RENATURATION OF BLOTTED ALLERGENS INCREASES THE SENSITIVITY OF SPECIFIC IGE DETECTION

Several authors have demonstrated that renaturation is an essential st ep for the appropriate recognition of blotted proteins. The use of non ionic detergents has been described as a useful alternative to enhance the antigenicity in immunoblotting, although elution from proteins by detergents has been observed. To measure the influence of different f actors on the sensitivity of specific IgE by immunoblotting, we used t wenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Non idet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing t his detergent, even at high concentrations (3%), and its use gave bett er sensitivity than methanol. On the other hand, we detected that meth anol possessed renaturing properties. A transfer method using diffusio n instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method we found that afte r NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effec ts.