Clinical Chemistry
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Clinical Chemistry 55: 1801-1808, 2009. First published August 6, 2009; 10.1373/clinchem.2009.127027
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(Clinical Chemistry. 2009;55:1801-1808.)
© 2009 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Development of an Immunoassay for the Quantification of Soluble LR11, a Circulating Marker of Atherosclerosis

Masanao Matsuo1, Hiroyuki Ebinuma1,a, Isamu Fukamachi1, Meizi Jiang2, Hideaki Bujo2 and Yasushi Saito3

1 Tsukuba Research Institute, Sekisui Medical, Ibaraki, Japan; 2 Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba, Japan; 3 Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba, Japan.

aAddress correspondence to this author at: Tsukuba Research Institute, Sekisui Medical, 3-3-1, Koyodai, Ryugasaki, Ibaraki 301-0852, Japan. Fax +81-297-62-8635; e-mail ebinuma002{at}sekisui.jp.

Background: Vascular smooth muscle cells (SMCs) migrate from the arterial media to the intima in the progression of atherosclerosis, and dysfunction of SMCs leads to enhanced atherogenesis. A soluble form of the LDL receptor relative with 11 ligand-binding repeats (sLR11) is produced by the intimal SMCs, and the circulating concentrations of sLR11 likely reflect the pathophysiological condition of intimal SMCs. Furthermore, polymorphism of the LR11 gene has been found to be related to the onset of Alzheimer disease. This study describes the development of a sandwich immunoassay for quantifying sLR11 in human serum and cerebrospinal fluid.

Methods: We used synthetic peptides or DNA immunization to produce monoclonal antibodies (MAbs) A2-2–3, M3, and R14 against different epitopes of LR11.

Results: sLR11 was immunologically identified as a 250-kDa protein in human serum and cerebrospinal fluid by SDS-PAGE separation, and was purified from serum by use of a receptor-associated protein and MAb M3. An immunoassay for quantification of sLR11 with a working range of 0.25–4.0 µg/L was developed using the combination of MAbs M3 and R14. Treatment of serum with 5.25% n-nonanoyl-N-methyl-d-glucamine reduced the matrix effects of serum on the absorbance detection in the ELISA system. The linear dynamic range of the ELISA spanned the variation of circulating sLR11 concentrations in individuals with atherosclerosis.

Conclusions: A sandwich ELISA was established for quantifying sLR11 in serum and cerebrospinal fluid. This technique provides a novel means for assessing the pathophysiology of atherosclerosis, and possibly neurodegenerative diseases.






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Copyright © 2009 by the American Association for Clinical Chemistry.