The preparation of copper-oxidized LDL for the measurement of oxidized LDLantibodies by EIA

In the present study we try to define the optimal conditions for preparatio n of copper-oxidized low-density lipoprotein (oxLDL) to be used for the ass ay of oxLDL antibodies by enzyme immunoassay (EIA). Oxidation of LDL was mo nitored by measuring the formation of conjugated dienes at 234 nm and the g eneration of fluorescent products with emission at 430 nm when excitation i s performed at 360 nm. The generation of immunogenic epitopes was evaluated by testing the reactivity of aliquots collected at different times during the oxidation process with human sera with high oxLDL antibody levels and w ith a purified human oxLDL antibody. The values of fluorescence emission at 430 nm correlated best with reactivity with oxLDL antibodies; strong react ivity was usually associated with values greater than 1.1 U. The time neede d for fluorescence emission to reach maximum levels varied between 6 and 14 h for most LDL, but it was considerably longer in a few LDL preparations. The maximal reactivity of oxLDL with oxLDL antibodies was observed when the LDL oxidation reaction was stopped 4 or more hours after the fluorescence readings reached their peak. At this stage of the oxidation reaction, apoli poprotein B fragmentation and aggregation were observed as shown by Western blot analysis. The CV for 13 EIA runs of two reference oxLDL antibodies re acting with four different pools of standardized oxLDL prepared according t o the stated guidelines was 14.5 and 3.9%, confirming the reproducibility o f our oxidation conditions. (C) 2000 Elsevier Science Ireland Ltd. All righ ts reserved.