A novel ELISA system for selective measurement of human adiponectin multimers by using proteases

BACKGROUND: Adiponectin, an antiatherogenic adipocyte-derived protein exists in human blood as multiple isoforms--trimeric low molecular weight (LMW), albumin-binding LMW (Alb-LMW), hexameric middle molecular weight (MMW), and high molecular weight (HMW) forms. We developed a novel ELISA system to detect total human adiponectin and the selective level of each adiponectin multimer for investigating the distribution of these levels in human blood. METHODS: Two monoclonal antibodies that were raised against human adiponectin were used to construct a sandwich ELISA to measure adiponectin levels. Adiponectin multimers were selectively measured after sample pretreatment with two proteases that specifically digested the trimeric forms or both the hexameric and trimeric forms. RESULTS: The ELISA had a dynamic range of 0.075-4.8 ng/ml. Intraassay variations (CV) were 5.3% (total adiponectin), 4.1% (MMW+HMW), and 3.3% (HMW). Comparison of the results of ELISA and quantitative western blot analysis of multimeric adiponectin in serum samples revealed good correlation (LMW+Alb-LMW, r=0.873; MMW, r=0.907; HMW, r=0.950). Each of the three forms of adiponectin multimer levels closely correlated with total adiponectin levels in healthy subjects. CONCLUSIONS: This ELISA system can be used to further investigate the physiological roles of human adiponectin multimers.