Canine factor VIII (FVIII) preparations isolated from cryoprecipitates by gel chromatography were pooled to provide one batch of antigen for simultaneous immunization of two rabbits and a goat. The goat and rabbit antisera had similar FVIII-neutralizing titers, but the latter had seven to ten times more precipitating titer for FVIII-related antigen (FVIII-RA). Absorption with material low in FVIII had little effect on the precipitating titer of the rabbit antibody, but it abolished the precipitating capacity of the goat antibody and caused a 20% reduction in the neutralizing titer of both antisera. Results obtained in the Laurell assay with the two different antisera were similar. This finding was true whether the FVIII-RA levels were reduced, normal, or elevated, as well as for heat-treated and frozen- thawed plasmas. Both antisera were neutralized by the same canine plasma to a similar extent. Analysis of FVIII concentrates by crossed immunoelectrophoresis suggested that canine FVIII-RA was heterogeneous, with slow-and fast-migrating components. The presence of more than one antigenic site on the FVIII complex was also supported by the disparity between the FVIII-neutralizing and -precipitating titers of goat antiserum and by the demonstration that FVIII-RA, FVIII-neutralizing antigen, and procoagulant activity varied independently.

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