title: Assay of factor VIII antigen (VIII:CAg) in 294 haemophilia A patients by a new commercial ELISA using monoclonal antibodies.
abstract: 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 > or = 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 > or = 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.
authors.affiliation: INSERM U.143, Hôpital de Bicêtre, Paris. girma@infobiogen.fr
authors.forename: J P
authors.initials: JP
authors.lastname: Girma
authors.forename: E
authors.initials: E
authors.lastname: Fressinaud
authors.forename: A
authors.initials: A
authors.lastname: Houllier
authors.forename: Y
authors.initials: Y
authors.lastname: Laurian
authors.forename: J
authors.initials: J
authors.lastname: Amiral
authors.forename: D
authors.initials: D
authors.lastname: Meyer
mesh_terms: D000911:Antibodies, Monoclonal; D000941:Antigens / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology*; D006801:Humans; D016014:Linear Models; D011933:Reagent Kits, Diagnostic; D014842:von Willebrand Diseases / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D011933:Reagent Kits, Diagnostic; D005169:Factor VIII