Characterization and experimental use of a monospecific antiserum to factor IX

Purified human factor IX was used to develop xenogeneic neutralizing and precipitating antibodies. The final antiserum (R2) neutralized only factor IX and was equivalent to 220 Bethesda-inhibitory units. It showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket respectively, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original antigen, and Hemophilia-B antigen-positive plasmas. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative contained only factor IX neutralizing and precipitating antibodies. Experiments with various factor IX concentrates revealed that the majority contained excess factor IX antigen compared to their coagulant activity. In addition, crossed antigen-antibody electrophoresis uncovered differences in the migration of the factor IX of Konyne preparations, when done in the presence of EDTA or calcium. This monospecific antiserum to human factor IX was subsequently used to investigate a large population of hemophilia B patients and carriers.