Shrimp Allergen Research Articles

Identification of the first major allergen of a squid (Todarodes pacificus)

Background: In Japan, squid is an important seafood, and some patients with food allergies are sensitive to squid. There has been no report, however, describing the major allergens of squid. Objective: To characterize squid allergens, we isolated a major allergen from the Pacific flying squid (Todarodes pacificus) and compared it with a major allergen from a shrimp (Penaeus orientalis). Methods: The major squid and shrimp allergens were isolated by column chromatography on diethylaminoethyl-Sepharose (Pharmacia, Uppsala, Sweden), hydroxylapatite, and Sephacryl S-300 (Pharmacia). The IgE reactivity of the isolated allergens was assessed by immunoblotting. The cross-reactivity between the squid and shrimp allergens was examined by use of mouse polyclonal and monoclonal antibodies to the major allergens. Amino acid sequence analyses of the isolated allergens were done. Results: The isolated squid allergen is a 38 kd, heat-stable protein. IgE antibody binding to the purified squid allergen was demonstrated by immunoblotting. Cross-reactivity between major squid and shrimp allergens was demonstrated with sera from patients allergic to squid or shrimp or with allergen-specific monoclonal antibodies. The amino acid sequence analysis of the major squid allergen showed a marked homology with tropomyosin from blood fluke planorbid (Biomphalaria glabrata), which is a common vector snail of Schistosoma mansoni. Conclusion: This 38 kd protein is a major allergen of the squid, Todarodes pacificus, and is believed to be squid muscle protein tropomyosin. We named it Tod p 1 according to International Union of Immunological Societies allergen nomenclature regulation. (J A LLERGY C LIN I MMUNOL 1996;98:948-53.)

IgE reactivity against a cross-reactive allergen in crustacea and mollusca: Evidence for tropomyosin as the common allergen

Background: Although cross-reactivity between mollusks and other crustaceans in shrimp-sensitive subjects has been reported, the mechanism of this allergenic cross-reactivity has not been studied in detail. Objective: To investigate this cross-reactivity in vitro, we have taken advantage of a complementary DNA that expresses tropomyosin, the immunodominant shrimp allergen. Methods: Serum IgE from nine patients with known anaphylaxis to shrimp and five normal volunteers were analyzed by immunoblotting against 13 distinct crustaceans and mollusks. As additional antigens, muscle preparations were isolated from grasshopper, cockroach, fruit fly, chicken, and mouse. Results: Sera from all patients, but not control subjects, reacted specifically with a 38 kd protein in all crustaceans and mollusks studied. In addition, 8 of 9 sera from patients, but from none of the normal control subjects, recognized proteins of various other molecular weights among the mollusk extracts studied. The 38 kd protein was identified as tropomyosin and was shown to share immunodominant epitopes among all species of crustaceans and mollusks tested by specific absorption studies. Moreover, sera from all nine subjects with shrimp allergy demonstrated IgE reactivities against grasshopper, cockroach, and fruit fly but not chicken or murine muscle. Conclusion: The allergic epitopes on tropomyosin are conserved among invertebrates including not only shellfish but also insects. This latter observation suggests that persons sensitive to shrimp should undergo further study for potential cross-reactive inhalant or ingested insect sensitivity. (J A LLERGY C LIN I MMUNOL 1996;98:954-61.)

603 Cross-reactivity of Pen a 1, the major shrimp allergen and mouse tropomyosin?

603 Cross-reactivity of Pen a 1, the major shrimp allergen and mouse tropomyosin?

IgE and monoclonal antibody reactivities to the major shrimp allergen Pen a 1 (tropomyosin) and vertebrate tropomyosins.

Pen a 1, the major shrimp allergen from brown shrimp Penaeus aztecus has been identified as the muscle protein tropomyosin. To identify Pen a 1 IgE binding sites, the reactivities of Pen a 1-specific monoclonal antibodies (mAbs) and shrimp-allergic subjects' IgE to shrimp and homologous mammalian tropomyosins were analyzed. Pen a 1, purified by preparative SDS-PAGE and commercially obtained porcine, bovine and rabbit tropomyosin were cleaved by CNBr or digested by endoproteinases Lys-C, Glu-C, trypsin, Arg-C and chymotrypsin. Reactivities of Pen a 1-specific mAbs and IgE to the resulting peptides were analyzed by dot blot and immunoblotting. The dot blot analysis showed that mAbs and IgE antibodies did not react with any of the mammalian tropomyosins. The immunoblot analysis showed that all Pen a 1 digests bound IgE or mAbs. However, not all peptides in each digest possessed an IgE binding site. IgE binding intensity and frequency varied by subject and peptide digest. IgE and mAb reactivity patterns were similar but no mAb reproduced the IgE binding patterns indicating that subject' IgE bound some epitopes that were not recognized by the Pen a 1-specific mAbs. These studies suggest that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule, Pen a 1 may have several similar epitopes, and that Pen a 1 epitopes do not appear to be located in the highly homologous parts of the tropomyosin molecule.

