Abstract
An immunochemical method for determining the concentration of plasma β-lipoprotein was developed. The assay was carried out by electrophoresis in an antibody-containing agarose gel (rocket immunoelectrophoresis or quantitative immunoelectrophoresis).
The antiserum was prepared in a rabbit injecting low-density lipoprotein (d. 1.019-1.063) which had been isolated by ultracentrifugation. 10ml of 1% agarose gel containing 2% (v/v) antiserum was spilled over a 84×94mm glass plate and gelled. On each plate 15 holes, 2.5mm in diameter, were punched out on its cathodal side. An aliquot of serum was incubated with an equal amount of 2M KCNO at 50°C for 20 minutes so the proteins were carbamylated. After adequate dilution, 4μl of the sample was applied to the hole. Four holes out of fifteen. on each plate were preserved for reference serum which was obtained from a healthy male person at the age of 39 years and processed equally as the sample serum. The concentration of β-lipoprotein in the reference serum was taken arbitrarily as 100 units/100ml. The electrophoresis was carried out with constant voltage at 2V/cm for 16 hours at 15°C. The height of the precipitin line was measured after staining with amidoblack 10B.
The average β-lipoprotein concentration for 20 normal subjects at the age of 18-22 years was 96±18 (mean±sd) units/100ml. Levels of β-lipoprotein for the subjects at the age of 36-65 years were 124±27 for 18 normal controls, 151±31 for 12 patients with an inactive form of chronic hepatitis 143±41 for 18 patients with an active form of chronic hepatitis, 113±37 for 33 cirrhotic patients and 187±61 for 22 subjects with cholestasis.
The reproducibility of the measurement was satisfactory. This method described here measures total apoprotein B and seems more sensitive than single radial immunodiffusion and easier to perform than radioimmunoassay.
