Allergen Tropomyosin Research Articles

Homologous tropomyosins from vertebrate and invertebrate: Recombinant calibrator proteins in functional biological assays for tropomyosin allergenicity assessment of novel animal foods.

BackgroundNovel foods may provide new protein sources for a growing world population but entail risks of unexpected food‐allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening.ObjectiveAs a proof of concept, to evaluate non‐/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi‐quantitative allergy risk assessment of novel TM‐containing animal foods with mealworm TM as an example.MethodsPurified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp‐allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells.ResultsShrimp and chicken TMs showed high sequence homology, both alpha‐helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp‐allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100‐ to 1000‐times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source.Conclusions & clinical relevanceAccording to current GM crops' allergenicity assessment, non‐allergenic chicken TM could falsely be considered an allergen on a weight‐of‐evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi‐quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity.

Development of ELISA Method for Detecting Crustacean Major Allergen Tropomyosin in Processed Food Samples

Crustacean aquatic products were recognized as allergenic food source and tropomyosin (TM) was identified as putative major allergen. At present, the most effective way to control allergenic incidence was avoidance of causative dietary source. This study reports the development of sandwich ELISA for effective detection of TM in suspected food sources by pairing two TM-specific polyclonal antibodies obtained from different species. The LOD was identified as 6.8 ng mL−1, with high specificity to the TM of shrimp and crab. No cross-reactivity was observed with the TM of fish, poultry, and mammalian. The mean recoveries in food matrices were range from 87.96 to 118.77%, by both intra- and inter-assay systems with coefficients of variation of < 10% and < 25%, respectively. The sandwich ELISA can be effectively applied for detection of TM in commercial products available in the market. It facilitates the allergen labeling and management for allergenic crustacean food products by both manufacturing and regulatory agencies.

Insight into the allergenicity of shrimp tropomyosin glycated by functional oligosaccharides containing advanced glycation end products

Tropomyosin (TM) is the main allergen of shrimp. Glycation reportedly reduced the allergenicity of TM, and the allergenicity reduction was heavily dependent upon the sources of saccharides. In this work we investigated, how glycation of tropomyosin by functional oligosaccharides affected the allergenicity. Compared to TM, the TM glycated by galacto-oligosaccharide (TM-GOS), mannan-oligosaccharide (TM-MOS) and maltopentaose (TM-MPS) had lower allergenicity and induced weaker mouse allergy responses. While the TM glycated by fructo-oligosaccharide (TM-FOS) had stronger allergenicity and induced severe mouse allergy symptoms, due to the generation of neoallergns that belonged to advanced glycation end products (e.g. CML). Therefore, GOS, MOS and MPS could be applied to desensitize shrimp TM-induced food allergy through glycation, while FOS was not suitable to reduce TM allergenicity. Glycation of TM by GOS, MOS and MPS, especially for MPS, significantly reduced allergenicity and alleviated allergy symptoms, which could be potentially explored for immunotherapy for shrimp-allergic patients.

Quantum-dot-based lateral flow immunoassay for the rapid detection of crustacean major allergen tropomyosin

Food allergy has become an important problem affecting people's health. As one of the most common allergenic food, crustaceans pose a dietary risk to allergic sensitizers. A rapid and sensitive detection method is needed for allergen monitoring. We developed a quantum-dot-based fluorescent lateral flow Immunoassay (LFIA) for detection of major crustacean allergen tropomyosin (TM). Polyclonal antibodies were labeled by quantum dots through covalent bonding and characteristics of combined probe were analyzed. Relevant conditions of strip preparation were optimized, with results of 0.8 mg/mL test line coating concentration and 1% Tween-20 10 mM PBS as flowing buffer. The detection limits of developed assay were 0.5 μg/mL visually and 0.05 μg/mL by instrument without complex operation. The LFIA showed good specificity and reproducibility, and the results for real food samples were consistent and comparable to validated ELISA kit. The novel developed detection strip would significantly improve applications for the on-site detection of TM in foods.

