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

• Published in: Journal of lipid research Vol. 45; no. 2; pp. 387 - 395
• Main Authors: Siggins, Sarah, Kärkkäinen, Minna, Tenhunen, Jukka, Metso, Jari, Tahvanainen, Esa, Olkkonen, Vesa M., Jauhiainen, Matti, Ehnholm, Christian
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2004; Elsevier
• Subjects: Adult; Aged; Antibodies, Monoclonal; Blood Proteins - chemistry; Carrier Proteins - blood; Carrier Proteins - genetics; Carrier Proteins - immunology; enzyme-linked immunosorbent assay; Enzyme-Linked Immunosorbent Assay - methods; high-activity form of phospholipid transfer protein; Humans; Isoenzymes - blood; Isoenzymes - genetics; Isoenzymes - immunology; lipid transfer protein; low-activity form of phospholipid transfer protein; Membrane Proteins - blood; Membrane Proteins - genetics; Membrane Proteins - immunology; Middle Aged; Phospholipid Transfer Proteins; Protein Denaturation; Recombinant Proteins - blood; Recombinant Proteins - genetics; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; high-activity form of phospholipid transfer protein; lipid transfer protein; enzyme-linked immunosorbent assay; low-activity form of phospholipid transfer protein
• ISSN: 0022-2275; 1539-7262
• DOI: 10.1194/jlr.D300023-JLR200

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.