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Originally published In Press as doi:10.1194/jlr.D700040-JLR200 on June 9, 2008

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Journal of Lipid Research, Vol. 49, 2259-2267, October 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

Vijay Paul*, Heinrich H. D. Meyer*, Katharina Leidl*,{dagger}, Soni Soumian§ and Christiane Albrecht1,*,**

* Physiology Weihenstephan, Technical University Munich, Weihenstephaner Berg 3, 85350 Freising, Germany
{dagger} Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany
§ MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College Hammersmith Hospital, Du Cane Road, London W12 0NN, United Kingdom
** Institute of Biochemistry and Molecular Medicine, University of Bern, Buehlstr. 28, 3012 Bern, Switzerland

Published, JLR Papers in Press, June 9, 2008.

The studies described in this manuscript were supported by grants of the Swiss National Science Foundation (320000-119984), the Novartis Stiftung für Medizinisch Biologische Forschung (07B44), and the Wolfermann-Nägeli-Stiftung.

1 To whom correspondence should be addressed: e-mail: christiane.albrecht{at}mci.unibe.ch

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9–1000 ng/well (0.09–10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97–0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 ± 0.82 vs. 17.03 ± 4.25 µg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Supplementary key words ATP binding cassette transporter A1 • ELISA • carotid plaques

Abbreviations: ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BCA, bicinchoninic acid assay; BLU, Boehringer light units; CEA, carotid endarterectomy; CV, coefficient of variation; hF, human fibroblasts; HRP, horseradish peroxidase; LXR, liver X receptor alpha; PBST, phosphate buffered saline-tween 20; RXR, retinoid X receptor; TMB, tetramethylbenzidine


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