Testing method and kit

Abstract

Provided is a method of testing an analyte in a sample. The method comprises: causing a first proximity probe and a second proximity probe to bind to an analyte in a sample in sequence to form an immune complex, the immune complex being captured on a solid-state matrix by means of binding of the first proximity probe to a capture probe, and adding a blocker after the first proximity probe binds to the capture probe and before the second proximity probe is added, the blocker binding to the capture probe to reduce non-specific binding of the capture probe to molecules other than the immune complex. Also provided is a blocker for the method.

Description

A detection method and a kit Technical Field

[0001] The present disclosure relates to the field of biological detection, and in particular to a detection method and a kit. Background Art

[0002] Enzyme-linked immunosorbent reaction (ELISA) is a classic method for qualitative and quantitative protein detection, but its detection limit is only at the ng / mL level, which cannot meet the needs of detecting ultra-trace biomolecules. Proximity ligation assay (PLA) is an in vitro protein detection technology with a sensitivity up to 1000 times that of ELISA. This technology uses nucleic acid aptamers or monoclonal / polyclonal antibody-oligonucleotide complexes as proximity ligation probes. When two proximity probes simultaneously recognize a target protein molecule, the oligonucleotides on the proximity probes are spatially close to each other, generating the so-called proximity effect. Under the action of specific linker oligonucleotides and ligase, the oligonucleotides on the two proximity probes connect to each other, forming a new oligonucleotide chain. This oligonucleotide chain is then detected by real-time fluorescence quantitative PCR using specific primers and probes, thus transforming protein detection into nucleic acid detection and enabling trace protein analysis.

[0003] Solid-phase PLA is a type of PLA technology primarily based on microspheres and test tubes. Nonspecific binding between free and non-free probes in the solid-phase PLA reaction system can lead to increased background signal, affecting the detection sensitivity of the PLA reaction.

[0004] Summary of the Invention

[0005] In order to solve the above technical problems, reduce the background signal in the solid-phase PLA technology detection process and further improve its detection sensitivity, the present invention provides a method that reduces the background signal of the PLA technology and improves the detection sensitivity by using a blocking agent that binds to the capture probe.

[0006] Specifically, the present disclosure provides a detection method, which includes:

[0007] 1) contacting a first proximity probe and a capture probe with a solid substrate in a solution, wherein the capture probe binds to the first proximity probe and the solid substrate, thereby capturing the first proximity probe on the solid substrate, thereby obtaining a first binding product, wherein the first proximity probe comprises a first nucleotide portion and a first binding portion for binding to an analyte in a sample to be tested;

[0008] 2) adding a blocking agent and a sample to be tested containing an analyte, wherein the blocking agent is used to block the capture probe, and the first proximity probe in the first binding product binds to the analyte in the sample to be tested, thereby obtaining a second binding product;

[0009] 3) adding a second proximity probe, so that the second proximity probe binds to the second binding product, wherein the second proximity probe comprises a second nucleotide portion and a second binding moiety for binding to an analyte in the test sample, and the second binding moiety and the first binding moiety bind to the same analyte to form an immune complex;

[0010] 4) allowing the first nucleotide portion of the first proximity probe to interact with the second nucleotide portion of the second proximity probe to produce a nucleic acid reporter molecule;

[0011] 5) Detecting the nucleic acid reporter molecule.

[0012] In certain embodiments, the blocker comprises an oligonucleotide sequence comprising or consisting of at least 15 consecutive nucleotides that complementarily hybridize with the capture probe; preferably, after the blocker binds to the capture probe, the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the capture probe is less than 7, preferably, less than 5.

[0013] In certain embodiments, the blocker is an oligonucleotide sequence comprising or consisting of at least 15-35 consecutive nucleotides that complementarily hybridize to the capture probe.

[0014] In certain embodiments, the oligonucleotide sequence comprises adenine deoxyribonucleotide A, guanine deoxyribonucleotide G, cytosine deoxyribonucleotide C and thymine deoxyribonucleotide T; preferably, one or more thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced by uracil deoxyribonucleotides U.

[0015] In certain embodiments, the 3' end of the oligonucleotide sequence has a modification that blocks the extension of the oligonucleotide sequence; preferably, the modification is a dideoxycytidine ddC modification.

[0016] In certain embodiments, the concentration ratio of the blocker to the capture probe is 1:1 to 5:1.

[0017] In certain embodiments, a washing step is further included between step 1) and step 2), between step 2) and step 3), between step 3) and step 4), and / or between step 4) and step 5); preferably, the washing step is performed 2-4 times; preferably, the washing buffer used in the washing step is: 0.05% Tween 20, 1× PBS, pH 7.0.

