Detection of recombinant Alt a1 in a two-site, IgM based, sandwich ELISA opens up possibilities of developing alternative assays for the allergen

Alternaria alternata is well known to induce IgE-mediated asthma in humans. Alt a1, a 29 kD glycoprotein doublet composed of 14.5 and 16 kD subunits, is the major allergen of this mould. Detection of Alt a1 relies on a two-site sandwich ELISA using the same IgG subclass immunoglobulin as primary and secondary antibody. In this study, we have compared two IgM monoclonal antibodies against recombinant and native Alt a1 in detecting the allergen in a two-site sandwich ELISA. Although both IgM clones detected the native and the recombinant allergen by SDS-PAGE immunoblotting and by the antibody-capture ELISA, only the IgM against recombinant Alt a1 was able to detect the corresponding, and not the native allergen, in a two-site sandwich ELISA. The IgM against native Alt a1 was unable to detect either allergen by this method. A combination of the two IgM clones and with a commercially available IgG failed to detect both allergens. However, atopic human IgE detected both forms of the allergen with the two IgM clones as primary antibody. This is the first time to demonstrate detection of Alt a1 in a two-site, IgM based, sandwich ELISA opening up possibilities for exploring novel detection methods, based on this approach.