Lupus anticoagulent testing

Abstract

The present invention relates generally to the field of diagnostic screening and diagnostic assays. In particular, the present invention provides an improved, rapid, and efficient method of screening for antiphospholipid antibodies, such as lupus anticoagulants (LA). The invention also relates to a kit for screening plasma levels for antiphospholipid antibodies in subjects in need thereof, such as those at risk for or suffering from, inter alia, antiphospholipid syndrome (APS) and systemic lupus erythromatosus (SLE).

1 . A kit for detecting antiphospholipid antibodies in a test plasma sample, comprising at least one control plasma sample, at least one procoagulant test reagent, and a high phospholipid substrate, wherein the kit is used with at least two coagulation assays. 
     
     
         2 . The kit of  claim 1 , wherein the coagulation assays are a dilute Russell Viper Venom Time assay (dRVVT), an activated partial thromboplastin time assay (aPTT), and/or a dilute prothrombin time assay (dPT), each assay comprising a screening step and a confirmatory step, wherein the confirmatory step of each assay uses the same high phospholipid substrate. 
     
     
         3 . The kit of  claim 1 , wherein the antiphospholipid antibodies are selected from the group consisting of lupus anticoagulants, anti-β2-GP1 antibodies, anti-protein C antibodies, anti-protein S antibodies, anti-complement antibodies, anticardiolipin antibodies, anti-thrombin antibodies, anti-prothrombin antibodies, anti-thrombomodulin antibodies, anti-tPA antibodies, anti-Factor XII antibodies, anti-kininogen antibodies, anti-annexin V antibodies and combinations thereof. 
     
     
         4 . The kit of  claim 1 , wherein the test plasma sample is derived from a subject at risk for or suffering from an autoimmune disorder. 
     
     
         5 . The kit of  claim 4 , wherein the autoimmune disorder is selected from the group consisting of antiphospholipid syndrome (APS), connective tissue diseases, immune thrombocytopenia purpura, Hashimoto's thyroiditis, rheumatoid arthritis, and systemic lupus erythromatosus (SLE). 
     
     
         6 . The kit of  claim 1 , wherein the at least one control plasma sample is a positive control plasma sample or a negative control plasma sample. 
     
     
         7 . The kit of  claim 1 , wherein the at least one procoagulant test reagent comprises a component selected from the group consisting of Russell's viper venom, Taipan snake venom, textarin, ecarin, thrombin, kaolin, silica, ellagic acid, tissue factor and combinations thereof. 
     
     
         8 . The kit of  claim 7 , wherein the at least one procoagulant test reagent contains a low level of phospholipids. 
     
     
         9 . The kit of  claim 1 , wherein the high phospholipid substrate comprises a component selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin, phosphatidic acid, dioleoyl phosphatidylethanolamine (DOPE), phospholipid coated beads, phospholipid suspensions, and combinations thereof. 
     
     
         10 . The kit of  claim 1 , further comprising a buffer containing calcium. 
     
     
         11 . A method of detecting antiphospholipid antibodies in a test plasma sample from a subject, the method comprising:
 a. contacting a first test plasma sample from the subject with a procoagulant test reagent containing a low level of phospholipids;   b. measuring the rate of coagulation or time of clot formation in the first test plasma sample;   c. contacting a second test plasma sample from the subject with the procoagulant test reagent and a high phospholipid substrate;   d. measuring the rate of coagulation or time of clot formation in the second test plasma sample; and   e. comparing the rates of coagulation from steps (b) and (d), wherein a decreased rate of coagulation in step (d), as compared with step (b), indicates the presence of antiphospholipid antibodies in the test plasma samples.   
     
     
         12 . The method of  claim 11 , wherein the antiphospholipid antibodies are selected from the group consisting of lupus anticoagulants, anti-β2-GP1 antibodies, anti-protein C antibodies, anti-protein S antibodies, anti-complement antibodies, anticardiolipin antibodies, anti-thrombin antibodies, anti-prothrombin antibodies, anti-thrombomodulin antibodies, anti-tPA antibodies, anti-Factor XII antibodies, anti-kininogen antibodies, anti-annexin V antibodies and combinations thereof. 
     
