COMPARISON BETWEEN ELISA AND GEL-FILTRATION ASSAY FOR THE QUANTITATION OF AIRWAY MUCINS

In this study, we developed immunoassay methods for the more convenien t and effective detection of rat tracheal mucin and the results were c ompared with those of [H-3]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifical ly recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0 similar to 2 mM) were applied to the primary rat tracheal surface epithelial (RT SE) cell culture which had been metabolically radiolabeled with [H-3]g lucosamine and the secretion of mucin was analyzed both by the immunoa ssay and the gel-filtration chromatography methods. For the immunoassa y, the following two procedures were employed. 1) Simple ELISA; the cu lture spent media were directly coated onto the assay plate and the im munoreactivity with mAbRT03 was assessed from the standard curve gener ated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP -stimulated culture spent media were added to inhibit the immunoreacti vity with mAbRT03. The contents of mucin in the sample were cal culate d from the standard inhibition curve which was generated with the puri fied rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present resu lt indicates that ELISA can be substituted for the laborious, time-con suming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.