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Journal of Lipid Research, Vol. 45, 387-395, February 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Methods

Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA

Sarah Siggins¹, Minna Kärkkäinen¹, Jukka Tenhunen, Jari Metso, Esa Tahvanainen, Vesa M. Olkkonen, Matti Jauhiainen and Christian Ehnholm²

Department of Molecular Medicine, National Public Health Institute, Biomedicum, FIN-00251 Helsinki, Finland

² To whom correspondence should be addressed. e-mail: christian.ehnholm{at}ktl.fi

Human plasma contains two forms of phospholipid transfer protein (PLTP), one catalytically active [high-activity PLTP (HA-PLTP)] and the other a low-activity (LA-PLTP) form. We present here a PLTP ELISA that allows not only for accurate measurement of PLTP concentration in plasma but also of the distribution of both LA- and HA-PLTP. To achieve similar immunoreactivity of the two PLTP forms, a denaturing sample pretreatment with 0.5% SDS was required. Distribution of LA- and HA-PLTP in plasma was assessed using size-exclusion chromatography, Heparin-Sepharose chromatography, anti-PLTP immunoaffinity chromatography, and dextran sulfate-CaCl₂ precipitation. All four methods demonstrated that 60% of plasma PLTP represents LA-PLTP and 40% represents HA-PLTP. According to the modified ELISA, the total serum PLTP concentration in a random Finnish population sample (n = 80) was 5.81 ± 1.33 mg/l (mean ± SD) (range, 2.78–10.06 mg/l) and the mean activity was 5.84 ± 1.39 µmol/ml/h (range, 3.21–11.15 µmol/ml/h).

To quantitate both forms of PLTP in sera from this sample, we combined dextran sulfate-CaCl₂ precipitation with the modified PLTP ELISA. The HA-PLTP mass (mean, 1.87 ± 0.85 mg/l) correlated significantly with serum PLTP activity, whereas that of LA-PLTP (mean, 3.94 ± 1.4 mg/l) showed no correlation with phospholipid transfer activity.

Abbreviations: HA-PLTP, high-activity PLTP; LA-PLTP, low-activity PLTP; MAb, monoclonal antibody; PLTP, phospholipid transfer protein

Supplementary key words lipid transfer protein • enzyme-linked immunosorbent assay • high-activity form of phospholipid transfer protein • low-activity form of phospholipid transfer protein

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