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Background: Allergic reactions to fish are a common cause of food allergy., Objective: We compared the binding of pediatric and adult fish-allergic patient IgE antibodies to fish proteins., Methods: Clinical histories of fish allergy were confirmed by prick skin tests, RAST and if possible, with blinded oral food challenges. The patients included five children with severe allergic reactions to catfish (4/5), cod (1/5), and tuna (1/5) and five adults with severe allergic reactions to catfish (5/5), cod (2/5), snapper (3/5), and tuna (2/5). Extracted proteins from catfish, cod, snapper, and tuna were separated with SDS-PAGE. IgE immunoblots and immunoblot inhibition studies were performed using serum sample from these patients., Results: Multiple fish proteins ranging from 12 to 45 kD from the four fish extracts were identified by SDS-PAGE. A major protein (12.5 kD) was present in all fish extracts except for raw tuna. Immunoblots using individual pediatric and adult serum samples revealed that the major IgE binding was to the 12.5-kD protein from catfish, cod, and snapper. The immunoblot with tuna using serum from a pediatric patient with isolated tuna anaphylaxis revealed an IgE binding protein band at 40 kD. Preincubation of serum samples from two separate fish-allergic patients with 1 mg of cod fish extract completely inhibited IgE binding to the 12.5-kD fish protein in subsequent immunoblots., Conclusions: Pediatric and adult fish-allergic patients have similar in vitro IgE binding to a 12.5-kD protein from fish extracts. This protein is immunochemically similar to Gad c I, the major allergen in cod.

Sixty-four unrelated patients with infantile Krabbe disease (globoid cell leukodystrophy, GLD) of Dutch (n = 41) or other European origin (n = 23) were screened for the presence of a large 30 kb deletion starting in intron 10 (IVS10del30 kb), a base substitution 1538T(T513M) and a polymorphism, 502T. The deletion and the T513M mutation were present in 52% and 8.5%, respectively, of the 82 GALC alleles of the Dutch patients. The 502T polymorphism, which had an allele frequency of 5.3% in a Dutch control panel, occurred in 65% of the GLD alleles. Analysis of patients and both parents in 26 of the families showed that del30 kb was invariably associated with 502T. However, 502T was also present on 40% of the GLD alleles with an as yet unidentified mutation, which is 7.5 times higher than its frequency in controls. This suggests that besides del30 kb at least one other relatively frequent mutation has arisen on the 502T GALC allele. A relatively high incidence of del30 kb was also found in 23 other European (non-Dutch) patients (allele frequency 35%), but T513M did not occur in this group. Practical examples described in this report illustrate the potential usefulness of mutation analysis in many families with Krabbe disease for heterozygote detection and prenatal diagnosis.

A major peanut allergen, Ara h 2, is recognized by serum IgE from > 90% of patients with peanut hypersensitivity. Biochemical characterization of this allergen indicates that it is a glycoprotein of approximately 17.5 kDa. Using N-terminal amino acid sequence data from purified Ara h 2, oligonucleotide primers were synthesized and used to identify a clone (741 bp) from a peanut cDNA library. This clone was capable of encoding a 17.5-kDa protein with homology to the conglutin family of seed storage proteins. The major linear immunoglobulin E (IgE)-binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from 15 peanut-sensitive patients. Ten IgE-binding epitopes were identified, distributed throughout the length of the Ara h 2 protein. Sixty-three percent of the amino acids represented in the epitopes were either polar uncharged or apolar residues. In an effort to determine which, if any, of the 10 epitopes were recognized by the majority of patients with peanut hypersensitivity, each set of 10 peptides was probed individually with serum IgE from 10 different patients. All of the patient sera tested recognized multiple epitopes. Three epitopes (aa27-36, aa57-66, and aa65-74) were recognized by all patients tested. In addition, these three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 protein. Mutational analysis of the Ara h 2 epitopes indicate that single amino acid changes result in loss of IgE binding. Two epitopes in region aa57-74 contained the amino acid sequence DPYSP that appears to be necessary for IgE binding. These results may allow for the design of improved diagnostic and therapeutic approaches to peanut hypersensitivity.

