Major Shrimp Allergen Research Articles

Penaeus monodon tropomyosin induces CD4 T-cell proliferation in shrimp-allergic patients

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

Gastrointestinal Digestion and Absorption of Pen j 1, a Major Allergen from Kuruma Prawn,Penaeus japonicus

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivo techniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed via the gastric mucosa prior to its digestion in the intestines.

Mapping IgE binding epitopes of major shrimp (Penaeus monodon) allergen with immunoinformatics tools

Tropomyosin, a myofibrillar protein, was described as the major allergen in crustaceans. The objective of this study was to screen and identify the linear IgE epitopes of tropomyosin in Penaeus monodon. Three immunoinformatics tools were used to predict the potential epitopes and the resultant epitopes were confirmed by dot-blot inhibition with sera from shrimp allergic subjects. As the result, 10 peptides were predicted and assembled by solid-phase synthesis. Eight epitopes were identified by dot-blot inhibition test, among which peptide 4 and 8 had not been reported by previous researches. Moreover, peptide 6 and 10 had IgE binding capability with all sera, while peptide 5, 8 and 9 could bind with 80% of the sera. Amino acid analysis demonstrated that Y, E, R, F and S presented more frequently in epitopes. Although further research should be done, it seems that immunoinformatics-driven epitope prediction can benefit epitope identification with high accuracy and efficiency.

Pulsed Ultraviolet Light Reduces Immunoglobulin E Binding to Atlantic White Shrimp (Litopenaeus setiferus) Extract

Pulsed ultraviolet light (PUV), a novel food processing and preservation technology, has been shown to reduce allergen levels in peanut and soybean samples. In this study, the efficacy of using PUV to reduce the reactivity of the major shrimp allergen, tropomyosin (36-kDa), and to attenuate immunoglobulin E (IgE) binding to shrimp extract was examined. Atlantic white shrimp (Litopenaeus setiferus) extract was treated with PUV (3 pulses/s, 10 cm from light source) for 4 min. Tropomyosin was compared in the untreated, boiled, PUV-treated and [boiled+PUV]-treated samples, and changes in the tropomyosin levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE binding of the treated extract was analyzed via immunoblot and enzyme-linked immunosorbent assay (ELISA) using pooled human plasma containing IgE antibodies against shrimp allergens. Results showed that levels of tropomyosin and IgE binding were reduced following PUV treatment. However, boiling increased IgE binding, while PUV treatment could offset the increased allergen reactivity caused by boiling. In conclusion, PUV treatment reduced the reactivity of the major shrimp allergen, tropomyosin, and decreased the IgE binding capacity of the shrimp extract.

Studies on the immunoreactivity of the major allergen tropomyosin in mantis shrimp (&lt;I&gt;Squilla oratoria&lt;/I&gt;)

Crustacean is one of the eight kinds of allergen sources in coastal areas,which causes the IgE-mediated hypersensitive reactions with clinical manifestations including urticaria,angioedema,asthma,and even fatal anaphylaxis. Tropomyosin (TM) is the major allergen of decapod crustaceans with highly conserved amino acid sequences. The purpose of this study is to confirm whether TM is a major cross-reactive allergen in mantis shrimp (Squilla oratoria) which is taxonomically distinct from decapods and largely consumed as a delicacy in China. Quantification of TM in different species of crustaceans was also conducted. Muscle sample from mantis shrimp was homogenized with phosphate buffer to prepare heated extracts and separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A protein band with molecular mass of about 36 ku was detected by IgE-immunoblotting by all of the sera with crustacean allergy,suggesting it is the major allergen of mantis shrimp. This protein was further purified to homogeneity by acetone powder preparation,isoelectric point precipitation,ammonium sulfate fractionation (40%-60% saturation),and heat treatment. Purified protein was demonstrated to be TM by Western blot using polyclonal antibody against TM from Chinese mitten crab (Eriocheir sinensis). Heated extracts from other crustaceans including mud crab (Scylla serrata),Pacific white shrimp (Penaeus vannamei) and short necked clam (Venerupis variegata) were also prepared,and the cross-reactivity of TM from mantis shrimp with TMs from these crustaceans was confirmed by inhibition immunoblotting using polyclonal antibody against TM and inhibition ELISA using sera with crustacean allergy. Quantification by ELISA using polyclonal antibody against TM revealed that TM content in mantis shrimp muscle is much lower than that in Pacific white shrimp and mud crab muscle,which was about 45 times lower in mantis shrimp muscle than that in Pacific white shrimp muscle. However,its allergenicity seems equivalent to other decapod crustaceans as showed by inhibition ELISA using sera with crustacean allergy. This may be due to the degradation of TM in mantis shrimp by its endogenous serine proteinases and cathepsins which destroyed the IgG-binding epitope but not the IgE-binding epitopes. In conclusion,this study demonstrated that allergenicity of mantis shrimp is almost equivalent to decapods and TM is the major allergen in mantis shrimp with high cross-reactivity to decapod crustaceans.

