Donald R. Hoffman

Immunochemical identification of the allergens in egg white

The allergenic activities of egg white components were tested by radioimmunoelectrophoresis (RIEP) and RAST with highly purified proteins. Sera from 33 patients who reacted to egg ingestion were used for both methods. Ovalbumin and ovomucoid were shown to be strong allergens by both techniques. Patients varied in relative reactivity to these two allergens. Conalbumin was found to be an important allergen by RAST, but its activity was underestimated by RIEP. Lysozyme was only a weak allergen. Two sera reacted by RIEP with other neutral egg white components. Both ovalbumin and ovomucoid were found in hard- and soft-boiled egg white in immunologically recognizable form, and small amounts of conalbumin were also detectable in cooked egg. There was no significant association of pattern of reactivity with age, sex, or presence of eczema. The sera of patients who experienced gastrointestinal symptoms from ingestion of egg white were less reactive with ovomucoid than sera from patients with other symptoms.

Allergens in hymenoptera venom. XXVI: The complete amino acid sequences of two vespid venom phospholipases.

The 2 major allergenic proteins in vespid venoms are antigen 5s and phospholipases (PLs). Vespid PLs have a molecular weight of about 34,000 and have been previously shown to have an A1B specificity, unlike the A2 specificity of bee PLs. The complete amino acid sequences of the venom PL from the yellow jacket, Vespula maculifrons, and the more acidic isoenzyme from the white faced hornet, Dolichovespula maculata, have been determined by sequencing overlapping peptides isolated from enzyme and chemical digests of the proteins. Ves m 1 is composed of 300 amino acids, with variants found at 3 positions. Dol m 1.02 is composed of 303 amino acids with variants at 2 positions. Comparison with the sequence of Dol m 1.01 determined by cDNA sequencing gave 66.7% identity with Dol m 1.01 with almost all variation in the first 131 positions. Ves m 1 showed 69% identity with Dol m 1.01 and 38.7% with Dol m 1.02. Comparison of the PL sequences with the Protein Identification Resource data base showed many similarities with the lipase family, but very little relationship to known PLs. The amount of structural similarity between Dol m 1 and Ves m 1 is similar to that found among antigen 5 molecules of different genera, and is sufficient to account for antigenic cross-reactivity. There appear to be a number of highly conserved regions of the PL molecules, especially in the C-terminal 168 residues. These regions may from common epitopes found in several species and genera of vespids. Antibodies against these common epitopes will not be able to distinguish among PLs from various vespids.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens in Hymenoptera venom. XXV: The amino acid sequences of antigen 5 molecules and the structural basis of antigenic cross-reactivity.

The complete amino acid sequences have been determined by solid-phase protein sequencing for eight different vespid venom antigen 5 molecules. These include five species of yellow jackets, Vespula squamosa, V. flavopilosa, V. germanica, V. pensylvanica and V. vidua, representing all three species groups; two variants from the European hornet, Vespa crabro; and a species of paper wasp, Polistes fuscatus, from a second subgenus. The new sequences were compared with the seven previously published sequences from yellow jackets, hornets, and wasps, and to that of Solenopsis invicta 3 allergen from imported fire ant venom. These comparisons provided structural evidence to support the observed high degree of cross-reactivity among the antigens of the common group of yellow jackets and among those of the two common North American subgenera of paper wasps studied. The antigen 5 of V. squamosa and of V. vidua were significantly different from those of the vulgaris group. Common features that could generate immunologic cross-reactivity were seen among the antigen 5 molecules of hornets of both genera and among those of yellow jackets, hornets, and paper wasps. The imported fire ant allergen has only minimal conserved areas in common with the vespid allergens, which explains the lack of observed IgE cross-reactivity. These results provide the structural basis for the cross-reactivity patterns observed in clinical practice and suggest that the commercial extracts of yellow jacket and paper wasp could be prepared with fewer carefully selected species.

Hymenoptera venom allergens.

Hymenoptera venoms each contain a variety of protein allergens. The major components have all been characterized, and most of the amino acid sequences are known. This article concentrates on the use of contemporary techniques including cloning, mass spectrometry and genomics in the characterization of venom allergens, and newer separation techniques for protein isolation. Examples of the use of these techniques with venom proteins are presented.

Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins

BACKGROUND Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. OBJECTIVE We sought to characterize the allergen biochemically and immunologically. METHODS The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. RESULTS The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. CONCLUSIONS Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

Allergens in Hymenoptera venom XXIV: the amino acid sequences of imported fire ant venom allergens Sol i II, Sol i III, and Sol i IV

The most common cause of insect venom allergy in the Southeastern United States is the imported fire ant. The allergens are among the most potent known, with nanogram doses causing sensitization and provoking anaphylaxis. The complete amino acid sequences of imported fire ant venom allergens, Sol i II, III, and IV, were determined by solid-phase protein sequencing of overlapping peptide fragments. Sol i II has a single sequence of 119 amino acids and a molecular weight of 13,217. It has seven cysteine residues, and in its native form is a disulfide-linked dimer. The highly purified molecule does not have phospholipase activity and is not structurally related to phospholipases or other known proteins. Sol i IV has 117 amino acids, for a molecular weight of 13,340. It has six cysteines and is a monomer. Its sequence is 35% identical to Sol i II, but it is not significantly related to other proteins. The Sol i IV sequence showed two amino acid variations. Sol i III was found to consist of 212 amino acids of molecular weight 24,040 in good agreement with 26,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence contained eight cysteine residues and was found to be 44% to 50% identical to five vespid venom antigen 5 molecules. IgE antibodies against Sol i III do not exhibit strong cross-reactivity with vespid antigen 5s. The sequence similarity is consistent with other data, suggesting that ants are related to wasps of the superfamily Vespoidea.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens in Hymenoptera venom: XXI. Cross-reactivity and multiple reactivity between fire ant venom and bee and wasp venoms