[0018] In certain embodiments, the analyte comprises a protein.

[0019] In certain embodiments, the solid matrix has a modification group a that binds to the capture probe; preferably, the solid matrix is ​​a magnetic bead; preferably, the modification group a is oligo-dT or streptavidin.

[0020] In certain embodiments, the capture probe is one or more single-stranded DNAs, the capture probe has a modification group b bound to the modification group a, and the capture probe is complementary hybridized with the first nucleotide portion of the first proximity probe; preferably, the modification group b is poly A or biotin; preferably, the capture probe is a single-stranded DNA, and the single-stranded DNA consists of 15-35 nucleotides.

[0021] In certain embodiments, the first binding portion and the second binding portion are each a nucleic acid aptamer, an antibody or an antigen-binding fragment of an antibody, the first nucleotide portion and the second nucleotide portion are each one or more single-stranded DNAs, and the one or more single-stranded DNAs of the first nucleotide portion contain a nucleotide sequence that hybridizes complementarily to the capture probe; preferably, the length of the one or more single-stranded DNAs is 30-100 nucleotides; preferably, one end of each of the one or more single-stranded DNAs has a modification group c selected from a thiol, an amino group, and an azide group, and the modification group c reacts with a coupling agent to couple the one or more single-stranded DNAs of the first nucleotide portion to the first binding portion and to couple the one or more single-stranded DNAs of the second nucleotide portion to the second binding portion; preferably, the coupling agent is selected from one or more of EMCS, sulfo-SMCC, Sulfo-LC-SPDP or SMPB; preferably, the first binding portion and the second binding portion respectively bind to different epitopes of the same analyte.

[0022] In certain embodiments, the immune complex consists of the first proximity probe, the second proximity probe, and the analyte.

[0023] In certain embodiments, in step 4), a connecting oligonucleotide and a ligase are added to connect the free end of the first nucleotide portion to the free end of the second nucleotide portion to form the nucleic acid reporter molecule; preferably, the ligase is selected from T4 DNA ligase, T7 DNA ligase, SplintR ligase, and Taq ligase.

[0024] In certain embodiments, the detection is performed by qPCR detection; preferably, UNG enzyme is added before or during the qPCR detection to degrade the blocker.

[0025] In certain embodiments, an elution step is further included between step 4) and step 5) to separate the immune complex from the solid phase matrix; preferably, the elution step includes adding enzyme-free sterile water and incubating at 70°C-95°C for 10-20 minutes.

[0026] In another aspect, the present disclosure further provides a blocking agent for use in the method of the present disclosure, wherein the blocking agent is used to block the capture probe.

[0027] In certain embodiments, the blocker comprises an oligonucleotide sequence comprising or consisting of at least 15 consecutive nucleotides that complementarily hybridize with the capture probe; preferably, after the blocker binds to the capture probe, the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the capture probe is less than 7, preferably, less than 5.

[0028] In certain embodiments, the blocker is an oligonucleotide sequence comprising or consisting of 15-35 consecutive nucleotides that complementarily hybridize to the capture probe.

[0029] In certain embodiments, the oligonucleotide sequence comprises adenine deoxyribonucleotide A, guanine deoxyribonucleotide G, cytosine deoxyribonucleotide C and thymine deoxyribonucleotide T; preferably, one or more thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced by uracil deoxyribonucleotides U.

[0030] In certain embodiments, the 3' end of the oligonucleotide sequence has a modification that blocks the extension of the oligonucleotide sequence; preferably, the modification is a dideoxycytidine ddC modification.

[0031] In another aspect, the present disclosure also provides a kit comprising the blocking agent of the present disclosure.

[0032] Compared with the prior art, the method provided by the present disclosure achieves the following technical effects:

[0033] The method disclosed herein reduces the background signal of PLA technology and improves detection sensitivity by using a blocker that binds to the capture probe. Specifically, the analyte is first bound to the first proximity probe, and then the second proximity probe is bound to the analyte to obtain an immune complex. The blocker provided by the present disclosure is added after the first proximity probe is bound to the capture probe and before the second proximity probe is added, so that the blocker binds to the capture probe to block the capture probe and block the nonspecific binding of the capture probe to other substances other than the immune complex, thereby reducing the background signal of PLA technology and improving detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 shows a schematic diagram of the use of a blocking agent in one embodiment of the detection method of the present disclosure.

[0035] FIG2 shows the amplification curves of sample numbers 1 to 8 of Example 1 of the present disclosure.

