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.
S A
SA
Ford
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ER
Tovey
B A
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Baldo
D000485:Allergens / Q000276:immunology; D001345:Autoradiography; D003101:Collodion; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D010936:Plant Extracts / Q000276:immunology*; D006109:Poaceae; D011058:Pollen / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D013997:Time Factors
D000485:Allergens; D010936:Plant Extracts; D012967:Sodium Dodecyl Sulfate; D007073:Immunoglobulin E; D003101:Collodion