Authentication of collagen VI antibodies

Abstract

Background: Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients. Methods: Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together. Results: Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains. Conclusions: This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.