Controls for proximity detection assays

Abstract

The present invention provides a method for detecting a plurality of analytes in a sample, comprising performing a multiplex proximity-based detection assay. The assay utilises pairs of proximity probes with shared hybridisation sites (i.e. hybridisation sites which are shared between different proximity probe pairs). Also provided is a product comprising a plurality of proximity probe pairs with shared hybridisation sites, which may be used in the method disclosed herein.

1 . A method for detecting a plurality of analytes in a sample, the method comprising performing a multiplex proximity-based detection assay, the assay comprising:
 (i) contacting the sample with a plurality of pairs of proximity probes, wherein each proximity probe pair comprises a first proximity probe and a second proximity probe, and each proximity probe comprises:
 (a) an analyte-binding domain specific for an analyte; and 
 (b) a nucleic acid domain, 
   wherein both probes within each pair comprise analyte-binding domains specific for the same analyte, and can simultaneously bind to the analyte; and each probe pair is specific for a different analyte;   wherein the nucleic acid domain of each proximity probe comprises an ID sequence and at least a first hybridisation sequence, wherein the ID sequence of each proximity probe is different; and wherein:   in each proximity probe pair, the first proximity probe and the second proximity probe comprise paired hybridisation sequences, such that upon binding of the first and second proximity probe to their analyte, the respective paired hybridisation sequences of the first and second proximity probes hybridise to each other or to a common splint oligonucleotide which comprises hybridisation sequences complementary to each of the paired hybridisation sequences of the first and second proximity probes;   and wherein at least one pair of hybridisation sequences is shared by at least two pairs of proximity probes;   (ii) allowing the nucleic acid domains of the proximity probes to hybridise to one another or to the splint oligonucleotide, to form a continuous or non-continuous duplex comprising the hybridisation sequence of a first proximity probe and a hybridisation sequence of a second proximity probe, wherein said duplex comprises at least one free 3′ end;   (iii) subjecting the duplex to an extension and/or ligation reaction to generate an extension and/or ligation product which comprises the ID sequence of the first proximity probe and the ID sequence of the second proximity probe;   (iv) amplifying the extension product or ligation product;   (v) detecting the extension product or ligation product, wherein detection of the extension product or ligation product comprises identification of the ID sequences therein, and determining the relative amounts of each extension product or ligation product; and   (vi) determining which analytes are present in the sample, wherein:
 (a) extension products and/or ligation products which comprise a first ID sequence from a first proximity probe belonging to a first proximity probe pair and a second ID sequence from a second proximity probe belonging to a second proximity probe pair are deemed background; and 
 (b) an extension product or ligation product which comprises a first ID sequence and a second ID sequence from a proximity probe pair, and which is present in an amount higher than the background, indicates that the analyte specifically bound by the proximity probe pair is present in the sample. 
   
     
     
         2 . The method of  claim 1 , wherein the analyte is or comprises a protein. 
     
     
         3 . The method of  claim 1  or  2 , wherein the analyte-binding domain is an antibody or fragment thereof. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein step (i) further comprises contacting the sample with one or more background probes which do not bind an analyte, said background probes comprising a nucleic acid domain comprising an ID sequence and a hybridisation sequence shared with at least one proximity probe;
 wherein an extension and/or ligation product generated as a result of an interaction between a background probe and a proximity probe is detected in step (v), and deemed background in step (vi). 
 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the ID sequences are barcode sequences. 
     
     
         6 . The method of any one of  claims 1  to  5 , wherein at least one pair of hybridisation sequences is unique to a single pair of proximity probes. 
     
     
         7 . The method of any one of  claims 1  to  6 , wherein no more than 10 proximity probe pairs share the same pair of hybridisation sequences. 
     
     
         8 . The method of  claim 7 , wherein no more than 5 proximity probe pairs share the same pair of hybridisation sequences. 
     
     
         9 . The method of any one of  claims 1  to  8 , wherein at least 25% of proximity probe pairs share their pair of hybridisation sequences with another proximity probe pair. 
     
     
         10 . The method of  claim 9 , wherein at least 50% of proximity probe pairs share their pair of hybridisation sequences with another proximity probe pair. 
     
     
         11 . The method of  claim 10 , wherein at least 75% of proximity probe pairs share their pair of hybridisation sequences with another proximity probe pair. 
     
     
         12 . The method of any one of  claims 1  to  11 , wherein the proximity-based detection assay is a proximity extension assay, wherein the nucleic acid domains of each proximity probe pair comprise complementary hybridisation sequences which hybridise to one another to form the duplex;
 and wherein the duplex is subjected to an extension reaction, said extension reaction comprising extending the at least one free 3′ end to generate an extension product comprising the ID sequence of the first proximity probe and the ID sequence of the second proximity probe. 
 
     
     
         13 . The method of any one of  claims 1  to  11 , wherein the proximity-based detection assay is a proximity ligation assay, wherein the nucleic acid domains of each proximity probe pair comprise paired hybridisation sequences which hybridise to the splint oligonucleotide to form the duplex, and wherein step (iii) comprises directly or indirectly ligating the nucleic acid domain of the first proximity probe to the nucleic acid domain of the second proximity probe to generate a ligation product comprising the ID sequence of the first proximity probe and the ID sequence of the second proximity probe. 
     
     
         14 . The method of  claim 12 , wherein in each proximity probe pair at least one nucleic acid domain is partially double-stranded. 
     
     
         15 . The method of  claim 14 , wherein in each proximity probe pair both nucleic acid domains are partially double-stranded. 
     