Identification of the major allergen in greasy-back shrimp (Metapenaeus ensis) by MALDI-TOF-MS

In order to better understand shrimp allergen, some basic characters of the major allergen of greasy-back shrimp (Metapenaeus ensis) were investigated. The major allergen was extracted and separated, and its peptide mass fingerprint (PMF) was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After screening in the NCBI database with Mascot searching engine, the results indicated that the major allergen of greasy-back shirmp was muscle tropomyosin. Database matching search showed that the top protein matched, i.e. the tropomyosin from giant tiger prawn (Penaeus monodon), had a Mowse value of 268. In addition, there were 27 queries matched with the allergen in greasy-back shirmp with an amino acid sequence coverage value of 65%. The matching scores and the sequence coverage values were also high with tropomyosins of other invertebrates, including Tyrophagus putrescentiae and Lepisma saccharina. These results indicated that the allergen of Metapenaeus ensis had high homology with other crustacean allergens, and provided molecular explanations for the high cross-reactivity of the major allergens between crustaceans and some other invertebrates.

Adsorption of tropomyosin from pink shrimp ( Pandalus eous) on stainless steel surface

Cross contamination of allergen to other food products is a serious problem in the food plant where many products shared the same equipment or processing lines. In order to decide a suitable cleaning method, understanding on adsorption of allergen onto the food-contact surface is required. In this study, the adsorption behavior of tropomyosin, a major allergen in shrimp, on stainless steel surface was investigated using shrimp extract as a sample food material. It was found that tropomyosin was adsorbed favorably on stainless steel surface than other proteins in the shrimp extract. Furthermore, the adsorption of proteins in the shrimp extract, including tropomyosin, was hardly removed by water.

Measurement of IgE antibodies to shrimp tropomyosin is superior to skin prick testing with commercial extract and measurement of IgE to shrimp for predicting clinically relevant allergic reactions after shrimp ingestion

Shrimp is a frequent cause of food allergy. Tropomyosin is the major allergen in shrimp, and it shares homology to tropomyosins from other crustaceans, dust mites, cockroach, and parasites. The aim of this study was to determine the value of detection of IgE to shrimp tropomyosin in the diagnosis of shrimp allergy. We have studied 35 patients with asthma, rhinitis, or both who were sensitized to Dermatophagoides pteronyssinus. All subjects underwent skin prick testing in addition to double-blind, placebo-controlled food challenges (DBPCFC); oral open challenges; or both with shrimp. Measurements of IgE to shrimp and shrimp tropomyosin were carried out by means of CAP and chimeric ELISA, respectively. Oral challenges confirmed the diagnosis of shrimp allergy in 7 patients. IgE measurement to shrimp tropomyosin was positive in 71.4% of the patients with shrimp allergy. Of the 28 patients without shrimp allergy, only 7.1% (2/28) had IgE to shrimp tropomyosin compared with 25% (7/28) who had IgE to shrimp and 35.7% (10/28) who had positive skin prick test responses to shrimp. Sensitivity was similar for all 3 methods (71.4%); in contrast, specificity of IgE to shrimp tropomyosin (92.8%) was greater than that of IgE to shrimp (75%) and skin prick testing (64.2%). With regard to diagnostic efficiency, measurement of IgE to shrimp tropomyosin was superior to measurement of IgE to shrimp and skin prick testing (88.5%, 74.2%, and 65.7%, respectively). Use of measurements of IgE to shrimp tropomyosin provided added value to the diagnosis of shrimp allergy.