[0036] FIG3 shows the amplification curves of sample numbers 9-16 of Example 1 of the present disclosure. DETAILED DESCRIPTION

[0037] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in the specification.

[0038] The present application is further described below. Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0039] In one aspect, the present disclosure provides a detection method, comprising:

[0040] 1) contacting a first proximity probe and a capture probe with a solid substrate in a solution, wherein the capture probe binds to the first proximity probe and the solid substrate, thereby capturing the first proximity probe on the solid substrate, thereby obtaining a first binding product, wherein the first proximity probe comprises a first nucleotide portion and a first binding portion for binding to an analyte in a sample to be tested;

[0041] 2) adding a blocking agent and a sample to be tested containing the analyte, wherein the blocking agent is used to block the capture probes, particularly the capture probes bound to the solid matrix but not bound to the first proximity probes, and the first proximity probes in the first binding product bind to the analyte in the sample to be tested, thereby obtaining a second binding product;

[0042] 3) adding a second proximity probe, so that the second proximity probe binds to the second binding product, wherein the second proximity probe comprises a second nucleotide portion and a second binding moiety for binding to an analyte in the test sample, and the second binding moiety and the first binding moiety bind to the same analyte to form an immune complex;

[0043] 4) allowing the first nucleotide portion of the first proximity probe to interact with the second nucleotide portion of the second proximity probe to produce a nucleic acid reporter molecule;

[0044] 5) Detecting the nucleic acid reporter molecule.

[0045] On the other hand, the present disclosure also provides a blocking agent for use in the method of the present disclosure, wherein the blocking agent is used to block the capture probe, in particular, to block the capture probe bound to the solid matrix but not bound to the first proximity probe.

[0046] In this article, term " blocking " generally refers to that blocker is combined with capture probe, makes the position that is combined with blocker in capture probe no longer be combined with other substances, thereby reduces or eliminates the non-specific binding of capture probe and other substances outside immune complex.In this article, " non-specific binding " can refer to any undesirable combination between molecule or substance.For example, in this article, expect capture probe to be combined with the first adjacent position probe, and do not expect capture probe to be combined with other substances (for example, the second adjacent position probe) outside the first adjacent position probe.

[0047] In this article, the term "detection" generally refers to a method for determining whether an analyte exists or any form of a measurement index of an analyte (such as expression abundance, expression amount change within a certain time, expression position, etc.). Therefore, "detection" may include determining, measuring, evaluating or analyzing the presence or absence of an analyte or the quantity or position of the presence in any way. This determination, measurement or evaluation can be relative, for example, when detecting two or more different analytes in a sample; or it can be absolute (i.e., determining the absolute amounts of different analytes respectively). Therefore, in the context of quantifying the target analyte in a sample, the term "quantitative" can mean absolute or relative quantification. Absolute quantification can be achieved by including one or more control analytes of known concentration and / or comparing the detected target analyte level with a known control analyte (e.g., generated by a standard curve). Alternatively, relative quantification can be achieved by comparing the level or amount detected between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative quantification relative to each other. In the method provided by the present disclosure, the purpose of detecting the analyte can be achieved by detecting a nucleic acid reporter molecule.

[0048] As used herein, the term "analyte" (also referred to as "target," "target analyte," or "object of interest") generally refers to any substance (e.g., a biochemical molecule) detected by the methods provided herein. The analyte is the target of the assay method provided herein and can be any biological molecule or compound to be detected, such as a biological macromolecule or small molecule (including organic and inorganic molecules). The analyte can be a cell or microorganism, including a virus, or a fragment or product thereof. The analyte can be any substance capable of simultaneously, directly or indirectly, binding to at least two "binding moieties of proximity probes." In certain embodiments, the analyte comprises a protein, i.e., the analyte contains a protein, protein fragment, polypeptide, or the like that can simultaneously, directly or indirectly, bind to at least two "analyte binding moieties." For example, the analyte is a complex formed by a protein, protein fragment, polypeptide, or other substance; for example, the analyte is a biological fragment (e.g., a fragment of a cell, virus, bacteria, or the like) containing a protein, protein fragment, or polypeptide component. In certain embodiments, the analyte can also be a single molecule or a complex comprising two or more molecular subunits, which may or may not be covalently bound to each other and may be the same or different. For example, the analyte detected by the method described herein can be a protein complex, which can be a homopolymer or a heteropolymer, or an aggregate of molecules (e.g., proteins). The aggregate analyte can be an aggregate of the same protein or different proteins. The analyte can also be a complex composed of a protein or peptide or a nucleic acid molecule (e.g., DNA or RNA). In certain embodiments, the analyte is a complex composed of a protein (e.g., a regulatory factor, a transcription factor) and a nucleic acid. In certain embodiments, the analyte is a complete protein molecule or a partial protein molecule. For example, Spyros et al., (2010) Molecular & Cellular Proteomics 9.2: 327-335 reported that solid phase PLA technology was used to detect some cytokines including interleukin 6 and interleukin 8 in plasma. In certain embodiments, the analyte is a protein or a polypeptide.

