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 - 16 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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,   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,   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.   
     
     
         36 . The assay of  claim 17 , wherein the analyte is selected from the group consisting of bacteria, viruses, toxins, prion agents, B-lactamases, hormones, immunoglobulins, tumour markers, growth factors, neurotransmitters, and metabolites of drugs of abuse. 
     
     
         37 . The assay of  claim 36 , wherein the analyte is a Gram-positive bacterium. 
     
     
         38 . The assay of  claim 37 , wherein the Gram-positive bacterium is  Staphylococcus aureus  (in particular antibiotic resistant strains such as MRSA),  Mycobacterium tuberculosis , or  Streptococcus pneumonia.    
     
     
         39 . The assay of  claim 17 , wherein the analyte is a virus selected from the group consisting of a rotavirus, a Norwalk virus (also known as Norovirus), a West Nile Virus, a measles virus, a mumps virus, a rubella virus, HIV, HPV and a hepatitis virus (all forms, especially Hepatitis C). 
     
     
         40 . The assay of  claim 17 , wherein the analyte is a toxin selected from the group consisting of botulinum toxin (including all botulinum serotypes A-G), tetanus toxin, anthrax toxins (including lethal toxin, oedema toxin and their component parts; lethal factor, oedema factor and protective antigen), ricin, mycotoxin, afalatoxin and superantigens. 
     
     
         41 . The assay of claim  5 , wherein the toxin is botulinum toxin, tetanus toxin, or a superantigen. 
     
     
         42 . The assay of  claim 17 , wherein the analyte is a prion protein (including PrP Sc  and PrP c ) or an agent responsible for causing Creutzfeld Jacob Disease (CJD) (including variant, familial, sporadic and iatrogenic forms), scrapie, Bovine Spongiform Encephalopathy (BSE), or Chronic Wasting Disease (CWD). 
     
     
         43 . The assay of  claim 17 , wherein the sample is selected from the group consisting of faeces (stool), vomitus, effluent samples, food, water, sewerage, and environmental samples, and wherein the analyte is selected from the group consisting of Gram-positive bacteria, botulinum toxin, tetanus toxin, rotavirus, or Norwalk virus. 
     
     
         44 . The assay of  claim 17 , wherein the sample comprises a blood component (including serum, plasma, whole blood, white blood cell fractions, buffy coat) and wherein the analyte is selected from the group consisting of superantigens, West Nile Virus, HIV, prion proteins and hormones. 
     
     
         45 . The assay of  claim 17 , wherein the sample is an airway sample (including sputum bronchoalveolar lavage, endotracheal aspirates, nasopharyngeal aspirates) and wherein the analyte is selected from the group consisting of  Mycobacterium tuberculosis, Streptococcus pneumonia  and immunoglobulins. 
     
     
         46 . The assay of  claim 17 , wherein the sample is an oral sample (including crevicular fluid, parotid saliva, whole saliva), and wherein the analyte is selected from the group consisting of a measles virus, a mumps virus, a rubella virus, and a hepatitis virus (all forms, especially Hepatitis C). 
     
     
         47 . The assay of  claim 17 , wherein the sample is cerebrospinal fluid and the analyte is selected from the group consisting of neurotransmitters, tumour markers and growth factors. 
     
     
         48 . The assay of  claim 17 , wherein the sample is pus or a swab sample (including those taken from throat, nose, ears, skin, and wounds), and wherein the analyte is  Staphylococcus aureus  (in particular antibiotic resistant strains such as MRSA).