CA-3172942-C - METHOD FOR DETECTING ANALYTES OF VARYING ABUNDANCE

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Abstract

The present invention provides a method of detecting multiple analytes in a sample, wherein said analytes have varying levels of abundance in the sample, said method comprising: (i) providing multiple aliquots from the sample; and (ii) in each aliquot, detecting a different subset of the analytes by performing a separate multiplex assay for each aliquot, wherein the analytes in each subset are selected based on their predicted abundance in the sample.

Inventors

Niklas NORDBERG
John BROBERG
Lotta WIK
Martin Lundberg

Assignees

OLINK PROTEOMICS AB

Dates

Publication Date: 20260505
Application Date: 20210326
Priority Date: 20200327

Claims (15)

Claims 1. A method of detecting multiple non-nucleic acid analytes in a sample of a sample type, said analytes having varying levels of abundance in the sample, said method 5 comprising: {i) providing multiple aliquots from the said sample; and {ii) performing a separate block of detection assays on each of said multiple aliquots, wherein each detection assay block detects a subset of analytes different to the subset which is detected by another detection assay block, and wherein each analyte in each 10 subset of analytes is assigned to the respective detection assay block based on predicted abundance of the analyte; wherein the predicted abundance of any analyte is predicted based on a normal concentration of that analyte in the same sample type or on measurements obtainable from the used detection assay; 15 wherein detection assays for analytes which are expected to be present in the sample at a similar concentration are included in the same detection assay block, and wherein detection assays for analytes which are expected to be present in the sample at different concentrations are included in different detection assay blocks; wherein one or more aliquots are diluted prior to performing the detection assays, by 20 an appropriate dilution factor in view of the predicted abundance of the analytes; and wherein the analytes are detected by detecting analyte-specific reporter nucleic acid molecules generated by the detection assays in each aliquot.
2. The method of claim 1, wherein: {a) an aliquot in which analytes are expected to be present at the lowest concentrations is undiluted or has the lowest dilution factor; {b) an aliquot in which analytes are expected to be present at the highest concentrations has the highest dilution factor; and {c) an aliquot in which analytes are expected to be present at concentrations in 30 between the lowest and the highest concentrations has a dilution factor in between the lowest and the highest dilution factors.
3. The method of claim 1 or 2, wherein the analyte is or comprises a protein. 35
4. The method of any one of claims 1 to 3, wherein an analyte is included in more than one subset if it has an expected concentration in between the expected concentrations of two subsets or if it is known to occur in the sample in a wide range of concentrations. 52 Date Rei;ue/Date Received 2024-04-12
5. The method of any one of claims 1 to 4, wherein the detection assays performed on each aliquot provide a relative or absolute quantitation of each analyte in the subset of analytes assigned to that aliquot.
6. The method of any one of claims 1 to 5, wherein: (i) the reporter nucleic acid molecules are amplified by PCR; or (ii) the reporter nucleic acid molecules are amplified by PCR and are detected by nucleic acid sequencing.
7. The method of claim 6, wherein at least one PCR reaction is run to saturation.
8. The method of any one of claims 1 to 7, wherein the reporter nucleic acid molecules, or the amplification products thereof, are pooled to create a first pool, and are amplified in 15 the first pool.
9. The method of any one of claims 6 to 8, wherein the PCR reaction comprises an internal control for each aliquot. 20
10. The method of any one of claims 1 to 9, wherein: (i) the reporter nucleic acid molecule is generated in a proximity probe detection assay;or (ii) the reporter nucleic acid molecule is generated in a proximity extension assay (PEA).
11. The method of any one of claims 1 to 8, wherein: (i) the reporter nucleic acid molecule comprises at least one barcode sequence, and detection of the reporter nucleic acid molecule comprises detecting the at least one barcode sequence;or 30 (ii) the reporter nucleic acid molecule comprises at least one barcode sequence, and detection of the reporter nucleic acid molecule comprises detecting the at least one barcode sequence, in conjunction with a sample index; or (iii) the reporter nucleic acid molecule comprises a combination of barcode sequences from the nucleic acid domains of a pair of proximity probes, and detection of the 35 reporter nucleic acid molecule comprises detection of the combination of barcode sequences; or 53 Date Rei;ue/Date Received 2024-04-12 (iv) the reporter nucleic acid molecule comprises a combination of barcode sequences from the nucleic acid domains of a pair of proximity probes, and detection of the reporter nucleic acid molecule comprises detection of the combination of barcode sequences, in conjunction with a sample index.
12. The method of any one of claims 1 to 11, wherein the sample is a plasma or serum sample.
13. The method of any one of claims 10 to 12, wherein the analytes are detected using 10 pairs of proximity probes, each proximity probe comprising: (i) an analyte-binding domain specific for an analyte; and (ii) a nucleic acid domain, wherein both probes within each pair comprise analyte-binding domains specific for the same analyte, and each probe pair is specific for a different analyte, and wherein each 15 probe pair is designed such that on proximal binding of the pair of proximity probes to their respective analyte the nucleic acid domains of the proximity probes interact to generate a reporter nucleic acid molecule; wherein at least 2 panels of proximity probe pairs are used, each panel being for the detection of a different group of analytes, and for each panel separate aliquots of the sample 20 are provided for the detection of a different subset of the analytes in the group; and wherein (a) within each panel, every probe pair comprises a different pair of nucleic acid domains; and (b) in different panels the probe pairs comprise the same pairs of nucleic acid domains. 25
14. The method of claim 13, for detecting analytes from different samples, wherein the PCR products generated by amplification of the reporter nucleic acid molecules generated for each sample are provided with a sample index; and wherein the PCR products generated from each different sample using the same panel of proximity probe pairs are pooled into a panel pool for nucleic acid sequencing, the 30 PCR products generated using each panel being pooled into separate panel pools; and wherein each panel pool is sequenced separately.
15. The method of any one of claims 6 to 14, wherein one or more adaptors for sequencing are added to the reporter nucleic acid molecules in one or more amplification 35 and/or ligation steps and wherein said nucleic acid sequencing is massively parallel DNA sequencing. 54 Date Rei;ue/Date Received 2024-04-12

