Controls for proximity detection assays

Abstract

A method for detecting a plurality of analytes in a sample comprises 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). A product comprising a plurality of proximity probe pairs with shared hybridisation sites may be used in the method disclosed herein.

1 . 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 sequences of each pair of proximity probes are barcode sequences which correspond to a particular analyte; and wherein in each proximity probe pair, the first proximity probe and the second proximity probe comprise paired hybridisation sequences; and   (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 the splint oligonucleotide; and wherein at least one pair of hybridisation sequences is shared by at least two pairs of proximity probes.   
     
     
         2 . The product of  claim 1 , wherein the protein-binding domain is an antibody or fragment thereof. 
     
     
         3 . The product of  claim 1 , 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. 
     
     
         4 . The product of  claim 1 , wherein at least one pair of hybridisation sequences is unique to a single pair of proximity probes. 
     
     
         5 . The product of  claim 1 , wherein no more than 10 proximity probe pairs share the same pair of hybridisation sequences. 
     
     
         6 . The product of  claim 1 , wherein at least 75% of proximity probe pairs share their pair of hybridisation sequences with another proximity probe pair. 
     
     
         7 . The product of  claim 1 , wherein in each proximity probe pair at least one nucleic acid domain is partially double-stranded. 
     
     
         8 . The product of  claim 7 , wherein in each proximity probe pair both nucleic acid domains are partially double-stranded. 
     
     
         9 . The product of  claim 7 , wherein the nucleic acid domains of each proximity probe pair comprise paired hybridisation sequences capable of hybridizing to the splint oligonucleotide to form the duplex, and 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, when subjecting the duplex to a ligation reaction. 
     
     
         10 . The product of  claim 7 , wherein the partially double-stranded nucleic acid comprises
 (i) a first oligonucleotide conjugated to the analyte-binding domain; and   (ii) a hybridisation oligonucleotide comprising, from 5′ to 3′, the first hybridisation sequence, the ID sequence and a second hybridisation sequence, and the ID sequence is located in a single-stranded part of the nucleic acid domain.   
     
     
         11 . The product of  claim 7 , wherein the nucleic acid domains of both proximity probes in a pair are conjugated to the protein-binding domain by their 5′ends. 
     
     
         12 . The product of  claim 11 , wherein one strand of the partially double-stranded nucleic acid domain has a free 3′-end. 
     
     
         13 . The product of  claim 1 , wherein the nucleic acid domains of the proximity probe pair hybridise to the splint oligonucleotide such that there is a gap between the 3′ terminus of one nucleic acid domain and the 5′ terminus of the other nucleic acid domain. 
     
     
         14 . The product of  claim 1 , further comprising a plurality of sample index oligonucleotides having nucleotide sequences identifying a source sample. 
     
     
         15 . The product of  claim 1 , wherein the nucleic acid domain is a DNA domain.