Assay of factor VIII antigen (VIII:CAg) in 294 Haemophilia A patients by a new commercial ELISA using monoclonal antibodies

Monoclonal antibodies (MoAbs 833 and D4H1) directed against human factor VIII (FVIII) have been produced on a large scale to measure VIII:CAg by two-site ELISA (Asserachrom VIII:CAg, Diagnostica Stago). F(ab’)₂ from MoAb 833 were used for coating and bound VIII:CAg was revealed with MoAb D4H1 coupled to peroxidase. Control plasma (100 VIII:CAg U dL^?1 by comparing with the International Standard) was used as reference. The assay sensitivity was 0.1 U dL^?1 VIII:CAg. No apparent effect of the plasma proteins was observed provided plasma dilution was 5. Thus this ELISA allowed us to estimate VIII:CAg levels of 0.5 U dL^?1 in plasma. Levels of VIII:CAg were similar to those of VIII:C (correlation coefficient r= 0.87) in plasma from normal individuals (32 cases) and in patients with von Willebrand disease of various types (30 cases). Among 294 patients with Haemophilia A (HA), 161 had severe HA (VIII:C < 1 U dL^?1). Among those patients, 124 were cross-reacting material (CRM) negative with undetectable VIII:CAg and 37 were CRM+ (VIII:CAg 1– 31 U dL^?1). In 42 patients with moderate HA (VIII:C 1– 5 U dL^?1), 33 were CRM reduced (VIII:CAg 0.5–8 U dL^?1) and nine were CRM+ with a VIII:CAg/VIII:C ratio of 6–91 (mean 34.3). In mild HA (91 cases with VIII:C 6 U dL^?1), 29 patients were classified as CRM+ (VIII:C 6–57 U dL^?1, VIII:CAg 17–130 U dL^?1 and VIII:CAg/VIII:C ratio 1.8–13.7 (mean 4.51)). In 62 CRM reduced patients there was a linear correlation between VIII:C (6–39 U dL^?1) and VIII:CAg (2–36 U dL^?1) levels (r= 0.88). In conclusion, this sensitive assay allows us to distinguish the quantitative CRM reduced and negative from the qualitative (CRM+) abnormalities in Haemophilia A.