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Analytical performance of a sandwich enzyme immunoassay for pre beta 1-HDL in stabilized plasma
Takashi Miida1, Osamu Miyazaki, Yasushi Nakamura
1Division 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. miida@med.niigata-u.ac.jp
Insights
A new method stabilizes pre beta 1-HDL (the initial acceptor of cellular cholesterol) for accurate immunoassay measurements. This pretreatment prevents significant changes in pre beta 1-HDL levels during storage, improving clinical utility.
Area of Science:
- Clinical Chemistry
- Lipid Metabolism
- Immunodiagnostics
Background:
- Pre beta 1-HDL is the primary acceptor of cellular cholesterol.
- Pre beta 1-HDL is inherently unstable, necessitating fresh plasma for accurate measurements.
- Existing methods for pre beta 1-HDL quantification are limited by sample stability issues.
Purpose of the Study:
- To develop and validate a stabilization method for pre beta 1-HDL.
- To evaluate the analytical performance of an immunoassay for pre beta 1-HDL using stabilized samples.
- To assess the clinical utility of the developed immunoassay for pre beta 1-HDL quantification.
Main Methods:
- Established an immunoassay utilizing a specific monoclonal antibody (MAb55201).
- Developed a pretreatment method involving dilution in 50% sucrose to stabilize pre beta 1-HDL.
- Stored plasma samples under various conditions (room temperature, 0°C, -80°C) with and without pretreatment, measuring pre beta 1-HDL concentrations over time.
Main Results:
- Non-pretreated pre beta 1-HDL levels significantly decreased at room temperature within 6 hours and showed altered levels after storage at 0°C and -80°C, particularly in hyperlipidemic individuals.
- Pretreated samples demonstrated stable pre beta 1-HDL concentrations across all tested storage conditions.
- Immunoassay results for pre beta 1-HDL showed a strong correlation with those obtained by native two-dimensional gel electrophoresis (r = 0.833, P < 0.05).
Conclusions:
- A novel pretreatment method effectively stabilizes pre beta 1-HDL, overcoming limitations associated with sample storage.
- The MAb55201 immunoassay, when used with pretreated plasma, provides a reliable and clinically useful method for measuring pre beta 1-HDL.
- This stabilized immunoassay facilitates accurate clinical assessment of pre beta 1-HDL, crucial for understanding cholesterol transport and lipid metabolism.
Abstract:
We have established an immunoassay for pre beta 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre beta 1-HDL is unstable during storage, fresh plasma must be used for pre beta 1-HDL measurements. In this study, we describe a method of stabilizing pre beta 1-HDL, and evaluate the analytical performance of the immunoassay for pre beta 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre beta 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre beta 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre beta 1-HDL was more stable at 0 degrees 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 degrees C, pre beta 1-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, pre beta 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre beta 1-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 pre beta 1-HDL.
