Shrimp Extract Research Articles

Generation of a comprehensive panel of crustacean allergens from the North Sea Shrimp Crangon crangon

BackgroundPublished data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies. MethodsThe North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP. ResultsSix recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract. ConclusionsWe identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies.

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.

Seafood hypersensitivity in mite sensitized individuals: is tropomyosin the only responsible allergen?

Seafood allergy has been related to mite sensitization, mainly mediated by the muscle protein tropomyosin. To determine the correlation between seafood hypersensitivity and mite sensitization (Dermatophagoides pteronyssinus and Chortoglyphus arcuatus, a highly prevalent storage mite in Spain) and to investigate the implication of tropomyosin in cross-reactivity. Patients from Northwest Spain were divided into 2 groups. The mite-seafood group contained 30 allergic mite individuals with a clinical history of food hypersensitivity. The mite group contained 40 individuals with positive skin prick test results to D pteronyssinus and C arcuatus but negative seafood test results. Specific IgE (sIgE) to whole mite and shrimp extracts, mite tropomyosin (rDer p 10), and shrimp tropomyosin (rPen a 1) were determined in each serum sample. Allergenic profiles were analyzed by immunoblot. Cross-reactivity studies were investigated by enzyme-linked immunosorbent assay and immunoblot inhibition studies. In the mite-seafood group, 71% of patients had positive sIgE results to shrimp and 55% of them to shrimp tropomyosin. A strong correlation was found between sIgE to shrimp tropomyosin and mite tropomyosin. Positive correlation was observed between sIgE to shrimp tropomyosin and severity of symptoms. In the mite group, none of the 20% of patients with sIgE to shrimp tested positive to shrimp tropomyosin. In the immunoblot inhibition experiment, the shrimp extract was totally inhibited by mite extract. These data suggest that primary sensitization is related to mite sensitization. Tropomyosin does not seem to be the main allergen involved in mite-seafood sensitization in mite sensitized individuals. High levels of sIgE to tropomyosin seem to be related to severity of symptoms.

Antimutagenicity and Antiproliferative Studies of Lipidic Extracts from White Shrimp (Litopenaeus vannamei)

An organic extract from fresh shrimp (Litopenaeus vannamei) was studied for antimutagenic and antiproliferative properties using Salmonella typhimurium tester strains TA98 and TA100 with metabolic activation (S9) and a cancer cell line (B-cell lymphoma), respectively. Shrimp extract was sequentially fractionated by thin layer chromatography (TLC) and each fraction was tested for antimutagenic and antiproliferative activities. Crude organic extracts obtained from shrimp reduced the number of revertants caused by aflatoxina B1, showing a dose-response type of relationship. Sequential TLC fractionation of the active extracts produced several antimutagenic and/or antiproliferative fractions. These results suggested that the lipid fraction of the tested species contained compounds with chemoprotective properties that reduce the mutagenicity of AFB1 and proliferation of a cancer cell line.

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.

Lack of neo-sensitization to Pen a 1 in patients treated with mite sublingual immunotherapy

BackgroundSome studies reported the possible induction of food allergy, caused by neo-sensitization to cross-reacting allergens, during immunotherapy with aeroallergens, while other studies ruled out such possibility.ObjectivesThe aim of this study was to evaluate the development of neo-sensitization to Pen a 1 (tropomyosin) as well as the appearance of reactions after ingestion of foods containing tropomyosin as a consequence of sublingual mite immunization.Materials and methodsSpecific IgE to Tropomyosin (rPen a 1) before and after mite sublingual immunotherapy in 134 subjects were measured. IgE-specific antibodies for mite extract and recombinant allergen Pen a 1 were evaluated using the immunoenzymatic CAP system (Phadia Diagnostics, Milan, Italy).ResultsAll patients had rPen a 1 IgE negative results before and after mite SLIT and did not show positive shrimp extract skin reactivity and serological rPen a 1 IgE conversion after treatment. More important, no patient showed systemic reactions to crustacean ingestion.ConclusionsPatients did not show neo-sensitization to tropomyosin, a component of the extract (namely mite group 10) administered. An assessment of a patient's possible pre-existing sensitisation to tropomyosin by skin test and/or specific IgE prior to start mite extract immunotherapy is recommended.Trial RegistrationThis trial is registered in EudraCT, with the ID number of 2010-02035531.