Peanut allergy is a significant health problem because of the prevelance and potential severity of the allergic reaction. Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the IgE-binding epitopes on the major peanut allergen, Ara h 1. At least twenty-three different linear IgE-binding epitopes, located throughout the length of the Ara h 1 protein, were identified. All of the epitopes were 6-10 amino acids in length, but there was no obvious sequence motif shared by all peptides. Four of the peptides appeared to be immunodominant IgE-binding epitopes in that they were recognized by serum from more than 80% of the patients tested and bound more IgE than any of the other Ara h 1 epitopes. Mutational analysis of the immunodominant epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgE-binding characteristics. The identification and determination of the IgE-binding capabilities of core amino acids in epitopes on the Ara h 1 protein will make it possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

Background: Low molecular weight allergens may be responsible for hypersensitivity reactions after the ingestion of wheat., Objective: The purpose of this investigation was to identify relevant, low molecular weight allergens after the ingestion of wheat protein., Methods: Serum samples were collected from seven children with wheat allergy and one adult with baker's asthma. Control serum samples were collected from wheat-tolerant patients. Wheat extracts were prepared and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 12.5% gels revealing numerous protein bands. IgE immunoblot analysis of crude wheat extracts identified multiple IgE-binding proteins. Wheat proteins were separated further with two-dimensional gel electrophoresis, which was followed by IgE immunoblotting investigations., Results: Immunoblot analysis identified a 15 kd wheat protein that bound IgE from all five children with wheat allergy who were evaluated. No IgE binding to this wheat protein was demonstrated in any of the control subjects. Samples representing the 15 kd wheat protein (isoelective point, 5.85) were selected. The N-terminal peptide sequence of this protein (residues 1 to 20) matched to a wheat alpha-amylase inhibitor., Conclusion: These data demonstrate that wheat alpha-amylase inhibitor is a relevant allergen in patients experiencing hypersensitivity reactions after the ingestion of wheat protein. This wheat protein, which has been implicated as an important allergen in patients with baker's asthma, represents a sensitizing allergen after both ingestion and inhalation.

Studies of the effects of yogurt on immunity and atopic diseases have suggested improvements in cytokine (interleukin-2 and interferon-gamma) responses and clinical scores in patients with allergic rhinitis. This study compares prospectively immune parameters of participants who received 16 oz of yogurt versus 16 oz of milk/day in a randomized cross-over design. Yogurt that contained live, active Lactobacillus bulgaricus and Streptococcus thermophilus or 2% milk was consumed for one month each. Twenty otherwise healthy adults with atopic histories documented by skin testing were enrolled. Immune studies were performed at the beginning and end of the two 1-month study phases, separated by a 2-week washout period. These studies included measurements of cellular, humoral, and phagocytic function. No adverse events were noted in either group. No significant improvements in any immune parameter were noted. The consumption of yogurt that contained the live active bacteria L bulgaricus and S thermophilus does not appear to enhance immune function in atopic individuals at the dosage and duration used in this study.

The current investigation focused on lymphoid cell populations of adult male Sprague-Dawley rats fed a total enteral nutrition diet in which ethanol provided 38% of the total calories. Rats received the National Research Council (NRC) recommended daily intake of nutrients 35 days. An evaluation of lymphocyte populations from peripheral blood demonstrated a decrease in the absolute number of B cells (p < or = 0.007) and absolute numbers of CD4 T cells (p < or = 0.06) in the ethanol-treated animals. Spleen and thymus weights were significantly reduced (p = 0.0001) in the ethanol-treated rats and the CD4/CD8 ratio of splenic lymphocytes decreased in the ethanol group (p < or = 0.03). Thymus T-cell recovery from the ethanol-treated group was significantly reduced with no apparent redistribution in subset numbers with the exception of a minor, yet significant, decrease (p < or = 0.05) in the CD4/CD8 ratio. These data are the first to demonstrate that chronic alcohol intake alters lymphoid cell populations in the peripheral blood and primary organs of the immune systems in the presence of adequate nutrition.

Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly (A)+ RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in E. coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general

Peanut allergy is a significant health problem because of the frequency, the potential severity, and the chronicity of the allergic sensitivity. Serum IgE from patients with documented peanut hypersensitivity reactions and a peanut cDNA expression library were used to identify clones that encode peanut allergens. One of the major peanut allergens, Ara h I, was selected from these clones using Ara h I-specific oligonucleotides and polymerase chain reaction technology. The Ara h I clone identified a 2.3-kb mRNA species on a Northern blot containing peanut poly A+RNA. DNA sequence analysis of the cloned inserts revealed that the Ara h I allergen has significant homology with the vicilin seed storage protein family found in most higher plants. The isolation of the Ara h I clones allowed the synthesis of this protein in Escherichia coli cells and subsequent recognition of this recombinant protein in immunoblot analysis using serum IgE from patients with peanut hypersensitivity. With the production of the recombinant peanut protein it will now be possible to address the pathophysiologic and immunologic mechanisms regarding peanut hypersensitivity reactions specifically and food hypersensitivity in general.

Background: Skin prick testing is a major tool for diagnosing food allergy. Food allergen extracts have not been standardized; this may lead to great variability in the predictive accuracy of skin prick tests., Methods: Six commercial peanut skin test extracts were compared in vitro with RAST inhibition assays, ELISA, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with sera from peanut-allergic adults and in vivo by skin prick testing., Results: ELISA showed that the content of peanut allergens Ara h I and Ara h II in the extracts ranged from 0.0015 to 0.0236 and 0.0001 to 0.0164 mg Eq/ml, respectively. RAST inhibition studies showed that the extracts produced curves of similar slope, suggesting conservation of allergenic epitopes. SDS-PAGE revealed differences in protein profiles because roasted extracts generally possessed the same number and proportion of major protein bands but raw extracts varied more in both respects. SDS-PAGE and immunoblotting showed that two of the extracts contained major IgE-binding protein bands that did not appear in the others. One roasted extract gave little protein banding and consequently little IgE binding., Conclusions: Skin testing results showed no differences in the ability of the extracts to provoke a positive skin test response in peanut-sensitive subjects.

The antigenic and allergenic structure of Ara h II, a major allergen of peanuts, was investigated with the use of four monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h II. Our previous studies with monoclonal antibodies generated to peanut allergens showed this method to be useful for epitope mapping. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive peanut challenge responses. The Ara h II monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified two different antigenic sites on Ara h II. In similar studies with pooled human IgE serum from patients with positive challenge responses to peanuts, we identified two closely related IgE-binding epitopes. These characterized monoclonal antibodies to Ara h II will be useful for future studies to immunoaffinity purify the Ara h II allergen and to use in conjunction with recombinant technology for determining structure-function relationships.

We evaluated the prevalence and characteristics of the principal foods implicated in 355 children diagnosed with IgE-mediated food allergy. Diagnosis was established on the basis of positive clinical history for the offending food, positive specific IgE by skin prick test and RAST, and open food challenge. Our results showed the principal foods involved in allergic reactions are: eggs, fish, and cow's milk. These are followed in frequency by fruits (peaches, hazelnuts and walnuts), legumes (lentils, peanuts and chick peas) and other vegetables (mainly sunflower seeds). The legumes demonstrated the highest degree of clinical cross-reactivity. Most patients with food allergy reacted to one or two foods (86.7%). Only 13.3% of patients reacted to 3 or more foods, mostly to legumes and fruits. We found that food allergy begins most frequently in the first (48.8%) and second (20.4%) years of life. Allergy to proteins of cow's milk, egg, and fish begins predominantly before the second year, demonstrating a clear relationship with the introduction of these foods into the child's diet. Allergy to foods of vegetable origin (fruits, legumes and other vegetables) begins predominantly after the second year.

Peanuts and soybeans are frequent causes of food hypersensitivity reactions in children. Sera from 12 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to peanut and sera from 5 patients with atopic dermatitis and a positive double-blind placebo-controlled food challenge to soybean were used to identify and characterize specific legume allergens. Identification of a minor allergen from peanut and a minor allergen from soybean was accomplished using various physicochemical techniques. The peanut fraction, peanut agglutinin, isolated by anion-exchange chromatography and electrolution and confirmed by amino acid sequencing, bound IgE in only 50% of the peanut challenge positive patients. The soybean fraction, soybean trypsin inhibitor, identified by gel filtration and electroelution and confirmed by amino acid sequencing, bound IgE in only 20% of the soy challenge positive patients. The identification of these two known legume proteins as minor allergens should allow further immunologic and structural investigations to compare the major and minor legume allergens.

