Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.

beta-Lactam drug allergens: fine structural recognition patterns of cephalosporin-reactive IgE antibodies.

Lack of experimental findings on the spectrum of cephalosporin allergenic determinants has hindered diagnosis of adverse reactions to these drugs and retarded understanding of allergenic cross-reactions between cephalosporins and between cephalosporins and penicillins. Subjects allergic to the widely used cephalosporin antibiotic cefaclor have serum immuno globulin (Ig) E antibodies that react with the drug. Quantitative hapten inhibition studies employing sera from subjects allergic to cefaclor revealed fine structural recognition differences between the combining site specificities of cefaclor-reactive IgE antibodies in the sera of different subjects. Unlike penicillins, where discrete side chain or thiazolidine ring determinants alone may be recognized, IgE binding determinants on cefaclor encompassed the entire molecule. Fine structural recognition specificity differences at positions R1 (side-chain) and R2 (substituent attached to dihydrothiazine ring) were detected between IgE antibodies in different sera. Some antibodies showed clear preferential recognition of the aminobenzyl group at position R1 and Cl at R2 while with others, a greater degree of recognition tolerance was seen at R1 where, for example, the aminohydroxybenzyl or aminodihydrobenzyl groups were recognized, and at R2 where a methyl or even an ester group was tolerated. As with the penicillins, cephalosporins as allergens cannot simply be considered as a group of compounds with a common allergenic determinant structure. IgE antibodies that bind to cefaclor show great heterogeneity indicated by clear, fine structural differences in recognition of the R1 and R2 groups on the drug.

Allergenic relationship between taxonomically diverse pollens.

BACKGROUND: Skin tests and tests for IgE antibodies show that subjects are usually sensitive to a number of different pollens, frequently from taxonomically diverse species which are assumed to be allergenically non-crossreactive. This suggests that the presence of IgE antibody-reactivity to an individual pollen may not necessarily have resulted from contact with that pollen or even with a taxonomically closely related species. OBJECTIVE: Since this has important consequences for allergen avoidance and desensitization of patients, we attempted to define allergenic relationships between diverse pollen species. METHODS: Sera from subjects were examined in direct IgE antibody binding experiments and by quantitative inhibition, protein blotting and adsorption and elution studies. RESULTS: Sera from subjects diagnosed as allergic to white cypress pine, Italian cypress, ryegrass or birch pollen were shown to have IgE antibodies that reacted with pollens from these four species and from cocksfoot, couch grass, lamb's quarter, wall pellitory, olive, plantain and ragweed. These reactions were confirmed in protein blotting and adsorption and elution studies where numerous IgE-binding bands were detected in all 11 different pollen extracts with sera from each of the different allergic categories. Further evidence of allergenic (i.e. IgE-binding crossreactivity between the different pollens was provided by inhibition studies in which clear-cut inhibitions of IgE binding to the different pollen allergen discs were obtained with comparable amounts of the different pollen extracts. CONCLUSION: We conclude that the presence of pollen reactive IgE antibodies may not necessarily be a true reflection of sensitizing pollen species.

Separation of jumper ant (Myrmecia pilosula) venom allergens: a novel group of highly basic proteins.

The sting of the jumper ant (Myrmecia pilosula) causes severe allergic reactions, including anaphylaxis in sensitized individuals. Two of the major allergens, Myr p I and Myr p II, have been cloned, immunocharacterized and nucleotide-sequenced and they encode 112 and 75 residue polypeptides, respectively. Both allergens are highly basic proteins having isoelectric point values greater than 10. However, electrophoretic analysis has generated conflicting results as to the actual sizes of the allergens in the native venom. Electrophoretic, immunological and N-terminal analyses suggested that these allergens undergo extensive post-translational processing to final forms of 45 and 27 residues, respectively. The results highlight the difficulties in the study of small, basic proteins and polypeptides by electrophoretic techniques.

Cypress pollen allergy. Identification of allergens and crossreactivity between divergent species.

