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.

Claims

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.

Description

BRIEF DESCRIPTION OF FIGURES

[0161] FIG. 1 shows a schematic representation of six different versions of proximity extension assays, described in detail above. The inverted ‘Y’ shapes represent antibodies, as an exemplary proximity probe analyte-binding domain.

[0162] FIG. 2 shows a comparison of results obtained for the level of expression of four analytes across six different samples, as determined by multiplex PEA, using either a traditional negative control or a shared hybridisation site negative control.

[0163] FIG. 3 shows a comparison of results obtained for the level of expression of five analytes across six different samples (same samples as used for FIG. 2), as determined by multiplex PEA, using either a traditional negative control or a shared hybridisation site negative control.

EXAMPLES

[0164] Plasma samples were obtained from 6 donors: 3 healthy subjects, one subject diagnosed with breast cancer, one diagnosed with rheumatoid arthritis (RA) and one diagnosed with inflammatory bowel disease (IBD).

[0165] A multiplex PEA was performed (using probes comprising antibodies conjugated to nucleic acid domains having the structure described in Version 6, above) to detect 9 proteins in the samples: NPDC1 (UniProt Q9NQX5); AHCY (UniProt P23526); TM (UniProt P07204); ANGPTL1 (UniProt O95841); LOX-1 (UniProt P78380); SEMA3F (UniProt Q13275); CDH2 (UniProt P19022); CANT1 (UniProt Q8WVQ1); and CA13 (UniProt Q8N1Q1). The probes targeted against NPDC1, AHOY, TM and ANGPTL1 all shared a pair of hybridisation sites; and the probes targeted against LOX-1, SEMA3F, CDH2, CANT1 and CA13 all shared a different pair of hybridisation sites. Each probe contained a unique barcode sequence. A negative control was also used, comprising phosphate buffered saline with 1% bovine serum albumin without sample.

[0166] The PEA was performed as described above. During amplification of the extension products, P5 and P7 sequencing adapters were added to each end of the products, along with a unique sample index for reporter nucleic acids from each different sample, and all extension products sequenced by massively parallel DNA sequencing, employing reversible dye terminator sequencing technique using an Illumina NovaSeq platform.

[0167] Background from standard negative control for a target was determined from the paired barcode interaction of probes for the target. Background from shared hybridisation sites for a target was determined from the mean value of the mismatched interactions (as determined by mismatched barcodes) between each respective probe of the pair of probes for the target and other probes within the group (i.e. probes that share hybridisation sites with the probes for the target), for each sample. In other words, for each target background from shared hybridisation sites was defined as non-specific interactions between each probe for the target and other probes having shared hybridisation sites. Non-specific interactions between probes, neither of which bind the target, were not included in the calculation of background.

[0168] The following results were obtained from the two groups of target analytes:

Group 1—Linear Analysis

[0169] Signal above background from negative control:

TABLE-US-00001 NPDC1 AHCY TM ANGPTL1 IBD Subject 4.744595 18.77055 24.26055 5.650493 RA Subject 24.15997 23.02676 31.97209 16.44529 Breast Cancer Subject 15.63025 5.763718 21.46488 16.13142 Healthy Control 1 10.11761 9.273049 18.64168 24.23299 Healthy Control 2 14.58207 2.398616 26.94637 17.11784 Healthy Control 3 26.35638 6.522163 38.96036 24.39238

[0170] Signal above background from shared hybridisation sites:

TABLE-US-00002 NPDC1 AHCY TM ANGPTL1 IBD Subject 6.587558 24.20433 33.56129 6.621035 RA Subject 30.17537 30.42173 36.67241 14.92188 Breast Cancer Subject 18.81776 6.457732 29.32031 14.772 Healthy Control 1 12.29969 10.44351 21.19694 21.6378 Healthy Control 2 14.15044 2.597143 28.14347 13.73367 Healthy Control 3 31.26646 7.661677 46.84286 21.24901

Group 1—Logarithmic Analysis (Base 2)

[0171] Signal above background from negative control:

