Assay for low molecular weight heparin
View Patent ↗A prothrombin time reagent for determination of low molecular weight heparin in frsh whole blood and in anti-coagulant treated blood is provided. The reagent is composed of recombinant animal tissue factor, and a mixture of synthetic phospholipids, which mixture includes a phosphatidylalcohol. A formulation buffer which includes a sensitivity adjuster is used in formulating the reagent. The recombinant animal tissue factor includes rabbit brain. The synthetic phospholipids of the mixture include palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylserine (POPS), and a phosphatidylalcohol. The phosphatidyl alcohol includes dioleoylphosphatidylethanol, dioleoylphosphatidylmethanol, dioleoylphosphatidylpropanol, dioleoylphosphatidylbutanol, and dioleoylphosphatidylinositol. The sensitivity adjuster included in the formulation buffer is γ-Cyclodextrin. The formulated reagent is air-dried and remains stable for at least 3 weeks at 37° C.
1. A prothrombin time reagent for determining the concentration of low molecular weight heparin in fresh whole blood and anti-coagulant-treated blood comprising:
a recombinant animal tissue factor;
a mixture of synthetic phospholipids including a selected phosphatidylalcohol, which mixture lipidates said recombinant animal tissue factor to form a lipidated tissue factor; and
a buffer solution comprising Hepes, sodium chloride; gamma-cyclodextrin; beta-alanine; serine; sorbitol; bovine serum albumin; phenol; and butylated hydroxytoluene, and wherein the reagent is exposed in an atmosphere of about 5% to about 10% relative humidity at about 20° C. to about 37° C. to thereby form an air-dried reagent.
2. The reagent according to claim 1 , wherein said reagent is stable for at least three weeks at a temperature of about 37° C.
3. A prothrombin time reagent for determining the concentration of low molecular weight heparin in fresh whole blood and anti-coagulant-treated blood comprising:
a recombinant animal tissue factor comprising rabbit brain; and
a mixture of synthetic phospholipids including a selected phosphatidylalchol, which mixture lipidates said recombinant animal tissue factor to form a lipidated tissue factor; and wherein said recombinant animal tissue factor comprising rabbit brain is present in an amount of about 4 μg/ml to about 6 μg/ml.
4. A protbrombin time reagent for determining the concentration of low molecular weight heparin in fresh whole blood and anticoagulant-treated blood, comprising:
a recombinant animal tissue factor;
a mixture of synthetic phospholipids including a selected phosphatidylalcohol, which mixture lipidates said tissue factor to form a liposome complex; and
a buffer solution comprising an aldehyde-free gamma-cyclodextrin sensitivity adjuster.
5. The reagent according to claim 4 , wherein said recombinant animal tissue factor comprises rabbit brain.
6. The reagent according to claim 5 , wherein said recombinant animal tissue factor comprising rabbit brain is present in an amount of about 4 μg/ml to about 6 μg/ml.
7. The reagent according to claim 4 , wherein said mixture of synthetic phospholipids further comprises palmitoyloleoylphosphatidylcholine (POPC) and palmitoyoleoylphosphatidylserine (POPS).
8. The reagent according to claim 7 , further comprising a concentration ratio of 70:10 to 20:10 to 20 for said mixture of POPC, POPS, and said selected phosphatidylalcohol.
9. The reagent according to claim 8 , wherein said selected phosphatidylalcohol is selected from the group consisting of:
dioleoylphosphatidylethanol, dioleoylphosphatidylmethanol, dioleoylphosphatidylpropanol, dioleoylphosphatidylbutanol, and dioleoylphosphatidylinositol.
10. The reagent according to claim 4 , wherein said buffer solution further comprises
Hepes, sodium chloride; beta-alanine; seine; sorbitol; bovine serum albumin; phenol; and butylated hydroxytoluene.
11. The reagent according to claim 4 , wherein the reagent is exposed in an atmosphere of about 5% to about 10% relative humidity at about 20° C. to about 37° C. for about two hours to thereby form an air-dried reagent.
12. The reagent according to claim 4 , wherein said sensitivity adjuster is present in an amount of about 1% by weight of the reagent.
13. The reagent according to claim 11 , wherein the air dried reagent remains stable for at least about three weeks at about 37° C.