The antigenic and allergenic structure of Ara h I, a major allergen of peanuts, was investigated with the use of seven monoclonal antibodies obtained from BALB/c mice immunized with purified Ara h I. Previous work with monoclonal antibodies produced to allergens has primarily been done with inhalant allergens. Only recently have the major allergens of various foods been determined so that investigations with monoclonal antibodies into the allergenic epitopes could begin. When used as a solid phase in an ELISA, these monoclonal antibodies captured peanut antigen, which bound human IgE from patients with positive results to challenges to peanuts. The Ara h I monoclonal antibodies were found to be specific for peanut antigens when binding for other legumes was examined. In ELISA inhibition studies with the monoclonal antibodies, we identified four different antigenic sites on Ara h I. In related studies with pooled human IgE serum from patients with positive results to challenges to peanuts, we identified three similar IgE-binding epitopes. As a means of purifying the Ara h I allergen, we prepared an immunoaffinity column with monoclonal antibody 8D9. We eluted from this column the allergen Ara h I, which had a mean molecular weight of 63.5 kd and which bound human IgE from individual and pooled serum of patients with peanut sensitivity.

Cockroach allergens have been established as important sensitizing agents in the induction of asthma. In the present investigation, we identified a 36-kD allergen from German cockroach (Blattella germanica) using fast protein liquid chromatography that reacted in the Bla g II monoclonal assay developed by Pollart et al. Defatted whole body German cockroaches were extracted in phosphate-buffered saline and fractionated using gel filtration (Superdex 75) and anion exchange (MonoQ) chromatography. The 36-kD allergen was isolated from this enriched allergen preparation by excision from modified preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroelution, dialysis and lyophilization. SDS-PAGE/Western immunoblot analysis using a serum pool of cockroach-sensitive individuals revealed a single band. The lyophilized 36-kD allergen was then analyzed for amino acid composition and digested for peptide amino acid sequencing.

The current study was undertaken to examine the potential role of T cells in the pathogenesis of peanut allergy. Peripheral blood mononuclear cells (PBMCs) from patients with peanut allergy, patients with asthma, and nonatopic normal control subjects were assessed for proliferation after stimulation with a 17 kd major peanut allergen (Ara h II), ovalbumin, casein, soy, and Candida albicans. We found that Ara h II and C. albicans induced significantly higher levels of proliferation than ovalbumin, casein, and soy. Because interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) play critical roles in IgE regulation, we assessed the production of these cytokines after stimulation with C. albicans and Ara h II. C. albicans stimulated similar levels of IFN-gamma in all three study groups. In contrast, after stimulation with Ara h II, culture supernatants from PBMCs of subjects with peanut allergy contained significantly lower levels of IFN-gamma than did the PBMCs of the two control groups (p = 0.02). More important, there was a significant (p = 0.05) inverse correlation between the serum IgE anti-Ara h II levels and IFN-gamma production by PBMCs from the respective peanut-allergic patients. IL-4 protein was not detected in culture supernatants of PBMCs stimulated with Ara h II. However, amplification of cytokine gene transcripts by polymerase chain reaction did demonstrate IL-4 expression in Ara h II-stimulated PBMCs from both patients with peanut allergy and control subjects. These data suggest that the level of IFN-gamma production in response to Ara h II may be an important factor in determining the development of peanut-specific IgE responses.

The Air Sentinel and polytetrafluoroethylene (PTFE) membranes were used to capture airborne particles over living colonies of German or American cockroaches. Silver-stained SDS-PAGE gels revealed protein bands at 80, 55, 36, and several bands below the 33-kD marker. SDS-PAGE/immunoblots of PTFE eluates from German cockroach colonies incubated with serum from cockroach-sensitive individuals revealed IgE-binding bands with apparent molecular weights of 36 and 80 kD. Only the 36-kD allergen and allergens below the 33-kD marker were evident in the American PTFE eluate. ELISA analysis with a monoclonal antibody assay identified the presence of both Bla g I and Bla g II in the German PTFE eluate. No Bla g I or Bla g II could be identified in the American PTFE eluate. These studies demonstrate that in addition to Bla g I and Bla g II, several other aerosolized allergens become airborne over cockroach colonies and may be important in the environment where cockroaches are abundant.

