Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA.

BACKGROUND: Among 13 allergens found in extracts of cooked brown shrimp (Penaeus aztecus) the 36 kd muscle protein tropomyosin has been identified as the only major shrimp allergen (Pen a 1). Cross-reacting molecules with similar molecular weights were detected in other crustacea species such as crab, lobster, and crawfish. Because Pen a 1 and Pen a 1-like allergens are important in crustacea allergy, the aim of this study was to develop a monoclonal antibody (mAb)-based sandwich ELISA to quantify Pen a 1 and to evaluate Pen a 1 levels in four commercial shrimp, crab, and lobster extracts. METHODS: Two Pen a 1-specific mAbs with different epitope specificities were selected. ELISA plates coated with captured mAb 3.2 were incubated with samples containing Pen a 1. Bound Pen a 1 was detected by a combination of biotinylated mAb 4.9.5 and alkaline phosphatase-labeled streptavidin. RESULTS: The optimized sandwich ELISA could detect Pen a 1 concentrations ranging from 4 to 125 ng/ml. Four commercial shrimp extracts demonstrated a 40-fold difference in Pen a 1 levels (24 to 920 microg/ml). Crab and lobster extracts contained detectable levels of Pen a 1-like proteins. No reactivity to cockroach, house dust mite, oyster, codfish, or peanut extracts was detected, which indicates that the developed assay is crustacea-specific. CONCLUSION: A sensitive sandwich assay was developed to quantify Pen a 1. This assay will be helpful to standardize shrimp extracts in regard to the content of the major allergen, Pen a 1, and to study cross-reactivities among and evaluate occupational exposure to different crustacea species.

Characterization of recombinant shrimp allergen Pen a 1 (tropomyosin).

Tropomyosin (Pen a 1) from brown shrimp, Penaeus aztecus, has been identified as the only major shrimp allergen. Since beef, pork and chicken are other tropomyosin-containing foods that are not very allergenic, tropomyosins can serve to investigate the contribution of the structural properties of a protein to its allergenicity. The aim of this study was to determine the primary structure of Pen a 1 and to identify IgE-binding epitopes. The screening of a unidirectional expression cDNA library from shrimp tail muscle with the Pen-a-1-specific monoclonal antibody 4.9.5 resulted in 4 positive Escherichia coli clones. Immunoblot analysis with human sera from shrimp-allergic subjects demonstrated IgE binding of all 4 recombinant shrimp proteins. Three of 4 expressed recombinant proteins have a molecular weight of approximately 36 kD, consistent with the molecular weight of natural Pen a 1. The DNA sequence analysis identified these recombinant shrimp proteins as tropomyosin and could be aligned with the sequence of greasyback shrimp (Metapenaeus ensis) tropomyosin (Met e 1). In order to characterize contiguous IgE-binding epitopes of Pen a 1, a peptide library (Novagen epitope mapping system) expressing 10-30 amino-acid-residue-long recombinant Pen a 1 peptides was constructed and screened with human IgE. Four recombinant, IgE-reactive Pen a 1 peptides were selected and sequenced. They show various degrees of sequence identity with tropomyosins of other arthropods, such as fruitfly and house dust mite, helminths and vertebrates.

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.

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.

Antigenic analysis (IgE and monoclonal antibodies) of the major shrimp allergen Pen a 1 (Tropomyosin) from Penaeus aztecus.

Pen a 1, the major shrimp allergen from the brown shrimp Penaeus aztecus was purified by preparative SDS-PAGE. Peptides were generated from Pen a 1 by CNBr cleavage and endoproteinase (Lys-C, Glu-C, trypsin, alkaline protease, Arg-C, chymotrypsin) digestion. The molecular weights of the resulting CNBr cleavage and enzymatic digestion products, separated by peptide SDS-PAGE, ranged from 1.5 to 20 kD. Following SDS-PAGE and semidry blotting, the analysis of monoclonal antibody (mAb) and subjects' IgE reactivities demonstrated that with the exception of alkaline protease, all cleavage procedures yielded IgE-binding peptides. However, since not all peptides of every digest bind IgE, it appears that IgE-binding epitopes are restricted to certain parts of the Pen a 1 molecule. mAbs bound to CNBr, Lys-C, trypsin, Glu-C and Arg-C peptides. Since mAbs reacted to several peptides from the same digest, Pen a 1 may have several similar epitopes. The comparison of IgE and mAb reactivities demonstrated similar but not identical binding patterns.