[0049] In this article, term " sample " can be any biological and clinical sample, comprise any cell or tissue sample of biology, or any liquid or preparation derived therefrom, such as cell culture, cell preparation, cell lysate etc.Sample also comprises environmental sample, such as, soil and water sample or food sample.Sample can be freshly prepared or previously processed in any appropriate manner (such as, for example, processed in order to store).Therefore, exemplary sample comprises any material containing biomolecule, or any other desired analyte or target analyte, comprises food and similar products, clinical and environmental sample.Described sample can be biological sample, comprise virus or cell material, comprise prokaryotic or eukaryotic cell, virus, bacteriophage, mycoplasma, protoplast and organelle.These biological materials comprise all types of mammalian and non-mammalian cells, plant cells, algae, comprise blue-green algae, fungi, bacteria, protozoa etc. Exemplary samples also include whole blood and blood-derived products such as plasma, serum and buffy coat, blood cells, urine, feces, cerebrospinal fluid or any other body fluid (e.g., respiratory secretions, saliva, milk, etc.), tissue, biopsy, cell culture, cell suspension, conditioned medium or other cell culture composition samples, etc. The sample can be pretreated in any appropriate or desired manner to prepare for use in the methods disclosed herein. For example, the sample can be processed by cell lysis or purification, separation, etc. of the analyte.

[0050] As used herein, the term "solid phase matrix" generally refers to any solid support that is suitable for connection to nucleic acids and facilitates the detection step. Examples of solid phase matrices include particulate materials (e.g., magnetic beads, gel beads), colloids, single interfaces, tubes, chips, multi-well plates, microtiter plates, membranes, gels, and resins. Exemplary solid phase matrices may include magnetic beads and microtiter plates. When the solid phase matrix is ​​a particulate material (e.g., magnetic beads), they can be distributed in the wells of a multi-well plate or in a centrifuge tube for batch parallel processing. In certain embodiments, the solid phase matrix has a modification group a that binds to the capture probe. For example, the modification group a includes carboxylate, amine, oligo-dT, streptavidin, protein A / G; in certain embodiments, the magnetic beads may be carboxylate magnetic beads, amine-blocked magnetic beads, oligo-dT magnetic beads, streptavidin magnetic beads, protein A / G magnetic beads, or silica-coated magnetic beads.