Electrochemical tropomyosin allergen immunosensor for complex food matrix analysis

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at −200 mV vs. Ag pseudo-reference electrode, using H2O2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL−1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients.The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.

Allergenicity suppression of tropomyosin from Exopalaemon modestus by glycation with saccharides of different molecular sizes

The allergenicity suppression of tropomyosin (TM) from Exopalaemon modestus by glycation with saccharides of different molecular sizes (glucose, maltose, maltotriose, maltopentaose and maltoheptaose) was investigated using immunoblotting, human colon epithelial cell line (Caco-2) and human basophil cell line (KU812). Glycation of TM by glucose, maltotriose, maltopentaose and maltoheptaose significantly destructed and masked TM epitopes to obtain lower allergenicity, while glycation of TM by maltose had insignificant suppression on TM allergenicity. In addition, the glycated TM by glucose, maltotriose, maltopentaose and maltoheptaose inhibited the proliferation and IL-8 secretion of Caco-2, and the CD63 and CD203c expression, MAPK signaling of KU812 basophils, while the glycated TM by maltose had insignificant suppression on the allergy reactivities of Caco-2 cells and KU812 basophils. Glycation of TM by saccharides with larger molecular sizes (such as maltoheptaose) could provide new insight into the desensitization of shrimp-induced food allergy.

Quantification of shellfish major allergen tropomyosin by SPR biosensor with gold patterned Biochips

Tropomyosin (TM), known as the major allergen in seafood, has been increasing concerns in industrialized countries. We proposed a simple, rapid and accurate analytical method using surface plasmon resonance (SPR) sensor with gold patterned biochips for detection and quantification of TM in shellfish. The limit of detection (LOD) was 1.0 μg mL−1 (S/N = 3), while the limit of quantitation (LOQ) was 2.5 μg mL−1 (S/N = 10). Moreover, the satisfactory recoveries (92.0–108.0%) was obtained for TM detection in food samples, showing the good accuracy of this method. Furthermore, the results also demonstrated that this developed method is suitable for the determination of TM from different shellfish species within 3 min. Therefore, the developed SPR sensor with gold patterned biochips will have positive significance on the rapid and accurate detection allergens in shellfish foodstuffs, related products and their production lines in the future.

A chiral assembly of gold nanoparticle trimer-based biosensors for ultrasensitive detection of the major allergen tropomyosin in shellfish

A biosensor based on a chiral assembly of polymer of gold nanoparticle (AuNP) trimers was developed for the detection and quantification of the major shellfish allergen tropomyosin (TROP). TROP and anti-TROP monoclonal antibodies (mAb) were immobilized on 20 nm and 30 nm 16-mercaptohexadecanoic acid (16-MHDA) functionalized AuNPs to assemble a trimer, which has a Circular dichroism (CD) signal. The free TROP from samples was quantified as an inhibitor for the formation of the AuNP trimer. The AuNP trimer-based biosensor allowed for the selective determination of TROP in the range of 0.1–15 ng mL−1 with the limit of detection (LOD) of 21 pg mL−1 (S/N = 3) and the limit of quantitation (LOQ) of 70 pg mL−1 (S/N = 10). The use of a AuNP trimer-based biosensor with simple sample preparation functions with specificity and accuracy; this highlights its applicability for the detection of allergens in shellfish products, related products and their production lines. Furthermore, based on the less conserved sequences of TROP in phylogenetically different species, this biosensor is currently being used to identify the adulteration of shellfish products using TROP as biomarker.

Transglutaminase-catalysed cross-linking eliminates Penaeus chinensis tropomyosin allergenicity by altering protein structure

ABSTRACT Transglutaminase (TG) is a major food processing enzyme that induces protein cross-linking. Tropomyosin (TM) is a major shellfish allergen that is responsible for at least 80% of the shrimp allergic incidents. The aim of this research was to investigate the effect of TG on TM allergenicity and reveal the involvement of TM structure alternation. After treated with TG for different time, the allergenicity of TM was assessed by indirect ELISA with shrimp allergic patients’ sera, and the structure of TM was characterized by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy and fluorescence spectroscopy. Results showed that the allergenicity slightly decreased at the early stage of reaction and dramatically decreased at the late stage, which is tightly correlated with the increment of β-turn content and UV absorption. In conclusion, TG modification is an effective and promising food processing method to eliminate TM allergenicity with significant prospect of industrial application.