The effect of simulated gastrointestinal digestion on shrimp Penaeus vannamei allergenicity

Pen a 1 is a major shrimp allergen that induces an allergy. The aim of this study is to assess the effect of simulated gastrointestinal fluids on the allergenicity of Pen a 1. Purified Pen a 1 from shrimp (Penaeus vannamei) was subjected to digestion in SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) for a set time. The allergenicity of digestive Pen a 1 was analyzed by immunoblotting and Ci-ELISA, using pool sera from patients with shrimp specific IgE. The results showed that Pen a 1 exhibited a decrease in allergenicity with increasing digestion time in the SGF and SIF. However, Pen a l exhibited strong resistance to digestive fluids, and all yielded fragments (33 kD, 23 kD, and 14 kD) showed allergic activity. Therefore, anti-digestion may be an important factor for Pen a 1 to induce an allergy.

Myosin light chain is a novel shrimp allergen, Lit v 3

Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. Few options are available for treatment, and avoidance is the only therapy recommended. We sought to identify relevant crustacean allergens for use as diagnostic and safe immunotherapeutic agents for subjects with shellfish allergy. Thirty-eight patients were selected with immediate allergic reactions to shrimp and increased shrimp-specific serum IgE levels. One-dimensional and 2-dimensional electrophoresis of shrimp extracts were followed by IgE immunoblotting. Protein identification was done with matrix-assisted laser desorption/ionization-mass spectrometry and Edman sequencing. A cDNA library was generated from white pacific shrimp (Litopenaeus vannamei) and screened with primers designed on the basis of internal sequences obtained from 2-dimensional tryptic digests. Full-length cDNA clones were isolated from the library and sequenced. Recombinant protein was expressed and tested with sera from patients with shrimp allergy. Immunoblotting demonstrated IgE binding to a 20-kDa shrimp protein by 21 (55%) of 38 sera. Tryptic digestion of the protein followed by matrix-assisted laser desorption/ionization-mass spectrometric analysis and Edman sequencing identified it as a myosin light chain (MLC). Screening of the shrimp cDNA library resulted in isolation of a novel protein cDNA. Open reading frame translation provided the amino acid sequence of a new allergenic shrimp protein with high similarity to Bla g 8 (cockroach MLC). Recombinant protein was recognized by 17 patients, confirming the allergenicity of shrimp MLC. We have identified and cloned a new major shrimp allergen, Lit v 3.0101, an MLC protein.

High level expression, purification and physico- and immunochemical characterisation of recombinant Pen a 1: A major allergen of shrimp

Well-characterised and immunologically active recombinant allergens are of eminent importance for improvement of diagnostic tools and immunotherapy of allergic diseases. The use of recombinant allergens has several advantages such as the more precise quantification of the active substance compared to allergen extracts and the reduced risk of contamination with other allergenic proteins compared to purified natural allergens. Optimised standard protocols for expression and purification and a detailed physico-chemical characterisation of such recombinant allergens are necessary to ensure consistent quality and comparability of results obtained with recombinant material. In this study the major allergen Pen a 1 of brown shrimp (Penaeus aztecus) was expressed in E. coli and purified in two steps by immobilised metal chelate-affinity chromatography (IMAC) and size-exclusion chromatography. Identity and purity were verified with N-terminal sequencing and peptide mass fingerprinting. Circular dichroism and NMR-spectroscopy indicated an alpha-helical flexible structure of rPen a 1 which is in accordance with the known structure of tropomyosins. Finally, the recombinant allergen proved to be immunologically reactive in IgE Western blot analysis and ELISA. This study provides a protocol for the preparation of recombinant shrimp tropomyosin in standardised quality.