Description

Method for Detecting Analytes of Varying Abundance Field The present invention provides a method of detecting multiple analytes in a sample, wherein the analytes have varying levels of abundance in the sample. In the method, s multiple aliquots of the sample are provided, and in each aliquot a subset of the analytes is detected, the subset of analytes being selected based on their predicted abundance in the sample. Also provided is a method of detecting an analyte in a sample, wherein the analyte is detected by detecting a reporter nucleic acid molecule specific for the analyte. In this method a PCR reaction is performed to amplify the reporter nucleic acid molecule, in which 10 PCR an internal control is used. The methods of the invention find particular utility in the context of a proximity extension assay (PEA). Background Modern proteomics methods require the ability to detect a large number of different 15 proteins (or protein complexes) in a small sample volume. To achieve this, multiplex analysis must be performed. Common methods by which multiplex detection of proteins in a sample may be achieved include proximity extension assays (PEA) and proximity ligation assays (PLA). PEA and PLA are described in WO 01/61037; PEA is further described in WO 03/044231, WO 2004/094456, WO 2005/123963, WO 2006/137932 and 20 WO 2013/113699. However, when, as is common, the proteins of interest are present in a wide concentration range, this presents a challenge, since the signal from proteins of high concentration may overwhelm the signal from proteins of low concentration, resulting in a failure to detect proteins present at lower concentrations. The present invention provides detection methods whereby analytes (e.g. proteins) 25 present in a sample at a wide range of concentrations may be reliably detected, improving the accuracy of multiplex detection methods. The methods of the invention may be applied to PEA or PLA as mentioned above, but may also be applied to any other technique used in multiplex analyte detection. PEA and PLA are proximity assays, which rely on the principle of "proximity probing". 30 In these methods an analyte is detected by the binding of multiple (i.e. two or more, generally two or three) probes, which when brought into proximity by binding to the analyte (hence "proximity probes") allow a signal to be generated. Typically, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction 35 between the nucleic acid moieties and/or a further functional moiety which is carried by the other probe(s). Thus signal generation is dependent on an interaction between the probes (more particularly between the nucleic acid or other functional moieties/domains carried by 1 CA 03172942 2022- 9- 22 them) and hence only occurs when the necessary probes have bound to the analyte, thereby lending improved specificity to the detection system. In PEA, nucleic acid moieties linked to the analyte-binding domains of a probe pair hybridise to one another when the probes are in close proximity (i.e. when bound to a 5 target), and are then extended using a nucleic acid polymerase. The extension product forms a reporter nucleic acid, detection of which demonstrates the presence in a sample of interest of a particular analyte (the analyte bound by the relevant probe pair). In PLA, nucleic acid moieties linked to the analyte-binding domains of a probe pair come into proximity when the probes of the probe pair bind their target, and may be ligated together, or alternatively 10 they may together template the ligation of separately added oligonucleotides which are able to hybridise to the nucleic acid domains when they are in proximity. The ligation product is then amplified, acting as a reporter nucleic acid. Multiplex analyte detection using PEA or PLA may be achieved by including a unique barcode sequence in the nucleic acid moiety of each probe. A reporter nucleic acid molecule corresponding to a particular analyte may be 15 identified by the barcode sequences it contains. The methods of the present invention find particular utility in multiplex PEA and PLA methods. The methods of the invention may be of utility in at least any field in which proteomics is used, in particular in diagnostics in the context of biomarker identification and quantification. Modern personalised medicine requires the ability to assess large panels of 20 biomarkers, e.g. in the field of oncology. As personalised medicine becomes ever more widespread, the ability to accurately identify and quantify a large number of biomarkers in a sample (across a range of concentrations) is increasing in importance. This need is addressed by the present invention. Summary of Invention To this end, in a first aspect the present invention provides a method of detecting multiple analytes in a sample, wherein said analytes have