Method for manufacturing a tissue factor-based prothrombin time reagent
First Claim
1. A method for manufacturing a tissue factor-based prothrombin time (PT) reagent, the method comprising:
- combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture, wherein the predetermined quantity of TF is based on a measured TF activity thereof; and
removing the detergent from the TF/phospholipid mixture to create an essentially detergent-free TF/phospholipid vesicle mixture, thereby creating a tissue factor-based PT reagent.
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Abstract
A method for manufacturing a tissue factor-based prothrombin-time reagent includes combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture. In this combining step, the quantity of TF is predetermined based upon its measured TF activity. Next, the detergent is removed from the TF/phospholipid mixture to produce an essentially detergent-free TF/phospholipid mixture that is useful as a tissue factor-based PT reagent. The method can include the use of hydrophobic resin that has been prescreened based on phospholipid recovery to accomplish the removal of the detergent.
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Citations
16 Claims
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1. A method for manufacturing a tissue factor-based prothrombin time (PT) reagent, the method comprising:
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combining predetermined quantities of tissue factor (TF), phospholipid and detergent-containing buffer to create a TF/phospholipid mixture, wherein the predetermined quantity of TF is based on a measured TF activity thereof; and
removing the detergent from the TF/phospholipid mixture to create an essentially detergent-free TF/phospholipid vesicle mixture, thereby creating a tissue factor-based PT reagent. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for manufacturing a recombinant tissue factor-based prothrombin time (PT) reagent, the method comprising:
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combining predetermined quantities of recombinant tissue factor (rTF), phospholipid and detergent-containing buffer to create a rTF/phospholipid mixture, by adding the predetermined quantity of rTF to a mixture of phospholipid and detergent-containing buffer, wherein;
the predetermined quantity of rTF is based on a measured rTF activity thereof, the rTF activity having been measured using a fluorogenic-based rTF assay employing a 6-peptidylamino-1-naphthalenesulfonamide fluorogenic substrate;
adding a prescreened hydrophobic resin to the rTF/phospholipid mixture;
incubating the rTF/phospholipid mixture and the added prescreened resin until the rTF/phospholipid mixture is essentially free of detergent; and
removing said hydrophobic resin from the rTF/phospholipid mixture, thereby providing an essentially detergent-free rTF/phospholipid vesicle mixture;
measuring the total phospholipid concentration of the essentially detergent-free rTF/phospholipid vesicle mixture; and
determining acceptability of the essentially detergent-free rTF/phospholipid mixture for use as a recombinant tissue factor-based PT reagent based on the measured total phospholipid concentration. - View Dependent Claims (14, 15, 16)
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Specification