Effects of Boiling on the IgE‐Binding Properties of Tropomyosin of Shrimp (Litopenaeus vannamei )

The thermal stability and IgE binding of raw and boiled shrimp extracts and the tropomyosins (TM) have not been reported. In this study, we compare the stability of raw and boiled shrimp TM of Litopenaeus vannamei and evaluate how boiling may alter the allergenicity of L. vannamei. Extracts were prepared from raw and boiled shrimp and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. The IgE-binding of the extracts was determined by western-blot and competitive inhibition enzyme-linked immunosorbent assay (iELISA). The TM was then purified from raw and boiled shrimp, the secondary structures analyzed by circular dichroism (CD) spectroscopy, and the IgE binding compared by slot blot analysis. The soluble protein content decreased and the higher molecular weight proteins increased in the extracts from boiled versus raw shrimp. Similar IgE binding characteristics were seen by extracts when using western blot analysis. Although iELISA results showed that extracts from raw shrimp bound higher IgE than extracts from boiled shrimp, dot-blot assay demonstrates higher IgE binding to purified TM from boiled shrimp than raw shrimp. The purified TM had a typical alpha-helical secondary structure and the stability of boiled TM was lower than that of raw TM. Extracts from boiled shrimp produce lower IgE binding than extracts from raw shrimp, which suggest that boiling can be used as a tool in attempting to reduce shrimp allergenicity. However, the purified TM from boiled shrimp, which shows enhanced IgE binding over that of raw shrimp, may be a more effective antigen in diagnosing shrimp allergy through immunoassay.

Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously

Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus ( Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus ( Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37 kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei.

Selective allergy to Venus antiqua clam

Prevalence of self reported mollusc allergy ranges from 0.15% (1) up to 0.4% (2). As the reactions elicited can be severe, even life threatening, molluscs have been included in the list of potential food allergens that must appear mandatory on the labelling of foods (3, 4). In this article, we present a case of isolated food allergy to King Broderip clam (Venus antiqua). A 68 year-old non-atopic man with a history of chronic bronchitis and high blood pressure, who had never worked in the food industry, presented 15–30 min after the intake of canned King Broderip clams, palpebral oedema, scalp and palmo-plantar pruritus, with shortness of breath that required treatment with antihistamines and corticosteroids in an emergency room. Skin prick tests with commercial extracts of clam, shrimp, mussel, oyster and squid were negative (Laboratorios Leti, Barcelona, Spain). Prick–prick tests with canned mussel, razor, cockle and Pullet carpet shell (Tapes pullastra, another variety of clam) were negative, but positive (7 · 5 mm) for King Broderip clam (Venus antiqua). Total IgE was 245 kU/l, and specific IgE to clam (Ruditapes spp), crab, shrimp, mussel, tropomyosin (rPen a1) and house dust mites were negative (CAP Phadia, Upsala, Sweeden). Oral challenges performed with canned mussel, razor and Pullet carpet shell were negative. An oral challenge was carried out with the same commercial brand of canned King Broderip clam to assess the implication of the single species and the high specificity of the reaction. Thirty minutes after the intake of the last portion (cumulative dose of 52 g), the patient developed generalized urticaria, facial oedema and mild bronchospasm with normal blood pressure that subsided quickly after the administration of intramuscular epinephrine, inhaled salbutamol and intravenous corticosteroids and antihistamines. To rule out the possibility of allergy to preservatives, a food challenge with canned cockle from the same brand as the canned King Broderip clam and containing the same additives was performed and no reaction was elicited. The aforementioned allergological study suggests a specific IgE mediated reactivity toKing Broderip clam.We have not detected sensitisation to other shellfish andwehave evenbeen able todemonstrate oral tolerance to other molluscs including other clam species and to crustacea. The patient was advised to avoid all clam species because of the difficulty of differentiating them, but was allowed to eat the remaining shellfish. Later on, he tolerated shrimps, prawns and oysters. Mollusc allergy is frequently associated with crustacean allergy and in these cases, tropomyosin is the culprit panallergen (3). However, when isolated allergy to molluscs or to a single species occurs, tropomyosin does not seem to have a relevant role, as in our patient. In such cases, molluscs specific allergens or even species specific, may be involved (3, 5, 6), but they have been poorly defined. Unfortunately,we failed to identify the allergen/s involved in an immunoblotting assay. We present a patient with a selective food allergy to King Broderip clam (Venus antiqua), who tolerated other molluscs including the closely related Pullet carpet clam (Tapes pullastra), which therefore suggests the existence of specific allergen(s) in this clam species.