604 A monoclonal antibody-based sandwich ELISA to quantify the major shrimp allergen Pen a 1

604 A monoclonal antibody-based sandwich ELISA to quantify the major shrimp allergen Pen a 1

Cloning, expression, and primary structure of Metapenaeus ensis tropomyosin, the major heat-stable shrimp allergen

Shrimp is a common cause of seafood hypersensitivity. To study the mechanism of seafood hypersensitivity at the molecular level, we have determined the primary structure of the major heat-stable allergen of shrimp by cloning, expression, nucleotide sequencing, and amino acid sequence determination of an IgE-reactive cDNA clone, Met e I, isolated from a Metapenaeus ensis expression library in λgt 11. We first constructed a cDNA library from the shrimp M. ensis in λgt 11. We then screened the library with sera from patients with hypersensitivity reactions to shrimp and identified a positive IgE-reactive clone, designated as Met e I. This cDNA was purified to homogeneity and subsequently expressed in the plasmid pGEX. Serum antibodies from patients with shrimp allergy demonstrated positive IgE reactivity by immunoblotting to a protein encoded by the clone Met e I; sera from nonallergic control subjects were not reactive. The nucleotide sequence of this cDNA clone revealed an open reading frame of 281 amino acid residues, coding for a protein of 34 kd. Comparison of the Met e I amino acid sequence with the Genbank database showed that Met e I is highly homologous to multiple isoforms of tropomyosin.

Identification of a cross-reactive allergen (presumably tropomyosin) in shrimp, mite and insects.

A monoclonal antibody to Dermatophagoides pteronyssinus is described that cross-reacts with an IgE-binding antigen present in insects, Crustacea (e.g. shrimp) and other invertebrates. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration and immunofluorescence it was shown that this monoclonal antibody presumably recognizes tropomyosin. Tropomyosin was shown to be involved in cross-reactivity between mite, shrimp and insects in shrimp-allergic patients.

Identification of the major brown shrimp (Penaeus aztecus) allergen as the muscle protein tropomyosin.

Shrimp, a major seafood allergen, was investigated as a model food allergen. Extracts from both shrimp (Penaeus aztecus) meat and cooking fluid contain a substantial and similar amount of allergenic activity. A 36-kD allergen, demonstrated in both extracts by SDS-PAGE/Western blot analysis, reacted with 28/34 (82%) sera from shrimp-sensitive, skin test and RAST-positive, individuals. This allergen, named Pen a I, was isolated by SDS-PAGE; its amino acid composition was rich in aspartic and glutamic acids. A 21-residue peptide, obtained from endoproteinase Lys-C digested Pen a I by high-performance liquid chromatography, demonstrated significant homology (60-87%) with the muscle protein tropomyosin from various species and origins. The greatest homology (87%) was noted with tropomyosin of the fruit fly (Drosophila melanogaster) reflecting the phylogenic relationship between these two arthropods. These studies demonstrate that tropomyosin is the major shrimp allergen. Although the amino acid sequence of this shrimp muscle protein shares considerable homology with tropomyosins of other species including man, significant differences remain in allergenic activity.

Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes.

Abstract The major heat-stable shrimp allergen (designated as Sa-II), capable of provoking IgE-mediated immediate type hypersensitivity reactions after the ingestion of cooked shrimp, has been shown to be a 34-kDa heat-stable protein containing 300 amino acid residues. Here, we report that a comparison of amino acid sequences of different peptides generated by proteolysis of Sa-II revealed an 86% homology with tropomyosin from Drosophila melanogaster, suggesting that Sa-II could be the shrimp muscle protein tropomyosin. To establish that Sa-II is indeed tropomyosin, the latter was isolated from uncooked shrimp (Penaeus indicus) and its physicochemical and immunochemical properties were compared with those of Sa-II. Both tropomyosin and Sa-II had the same molecular mass and focused in the isoelectric pH range of 4.8 to 5.4. In the presence of 6 M urea, the mobility of both Sa-II and shrimp tropomyosin shifted to give an apparent molecular mass of 50 kDa, which is a characteristic property of tropomyosins. Shrimp tropomyosin bound to specific IgE antibodies in the sera of shrimp-sensitive patients as assessed by competitive ELISA inhibition and Western blot analysis. Tryptic maps of both Sa-II and tropomyosin as obtained by reverse phase HPLC were superimposable. Dot-blot and competitive ELISA inhibition using sera of shrimp-sensitive patients revealed that antigenic as well as allergenic activities were associated with two peptide fractions. These IgE-binding tryptic peptides were purified and sequenced. Mouse anti-anti-idiotypic antibodies raised against Sa-II specific human idiotypic antibodies recognized not only tropomyosin but also the two allergenic peptides, thus suggesting that these peptides represent the major IgE binding epitopes of tropomyosin. A comparison of the amino acid sequence of shrimp tropomyosin in the region of IgE binding epitopes (residues 50-66 and 153-161) with the corresponding regions of tropomyosins from different vertebrates confirmed lack of allergenic cross-reactivity between tropomyosins from phylogenetically distinct species.

