PREPARATION OF A HUMAN STANDARD FOR DETERMINATION OF THE LEVELS OF ANTIBODIES TO OXIDATIVELY MODIFIED LOW-DENSITY LIPOPROTEINS

As part of our ongoing effort to develop a standardized competitive en zyme immunoassay for human autoantibodies to oxidized low-density lipo protein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of t he standard involved purification of human anti-oxLDL antibodies by af finity chromatography using immobilized oxLDL. The total concentration of antibody in this purified human oxLDL antibody was established by adding the concentrations of immunoglobulin G (IgG), IgA, and IgM dete rmined in the standard by radial immunodiffusion. The isolated antibod y was used to calibrate a whole human serum standard, which was used t o calibrate the assays to detect antibody in serum samples. We also re visited the general conditions for performance of our competitive assa y. We determined that 1/20 was the ideal dilution for performing the a bsorption step, and that 1/20 and 1/40 were optimal dilutions to assay oxLDL antibody in unknown serum samples. We also established that the optimal concentration of oxLDL for absorption of free antibody in ser um samples was 200 mu g of oxLDL/ml; no significant decrease in the re activity of samples with immobilized oxLDL was observed when higher co ncentrations of oxLDL were used for absorption. The minimum detection level of the assay is 0.65 mg/liter. Because serum samples are diluted 1/20 and 1/40 for the assay, the minimal concentration of antibody de tectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of thre e samples containing antibody concentrations of 240, 340, and 920 mg/l iter ranged from 8 to 6.1%. The interassay coefficients of variation f or sera with antibody levels of 100 to 594 mg/liter varied from 9.2 to 7.0%, and for isolated antibodies with concentrations of 52 to 111 mg /liter, the coefficients varied from 5.8 to 3.9%.