Micromethods for the analysis of tear proteins in pharmacological studies

Although it is well established that ocular mucins and other proteins are e ssential for tear film stability, whether certain drugs, like non steroidal antiinflammatory drugs (NSAIDs), could cause ocular dryness by inhibiting their secretion is not known. To perform these and other studies of pharmac ological interest, we evaluated several micromethods for the analysis of te ar samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, la ctoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 mu l of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase as says based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or ny lon membranes, prepared by an ordinary paper punch, which were coated with PGM and stained by Alcian blue or the periodic acid Schiff's reagent. The d ensitometric analysis was carried out using an ordinary flat scanner contro lled by a personal computer equipped with an inexpensive software. The sens itivity of these simple assays was low (100-500 mu g) but considered suffic ient for certain studies. A more sensitive assay (5-20 mu g) was developed by immobilizing PGM in small agarose gels (100 mu l), prepared in the wells of 96-well microplates, which could by stained by stains-all and analyzed by an automatic plate reader at 595 nm.