A direct sandwich enzyme-linked limmunosorbent assay for human serum apolipoprotein A-I

Abstract

Apolipoprotein (apo) A-I, an abundant protein in high-density lipoproteins (HDL), has received considerable clinical attention as a negative predictor of coronary artery disease, because the plasma concentration of apo A-I has been shown to be inversely correlated with the incidence of coronary artery disease. A direct sandwich enzyme-linked immunosorbent assay (ELISA) for determination of the level of human serum apo A-I was developed using highly purified anti-apo A-I polyclonal antibodies raised in rabbits as the capturing antibody, and as the detecting antibody as well. Microtiter plates were coated with purified anti-apo A-I polyclonal antibodies. The concentration of apo A-I in samples was determined from the peroxidase color reaction. The sensitivity of the assay was determined to be 10 ng of apo A-I per ml of serum. The working range, where the standard curve showed a linearity, was between 10 and 100 ng/ml. The mean intra-assay and inter-assay coefficients of variation (CV) were <5% and <7%,respectively. No cross reactivities with serum albumin, apo A-II, B-100, C-I, C-II and E were detected. For the evaluation of validity of the assay, 20 serum samples of domestic subjects were employed, and the serum apo A-I measurement by the sandwich ELSA of this report was compared with those of Beckman's and Kallestad's immunonephelometry. The results indicated that there is a good correlation between the data obtained by the present ELISA method and by the two commercially available immunonephelometric assay methods (r=0.95 and 0.84, respectively).