TABLE-US-00003 NPDC1 AHCY TM ANGPTL1 IBD Subject 2.246285 4.230399 4.60054 2.498377 RA Subject 4.594547 4.52524 4.998741 4.039603 Breast Cancer Subject 3.966269 2.527 4.423906 4.011802 Healthy Control 1 3.338797 3.213044 4.22046 4.598901 Healthy Control 2 3.866123 1.262202 4.752019 4.097428 Healthy Control 3 4.72008 2.70535 5.283935 4.608359

[0172] Signal above background from shared hybridisation sites:

TABLE-US-00004 NPDC1 AHCY TM ANGPTL1 IBD Subject 2.719744 4.597194 5.068726 2.727057 RA Subject 4.915299 4.92703 5.196623 3.899357 Breast Cancer Subject 4.234023 2.691028 4.873829 3.884793 Healthy Control 1 3.62055 3.384535 4.405784 4.435482 Healthy Control 2 3.822775 1.376925 4.814728 3.779645 Healthy Control 3 4.966544 2.93766 5.549757 4.409324

[0173] The logarithmic results are shown in the graph of FIG. 2.

Group 2—Linear Analysis

[0174] Signal above background from negative control:

TABLE-US-00005 LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 93.75019 7.710203 11.65482 30.88921 22.95267 Subject RA 51.94867 15.51322 13.56623 46.84155 304.8523 Subject Breast 12.1141 15.45434 16.45051 36.89154 104.631 Cancer Subject Healthy 23.56257 8.300925 8.070123 29.4027 4.299637 Control 1 Healthy 18.14679 6.530255 17.95702 36.27412 14.72176 Control 2 Healthy 25.83432 13.76144 13.69109 34.4381 5.858678 Control 3

[0175] Signal above background from shared hybridisation sites:

TABLE-US-00006 LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 141.2799 18.21138 16.84211 31.75165 31.70223 Subject RA 65.79012 31.66038 15.88331 39.08923 338.9779 Subject Breast 14.1692 29.58474 20.04621 31.2304 117.9876 Cancer Subject Healthy 33.38638 17.40281 10.59963 26.28186 5.242938 Control 1 Healthy 23.59205 11.80547 21.32377 31.38729 17.40023 Control 2 Healthy 32.05737 26.6478 16.27754 27.05954 6.934537 Control 3

Group 2—Logarithmic Analysis (Base 2)

[0176] Signal above background from negative control:

TABLE-US-00007 LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 6.55075 2.946769 3.542855 4.949031 4.52059 Subject RA 5.699015 3.955426 3.761948 5.549717 8.251967 Subject Breast 3.598615 3.94994 4.04006 5.205218 6.709166 Cancer Subject Healthy 4.558425 3.053272 3.012591 4.877877 2.104215 Control 1 Healthy 4.181643 2.707139 4.166476 5.180869 3.879879 Control 2 Healthy 4.691217 3.782559 3.775165 5.105934 2.550575 Control 3

[0177] Signal above background from shared hybridisation sites:

TABLE-US-00008 LOX-1 SEMA3F CDH2 CANT1 CA13 IBD 7.142412 4.186769 4.074001 4.988759 4.986513 Subject RA 6.039799 4.984607 3.989439 5.288699 8.405047 Subject Breast 3.824686 4.886781 4.325258 4.964879 6.882491 Cancer Subject Healthy 5.061188 4.121249 3.405942 4.715995 2.390375 Control 1 Healthy 4.560229 3.561384 4.41439 4.972109 4.121035 Control 2 Healthy 5.002584 4.735944 4.024811 4.758066 2.7938 Control 3

[0178] The logarithmic results are shown in the graph of FIG. 3.

[0179] While for both groups of analytes, the actual values of the signals above background may differ between the two types of control (negative control and shared hybridisation sites control), the values shift approximately equally for each analyte for each sample (i.e. there is a parallel shift). This is demonstrated by the high R.sup.2 values for the results obtained with each analyte, which indicate a very high correlation between the degree of signal above background as determined by each of the two methods. These results demonstrate that the use of shared hybridisation sites is a valid alternative to a standard negative control since the relative signal levels of an analyte is retained between samples. The results from the background determination from shared hybridisation sites show similar discrimination between samples as when using a standard negative control.