A Monoclonal Antibody To Factor IX

We describe a mouse monoclonal antibody with high affinity for a functional site on coagulation factor IX.

Mice were hyperimmunised to factor IX by repeated intraperitoneal injections of between 15 and 20 µg of purified factor IX in complete Freunds adjuvant, over a period of 5 months. Three days after a final boost with 15 /ug of factor IX in saline, the spleen was removed from a mouse that had a serum anti factor IX activity of 200 NIH units measured in the standard coagulation assay. The spleen cells were fused with the HAT sensitive, nonsecreting mouse myeloma cell line, P3-NSI/1-Ag4-1 (donated by C. Milstein) using polyethylene glycol 1500 (40% w/w). After plating at 1 × 10⁶ cells/ml in 2 ml multiwell culture plates (211 cultures) hybrid cells were selected from the mixed cell population by culturing in the presence of HAT medium for a period of 33 days. Hybrid clones of cells appeared from day 10 onwards and culture supernatants were tested for the production of antibody to factor IX in a coagulation assay.

One of the cultures that produced an inhibitory effect in this assay has been subcloned twice and established as a monoclonal cell line that can be maintained in continuous culture or can be grown as an ascites tumour in syngeneic mice. This.cell line secretes an IgG_1(k) antibody designated RFF-IX/I, that binds to the coagulation site of factor IX. RFF-IX/I inhibits only factor IX and no other coagulation factor. A solid-phase radiometric assay is also described that detects the presence of RFF-IX/Ia by its ability to bind to factor IX linked to polystyrene beads and that can be used to screen for production of similar antibodies by hybrid cell lines.