Binding assay analysis

Abstract

Methods for determining a sample concentration of target entities in a sample, for example, determining a concentration of target antigens or antibodies in a blood sample or other biological sample.

1 . A method for determining a sample concentration of target entities in a sample, the method comprising:
 obtaining assay data comprising data points of respective local measurements indicative of a local concentration of the target entities at each of a plurality of assay areas of an assay assembly, wherein the assay areas are connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being, arranged to bind to the target entities in the sample, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next;   modeling, the assay data with a parameterized function of the local measurements against a quantity indicative of the position of the respective assay areas in the sequence, wherein one or more of the parameters are dependent on the sample concentration; and   determining a value indicative of the sample concentration based on at least one of the one or more parameters.   
     
     
         2 . A method according to  claim 1 , wherein the parameterized function is derived from assay data sets obtained for a range of sample concentrations. 
     
     
         3 . A method according to  claim 1 , wherein one of the one or more parameters is indicative of an offset amount which offsets the quantity indicative of the position of the assay area in the sequence such that the parameterized function is a function of the local measurements against the quantity indicative of the position of the assay area in the sequence, offset by the offset amount. 
     
     
         4 . A method according to  claim 3 , wherein the offset amount is determined by minimizing a difference between the respective local measurement and a corresponding value of the parameterized function for each assay area of the assay assembly. 
     
     
         5 . A method according to  claim 1 , wherein the parameterized function is characteristic of the assay assembly. 
     
     
         6 . A method according to  claim 5 , wherein the parameterized function is at least in part defined by one or more fixed parameters characteristic of the assay assembly. 
     
     
         7 . A method according to  claim 6 , wherein the one or more fixed parameters are determined from data sets of local measurements against the quantity obtained for respective sample target concentrations spanning a range of sample target concentrations. 
     
     
         8 . A method according to  claim 1 , wherein the parameterized function is a logistic function. 
     
     
         9 . A method according  claim 8 , wherein the parameterized function is proportional to 
       
         
           
             
               
                 DP 
                 
                   ma 
                    
                   
                       
                   
                    
                   x 
                 
               
               
                 1 
                 + 
                 
                   exp 
                    
                   
                     [ 
                     
                       Shape 
                       × 
                       
                         ( 
                         
                           
                             DZ 
                             i 
                           
                           + 
                           Offset 
                         
                         ) 
                       
                     
                     ] 
                   
                 
               
             
           
         
       
       wherein DP max  and Shape are fixed parameters characteristic of the assay assembly, Offset is a parameter dependent on a sample target concentration, and DZ i  is indicative of the position of the respective assay area, i, in the sequence. 
     
     
         10 . A method according to  claim 9 , including fitting the parameterized function to the assay data by adjusting the value of Offset, and determining the value indicative of the concentration of target entity in the sample based on Offset. 
     
     
         11 . A method according to  claim 1 , comprising determining the value based on at least one of the one or more parameters using a calibration function, wherein the calibration function comprises a first function for use at sample concentrations of target entities above a given value, and a second function for use at sample concentrations of target entities below the given value. 
     
     
         12 . A method according to  claim 9 , wherein the value indicative of the sample concentration is determined using a calibration function and wherein the calibration function comprises a first function for use at sample concentrations of target entities above a given value, and a second function for use at sample concentrations of target entities below the given value, wherein the first function is a function of Offset, and the second function is a function of: 
       
         
           
             
               
                 DP 
                 
                   ma 
                    
                   
                       
                   
                    
                   x 
                 
               
               
                 1 
                 + 
                 
                   exp 
                    
                   
                     [ 
                     
                       Shape 
                       × 
                       Offset 
                     
                     ] 
                   
                 
               
             
           
         
       
     
     
         13 . A method according to  claim 1 , wherein each local measurement is indicative of a variation in a refractive index at the surface of the respective assay area. 
     
     
         14 . A method according to  claim 13 , wherein the refractive index at the surface of the respective assay area is determined based on the detection of a change in Surface Plasmon Resonance. 
     
     
         15 . A method according to  claim 13 , wherein the variation in the refractive index at the surface of the respective assay area is amplified by an amplifier solution flowing past the respective assay area, wherein the amplifier solution is arranged to interact with target entities bound to the surface of the assay area such that the variation in the refractive index at the respective assay area is amplified when the amplifier has interacted with the hound target entities. 
     