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.

The relationships among shrimp-specific IgG subclass antibodies and immediate adverse reactions to shrimp challenge.

High levels of shrimp-specific IgE, in association with a positive prick test, are not always predictive of a positive, immediate response to double-blind, placebo-controlled, food challenge (DBPCFC) with shrimp. The observation that shrimp-sensitive individuals in general have increased levels of circulating shrimp-specific IgG is of interest because antigen/allergen-specific IgG subclasses have been associated with adverse reactions to foods. Therefore, this current study measured shrimp-specific IgG subclass and IgE antibodies in 31 individuals with histories of immediate, adverse reactions to shrimp immediately before DBPCFC and 20 shrimp-tolerant subjects. Individuals with a history of shrimp sensitivity had significantly raised shrimp-specific IgG2 and IgG4 compared to shrimp-tolerant individuals. Challenge-positive subjects were distinguished from subjects with negative or equivocal responses by an increased IgG2 (p less than or equal to 0.001). Specific IgG4 was not raised (p less than or equal to 0.065). These studies indicate that some shrimp-specific IgG subclass levels are increased in shrimp-sensitive subjects. However, none of the subclass responses were significantly predictive of a positive response to DBPCFC and therefore were not diagnostic of shrimp intolerance.

The natural history of shrimp hypersensitivity.

Sera collected sequentially during a 24-month interval from 11 individuals with shrimp hypersensitivity and 10 nonhypersensitive control subjects were evaluated for shrimp-specific IgE, IgG, IgM, and IgA reactivity. Shrimp-hypersensitive subjects underwent double-blind, placebo-controlled shrimp challenges; seven exhibited positive challenges, and four subjects reported the subjective symptom of oropharyngeal pruritus. Shrimp-specific IgE levels in all subjects were relatively constant during the 24 months of this study and not affected by shrimp challenge, although some fluctuation in the shrimp-specific IgG, IgM, and IgA reactivity were noted, apparently unrelated to shrimp challenge. Shrimp-specific IgE and IgG, but not IgM and IgA, were significantly higher in the group with shrimp hypersensitivity as compared to the control subjects. Moreover, the challenge-positive subjects had higher levels of both shrimp-specific IgE and IgG than subjects reporting pruritus. The levels of shrimp-specific IgG correlated directly with shrimp-specific IgE reactivity. These studies indicate that serum levels of shrimp-specific IgE are significantly elevated in shrimp-hypersensitive subjects who exhibit positive food challenges, and these baseline levels did not appear to be altered long term by isolated shrimp challenge. Furthermore, baseline shrimp-specific antibody (IgG, IgM, and IgA) levels noted in normal subjects were not markedly affected by frequent ingestion of shrimp.

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.

Food specific skin-test reactivity in atopic subjects.

It has been proposed that a permanent or transitory increase in gut permeability is an important facet in the development of food allergy. If this occurs, then individuals with a history of a specific food allergy should have a higher incidence of immunological reactivity to other food allergens as compared to food tolerant subjects. To test this hypothesis, we evaluated the prevalence of food-specific IgE responses by skin-prick testing in 60 individuals. Subjects were classified by a history of food allergy (shrimp hypersensitivity) and atopic status. Prevalence of skin-prick test reactivity to shrimp and a panel of nine other food extracts was determined. Skin-test reactivity to shrimp was related both a history of shrimp sensitivity and atopic status. However, the prevalence of skin-test reactivity to other foods was not related to clinical history of shrimp allergy, although it was related to atopy. A subset of subjects with shrimp allergy had multiple positive skin reactions to many of the nine other foods. This reactivity was associated with a history of pulmonary symptoms following shrimp ingestion. In general our results do not support non-specific increases in gut permeability being important in the development of food allergy.

