Quantification of lipoprotein lipase (LPL) by dissociation-enhanced lanthanide fluorescence immunoassay. Comparison of immunoreactivity of LPL mass and enzyme activity of LPL.
Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and in very low density lipoproteins. In this study, a new sensitive enzyme immunoassay, the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), for the quantification of immunoreactive LPL mass in biological specimens was developed. In the indirect sandwich DELFIA assay polyclonal anti-human or anti-bovine LPL IgGs were used as capture antibodies, monoclonal antibody (mAb) 5D2 and Eu(3+)-labelled goat anti-mouse IgG were used as detection antibodies. In the direct sandwich DELFIA assay, mAb 5D2 was used as capture and Eu(3+)-labelled mAb 5D2 as detection antibodies. Both purified bovine and human LPL proteins served as standards in the indirect and the direct DELFIA assay. Standard curves were linear between 0.1 and 1000 ng LPL/ml, assuring the sensitivity of the DELFIAs within this range. Mean values for immunoreactive LPL mass in normal individuals were found to be 40.3 +/- 14.4 ng/ml preheparin plasma and 334.1 +/- 71 ng/ml postheparin plasma. In patients affected with type I hyperlipoproteinemia 82.4 +/- 29.3 ng/ml (postheparin plasma) were determined. Coefficients of inter- and intra-assay variation were 4.3% and 6.2% on average. The correlation coefficient between the indirect and the direct DELFIA technique was 0.9694. The correlation coefficient between immunoreactive LPL mass (estimated by DELFIA) and LPL activity (estimated by the LPL activity assay) was 0.9345. Our data are consistent with the concept that LPL is active as a dimer. Dissociation of the LPL dimer into monomers is tightly coupled to both loss of immunoreactivity and enzyme activity of LPL.
Wicher
I
I
Karl-Franzens University Graz, Institute of Medical Biochemistry, Austria.
Sattler
W
W
Ibovnik
A
A
Kostner
G M
GM
Zechner
R
R
Malle
E
E
D000818:Animals; D000937:Antigen-Antibody Reactions; D002417:Cattle; D004789:Enzyme Activation / Q000276:immunology; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000032:analysis; D008071:Lipoprotein Lipase / Q000032:analysis* / Q000276:immunology; D008674:Metals, Rare Earth
D007106:Immune Sera; D007074:Immunoglobulin G; D008674:Metals, Rare Earth; D008071:Lipoprotein Lipase