Monoclonal antibody to an epitope on the heavy chain of factor IX missing in three hemophilia-B patients.

A murine hybridoma cell line that produces a monoclonal IgG1 antibody to human factor IX was established to provide a conformational probe for the clotting factor and its genetic variants. The antibody inhibited factor IX procoagulant activity, but did not appreciably interfere with the cleavage of factor IX by factor XIa nor with the binding of antithrombin-III-heparin complex to factor IXa. The antigen-solid-phase-antibody complex could be readily dissociated by relatively low concentrations of guanidine or sodium dodecyl sulfate, but only partially by high concentrations of urea. After gel electrophoresis and blotting of reduced samples of factor IXa, the antibody bound exclusively to the heavy chain. Sensitive immunoradiometric assays were developed using insolubilized monoclonal or polyclonal antibodies. Bovine factor IX had little cross-reactivity with the monoclonal antibody. Of 55 patient samples representing different pedigrees with hemophilia-B, antigen levels by the two assays were in excellent agreement in 49. There were 2 severely affected patients whose levels were too low to quantitate in the monoclonal antibody assay. A third, who had the lowest level of all by polyclonal antibody testing, and 3 less severely affected patients had no detectable antigen in the monoclonal antibody assay system (less than 0.03 U/dl). The latter 3 had at least 100-500 times as much antigen by polyclonal antibody testing. It is proposed that these 3 individuals have structural defects involving the epitope recognized by the monoclonal antibody and that they are due to amino acid substitutions between residues 188 through 359. Furthermore, it is suggested the substitutions lead to abnormal kinetic properties.