Studies of spontaneous proliferation of mononuclear cells in human immunodeficiency virus (HIV-1)-infected individuals.

We evaluated the ability of mononuclear cell populations from 56 hemophiliac subjects, 69 homosexuals, and 32 control subjects to proliferate spontaneously in vitro. Levels of spontaneous proliferation (SP) were not significantly different among homosexuals or hemophiliac subjects when they were grouped as asymptomatic human immunodeficiency virus (HIV) seronegative, asymptomatic seropositive, persistent generalized lymphadenopathy, acquired immunodeficiency syndrome-related complex, or acquired immunodeficiency syndrome groups. There was, however, a stepwise increase in levels of SP corresponding with progression of disease in both patient groups. No correlations were noted between SP and CD8+ cell populations or mitogenic responses in study subjects, but SP correlated inversely to percentages and numbers of CD4+ lymphocytes. Depletion of CD4+ cells from mononuclear cell populations of HIV-infected subjects decreased SP. Thus, in HIV-1-infected subjects, SP increases as HIV-1-related disease progresses and appears to be CD4 dependent.

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% (23/30) of the subjects; one individual reacted to brown shrimp extract only. Elevated RASTs to both extracts were observed in 16/31 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.

T cells expressing high levels of non-major histocompatibility complex (MHC)-restricted cytotoxicity are present in early and late clinical phases of human immunodeficiency virus 1 (HIV-1) infection.

Cytotoxic cells appear to play an important role in host defense against viral infection. In HIV-1 infection there is an expansion of the Leu7-positive lymphocyte population which is associated with cytotoxic activity. Since a form of non-MHC-restricted T-cell cytotoxicity [lectin-dependent cell cytotoxicity (LDCC)] has been reported to be mediated by CD3+Leu7+ cells, we evaluated LDCC and Leu7-positive lymphocyte populations in HIV-1-infected subjects and healthy controls. Both LDCC and percentages of Leu7+CD3+ and Leu7+CD2+ cells were increased in HIV-1-infected individuals as compared to controls. However, the CD3+Leu7+ lymphocyte population was increased to a greater degree than the CD8+Leu7+ population and a minor Leu7+ cell population (Leu7+CD4+) was expanded in the early stages of infection. Lectin-dependent cell cytotoxicity was positively correlated with the percentages of Leu7+CD3+ cells. Thus T-cells with the capacity to mediate high levels of non-MHC-restricted cytotoxicity are present in increased proportions in HIV-1-infected individuals and persist in advanced disease. Further studies are required to see if these cells participate in HIV-specific cytotoxicity or reflect an aberrant, ineffective, or immunologically detrimental response to the virus.

Provocation-challenge studies in shrimp-sensitive individuals.

Thirty individuals with history of immediate, objective, adverse reactions after shrimp ingestion underwent double-blind, placebo-controlled shrimp-food challenges. All individuals who did not exhibit a positive response (reproduction of objective symptoms) were administered an open challenge of 16 whole cooked shrimp. Positive challenge responses occurred in 9/30 subjects (30%); six of these subjects experienced a positive response during the double-blind phase. Of the 21 remaining subjects, 12 experienced generalized pruritus as their only symptom, whereas nine subjects had completely negative challenge responses. All placebo challenges were negative. Although a positive skin test was strongly associated with challenge symptoms (p less than 0.001), the shrimp prick skin test titration end points were not different among the challenge groups. The serum shrimp RAST percent was significantly higher in the positive challenge group (p less than 0.02). Mean levels of shrimp-specific serum IgG, IgA, and IgM levels were not different among the challenge groups. Although no single immunologic variable could be consistently used to identify subjects more likely to exhibit a positive challenge response, the composite of a positive shrimp prick skin test and elevated serum shrimp-specific IgE (RAST percent label bound greater than 11%) demonstrated a correct predictive value of 87% in this group of shrimp-sensitive subjects.

Human immunodeficiency virus infection in hemophiliac patients. A three-year prospective evaluation.

