Measurement of human factor VIII by avidin-biotin dot immunobinding ELISAs

Hemophilia A is a congenital bleeding disorder which is characterized by a functional deficiency of the coagulation protein factor VIII. We have developed sensitive enzyme-linked immunosorbent assays (ELISAs) for measuring the antigenic reactivity of factor VIII. The assays utilize dot immunobinding techniques, commercial monoclonal antibodies, and a detection system enhanced by the interaction of avidin and biotin. The dot immunobinding ELISAs were optimized for measuring factor VIII in normal and hemophilic plasma, and in partially and highly purified preparations of factor VIII. Linear standard curves were established for all samples, defining the range for accurate measurement. Factor VIII was detected at concentrations as low as 0.0005 U/ml, which represents 0.1 pg of protein.