Immunoradiometric Assay Of VIII:CAG, A Potential Tool To Detect Human Anti-VIII:C Antibodies

1981 ◽  
Author(s):  
J A Hellings ◽  
J Over ◽  
F R van Leeuwen ◽  
J A van Mourik
Keyword(s):  
Factor Viii ◽  
Solid Phase ◽  
High Titer ◽  
Test Sample ◽  
Test System ◽  
Human Antibody ◽  
Immunoradiometric Assay ◽  
Plasma Samples ◽  
Heterologous Antiserum ◽  
High Dilutions

A modification of a two-site, solid-phase immunoradiometric assay (IRMA) for Factor VIII coagulant antigen (VIII: CAg) has been evaluated for its potential to detect antibodies against Factor VIII coagulant activity (VIII: C) in patient plasma samples. For this purpose the assay system comprised four steps: 1) coating of test tubes with human anti —VIII:C , 2) incubation with normal Factor VIII— VWF complex, 3) incubation with test sample, and 4) binding of radiolabeled human anti–VIII: C as marker.Of eight hemophilic plasma samples containing antibodies against VIII: C (as detected in a clotting assay) five were able to prevent binding of radiolabel partially and three prevented this completely. One of these three (which actually was the antibody used in the IRMA), was effective even at very high dilutions. Two hemophilic plasma samples without detectable antibodies in the clotting assay, a severe Von Willebrand’s disease plasma and normal plasma samples showed no significant interference with binding of radiolabeled human anti-VIII: C. Also, a plasma sample containing a high titer of spontaneous human antibody to VIII: C as well as a heterologous antiserum against Factor VIII—VWF complex did not interfere with binding of radio-activity.It is concluded that the test system described is a sensitive tool to detect antibodies of the same specificity as those used in the IRMA. It may also detect antibodies of differing specificity. The lack of crossreactivity with some antibodies points to interindividual differences in specificity of antibodies against VIII: C.

Measurement Of Inhibitors To Procoagulant Factor VIII (VIIIC) By Immunoradiometric Assay (IRMA)

1981 ◽  
Author(s):  
R A Furlong ◽  
I R Peake ◽  
A L Bloom
Keyword(s):  
Factor Viii ◽  
Solid Phase ◽  
Factor Viii Inhibitor ◽  
Immunoradiometric Assay ◽  
Plasma Samples ◽  
Antigenic Sites ◽  
Increased Sensitivity ◽  
Viii Inhibitor ◽  
Dilution Curves

Antibodies against VIIIC (VIIICAg) were assayed using a modification of a two-site solid phase IRMA for factor VIII clotting antigen (VIIICAg). Anti-VIIICAg antibodies obtained from a multi- transfused haemophiliac were separated as IgG and labelled with I125. This was used to test plasma from patients with factor VIII inhibitor by competetive binding to common antigenic sites on immunoimobilised VIIIC. A haemophilic inhibitor assessed as 225u by the Bethesda method was used as standard. Results of inhibitor assay using the IRMA in 19 plasma samples from 15 severe haemophiliacs were similar to those obtained by the coagulation method. The increased sensitivity by IRMA of 0.01 u/ml enabled measurement of a haemophilic inhibitor undetectable by clotting assay. Anti VIIICAg activity was also detectable in plasmas from three individuals with acquired inhibitors against VIIIC. These plasmas which also had measurable residual VIIICAg gave dilution curves non-parallel to the standard haemophilic plasma curve. Measurement of haemophilic inhibitors using three IRMAs each employing different I125 labelled haemophilic anti VIIICAg antibodies showed that there was no difference in the sensitivity of the three assays but in some plasmas the results were discrepant indicating different specificities of the labelled antibodies.

Carrier Detection In Haemophilia A By Measurement Of Factor VIII Clotting Antigen (VIIICAg) And Factor VIII Related Antigen (VIIIRAg

1981 ◽  
Author(s):  
I R Peake ◽  
R G Newcombe ◽  
B L Davies ◽  
R A Furlong ◽  
C A Ludlam ◽  
...  
Keyword(s):  
Factor Viii ◽  
Laboratory Data ◽  
Carrier Detection ◽  
Clotting Factor ◽  
Haemophilia A ◽  
Immunoradiometric Assay ◽  
Plasma Samples ◽  
Severe Haemophilia ◽  
Likelihood Ratios ◽  
Unequal Variance

