A Monoclonal Anti Human Factor Ix Produced By A Mouse Hybridoma

Spleen cells of a Balb-c mouse immunized with purified human FIX were fused with mouse myeloma cells (MOPC-21 derivative]. Among the fusion products one hybridoma was found producing an inhibitor of factor IX procoagulant activity. After sub- clonation (5 x) a stable hybridoma was obtained. In vitro cell culture of the hybridoma cells gives anti-FIX titers (Bethesda units] of about 0.8 units/ml. Injection of the hybridoma cells in pristane pretreated mice results in antiFIX titers of 600-1000 units/ml ascites. Analysis of the produced immunoglobulin demonstrated the presence of one main band after iso-electric focussing, which contained heavy chains both of IgG₁ and IgG_2B subclass.

Monoclonal anti-factor IX was isolated from ascites liquid using affinity chromatography on protein-A-Sepharose. Using the purified antibody a radioimmunoassay was developed for the epitope on factor IX, against which this antibody is directed. The epitope is present both on FIX and activated FIXa; however, the affinity of the antibody for binding to FIXa is at least 10 times less. The antibody has no significant affinity for binding to the isolated heavy and light chain of FIXa.

About 27 different genetic variants of factor IX [from haemophilia B patients were tested for the presence of this epitope. All FIX-variants possessed the epitope. At least 2 variants demonstrated a reduced affinity for binding to the antibody.