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US 2023/0107654 A1Published application
Controls for proximity detection assays
AssigneeOlink Proteomics AB
InventorJohn BROBERG
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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.
Claims37
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.
16 more elements
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).
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.
9 more elements
Dependencies are inferred from the claim text.