Assay with reduced background

Abstract

In an assay, an analyte in a sample is contacted with a thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, wherein a complex is formed. ADP is added, and then formation of ATP is monitored. Prior to the addition of ADP, endogenous kinase and uncomplexed thermostable reporter adenylate kinase is substantially removed by washing and residual-endogenous kinase is inactivated by heating. Prior to contacting the analyte with the thermostable reporter adenylate kinase, the sample has a background activity of at least 300,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.

1 . An assay for an analyte in a sample, comprising contacting the analyte with a thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, wherein a complex is formed, adding ADP and testing for the formation of ATP, wherein, prior to the addition of ADP, endogenous kinase and uncomplexed thermostable reporter adenylate kinase is substantially removed by washing and residual endogenous kinase is inactivated by heating, and wherein the amount of ATP correlates to the concentration of the analyte, and wherein prior to contacting said analyte with said thermostable reporter adenylate kinase, the sample has a background activity of at least 300,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.  
     
     
         2 . The assay of  claim 1 , wherein prior to contacting said analyte with said thermostable reporter adenylate kinase the sample has a background activity of at least 400,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.  
     
     
         3 . The assay of  claim 1 , wherein prior to contacting said analyte with said thermostable reporter adenylate kinase the sample has a background activity of at least 500,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.  
     
     
         4 . The assay of  claim 1 , wherein the amount of thermostable reporter adenylate kinase complexed with the analyte is substantially proportional to the amount of analyte.  
     
     
         5 . The assay of  claim 1 , wherein formation of ATP is measured by luciferin/luciferase.  
     
     
         6 . The assay of  claim 1 , further comprising adding an ATPase to the analyte and removing the ATPase from the analyte prior to adding ADP.  
     
     
         7 . The assay of  claim 6 , wherein the ATPase is inactivated by heating the ATPase.  
     
     
         8 . The assay of  claim 1 , wherein after the washing and heating steps, the sample has a reduced background activity when measured in the presence of luciferin/luciferase in a luminometer, said reduced background activity having a Relative Light Unit value that is 20-fold to 1000-fold lower than said background activity.  
     
     
         9 . The assay of  claim 8 , wherein said reduced background activity has a Relative Light Unit value that is 50-fold to 800-fold lower than said background activity.  
     
     
         10 . The assay of  claim 1 , wherein said analyte is present at a concentration of less than 10 ng/ml.  
     
     
         11 . The assay of  claim 1 , wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         12 . The assay of  claim 1 , wherein the sample is selected from the group consisting of faeces (stool), serum and whole blood.  
     
     
         13 . The assay of  claim 8 , wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         14 . The assay of  claim 8 , wherein the sample is selected from the group consisting of faeces (stool), serum and whole blood.  
     
     
         15 . The assay of  claim 10 , wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         16 . The assay of  claim 10 , wherein the sample is selected from the group consisting of faeces (stool), serum and whole blood.  
     
     
         17 . An assay for an analyte in a sample, comprising contacting the analyte with a thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, wherein a complex is formed, adding ADP and testing for the formation of ATP, wherein, prior to the addition of ADP, endogenous kinase and uncomplexed thermostable reporter adenylate kinase is substantially removed by washing and, residual endogenous kinase is inactivated by heating, wherein the amount of ATP correlates to the concentration of the analyte, and wherein the analyte is present in the sample at a concentration of less than 10 ng/ml.  
     
     
         18 . The assay of  claim 17 , wherein said analyte is present in the sample at a concentration of less than 1 ng/ml.  
     
     
         19 . The assay of  claim 17 , wherein said analyte is present in the sample at a concentration of less than 100 pg/ml.  
     
     
         20 . The assay of  claim 17 , wherein said analyte is present in the sample at a concentration of less than 1 pg/ml.  
     
     
         21 . The assay of  claim 17 , wherein said analyte is present in the sample at a concentration of less than 100 fg/ml.  
     
     
         22 . The assay of  claim 17 , wherein said analyte is present in the sample at a concentration of less than 10 fg/ml.  
     
     
         23 . The assay of  claim 17 , wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         24 . The assay of  claim 17 , wherein the sample is selected from the group consisting of faeces (stool), serum and whole blood.  
     
     
         25 . An assay for an analyte in a sample, comprising contacting the analyte with a thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, wherein a complex is formed, adding ADP and testing for the formation of ATP, wherein, prior to the addition of ADP, endogenous kinase and uncomplexed thermostable reporter adenylate kinase is substantially removed by washing and, residual endogenous kinase is inactivated by heating, wherein the amount of ATP correlates to the concentration of the analyte, and wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         26 . The assay of  claim 25 , wherein the sample is selected from the group consisting of faeces (stool), serum and whole blood.  
     
     
         27 . An assay for determining the presence and/or amount of an analyte in a sample, comprising exposing the sample to thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, so that the reporter adenylate kinase is specifically associated with any analyte present in the sample via the binding agent; removing the thermostable reporter adenylate kinase that is not bound to the analyte; exposing said thermostable reporter adenlyate kinase bound to the analyte to ADP; and testing for the formation of ATP, wherein prior to the addition of ADP, residual kinase other than thermostable reporter adenylate kinase is substantially removed by heating, and wherein prior to exposing the sample to said thermostable reporter adenylate kinase, the sample has a background activity of at least 300,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.  
     