214 Isolation and characterization of an important 36 kd shrimp allergen

214 Isolation and characterization of an important 36 kd shrimp allergen

Characterization of water-soluble shrimp allergens released during boiling

Water-soluble shrimp allergens released during boiling (shrimp water) were characterized and compared to allergen extracts from boiled shrimp (shrimp meat). Both shrimp extracts contained acidic proteins (isoelectrofocusing) and demonstrated similar allergenic activity (RAST and RAST inhibition). Shrimp-water extract was analyzed further by immunoprinting with sera from 14 shrimp-sensitive, RAST-positive subjects, and six nonsensitive, RAST-negative individuals. Although none of the sera from shrimp-tolerant individuals reacted, 12 14 sera (85.7%) from shrimp-sensitive subjects reacted with shrimp-water proteins with acid isoelectric points. Shrimp-water extract was fractionated by chromatofocusing with pH and NaCl gradients. A number of eluted ultraviolet-absorbing peaks contained allergens as determined by RAST inhibition. Isoelectrofocusing demonstrated many protein bands present in these peaks, some of which bound IgE from a RAST-positive sera pool. These studies indicate that shrimp water is an excellent source of shrimp allergens, that chromatofocusing is a useful method for fractionation of shrimp allergens, and that shrimp allergens are generally protein molecules with acid isoelectric points.

Species-specific shrimp allergens: RAST and RAST-inhibition studies

Two edible shrimp species are widely available in Louisiana, Penaeus setifecus (white shrimp) and Penaeus aztecus (brown shrimp). Some sensitive individuals report only occasional allergic symptoms after shrimp ingestion, suggesting that there may be species-specific allergens. To investigate this possibility, we evaluated shrimp species-specific reactivity in 31 individuals with a history of immediate hypersensitivity reactions after shrimp ingestion with skin prick tests and RASTs with white and brown shrimp extracts. On selected individuals, RAST-inhibition studies were performed with white shrimp and/or brown shrimp-coupled disks, with white and/or brown shrimp extracts as inhibiting allergen. Positive skin tests to both types of extract were observed in 77% (23130) of the subjects; one individual reacted to brown shrimp extract only. Elevated RASTs to both extracts were observed in 16131 study participants; one subject reacted only to white shrimp extracts and two subjects to brown shrimp extract alone. Sera from two individuals tested by RAST inhibition recognized qualitatively different allergens in brown and white shrimp extracts, supporting the hypothesis that there are species-specific shrimp allergens. Species specificity is important because it may explain the intermittent symptoms of some study subjects. The percentage of shrimp-sensitive subjects testing positive by skin test and RAST can be increased by use of extracts from more than one species of shrimp.

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies.

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.

Identification of a shrimp-derived allergen as tRNA.

Abstract During an attempt to isolate shrimp allergens, evidence was obtained that shrimp ribonucleic acid was capable of eliciting a specific IgE response in man and an experimental animal model system. The shrimp ribonucleic acid was extracted from boiled whole shrimp (Peneaus indicus), and was isolated by salt precipitation and sequential chromatography over DEAE-Sephacel and BioGel P-100. The allergenic material was identified as a ribonucleic acid based on the following criteria: a maximal absorption at 258 nm, failure to stain positively with Coomassie Brilliant Blue on slab gel electrophoresis, positive staining with ethidium bromide, co-migration with yeast tRNA on submerged gel electrophoresis in 1.5% Agarose M, and sensitivity to ribonuclease T2 and 0.3 M NaOH. Treatment with protease did not alter its allergenic activity. The RNA was capable of binding allergen-specific IgE in sera from two shrimp-sensitive patients, as demonstrated by microELISA and solid-phase radioimmunoassay (SPRIA) using antigen-coated nitrocellulose filter paper discs and purified 125I-labeled goat anti-human IgE. RNA isolated from shrimp by a conventional tRNA isolation procedure also had the ability to specifically bind IgE in the sera of shrimp-sensitive patients. IgE antibodies to shrimp RNA did not recognize yeast tRNA or salmon testes DNA, and were not detected in sera of other subjects. The shrimp-derived RNA was further able to induce a reaginic response in mice. A combination of in vitro aminoacylation of shrimp tRNA and SPRIA resulted in the identification of the allergenic tRNA as tRNA(Tyr) and tRNA(Arg). Thus, shrimp tRNA is capable of inducing a specific IgE response in man.

172 Identification of shrimp allergens by immunoelectrophortic methods

172 Identification of shrimp allergens by immunoelectrophortic methods