Studies on a monoclonal antibody to human factor viii coagulant activity, with a description of a facile two-site factor VIII coagulant antigen assay.

IgG was purified from the ascites tumor fluid obtained from mice injected with a monoclonal cell line secreting antibody that inhibited VIII:C. With a modified Bethesda assay method (18 hr, 4 degrees C), the titer of the purified IgG was 14,000 U/mg. In a fluid-phase IRMA for VIII:CAg utilizing the Fab fragment prepared from the monoclonal IgG, two high-titer human anti-VIII:C inhibitors (IgG fractions) showed no demonstrable competition for the monoclonal VIII:CAg binding site. Conversely, neither human antibody (125I-Fab fragment) was displaced from its VIII:CAg binding site by the monoclonal IgG molecule. When the monoclonal antibody was used in a fluid-phase IRMA, slightly decreased VIII:CAg levels were found in serum. A facile one-step, two-site IRMA using Sepharose-bound human anti-VIII:C and labeled monoclonal IgG was designed. With this assay, in contrast to the finding with the fluid-phase IRMA, both the rate and apparent level of binding of VIII:CAg "sandwiched" between the two antibodies were increased approximately twofold in serum compared to the native plasma. A similar increase in rate and apparent level of binding was also found after thrombin treatment of VIII:C/vWf relative to the untreated control preparation.

The effect of naturally occurring antibodies to factor VIII on an immunoradiometric assay for factor VIII coagulant antigen. Observation on a cross-reacting material-positive (CRM+) hemophiliac with a factor VIII inhibit.

An IRMA for VIII:CAg was performed on plasma samples from 13 hemophilic and four nonhemophilic subjects, containing naturally occurring antibodies against factor VIII. The VIII:C binding ligand was an iodinated Fab fraction of a high-titer factor VIII antibody arising in a hemophiliac. Since the IRMA is an equilibrium binding assay, the inclusion of an antibody against VIII:C in the test system would be expected to compete with the ligand for the available antigenic sites on the VII:C molecule: the higher the titer of the antibody expressed in BIU, the greater the degree of inhibition of the IRMA measuring VIII:CAg. In a mixture of equal parts of normal and inhibitor plasmas, the amount of residual VIII:C remaining after 2 hr incubation at 37 degrees C differed from the residual VIII:CAg in half (three of six) of the high-titer inhibitors (> 30 BIU) so studied. In the inhibitors of lower titer (< 20 BIU), correlation between BIU and the percentage of VIII:CAg neutralized was not observed. Three of these inhibitor plasmas did not significantly compete with the ligand. With this competitive protein-binding technique the four plasmas with spontaneous inhibitors could not be distinguished from those arising in hemophiliacs. In one hemophilic plasma the titer of VIII:C inhibitor over a 6-day span increased from 4 to 500 BIU as the VIII:CAg level dropped from 84% to 2%. The finding of circulating VIII:CAg in the presence of an VIII:C inhibitor further substantiates the heterogeneity of the naturally occurring VIII:C antibody and is evidence that factor VIII inhibitors can occur in CRM+ hemophilia.

The action of immobilized thrombin on factor VIII, fibrinogen and a synthetic tripeptide.

Bovine thrombin was insolubilized by attachment to cyanogen bromide-activated Sepharose (Sepharose-thrombin) or to activated (Affi-Gel 10) agarose containing a 10 A long arm (Affi-Gel-thrombin). Coupling in both instances approximated 7,000 units of thrombin per ml packed gel as determined by 125I-thrombin incorporation. The thrombin beads hydrolyzed the synthetic tripeptide Bz-Phe-Val-Arg-pNA (S-2160) at different rates, with the Sepharose-thrombin more active (220 esterase units per ml) than Affi-Gel thrombin (20.4 units per ml). The Km was significantly higher for the insolubilized thrombins (2 X 10(-3) M) than uncoupled thrombin (Km = 8 X 10(-5) M). The Sepharose-thrombin activated factor VIII significantly more rapidly than Affi-Gel-thrombin. Neither matrix-bound thrombin clotted a fibrinogen solution or liberated significant amounts of fibrinopeptides over 48 hr. This data indicates that a proteolysis of factor VIII, rather than a complex with thrombin, is the method of activation of factor VIII and that factor VIII is more accessible to the action of immobilized thrombin than is fibrinogen.