Effect of reduction and heat on the detection of house dust mite (Dermatophagoides pteronyssinus) allergen Der p I by protein blotting.

Abstract

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.