Assay methods

Abstract

The present invention is directed to methods for reducing cross-reactivity between species employed in multiplexed immunoassays.

1 . (canceled) 
     
     
         2 . A method of detecting an analyte of interest in a sample comprising:
 a. contacting a complex on a surface comprising the analyte, a binding reagent for the analyte, a first detection reagent for the analyte comprising a first proximity probe, and a second detection reagent for the analyte comprising a second proximity probe, with one or more connector oligonucleotides comprising a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the contacting is performed under conditions sufficient for the first circularization probe and the second circularization probe to form a circular DNA template, and wherein the surface further comprises an anchoring reagent comprising an anchoring sequence;   d. amplifying the circular DNA template to generate an amplicon comprising a plurality of detection sequences and an anchoring sequence complement;   c. hybridizing the anchoring sequence to the anchoring sequence complement;   d. hybridizing a plurality of detection probes to the plurality of detection sequences; and   e. measuring the amount of analyte bound to the surface.   
     
     
         3 . The method of  claim 2  wherein the first and second detection reagents are each antibodies and the analyte is an antigen recognized by the antibodies. 
     
     
         4 . The method of  claim 2  wherein anchoring reagent is selected from biotin, avidin, streptavidin, or albumin. 
     
     
         5 . The method of  claim 2  wherein the measuring comprises measuring optical absorbance, fluorescence, phosphorescence, chemiluminescence, light scattering, magnetism, electrochemiluminence (ECL), or combination thereof. 
     
     
         6 . The method of  claim 2 , wherein each of the plurality of detection probes comprise a detectable label. 
     
     
         7 . The method of  claim 6 , wherein the detectable label is an ECL label, and the measuring comprises measuring an ECL signal, thereby measuring the amount of analyte in the sample. 
     
     
         8 . The method of  claim 7 , wherein the surface is an electrode, and the measuring further comprises applying a voltage waveform to the electrode to generate the ECL signal. 
     
     
         9 . The method of  claim 2 , wherein the surface comprises a binding domain, and the binding reagent and the anchoring reagent are each bound to the binding domain. 
     
     
         10 . The method of  claim 2 , wherein the surface comprises a first and second binding domain, wherein the first and second binding domains are adjacent, and the binding reagent is bound to the first binding domain and the anchoring reagent is bound to the second binding domain. 
     
     
         11 - 23 . (canceled) 
     
     
         24 . A method of detecting a plurality of analytes of interest in a sample comprising:
 a. forming a plurality of complexes on a surface comprising a plurality of discrete binding domains, each complex comprising an analyte of the plurality of analytes, a binding reagent for the analyte, a first detection for the analyte comprising a first proximity probe, and a second detection reagent for the analyte comprising a second proximity probe, wherein each complex is in a discrete binding domain of the surface, and wherein each binding domain comprises an anchoring reagent comprising an anchoring sequence;   b. contacting each complex of the complex of the plurality of complexes with one or more connector oligonucleotides comprising: a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the contacting is performed under conditions sufficient for the first circularization probe and the second circularization probe to form a circular DNA template, thereby forming a plurality of circular DNA templates;   c. amplifying the circular DNA template of each complex to generate a plurality of amplicons each comprising a plurality of detection sequences and an anchoring sequence complement;   d. hybridizing the anchoring sequence to the anchoring sequence complement in each binding domain;   e. hybridizing a plurality of detection probes to the plurality of detection sequences in each binding domain; and   f. measuring the amount of the plurality of analytes bound to the surface.   
     
     
         25 - 30 . (canceled) 
     
     
         31 . The methond of  claim 24 , further comprising:
 exposing the sample to the plurality of binding domains under conditions so that at least one analyte of the plurality of analytes is immobilized in at least some of the plurality of binding domains, wherein each binding domain is positioned within a well of a multi-well plate;   determining the presence or absence of analyte in each binding domain so as to identify the number of binding domains that contain analyte and/or the number of binding domains that do not contain analyte; and   determining the concentration of analyte in the sample from the number of binding domains that contain and/or do not contain analyte.   
     
     
         32 - 38 . (canceled) 
     
     
         39 . The method of  claim 24 , further comprising:
 partitioning at least a portion of the plurality of analytes in the sample into the plurality of binding domains, so that, for substantially all binding domains, each binding domain contains either no analytes or a single analyte;   determining the presence or absence of analyte in the plurality of binding domains to provide a fraction of binding domains that contain analyte; and   determining the concentration of analytea in the sample from the fraction of binding domains that contain analyte.   
     
     
         40 - 55 . (canceled) 
     
     
         56 . A kit for the measurement of an analyte of interest in a sample, the kit comprising:
 a. a surface comprising a binding reagent for the analyte and an anchoring reagent comprising an anchoring sequence; and   b. in one or more containers, compartments, or vessels:   i. a first detection reagent for the analyte and a second detection reagent for the analyte, wherein the first and second detection reagents comprise a first proximity probe and a second proximity probe, respectively;   ii. one or more connector oligonucleotides comprising a first circularization probe complementary to a first region of the first proximity probe and a first region on the second proximity probe, and a second circularization probe complementary to a second non-overlapping region of the first proximity probe and a second non-overlapping region of the second proximity probe, wherein the one or more connector oligonucleotides further comprises a detection sequence complement; and   iii. one or more detection probes complementary to the detection sequence complement.   
     
     
         57 . The kit of claim  35 , further comprising DNA polymerase. 
     
     
         58 . The kit of claim  35 , wherein each of the one or more detection probes comprise a detectable label. 
     
     
         59 . The kit of claim  37 , wherein the detectable label is an ECL label, and the kit further comprises an ECL co-reactant. 
     
     
         60 - 143 . (canceled) 
     
     
         144 . The method of  claim 2 , wherein the complex is formed by: (i) contacting the sample with the surface comprising the binding reagent and the anchoring reagent to form a surface-bound complex; and (ii) contacting the surface-bound complex with the first detection reagent and the second detection reagent. 
     
     
         145 . The method of  claim 2 , wherein the complex is formed by: (i) contacting the sample with the first detection reagent and the second detection reagent to form a detection complex; and; and (ii) contacting the detection complex with the surface comprising the binding reagent and the anchoring reagent. 
     
     
         146 . The method of  claim 2 , wherein the surface comprises a multi-well assay plate comprising a plurality of wells, wherein each well comprises one or more binding domains. 
     
     
         147 . The method of  claim 146 , wherein the multi-well plate is a 4 well plate, 6 well plate, 24 well plate, 96 well plate, 384 well plate, 1536 well plate, 6144 well plate, or 9600 well plate.