     
         13 . The method of  claim 11 , wherein the procoagulant comprises a component selected from the group consisting of Russell's viper venom, Taipan snake venom, textarin, ecarin, thrombin, kaolin, silica, ellagic acid, tissue factor and combinations thereof. 
     
     
         14 . The method of  claim 11 , wherein the high phospholipid substrate comprises a component selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin, phosphatidic acid, dioleoyl phosphatidylethanolamine (DOPE), phospholipid coated beads, phospholipid suspensions, and combinations thereof. 
     
     
         15 . The method of  claim 11 , wherein the detection uses spectrophotometry. 
     
     
         16 . A method of diagnosing an autoimmune disorder in a subject, wherein the autoimmune disorder is characterized by the presence of antiphospholipid antibodies and a decreased rate of coagulation in a test plasma sample from the subject, the method comprising:
 a. contacting a first test plasma sample from the subject with a first procoagulant test reagent containing a low level of phospholipids;   b. measuring the rate of coagulation or time of clot formation in the first test plasma sample;   c. contacting a second test plasma sample from the subject with the first procoagulant test reagent and a high phospholipid substrate;   d. measuring the rate of coagulation or time of clot formation in the second test plasma sample;   e. comparing the rates of coagulation from steps (b) and (d),   f. repeating steps (a) through (e) with a second procoagulant test reagent; and   g. repeating steps (a) through (e) with a third procoagulant test reagent;   wherein a decreased rate of coagulation in step (d) of each test, as compared with step (b) of each test, indicates the autoimmune disorder.   
     
     
         17 . The method of  claim 16 , wherein the autoimmune disorder is selected from the group consisting of antiphospholipid syndrome (APS), connective tissue diseases, immune thrombocytopenia purpura, Hashimoto's thyroiditis, rheumatoid arthritis, and systemic lupus erythromatosus (SLE). 
     
     
         18 . The method of  claim 16 , wherein the high phospholipid substrate comprises a component selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin, phosphatidic acid, dioleoyl phosphatidylethanolamine (DOPE), phospholipid coated beads, phospholipid suspensions, and combinations thereof. 
     
     
         19 . The method of  claim 16 , wherein the first, second, and third procoagulant test reagents are independently selected from the group consisting of Russell's viper venom, Taipan snake venom, textarin, ecarin, thrombin, kaolin, silica, ellagic acid, tissue factor and combinations thereof. 
     
     
         20 . A method of decreasing the likelihood of false positives in one or more coagulation assays used in detecting the presence of antiphospholipid antibodies and a decreased rate of coagulation in a test plasma sample, wherein the coagulation assay comprises a screening step and a confirmatory step, the method comprising:
 a. contacting a test plasma sample with a procoagulant containing a low level of phospholipids in the screening step;   b. measuring the rate of coagulation or time of clot formation in the test plasma sample; and   c. confirming the rate of coagulation in the screening step by contacting the test plasma sample with the procoagulant and a high phospholipid substrate,   wherein the same procoagulant is used in the screening step and the confirmatory step, thereby decreasing the likelihood of false positives in the one or more coagulation assays.   
     
     
         21 . The method of  claim 20 , wherein the one or more coagulation assays comprise an activated partial thromboplastic time assay (aPTT), a dilute prothrombin time assay (dPT), or a dilute Russell's viper venom time assay. 
     
     
         22 . The method of  claim 20 , wherein the procoagulant comprises a component selected from the group consisting of Russell's viper venom, Taipan snake venom, textarin, ecarin, thrombin, kaolin, silica, ellagic acid, tissue factor and combinations thereof. 
     
     
         23 . The method of  claim 20 , wherein the high phospholipid substrate comprises a component selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cardiolipin, phosphatidic acid, dioleoyl phosphatidylethanolamine (DOPE), phospholipid coated beads, phospholipid suspensions, and combinations thereof.