Peanuts are frequently a cause of food hypersensitivity reactions in children. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used in the process of identification and purification of the peanut allergens. Identification of a second major peanut allergen was accomplished with use of various biochemical and molecular techniques. Anion exchange chromatography of the crude peanut extract produced several fractions that bound IgE from the serum of the patient pool with positive challenges. By measuring antipeanut specific IgE and by IgE-specific immunoblotting we have identified an allergic component that has two closely migrating bands with a mean molecular weight of 17 kd. Two-dimensional gel electrophoresis of this fraction revealed it to have a mean isoelectric point of 5.2. According to allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature this allergen is designated, Ara h II (Arachis hypogaea).

Peanuts and soybeans are two of the six most common foods to cause food hypersensitivity reactions in children. We used the serum of 10 patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to peanut and two patients with atopic dermatitis and positive double-blind, placebo-controlled, food challenges to soybean to investigate the change in IgE-specific and IgG-specific binding to these proteins altered by either chemical or thermal denaturation. We used IgE- and IgG-specific ELISA-inhibition analyses to compare these effects on the crude peanut and crude soy extracts, as well as on the major allergenic fractions of both proteins. Heating the soy proteins at various temperatures and time intervals did not significantly change the IgE- or IgG-specific binding of the soy positive pooled serum. When the peanut proteins were subjected to similar heating experiments, the IgE- and IgG-specific binding did not change. When these same proteins were treated with enzymes in the immobilized digestive enzyme assay system used to mimic human digestion, the binding of IgE to the crude peanut and crude soy extracts was reduced; 100-fold for peanut and 10-fold for soybean. Therefore it appears that thermal denaturation of peanut and soybean protein extracts does not enhance or reduce IgE- and IgG-specific binding activity. Chemical denaturation appears to minimally reduce the binding of these proteins.

Peanuts are among the most common causes of immediate hypersensitivity reactions to foods. Serum from nine patients with atopic dermatitis and a positive double-blind, placebo-controlled, food challenge to peanut were used to begin the process of identification and purification of the major peanut allergens. Identification of a major peanut allergen was accomplished by use of anion-exchange column chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ELISA, thin-layer isoelectric focusing, and IgE-specific immunoblotting. Anion-exchange chromatography revealed several fractions that bound IgE from the serum of the challenge-positive patient pool. By measuring antipeanut-specific IgE in the ELISA and in IgE-specific immunoblotting, we identified an allergenic component with two Coomassie brilliant blue staining bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a mean molecular weight of 63.5 kd. Examination of this fraction by the IgE antipeanut ELISA with individual serum and by the ELISA-inhibition assay with pooled serum, we identified this fraction as a major allergen. Thin-layer isoelectric focusing and immunoblotting of this 63.5 kd fraction revealed it to have an isoelectric point of 4.55. Based on allergen nomenclature of the IUIS Subcommittee for Allergen Nomenclature, this allergen is designated, Ara h I (Arachis hypogaea).

Asthma epidemics in Barcelona, Spain, have been attributed to dust generated by the unloading of soybeans in the harbor. Sera of four different groups of 10 subjects in each group were studied: (1) patients attending an emergency room in Barcelona for an asthma attack on epidemic days, group A, (2) patients attending an emergency room for an attack on nonepidemic days, group B, (3) patients with asthma from other cities, group C, and (4) patients without asthma from Barcelona matched by age and sex with group A, group D. All subjects in group A had IgE to allergens in extracts of various soybean samples. In contrast, only one of the 10 subjects in each of groups B and C and none of those subjects in group D had IgE to uncleaned bean and hull extracts. Radioimmunoassay demonstrated that in sera from patients with asthma during an asthma outbreak reacted primarily to soybean hull and dust extracts. Sodium dodecyl sulfate-polyacrylamide and gel electrophoresis thin-layer isoelectrofocusing demonstrated protein bands of 97.4 to less than 14.4 kd and isoelectric point between 6 and 3.5. By Western blot and thin layer isoelectrofocusing/blotted radioimmunoisoelectrofocusing, IgE of patients with asthma during an asthma outbreak reacted weakly to two protein bands of molecular weight ranging from 42 to 21 kd, strongly to glycoprotein bands with molecular weight less than 14.4 kd, and isoelectric point less than 6, which appeared to be the major allergens.

