Methodological considerations when using nitrocellulose immunoblotting from polyacrylamide gels to study the mould allergens Aspergillus fumigatus and Alternaria alternata.

The effects of sample treatments, separation conditions, and the possible presence of antibody interference phenomena in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) followed by nitrocellulose immunoblotting (IB) were studied using allergens from Alternaria alternata (Alt.a.) and Aspergillus fumigatus (Asp.f.) as model systems. In order to obtain good resolution in the IB method the mould allergens were separated in gradient gels under dissociating conditions (SDSgPAGE) including sample treatment with boiling, SDS and 2-mercaptoethanol (ME). These treatments all reduced the IgE binding capacities of the Asp.f. and Alt.a. extracts studied. Nevertheless, a great variety of IgE-binding components were detected after IB, and this could probably be explained by refolding of allergens during the blotting procedure where SDS was partly removed. A comparison of IgG-enriched and IgG-reduced serum fractions in IB revealed small differences in the IgE-binding patterns suggesting that IgG interference is of minor importance in the system studied. The IB method must be individually optimized for each new allergen studied using well characterized sera, preferably monospecific for the major components. Taking these facts into consideration, the IB technique is a valuable complement to other methods in the study of mould allergens.