolated and purified from cell extracts of Proteus vulgaris recovered from spoiling cod fish (Gadus morhua). The molecular weight and isoelectric point of the enzyme were 120,000 +/- 2,000 and pH 6.8. The Michaelis constant for inosine as substrate was 3.9 x 10(-5). Guanosine also served as a substrate (Km = 2.9 x 10(-5). However, the enzyme was incapable of phosphorylizing adenosine. Adenosine proved to be useful as a competitive inhibitor and was used as a ligand for affinity chromatography of purine nucleoside phosphorylase following initial purification steps of gel filtration and ion-exchange chromatography.     

A two-centre evaluation of the human organotypic skin explant culture model for screening contact allergens

Animal models are considered to be the "gold standard" for determining the potential contact allergenicity of low molecular weight chemicals. However, governmental regulations and ethical considerations limit the use of animals for such purposes. There is therefore a need for in vitro alternative models. The human organotypic skin explant culture (HOSEC) model is reported to be a promising alternative method for the predictive testing of contact allergens. The accelerated migration of Langerhans cells from the epidermis upon exposure to contact allergens is used to identify chemicals that are potentially capable of inducing a delayed-type hypersensitivity. In the study described in this paper, the model was further refined, and used, in two independent laboratories, to screen 23 low molecular weight compounds of known classification for their allergenicity. Each laboratory was able to accurately detect the contact allergens, despite small variations in the protocols used. However, the classification of dermal irritants, which have often been falsely classified as allergens, varied between the two laboratories. Despite the current limitations of the HOSEC model, the accuracy of the predictions made (sensitiser or non-sensitiser) compare favourably with classifications obtained with commonly used animal models. The HOSEC model has the potential to be developed further as an in vitro alternative to animal models for screening for contact allergens.     

家蚕30 kD特异性变应原的分析、纯化与质谱鉴定

以Coca's提取液分别提取到不同时期家蚕Bombyx mori的粗浸液,利用SDS-PAGE和Western blotting鉴定其特异性变应原,然后用DEAE-52离子交换层析及切胶纯化出30 kD的特异性变应原,再经MALDI-TOF在线联机分析,所得质谱数据进入网站搜索分析。结果显示：1～5龄家蚕均有20条左右蛋白带,其中 5龄家蚕有23条蛋白带,主带有11条（82、79、60、51、46、38、32、30、28、24和18 kD）。选用家蚕过敏患者阳性血清进行免疫印迹,1～4龄家蚕均显示出82和79 kD的特异性变应原;但只有5龄家蚕的30 kD蛋白为特异性变应原,通过离子交换层析和经切胶纯化出30 kD蛋白,再经MALDI-TOF-MS鉴定该蛋白为外膜蛋白。提示家蚕不同时期抗原成分有所变化,5龄家蚕新出现的30 kD蛋白为特异性变应原。