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 IgG1(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.