[0051] As used herein, the term "proximity probe" generally refers to a compound molecule that is capable of specifically binding to the same analyte and thereby spatially approaching each other, thereby further producing a reporter molecule that can be used for detection of the analyte. A pair of proximity probes (i.e., a first proximity probe and a second proximity probe) that recognize and bind to the same target analyte is also referred to as a proximity probe pair. In the method provided by the present disclosure, more than one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 50, 100 or more) proximity probes can be used simultaneously to detect different analytes in a sample, and the upper limit of the number of species of the antibody pairs used simultaneously is not particularly limited, and the number of species of proximity probes used can be determined according to the specific detection purpose, for example, less than 10,000 species, less than 5,000 species, less than 1,000 species, less than 500 species, less than 300 species, less than 100 species, less than 50 species, less than 30 species, less than 20 species, less than 10 species, less than 5 species, less than 3 species. Wherein, each probe pair can identify a specific target analyte, and different types of probe pairs identify different analytes to achieve simultaneous detection of multiple analytes. The types of probe pairs can be defined by the binding specificity for different analytes, the barcode sequence for a specific target analyte carried in the probe pair (the first proximity probe and / or the second proximity probe in a probe pair carry a barcode sequence for the same analyte). In this article, the first proximity probe comprises a first nucleotide portion and a first binding portion for binding to the analyte in the sample to be tested, the second proximity probe comprises a second nucleotide portion and a second binding portion for binding to the analyte in the sample to be tested, the second binding portion and the first binding portion bind to the same analyte, and the binding can be direct or indirect, for example, the first binding portion and the second binding portion can directly bind to the target analyte (correspondingly, the immune complex formed thereby consists of the first proximity probe, the second proximity probe and the analyte), or indirectly bind to the analyte through other intermediate molecules (e.g., a linker molecule (e.g., a bispecific antibody) that simultaneously binds to the first binding portion or the second binding portion and the analyte) (correspondingly, the immune complex formed thereby comprises the first proximity probe, the second proximity probe and the analyte and the linker molecule).In certain embodiments, the first binding moiety and the second binding moiety are each a nucleic acid aptamer (Fredriksson et al. (2002) Nat Biotech 20: 473-477), an antibody or an antigen-binding fragment, derivative or mimetic of an antibody, the first nucleotide portion and the second nucleotide portion are each one or more single-stranded DNAs, the one or more single-stranded DNAs of the first nucleotide portion comprise a nucleotide sequence that hybridizes complementarily to the capture probe; in some embodiments, the one or more single-stranded DNAs are 30-100 nucleotides in length; in some embodiments, one end of each of the one or more single-stranded DNAs has a modification group c selected from a thiol, an amino group, and an azide group, and the modification group c reacts with a coupling agent to couple the one or more single-stranded DNAs of the first nucleotide portion to the first binding portion and the one or more single-stranded DNAs of the second nucleotide portion to the second binding portion; in some embodiments, the coupling agent is selected from EMCS, sulfo-SMCC, Sulfo-LC-SPDP or SMPB; in some embodiments, the first binding portion and the second binding portion respectively bind to different epitopes of the same analyte; in some embodiments, the first nucleotide portion and the second nucleotide portion comprise a barcode sequence for a specific target analyte and / or a barcode sequence for a specific sample. In certain embodiments, the first nucleotide portion and the second nucleotide portion are each a single-stranded DNA (e.g., a single-stranded DNA consisting of 30-100 nucleotides), and the single-stranded DNA of the first nucleotide portion contains a nucleotide sequence that complementarily hybridizes with the capture probe; in certain embodiments, the second nucleotide portion is a single-stranded DNA (e.g., a single-stranded DNA consisting of 30-100 nucleotides), and the first nucleotide portion is two or more single-stranded DNAs (e.g., a single-stranded DNA consisting of 30-100 nucleotides), one of which interacts with the single-stranded DNA of the second nucleotide portion to produce a reporter molecule, and any one or more of the other single-stranded DNAs can complementarily hybridize with the capture probe.

[0052] Antigen-binding fragments, derivatives, or mimetics of the above antibodies can be prepared using any convenient method, such as those disclosed in US Patents 5,851,829 and 5,965,371.

[0053] The proximity probes used in the methods of the present disclosure can be prepared using any conventional method. For example, methods for preparing nucleic acid / antibody conjugates are well known to those skilled in the art, for example, see, for example, US Pat. No. 5,733,523.

[0054] In this article, the term "capture probe" generally refers to a compound molecule (e.g., single-stranded DNA) that can bind to the first proximity probe to capture the immune complex formed by the first proximity probe, the second proximity probe, and the analyte as described herein to a solid phase matrix. In some embodiments, the capture probe is more than one single-stranded DNA, in some embodiments, the capture probe is a single-stranded DNA (e.g., a single-stranded DNA consisting of 15-35 nucleotides), and the capture probe is partially complementary to the first nucleotide of the first proximity probe; in some embodiments, the first nucleotide portion of the first proximity probe is more than two single-stranded DNAs, and the capture probe can be complementary to any of the single-stranded DNAs. In this article, the nucleotide sequence of the capture probe can be completely complementary to the first nucleotide portion of the first proximity probe, or partially complementary to the first nucleotide portion of the first proximity probe, as long as the purpose of capturing the immune complex to a solid phase matrix can be achieved. In this article, the capture probe can be coupled to the solid phase matrix by any means known in the art, for example, by covalent bonds (e.g., chemical cross-linking) or by non-covalent binding. For example, the capture probe has a modification group b that is bound to the modification group a; for example, the modification group b is poly A or biotin. In certain embodiments, the capture probe can be two or more single-stranded DNAs (e.g., two, three, or four strands), which can partially hybridize and pair with each other to form a nucleotide sequence having a double-stranded structure and a single-stranded structure, wherein the single-stranded structure of the nucleotide sequence (e.g., the single-stranded structure at the end not coupled to the solid phase substrate) is complementary to the first nucleotide portion of the first proximity probe, and the length of the single-stranded structure is sufficient to capture the immune complex on the solid phase substrate. In the methods provided herein, more than one (e.g., one, two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, twenty-five, thirty, fifty, or even one hundred) capture probes can be used simultaneously for detection. The upper limit of the number of capture probes used simultaneously is not particularly limited, and the number of capture probes used can be determined according to the specific purpose of detection, for example, less than 10,000, less than 5,000, less than 1,000, less than 500, less than 300, less than 100, less than 50, less than 30, less than 20, less than 10, less than 5, or less than 3. The more than one capture probes can have different nucleotide sequences (e.g., different lengths or different base compositions), and these different capture probes can capture different immune complexes, respectively, or can complementarily hybridize with different positions of the single-stranded DNA of the first nucleotide portion in the same immune complex.

