Summary
Factor VIII coagulant antigen (VIII : CAg) was measured in a sandwich-ELISA. Microplates
were used as solid phase and peroxidase conjugated F(ab’)2 fragments of IgG isolated
from inhibitor plasma was used as label without affinity purification. The capacity
of the assay was high and the sensitivity for VIII : CAg was 0.002 U/ml. Using this
assay it was possible to measure coagulation inactive VIII : CAg in samples from purification
studies. Below 0.05 VIII : CAg U/ml these samples responded in parallel with standard
plasma.
Seven of 7 inhibitor antibodies tested were able to inhibit binding of peroxidase-conjugate
in the VIII : CAg assay, and the inhibitory capacity correlated with coagulation inhibition
as measured by the Bethesda method. Using the highest titered antibodies bound to
a solid phase, VIII : CAg was isolated and identified in SDS-PAGE as a doublet with
a molecular weight of 77-80 kD.
Keywords
Factor VIII : CAg - Solid phase immunoisolation - ELISA - Haemophilia A inhibitor
antibodies