Glycation by saccharides of different molecular sizes affected the allergenicity of shrimp tropomyosin via epitope loss and the generation of advanced glycation end products.

Tropomyosin is the most potent allergen of shrimp that can cause severe food allergy. However, to date, an effective approach to eliminate this allergenicity is still lacking. Glycation is a promising approach that can reduce the allergenicity of shrimp tropomyosin by destroying the epitopes; however, advanced glycation end products (AGEs) are also generated during glycation, which can function as neoallergens to strengthen the allergenicity; therefore, it is hard to tell how the glycation of an allergen with different saccharides affects the allergenicity via epitope loss and neoallergen generation. This study was aimed at the elucidation of how the glycation of tropomyosin (TM) with saccharides of different molecular sizes (glucose, maltose, and maltotriose) affected the allergenicity through epitope loss and the generation of neoallergns that belonged to advanced glycation end products (AGEs). Saccharides of higher molecular sizes (maltotriose) could lead to higher glycated TM than saccharides of smaller molecular sizes (glucose and maltose). Compared with TM, the TM glycated by glucose (TM-G) and maltotriose (TM-MTS) had lower allergenicity and contributed to weaker mouse allergy symptoms; on the other hand, the TM glycated by maltose (TM-M) had no significant impact on the allergenicity due to the generation of AGE-related neoallergens, which might offset the glycation-induced epitope loss. The glycation of TM by maltotriose led to lower generation of AGE neoallergens (e.g. CML) than that in the cases of glucose and maltose; therefore, maltotriose could be applied to desensitize TM-induced food allergy through glycation, and this could be a potential immunotherapy for shrimp allergy patients.

Cross-linking of shrimp tropomyosin catalyzed by transglutaminase and tyrosinase produces hypoallergens for potential immunotherapy.

Tropomyosin (TM) is the major allergen in shellfish, which causes severe food allergy worldwide and shows wide cross-reactivity in different species. Transglutaminase (TG) and tyrosinase (Tyr) are protein modification enzymes that catalyze substrate cross-linking and change protein structures and properties. The aim of this work was to investigate the effect of TG and Tyr on TM. After TG/Tyr treatment, the cross-linking and protein structural change were observed by SDS-PAGE, scanning electron microscopy and circular dichroism. Furthermore, ELISA and mast cell degranulation assay with allergic sera demonstrated the allergenicity reduction after treatment. An in vivo TM-sensitization mouse model showed that both TG and Tyr treatments reduced the IgE-induction capacity of TM but maintained the IgG-induction ability, making the treated TM a potential hypoallergen. Moreover, TG-but-not-Tyr-treated TM promoted regulatory T cell proliferation and thus may facilitate the establishment of oral tolerance. This work provided an effective method to reduce TM allergenicity and produce hypoallergens, which has broad application prospects in low allergenic food production and allergen-specific immunotherapy.

Comment on 'Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one compound peptide libraries'.

Comment on 'Screening and identification of mimotopes of the major shrimp allergen tropomyosin using one-bead-one compound peptide libraries'.

Conformation, allergenicity and human cell allergy sensitization of tropomyosin from Exopalaemon modestus: Effects of deglycosylation and Maillard reaction.

Tropomyosin (TM) from Exopalaemon modestus was glycoprotein, the effects of deglycosylation (enzyme and chemical regents) and Maillard reaction on the conformation, allergenicity and human cell sensitization of TM were investigated. TM had both N-glycan and O-glycan. After deglycosylation and glycation, the α-helix of TM reduced, while other secondary structures promoted. Deglycosylation enhanced TM allergenicity and digestibility, contributed to digests with lower allergenicity, glucose glycation weakened the allergenicity and digestibility of TM, led to digests with weaker allergenicity. Gastric digestion partly reduced allergenicity of digests, intestinal digestion highly reduced digests' allergenicity, gastrointestinal digestion most efficiently weakened the allergenicity of digests. After deglycosylation, the deglycosylated TM exerted stronger activation of basophil (KU812), while glucose glycated TM weakened KU812 activation. Deglycosylation exerted inhibition on colon cell (Caco-2) proliferation and increase of IL-8 release, while glucose glycated TM suppressed Caco-2 proliferation and IL-8 release.