Comparison of Raw and Boiled Shrimp Extracts for Skin Prick Test at Different Storage Time

RATIONALE: The difference of stability between raw and boiled shrimp extracts has never been reported. The aim of the study was to compare the stability of raw and boiled shrimp extracts of two species; Penaeus monodon (seawater shrimp) and Macrobrachium rosenbergii (freshwater shrimp) at different storage time by using skin test reactivity.

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.

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.

Specific allergy to Penaeus monodon (seawater shrimp) or Macrobrachium rosenbergii (freshwater shrimp) in shrimp‐allergic children

Allergy to specific shrimp species has not been studied systematically by oral challenges. A comparison of allergy to different shrimp species, especially seawater or freshwater varieties treatment, would be useful in testing shrimp-allergic subjects. The aim of the study was to identify cases of specific allergy to seawater shrimp, Penaeus monodon (Pm), or freshwater shrimp, Macrobrachium rosenbergii (Mr), among shrimp-allergic children. Comparisons of skin tests using commercial and crude shrimp extracts plus the prick-to-prick (PTP) method were investigated. Sixty-eight children with a history of shrimp allergy and skin tests positive to shrimp were orally challenged to both shrimp species. Reactivity to skin prick tests using extracts of Pm (PmSPT), Mr (MrSPT), commercial shrimp (ComSPT), and PTP tests (PmPTP, MrPTP) was compared. Food challenges identified specific allergy to Pm and Mr in 17.65% and 23.53% of the subjects, respectively. Positive and negative challenges to both shrimp species were found in 47.06% and 11.76% of the subjects, respectively. Correlations between the mean weal diameter (MWD) from ComSPT-PmSPT, ComSPT-PmPTP, ComSPT-MrPTP, PmSPT-PmPTP and MrSPT-MrPTP, but not ComSPT-MrSPT, were observed. The MWD from PmSPT and PmPTP were significantly larger in patients with positive than negative challenges to P. monodon (P<0.05). There was a trend that MWD from MrSPT were larger in patients with positive than negative challenges to M. rosenbergii (P=0.058). In the Pm allergy group, PmSPT with an MWD of 30 mm provided 80% predictive probability for positive challenges. PmPTP and ComSPT with an MWD of 22.5 and 20 mm provided 95% predictive probability, respectively. In the Mr allergy group, MrSPT with an MWD of 30 mm provided 95% predictive probability. Specific allergy to Pm or Mr was confirmed by food challenges. SPT using crude extracts and the PTP test are useful tools for screening shrimp sensitization before a food challenge. The predictive probability of SPT is helpful where a food challenge is not feasible.

Comparison of Skin Prick Test to Crude Shrimp Extract with Prick-to-prick Skin Test to Cooked Shrimp for the Diagnosis of Shrimp Allergy in Children

Shrimp is a major cause of seafood sensitivity among Thai children. The aim of the study was to compare the diagnostic value of skin prick test (SPT) to crude shrimp extract with prick-to-prick skin test (PTP) to cooked shrimp using the outcome of oral food challenges as the gold standard.

COMPOSITION, ANTIOXIDATIVE AND OXIDATIVE STABILITY OF MUNGOONG, A SHRIMP EXTRACT PASTE, FROM THE CEPHALOTHORAX OF WHITE SHRIMP

ABSTRACT The chemical composition and antioxidative activity of mungoong, a shrimp extract paste from the cephalothorax of white shrimp (Litopenaeus vannamei), were studied. Mungoong contained 42.3% polyunsaturated and 29.59% saturated fatty acid. It was rich in C20:5 n‐3 (eicosapentaenoic acid) (4.31 g/100 g) and C22:6 n‐3 (docosahexaenoic acid) (7.07 g/100 g). Mungoong consisted of Na (15.3 g/kg) and Ca (8.07 g/kg) as the major minerals. Fe and Cu were found at very low content. Glutamine was the most abundant amino acid, while lysine, alanine and asparagine were predominant in mungoong. Mungoong water extract at a level of 1,000 ppm exhibited antioxidative activity in a β‐carotene–linoleic acid, lecithin liposome and comminuted fish model system. Antioxidative potential determined by 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) and 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging activities and ferric‐reducing antioxidant power was quite stable during 8 weeks of storage at 4C and room temperature (28–30C). Antioxidative activities determined by all assays remained constant within the first week (P &gt; 0.05). Thereafter, only slight decreases in activities were noticeable. During storage at both temperatures, mungoong's 2‐thiobarbituric acid (TBA) values increased during the first 2 weeks of storage (P &lt; 0.05), followed by the gradual decrease of 8 weeks. Nevertheless, no marked changes in TBA values were observed between samples kept at both storage temperatures. Thus, antioxidative peptides might contribute to the retardation of lipid oxidation of mungoong during the extended storage.PRACTICAL APPLICATIONSMungoong, which is an extract paste from the cephalothorax of white shrimp, was demonstrated to be a good source of protein, n‐3 fatty acids as well as minerals. Additionally, mungoong could serve as a novel natural antioxidant with high stability. Therefore, mungoong produced from shrimp‐processing by‐products can be used as a neutraceutical product. This will lead to the full utilization of shrimp‐processing waste and would reduce environmental problems associated with improper disposal of such wastes.

