Radioimmunoassay of rat apolipoprotein B peptides in lipoproteins and tissues.
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) separates rat apolipoprotein B (apoB) into one lower and two higher molecular weight components. Of the latter, peptide I (PI) corresponds to human B-100, while the slightly faster-migrating peptide II (PII) lacks a human counterpart; the smaller species peptide III (PIII) corresponds to human B-48. We describe here a competitive radioimmunoassay which separately measures the amounts of total (i.e., PI + PII + PIII) and larger (i.e., PI + PII) rat apoB peptides, with the amounts of PIII obtained by difference. Standard rat PIII and combined PI + PII (PI,II) were isolated by high-pressure gel filtration liquid chromatography in the presence of SDS, and the PI,II was used as an immunogen to raise rabbit antisera which were capable of binding all three forms of rat apoB. However, Scatchard analysis showed this binding to represent two distinct types of antibodies: one high-affinity class which bound only PI,II and a second class which bound all apoB peptides with equal but lower affinity. Thus, since 125I-labeled PIII was displaced equally effectively by PI,II and PIII, but 125I-labeled PI,II was displaced only by PI,II, the unabsorbed antiserum could be used to measure either total apoB or PI,II alone, depending on the choice of labeled ligand. The validity of the assay for apoB peptides in very-low-density and low-density lipoproteins and in liver microsomes was verified by comparison with peptide determinations by SDS-PAGE.
D000818:Animals; D001055:Apolipoproteins B / Q000032:analysis*; D002850:Chromatography, Gel; D007106:Immune Sera / Q000276:immunology; D008074:Lipoproteins / Q000032:analysis*; D008099:Liver / Q000032:analysis; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D001055:Apolipoproteins B; D007106:Immune Sera; D008074:Lipoproteins; D010446:Peptide Fragments