Quantitative Sandwich ELISA for the Determination of Tropomyosin from Crustaceans in Foods

The ubiquitous muscle protein tropomyosin has been identified as the major shrimp allergen and is suggested to be a cross-reacting allergen. Previously, only a few methods for the detection of tropomyosin in food have been published. A quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of tropomyosin from crustaceans in foods has been developed and validated. A polyclonal rabbit antitropomyosin capture antibody and the biotinylated conjugate of the same antibody for detection were the basis for the ELISA, which was specific for crustaceans. The ELISA was able to quantitate tropomyosin in various food matrixes, had a detection limit of 1 microg/g, and cross-reacted to some extent with cockroach. Recoveries ranged from 63 to 120%, and the intra and interassay coefficients of variation were <6 and <14%, respectively.

Influence of ultrasonic treatment on the allergenic properties of Shrimp (Penaeus vannamei) Allergen

The present study was undertaken to determine whether high intensity ultrasound could reduce the allergic properties of shrimp allergens. Reducing the allergenic properties of these allergens will be beneficial to allergic individuals. Samples of shrimp protein extract and shrimp muscle were treated by high-intensity ultrasound with water bathing at 0℃ or 50℃ for different time periods. The treated and untreated samples were then analyzed by SDS-PAGE, Western blots and competitive inhibition ELISA (Ci-ELISA) to determine the shrimp allergenicity. The results show that high-intensity ultrasound has no effect on allergenicity when the extracts were treated at 0℃. However, a significant decrease was observed in the level of the major shrimp allergen, Pen a 1, when the samples were treated at 50℃. In the determination of allergenicity with Ci-ELISA, a reduction in IgE binding was also observed.

Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus

Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen-specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp-specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody-binding capacity for specific immunotherapy. The aim was to clone, express and characterize a full-length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE-mediated mediator release. Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5' RACE) was performed to obtain full-length cDNA coding for Pen a 1. Using a gene-specific primer, PCR was performed and full-length cDNA was cloned and sequenced. Recombinant His-tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody-binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE-mediated type I allergy. The deduced amino-acid sequence was 284 residues long and amino-acid sequence identities with allergenic and non-allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had alpha-helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild-type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation. Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component-resolved diagnosis and the generation of modified shrimp tropomyosin for allergen-specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp extract in combination with cholera toxin as adjuvant may be a suitable model to study shrimp allergy.

Reduction of allergenic properties of shrimp (Penaeus Vannamei) allergens by high intensity ultrasound

The tropomyosin fraction of shrimp proteins is potentially responsible for an allergic reaction in individuals with a genetic predisposition to allergy. However, there are no efficient and safe methods to reduce its allergenicity. High-intensity ultrasound is known to change the structure of proteins. The aim of this study was to assess the effect of high-intensity ultrasound on the IgE-binding capacity of shrimp protein extracts (PE) and of major shrimp allergen Pen a 1(tropomyosin). Peeled shrimp (Penaeus vannamei) muscle was sealed in a plastic bag and treated with high-intensity ultrasound (30 Hz, 800 W) for 1.5 h at 0 and 50 °C, respectively; others were treated with boiling water for 15 min. Then PE and Pen a 1 were made with treated shrimp muscle. The allergenicity of PE and purified allergen from different-treated shrimp were analyzed by the enzyme allergosorbent test (EAST) and competitive inhibition ELISA (Ci-ELISA) using sera of 15 shrimp-allergic patients. PE from high-intensity ultrasonic treatment at 50 °C (treated 2) shrimp was 2.2-fold, 2.5-fold lower respectively than that of untreated shrimp and high-intensity ultrasonic treatment at 0 °C (treated 1) shrimp. While PE from heated (treated 3) shrimp was 1.2-fold lower than that of untreated shrimp. The (Concentration of the different-treated sample that inhibit 50% of the IgE binding to coated untreated sample) IC50 ratios of Pen a 1 prepared from treated 2 shrimp, treated 1 shrimp and treated 3 shrimp to untreated shrimp were 1/15, 1/1.1 and 1/1, respectively. The results suggest that high-intensity ultrasound at 50 °C may reduce the allergenicity of shrimp.

