Isolation and purification of adiponectin multimers from human plasma and detection of their bioactivity

AIM: To isolate adiponectin isoforms from human plasma, establish a method to detect human plasma adiponectin isoform biological activity and provide antigen to prepare adiponectin isoform-specific antibody. METHODS: Human plasma stored at -80℃ was thawed and diluted. Plasma adiponectin multimers were isolated by ammonium sulfate precipitation, anion-exchange chromatography and gel filtration chromatography. Adiponectin purity and recovery rate were detected by polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). Adiponectin bioactivity was detected by incubating human umbilical vein endothelial cells with purified adiponectin isoform and determining the phosphorylation level of AMP-activated protein kinase. RESULTS: The three adiponectin isoforms isolated from plasma all activated AMPK and the high-molecular-weight form was the most potent. Adiponectin activity recovery rate was about 40%. CONCLUSION: Our method efficiently purifies adiponectin multimers from human plasma and preserves their bioactivity, which establishes the basis for further study on adiponectin.