Use of antibody specificity to study the surface disposition of apoprotein A-I on human high density lipoproteins.

Only -10% of the apoprotein A-I (ape-A-I) in intact high density lipoprotein 2 (HDL*, HDL,-apo-A-I) is detected by radioimmunoassay, suggesting that not all of HDL,-apo-AI is immunologically reactive. Previous work had indicated that the COOH-terminal region of HDL,-apo-A-I was immunologically more reactive than the NH, region, whereas the two terminal regions of the isolated apo-A-I molecule was equally reactive. To confirm these immunologic differences, apo-A-I and HDL2 were used as immunogens and the specificities of antisera were compared. In addition, an anti-apo-A-I antiserum was absorbed with HDLZ and the binding capacity of the absorbed antiserum for the COOHand NH,-terminal fragments of apo-A-I was ascertained. HDL, was isolated by ultracentrifugation (d 1.083 to 1.124) and apo-A-I was obtained from delipidated HDL, by column chromatography (Sephadex G-200, 0.01 M Tris, pH 8.6, 8 M urea). CNBr fragments were prepared corresponding to the COOHand NH,-terminal regions (CNBr I and CNBr II, respectively). APO-A-I and the fragments were iodinated with Na”“I using lactoperoxidase. The maximal binding of these purified 12JI-labeled proteins by the antisera was assessed in double antibody radioimmunoassays. Anti-apo-A-I antisera bound -90% of Y-apo-A-I (-100 pmol) and -60% of ‘291-COOH-terminal (-150 pmol) and ‘““I-NH,-terminal fragments (-80 pmol). Anti-HDL, antisera bound similar amounts of lzzI-apo-A-I and ‘*“I-COOHterminal fragment but they bound only -10% of the ‘*“INH,-terminal fragment (-12 pmol). Antisera produced against the isolated purified fragments bind only the appropriate ‘““I-labeled fragments and -60% of ‘*“I-apo-A-I. Thus, apo-A-I stimulated the production of antisera which contained antibodies directed against both ends of the molecule; HDL,-apo-A-I by contrast stimulated antisera directed preferentially against the COOH-terminal area. In the antiserum absorption experiments, when increasing amounts of HDL, protein (27 to 1500 pglml of antiserum) were added to the anti-apo-A-I antiserum, the capacity of the antiserum to bind both ‘Z”I-COOHand lzSI-NH,terminal fragments diminished. However, after addition of