Isolation and Fractionation of Apolipoprotein-C Using Pooled Serum from Normolipemic and Mild Hyperlipemic Individuals and Preparation of Monospecific Antisera Against Apoliporotein-C Families

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Apo-C has been isolated from VLDL of severe hypertriglyceridemic serum in western contries where this type of disease is rather common. Because of a very low incidence of hypertriglyceridemia among Japanese population, we developed a method to isolate Apo-C from normolipemic or mild hyperlipemic serum. Pooled serum was spun at d, 1.125 for 2.67 × 10<SUP>8</SUP> g-min twice and d <1.125 fraction was isolated and delipidated. Apoprteins were disolved in Tris-HC1 buffer of pH 8.2 containing 6M urea and subjected to chromatography on Sephadex G-150 column to separate Apo-C fraction. Subsequently, Apo-C fraction was rechromatographed to remove the contaminating Apo-A and fractionated on DEAE Cellulose column with a density gradient of TrisHC1 of pH 8.2, rainging from 0.01 to 0.12M. Reasonably purified C-I, C-II, and C-III-2 fractions were obtained. They were further purified by polyacrylamide gel electrophoresis and visualized under ultraviolet light after staining with fluorescent dye. Antisera were raised in rabbits by the administration of the fluorescent bands which were cut from the gels and emulsified with Freund's complete adjuvant.