Characterisation of a polyclonal antibody to a major mucin fraction in canine ocular surface aspirates

Purpose. Mucins, which are glycosylated in a tissue specific manner, present a multiplicity of peptide and carbohydrate epitopes. Some of these may be shared with mucins at other sites, and with other non-mucin glycoconjugates (e.g. blood group antigens). We are investigating the ocular mucin secretory system in the dog, and have raised a potyclonal antibody to a major mucin fraction purified from canine ocular surface aspirates. Here we investigate the specificity of this antibody. Methods. Ocular mucus was aspirated from the ocular surface of 28 normal dogs; dispersed in a cocktail of proteinase inhibitors; and secreted mucins fractionated and purified by CsCl density gradient centrifugation, followed by gel filtration. A rabbit polyclonal antibody was raised to purified ocular mucin in the density range 1.361.47g/ml: representing a fraction against which we currently have no antibody. The specificity of the antibody was examined histologically, and on slot blots of purified mucins. Results. On sections the antibody specifically binds, at high titre, to goblet cells in the conjunctiva, stomach, and respiratory tract. Chemical and enzymic treatment indicates that most of the immunoreactivity relates to accessible peptide epitopes. Slot blots show binding to all purified fractions of canine secreted ocular mucins, but not to a range of other glycoconjugates. The antibody also binds to human ocular goblet cells on cytological impressions. Conclusions. Our results suggest that this antibody primarily recognises peptide epitopes which are specific to mucins and not carbohydrate structures shared with other glycoconjugates. As such, it is a useful reagent for the detection and quantification of ocular mucin. Supported by the Wellcome Trust Project Grant 037530/Z/93/Z/1.5/WRE/JL. None.