Comparison between ELISA and gel-filtration assay for the quantitation of airway mucins

Abstract

In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [H-3]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically 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 (RTSE) cell culture which had been metabolically radiolabeled with [H-3]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated 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 immunoreactivity with mAbRT03. The contents of mucin in the sample were cal culated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.