Binding assay using binding agents with tail groups

Abstract

The present invention discloses methods and kits for the determination of the concentration of one or more analytes in a liquid sample using capture agents immobilised on a solid support and binding agents for binding the analyte(s), the binding agents having tail groups capable of binding to the respective capture agent. Preferably, the capture agents and binding agents are complementary oligonucleotides, and the capture agents are immobilised in the form of microspots. The use of the tail groups and capture agents can allow the binding of the analyte(s) to the binding agent(s) to take place in solution, rather than at a surface, improving the kinetics associated with this process. In addition, the user of the assay can customise any suitable binding agents for use with a universal support, by attaching tail groups them.

1 . A method for determining the concentration of one or more analytes in a liquid sample comprising: 
 (a) immobilising one or more capture agents on a solid support, each capture agent being capable of specifically binding a given binding agent;    (b) contacting the liquid sample with one or more binding agents, each binding agent having binding sites specific for a given analyte so that a fraction of the binding sites become occupied by the analyte, and having a tail group adapted to bind to the corresponding capture agent;    (c) contacting the liquid sample, either simultaneously or sequentially with the step (b), with the immobilised capture agents so that the binding agents become bound to their respective capture agents; and,    (d) determining a value representative of the fraction of the binding sites of a given binding agent occupied by an analyte whereby to determine the concentration of the analyte in the liquid sample    wherein the capture agents are oligonucleotides having sequences which can hybridise to a complementary sequence on the tail group of the corresponding binding agent.    
     
     
         2 . A method of immobilising one or more binding agents on a support, each binding agent having binding sites specific for a given analyte and having a tail group adapted to bind to a capture agent, comprising: 
 (a) immobilising one or more capture agents on a support each capture agent being capable of binding to the tail group of a given binding agent; and,    (b) contacting the binding agents with the support having the capture agents immobilised thereon so that the binding agents become specifically bound to their respective capture agents through their tail groups;    wherein the capture agents are oligonucleotides having sequences which can hybridise to a complementary sequence on the tail group of the corresponding binding agent.    
     
     
         3 . A method according to  claim 2  wherein the method additionally comprises the step of attaching the tail groups to the binding agents prior to exposing them to the capture agents immobilised on the support.  
     
     
         4 . A method according to any one of  claims 1  to  3  wherein the oligonucleotides are between 8 and 30 bases long.  
     
     
         5 . A method according to any one of the preceding claims wherein the binding agent is an antibody having binding sites specific for an analyte.  
     
     
         6 . A method according to any one of the preceding claims wherein a small amount of each binding agent is used so that the ambient concentration of the analyte for which the binding agent is specific is not significantly disturbed.  
     
     
         7 . A method according to  claim 6  wherein the small amount of binding agent is less than 0.1 V/K moles, where V is the volume of the sample and K is the effective affinity constant for the analyte binding to the binding agent.  
     
     
         8 . A method according to any one of the preceding claims wherein each capture agent is used in excess to bind substantially all of a given binding agent.  
     
     
         9 . A method according to any one of the preceding claims wherein the capture agent(s) are immobilised on a support at discrete locations.  
     
     
         10  A method according to  claim 9  wherein the discrete locations are microspots.  
     
     
         11 . A method according to any one of the preceding claims wherein a given capture agent is immobilised on the support at a plurality of locations so that a series of measurements of the concentration of a given analyte can be made simultaneously.  
     
     
         12 . A method according to any one of the preceding claims wherein the value representative of the fraction of the binding sites occupied by the analyte is determined using developing agents in a competitive and/or non-competitive method, the developing agents being labelled with markers.  
     
     
         13 . A method according to  claim 12  wherein the marker are fluorescent or chemiluminescent markers.  
     
     
         14 . A kit for determining the concentrations of one or more analytes in a liquid sample in a method according to any one of  claims 1  to  13 , the kit comprising: 
 (a) a solid substrate having attached thereto at a plurality of locations capture agent capable of specifically binding a given binding agent;  
 (b) one or more binding agents, each binding agent having binding sites specific for an analyte, and having a tail group adapted to bind one or more capture agents; and  
 (c) one or more developing agents having markers capable of binding to occupied binding agent binding sites or analyte bound to binding agent or unoccupied binding agent binding sites;  
 wherein the capture agents are oligonucleotides having sequences which can hybridise to a complementary sequence on the tail group of the corresponding binding agent.  
 
     
     
         15 . A kit for customising an assay for the determination of the concentration of one or more analytes in a liquid sample, the kit comprising: 
 (a) one or more tail groups, each tail group being for attachment to a binding agent;    (b) a solid substrate having attached thereto at a plurality of locations one or more capture agents capable of specifically binding to a tail group;    wherein the capture agents are oligonucleotides having sequences which can hybridise to a complementary sequence on the tail group of the corresponding binding agent and the user of the assay attaches the tail groups to the binding agents, thereby providing binding agents which can be used in conjunction with the solid substrate to which the capture agents are attached in a method according to any one of  claims 1  to  13 .    
     
     
         16 . A solid support for use in determining the concentration of one or more analytes in a liquid sample, the solid support having immobilised thereon one or more binding agents, each binding agent having binding sites specific for a given analyte and having a tail group, the binding agents being-specifically attached to the support by capture agents wherein the capture agents are oligonucleotides having sequences which are hybridised to a complementary sequence on the tail group of the corresponding binding agent.