Development and validation of a lectin-based assay for the quantitation of rat respiratory mucin.
The significance of a mucus hypersecretory phenotype to the pathogenesis of airways disease is controversial, but increasing evidence suggests that it can negatively impact upon patient health status. A critical aspect of developing our understanding of the role of mucus hypersecretion in disease is the development and appropriate use of methods permitting detection and quantitation of mucins in biological samples. However, the unique biophysical and biochemical properties of this class of glycoproteins do not lend themselves to routine quantitation. Individual pure mucins are not commercially available, the reactivity profile of commonly used reagents is generally not well characterized and assay development and validation is rarely covered adequately in the literature. Therefore quantitation of mucin in biological samples relies upon careful histochemical and biochemical characterization and partially purified mucin preparations. The absence of tools considered essential for assay development in other areas means that this characterization process does not generally lead to proof of selective detection of mucin, but rather to a level of confidence that mucin is detected and defined contaminants are not. This chapter provides an example of the process of development and validation of a lectin-based assay for quantitation of mucin in untreated complex biological samples.
Jackson
Alan
A
Novartis Respiratory Research Centre, Wimbleburst Road, Horsham, West Sussex RH125AB, UK.
Kemp
Phil
P
Giddings
June
J
Sugar
Rosemary
R
D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019765:Chondroitin ABC Lyase / Q000378:metabolism; D002850:Chromatography, Gel; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000737:chemistry; D008970:Molecular Weight; D009077:Mucins / Q000032:analysis* / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism*; D051381:Rats; D012137:Respiratory System / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D013183:Sputum / Q000737:chemistry; D013379:Substrate Specificity
D008070:Lipopolysaccharides; D009077:Mucins; D037121:Plant Lectins; C036850:Ulex europaeus lectins; D006821:Hyaluronoglucosaminidase; D019765:Chondroitin ABC Lyase