The Effect Of Coagulation On The Association Of VIII:CAg With VIIIR:Ag

1981 ◽  
Author(s):  
J A van Mourik ◽  
P H G Lantinga ◽  
J A Hellings
Keyword(s):  
Factor Viii ◽  
Whole Blood ◽  
Calcium Concentration ◽  
Solid Phase ◽  
Binding Capacity ◽  
The Other ◽  
Void Volume ◽  
Concomitant Increase ◽  
Initial Value ◽  
Factor Viii Related Antigen

Solid-phase immunoradiometric assays specific for Factor VIII coagulant antigen (VIII:CAg) and Factor VIII related antigen (VIIIR:Ag) have been used to assay the immunoreactivity of these antigens in plasma and whole blood during coagulation at physiological calcium concentration. When non-anticoagulated plasma, prepared from blood immediately after venipuncture, was incubated at 37°C, the concentration of VIII:CAg and VIlIR:Ag did not change. However, when whole blood, collected withoutanticoagulant, was incubated, the concentration of VIII:CAg gradually decreased to 50% of the initial value whereas the concentration of VIIIR:Ag remained unchanged. Gelchromatographic analyses revealed that coagulation of plasma leads to progressive dissociation of VIII:CAg from the factor VIII:VWF complex. When plasma was chromatographed before the onset of coagulation, VIII:CAg was eluted at the void volume together with VIIIR:Ag whereas after coagulation of the plasma VIII:CAg devoid of VIIIR:Ag was eluted after the void volume. Similarly, when the supernatant plasma from blood was chromatographed before the onset of coagulation, VIII:CAg together with VIIIR:Ag was eluted at the void volume whereas during and after coagulation the amount of VIII:CAg associated with VIIIR:Ag gradually decreased. However, no concomitant increase of the concentration of dissociated VIII:CAg was noted under the latter conditions. It seems likely, therefore, that adherance of dissociated VIII:CAg to cellular constituents accounts for the loss of VIII:CAg during coagulation of blood. On the other hand, it can not be excluded that cellular enzymes, extruded during coagulation, affect the antibody-binding capacity of VIII:CAg.Further studies indicate that, at least in part, dissociation of the factor VIII:VWF complex during coagulation is mediated by thrombin.

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 Isolation of Low Abundance Proteins and Cells Using Buoyant Glass Microbubble Chromatography

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Steingrimur Stefansson ◽  
Daniel L. Adams ◽  
Cha-Mei Tang
Keyword(s):  
Protein Binding ◽  
Biological Samples ◽  
Magnetic Beads ◽  
Solid Phase ◽  
Binding Capacity ◽  
Protein A ◽  
The Other ◽  
Neurofilament Protein ◽  
Surface Areas ◽  
Fast Flow

Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining them in situ.

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.

Immunologie Studies in von Willebrand’s Disease

1976 ◽  
Vol 35 (01) ◽  
pp. 110-119 ◽  
Author(s):  
Y Sultan ◽  
J Simeon ◽  
P Maisonneuve ◽  
J. P Caen
Keyword(s):  
Factor Viii ◽  
Bleeding Time ◽  
The Other ◽  
Von Willebrand's Disease ◽  
Factor Viii Related Antigen ◽  
Von Willebrand’S Disease ◽  
Short Period ◽  
Von Willebrand ◽  
Immunologic Properties

SummaryTwo patients with a severe von Willebrand’s disease characterized by no detectable factor VIII related antigen in their plasma received transfusions of cryoprecipitate. The bleeding time was corrected for a short period of time and returned to its pretransfusional value although the other parameters of the disease were still corrected. Electrophoretic and immunologic properties of factor VIII related antigen infused were determined serially after transfusion. Modifications of these properties occurred progressively after transfusion. The half disappearance time of F. VIIIR. A. was determined and found to be considerably shorter than in hemophilic recipients. This study suggests an alteration in vivo of F. VIIIR. A. infused into von Willebrand recipients.

Use of monoclonal antibodies in an enzyme immunoassay for factor VIII-related antigen.

1984 ◽  
Vol 30 (1) ◽  
pp. 87-92 ◽  
Author(s):  
L A Bradley ◽  
E L Franco ◽  
H M Reisner
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Solid Phase ◽  
Mammalian Species ◽  
Test Sample ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Curves ◽  
Factor Viii Related Antigen ◽  
Assay Precision

Abstract Two monoclonal antibodies (MAb 53, MAb D7) were produced, each having specificity for Factor VIII-related antigen (FVIIIR:Ag), but exhibiting no inhibitory effect on either procoagulant activity or the ability of von Willebrand factor to agglutinate platelets in the presence of the antibiotic ristocetin. For quantification of FVIIIR:Ag, we used the antibodies in a competitive enzyme-linked immunosorbent assay (ELISA). Binding of either of the MAb's to solid-phase antigen was inhibited by free FVIIIR:Ag in the test sample. Dose-response curves for the reference standards were consistently linear (r2 greater than 0.990) and reproducible. The normal range of FVIIIR:Ag detected in plasma (normal defined as 1000 units/L) was similar to that reported for polyclonal heterologous antibodies in similar ELISA or immunoradiometric (IRMA) systems, and the assay was sensitive to 10 units of FVIIIR:Ag per liter. Inter- and intra-assay precision was good, coefficients of variation being less than 11%. Studies on patients showed good correlations between values measured by MAb ELISAS and IRMA (polyclonal rabbit antibody) over FVIIIR:Ag concentrations ranging from less than 10 to 2700 units/L (r = 0.971, p less than 0.001 for MAb 53; r = 0.938, p less than 0.001 for MAb D7). Both ELISAS could be used to quantify FVIIIR:Ag in other mammalian species. The assay is inexpensive and simple, and all reagents required for it are commercially available.