In order to assess the value of measurement of VIIICAg in the detection of carriers of haemophilia A, plasma samples were obtained on three separate occasions from each of 23 obligate carriers of mild and severe haemophilia, and 26 normal females. At each visit each sample was divided into three and each aliquot was then assayed for VIIICAg (immunoradiometric assay), clotting factor VIII (VIIIC) (two stage assay) and VIIIRAg (Laurell immu noelectrophoresis). After calculating median values at each visit, and for the three visits, a comparison of the ratios VIIIC/VIIIRAg and VIIICAg/VIIIRAg was made. Likelihood ratios (of being a carrier) were calculated using an unequal variance predictive method for both ratios. These showed that laboratory data calculated on the median of the three-visit medians had greater discriminatory power than a single-visit median value. Using the median of three visits both VII IC/VIIIRAg and VIIICAg/VIIIRAg gave the same proportional misclassification of carriers as normals (4 of 23- 17%). However the ratios VII ICAg/VIIIRAg were more discriminatory due to the greater reproducibility between visits of VIIICAg results than those of VIIIC. There was no statistically significait difference between VII ICAg/VIIIRAg (or VII IC/VIIIRAg) ratios obtained from carriers of mild or severe haemophilia. The ratio VII ICAg/VIIIRAg was therefore shown to be the method of choice for carrier detection except theoretically in the rare CRM+ families.

The Influence of Thrombin on the Clotting Activity of Factor VIII. A Study with Insolubilized Thrombin

1978 ◽  
Vol 39 (03) ◽  
pp. 600-606 ◽  
Author(s):  
Th Vukovich ◽  
E Koller ◽  
W Doleschel
Keyword(s):  
Factor Viii ◽  
Test Sample ◽  
Test System ◽  
Factor Viii Activity ◽  
Two Stage ◽  
One Stage ◽  
Analytical Procedures ◽  
The One

SummaryAn investigation of the influence of thrombin on the clotting activity of factor VIII was made. Purified factor VIII and different amounts of thrombin complexed to Sepharose 4 B were mixed and incubated for various periods of time. The factor VIII activities of these incubation mixtures were determined by the one- and two-stage analytical procedures in the presence of the thrombin-sepharose and in its absence following the latter removal from the test sample by filtration. The results so obtained confirm the view that thrombin inactivates factor VIII. Evidences for a thrombin-induced potentiation of the factor VIII activity, seen only in the thrombin-sepharose containing test samples analyzed by the one-stage method, are here interpreted as thrombin-effects peculiar to this factor VIII test system and not as potentiation by thrombin of the factor itself.

Detection of hemophilia A carriers by use of frozen plasma samples.

1978 ◽  
Vol 24 (9) ◽  
pp. 1628-1630 ◽  
Author(s):  
H C Yang ◽  
J Hardin ◽  
C Vaudreuil
Keyword(s):  
Regression Analysis ◽  
Factor Viii ◽  
Hemophilia A ◽  
Carrier State ◽  
Immunoradiometric Assay ◽  
Plasma Samples ◽  
Factor Viii Activity ◽  
Factor Viii Antigen ◽  
Normal Women ◽  
Frozen Plasma

Abstract The efficacy of using promptly frozen plasma samples in the diagnosis of the carrier state for hemophilia A was evaluated by simultaneous measurement of factor VIII acitivity and antigen in 20 normal women and 20 obligate carriers. Factor VIII antigen was measured by two methods, electroimmunoassay and immunoradiometric assay. When the factor VIII activity and antigen data were evaluated by regression analysis, 94% of the carriers were correctly identified at the 95% confidence level.

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).

A Rapid One-Step Immunoradiometric Assay for Factor VIII-Procoagulant Antigen Utilizing Monoclonal Antibodies

1983 ◽  
Vol 50 (04) ◽  
pp. 860-863 ◽  
Author(s):  
H V Stel ◽  
E C I Veerman ◽  
J G Huisman ◽  
M C Janssen ◽  
J A van Mourik
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Lower Limit ◽  
Solid Phase ◽  
Immunoradiometric Assay ◽  
Step Procedure ◽  
One Step

SummaryA two-site immunoradiometric assay for factor VIII-procoagulant antigen (VIIICAg) that relies completely on monoclonal antibodies has been developed. By selecting an appropriate combination of these antibodies, it was possible to develop an assay in which the radiolabelled monoclonal antibody did not inhibit the binding of antigen to the solid-phase monoclonal antibodies. Thus, the entire test could be carried out as a one-step procedure. With this one-step assay, an amount of 0.0025 U VIIICAg/ml plasma could be detected after 4 hr of incubation, whereas 18 hr of incubation resulted in a lower limit of sensitivity of 0.0005 U VIIICAg/ml. The use of a one-step assay provides a significant advantage over the conventional two-step assay by simplifying, shortening and rendering the performance of the assay more convenient.