     
         16 . A method according to  claim 15 , wherein each local measurement comprises a difference between a pre-amplification signal and post-amplification signal, wherein the pre-amplification signal has been detected after interaction of the sample with the respective assay area and before interaction of the amplifier solution with target entities bound to the respective assay area, and the post-amplification signal has been detected after interaction of the amplifier solution with target entities bound to the respective assay area. 
     
     
         17 . A method according to  claim 16 , wherein modeling the assay data comprises using an adjustment term to account for a bulk refractive index of the sample. 
     
     
         18 . A method according to  claim 17 , wherein the adjustment term is determined based on a difference between a baseline signal detected prior to interaction of the sample with the assay area and the pre-amplification signal. 
     
     
         19 . A method according to  claim 15 , wherein each local measurement is indicative of a rate at which the amplifier solution interacts with the respective assay area. 
     
     
         20 . A method according to  claim 15 , wherein each local measurement comprises a measurement indicative of the time taken from introduction of the amplifier solution into the respective assay area to detection of a signal feature, for example a maximum or threshold signal amplitude. 
     
     
         21 . A method according to  claim 1 , wherein an amount of probe entities with target entities between each pair of the plurality of assay areas is substantially constant. 
     
     
         22 . A method according to  claim 1 , where the quantity indicative of the position of the assay area in the sequence is indicative of an amount of probe entities upstream of the position of the assay area. 
     
     
         23 . A method according to  claim 1 , wherein each assay area is connected to the next, assay area in the sequence via a conduit. 
     
     
         24 . A method according to  claim 1 , wherein obtaining assay data comprises:
 introducing a sample into the assay assembly;   causing the sample to flow through the assay assembly; and   carrying out local measurements at each assay area.   
     
     
         25 . A system for determining a sample concentration, of target entities in a sample, the system comprising, a processor arranged to:
 obtain assay data comprising data points of respective local measurements indicative of a local concentration of the target entity at each of a plurality of assay areas of an assay assembly, wherein the assay areas are connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized, at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next;   model the assay data with a parameterized function of the local measurements against a quantity indicative of the position of the respective assay areas in the sequence, wherein one or more of the parameters are dependent on the sample concentration; and   determine a value indicative of the sample concentration based on at least one of the one or more parameters.   
     
     
         26 . A system according to  claim 26  arranged to carry Out the method of  claim 1 . 
     
     
         27 . A system according to  claim 25 , the system further comprising:
 an assay assembly comprising a plurality of assay areas connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being, arranged to bind to the target entities, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next; and   at least one detector arranged to carry out local measurements at each assay area to obtain assay data comprising data points of respective local measurements indicative of a local concentration of the target entities at each of the plurality of assay areas; wherein the processor is arranged to obtain assay data from the at least one detector.   
     
     
         28 . A method for determining a sample concentration of target entities in a sample, the method comprising:
 obtaining assay data comprising a local measurement indicative of a local concentration of the target entity at an assay area of an, assay assembly, wherein the assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to target entities, wherein the local measurement is based on signals indicative of a variation in a refractive index at the surface of the assay area, such variation being amplified following interaction of an amplifier solution with target entities bound to the surface of the assay area, and wherein the local measurement comprises a difference between a pre-amplification signal and post-amplification signal, wherein the pre-amplification signal is detected after interaction of the sample with the assay area and before interaction of the amplifier solution with target entities bound to the assay area, and the post-amplification signal is detected after interaction of the amplifier solution with target entities bound to the assay area;   adjusting the local measurement using an adjustment term such that a bulk refractive index, of the sample is taken into account;   using, the adjusted local measurement to determine a value indicative of the sample concentration.   
     
     
         29 . A method according to  claim 28 , wherein the adjustment term is determined based on a difference between a baseline signal detected prior to interaction of the sample with the assay area and the pre-amplification signal. 
     
     
         30 . An assay assembly for determining a sample concentration of target entities in a sample, the assay assembly comprising a plurality of assay areas serially connected such that a sample flowing through the assay assembly flows past each assay area in sequence, wherein:
 each assay assembly comprises an inlet and an outlet;   for each pair of assay areas in the plurality of assay areas, the outlet of a first assay area is coupled to the inlet of a second assay area by a coupling portion, such that a sample flowing through the assay assembly flows from the first assay area to the second assay area via the coupling portion for each pair of assay areas in the plurality of assay areas; and   each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is detectably depleted as the sample flows from one of the assay areas to the next.   
     
     
         31 . A microfluidic device comprising an assay assembly according to  claim 30 .