Quantification of human serum apolipoprotein AI by enzyme immunoassay.

Abstract

Abstract We developed a quantitative assay for apolipoprotein AI (apo AI) in human serum, using a "sandwich"-type enzyme-linked immunosorbent assay. Diluted serum samples were pipetted into the wells of polystyrene microtiter plates that had been previously coated with purified rabbit anti-human apo AI antibodies. After incubation for 2 h and washing, antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) were added and incubated for 2 h; after further washing, the bound enzyme was assayed by oxidation of o-phenylenediamine. Assay conditions were optimized for the incubation time and the amounts of coating antibodies and conjugate. Assay sensitivity is about 0.5 ng of apo AI, with a working range of 1 to 14 ng, similar to that of radioimmunoassays for human apo AI. The standard curves for apo AI in serum or HDL and for purified apo AI were parallel. Delipidation, heat treatment, or addition of detergents did not affect the amount of immunoassayable apo AI in human serum. The intra- and interassay CVs were 4 and 8%, respectively. Results for 100 serum samples compared well with those by immunonephelometry (r = 0.94).