Risk assessment for Chronic Heart Disease (CHD) uses indirect estimates of LDL cholesterol from total cholesterol, HDL and VLDL estimates. No clinical assay exists to quantitate LDL DIRECTLY from serum. We propose a model competitive binding assay using PKH26 linked LDL particles (LDLs) to quantify LDLs directly and taking advantage of the recent interest in relating apolipoproteins measurements to CHD. Phase I specific aims are: 1) Comparison of Zyn-linked (PKH26) LDLs to commercially available LDLs using physical properties (density, stability, dye to particle ratio) and functional properties (LDL receptor binding); 2) Selection of a commercially available anti Apo-B antibody based on: specificity and affinity (to normal and mutant LDLs); 3) Generation of magnetic beads: anti Apo-B antibody reagents; and 4) Creation of a working assay to directly measure LDLs. Patient LDLs will compete with Zyn-linked LDLs for binding sites on Apo-B specific magnetic bead coupled antibodies, which will be used in the separation for LDLs. Signal will be quantitated following release from bound particles recovered by magnetic separation. Transformation of the Phase I model assay to a clinically useful test will involve depletion of other non-LDL lipoprotein particles, followed by comparison with current clinical assays.
