Assay with reduced background
View Patent ↗In as assay, an analyte is specifically associated with a reporter adenylate kinase, ADP is added and then formation of ATP is monitored. Prior to addition of ADP, adenylate kinase other than reporter adenylate kinase is removed. Assay apparatus comprises a solid phase on which is immobilised the analyte or an antibody specific for the analyte, a reporter composition comprising a thermostable adenylate kinase coupled to an antibody specific for the analyte, and ADP plus associated reagents for conversion of ADP into ATP by thermostable adenylate kinase.
1. An assay for an analyte, 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 formation of ATP wherein, prior to the addition of ATP, 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 said ATP formed correlates to the concentration of the analyte.
2. 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.
3. The assay of claim 1 , wherein formation of ATP is measured using luciferin/luciferase.
4. An assay for determining the presence and/or amount of an analyte in a sample, comprising exposing the sample to a 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 thermostable reporter adenylate that is not bound to analyte; exposing said thermostable reporter adenylate kinase bound to the analyte to ADP; and testing for formation of ATP, wherein prior to addition of ADP, residual kinase other than thermostable reporter adenylate kinase is substantially removed by heating.
5. The assay of claim 1 , comprising further adding an ATPase to the analyte and removing the ATPase from the analyte prior to adding ADP.
6. The assay of claim 5 , wherein the ATPase is inactivated by heating the ATPase.
7. An assay for determining 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 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; therein prior to adding the substrate non-thermostable enzymes are destroyed by application of heat.
8. The assay of claim 7 , wherein the enzyme is adenylate kinase and the substrate is ADP, the ADP is converted into ATP by the thermostable enzyme.
9. The assay of claim 8 , wherein background ATP compound is removed by the addition of ATPase prior to adding ADP.
10. An assay for an analyte, comprising the steps:
(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.