Studies employing sera from 34 subjects allergic to white cypress pine (Callitris glaucophylla) pollen identified 18 IgE antibody-binding components in the pollen of this species, five of which (MWs approximately 94, 68, 64, 43 and 34 kDa) were recognized by all of the sera. Protein blotting and quantitative inhibition studies revealed clear cross-reactivity between C. glaucophylla and Cupressus sempervirens pollen proteins and striking similarities in the IgE recognition band patterns of the two pollens. Inhibition experiments with other pollen extracts revealed that sera from C. glaucophylla pollen-allergic subjects can be divided into two groups--those inhibited only by extracts from the two Cupressaceae pollens and those inhibited both by these pollen proteins and by pollen extracts from other species. Most of the crossreactions in the latter group cannot be explained on the basis of taxonomic relationships or separate sensitizations. As with previous studies on birch and olive pollens, we conclude that pollen allergenic crossreactivity is much more wide-ranging than generally believed.

Immunoaffinity analysis of cross-reacting allergens by protein blotting.

IgE antibodies from sera having reactivity against ryegrass pollen protein allergens, wheat endosperm protein allergens and also several other cereal protein allergens were adsorbed with either ryegrass pollen or the wheat/globulin fraction immobilised on solid phases and subsequently eluted with low pH buffer. The eluted antibodies were reacted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) blots of the different allergens. Antibodies adsorbed and subsequently eluted from the two allergen sources recognised different spectra of proteins in the ryegrass pollen and cereal allergen sources and indicated the degree of immunological cross-reactivity. Intra-species cross-reactivity of IgE antibodies was demonstrated employing similar methods to those used for the pollen and cereal allergens by using a recombinant allergen from the venom of the ant Myrmecia pilosula as the immunoadsorbent protein on the solid phase.

Olive (Olea europea) and privet (Ligustrum vulgare) pollen allergens. Identification and cross-reactivity with grass pollen proteins.

Protein blotting studies showed that three olive pollen components with mol. wts approximately 18-19, 20 and 40 kD can be considered to be major allergens. For privet pollen, the highest recognition frequencies were for allergens of mol. wts approximately 20, approximately 19, approximately 40 and approximately 70 kD. When results with the 62 subjects examined were separated into groups corresponding to their geographical locations, viz. Italy, France and Australia, subjects sensitized to olive, but not other pollens (some Italian subjects), were found to show higher frequencies of recognition of major olive allergens than subjects sensitized to olive pollen via cross-reacting allergens from unrelated pollen sources (the Australian and French subjects). Blotting, adsorption and elution and inhibition studies clearly demonstrated allergenic cross-reactivity (that is, antigenic cross-reactivity detected by IgE antibodies) between olive, privet, ryegrass (Lolium perenne) and couch grass (Bermuda grass: Cynodon dactylon) pollen components. As with our previous findings with birch pollen, we conclude that the presence of pollen-reactive IgE antibodies may not necessarily be a true reflection of the sensitizing pollen species.

Cypress (Cupressus sempervirens) pollen allergens: identification by protein blotting and improved detection of specific IgE antibodies.

On the basis of results of an investigation of the effects of different treatments employed, a dialysed and reduced extract of Cupressus sempervirens was separated electrophoretically on sodium dodecylsulphate-polyacrylamide gels before being transferred and then fixed with glutaraldehyde to nitrocellulose membrane. Probing with sera from 91 subjects allergic to C. sempervirens pollen followed by detection of bound IgE antibodies with [125I]-labelled anti-human IgE revealed 17 IgE-binding proteins in the molecular weight range 14-96 kilodaltons (kDa). One component, of molecular weight approximately 42 kDa, reacted with IgE antibodies in the sera of 81.3% of the allergic subjects and, for each of the subjects, this component bound the greatest quantity of IgE. Almost 50% of the sera recognized only the approximately 42 kDa component, reinforcing the conclusion that this component is the major allergen of C. sempervirens pollen. A comparative study employing C. sempervirens pollen allergen discs prepared commercially or in the laboratory showed that values of the uptakes of [125I]-anti-IgE indicating the presence of pollen-reactive IgE antibodies obtained with the latter discs were consistently higher (means 4.5 vs. 0.88), and that false-negative results were obtained when many sera were used with the commercial discs. The results of this study provide an essential basis for the production of standardized, safe and effective C. sempervirens pollen extract applicable to diagnosis and therapy of cypress pollen allergy.

