Enzyme‐linked immunosorbent assay to measure apolipoproteins Al and B secreted by a human hepatic carcinoma cell line (Hep G2)

Abstract

We describe an enzyme-linked immunosorbent assay (ELISA) to measure apolipoproteins AI and B secreted by Hep G2 cells and in cell homogenates. These assays utilize commercially available polyclonal antibodies, affinity-purified to improve their specificity, thereby achieving a dramatic increase in the sensitivity of the assay. These affinity-purified antibodies were also more sensitive than a series of monoclonal antibodies tested. We achieved a sensitivity of 0.4 ng in the apo AI assay, and a sensitivity of 5 ng in the apo B assay. By these methods, we measured secretion rates by Hep G2 cells of 358 +/- 41 ng/mg cell protein/hr for apo B and 137 +/- 8 ng/mg cell protein/hr for apo AI. These assays also allowed the measurement of intracellular apolipoproteins and thus can be used to facilitate investigations of human lipoprotein metabolism in cell culture systems.

Measurement of apolipoprotein B in various cell lines: Correlation between intracellular levels and rates of secretion

“…The intracellular levels (0.43 ± 0.08 pmol apoB/mg cell proteins) of apoB found in the present study were similar to those reported by Hahn et al (26) but were significantly higher than those reported by Byrne et al (29). The lower values in the later studies may be due to the use of a detergent that might not have solubilized all the intracellular apoB.…”

“…The lower values in the later studies may be due to the use of a detergent that might not have solubilized all the intracellular apoB. In contrast, the secretion rates (0.11 ± 0.01 pmol/mg/h) observed in the present study are similar to those of Byrne et al (29) but significantly different from those of Hahn et al (26). In the later studies, secretion rates were studied in the first 6 h after a media change and thus may not represent steady-state rates.…”

“…This procedure has been used to measure apoB levels in plasma and various lipoprotein fractions (data not shown). The method described here is more sensitive, does not require affinity purification of antibodies, and is rapid compared to a recently described ELISA method for apoB detection (26). Hahn et al (26) evaluated the use of monoclonal antibodies as capture antibodies and found that they were less effective than affinity-purified polyclonal antibodies.…”

Impaired cell functions of hepatocytes incubated with plasma of septic patients

“…It exhibits biosynthetic capabilities similar to those of normal hepatocytes [36], retains cell surface receptors [29,37], processes mediators like interleukin 6 [38] and it produces most of the plasma proteins [31,[39][40][41]. The cells also have been shown to retain cytochrome P450-dependent monooxygenase enzymes [20,42] and glucuronic-and sulfate-conjugation abilities [35,43].…”

Modulation of cholesterol metabolism by Ginkgo biloba L. nuts and their extract

“…Apolipoprotein B was determined by Enzyme-Linked Immunosorbant Assay (Hahn, Parkes, & Goldberg, 1992). Ninety six well microtiter plates were coated with primary antibody (sheep anti-human apolipoprotein B, IgG fraction, Binding Site Ltd., Birmingham, UK) and unbound sites were blocked with bovine serum albumin.…”