A Simplified Immunoradioactive Assay For Human Factor VIII Coagulation Antigen

Normal human factor VIII (FVIII) is a high molecular weight glycoprotein expressing two measurable biological activities, FVIII procoagulant activity (VIII:C) and von Willebrand factor activity (vWf). The corresponding antigenic moieties are referred to respectively as FVIII coagulation antigen (VIII:CAg) and FVIII related antigen (VIII: RAg). Since VIII:C is extremely labile measurement of VIII: CAg is of importance for pre-natal diagnosis of haemophilia where VIII:C is reduced and von Willebrand’s disease (vWd) where all measurable parameters of FVIII are reduced.

Current immunoradiometric assays for VIII:CAg depend on both the availability of high titre human antibodies directed against VIII:C and the possibility of preparing highly specific ¹²⁵I-IgG from human antiserum

We have developed a solid phase immunoradiometric assay for VIII:CAg. FVIII in a test sample binds to human anti- VIII:C IgG immobilised on the surface of a polystyrene tube. The bound FVIII is detected using rabbit ¹²⁵I-anti human FVIII-IgG. The standard curve was linear for dilutions of normal pooled plasma (NPP) of 1/5 to 1/500. The lower limit of the assay was 0.002U/ml of NPP. Using this assay the concentration of VIII:CAg correlated well with the VIII:C levels in the plasma from normal individuals (r=0.84, n=15). Homozygote vWd patients had undetectable levels of VIII:CAg, VIII:C, VIII:RAg or vWf activities. Patients with haemophilia A with less than O.OlU/ml VIII:C could be divided into two groups on the basis of the VIII:CAg assay, CRM and CRMf. Markedly reduced levels of VIII:CAg were detected in serum.

The advantage of this assay over the previously available assays for VIII:CAg are that smaller volumes of high titre human anti VIII IgG are required and the time consuming step of preparing ¹²⁵I-human anti VIII-IgG is eliminated. The rabbit ¹²⁵I-IgG specific for human VIII is the same as used in routine immunoradiometric assays for FVIII:RAg