Determination of analyte concentration using two labelling markers

Abstract

In order to measure the concentration of an analyte in a liquid sample the sample is contacted with a receptor molecule having binding sites for the analyte and labelled with a first marker, whereby a fraction of the binding sites on the receptor molecule become occupied by the analyte, the sample being contacted with such a small amount of the receptor, having regard to its affinity constant with the analyte, that only an insignificant fraction of the analyte becomes bound to the receptor. The receptor having fractionally occupied binding sites is then back-titrated by means of a back-titration technique involving a system including a second marker different from the first, and the relative strengths of the two signals produced by the two markers are measured to provide a value representative of the fractional occupancy of the binding sites on the receptor molecule by the analyte. This value is compared with one or more corresponding values obtained in the same way using one or more standard liquid samples of known analyte concentration. An analytical device suitable for use in such a method comprises an extended solid substrate ( 20 ) bearing at a plurality of spaced-apart locations ( 21 ) a plurality of different receptors ( 22 ) each having binding sites on their molecule for an analyte, the receptor at any one location having binding sites specific for an analyte different from the analyte for which the receptor at one or more other locations has specific binding sites, each of the receptors being optionally labelled with a marker This device can be used as part of a kit for the method.

1 . A method of determining the concentration of an analyte in an unknown liquid sample comprising contacting the sample with a receptor molecule having binding sites for the analyte and labelled with a first marker, whereby a fraction of the binding sites on the receptor molecule become occupied by the analyte, back-titrating the receptor having fractionally occupied binding sites by means of a back-titration technique involving a system including a second marker different from the first, measuring the relative strengths of the two signals produced by the two markers to provide a value representative of the fractional occupancy of the binding sites on the receptor molecule by the analyte, and comparing that value with one or more corresponding values obtained in the same way using one or more standard liquid samples of known analyte concentration, characterised in that the unknown and standard liquid samples are each contacted with such a; small amount of the receptor, having regard to its affinity constant with the analyte, that only an insignificant fraction of the analyte becomes bound to the receptor.  
     
     
         2 . A method as claimed in  claim 1 , characterised in that the markers are fluorescent markers.  
     
     
         3 . A method as claimed in  claim 1  or  2 , characterised in that the back-titration technique involves a competitive system including a back-titration reagent selected from the analyte labelled with a marker and another material able to occupy only the unoccupied analyte binding sites on the receptor molecule and labelled with a marker.  
     
     
         4 . A method as claimed in  claim 1  or  2 , characterised in that the back-titration technique involves a non-competitive system including a back-titration reagent which is able to bind with the bound analyte or only with the receptor molecule binding sites occupied by the bound analyte and which is already labelled or is subsequently labelled with a marker.  
     
     
         5 . A method as claimed in any of  claims 1  to  4 , characterised in that the insignificant fraction is less than 2% of the total amount of the analyte present.  
     
     
         6 . A method as claimed in any of  claims 1  to  5 , characterised in that the receptor is an antibody for the analyte.  
     
     
         7 . A method as claimed in any of  claims 1  to  6 , characterised in that the analyte is a hormone.  
     
     
         8 . A method as claimed in any of  claims 1  to  7 , characterised in that the sample containing the analyte does not contain the analyte reversibly bound to a binding agent.  
     
     
         9 . A method as claimed in any of  claims 2  to  8 , characterised in that the markers are different lanthanide chelate becoming fluorescent only on activation and the fluorescence is estimated by time-resolved pulse fluorescence involving scanning with a high-intensity light beam.  
     
     
         10 . An analytical device comprising an extended solid substrate bearing at a plurality of spaced-apart locations a plurality of different receptors each having binding sites on their molecule for an analyte, the receptor at any one location having binding sites specific for an analyte different from the analyte for which the receptor at one or more other locations has specific binding sites, each of the receptors being optionally labelled with a marker.  
     
     
         11 . A kit for use in a method determining the concentration of an analyte in a liquid sample, said kit comprising a solid substrate having attached thereto molecules of a receptor labelled with a first, preferably fluorescent marker and having binding sites for an analyte, the amount of receptor on the substrate being selected according to the size of the liquid sample so that only an insignificant fraction of the analyte in the sample becomes bound to the receptor, one or more standard solutions containing known concentrations of the analyte and a back-titration reagent labelled with a second, preferably fluorescent marker different from the first and able to bind directly or indirectly either with the bound analyte or with the binding sites occupied by the bound analyte or to bind with the binding sites not occupied by the bound analyte.  
     
     
         12 . A kit as claimed in  claim 11 , characterised in that it includes an analytical device as claimed in claim  10 .