     
         28 . An assay for determining the presence and/or amount of an analyte in a sample, comprising exposing the sample to thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, so that the reporter adenylate kinase is specifically associated with any analyte present in the sample via the binding agent; removing the thermostable reporter adenylate kinase that is not bound to the analyte; exposing said thermostable reporter adenlyate kinase bound to the analyte to ADP; and testing for the formation of ATP, wherein prior to the addition of ADP, residual kinase other than thermostable reporter adenylate kinase is substantially removed by heating, and wherein the analyte is present in the sample at a concentration of less than 10 ng/ml.  
     
     
         29 . An assay for determining the presence and/or amount of an analyte in a sample, comprising exposing the sample to thermostable reporter adenylate kinase coupled to a binding agent specific for the analyte, so that the reporter adenylate kinase is specifically associated with any analyte present in the sample via the binding agent; removing the thermostable reporter adenylate kinase that is not bound to the analyte; exposing said thermostable reporter adenlyate kinase bound to the analyte to ADP; and testing for the formation of ATP, wherein prior to the addition of ADP, residual kinase other than thermostable reporter adenylate kinase is substantially removed by heating, and wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         30 . An assay for determining the presence and/or amount of an analyte in a sample comprising, exposing the sample to a detector compound, the detector compound comprising an antibody specific to the analyte coupled to a thermostable enzyme; isolating (i) detector compound that has specifically bound to analyte from (ii) detector compound that has not specifically bound to analyte; determining the presence of and/or amount of detector compound that has bound to analyte by adding a substrate for the thermostable enzyme and measuring a product formed by conversion of said substrate to said product by said thermostable enzyme; wherein prior to the addition of substrate non-thermostable enzymes are destroyed by application of heat, and wherein prior to exposing the sample to said detector compound, the sample has a background activity of at least 300,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.  
     
     
         31 . An assay for determining the presence and/or amount of an analyte in a sample comprising, exposing the sample to a detector compound, the detector compound comprising an antibody specific to the analyte coupled to a thermostable enzyme; isolating (i) detector compound that has specifically bound to analyte from (ii) detector compound that has not specifically bound to analyte; determining the presence of and/or amount of detector compound that has bound to analyte by adding a substrate for the thermostable enzyme and measuring a product formed by conversion of said substrate to said product by said thermostable enzyme; wherein prior to the addition of substrate non-thermostable enzymes are destroyed by application of heat, and wherein the analyte is present in the sample at a concentration of less than 10 ng/ml.  
     
     
         32 . An assay for determining the presence and/or amount of an analyte in a sample comprising, exposing the sample to a detector compound, the detector compound comprising an antibody specific to the analyte coupled to a thermostable enzyme; isolating (i) detector compound that has specifically bound to analyte from (ii) detector compound that has not specifically bound to analyte; determining the presence of and/or amount of detector compound that has bound to analyte by adding a substrate for the thermostable enzyme and measuring a product formed by conversion of said substrate to said product by said thermostable enzyme; wherein prior to the addition of substrate non-thermostable enzymes are destroyed by application of heat, and wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.  
     
     
         33 . An assay for an analyte comprising the steps of: 
 (a) specifically binding the analyte with a thermostable reporter kinase which has been coupled to a binding agent specific for the analyte forming a complex;    (b) washing to remove endogenous non-thermostable kinase and thermostable reporter kinase not bound to analyte;    (c) heating to inactivate endogenous kinase not removed by step (b); and    (d) adding ADP and testing for formation of ATP,    and wherein, prior to step (a), the sample has a background activity of at least 300,000 Relative Light Units per mg protein per ml sample when measured in the presence of luciferin/luciferase by a luminometer.    
     
     
         34 . An assay for an analyte comprising the steps of: 
 (e) specifically binding the analyte with a thermostable reporter kinase which has been coupled to a binding agent specific for the analyte forming a complex;    (f) washing to remove endogenous non-thermostable kinase and thermostable reporter kinase not bound to analyte;    (g) heating to inactivate endogenous kinase not removed by step (b); and    (h) adding ADP and testing for formation of ATP,    and wherein the analyte is present in the sample at a concentration of less than 10 ng/ml.    
     
     
         35 . An assay for an analyte comprising the steps of: 
 (i) specifically binding the analyte with a thermostable reporter kinase which has been coupled to a binding agent specific for the analyte forming a complex;    (j) washing to remove endogenous non-thermostable kinase and thermostable reporter kinase not bound to analyte;    (k) heating to inactivate endogenous kinase not removed by step (b); and    (l) adding ADP and testing for formation of ATP,    and wherein the sample is selected from the group consisting of urine, faeces (stool), vomitus, blood components (including serum, plasma, whole blood, white blood cell fractions, buffy coat), airway samples (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates), oral samples (including crevicular fluid, parotid saliva, whole saliva), cerebrospinal fluid (CSF), tissue homogenates (including brain homogenate, tonsil homogenate), pus, swab samples (including those taken from throat, nose, ears, skin, and wounds), effluent samples, surface swabs, food, water, beverages, soil, air sampling and sewerage.