Isolation of apolipoprotein-B-48 from chylomicrons by high-performance liquid chromatography

Abstract Apolipoprotein-B-48 (B-48) is a component of chylomicrons and chylomicron remnants and originates in the human gut wall. Its molecular weight represents 48% of the hepatic apolipoprotein-B-100 (B-100)/ So far, immunochemical assays are only available for B-100 and do not permit the specific determination of B-48. As a convenient immunochemical method is necessary for clinical and biochemical investigations of chylomicron remnants, we isolated B-48 in amounts sufficient to permit the production of monoclonal antibodies. After delipidation of the chylomicron fraction from 3 1 of human chylous ascites, size-exclusion chromatography was performed by using two serially connected TSK columns. B-48 was eluted as a second peak at about 35% of the total volume, well separated from B-100 (eluted as a first peak). After detection by UV absorption at 280 nm, the fractions collected from 25 automated runs were combined, ultrafiltered and examined for their protein composition by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This indicated that we were able to isolate rapidly 3.4 mg of B-48 in a purity sufficient to perform immunization.