Partial Purification and Characterization of Factor VIII-Activity Present in the Supernatant of Cryoprecipitates

1975 ◽  
Author(s):  
J. Over ◽  
I. Bakker-Woudenberg ◽  
B.N. Bouma ◽  
J. A. van Mourik ◽  
J.J. Sixma
Keyword(s):  
Factor Viii ◽  
Void Volume ◽  
Partial Purification ◽  
Factor Viii Activity ◽  
Purification And Characterization ◽  
Factor Viii Related Antigen ◽  
Elution Pattern ◽  
Normal Plasma ◽  
Von Willebrand

A relative higher percentage of the initial factor VIII activity is found in the supernatant of plasma from patients with von Willebrand’s Disease than in that from normals after cryoprecipitation. Moreover no factor VIII related antigen can be demonstrated in the supernatant after cryoprecipitate. A further study of the characteristics of this cryo-supernatant-factor VIII seemed therefore of interest.Cryosupernatant-factor VIII was partially purified by poly ethyl enegly col and ammoniumsulfate precipitation, followed by Sepharose 6B gelfiltration. Cryosupernatant-factor VIII was eluted at the void volume of Sepharose 6B columns, but in contrast to cryoprecipitated factor VIII it was retarded on Sepharose 2B columns.Cryosupernatant-factor VIII has antigenic determiniant(s) in common with normal factor VIII.Von Willebrand plasma’s obtained 6 to 40 hours after transfusion with normal cryoprecipitate were applied to gelchromatography on Sepharose 6B columns. The factor VIII activity was eluted at the void volume. On Sepharose 2B the elution pattern of the factor VIII activity was comparable to that of normal plasma.

Comparison of Factor XIII Concentration in Cryoprecipitated Plasma Versus Fresh Frozen Plasma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4169-4169
Author(s):  
J. S. Caudill ◽  
W. L. Nichols ◽  
E. A. Plumhoff ◽  
S. L. Schulte ◽  
J. L. Winters ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xiii ◽  
Solid Phase ◽  
Control Measure ◽  
Coagulation Factor ◽  
Fresh Frozen Plasma ◽  
Binding Activity ◽  
The Other ◽  
Fresh Frozen ◽  
Frozen Plasma

Abstract BACKGROUND: Coagulation factor XIII deficiency, in either its acquired or inherited form, is a rare cause of abnormal bleeding. In patients with Factor XIII (F XIII) deficiency, recommended means of factor replacement include infusion of fresh frozen plasma (FFP), cryoprecipitated plasma (Cryo), or F XIII concentrates1. Comparisons of F XIII concentration in FFP and Cryo are not well defined. To aid management of F XIII deficiency, we measured F XIII activity and antigen in FFP and Cryo. In addition, we determined concentrations of fibrinogen, factor VIII (F VIII) and von Willebrand factor (VWF), including antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and collagen binding activity (VWF:CB). STUDY DESIGNS AND METHODS: 10 bags each of FFP and of Cryo from blood group O donors obtained from the Mayo Clinic Division of Transfusion Medicine blood bank, were analyzed. F XIII activity was assayed by ELISA detecting activated F XIII-mediated amine incorporation into solid phase fibrinogen (Pefakit, Pentapharm). F XIII antigen was measured using radial immunodiffusion (The Binding Site). Fibrinogen was measured by Clauss kinetic clotting assay (Fibriquik, Biomerieux) and also by endpoint delta OD derived from the prothrombin time (ACL, Instrumentation Lab). F VIII was measured by 1-stage APTT-based assay; VWF:Ag by automated LIA (Diagnostica Stago); VWF:RCo by aggregometry of washed platelets; and VWF:CB by ELISA (Corgenix). RESULTS: Mean concentrations of F XIII activity were 60 U/bag (+/−30) in Cryo and 288 U/bag (+/− 77) in FFP. F XIII antigen concentrations paralleled the activity results and are depicted in Table 1, which also includes results for the other analytes. The mean fluid volumes of Cryo and FFP were 21.3 ml/bag (+/− 2.7) and 245 mL/bag (+/− 29), respectively. Table 1. Comparison of Hemostatic Components in Cryoprecipitated Plasma (Cryo) versus Fresh Frozen Plasma (FFP) Cryoprecipitate Fresh Frozen Plasma Component Mean (SD) Mean (SD) Factor XIII activity 60 U/bag (+/− 30) 288 U/bag (+/− 77) Factor XIII antigen 0.65 mg/bag (+/− 0.23) 2.36 mg/bag (+/− 0.76) Factor VIII 133 U/bag (+/− 36.5) 265 U/bag (+/− 83) Fibrinogen Clauss 184 mg/dl (+/− 44) 725 mg/dl (+/− 199) Fibrinogen ACL 319 mg/dl (+/− 76) 864 mg/dl (+/− 327) VWF:Ag 181 U/bag (+/− 53) 218 U/bag (+/− 70) VWF:RCo 168 U/bag (+/− 34) 221 U/bag (+/− 65) VWF:CB 165 U/bag (+/− 40) 208 U/bag (+/− 71) Bag volume 21.3 ml/bag (+/− 2.7) 245 ml/bag (+/− 29) CONCLUSION: In contrast to some of the other cryoprecipitable coagulation proteins, F XIII is only mildly enriched in Cryo when compared with FFP (about 2- to 3-fold). Although both products are practical means of F XIII replacement, and F XIII is somewhat enriched in Cryo, FFP may be a preferred product for F XIII replacement when infusion volume is not a major consideration (eg, in adults versus small children) and risks of exposure to multiple donors are considered. While Cryo is no longer recommended as the product of choice for VWF (or F VIII) replacement, our studies also demonstrate that Cryo is significantly enriched in VWF (which is not routinely assayed as a quality control measure for Cryo, in contrast to requirements for F VIII and fibrinogen content).

