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 [3H]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throught in this study. To induce mucin secretion, varying concentrations of ATP (0∼2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [3H]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 calculated 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.
Similar content being viewed by others
References Cited
Adler, K. B., Mucin secretion by explants of respiratory tissuein vitro in Schiff, L. J. (Ed.),In Vitro Models of Respiratory Epithelium. CRC press, Boca Raton, FL, pp. 27–50, 1986.
Adler, K. B., Cheng, P. W. and Kim, K. C., Characterization of guinea pig tracheal epithelial cells maintained in biphasic organotypic culture: Cellular composition and biochemical analysis of release glyco-conjugates.Am. J. Respir. Cell Mol. Biol., 2, 145–154 (1990).
Baker, A. P., Hillegass, L. M., Holden, D. A. and Smith, W. J., Effect of kallidin, substance P and other basic polypeptides on the production of respiratory macromolecules.Am. J. Respir. Dis., 115, 811–817 (1977).
Basbaum, C. B., Chow, A., Macher, B. A., Finkbeiner, W. E., Veissiere, D. and Forsberg, S., Tracheal carbohydrate antigens identified by monoclonal antibodies.Arch. Biochem. Biophys., 249, 363–373 (1986).
Boat, T. F., Cheng, P. W. and Leigh, M., Biochemistry of mucus, In Takashima, T. and Shimura, S. (Eds.).Biochemistry of mucus in airway secretions. Marcel Dekler, Inc., New York, pp. 217–282, 1993.
Cheng, P. W., Sherman, J. M., Boat, T. F. and Bruce, M., Quantitation of radiolabeled mucous glycoproteins secreted by tracheal explants.Anal. Biochem., 117, 301–306 (1981).
Coles, S. J. and Reid, L., Inhibition of glycoconjugates secretion by colchicin and cytochalasin B.Cell Tissue Res., 214, 107–118 (1981).
Davis, C. W., Dowell, M. L., Lethem, M. and van Scott, M., Goblet cell degranulation in isolated canine tracheal epithelium: response to exogeneous ATP, ADP, and adenosine.Am. J. Physiol., 262, C 1313–1323 (1992).
Kim, K. C., Mucin-like glycoproteins secreted from cultured hamster tracheal surface epithelial cells: Their hydrophobic nature and amino acid composition.Exp. Lung Res., 17, 65–76 (1991).
Kim, K. C., Opaskar-Hincman, H. and Bhaskar, K. R., Secretions from primary hamster tracheal surface epithelial cells in culture: Mucin-like glycoproteins, proteoglycans and lipids.Exp. Lung Res., 15, 299–314 (1989).
Kim, K. C., Park, H. R., Shin, C. Y., Akiyama, T. and Ko, K. H., Nucleotide-induced mucin release from airway epithelial cells involves the P2U purinoceptor.Eur. Respir. J., 9, 542–548 (1996).
Kim, K. C., Rearick, J. I., Nettesheim, P. and Jetten, A. M., Biochemical characterization of mucous glyco-proteins synthesized and secreted by hamster tracheal epithelial cells in primary culture.J. Biol. Chem., 260, 4021–4027 (1985).
Kim, K. C., Wasano, K., Niles, R. M., Schuster, J. E., Stone, P. J. and Brody, J. S., Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.Proc. Natl. Acad. Sci. USA., 84, 9304–9308 (1987).
Kler, G. and Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity.Nature, 256, 495–497 (1975).
Lin, H., Carlson, D. M., St. George, J. A., Plopper, C. G. and Wu, R., An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.Am. J. Respir. Cell Mol. Biol., 1, 41–48 (1989).
Logun, C., Mullol, J., Rieves, D., Hoffman, A., Johnson, C., Miller, R., Goff, M., Kaliner, M. and Shelhamer J. H., Use of a monoclonal antibody enzyme-linked immunosorbent assay to measure human respiratory glycoprotein productionin vitro.Am. J. Respir. Cell Mol Biol., 5, 71–79 (1991).
Rearick, J. I., Deas, M. and Jetten, A. M., Synthesis of mucous glycoproteins by rabbit tracheal cellsin vitro. Modulation by substratum, retinoids and cyclic AMP.Biochem. J., 242, 19–25 (1987).
Reid, L., Bhaskar, K. and Coles, S., Control and modulation of airway epithelial cells and their secretions.Exp. Lung. Res., 4, 157–170 (1983).
Samet, J. M. and Cheng, P. W., The role of mucus in pulmonary toxicology.Environ. Health Persp., 102, 89–103 (1994).
Shin, C. Y., Choi, E. Y., Kim, K. C. and Ko, K. H., Production and characterization of monoclonal antibodies against rat tracheal mucins.Hybridoma 1998 (in press).
Slayter, H. S., Lambling, G. and Letreut, A. S., Complex structure of human bronchial mucus glycoprotein.Eur. J. Biochem., 142, 209–218 (1984).
Spicer, S. S. and Martinez, J. R., Mucin biosynthesis and secretion in the respiratory tract.Environ. Health Perspect., 55, 193–204 (1984).
Strous, G. J. and Dekker, J., Mucin-type glycoproteins.Crit. Rev. Biochem. Mol. Biol., 27, 57–59 (1992).
Van Scott, M. R., Lee, N. P. and Yankaskas, J. R., Effect of hormones on growth and function of cultured canine tracheal epithelial cells.Am. J. Physiol., 255, C237-C245 (1988).
Wasano, K., Kim, K. C., Niles, R. M. and Brody, J. S., Membrane differentiation markers of airway epithelial secretory cells.J. Histochem. Cytochem., 36, 167–178 (1988).
Whitcutt, M. J., Adler, K. B. and Wu, R., A biphasic culture system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.In Vitro Cell Dev. Biol., 24, 420–428 (1988).
Wu, R., Martin, W. R., Robinson, C. B., George, J. A., Plopper, C. G., Kurland, G., Last, J. A., Cross, C. E., McDonald, R. J. and Boucher, R., Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture.Am. J. Respir. Cell Mol. Biol., 3, 467–478 (1990).
Wu, R., Nolan, E. and Turner, C., Expression of tracheal differentiated function in serum-free hormone-supplemented medium.J. Cell Physiol., 125, 167–181 (1985).
Wu, R., Plopper, C. G. and Cheng, P. W., Mucin-like glycoproteins secreted by cultured hamster tracheal epithelial cells.Biochem. J., 277, 713–718 (1991).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shin, C.Y., Kang, SJ., Kim, K.C. et al. Comparison between ELISA and gel-filtration assay for the quantitation of airway mucins. Arch. Pharm. Res. 21, 253–259 (1998). https://doi.org/10.1007/BF02975284
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF02975284