Immunoradiometric Assay of Factor VIII: Coagulant Antigen using Four Human Antibodies. Study of 27 Cases of Haemophilia A

Abstract

Immunoradiometric assay (IRMA) of VIII:C antigen was performed using either IgG or monovalent Fab fragments from four antibodies arisen in polytransfused haemophilia A patients (titre between 100 and 1500 U/ml). Using IgG isolated by a solid or a liquid phase system, only the high titre (greater than or equal to 1000 U/ml) antibodies could be used for IRMA, with a sensitivity of 0.2% VIII:CAg. Using Fab fragments isolated by liquid phase, high and low (less than or equal to 150 U/ml) titre antibodies could be used and the IRMA was significantly improved with a 10-fold higher sensitivity. The affinity of the antibodies for VIII:CAg, studied by displacement curves using a modification of the IRMA, was found not to depend upon the titre of the antibody. Comparative levels of VIII:C and VIII:CAg in 27 cases of haemophilia A emphasize the heterogeneity of this disorder, two types of severe and three types of mild haemophilia being observed.

In vitro and in vivo Evaluation of a Factor VIII Concentrate Heat-Treated to Inactivate HTLV-III/LAV Viruses

“…FVIII: C was measured in concentrates by both one-stage [2] and two-stage [3] Factor VIII antigen (FVIII: Ag) assay was performed with an IRMA method [4], Von Willebrand factor antigen (vWLAg) was estimated by an enzyme linked-immunoassay already described [5]. Ristocetin cofactor activity was assayed by aggregometry using fresh washed platelets [6].…”

Chromatographic Preparation of a Therapeutic Highly Purified von Willebrand Factor Concentrate from Human Cryoprecipitate

“…The CB activity (CBA) was quantified by ELISA [18] using a mono clonal capture antibody specifically directed to the CB domains, and calf-skin type I collagen. FVIILc was determined by a chromogenic substrate (CBS 4803) from Stago and FVIII antigen by IRMA [19]. The reference corresponded to a pool of normal plasma calibrated against the 2nd International Standard for FVIII concentrates (plas ma 87/718) provided by the NIBSC.…”

Direct characterization of factor VIII in plasma: detection of a mutation altering a thrombin cleavage site (arginine-372----histidine).

“…Although reduced or absent factor VIII procoagulant activity is consistently identified, hemophilia A is a heterogeneous disorder when characterized immunologically using human antibodies to factor VIII (1)(2)(3)(4)(5)(6)(7)(8)(9) and when the specific molecular defects are identified (10)(11)(12)(13)(14)(15)(16).…”

“…Almost all plasmas of patients with severe hemophilia A lack factor VIII protein by both procoagulant and immunologic assays (4)(5)(6)(7)(8)(9). In some cases, these individuals have been found to have short deletions or point mutations leading to nonsense codons that appear to prevent factor VIII synthesis (10)(11)(12).…”

“…These plasmas are termed crossreacting material positive (CRM+). The remaining hemophilic plasmas are either CRM-reduced, in which case the reduction in immunologically detectable protein is comparable to the reduction in functional activity, or CRM-negative (CRM-)-i.e, there is no detectable factor VIII by sensitive immunoassays (1)(2)(3)(4)(5)(6)(7)(8)(9). Although the nature of the molecular defect has been established in several mild CRM-reduced individuals (14)(15)(16) and in one severe CRM+ patient (13), the properties of their plasma factor VIII protein have not yet been characterized.…”