As part of our ongoing effort to develop a standardized competitive enzyme immunoassay for human autoantibodies to oxidized low-density lipoprotein (oxLDL), we have generated a reference human antibody standard and revised some of the conditions of the assay. The preparation of the standard involved purification of human anti-oxLDL antibodies by affinity 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 determined in the standard by radial immunodiffusion. The isolated antibody was used to calibrate a whole human serum standard, which was used to calibrate the assays to detect antibody in serum samples. We also revisited the general conditions for performance of our competitive assay. We determined that 1/20 was the ideal dilution for performing the absorption 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 serum samples was 200 microgram of oxLDL/ml; no significant decrease in the reactivity of samples with immobilized oxLDL was observed when higher concentrations 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 detectable in serum is 20-fold higher, i.e., 13 mg/liter. The intraassay coefficient of variation calculated from seven determinations of three samples containing antibody concentrations of 240, 340, and 920 mg/liter ranged from 8 to 6.1%. The interassay coefficients of variation for 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%.
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