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Journal of Lipid Research, Vol. 44, 645-650, March 2003
Copyright © 2003 by Lipid Research, Inc.

Methods

Analytical performance of a sandwich enzyme immunoassay for preß1-HDL in stabilized plasma

Takashi Miida^1,*, Osamu Miyazaki, Yasushi Nakamura, Satoshi Hirayama, Osamu Hanyu, Isamu Fukamachi and Masahiko Okada^*

^* Division of Clinical Preventive Medicine, Department of Community Preventive Medicine, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Niigata, Niigata 951-8510, Japan
Daiichi Pure Chemicals, Diagnostics Research Laboratories, Tokai Research Group, Muramatsu 2117, Tokai, Ibaraki 319-1182, Japan
Division of Endocrinology and Metabolism, Department of Homeostatic Regulation and Developments, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi 1-757, Niigata, Niigata 951-8510, Japan

¹ To whom correspondence should be addressed. e-mail: miida{at}med.niigata-u.ac.jp

We have established an immunoassay for preß1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because preß1-HDL is unstable during storage, fresh plasma must be used for preß1-HDL measurements. In this study, we describe a method of stabilizing preß1-HDL, and evaluate the analytical performance of the immunoassay for preß1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Preß1-HDL concentration was measured by immunoassay. In nonpretreated samples, preß1-HDL decreased significantly from the baseline after 6 h at room temperature. Although preß1-HDL was more stable at 0°C than at room temperature, it increased from 30.2 ± 8.5 (SE) to 56.5 ± 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 ± 1.2 to 37.9 ± 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at -80°C, preß1-HDL increased from 29.0 ± 4.0 to 38.0 ± 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, preß1-HDL concentration did not change significantly under any of the above conditions. Moreover, preß1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05).

An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of preß1-HDL.

Abbreviations: 2D-gel, two-dimensional gel; DTNB, 5,5'-dithio-bis(2-nitrobenzoic acid)

Supplementary key words hyperlipidemia • lecithin:cholesterol acyltransferase • apolipoprotein A-I • two-dimensional gel electrophoresis

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