Africanized honeybees (HBs) pose a hazard to both normal and sting-sensitive subjects in certain areas of Central and South America, and it is predicted that they will soon be present in the southern United States as well. Using an electrical stimulation device, we collected Africanized HB venom (AHV) in Venezuela and European HB venom (EHV) in Louisiana. These venoms, along with commercial European HB venom (CHV), were compared by thin-layer isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Coomassie brilliant blue and silver-stained banding patterns of AHV and EHV were essentially identical to CHV. Western blots were prepared from SDS-PAGE gels and tested with pooled sera from EHV-sensitive subjects and then radiolabeled antihuman IgE. The resulting autoradiographs revealed similar banding patterns among EHV, AHV, and CHV. RAST-inhibition studies were performed with solid-phase CHV and pooled sera from EHV-sensitive subjects. The specific allergenic activities of the three HB venoms (allergy units per milligram of protein) were comparable. By RAST-inhibition assay with solid-phase, highly purified individual venom components, AHV and EHV both contained phospholipase A2, hyaluronidase, and high-molecular-weight allergens. The increased morbidity after Africanized HB stings is likely related to their more defensive behavior during which many bees react by stinging rather than to biochemical or allergenic differences between AHV and EHV.

Whole-body extracts of the feral and peridomestic Asian cockroach (Blattella asahinai) and the three domestic cockroach species, German (Blattella germanica), American (Periplaneta americana), and Oriental (Blatta orientalis), were compared allergenically using an IgE serum pool from 4 German cockroach sensitive individuals. In crossover radioallergosorbent inhibition analysis, the Asian cockroach shared allergenic activity primarily with the German cockroach polymer and to a lesser extent with either the American or Oriental cockroach polymers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thin-layer isoelectric focusing analysis of the extracts showed similar although varying intensities of Coomassie blue stained banding patterns among five extracts analyzed. Electroblotting analysis with 12.5% SDS-PAGE of the whole-body German cockroach extract and IgE serum from individuals sensitive to German cockroach revealed eight allergenic components with apparent molecular weights of 92, 80, 67, 48, 36, 27, 25 and 18 kD. Five components could be identified in the whole-body extract of the Asian cockroach corresponding to apparent molecular weights of 92, 67, 48, 40, and 32 kD. Analysis of individual serum by immunoblot analysis with each of the cockroach extracts showed considerable heterogenicity in the IgE-binding pattern. Although the Asian cockroach demonstrated considerable cross-reacting allergenic components to German and relatively fewer cross-reacting allergenic components to either the Oriental or American, it is too early to establish genus- or species-specific cockroach allergens. It is important to point out that German cockroach sensitive individuals should be made aware of the potential exposure of Asian cockroach aeroallergens both indoors and outdoors in areas with high infestations of Asian cockroaches.

Receptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of various anti-receptor antisera, and characterized by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Most antisera were shown to react with a receptor designated H and which, as shown previously, could be isolated by IgE-Sepharose but not by a combination of IgE and anti-IgE. Only two highly purified anti-receptor antibody preparations, which had been purified by adsorption to and elution from rat basophilic leukaemia cells, reacted primarily with a second kind of receptor molecule which had previously been designated R. Some indirect evidence was obtained which suggests that H is a highly immunogenic molecule. It was confirmed that the apparent molecular weight of H-like molecules varied on different RBL cell lines.