[0055] As used herein, the term "binding" generally refers to an interaction that forms a complex between molecules (e.g., a binding moiety and an analyte, a capture probe and a first adjacent probe, or a blocker and a capture probe). The interaction can be a non-covalent interaction (including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions). In this article, the binding associated with the analyte can be direct binding (i.e., direct interaction between the binding moiety and the analyte) or indirect binding (e.g., the binding moiety and the analyte are indirectly bound together by binding to a linker molecule (e.g., a bispecific antibody); an "analyte binding moiety" associated with the analyte, for example, as used herein, refers to any substance capable of binding to the analyte. In certain embodiments, the analyte binding moiety specifically binds to its corresponding target analyte, i.e., the analyte binding moiety binds to its corresponding target analyte with an affinity greater than that of other components in the sample. In certain embodiments, the binding of the analyte binding moiety to the target analyte can be different from the binding to the non-target analyte, wherein the analyte binding moiety does not bind to the non-target analyte, or binds to the non-target analyte negligibly or undetectably, or any such non-specific binding (if any) is at a relatively low level that can be distinguished. The binding between the target analyte and its corresponding analyte binding moiety is generally non-covalent. In this article, "binding" between nucleotide sequences can be achieved by complementary hybridization.

[0056] In certain embodiments, in the method disclosed herein, a washing step is further included between step 1) and step 2), between step 2) and step 3), and / or between step 3) and step 4); for example, the washing step is 2-4 washes. Suitable washing buffers are known in the art, such as tris (hydroxymethyl) aminomethane (Tris)-based buffers (e.g., Tris-buffered saline, TBS) or phosphate buffered saline (e.g., phosphate-buffered saline, PBS). The washing buffer may also include a detergent, such as an ionic or non-ionic detergent; for example, the washing buffer used in the washing step is: 0.01% to 0.1% Tween 20, 0.5 to 1× PBS, pH 7.0 to 7.5; for example, the washing buffer is: 0.05% Tween 20, 1× PBS, pH 7.0.

[0057] In one embodiment, the method of the present disclosure sequentially contacts a first proximity probe and a second proximity probe with an analyte (i.e., the first proximity probe is first bound to the analyte and then bound to the second proximity probe, thereby forming an immune complex consisting of the first proximity probe, the second proximity probe, and the analyte), and washes after the first proximity probe binds to the analyte and after the second proximity probe binds to the analyte to remove unbound molecules (e.g., capture probes, proximity probes, etc.), thereby reducing nonspecific binding, lowering background signals, and improving detection sensitivity.

[0058] In one embodiment, in step 2) of the method of the present disclosure, the blocking agent may be added before or after the addition of the sample to be tested containing the analyte, or may be added simultaneously with the sample to be tested containing the analyte.

[0059] The blocker in the method disclosed herein (the blocker provided by the present disclosure) can be any compound molecule that can bind to the capture probe to reduce the non-specific binding of the capture probe to other substances outside the immune complex. The compound molecule can, for example, be a small molecule compound, protein, polypeptide, peptide nucleic acid, nucleic acid molecule, etc. that can specifically recognize and bind to the capture probe, or a hybrid thereof. In certain embodiments, the blocker herein can be a compound molecule comprising an oligonucleotide sequence; in certain embodiments, the blocker herein can be an oligonucleotide sequence; on the one hand, the oligonucleotide sequence can be complementary hybridized with the capture probe to form a stable double-stranded structure (e.g., a double-stranded structure with a length of at least 15 nucleotide pairs) to block the capture probe, and on the other hand, after the oligonucleotide sequence is complementary hybridized with the capture probe, the number of continuous nucleotides exposed at the 3' end and / or 5' end of the nucleotide sequence of the capture probe (also referred to as the length of the single-stranded structure) is insufficient to produce non-specific binding with other molecules, thereby achieving the purpose of the present invention: blocking the capture probe, thereby reducing the non-specific binding of the capture probe to other molecules outside the immune complex, thereby reducing the background signal and improving the detection sensitivity. Therefore, those skilled in the art can determine the length of the oligonucleotide sequence and the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the nucleotide sequence of the capture probe after complementary hybridization with the capture probe by conventional experimental means according to the length, nucleotide composition and detection purpose of the specific capture probe used to achieve the purpose of the present invention, without being limited by the embodiments shown in this disclosure for illustrative purposes.

