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Mite allergens, fractionated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 45 mite atopic sera and an enzyme immunostaining assay for IgE. More than 20 different mite components bound IgE and almost every serum showed a different pattern of binding. 7 of the 20 components were bound by half of the sera and 70% were bound by at least 20% of the sera. These results demonstrate a greater number of house dust mite allergens and a far greater diversity of the IgE antibody response to mite allergens than has previously been described.

Components of an extract of Dermatophagoides pteronyssinus were electrophoretically separated, transferred to nitrocellulose membranes, and then probed with a variety of antibodies and lectins. Using mite-allergic human sera as the first antibody source, IgE-binding components were identified. Binding patterns by monoclonal and heterologous sera were also obtained. The method is suitable for the qualitative, and ultimately the quantitative, characterization of allergen extracts. The electroblotting procedure employing specific probes, therefore, promises to be a valuable aid for allergen standardization.

Employment of reducing conditions during sample preparation alters the mobility of the house dust mite (Dermatophagoides pteronyssinus) allergen Der p I as determined by sodium dodecylsulphate-poly-acrylamide gel electrophoresis (SDS-PAGE) from an apparent MW of approximately 25 kD (unreduced) to an apparent MW of approximately 30 kD. Probing of nitrocellulose transfers with sera from subjects allergic to D. pteronyssinus showed that reduction of Der p I was accompanied by a substantial loss of IgE-antibody-binding capacity by this allergen. An important consequence of the effect of reduction on Der p I is that the electrophoretic mobility of this protein becomes very similar to a closely spaced pair of protein-staining bands, probably Der p III, of MWs 30-31 kD. These bands bind IgE antibodies strongly and with high frequency and exhibit the same electrophoretic mobilities under both reducing and non-reducing conditions. Thus, for the clear resolution of allergen Der p I from other IgE-binding components in the same MW region, including Der p III, house dust mite samples for analysis by SDS-PAGE and blotting should not be reduced.

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.