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

¹ Diagnostics Research Laboratories, Daiichi Pure Chemicals Co. Ltd., Ibaraki, Japan; ² Division of Clinical Preventive Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;³ Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan;

^aaddress correspondence to this author at: Division of Clinical Preventive Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Chuo-ku, Niigata, Niigata 951-8510, Japan; fax 81-25-223-0996, e-mail: miida{at}med.niigata-u.ac.jp

Abstract

Background: 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.

Methods: 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.

Results: The ELISA system measured adiponectin in the range of 1.0–167 µg/L. The between-assay imprecision estimates (CVs) were 6%–17% for the 3 forms. The mean total CSF adiponectin concentration (7.2 µg/L) was 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.

Conclusions: The modified ELISA system measures the 3 multimers separately and is sufficiently sensitive to measure adiponectin in CSF.

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