Preparation of dried synthetic prothrombin time reagents

• US 6,183,979 B1
• Filed: 03/24/1999
• Issued: 02/06/2001
• Est. Priority Date: 03/24/1999
• Status: Expired due to Term

• Current Assignee: International Technidyne Corporation (Sphingotec GMBH)
• Original Assignee: International Technidyne Corporation (Sphingotec GMBH)
• Inventors: DʼAgostino, Paul Michael, Lee, Ted C. K., Gorman, Anne Jessica
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Moran, Marjorie A.
• Application Number:
  US09/275,338
• Time in Patent Office: 685 Days
• Field of Search: 435/13, 554/79
• US Class Current: 435/13
• CPC Class Codes: G01N 2405/04 Phospholipids, i.e. phosphoglycerides
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

A method for preparation of an air-dried prothrombin time (PT) reagent which uses a recombinant protein and synthetic phospholipids is described. The source for the recombinant protein is rabbit brain, and the phospholipids employed are palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS). The particular formulation buffer used to dilute the lipidated tissue factor provides a reagent that is dried without lyophilization and remains stable for at least 2 weeks at 37 C. A method for preparing the improved PT reagent and a method of using the reagent to analyze blood PT is also provided.

Claims

1. A method for preparing a non-lyophilized air dried prothrombin time reagent used for determining prothrombin time, the method comprising the steps of:

combining a recombinant fusion protein of bacterial thioredoxin and rabbit brain tissue factor with a mixture of synthetic phospholipids in an amount sufficient to activate said protein; and

air-drying said protein and mixture combination at a temperature above about 10°

C. thereby forming the reagent;

wherein the air dried reagent remains stable for at least 2 weeks at 37°

C.

2. The method of claim 1, wherein the bacterial thioredoxin comprises sequences obtained from Escherichia coli.

3. The method of claim 1, wherein the mixture of synthetic phospholipids comprises palmitoyloleoylphosphatidylcholine and palmitoyloleoylphosphatidylserine.

4. The method of claim 1, wherein the mixture includes a HEPES buffer solution having a pH in the range of about 7.0 to about 7.8.

5. The method of claim 1, wherein the mixture includes a calcium compound which provides a source of calcium ions for calcium dependent coagulation factors.

6. The method of claim 1, wherein the reagent has an International Sensitivity Index of substantially 1.0.

7. The method of claim 1, wherein the mixture includes an amino acid chelating agent, a carrier protein, a humectant, and a biocide.

8. The method of claim 7, wherein the amino acid chelating agent includes serine, the carrier protein includes BSA, the humectant includes sorbitol and glycerol, and the biocide includes phenol.

9. The method of claim 5, wherein the calcium ions source is calcium gluconate.

10. A method of preparing a non-lyophilized air dried reagent which is used for determining prothrombin time, the method comprising the steps of:

adding synthetic phospholipids consisting of palmitoyloleoyl-phosphatidylcholine and palmitoyloleoylphosphatidylserine in a molar ratio range of 6;

4 to 8;

2 respectively to a solution containing an antioxidant, a buffer, a salt, an amino acid chelating agent, a detergent, and a humectant;

adding a recombinant fusion protein of bacterial thioredoxin and recombinant rabbit brain tissue factor to said phospholipid solution;

incubating said protein solution for a selected time at a selected temperature to form a homogeneous mixture;

dialyzing the mixture of lipid and tissue factor in a selected dialysis medium to remove said detergent and to form lipid vesicles having said tissue factor inserted therein;

diluting the lipidated tissue factor with a formulation buffer;

air-drying the formulated lipidated tissue factor at a temperature above about 10°

C. and at a selected relative humidity, wherein the air dried reagent remains stable for at least two weeks at 37°

C.

11. The method of claim 10, wherein said formulation buffer comprises a buffer, a salt, an amino acid chelating agent, a humectant, a calcium compound, a carrier protein, a biocide, and an antioxidant, and wherein the formulation buffer has a pH in the range of 7.0 to 7.8.

12. The method of claim 10, wherein said selected dialysis medium comprises a first buffer solution containing glycine and a second buffer solution containing serine.

13. The method of claim 10, wherein said reagent has an International Sensitivity Index of substantially 1.0.

14. The method of claim 10, wherein said palmitoyloleoylphosphatidylcholine and palmitoyloleoylphosphatidylserine are blended in a molar ratio range of about 7 to about 3 respectively.

15. The method of claim 10, wherein the antioxidant includes butylated hydroxytoluene, the buffer includes Hepes, the isotonic reagent includes sodium chloride, the amino acid chelating agent includes serine, and the humectant includes sorbitol and glycerol;

and the carrier protein includes bovine serum albumin.

16. The method of claim 10 wherein the dialyzed mixture of lipid and tissue factor is incubated at ambient temperature for a selected time.

17. The method of claim 10 wherein the detergent includes octyl glucoside.

18. The method of claim 10 wherein a hydrophobic resin is placed in the dialysis medium.

19. The method of claim 10 where the solution is dried in air in the temperature range of about 10 to about 50°

C. and a relative humidity range of about 2 to about 10% R.H.