[0060] In the methods provided herein, more than one (e.g., one, two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen, twenty, twenty-five, thirty, fifty, or one hundred or more) blocking agents may be used simultaneously for detection. The upper limit of the number of blocking agents used simultaneously is not particularly limited and can be determined based on the specific purpose of detection, for example, less than 10,000, less than 5,000, less than 1,000, less than 500, less than 300, less than 100, less than 50, less than 30, less than 20, less than 10, less than 5, or less than 3. The one or more blocking agents may be blocking agents that bind to different capture probes or blocking agents that bind to different positions of the same capture probe.

[0061] In certain embodiments, the blocker comprises an oligonucleotide sequence (or the blocker is an oligonucleotide sequence), which comprises or consists of at least 15 consecutive nucleotides that complementarily hybridize to the capture probe, for example, the at least 15 consecutive nucleotides can be at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34 or at least 35 consecutive nucleotides; the number of the consecutive nucleotides that complementarily hybridize to the capture probe can be less than 50, for example, less than 45, less than 40, less than 35, less than 30, less than 25, or less than 20. In certain embodiments, when the blocker is bound to the capture probe, the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the capture probe is 7 or less, for example, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. For example, the blocker complementarily hybridizes with the entire single-stranded nucleotide sequence of the capture probe. In certain embodiments, when the blocker is bound to the capture probe, the total number of consecutive nucleotides exposed at the 3' end and the 5' end of the capture probe is 14 or less, for example, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less.

[0062] As used herein, the term "contiguous nucleotides" generally refers to nucleotides linked sequentially by phosphodiester bonds in a nucleotide sequence. The term "exposed contiguous nucleotides" refers to contiguous nucleotides in a single-stranded form in the capture probe after the blocker and capture probe are paired and bound.

[0063] In certain embodiments, the blocker is an oligonucleotide sequence comprising or consisting of 15-35 consecutive nucleotides that complementarily hybridize to the capture probe, for example, the 15-35 consecutive nucleotides include 16-34, 17-33, 18-32, 19-31, 20-30, 21-29, 22-28, 23-27, 16-32, 16-30, 16-28, 16-26, 17-35, 17-34, or 28-30 consecutive nucleotides.

[0064] In certain embodiments, the oligonucleotide sequence comprises an adenine deoxyribonucleotide A, a guanine deoxyribonucleotide G, a cytosine deoxyribonucleotide C, and a thymine deoxyribonucleotide T; in certain embodiments, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced with uracil deoxyribonucleotides U. In certain embodiments, all thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced with uracil deoxyribonucleotides U.

[0065] In certain embodiments, the 3' end of the oligonucleotide sequence has a modification that blocks the extension of the oligonucleotide sequence, such as 3' end phosphorylation, Spacer C3, amino or dideoxycytidine ddC modification; for example, preferably, the modification is dideoxycytidine ddC modification.

[0066] In certain embodiments, the concentration ratio of the blocker to the capture probe is 1:1 to 5:1, e.g., 1.5:1 to 4.5:1, 2:1 to 4:1, 2.5:1 to 3.5:1, 1:1 to 3:1, 3:1 to 5:1, 2:1 to 5:1, or 2:1 to 3:1.

[0067] In certain embodiments, the solution in step 1) is a binding buffer. In certain embodiments, steps 1), 2), and / or 3) are performed in a binding buffer. The binding buffer can be any suitable buffer known in the art (e.g., SSC buffer, Tris, HEPES, Bis-Tris, PBS, or MOPS). For example, the binding buffer comprises: 0.1% to 0.5% BSA, 0.01% to 0.1% Tween 20, 0.5 to 2×PBS, with a pH of 7.0 to 7.5; for example, the binding buffer comprises: 0.2% bovine serum albumin (BSA), 0.1% Tween 20, 2×PBS (200 mM phosphate, 300 mM NaCl), with a pH of 7.0 to 7.5.