The use of raw or boiled crustacean extracts for the diagnosis of seafood allergic individuals

Seafood plays an important role in human nutrition and is responsible for severe hypersensitivity reactions. To evaluate how the cooking process may alter the in vivo and in vitro allergenicity of these extracts. Raw and boiled extracts of shrimps and 2 types of lobsters were manufactured. Boiled extracts were prepared after the raw material was boiled for 15 minutes in phosphate-buffered saline. Raw and boiled extracts were homogenized and extracted for 4 hours. Afterward, the extracts were centrifuged, dialyzed, filtered, and freeze-dried. Seventy-eight patients were skin prick tested with these raw and boiled extracts. Specific IgE against the 6 extracts was measured by enzyme-linked immunosorbent assay (ELISA). Immunoblots and ELISA inhibition studies were performed with a pool of sera. In vivo results showed that boiled extracts induced statistically significant larger wheals than raw extracts. More patients with positive results were also detected with boiled extracts. In vitro experiments by direct ELISA confirmed the in vivo results. The protein content in the boiled extracts decreased, and important differences were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-reactivity experiments showed that both types of extracts retained similar allergenic characteristics, even if the immunoblots revealed some differences in IgE binding. More patients were identified using boiled extracts of shrimp and American and spiny lobsters than with raw extracts. The wheal sizes of the skin test reactions and specific IgE levels were also significantly greater using boiled extracts. The use of boiled extracts seems to be more effective in diagnosing seafood allergy.

Sample clean-up by sol-gel immunoaffinity chromatography for determination of chloramphenicol in shrimp

The paper describes the characterisation and application of sol-gel columns prepared by entrapping anti-chloramphenicol (CAP) antibodies. Retention of CAP in the column was caused by specific interactions with the anti-CAP antibodies and not by non-specific adsorption to the sol-gel glass. After optimising important operation conditions, e.g. feeding medium, feeding flow-rate, elution medium, elution flow-rate and elution volume, the sol-gel columns were included in a clean-up procedure developed to determine CAP in shrimp. The selectivity of the columns was high enough to efficiently remove interfering matrix compounds. Due to the chromatographic conditions applied retention of cross-reacting substances in the immunoaffinity column did not pose a problem. CAP recovery of the analytical method was 68% with a relative standard deviation of 4% (n=4). In spite of applying highly complex shrimp extracts the columns could be used for clean-up of at least 12 samples. However, when detection of CAP is carried out with an UV detector the analytical method has a relatively poor sensitivity (LOD=1.8 ng/g, S/N=3). The most obvious way is to replace the UV detector by a detector based on an inherently more sensitive and selective detection principle, like a mass spectrometer.

Molecular cloning of a C-type lectin (LvLT) from the shrimp Litopenaeus vannamei: Early gene down-regulation after WSSV infection

C-type lectin is one of the pattern-recognition proteins of the non-self innate immune system in the invertebrates. In this study, a lectin-like cDNA (LvLT) of Litopenaeus vannamei was cloned and characterized. LvLT cDNA consists of 1035 nt encoding for a protein with 345 amino acid residues. The deduced LvLT consists of two putative carbohydrate-recognition domains (CRDs) as found in most C-type lectins. The first CRD consists of an amino acid motif (QPD) for the binding of galactose and the other CRDs consist of amino acid motifs (EPN) for the binding of mannose. Except for some conserved amino acid residues, the CRD of LvLT shared an overall low amino acid sequence identity with CRDs of other lectins. Unlike other shrimp lectins, LvLT is expressed only in the hepatopancreas but not in the hemocytes as revealed by RT-PCR. When juvenile shrimp were challenged with shrimp extracts containing white spot syndrome virus (WSSV), the expression levels of LvLT decreased initially in the first 2 h and then increased to a much higher level after 4 h. The results suggest that the initial reduction in LvLT transcript level may be related to the WSSV infection in shrimp.

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.

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.