Purification of F.VIII:C by antigen-antibody chromatography

Holmberg, Lars; Ljung, Rolf

doi:10.1016/0049-3848(78)90256-6

Cited by 50 publications

(14 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It appears that the adsorption of pro teins other than factor VIII results from nonspecific hydrophobic and ionic interac tions with insoluble antibodies; the affinity between the soluble proteins and agaroselinked immunoglobulin was observed also by other authors [13,14]. We have found no measurable VIIIR:AG concentration in the dialyzed fractions of the NaSCN-dissociated protein peak, in agreement with the observation of Holmberg and Ljung [6], Factor VUI-depleted plasma was recon stituted with human fibrinogen and the cali bration curve for the one-stage assay of VIII:C was constructed using this artificial reagent as substrate. The resulting curve is parallel to that obtained with the hemophilic plasma and is a straight line in the log-log plot against VIII:C concentration range be tween 10 and 100%.…”

Section: Resultssupporting

confidence: 81%

Preparation of Factor VIII-Deficient Plasma by Immunoadsorption

Furlan¹,

Felix²,

Beck³

1979

Vox Sanguinis

View full text Add to dashboard Cite

An immunoadsorbent was prepared by coupling rabbit and human antibodies against human factor VIII to Sepharose CL-2B. The resulting insoluble antibodies completely removed factor VIII and fibrinogen from normal human citrate plasma. Other coagulation factors were satisfactorily recovered in the eluted plasma. Following addition of fibrinogen, the factor VIII-deficient plasma was used for calibration of the one-stage factor VIII assay and compared with the hemophilic plasma. Parallel straight lines were obtained in the log-log plot against VIII:C, indicating that the artificial reagent can be used as a substitute for hemophilic plasma in determination of factor VIII procoagulant activity. The immunoadsorbent can be regenerated and repeatedly used for affinity chromatography binding of factor VIII.

show abstract

Section: Resultssupporting

confidence: 81%

Preparation of Factor VIII-Deficient Plasma by Immunoadsorption

Furlan¹,

Felix²,

Beck³

1979

Vox Sanguinis

View full text Add to dashboard Cite

show abstract

“…The conventional assay is a sandwich technique in which rabbit antibodies are used both for the immobilization of V1IIR:Ag and for radiolabelling [6]. The method was modified by using radiolabelled monoclonal antibodies for measuring bound V1IIR:Ag and either monoclonal antibodies or rabbit antibodies for VII1R:Ag immobilization.…”

Section: Methodsmentioning

confidence: 99%

“…The introduction of monoclonal antibodies in these tests has several advantages; practically unlimited access to the same specific antibody; time-consuming affinity purification of the radiolabelled rabbit antibody, entailing two gel filtration steps, can be avoided and the radioactivity handled can be reduced, as only 2.2% of the radioactivity will h e recovered after affinity purification [6]. …”

Section: Monoclonal Antibody In Radiocrossed Immunoelectrophoresismentioning

confidence: 99%

The use of monoclonal antibodies in measuring factor Vlll/von Willebrand factor

Lamme¹,

Wallmark²,

Holmberg³

et al. 1985

Scandinavian Journal of Clinical and Laboratory Investigation

View full text Add to dashboard Cite

Here we report the production of four different monoclonal antibodies against factor VIII/von Willebrand factor (F VIII/vWF) and the use of these antibodies in immunoradiometric assay (IRMA), crossed immunoelectrophoresis (CIE) and multimeric sizing (MS) for analysing the various types of von Willebrand's disease. None of the antibodies inactivated factor VIII coagulant activity and one (R1) of them partly inhibited the ristocetin co-factor activity. One monoclonal antibody (R2) was radiolabelled and compared with 125I rabbit affinity purified antibody against F VIII/vWF. The two IRMA techniques gave similar results in 26 normals and 22 samples representing all variants of von Willebrand's disease. This monoclonal antibody could also be used in multimeric sizing and not only produced patterns identical to those obtained with the rabbit affinity purified antibody, but also gave better resolution. Further advantages of using monoclonal antibodies in these tests are: practically unlimited access to the same specific antibody, time-consuming affinity purification of the rabbit antibody can be avoided and the overall use of radioactivity reduced. This study demonstrates that one (R2) of the four monoclonal antibodies is suitable for routine analysis of F VIII/vWF and the use of this antibody simplifies the laboratory work in classifying von Willebrand's disease.

show abstract

“…The two moieties of the factor-VIII complex can be dissociated in vitro by means of high ionic strength buffers [4][5][6][7]. When citrated plasma is chromatographed on agarose gels with high ionic strength buffers, the HMW moiety elutes in the void volume, while the coagulant moiety elutes later [8].…”

mentioning

confidence: 99%

Dissociation of the factor‐VIII complex during clotting: role of thrombin and phospholipids

Muntean

Rothwangl

1984

Eur J Clin Investigation

View full text Add to dashboard Cite

To study the dissociation of the two moieties of the factor-VIII complex during clotting, plasma, concentrate and serum were chromatographed on 4% agarose. In plasma and concentrate factor-VIII coagulant activity (VIII C), factor-VIII coagulant antigen (VIII C:Ag), and factor-VIII-related antigen (VIII R:Ag) eluted together in the void volume, but some VIII C:Ag eluted after the void volume. The amount of VIII C:Ag eluting after the void volume was made smaller by adding proteinase inhibitors. In serum nearly all VIII C:Ag eluted after the void volume. From factor-VIII complex immunoadsorbed by means of an antibody against VIII R:Ag no VIII C:Ag was dissociated by thrombin or by thrombin and physiologic CaCl2 concentrations. Radiolabelled human thrombin did not bind to the VIII C:Ag of immunoadsorbed factor-VIII complex. VIII C:Ag displaying VIII C was dissociated from immunoadsorbed factor-VIII complex by human brain thromboplastin or by phosphatidyl-serine. Our results suggest that VIII C:Ag and VIII R:Ag dissociate during clotting. This dissociation seems not to be mediated by thrombin, but may be mediated by phospholipids.

show abstract

Purification of F.VIII:C by antigen-antibody chromatography

Cited by 50 publications

References 12 publications

Preparation of Factor VIII-Deficient Plasma by Immunoadsorption

Preparation of Factor VIII-Deficient Plasma by Immunoadsorption

The use of monoclonal antibodies in measuring factor Vlll/von Willebrand factor

Dissociation of the factor‐VIII complex during clotting: role of thrombin and phospholipids

Contact Info

Product

Resources

About