Recombinant rabbit tissue factor based prothrombin time reagent
First Claim
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1. A method of preparing a liquid or a lyophilized reagent for determining prothrombin time or fibrinogen levels in a plasma sample, the method comprising the steps of:
- (a) providing a solution, suspension or dispersion containing a phospholipid vesicle;
wherein said solution, suspension or dispersion is surfactant free;
(b) admixing surfactant solubilized tissue factor protein with said solution, suspension or dispersion; and
(c) incubating the mixture produced in step (b) under conditions and for a time sufficient to permit said surfactant-solubilized tissue factor protein to associate with said phospholipid vesicle to produce a reagent capable of inducing coagulation in the plasma sample without removal of the surfactant.
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Abstract
Liquid and lyophilized reagents for determining prothrombin time and/or fibrinogen levels in a plasma sample are disclosed. The reagents preferably are based on recombinant rabbit tissue factor. Also disclosed is a purified preparation of recombinant rabbit tissue factor having unique properties, and a novel method for making PT reagents.
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19 Claims
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1. A method of preparing a liquid or a lyophilized reagent for determining prothrombin time or fibrinogen levels in a plasma sample, the method comprising the steps of:
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(a) providing a solution, suspension or dispersion containing a phospholipid vesicle;
wherein said solution, suspension or dispersion is surfactant free;(b) admixing surfactant solubilized tissue factor protein with said solution, suspension or dispersion; and (c) incubating the mixture produced in step (b) under conditions and for a time sufficient to permit said surfactant-solubilized tissue factor protein to associate with said phospholipid vesicle to produce a reagent capable of inducing coagulation in the plasma sample without removal of the surfactant. - View Dependent Claims (2, 3, 4)
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5. A purified preparation of recombinant rabbit tissue factor, said preparation comprising:
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recombinant rabbit tissue factor comprising an amino acid sequence defining from an N-terminus to a C-terminus, (a) an extracellular domain, (b) a lipid binding domain, (c) an intracellular domain, and (d) an affinity tag for use in selectively enriching said recombinant rabbit tissue factor; wherein the purified preparation is enriched for the recombinant rabbit tissue factor protein having the lipid binding domain relative to a recombinant rabbit tissue factor without the affinity tag. - View Dependent Claims (6, 7, 8, 9, 10)
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11. A liquid or lyophilized reagent for determining prothrombin time or fibrinogen levels in a plasma sample, the reagent consisting essentially of:
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(a) a surfactant-solubilized recombinant rabbit tissue factor protein; (b) a phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylserine, and a combination thereof; and (c) a buffer system; wherein the phospholipid contains a mixture of phosphatidylserine and phosphatidylcholine in a ratio from about 20;
80 to abut 30;
70. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18, 19)
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Specification