Reduced Allergenic Potency of VR9-1, a Mutant of the Major Shrimp Allergen Pen a 1 (Tropomyosin)

The major shrimp allergen, tropomyosin, is an excellent model allergen for studying the influence of mutations within the primary structure on the allergenic potency of an allergen; Pen a 1 allows systematic evaluation and comparison of Ab-binding epitopes, because amino acid sequences of both allergenic and nonallergenic tropomyosins are known. Individually recognized IgE Ab-binding epitopes, amino acid positions, and substitutions critical for IgE Ab binding were identified by combinatorial substitution analysis, and 12 positions deemed critical were mutated in the eight major epitopes. The mutant VR9-1 was characterized with regard to allergenic potency by mediator release assays using sera from shrimp-allergic subjects and sera from BALB/c, C57BL/6J, C3H/HeJ, and CBA/J mice sensitized with shrimp extract using alum, cholera toxin, and Bordetella pertussis, as adjuvants. The secondary structure of VR9-1 was not altered; however, the allergenic potency was reduced by 90-98% measuring allergen-specific mediator release from humanized rat basophilic leukemia (RBL) cells, RBL 30/25. Reduced mediator release of RBL-2H3 cells sensitized with sera from mice that were immunized with shrimp extract indicated that mice produced IgE Abs to Pen a 1 and to the same epitopes as humans did. In conclusion, data obtained by mapping sequential epitopes were used to generate a Pen a 1 mutant with significantly reduced allergenic potency. Epitopes that are relevant for human IgE Ab binding are also major binding sites for murine IgE Abs. These results indicate that the murine model might be used to optimize the Pen a 1 mutant for future therapeutic use.

Recombinant tropomyosin from Penaeus aztecus (rPen a 1) for measurement of specific immuno‐ globulin E antibodies relevant in food allergy to crustaceans and other invertebrates

Immunoglobulin E (IgE)-mediated food allergy to crustaceans and mollusks is relatively common and affected individuals typically react to a range of different species. The only known major allergen of shrimp was first described over 20 years ago and later identified as the muscle protein tropomyosin. This protein may be useful as a defined and relevant diagnostic marker for allergic sensitization to invertebrate foods. In order to generate an assay reagent suitable for this purpose, tropomyosin from the shrimp Penaeus aztecus (Pen a 1) was produced as a recombinant protein in Escherichia coli and characterized with respect to IgE antibody binding properties in comparison to natural shrimp tropomyosin. Hexahistidine-tagged rPen a 1 accumulated as a predominantly soluble protein in the E. coli expression host and a two-step chromatographic procedure provided a high yield of pure and homogeneous protein. rPen a 1 displayed chromatographic and folding characteristics similar to those of purified natural shrimp tropomyosin. Serum preincubation with serial protein dilutions revealed similar capacity of recombinant and natural tropomyosin to compete with immobilized shrimp extract for IgE binding. rPen a 1 was further shown to extensively and specifically compete for IgE binding to extracts of other crustacean species, house dust mite and German cockroach.

An animal model for shrimp allergy: The influence of genetic background and adjuvant on the allergen-specific IgE response