Rapid on-site detection of shrimp allergen tropomyosin using a novel ultrafast PCR system.

Shrimp is seafood that can commonly trigger allergic reactions. In this study, the ultrafast real-time PCR assay with portable device was developed to detect a shrimp-derived major allergen, tropomyosin, without complicated DNA extraction. For shrimp allergen detection, a specific primer pair was designed based on the shrimp tropomyosin gene and 18S ribosomal RNA gene as internal control. Primer specificity was assessed using 8 common seafood species. Serially diluted shrimp DNA was used to determine the limit of detection of the ultrafast PCR system, which was approximately 3.2pg. Twenty-three food samples containing shrimp were evaluated to verify the applicability of a direct ultrafast PCR method for detecting shrimp allergens without DNA isolation. It took less than 30min from sample preparation-to-result analysis to detect shrimp DNA in raw and processed samples. Therefore, this PCR system can be effectively and conveniently utilized in the field to detect shrimp in various food products.

Maillard reaction with ribose, galacto-oligosaccharide or chitosan-oligosaccharide reduced the allergenicity of shrimp tropomyosin by inducing conformational changes

Tropomyosin (TM), the predominant shellfish allergen, has induced severe food allergy worldwide, but efficient prevention and cure measures are limited nowadays. In the present study, we introduced an approach to eliminate Penaeus chinensis TM by Maillard reaction with reducing sugars including ribose, galacto-oligosaccharide (GOS) and chitosan-oligosaccharide (COS), which reduced the allergenicity by up to 60%. We also evaluated the conformational changes of TM during reaction by far-UV circular dichroism, and found the reaction-induced alternation from α-helix to β-sheet. We further determined the effect of Maillard reaction on TM allergenic epitopes to investigate the detailed mechanisms, and revealed the structure preference of different sugars. Correlation analysis with experimental data revealed that α-helix mediated the allergenicity elimination by Maillard reaction. There results provided theoretical foundations for the improvement and application of Maillard reaction in allergenic food processing.

Structural changes of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) treated shrimp tropomyosin decrease allergenicity

The aim of this study was to analyze the effect of AAPH on the conformational structure and allergenicity of shrimp tropomyosin (TM). The structure of AAPH-TM was evaluated by SDS-PAGE, fluorescence, circular dichroism (CD) and ultraviolet light (UV), and the allergenicity was evaluated by in vivo and in vitro methods. Results showed that AAPH can induce structural changes through TM aggregations. These aggregations can decrease the IgG/IgE binding capacity on immunoblot analysis. Further competitive inhibition ELISA (ciELISA) results showed the IC50 of AAPH-TM (AAPH 0–25 mmol/l) changed from 0.086 to 46.2 μg/ml, which correlated with TM structural changes. An RBL-2H3 cell assay showed that release rate of β-hexosaminidase and histamine decreased by 51.6% and 68.1% with AAPH (5 mmol/l) treatment, respectively. A mouse model showed AAPH-TM could decrease levels of IgE/IgG1, release of histamine and mMCP-1 in sera. In conclusion, AAPH induced TM aggregation can cause structural changes and decrease the allergenicity.

Effect of Heat Processing on IgE Reactivity and Cross-Reactivity of Tropomyosin and Other Allergens of Asia-Pacific Mollusc Species: Identification of Novel Sydney Rock Oyster Tropomyosin Sac g 1.

ScopeShellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross‐reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia‐Pacific molluscs are characterized: Sydney rock oyster (Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop (Amusium balloti), and southern calamari (Sepioteuthis australis), examining cross‐reactivity between species and with blue swimmer crab tropomyosin, Por p 1.Methods and resultsIgE ELISA showed that cooking increased IgE reactivity of mollusc extracts and basophil activation confirmed biologically relevant IgE reactivity. Immunoblotting demonstrated strong IgE reactivity of several proteins including one corresponding to heat‐stable tropomyosin in all species (37–40 kDa). IgE‐reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross‐reactivity with other molluscs, while mussel cross‐reactivity was weakest. Inhibition immunoblotting demonstrated high cross‐reactivity between tropomyosins of mollusc and crustacean species.ConclusionThese findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy.

Influence of thermal treatment on the characteristics of major oyster allergen Cra g 1 (tropomyosin).

Shellfish, including oysters, often cause allergic reactions in adults. Thermal treatment is one of the most common technologies for dealing with seafood, which may affect biological properties. The present study aimed to evaluate the impact of heating on the conformation and potential allergenicity of oyster-derived tropomyosin (Cra g 1). Sodium dodecylsulphate-polyacrylamide gel electrophoresis showed that there was an apparent band at 35 kDa of raw tropomyosin after purification and more significant polymers appeared in the heated protein. Interestingly, obvious changes in the intensity of the circular dichroism signal and 1-anilino-8-naphthalene sulfonate-binding fluorescence were observed especially in the case of the roasted form, which was associated with an increase in antibody reactivity. The degree of immunoglobulin (Ig)E binding of this treatment was demonstrated in the order roasted>boiled>raw. Furthermore, sequence alignment and amino acid composition revealed that Cra g 1 shared relatively high homology to tropomyosins from other shellfish and was also abundant in lysine that was apt to be modified by reducing sugars during heating. Heated Cra g 1 produces higher IgE reactivity than the raw form as a result of the denaturation and formation of polymers. These findings will benefit the diagnosis and management of potential allergenicity as a result of shellfish. © 2018 Society of Chemical Industry.

Effects of gamma irradiation on tropomyosin allergen, proximate composition and mineral elements in giant freshwater prawn (Macrobrachium rosenbergii).

Effects of food irradiation on allergen and nutritional composition of giant freshwater prawn are not well documented. Thus, this study aimed to investigate the effects of gamma irradiation on tropomyosin allergen, proximate composition, and mineral elements in Macrobrachium rosenbergii. In this study, prawn was peeled, cut into small pieces, vacuum packaged and gamma irradiated at 0, 5, 7, 10 and 15kGy with a dose rate of 0.5kGy/h using cobalt-60 as the source, subsequently determined the level of tropomyosin, proximate composition and mineral elements respectively. The results showed that band density of tropomyosin irradiated at 10 and 15kGy is markedly decreased. Proximate analysis revealed that moisture, protein, and carbohydrate content were significantly different as compared with non-irradiated prawn. Meanwhile, gamma irradiated M. rosenbergii at 15kGy was observed to be significantly higher in nickel and zinc than the non-irradiated prawn. The findings provide a new information that food irradiation may affect the tropomyosin allergen, proximate composition and mineral elements of the prawn.

Identification of Allergenic Epitopes and Critical Amino Acids of Major Allergens in Chinese Shrimp ( Penaeus chinensis) by Immunoinformatics Coupled with Competitive-Binding Strategy.

Chinese shrimp ( Penaeus chinensis) is widely cultured and consumed in Asia but is also a major food allergen locally. Although they may be the foundation for preventing and treating allergies, the allergenic epitopes of the major allergens tropomyosin (TM) and arginine kinase (AK) in Penaeus chinensis have not been identified. Here, we applied Immunoinfo-CB (immunoinformatics coupled with competitive-binding strategy) to address the point. Potential allergenic epitopes of TM and AK were predicted by multiple immunoinformatics tools, followed by validating with inhibitory dot-blot assay, indirect competition ELISA, and mast cell degranulation assay. Furthermore, critical amino acids in allergenic epitopes were also identified by Immunoinfo-CB. Our findings provide new insight into allergenic epitopes and critical amino acids of TM and AK responsible for the anaphylactic response. The Immunoinfo-CB therefore offers promises for characterization of IgE-binding epitopes that might be used as new targets for immunotherapy of food allergy.