A Monoclonal Antibody To VIII:C Produced By A Mouse Hybridoma

Spleen cells of a Balb-c mouse immunized with VIII:C (isolated by affinity chromatography) were fused with mouse myeloma cells (MOPC-21 derivative). After the fusion 12/32 wells produced an inhibitor to VIII:C. After subclonation (3 x) a stable hybridoma line was obtained. The antibody in the supernatant was detected with a modified VIII: Cinhibitor technique. The supernatant of in vitro cell cultures of the hybridoma cells contained anti-VIII:C titers (Bethesda) of about 0.3-1.0 units/ml. Injection of the hybridoma cells in pristane pretreated Balb-c mice results in anti-VIII:C titers of 5,000-10,000 units/ml ascites.

Analysis of the produced immunoglobulin demonstrated the presence of one band after isoelectric focussing, which contained heavy chains both of IgG₁ and IgG_2B subclass. Because of the unusual kinetics of the monoclonal antibody with VIII:C extensive characterisation of the nature of its VIII: C neutralising properties was necessary.

The monoclonal antibody does not bind ¹²⁵I-fibrinogen or isolated VIIIR:AG, it reacts with isolated VIII:C and can be used in a two-site immunoradiometric assay for VIIICAG. The epitope against which the antibody is directed is not present on ‘serum-VIIICAG’.