A monoclonal antibody against hamster tracheal mucin, which recognizes N-acetyl-galactosamine containing carbohydrate chains as an epitope.

Abstract

Airway mucin that is present in airway secretion, plays an important role in host-defense by trapping airborne particles and removing them by mucociliary transport system. For the study of mucin, it is crucially important to have antibodies specific against mucin because other commonly used methods such as histologic stain for the detection of mucin usually suffer from varying levels of nonspecificity. In this study, we produced a monoclonal antibody (MAb) against hamster airway mucin, which is one of the most commonly used animal species for the study of mucin in vitro, and characterized its immunological properties along with the determination of the epitope it recognizes. The MAb, which was named MAb HTA, was IgM isotype and specific against mucin from both in vitro cell culture and in vivo airway secretion. In Western blot, MAb HTA specifically recognized high molecular weight airway mucin, which was also confirmed by the appearance of peak profile of immunological signal only on void volume fraction in Sepharose CL-4B gel filtration chromatography. It also immunoprecipitated high molecular weight hamster airway mucin with the aid of antimouse IgM agarose. In immunohistochemical stain of hamster trachea, it showed strong signal on airway epithelium and also on the mucin secreting goblet cell granules. The immunological signal was greatly increased by the treatment of endotoxin, which has been reported to cause airway secretory cell metaplasia. The MAb HTA recognized carbohydrate chains containing N-acetyl-galactosamine, one of the linking sugars of airway mucin, as an epitope. Treatment of mucin with N-acetyl-galactosaminidase caused great reduction of immunological signal. To the best of our knowledge, this is the first to report a MAb that recognizes N-acetylgalactosamine, a linking sugar of airway mucin. The specificity of MAb HTA against airway mucin and the clear demonstration of the epitope it recognizes should greatly aid the pharmacological and biochemical study of mucin in various physiological and pathological situations.