Thirty-seven heterosexual hemophiliac patients underwent prospective evaluation with clinical examinations, serologic studies for antibody to human immunodeficiency virus (HIV), and tests of immune function for an average of 37 months. At the time of entry into the study in 1982 to 1983, 18 subjects (49 percent) were already seropositive for HIV and 11 (30 percent) had persistent generalized lymphadenopathy. Seventy percent of the total population were clinically asymptomatic. In nine subjects, seroconversion occurred during the study such that 81 percent of the population was seropositive at the conclusion. During the same period, lymphadenopathy developed in six subjects, there was progression to AIDS-related complex (ARC) in four, and acquired immunodeficiency syndrome (AIDS) developed in one patient. Thus, at the end of the study, 54 percent were clinically asymptomatic, 32 percent had persistent lymphadenopathy, and 11 percent had ARC. Subjects who remained seronegative had received less factor concentrate than seropositive subjects, remained asymptomatic, and had normal results on tests of immune function. In those who had experienced seroconversion, there were decreased absolute numbers of CD4+ lymphocytes prior to seroconversion, and abnormalities of lymphocyte function developed after seroconversion. The development of persistent generalized lymphadenopathy was associated temporally with seroconversion. The presence of persistent generalized lymphadenopathy did not appear to be associated with an increased risk for AIDS in seropositive persons, since the condition of most hemophiliac patients with persistent generalized lymphadenopathy at the time of initial evaluation remained clinically and immunologically stable. In contrast to patients with persistent generalized lymphadenopathy, asymptomatic seropositive subjects had progressive abnormalities of lymphocyte function over time that were independent of the numbers of CD4+ cells in the peripheral blood.

Seroconversion to human immunodeficiency virus (HIV) in hemophiliacs. Relation to lymphadenopathy.

The authors studied the natural history of human immunodeficiency virus (HIV) exposure in 187 hemophiliacs followed for an average of 45 months. Overall, 55 percent developed antibody specific for HIV and 21 percent developed persistent generalized lymphadenopathy. Most patients seroconverted sometime between early 1982 and the end of 1984. Four patients developed acquired immune deficiency syndrome (AIDS) and four seropositive patients developed idiopathic thrombocytopenia (ITP). One of the four patients who developed AIDS and three of the four with ITP had preexisting lymphadenopathy. None of the 10 patients with lymphadenopathy or the 20 asymptomatic patients was seropositive for human T-lymphotropic virus, type I. Although seropositivity and lymphadenopathy have been found in many of the authors' patients, few have developed clinical disease that can be related to HIV infection.

Immunologic evaluation of shrimp-allergic individuals.

Thirty-three individuals with a history of immediate hypersensitivity reactions after shrimp ingestion and 29 nonshrimp-sensitive control subjects were evaluated for evidence of crustacea-specific immunity by skin prick test titration end point, RAST, and ELISA, with extracts of shrimp, crab, crayfish, and lobster. Individuals were categorized as either atopic or nonatopic on the basis of history and skin test reactivity to common inhalant allergens. Most (28/33) shrimp-sensitive subjects had positive skin prick tests to shrimp extract, whereas skin tests were negative in 27/29 control subjects. Eighty-one percent of atopic and 41% of nonatopic shrimp-sensitive subjects had elevated shrimp-RAST ratios. The RAST ratios of atopic individuals were significantly higher than ratios of nonatopic individuals, and there was a significant correlation between shrimp-RAST ratios and historical clinical symptom scores. RAST determinations of all control subjects were negative. Shrimp-sensitive subjects also had significantly elevated serum levels of shrimp-specific IgG and IgA as compared to control individuals. Both IgG and IgA shrimp-specific reactivity demonstrated a significant positive correlation with shrimp-RAST ratios. These studies indicate that IgE-mediated, type I mechanisms, detected by positive shrimp skin tests and RASTs, appear to be operative in crustacea-sensitive individuals, particularly those with concurrent respiratory allergy. Although the role of shrimp-specific IgG and IgA antibodies in the immunopathogenesis of crustacea allergy remains unclear, such antibodies appear to represent increased immunologic recognition of shrimp allergens/antigens in shrimp-sensitive subjects.