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Clinical Chemistry 56: 1320-1328, 2010. First published June 8, 2010; 10.1373/clinchem.2010.146118
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Endocrinology and Metabolism

A Multiplex Immunoassay for Human Adipokine Profiling

Henk S. Schipper1,2,5, Wilco de Jager2, Mariska E.A. van Dijk2, Jenny Meerding2, Pierre M.J. Zelissen3, Roger A. Adan4, Berent J. Prakken2 and Eric Kalkhoven1,2,5,*

1 Department of Metabolic and Endocrine Diseases,
2 Department of Pediatric Immunology,
3 Department of Endocrinology, and
4 Rudolph Magnus Institute of Neurosciences, Department of Neuroscience and Pharmacology, UMC Utrecht, the Netherlands;
5 Netherlands Metabolomics Center, the Netherlands.

* Address correspondence to this author at: Department of Metabolic and Endocrine Diseases, UMC Utrecht, Room KE.03.139.2, Lundlaan 6, 3584 EA Utrecht, The Netherlands. Fax +31-30-2504295; e-mail e.kalkhoven{at}umcutrecht.nl.

BACKGROUND: Adipose tissue secretory proteins, called adipokines, play pivotal roles in the pathophysiology of obesity and its associated disorders such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Because methods for comprehensive adipokine profiling in patient plasma and other biological samples are currently limited, we developed a multiplex immunoassay for rapid and high-throughput measurement of 25 adipokines in only 50 µL of sample.

METHODS: (Pre)adipocyte and ex vivo cultured adipose tissue supernatants were generated and together with plasma from 5 morbidly obese patients and 5 healthy and normal weight controls used to develop the adipokine multiplex immunoassay and test its usefulness in biological samples. We assessed adipokine dynamic ranges, lower limits of detection and quantification, cross-reactivity, intra- and interassay variation, and correlation with adipokine ELISAs.

RESULTS: The limits of quantification and broad dynamic ranges enabled measurement of all 25 adipokines in supernatants and patient plasmas, with the exception of TNF-{alpha} in plasma samples. Intraassay variation was <10% for all adipokines; interassay variation was <15%. The multiplex immunoassay results correlated significantly with ELISA measurements. Plasma adipokine profiling showed significantly higher concentrations of the novel adipokines cathepsin S (5.1 x 104 vs 4.3 x 104 ng/L, P = 0.003) and chemerin (4.1 x 105 vs 2.7 x 105 ng/L, P = 0.0008) in morbidly obese patients than normal weight controls, besides the established differences in adiponectin and leptin concentrations.

CONCLUSIONS: Our findings underscore the relevance of the novel adipokines cathepsin S and chemerin, but foremost the potential of this novel method for both comprehensive adipokine profiling in large patient cohorts and for biological discovery.