IMMUNOCHEMICAL ANALYSIS OF RABBIT ANTIHUMAN FIBRINOPEPTIDE-B ANTIBODIES

The serologic immune response to human fibrinopeptide B (hFPB), a unique thrombin proteolytic product of the fibrinogen Bβ chain (Bβ1-14), was studied as a basis for developing immune probes for sequential regions in the fibrinogen molecule. Outbred rabbits hyperimmunized with hFPB analogue-bovine albumin conjugates produced antisera specific for native hFPB, as measured by radioimmunoassay using [125I]Tyr-hFPB analogue as a tracer. By use of this assay system, these sera were found capable of distinguishing free hFPB from the peptide bound to its parent fibrinogen molecule. Immunoreactive sites seen by these sera were characterized in terms of displacement of bound radiolabeled tracer by synthetic homologues of the hFPB sequence. In four immune rabbit sera, displacement of binding comparable on a molar basis using hFPB as inhibitor was obtained with fragments Bβ3-14, Bβ5-14, and Bβ7-14 but not with fragment Bβ9-14. In an additional three sera, inhibition comparable to that of intact hFPB occurred only with fragments Bβ3-14 and Bβ5-14. In five of the seven sera tested, Arg14 seemed to contribute critically to hFPB antigenicity. Isoelectric focusing experiments showed that the immune response of each sera was limited to 8-10 discrete bands, indicating that these functional restrictions in site specificity were associated with a limited structural heterogeneity. Our data suggest that the region comprising the COOH-terminal 8-10 residues of hFPB is immunologically hindered by its attachment to the Bβ chain of its parent fibrinogen molecule. These findings are analogous to those of earlier studies on the immunochemistry of human fibrinopeptide A (hFPA) wherein immunogenic determinants in hFPA which were characteristic of the free peptide in solution could be localized to the COOH-terminal decapeptide sequence [Wilner, G. D„ Nossel, H. L., Canfield, R. E., & Butler, V. P., Jr. (1976) Biochemistry 15, 1209], Taken together, these results suggest that antisera which are specific for free fibrinopeptides in solution may be directed against conformationally ordered regions shown to be present in the COOH-terminal halves of these peptides [Huseby, R. M. (1973) Physiol. Chem. Phys. 5, 1]. It is concluded that antibodies of limited heterogeneity may be produced by immunization with peptide haptens possessing limited numbers of determinants and that antibodies so produced are potentially useful as sequence-specific immune probes. © 1979, American Chemical Society. All rights reserved.