Quantification of apolipoproteins in rat serum and in cultured rat hepatocytes by enzyme-linked immunosorbent assay

Abstract

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure apolipoproteins in rat serum. Nondelipidated whole serum was heat-treated at 52 degrees C for 3 h in phosphate-buffered saline containing 0.1% Tween-20 before assay. Monospecific rabbit anti-rat apolipoprotein antibodies were added to 96-well polystyrene microtiter plates which had been coated with purified rat serum apolipoproteins or unknown samples. After incubation and washing, goat anti-rabbit serum antibodies conjugated with horseradish peroxidase were added to the plates and incubated. The bound peroxidase activity was assayed after further washing. Serum apolipoprotein concentrations were calculated by comparison against purified standards that were assayed simultaneously with the unknown samples. The intraassay coefficients of variation for apolipoprotein AI, E, and AIV (Apo AI, E, and AIV) were 2.3, 4.4, and 5.3%, and interassay coefficients of variation were 6.1, 5.5, and 7.9%, respectively. The ELISA assay is sensitive to nanogram quantities of rat serum apolipoproteins and the results agree well with those measured by densitometry. The serum concentrations of Apo AI, E, and AIV of a normal fed rat were found to be 504 +/- 8, 413 +/- 20, and 262 +/- 20 micrograms/ml, respectively. When cultured as monolayers in Waymouth's medium for 1 day, rat hepatocytes secreted Apo AI, E, and AIV at rates of 2.51, 61.8, and 48.9 ng protein/mg cell protein/h.