The sensitivity, specificity, and precision of immunoassays for quantitation of IgE in cell-culture supernatants were tested in a multicenter trial involving 22 laboratories. Fourteen coded test samples included cell-culture medium alone, culture medium with varying concentrations of polyclonal or myeloma IgE, and medium from unstimulated or pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cell (PBMC)-culture supernatants. Two laboratories reported measurable IgE in non-IgE-containing control samples. Although the IgE content of a single 0.50 ng/ml polyclonal IgE sample should have been measured easily by the claims of all laboratories, only 13 laboratories measured IgE in this sample in 22 of the 48 assays. Most laboratories could measure both polyclonal and myeloma IgE at 5.0 ng/ml; however, the IgE determinations for the myeloma proteins were nearer the predicted value. Although 15 laboratories found measurable IgE in the PWM-stimulated PBMC-culture supernatants from a single nonatopic donor, the levels did not differ significantly from that measured in unstimulated PBMC-culture supernatants. Three laboratories reported considerably higher IgE levels in the PWM-stimulated PBMC-culture supernatants than in other PBMC-culture supernatants. Only 13 laboratories could quantitate IgE in each of coded duplicate samples containing 0.50 ng/ml polyclonal IgE. These findings indicate a wide variability in the sensitivity, specificity, and precision of assays used to quantitate low levels of IgE protein. Investigators should be encouraged to make greater use of existing national and international reference materials in the standardization and performance of their IgE immunoassays.

Surface proteins and glycoproteins of RBL cells were labelled enzymatically with 125I and then solubilized with Nonidet P-40. Analysis by polyacrylamide-gel electrophoresis in SDS on 10% gel revealed 10 distinctive peaks ranging in molecular weights from 17,000 to 200,000 daltons. Mainly components of higher molecular weights were bound by lentil-lectin Sepharose and could be eluted with alpha-methyl mannoside. The receptor for IgE was clearly shown to bind to the lentil-lectin. A second cell surface component which previously had been shown to bind to IgE-Sepharose as well, was found to bind only slightly to lentil-lectin. Thus, it can be concluded that the receptor for IgE is a glycoprotein with mannose and/or N-acetylglucosamine in the carbohydrate moiety(s).

A lyophilized candidate International Reference Standard of short ragweed pollen extract was prepared by use of defined source material. In preliminary experiments, this extract was demonstrated by RAST inhibition and crossed radioimmunoelectrophoresis assays to contain several well-characterized ragweed allergens and to contain multiple antigenic bands by crossed immunoelectrophoresis analysis. In a subsequent multinational collaborative study involving 12 laboratories in five countries, the candidate extract was compared with existing national reference or commercial ragweed extracts by a variety of immunochemical, biochemical, and physicochemical procedures. The candidate extract could be used to assign relative orders of potency to the comparison-test extracts. In separate studies, the candidate extract was demonstrated to be stable when it was stored at either -20 degrees C or +5 degrees C for at least 2 yr. The candidate extract has been accepted as an International Reference Standard with an assigned arbitrary potency of 100,000 units per ampule .

It has previously been shown that two receptors, H and R, having apparent mol. wt of 55,000 and 45,000 daltons, can be isolated from surface iodinated RBL cells. In addition, small amounts of a third molecule of 71,000 daltons (71 K) can now be detected in receptor preparations isolated by means of IgE-Sepharose. The SDS-PAGE patterns show that the three surface molecules were not significantly altered when cell solubilization was carried out in the presence of protease inhibitors. The same three molecules were also observed when cells were biosynthetically labelled with [3H]-fucose of [3H]-galactose. When cells were labelled with [3H]-leucine or [3H]-tyrosine, several additional molecules were observed in IgE-Sepharose purified preparations. When cells were exposed to free IgE- before solubilization, significant inhibition of binding to IgE-Sepharose could only be seen among molecules corresponding to H, R and 71 K. When receptors were isolated by means of DNP-IgE and anti-DNP-Sepharose, fewer molecules were found. One, which was frequently observed, had a mol. wt of 26,000 daltons. This molecule may be non-covalently associated with receptors for IgE.

A lyophilized candidate extract of Bermuda-grass (Cynodon dactylon) pollen, intended for use as an International Reference Standard, was prepared by pooling four individual candidate extracts. In preliminary investigations, the four candidate extracts encompassed a variety of extraction methods. The collaborative testing program simultaneously analyzed the proposed reference and the individual extracts and included 11 laboratories performing RAST inhibition, histamine release, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, isoelectric focusing, sodium dodecyl sulfate gel electrophoresis, and protein determinations with a variety of reagents and methods. The four candidate extracts and the pooled reference were found to be equivalent. The stability of this extract has also been studied. This International Reference Preparation of Bermuda grass-pollen extract should be useful for research and industry.