Rationale Shrimp allergy is characterized by severe symptoms, yet no specific immunotherapy (SIT) is available due to expected severe adverse reactions. Our aim was to develop a mouse model of shrimp allergy that mimics the immune response of human shrimp-allergic individuals. Methods Mice of the strains Balb/c, C3H/HeJ, C57Bl/6J, and CBA/J were sensitized with extract of brown shrimp (Penaeus aztecus) using cholera toxin (oral), alum (i.p.), and B. pertussis (i.p.) as adjuvants. The IgE responses were determined by sensitizing rat basophilic leukemia (RBL-2H3) with mouse sera and measuring the beta-hexosaminidase release upon allergen challenge with shrimp extract and recombinant Pen a 1, the major shrimp allergen. Results Of the four mouse strain tested the strongest IgE response was observed in C3H/HeJ and CBA/J mice, with some activity in sera from sensitized Balb/c mice; C57Bl/6J mice did not produce appreciable amounts of specific IgE antibodies with any of the adjuvants used. Alum and cholera toxin were the most effective adjuvants to induce IgE antibodies to shrimp extract, however, only C3H/HeJ and CBA/J mice that received cholera toxin as adjuvant produced IgE antibodies to Pen a 1 as measured by mediator release assay. Conclusions Sensitizing C3H/HeJ orally with shrimp extract using cholera toxin as adjuvant produces an immune response that is characterized by high amounts of specific IgE to shrimp. In comparison with CBA/J mice, only C3H/HeJ produced Pen a 1-specific IgE with both cholera toxin and alum as adjuvants, and, thus, C3H/HeJ mice plus cholera toxin may be to better model to study shrimp allergy.

IgE and IgG antibody response to natural and recombinant Pen a 1 measured by ELISA and mediator release assay

Rationale To assess mouse reactivity to the major shrimp allergen, nPen a 1 and rPen a 1 were used to analyze murine IgG and IgE antibody responses by ELISA, and mediator release in rat basophilic leukemia (RBL) cells. Methods C3H/HeJ mice were orally immunized with shrimp extract, nPen a 1, and rPen a 1 (0.1–10ug) using cholera toxin as adjuvant. IgG and IgE responses were quantified by ELISA. RBL cells were sensitized with IgE-containing mouse sera; mediator release was measured after challenging the cells with Pen a 1. Results Higher doses of shrimp extract, nPen a 1, and rPen a 1 stimulated greater IgG and IgE antibody production. rPen a 1 induced the highest antibody response followed by nPen a 1 and shrimp extract, respectively. Comparison of 10ug rPen a 1 to 10ug shrimp extract demonstrated a two-fold greater IgG response and a three-fold greater IgE response. However, 0.1ug of rPen a 1 induced a higher IgE response than 1.0ug rPen a 1. Serum from Pen a 1 immunized mice also reacted to crawfish, snowcrab, and lobster extracts. In the RBL assay, Pen a 1 showed comparable allergenic potency as shrimp; this is similar to shrimp allergy in humans. Conclusions This study suggests that rPen a 1 yields an antibody response superior to both shrimp extract and nPen a 1 in mice. Pen a 1 accounts for most of the allergenic potency of shrimp. The data suggests this model is a suitable tool to study novel immunotherapy treatments for shrimp allergy.

Comparison of IgE reactivity of recombinant and natural pen a 1 by RAST and mediator release

Rationale Pen a 1 is the major shrimp allergen of Penaceus aztecus and has been identified as the muscle protein tropomyosin. It is responsible for the majority of IgE-mediated hypersensitivity to shrimp. Since recombinant proteins may be useful in diagnosis and treatment of food allergic individuals, recombinant Pen a 1 (rPen a 1) was cloned and expressed in E. coli. This study was performed to compare the immunological activity of rPen a 1 and natural Pen a 1 (nPen a 1) by RAST and mediator release. Methods Sera from six shrimp-allergic individuals with positive skin tests to shrimp extract were tested. RAST was performed using discs coated with whole shrimp extract, nPen a 1 (purified by preparative SDS-PAGE), and rPen a 1 at different concentrations (10.0 - 0.01 μg per disc). Peanut-coated discs were used as controls. Mediator release was performed by sensitizing humanized rat basophilic leukemia cells with human IgE from shrimp-allergic individuals. Results All six of the shrimp-allergic sera had RAST reactivity at all concentrations to both nPen a 1 and rPen a 1. Non-allergic control sera did not show reactivity. Linear regression analysis demonstrated identical IgE antibody binding capacity of rPen a 1 and nPen a 1. This was confirmed by the mediator release assay. Conclusion The use of rPen a 1 may have a role in determining shrimp-allergic individuals. Since this study is small, further serological testing should be conducted to confirm these results and skin testing with rPen a 1 should be performed.