Immunoradiometric Assay of Factor VIII Related Antigen

1975 ◽  
Author(s):  
Z. M. Ruggeri ◽  
P. M. Mannucci ◽  
S. L. Jeffcoate ◽  
G. I. C. Ingram
Keyword(s):  
Factor Viii ◽  
Solid Phase ◽  
Rabbit Antiserum ◽  
Normal Subjects ◽  
Immunoradiometric Assay ◽  
Factor Viii Related Antigen ◽  
Highly Correlated ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Specific Rabbit

The development of a solid phase non-competitive immunoradiometric assay (two-site assay) has allowed us to measure factor VIII related antigen (VIIIAGN) in normal plasma diluted up to 2500 times (4.10-4 U/mg). The assay is based on the extraction of VIIIAGN from plasma by means of polystyrene tubes coated with a specific rabbit antiserum and subsequent labelling of the extracted protein with 125I labelled rabbit anti-VIIIAGNIgG. The plasma values obtained in 32 normal subjects were highly correlated with those obtained by means of rocket Immunoelectrophoresis (r = 0.94). A positive correlation was also shown with factor VIII procoagulant activity (VIIIAHF) (r = 0.61), and with von Willebrand factor (VIIIVWF) (r = 0,64). In 14 patients with severe von Willebrand’s disease (vWd), VIIIAGN was not detectable (< 4.10-1 U/ml) in 8 cases or measurable in trace amounts (6.10-4–10-2 U/ml) in 6 cases. Measurable levels could also be obtained (5.10-2–10-1 U/ml) in 14 additional cases of vWd in which VIIIAGN was below the sensitivity of the rocket Immunoelectrophoresis technique (10-1 U/ml).

scholarly journals Selective absence of large forms of factor VIII/von Willebrand factor in acquired von Willebrand's syndrome. Response to transfusion

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 600-606 ◽  
Author(s):  
D Meyer ◽  
D Frommel ◽  
MJ Larrieu ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Procoagulant Activity ◽  
Factor Viii Related Antigen ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

Abstract A previously healthy elderly man with mucocutaneous bleeding was found to have a benign monoclonal IgG gammapathy associated with criteria for severe von Willebrand disease (Factor VIII procoagulant activity, Factor-VIII-related antigen, and ristocetin cofactor activity, less than 10% of normal). Associated qualitative abnormalities of factor VIII/von Willebrand factor were demonstrated by radiocrossed immunoelectrophoresis and immunoradiometric assay. The late clinical onset and negative family history are in favor of an acquired form of vWD. The monoclonal gammapathy and abnormalities of factor VIII/von Willebrand factor have been stable over a 10-yr period. No inhibitor to Factor VIII procoagulant activity, ristocetin cofactor activity, or Factor-VIII-related antigen could be demonstrated. Following transfusion of cryoprecipitate (with a normal cross immunoelectrophoretic pattern), there was a rapid removal of the large forms of Factor.-VIII-related antigen, paralleled by a decay of ristocetin cofactor activity. The transfusion study of this patient with acquired von Willebrand disease type II (variant of von Willebrand disease) serves to emphasize the relationship between polydispersity of Factor VIII/von Willebrand Factor and functional heterogeneity.

Monoclonal Antibodies As Probes Of Factor VIII/Von Willebrand Factor

1981 ◽  
Author(s):  
D Meyer ◽  
T S Zimmerman ◽  
T S Edgington
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Significant Inhibition ◽  
Myeloma Cells ◽  
Close Proximity ◽  
Antibody Titers ◽  
Von Willebrand ◽  
Willebrand Factor

Monoclonal antibodies are essential for the isolation and specific assay of the various components of Factor VIII/von Willebrand Factor (F.VIII/vWF) and for the immunochemical analysis of the surface epitopes of this protein. In this study, we have produced and characterized a panel of thirty-eight antibody synthesizing hybridomas. Mice were hyperimmunized with human F.VIII/vWF and three days after a 10-20 jig booster dose, spleen cells were harvested and fused with P3X63Ag8 or SP2/0 myeloma cells. All antibodies reacted by solid phase ELISA with purified F.VIII/ vWF and normal cryoprecipitate and did not bind to cryoprecipitate from severe vWD, fibrinogen, or fibronectin. Antibody titers (20 μl) varied from lO2 to 106. Seven antibody producing hybridomas derived from P3X63Ag8 myeloma cells were cloned, recloned, and were stable in ascites growth. Two of these partially blocked VIIIR:RCo while the mixture of the seven exhibited significant inhibition, suggesting a cooperative effect in steric hindrance or induction of allosteric effects on the VIIIR:RCo function of the molecule. Three out of thirty-one hybridomas derived from SP2/0 myeloma cells blocked Factor VIII activity. A spatial map of the epitopes at the surface of F.VIII/vWF was derived from competitive displacement studies of the monoclonal antibodies on immobilized F.VIII/vWF. Three types of results were observed : no displacement, indicating independence of two epitopes; partial displacement, indicating steric proximity; and complete displacement, indicating epitope identity or extremely close proximity.

scholarly journals Localization of a factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1679-1682 ◽  
Author(s):  
Y Takahashi ◽  
M Kalafatis ◽  
JP Girma ◽  
K Sewerin ◽  
LO Andersson ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Binding Domain ◽  
Phase System ◽  
Dependent Manner ◽  
Terminal Portion ◽  
Binding Domains ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (F.VIII) was tested for its ability to bind in solid phase system to von Willebrand Factor (vWF) or fragments obtained with Staphylococcus aureus V-8 protease, ie, SpIII (N-terminal), SpI (central), and SpII (C-terminal). Bound F.VIII was estimated in situ by clotting and chromogenic assays. F.VIII bound in a dose-dependent manner to immobilized vWF and SpIII but not to SpII or SpI. Binding was inhibited by 0.25 mol/L CaCl2 as well as by an excess of vWF or SpIII. Accordingly, immobilized F.VIII specifically bound 125I-vWF and SpIII but not SpII or SpI. Twelve monoclonal antibodies (MoAbs) directed towards SpIII, specifically blocking binding of F.VIII to vWF or SpIII, were used for the mapping of plasmic or tryptic fragments of vWF or SpIII. We thus established that a F.VIII binding domain of vWF is located on a 34 kilodalton (kd) fragment of the N-terminal portion of vWF, between residues 1 and 910, and that it is distinct from the GPIb and collagen binding domains.

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