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US 5,308,756 AGranted
Protein S chromogenic assay
AssigneeBaxter Diagnostics Inc.
InventorsPiet van de Waart, Barry J. Woodhams
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Abstract
A chromogenic assay for determination of Protein S, using an indicator of Factor Xa as a measure of Protein S concentration. This technique utilizes the dependency of activated Protein C on Protein S in order to inactivate a known portion of Factor VIII. Residual Factor VIII is then activated and acts with Factor IXa to activate Factor X. The chromogenic substrate is then cleaved by Factor Xa and the rate of conversion of the indicator molecule is an indirect indicator of free Protein S.
Claims29
A method for determining levels of free or functional Protein S in a Protein S containing sample the method comprising:
- a. eliminating the effects of von Willebrands Factor;
- b. incubating the sample with activated Protein C, Factor VIII, phospholipids and calcium;
- c. incubating the mixture with factors IIa, IXa, and X;
- d. adding to the incubation mixture a factor Xa specific substrate;
- and
- e. determining the amount of indicator formed from cleavage of the substrate as a measure of functional Protein S.
3 more elements
A method for determining the levels of Protein S in a Protein S containing sample comprising:
- a. combining the Protein S containing sample with (1) a reagent to eliminate the effect of substantially all von Willebrands Factor present in the Protein S containing sample, (2) activated Protein C in an amount sufficient to inactivate a portion of a fixed amount of Factor VIII, (3) phospholipids and (4) calcium ion in amounts sufficient to facilitate the inactivation of Factor VIII;
- b. incubating the mixture for a time sufficient to eliminate the effects of the von Willebrands factor;
- c. combining the incubation mixture with a fixed amount of Factor VIII;
- d. incubating the mixture for a time sufficient to inactivate a portion of the Factor VIII;
- e. combining the incubation mixture with Factor IIa, Factor IXa, Factor X and calcium ions whereby there are sufficient amounts of Factor IIa and calcium ion to activate the remaining Factor VIII, and a sufficient amount of Factor IXa to activate the Factor X in the presence of the activated Factor VIII;
- f. incubating the mixture until a portion of the Factor X is converted to Factor Xa;
- g. adding to the incubation mixture a sufficient amount of thrombin inhibitor to inhibit thrombin activity without affecting Factor Xa activity;
- h. adding to the incubation mixture an indicator agent capable of reacting with Factor Xa;
- and
- i. measuring the signal molecule.
7 more elements
A method for determining the levels of Protein S in a Protein S containing sample comprising:
- a. combining the Protein S containing sample with (1) a reagent to eliminate the effects of substantially all von Willebrands Factor present in the sample and (2) Protein C in an amount which when activated is sufficient to inactivate a portion of a predetermined amount of Factor VIII and (3) phospholipids and (4) calcium ion in amounts sufficient to facilitate the inactivation of Factor VIII;
- b. incubating the mixture for a time sufficient to eliminate the effects of the von Willebrands Factor on Factor VIII;
- c. combining the mixture with a sufficient amount of snake venom activator in an amount sufficient to activate the Protein C;
- d. incubating the mixture for a time sufficient to activate the Protein C;
- e. combining the incubation mixture with a fixed amount of Factor VIII;
- f. incubating the mixture for a time sufficient to inactivate a portion of the Factor VIII;
- g. combining the incubation mixture with Factor IIa, Factor IXa, Factor X and calcium ions whereby there are sufficient amounts of Factor IIa and calcium ion to activate the remaining Factor VIII, and a sufficient amount of Factor IXa to activate the Factor X in the presence of the activated Factor VIII;
- h. Incubating the mixture until a portion of the Factor X is converted to Factor Xa;
- i. adding to the incubation mixture whereby to release a signal molecule and a sufficient amount of thrombin inhibitor to inhibit thrombin activity without affecting Factor Xa activity;
- j. adding to the incubation mixture an indicator agent capable of reacting with Factor Xa;
- and
- k. measuring the signal molecule.
9 more elements
A kit for performing a Protein S assay on a sample comprising:
- a. a first vessel containing:
- a sufficient amount of an agent to eliminate the effects on Factor VIII of substantially all of the von Willebrands Factor in the sample and a sufficient amount of Protein C to inactivate a portion of a fixed amount of Factor VIII, and sufficient amount of phospholipid and calcium ion to facilitate the inactivation of the Factor VIII;
- b. a second vessel containing:
- a fixed amount of Factor VIII;
- c. a third vessel containing:
- a sufficient amount of Factor IIa and calcium ion to activate the remaining Factor VIII, and sufficient amounts of Factor X and Factor IXa so as to activate the Factor X to Factor Xa in the presence of the activated Factor VIII;
- d. a fourth vessel containing a sufficient quantity of an indicator agent capable of reacting with Factor Xa, and a sufficient amount of a thrombin inhibitor to inhibit thrombin activity without affecting Factor Xa activity.
4 more elements
A kit for performing a Protein S assay on a sample comprising:
- a. A first vessel containing:
- a sufficient amount of an agent to eliminate the effects of substantially all of the von Willebrands factor in the sample and a sufficient amount of Protein C to inactivate a portion of a fixed amount of Factor VIII, and sufficient amount of phospholipid and calcium ion to facilitate the inactivation of the Factor VIII;
- b. a second vessel containing:
- a fixed amount of Factor VIII;
- c. a third vessel containing:
- a sufficient amount of Factor IIa and calcium ion to activate the remaining Factor VIII, and sufficient amounts of Factor IXa so as to activate the Factor X to Factor Xa in the presence of the activated Factor VIII;
- d. a fourth vessel containing a sufficient quantity of a quenching agent to stop the conversion of Factor X to Factor Xa;
- and
- e. a fifth vessel containing a sufficient quantity of an indicator agent capable of reacting with Factor X and a sufficient amount of a thrombin inhibitor to inhibit thrombin activity without affecting Factor Xa activity.
6 more elements
Dependencies are inferred from the claim text.