Micromethods for the analysis of tear proteins in pharmacological studies

Although it is well established that ocular mucins and other proteins are essential 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 pharmacological interest, we evaluated several micromethods for the analysis of tear samples. The major proteins of the tear fluid collected in capillaries, i.e. IgA, lactoferrin, tear specific prealbumin and lysozyme, were analyzed by SDS-PAGE and gel permeation HPLC, using 2.5-5 microliters of sample. Gastric mucin (PGM), examined as a standard, was analyzed by solid phase assays based on previously described histochemical staining methods: dot blot assays were performed using small disks of polyvinylidene difluoride or nylon 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 densitometric analysis was carried out using an ordinary flat scanner controlled by a personal computer equipped with an inexpensive software. The sensitivity of these simple assays was low (100-500 micrograms) but considered sufficient for certain studies. A more sensitive assay (5-20 micrograms) was developed by immobilizing PGM in small agarose gels (100 microliters), 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.