The relationships between fire ant venom and bee and wasp venoms were explored by studying sera from five groups of subjects. Group 1 included adults not allergic to any venoms and who were not exposed to fire ants. Group 2 included adults with fire ant exposure who were not allergic to venoms. Group 3 included patients with recent systemic reactions to fire ant venom. Group 4 included patients allergic to bee and vespid venoms with no fire ant exposure. Last, group 5 included patients allergic to bee and vespid venoms with fire ant exposure. None of the serum samples from group 1 was RAST reactive to fire ant venom, but 24% of those from group 2 were fire ant positive, as were 100% of those from group 3, 51% of those from group 4, and 87% of those from group 5. The RAST-positive patients in groups 2 and 5 were also skin test positive. RAST inhibition studies demonstrated cross-reactivity in some cases and multiple reactivity in others. The serum samples were further investigated via nondenaturing electrophoretic immunoblot studies and RAST with highly purified allergens. Serum samples from group 4 reacted to a single band on immunoblots and with only one of the four purified allergens from fire ant venom (Solenopsis invicta I, or Sol i I). Serum samples from groups 2, 3, and 5 showed various patterns of allergen reactivity. All serum samples from patients allergic to fire ant venom who also reacted to bee and/or vespid venoms by RAST contained IgE antibodies binding to Sol i I.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens in Hymenoptera venom: XX. Isolation of four allergens from imported fire ant (Solenopsis invicta) venom

Commercial Solenopsis invicta (Sol i) venom was fractionated by gel filtration and high-performance cation exchange chromatography. Four proteins were isolated and purified to homogeneity. The four proteins were tested with a panel of sera from patients allergic to fire ant venom; all proteins had significant allergenic activity. These proteins corresponded to four of the bands we previously reported to be allergenic by immunoblot analysis. Sol i I has an apparent molecular weight of 37,000 daltons and yields bands of 18,000, 16,500 and 14,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; cation-exchange chromatography indicates that there are three charge forms. Sol i II has a native molecular weight of 28,000 daltons and appears to be easily cleaved into half molecules; it is a phospholipase structurally unlike either bee or wasp phopholipases. Sol i III has a native and denatured molecular weight of 26,000 daltons. Sol i IV has an apparent native molecular weight of 20,000 daltons and gives a single chain of 15,000 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sol i II and III are the major proteins in the venom; there are only small amounts of Sol i I and IV. All are significant allergens, and patients are found who react most strongly with each. Regression analysis of RAST data with highly purified allergens indicated that the IgE responses to the allergens were not related to each other. Amino acid compositions indicated that the four allergens were distinct and that the allergens were structurally different from each other. Four proteins identical to Sol i I to IV were isolated from hand-milked pure venom.

The natural history of exposure to the imported fire ant (Solenopsis invicta).

BACKGROUND Imported fire ants (IFA) are a common cause of insect venom hypersensitivity in the southeastern United States. The purpose of this study was to determine the sting attack rate and development of specific IgE in an unsensitized population. METHODS Study participants consisted of 137 medical students with limited exposure to IFA-endemic areas who were temporarily training in San Antonio, Tex. Subjects were surveyed for prior IFA exposure with a questionnaire, and IFA-specific IgE was evaluated with RAST and intradermal skin testing. Evaluations were performed on arrival and reported at departure from the endemic area 3 weeks later. RESULTS One hundred seven subjects completed the study. Field stings were reported in 55 subjects, resulting in a sting attack rate of 51%. In these 55 subjects 53 (96%) reported a pustule or a small local reaction at the sting site, one (2%) reported an isolated large local reaction, and none reported a systemic reaction. At the 3-week follow-up skin test and RAST conversions occurred in seven subjects (13%) and in one subject (1.8%), respectively. CONCLUSIONS Even brief exposures to IFA-endemic areas result in significant sting rates and concurrent rapid development of IFA-specific IgE in 16% of stung subjects.

Allergens in Hymenoptera venom XI. Isolation of protein allergens from Vespula maculifrons (yellow jacket) venom

Pure Vespula maculifrons venom was demonstrated to contain five major allergenic proteins, which were all isolated from commercial venom sac extract. The five proteins: Vmacl, MW 97,000; hyaluronidase, MW 46,000; Vmac3, MW 39,000; phospholipase A and B, MW 34,000; and antigen 5, MW 22,000 were all demonstrated to be biochemically and immunologically distinct. All five proteins had significant allergenic activity, with phospholipase and hyaluronidase demonstrating the most IgE binding with 39 sera from allergic patients. Sera from honeybee-reactive patients, who had weak cross-reactions with yellow jacket venom, demonstrated strong IgE binding to purified V. maculifrons hyaluronidase and Vmacl. Dose-dependent inhibition of RAST was observed by use of honeybee hyaluronidase and high-molecular-weight fraction to inhibit the binding to the corresponding yellow jacket allergen.