     
         16 . The method of  claim 14  or  15 , wherein the partially double-stranded nucleic acid domain comprises:
 (i) a first oligonucleotide conjugated to the analyte-binding domain; and 
 (ii) a hybridisation oligonucleotide comprising the first hybridisation sequence, the ID sequence and a second hybridisation sequence, the first hybridisation sequence being located at the 3′ end of the hybridisation oligonucleotide; 
 wherein the double-stranded part of the nucleic acid domain comprises a duplex between the second hybridisation sequence of the hybridisation oligonucleotide and the first oligonucleotide, and the single-stranded part of the nucleic acid domain comprises the first hybridisation sequence of the hybridisation oligonucleotide. 
 
     
     
         17 . The method of  claim 16 , wherein the hybridisation oligonucleotide comprises, from 5′ to 3′, the second hybridisation sequence, the ID sequence and the first hybridisation sequence, and the ID sequence is located in the single-stranded part of the nucleic acid domain. 
     
     
         18 . The method of  claim 16  or  17 , wherein at least one hybridisation oligonucleotide is extended in step (iii) to generate the extension product. 
     
     
         19 . The method of any one of  claims 16  to  18 , wherein in each proximity probe pair both nucleic acid domains are partially double-stranded, and one or both hybridisation oligonucleotides are extended in step (iii) to generate the extension product. 
     
     
         20 . The method of any one of  claims 1  to  19 , wherein the nucleic acid domain is a DNA domain. 
     
     
         21 . The method of any one of  claims 1  to  20 , wherein in step (iv) the extension product or ligation product is amplified by PCR. 
     
     
         22 . The method of any one of  claims 1  to  21 , wherein the extension product or ligation product is detected by nucleic acid sequencing. 
     
     
         23 . The method of  claim 22 , wherein prior to sequencing one or more sequencing adapters is attached to the extension product or ligation product in one or more amplification and/or ligation steps. 
     
     
         24 . The method of  claim 23 , wherein in step (iv) the extension product or ligation product is amplified in a first PCR reaction wherein a first sequencing adapter is added to one end of the extension product or ligation product;
 and the product of the first PCR reaction is amplified in a second PCR reaction wherein a second sequencing adapter is added to the other end of the extension product or ligation product.   
     
     
         25 . The method of  claim 24 , wherein the first PCR reaction is performed with a nucleic acid polymerase that also has 3′ to 5′ exonuclease activity, and the second PCR reaction is performed with a nucleic acid polymerase that lacks 3′ to 5′ exonuclease activity. 
     
     
         26 . The method of any one of  claims 24  to  27 , wherein prior to sequencing a sample index sequence is attached to the extension product or ligation product in an amplification or ligation step, preferably wherein the sample index sequence is added to the extension product or ligation product during PCR amplification in step (iv). 
     
     
         27 . The method of any one of  claims 22  to  26 , wherein the nucleic acid sequencing is massively parallel DNA sequencing. 
     
     
         28 . The method of any one of  claims 1  to  27 , wherein the sample is a plasma or serum sample. 
     
     
         29 . A product comprising:
 (i) a plurality of proximity probe pairs, wherein each proximity probe pair comprises a first proximity probe and a second proximity probe, and each proximity probe comprises:
 (a) a protein-binding domain specific for a protein; and 
 (b) a nucleic acid domain, 
   wherein both probes within each pair comprise protein-binding domains specific for the same protein, and can simultaneously bind to the protein; and each probe pair is specific for a different protein;   wherein the nucleic acid domain of each proximity probe comprises an ID sequence and at least a first hybridisation sequence, wherein the ID sequence of each proximity probe is different; and wherein in each proximity probe pair, the first proximity probe and the second proximity probe comprise paired hybridisation sequences; and, optionally   (ii) a plurality of splint oligonucleotides, each splint oligonucleotide comprising hybridisation sequences complementary to each of the paired hybridisation sequences of a proximity probe pair;   wherein the hybridisation sequences of each proximity probe pair are configured such that upon binding of the first and second proximity probe to their protein, the respective paired hybridisation sequences of the first and second proximity probes hybridise to each other or to a splint oligonucleotide;   and wherein at least one pair of hybridisation sequences is shared by at least two pairs of proximity probes.   
     
     
         30 . The product of  claim 29 , wherein the protein-binding domain is an antibody or fragment thereof. 
     
     
         31 . The product of  claim 29  or  30 , further comprising one or more background probes which do not bind an analyte, said background probes comprising a nucleic acid domain comprising an ID sequence and a hybridisation sequence shared with at least one proximity probe. 
     
     
         32 . The product of any one of  claims 29  to  31 , wherein the ID sequences are barcode sequences. 
     
     
         33 . The product of any one of  claims 29  to  32 , wherein at least one pair of hybridisation sequences is unique to a single pair of proximity probes. 
     
     
         34 . The product of any one of  claims 29  to  33 , wherein no more than 10 proximity probe pairs share the same pair of hybridisation sequences. 
     
     
         35 . The product of any one of  claims 29  to  34 , wherein at least 75% of proximity probe pairs share their pair of hybridisation sequences with another proximity probe pair. 
     
     
         36 . The product of any one of  claims 29  to  35 , wherein the nucleic acid domains of each proximity probe pair comprise complementary hybridisation sequences capable of hybridising to one another to form a duplex. 
     
     
         37 . The product of any one of  claims 29  to  36 , wherein the proximity probe pairs are as defined in any one of  claim 14  to  17  or  20 .