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.

Claims

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.

Description

BRIEF DESCRIPTION OF FIGURES

[0164] 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.

[0165] 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.

[0166] 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

[0167] 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).

[0168] 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); AHOY (UniProt P23526); TM (UniProt P07204); ANGPTL1 (UniProt 095841); 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.

[0169] 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 IIlumina NovaSeq platform.

[0170] 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.

[0171] The following results were obtained from the two groups of target analytes:

[0172] Group 1—Linear Analysis

TABLE-US-00001 NPDC1 AHCY TM ANGPTL1 Signal above background from negative control: 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 Signal above background from shared hybridisation sites: 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

[0173] Group 1—Logarithmic Analysis (Base 2)

TABLE-US-00002 NPDC1 AHCY TM ANGPTL1 Signal above background from negative control: 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 Signal above background from shared hybridisation sites: 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

[0174] The logarithmic results are shown in the graph of FIG. 2.

[0175] Group 2—Linear Analysis

TABLE-US-00003 LOX-1 SEMA3F CDH2 CANT1 CA13 Signal above background from negative control: IBD Subject 93.75019 7.710203 11.65482 30.88921 22.95267 RA Subject 51.94867 15.51322 13.56623 46.84155 304.8523 Breast Cancer Subject 12.1141 15.45434 16.45051 36.89154 104.631 Healthy Control 1 23.56257 8.300925 8.070123 29.4027 4.299637 Healthy Control 2 18.14679 6.530255 17.95702 36.27412 14.72176 Healthy Control 3 25.83432 13.76144 13.69109 34.4381 5.858678 Signal above background from shared hybridisation sites: IBD Subject 141.2799 18.21138 16.84211 31.75165 31.70223 RA Subject 65.79012 31.66038 15.88331 39.08923 338.9779 Breast Cancer Subject 14.1692 29.58474 20.04621 31.2304 117.9876 Healthy Control 1 33.38638 17.40281 10.59963 26.28186 5.242938 Healthy Control 2 23.59205 11.80547 21.32377 31.38729 17.40023 Healthy Control 3 32.05737 26.6478 16.27754 27.05954 6.934537

[0176] Group 2—Logarithmic Analysis (Base 2)

TABLE-US-00004 LOX-1 SEMA3F CDH2 CANT1 CA13 Signal above background from negative control: IBD Subject 6.55075 2.946769 3.542855 4.949031 4.52059 RA Subject 5.699015 3.955426 3.761948 5.549717 8.251967 Breast Cancer Subject 3.598615 3.94994 4.04006 5.205218 6.709166 Healthy Control 1 4.558425 3.053272 3.012591 4.877877 2.104215 Healthy Control 2 4.181643 2.707139 4.166476 5.180869 3.879879 Healthy Control 3 4.691217 3.782559 3.775165 5.105934 2.550575 Signal above background from shared hybridisation sites: IBD Subject 7.142412 4.186769 4.074001 4.988759 4.986513 RA Subject 6.039799 4.984607 3.989439 5.288699 8.405047 Breast Cancer Subject 3.824686 4.886781 4.325258 4.964879 6.882491 Healthy Control 1 5.061188 4.121249 3.405942 4.715995 2.390375 Healthy Control 2 4.560229 3.561384 4.41439 4.972109 4.121035 Healthy Control 3 5.002584 4.735944 4.024811 4.758066 2.7938

[0177] The logarithmic results are shown in the graph of FIG. 3.

[0178] 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.