Crossreactivity of IgE antibodies from sera of subjects allergic to both ryegrass pollen and wheat endosperm proteins: evidence for common allergenic determinants.

Positive RAST (greater than 5% radioactive uptakes) to wheat endosperm proteins were found in approximately one-quarter of subjects who had both a positive skin prick and RAST (greater than 10% radioactive uptake) to ryegrass pollen proteins. Immunoblotting of proteins electrophoretically transferred to nitrocellulose membrane after SDS-PAGE of ryegrass pollen and wheat endosperm proteins confirmed the crossreactive properties of the sera identified by RAST testing. Immunoadsorption of serum IgE onto nitrocellulose membrane, to which ryegrass pollen or wheat endosperm proteins had been adsorbed, removed IgE from crossreactive sera reactive to both ryegrass pollen and wheat endosperm proteins. Elution of the adsorbed IgE from the nitrocellulose membrane after immunoadsorption and probing blotted strips of both ryegrass pollen and wheat endosperm proteins supported the results obtained from the immunoadsorption experiments. This data provides evidence that the crossreactivity of IgE antibodies in sera reacting with both ryegrass pollen and wheat endosperm proteins involves common or related determinants and has implications for the clinical management of these allergic subjects.

Anaphylaxis following administration of papaveretum. Case report: Implication of IgE antibodies that react with morphine and codeine, and identification of an allergenic determinant.

IgE antibodies that reacted with morphine and codeine were detected in the serum of a subject who experienced a life-threatening anaphylactic reaction following the administration of Omnopon-Scopolamine (papaveretum-hyoscine). Hapten inhibition studies with morphine and a number of structurally-related analogues revealed that morphine and codeine were the most potent inhibitors of IgE binding to a morphine-solid phase. Nalorphine, meperidine, and methadone were also good inhibitors of IgE binding, but naltrexone, buprenorphine, and fentanyl proved to be poor inhibitors. From a detailed examination of structure-activity relationships, the authors conclude that the important structural features of the morphine allergenic (that is, IgE binding) determinant comprises the cyclohexenyl ring with a hydroxyl group at C-6 and, most important of all, a methyl substituent attached to the N atom. The authors' findings suggest that morphine analogues administered to such a patient may provoke clinical anaphylaxis. Hyoscine reacted weakly with IgE antibodies in the subject's serum, but this was thought to be due to weak cross-reaction between this compound and morphine.

Allergic response to birch and alder pollen allergens influenced by geographical location of allergic subjects.

A detailed analysis was made of the reactivity patterns of birch pollen-allergic subjects from Norway and Australia to the various IgE-binding components of pollens from several different birch and alder species. Typically, with each of the pollens examined, the Norwegian subjects exhibited a specific limited response pattern in which only a few of the 18 possible allergenic bands were recognized. A single, major IgE-binding band of approximate molecular weight 16-17 kD was recognized in each pollen by all the Norwegian patients used in the study. In contrast to this situation, there was no 'typical' recognition pattern amongst the Australian patients. Although recognition of the 16- to 17-kD components occurred in some cases, it was not dramatically superior to any other single component. Recognition of higher molecular weight components occurred more frequently than with the Norwegian group. Reasons for the differences in the response patterns of the Australian and Norwegian groups are discussed with reference to the exposure of the populations to different allergenic sources and genetic variation.

Standardization of rye-grass pollen (Lolium perenne) extract. An immunochemical and physicochemical assessment of six candidate international reference preparations.

Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.

Cytochrome c allergens isolated from the pollens of the dicotyledons English plantain (Plantago lanceolata) and Paterson's curse (Echium plantagineum).

Two cytochrome c allergens were isolated from extracts of the pollens of the dicotyledons English plantain (Plantago lanceolata) and Paterson's Curse (Echium plantagineum) by ion exchange chromatography, gel filtration and preparative isoelectric focusing. They were characterized by their absorption spectra, mol. wt, pI and amino acid composition. The cytochromes c bound specific IgE in the sera of hypersensitive patients by RAST. Preliminary evidence for allergenic cross-reactivity between them was obtained by RAST inhibition.

A comparison of the antigenic and allergenic components of birch and alder pollens in Scandinavia and Australia.

Allergens in birch (Betula) pollens from B. pendula grown in Australia and Norway, B. davurica and B. populofolia and from alder (Alnus incana) were identified by electroblotting, following separation by SDS-PAGE, transfer to nitrocellulose membranes and incubation with sera from birch pollen-allergic subjects. Of 42 antigenic components detected by protein staining in the pollen extract from B. pendula grown in Norway, 17 bound IgE. The allergenic components included those already reported in the literature at MWs of 40, 29, 25, 17 and 10-12 kd, as well as previously undescribed components at MWs of 90, 79, 60, 50, 38, 35, 31, 27, 23, 16, 15 and 14 kd. The major IgE-binding components were located in the low MW region 10-17 kd for all species of birch and alder pollen proteins studied. Results reported here provide the first evidence of birch and alder pollen allergies in Australia. Extensive heterogeneity was observed amongst the sera from birch pollen-allergic subjects in both Norway and Australia. Cross-reactivity appears to exist among the proteins present in pollen from the various birch species and from alder.

Identification of Bermuda grass (Cynodon dactylon)--pollen allergens by electroblotting.

Bermuda grass-pollen proteins were electrophoretically separated on polyacrylamide gels and transferred to nitrocellulose where IgE-binding components were detected by reaction with individual patient's serum and 125I-labeled antihuman IgE. Seventeen pollen components (in the molecular weight (MW) range of 8000 to 94,000 daltons), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, bound IgE antibodies from a panel of 44 sera from allergic patients. The spectrum of Bermuda grass-pollen IgE-binding components detected is greater in number and wider in MW range than has previously been described. A component of MW 34,000 daltons (fraction 9) bound IgE from 100% of atopic sera tested. This component also bound the greatest quantity of IgE. Electrophoresis and transfer under nondissociating conditions revealed a component of MW 100,000 daltons that also bound IgE antibodies in all 44 sera tested. This component may be an aggregated form of fraction 9. A comparison of the electroblotting results obtained under dissociating and nondissociating conditions suggests once again that allergenic proteins in crude extracts may aggregate or associate during in vitro studies. Electrophoretic transfer analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis may thus be the method of choice for allergen separation and identification.

Protein blotting on nitrocellulose: some important aspects of the resolution and detection of antigens in complex extracts.

The resolution and detection of individual components in complex extracts by protein blotting have been investigated. By probing nitrocellulose transfers with monospecific and multispecific antisera, it was demonstrated that dissociating conditions were required for the maximum resolution of antigens by polyacrylamide gel electrophoresis, a conclusion reinforced by results from 2-D electrophoresis. The dissociating and reducing treatments employed, however, were both shown to be responsible for some loss of total antigenicity and included the complete loss of at least one important antigen. Assays with nitrocelluloses of different pore sizes demonstrated that both higher protein-binding capacities and higher backgrounds were associated with the use of the smallest pore size, while the sensitivity of the assay was greatest when a non-ionic detergent, and not proteins, were used for blocking. Nitrocellulose-bound proteins may be stained with amido black, India ink, toluidine blue, Ponceau S or a gold sol, but these agents do not always give identical staining patterns. While detection of components with immuno-enzyme staining methods had some advantages, problems with non-specific binding were encountered. These did not occur with affinity purified radiolabelled second antibodies, which in combination with scanning of autoradiographs allowed a quantitative approach to be adopted.

