Quantitation of the active and low-active forms of human plasma phospholipid transfer protein by ELISA.
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 approximately 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 micromol/ml/h (range, 3.21-11.15 micromol/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.
Siggins
Sarah
S
Department of Molecular Medicine, National Public Health Institute, Biomedicum, FIN-00251 Helsinki, Finland.
Kärkkäinen
Minna
M
Tenhunen
Jukka
J
Metso
Jari
J
Tahvanainen
Esa
E
Olkkonen
Vesa M
VM
Jauhiainen
Matti
M
Ehnholm
Christian
C
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D001798:Blood Proteins / Q000737:chemistry*; D002352:Carrier Proteins / Q000097:blood* / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000235:genetics / Q000276:immunology; D008565:Membrane Proteins / Q000097:blood* / Q000235:genetics / Q000276:immunology; D008875:Middle Aged; D049294:Phospholipid Transfer Proteins; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000097:blood / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D011994:Recombinant Proteins