Renaturation of blotted allergens increases the sensitivity of specific IgE detection.
Several authors have demonstrated that renaturation is an essential step for the appropriate recognition of blotted proteins. The use of nonionic 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 factors on the sensitivity of specific IgE by immunoblotting, we used twenty human sera from atopic patients who were allergic or nonallergic to a common, reliable allergen (grass pollen mixture). The use of Nonidet-P40 was found to be a useful alternative for the renaturation of the allergens. No elution from the membrane was found when employing this detergent, even at high concentrations (3%), and its use gave better sensitivity than methanol. On the other hand, we detected that methanol possessed renaturing properties. A transfer method using diffusion instead of electric transfer gave the best results and two membranes could be obtained from each gel. Using this method, we found that after NP-40 incubation of the membrane, the use of bovine albumin could be omitted as blocking agent and that its use had even deleterious effects.
Muro
M D
MD
Department of Immunology, Health Institute Carlos III, Madrid, Spain.
Fernández
C
C
Moneo
I
I
D000485:Allergens / Q000276:immunology*; D000918:Antibody Specificity; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis*; D007073:Immunoglobulin E / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000485:Allergens; D007073:Immunoglobulin E