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.

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.