Quantitation of Plasma Apolipoprotein A-I with a Sandwich Type Enzyme-Linked Immunosorbent Assay Using Monoclonal Antibodies

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for the quantification of human apolipoprotein A-I (apoA-I) was developed using monoclonal antibodies. For this assay, we used three monoclonal antibodies to trap and detect apo A-I. HDAI16 and HDA15 monoclonal antibodies were used for trapping apoA-I and HDAI8 monoclonal antibody was for detecting apoA-I. These three monoclonal antibodies were produced by immunizing mice with high density lipoprotein (HDL) isolated from human plasma. By immunoblot analysis, these three monoclonal antibodies were specific to apoA-I and showed no cross-reactivities with other plasma proteins. The results of competition assays for epitope cross-reactivity test also verified that these monoclonal antibodies identified separate and distinct epitopes on HDL and apoA-I. Affinity constants of monoclonal antibodies were measured by ELISA. Their association constants ranged from $10^7$ to $10^8$ $M^{-1}$. For this assay, pure apoA-I was isolated by affinity chromatography using monoclonal antibodies. In this sandwich assay, the amount of HRP-labeled HDAI8 bound to apoA-I trapped by HDAI16 and HDAI5 was proportional to apoA-I concentration in the range of 0 to 500ng/ml. ApoA-I concentration in plasma was calculated from the linear regression equation of standard curve. The precision and reliability of the assays are reflected in the low intra-and interassay coefficients of variation that averaged 3.25% and 4.30%, respectively. This assay is sensitive, simple, reproducible, convenient in incubation interval, and does not use radioisotope: thus it can be widely applied in clinical laboratories.