Two receptors for IgE, having apparent molecular weights of 55,000 and 45,000 daltons were isolated from rat basophilic leukaemia cells by means of IgE-Sepharose. Both molecules were bound by concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin (castor bean lectin). The lectins of pea and gorse origin only bound the 45,000 dalton receptor.

Peanuts are a common cause of food allergy, but they have infrequently been documented as causing fatal anaphylactic reactions. We review five previously reported fatalities caused by peanut allergy, along with data on IgE binding proteins in extracts of a commercial product containing peanuts that have been deflavored and reflavored and colored to resemble walnuts, pecans, or almonds. Ingestion of this product may pose hazards to peanut-sensitive persons. Finally, we identify several factors that may contribute to the severity and possible lethality of food-induced anaphylaxis.

REceptors for IgE of rat basophilic leukaemia (RBL) cells, maintained in different laboratories were isolated by means of IgE-Sepharose or IgE and anti-IgE, and characterized by SDS-polyacrylamide gel electrophoresis. All cell lines were found to be associated with a receptor molecule (R) which could be isolated either with IgE-Sepharose or IgE and anti-IgE and a second receptor (H) which could only be isolated with the aid of IgE-Sepharose. The relative amounts of these two molecules, as isolated from surface iodinated cells, varied from the RBL cell line to the other and their apparent molecular weights were not identical on all cell lines. Since comparisons were made on the same gel using receptors isolated from cells labelled with different isotopes of iodine, differences in molecular weight must be considered as being intrinsic and not due to methodological variations. These results provide an explanation why differences were observed among receptors for IgE as characterized in various laboratories. In spite of the fact that the various RBL cell lines originated from the same chemically-induced tumour they have, over the years, undergone changes which are reflected in the receptors for IgE.

To further study the immunology of insect hypersensitivity, we identified and partially characterized the principal allergens in whole body German cockroach (WBGCR) (Blattella germanica) and compared this extract to whole body antigens prepared from other insects. WBGCR extract was fractionated over a calibrated Sephadex G-200 column; peak allergenic activity was contained in fraction 3 (GCR3), containing components with apparent molecular weights ranging from 12,500 to 75,000 daltons. The antigenicity, allergenicity, and specificity of GCR3 components were tested by using rabbit antisera raised to GCR3 or true armyworm (Pseudaletia unipuncta). Radioimmunoassay, cross-inhibition and immunoblot studies revealed, particularly in the IgE system, that GCR3 contained an allergen with a pI of 6.7 and MW of 36,000 daltons that was unique to WBGCR extracts and not present in other insect species, including true armyworm, caddis fly, lakefly, yellow jacket, or honeybee. This GCR3 component may represent a specific marker for the diagnosis of cockroach hypersensitivity in an insect-sensitive population of individuals.

As part of a program to establish international standards of selected allergens, 6 coded extracts of Alternaria were assessed in 6 laboratories by immunochemical, biochemical and physicochemical procedures. Direct RAST, RAST inhibition, quantitative skin tests and leukocyte histamine release were used to assign relative orders of potency to the 6 extracts. The composition and major allergen content was tested by thin-layer isoelectric focusing and quantitative immunoelectrophoresis (crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis). Three laboratories determined the quantity of purified allergens in each of the preparations. In addition, source materials were sent to an expert Alternaria taxonomist for independent identification. The results showed considerable variation with respect to total allergenic potency and content of individual allergens. Source materials could not be confirmed as Alternaria in some instances. Based on fulfillment of written specifications and assay results, extract No. 6 was recommended by the Alternaria Working Group as the candidate international standard to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies.

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.

A lyophilized Alternaria extract prepared from a defined eight-strain source material was compared with three other Alternaria extracts to assess its potential suitability as an international standard (IS). Twenty-two laboratories in 12 different countries collaborated. Assay methods included RAST inhibition, quantitative immunoelectrophoresis, thin-layer isoelectric focusing, skin testing, leukocyte histamine release, and various other methods for thorough characterization of the extracts. In addition, three laboratories quantitated specific allergen in each extract. The proposed IS extract could be used to assign a relative potency to other test extracts. In separate studies, the proposed IS extract was demonstrated to be stable for at least 21 months when it is stored at -70 degrees C, -20 degrees C, and 4 degrees C.