scholarly journals Isolation of Specific Homologous Antibodies to Factor VIII by Immunoadsorption

1977 ◽  
Author(s):  
J.-M. Lavergne ◽  
D. Meyer ◽  
J. Koutts ◽  
N. Ardaillou ◽  
J. P. Girma ◽  
...  
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Solid Phase ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
The Third ◽  
Human Factor Viii ◽  
Subsequent Elution ◽  
Willebrand Factor

Current immunological studies of Factor VIII use heterologous antibodies which predominantly measure Willebrand Factor (WF) and give little information on Factor VIII procoagulant activity (VIII:C). Purification of homologous antibodies specific for VIII:C has been hampered by the fact that they do not form immune precipitates. We have attempted to isolate such antibodies by solid phase immunoadsorption and subsequent elution. Human Factor VIII was specifically bound to goat anti-human Factor VIII IgG previously immobilized onto Sepharose 6 B beads. IgG isolated from a Haemophiliac with a high titer anti-VIII:C antibody (700 Oxford U/ml) was labeled with 125-I and reacted for 72 hours with these beads. The column was then washed with 0.1 M glycine, 0.5 M NaCl, pH 10 buffer to remove non specifically adsorbed material. Specifically adsorbed material was then eluted with 2.5 M MgCl2, pH 7.3, and the peak of radioactivity was filtered on Biogel A-5 m. Both anti-VIII:C and radioactivity were recovered in three distinct peaks. The third peak, corresponding to IgG, contained anti-VIII:C activity with a 10 fold purification as estimated by specific radioactivity. The second peak, eluting just in front of the IgG, had half the specific radioactivity of the third peak. The first peak, corresponding to the Vo, contained Factor VIII related antigen and very little anti-VIII:C activity. The pattern by SDS-polyacrylamide gel electrophoresis is compatible with the existence of Factor VIII (WF-VIII:C)-anti-VIII:C complexes in the first peak; VIII:C-anti-VIII:C complexes in the second, and free anti-VIII:C IgG in the third one. Thus the method leads to the formation of stable VIII:C-anti-VIII:C complexes, allowing the purification of specific human anti-VIII:C antibodies.

Enzyme immunoassay for factor VIII-related antigen.

1982 ◽  
Vol 28 (6) ◽  
pp. 1356-1358 ◽  
Author(s):  
J Cejka
Keyword(s):  
Regression Analysis ◽  
Factor Viii ◽  
Enzyme Immunoassay ◽  
Immunosorbent Assay ◽  
Present Method ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Factor Viii Related Antigen ◽  
Elisa Technique

Abstract I describe a simple enzyme-linked immunosorbent assay (ELISA) for the quantitation of Factor VIII-related antigen in plasma with use of commercially available peroxidase-labeled antiserum and solid-phase support. Regression analysis of 85 plasma samples analyzed by this technique (y) and by a commonly used electroimmunoassay (Anal. Biochem. 15: 45-52, 1966) (x) gave the equation y = 0.223 + 0.77x (r = 0.973). The present method was also compared with enzyme immunoassay in which a phosphatase-labeled antiserum prepared in our laboratory was used; the correlation between the two assays was very good. The simplicity and specificity of the ELISA technique should make it a useful alternative to the more difficult and time-consuming Laurell method.

The Application of a Monoclonal Antibody to Factor VIII Related Antigen (VIIIRAg) in Immunoradiometric Assays for Factor VIII

1985 ◽  
Vol 53 (01) ◽  
pp. 143-147 ◽  
Author(s):  
J E Thomas ◽  
I R Peake ◽  
J C Giddings ◽  
A N Welch ◽  
A L Bloom
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Inhibitory Effect ◽  
Immunoradiometric Assay ◽  
Strong Inhibitory Effect ◽  
Cofactor Activity ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

SummaryA two site solid phase immunoradiometric assay (IRMA) for VIIIRAg has been developed using a monoclonal antibody as both the solid phase ligand and the radiolabelled antibody. A range of normal plasmas and von Willebrand’s disease (vWd) plasmas has been assayed for VIIIRAg by this method and compared with VIIIRAg values measured by an IRMA using polyclonal antibodies and by immunoelectrophoresis and with ristocetin cofactor activity (VIIIRiCoF). It was found that the monoclonal IRMA correlated well with the polyclonal IRMA and the immunoelectrophoretic assay, but the correlation with the VIIIRiCoF assay was relatively poor, in spite of the strong inhibitory effect of the antibody on VIIIRiCoF activity.

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