A re-examination of ryegrass (Lolium perenne) pollen allergens.

Electroblotting of crude ryegrass pollen extracts and purified group I, II and III allergens identified 14 IgE-binding components, 8 of which were previously unrecognized. In addition to allergen groups I, II and III, which are already regarded as clinically important, and on the basis of the frequencies and intensities of IgE binding with sera from 42 ryegrass pollen-allergic patients, proteins with molecular weights (MWs) of 60, 32, 30 and 28 kD were identified as allergens of possible major clinical importance. Six other pollen components with MWs ranging from 23 to 80 kD and which reacted with IgE antibodies in the sera of 33-50% of patients, should also be viewed as proteins with potential clinical relevance for at least a proportion of the patients. The electrophoretic separation patterns of ryegrass pollen extracts in both alkaline and acid gels and IgE-probed membrane transfers produced in this study should serve as useful reference patterns for standardization purposes. In addition, the identification of the complete allergen recognition pattern by individual patients will permit safer and more effective diagnosis and therapy of ryegrass pollen sensitivities.

Identification of Parietaria judaica pollen allergens.

Parietaria judaica pollen allergens, fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 52 sera collected in Australia and Sicily from P. judaica pollen-allergic patients. IgE-binding pollen components transferred to nitrocellulose were detected by reaction with 125I-anti-human IgE and autoradiography. Nine pollen components, ranging in molecular weight (MW) from approximately 10,000 to 80,000 daltons, bound IgE antibodies but the two fastest migrating components sometimes each separated into two very closely migrating bands. The faster of the two components exhibiting doublet formation (MW approximately 10,000 daltons) showed by far the highest frequency of IgE binding, being recognised by 50 of the 52 sera examined. Although patients' IgE reaction patterns to P. judaica allergens were heterogeneous, the degree of heterogeneity was much less than that observed with house dust mite and other pollen extracts studied by electrophoretic transfer analysis. Results with gradient gel-nitrocellulose transfer experiments which showed no IgE-binding components with MWs less than 70,000 daltons, and comparisons of our electroblotting results with crossed radioimmunoelectrophoresis results of others, suggested that the doublet proteins with MWs of approximately 10,000 probably bind to higher MW proteins in P. judaica pollen extracts.

Identification of orchard grass (Dactylis glomerata) pollen allergens following electrophoretic transfer to nitrocellulose.

Orchard grass (cocksfoot) pollen extracts, fractionated by polyacrylamide gradient electrophoresis or SDS gel electrophoresis were electroblotted onto nitrocellulose membranes and probed with sera from orchard grass pollen-allergic patients and 125I-anti-human IgE. The IgE-binding components of the pollen were detected by autoradiography. Elution studies showed that allergens could be extracted immediately and continuously over a 3-hour period. Two fractions of MWs 28,000 and 30,000 could be detected only after 20 min extraction. SDS-PAGE separations gave the better resolution revealing 19 electrophoretically-separate components, 13 of which bound human IgE. All of the IgE-binding components had MWs in the range 14,000 - 70,000. Three of the bands bound IgE from more than 85% of the serum samples. Following gradient gel electrophoresis, IgE binding was exhibited by 10 bands in the range MW 5,000 to greater than 669,000. The technique used allows one to quantitatively examine patients' sera for allergen-specific IgE antibodies and to identify the clinically important allergens. Results revealed numerous allergenic components over a wide MW range while patterns of IgE binding with different patients' sera demonstrated a great diversity of IgE antibody responses. This study demonstrates the suitability of the electroblotting technique combined with autoradiography for the investigation of allergenic components of grass pollen extracts and hence has application to extract standardization and immunotherapy. Such studies can be carried out rapidly, economically and with a high degree of sensitivity.