Enzyme-linked immunosorbent assay to measure apolipoproteins AI and B secreted by a human hepatic carcinoma cell line (Hep G2).
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.
Hahn
S E
SE
Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
Parkes
J G
JG
Goldberg
D M
DM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016632:Apolipoprotein A-I / Q000032:analysis / Q000378:metabolism* / Q000592:standards; D001055:Apolipoproteins B / Q000032:analysis / Q000378:metabolism* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000378:metabolism
D000596:Amino Acids; D016632:Apolipoprotein A-I; D001055:Apolipoproteins B