Improved ELISA for Selective Measurement of Adiponectin Multimers and Identification of Adiponectin in Human Cerebrospinal Fluid

Abstract

Human serum adiponectin exists in 3 multimer forms: high molecular weight (HMW), middle molecular weight, and low molecular weight (LMW), with some of the latter bound to albumin (Alb)-LMW. Some studies have suggested that adiponectin crosses the blood-brain barrier and plays a central role in energy homeostasis. To determine cerebrospinal fluid (CSF) adiponectin at extremely low concentrations, we modified the protocol of the ELISA system used to assay serum adiponectin. The 3 multimers of adiponectin were measured separately by pretreating CSF with 2 proteases. We measured the CSF adiponectin concentrations in anonymous human samples (n = 19). The molecular sizes of adiponectin in CSF pretreated with proteases or untreated were determined by use of native PAGE and immunoblotting. The ELISA system measured adiponectin in the range of 1.0-167 microg/L. The between-assay imprecision estimates (CVs) were 6%-17% for the 3 forms. The mean total CSF adiponectin concentration (7.2 microg/L) was approximately 1/1000 of the mean concentration in serum. Unlike serum adiponectin, the LMW and Alb-LMW forms predominated in all of the CSF samples. Immunoblotting analysis revealed that most LMW forms were bound to Alb, although the HMW form was detected in some samples. The modified ELISA system measures the 3 multimers separately and is sufficiently sensitive to measure adiponectin in CSF.