Substitution of Four Residues Revealed by High-Throughput Screening of Human Factor VIII A2 Domain Generated a Recombinant Molecule That Escapes to Anti-Factor VIII Antibodies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3067-3067
Author(s):  
Jean-Luc Plantier ◽  
Didier Saboulard ◽  
Marc Delcourt ◽  
Nathalie Enjolras ◽  
Claude Negrier
Keyword(s):  
Monoclonal Antibody ◽  
Factor Viii ◽  
High Throughput ◽  
Solid Phase ◽  
Binding Capacity ◽  
Residual Activity ◽  
Wild Type ◽  
Human Factor Viii ◽  
Inhibitory Antibodies ◽  
A2 Domain

Abstract Using Massive Mutagenesis technique® we performed a high-throughput alanine substitution of 206 residues between aminoacids 376 to 649 from factor VIII (FVIII) A2 domain. The pattern of activity and the levels of production of FVIII mutants were assessed following transient expression in COS-1 cells. FVIII mutants that kept at least 50% of wild-type activity were then screened in an inhibitor assay against total immunoglobulin G (IgG) fractions from patients with severe hemophilia A who had developed inhibitory antibodies (n=4; range 6–15 BU/mL) or a non immune IgG as control. In this assay, the cell culture supernatants containing FVIII were incubated in a volume of FVIII-depleted plasma for 1h30 in the presence of IgG. The residual activity was then measured in a chronometric assay. No single mutations were able to significantly allow FVIII to escape inhibitors. Four mutations (S409A, L462A, E507A, L629A) having a tendency to resist to inhibitors were selected and recombined two by two leading to a significant but insufficient resistance to anti-FVIII antibodies. The effect of the mutations was additive since a molecule (FVIII-4A2) combining the 4 substitutions significantly resisted to the inhibitory antibodies. Residual activity of FVIII-4A2 ranged from 8% to up to 82% of the initial activity depending on the inhibitor plasma whereas this residual activity never exceeded 30% for control wild-type FVIII. Following production by CHO cells, purified FVIII-4A2 demonstrated a similar pattern of resistance to the four IgG fractions already assayed. FVIII-4A2 was then assayed against 11 additional unrelated inhibitors (range 3–2662 BU/mL) and displayed also a resistance against 10 out of the 11 IgG fractions. The resistance was in all case only partial in relation with the likely presence of anti-C2 and/or anti-A3-C1 inhibitors within the IgG fractions. As detected in a solid-phase assay, the decrease in inhibitory effect was for some of the IgG fractions partly related to a decrease in their binding capacity. As a control experiment, FVIII-4A2 was poorly recognized by the monoclonal antibody GMA012 directed against the A2 domain. In contrast, the binding to ESH4, an anti-C2 monoclonal antibody was not affected. Such combination of mutations opened the perspective for the generation of a recombinant FVIII molecule that can be used as an effective substitutive FVIII therapy in patients with inhibitors.

Hypersensitivity to Thromboxane A2 in Cholesterol-Rich Human Platelets

1990 ◽  
Vol 64 (04) ◽  
pp. 594-599 ◽  
Author(s):  
Takuya Tomizuka ◽  
Kyohei Yamamoto ◽  
Aizan Hirai ◽  
Yasushi Tamura ◽  
Sho Yoshida
Keyword(s):  
Calcium Concentration ◽  
Binding Capacity ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Cholesterol Content ◽  
Human Platelets ◽  
Dissociation Rate ◽  
Platelet Membrane ◽  
Maximal Concentration ◽  
Equilibrium Dissociation

SummaryThe effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,ll-epithio-ll,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

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