Analysis of pigeon intestinal mucin allergens using a novel dot blot assay

Many glycoproteins contain multiple glycosylation sites that can present multi-valent epitopes for antigenic recognition. Release of the oligosaccharides results in loss of avidity of antibody binding, which has been overcome by reforming clustered ligands, usually by reductive amination of free reducing oligosaccharides to poly-amine groups. We have adapted this approach to hydrazinolytic release of O-linked chains of mucin glycoproteins and 'one-pot' microscale coupling to poly-L-lysine (PLL). The conjugated PLL adheres to nitrocellulose membranes through washing procedures required for antibody or lectin overlay and detection. We show evidence for the applicability of this technique using lectin and antibody reactivity to the oligosaccharides of pigeon intestinal mucins, which have been implicated in the allergic disease pigeon fanciers' lung.