[0068] In step 4) of the disclosed method, the first nucleotide moiety and the second nucleotide moiety interact to produce a nucleic acid reporter molecule. It will be appreciated by those skilled in the art that any nucleic acid reporter molecule production method known in the art (i.e., the mode in which the first nucleotide moiety and the second nucleotide moiety interact) can be used in this step, including ligation, polymerization extension or cooperative hybridization. Adjacent position connection (PLA) and adjacent position extension (PEA) are all known in the art. In this article, the term "nucleic acid reporter molecule" generally refers to a single-stranded nucleotide sequence (e.g., formed by connection) or a double-stranded nucleotide sequence (e.g., formed by complementary hybridization) comprising a portion or all of the nucleotide sequence of the first nucleotide moiety and the portion or all of the nucleotide sequence of the second nucleotide moiety that interacts simultaneously, therefore, a single adjacent probe that is not a part of an immune complex will not produce a reporter molecule, thereby avoiding the background of the binding agent from a single non-specific binding.

[0069] In certain embodiments, in step 4) of the disclosed method, a nucleic acid reporter molecule is produced by adjacent ligation, wherein the first nucleotide portion and the second nucleotide portion are sufficiently adjacent to connect to form a nucleic acid reporter molecule for the next step of detection. In certain embodiments, in step 4) of the disclosed method, the nucleic acid reporter molecule is produced by adjacent extension, wherein the first nucleic acid portion and the second nucleotide portion are sufficiently adjacent to interact with each other to form a double-stranded structure (nucleic acid reporter molecule) for the next step of detection; the 3' end of at least one nucleic acid tag of the double-stranded structure can be extended to produce an extension product. In certain embodiments, in step 4) of the disclosed method, the nucleic acid reporter molecule is produced by cooperative hybridization, wherein the first nucleic acid portion and the second nucleotide portion are sufficiently adjacent to interact with each other to form a hybridization product for the next step of detection.

[0070] In certain embodiments, in step 4), a connecting oligonucleotide and a ligase / polymerase are added to connect the free end of the first nucleotide portion to the free end of the second nucleotide portion to form a template sequence; preferably, the ligase is selected from T4 DNA ligase, T7 DNA ligase, SplintR ligase, and Taq ligase.

[0071] As used herein, the term "linking oligonucleotide" (also referred to as a splint sequence) generally refers to a nucleotide sequence that is complementary to and hybridizes with both the free end nucleotide sequence of the first nucleotide portion and the free end nucleotide sequence of the second nucleotide portion. After the linking oligonucleotide is bound to the first nucleotide portion and the second nucleotide portion, adjacent ligation of the first nucleotide portion and the second nucleotide portion can be achieved under the action of a ligase or a polymerase. For example, the linking oligonucleotide is complementary to the nucleotide sequences on two adjacent probes bound to the same analyte, forming a partially double-stranded DNA structure with a gap, and the gap is filled under the action of a ligase, thereby forming a specific nucleic acid reporter molecule.

[0072] As used herein, the terms "hybridization," "hybridizable," or "complementary," and "complementary hybridization" generally refer to the ability of a nucleic acid (e.g., RNA, DNA) to specifically and non-covalently bind to another nucleic acid sequence under suitable in vitro and / or in vivo conditions of temperature and solution ionic strength (i.e., to form Watson-Crick base pairs and / or G / U base pairs (also referred to as complementary pairing)). Watson-Crick base pairing includes: adenine / adenosine (A) pairing with thymidine / thymine (T), A pairing with uracil / uridine (U), and guanine / guanosine (G) pairing with cytosine / cytidine (C). In certain embodiments, in hybridization between two RNA molecules (e.g., dsRNA), or hybridization between a DNA molecule and an RNA molecule (e.g., when the target recognition sequence of a probe is paired with mRNA, etc.), G may also pair with U bases. Hybridization requires that the two nucleic acids contain complementary sequences, but possible mismatches between bases cannot be ruled out. The conditions applicable to hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the hybrid of nucleic acids having these complementary sequences. In this article, when describing the complementary hybridization of two nucleic acid sequences, it can refer to the complete complementary pairing of the two nucleic acid sequences, or it can refer to the complementary pairing of the full length of one nucleic acid sequence with a portion of the other nucleic acid sequence, or it can refer to the complementary pairing of a portion of one sequence with a portion of the other sequence.

[0073] In certain embodiments, salmon sperm DNA is added to step 1), step 2), step 3) and / or step 4).

[0074] The nucleic acid reporter molecules generated in step 4) can be detected using any existing nucleic acid detection technology, including but not limited to quantitative PCR (qPCR), digital PCR (dPCR), or next-generation sequencing (NGS). In some embodiments, the detection is qualitative. In some embodiments, the detection is quantitative.

[0075] In addition to the common techniques described above, other nucleic acid amplification / detection methods may be used, including but not limited to: rolling circle amplification (RCA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Other detection technologies also include but are not limited to: QuantiGene assay from ThermoFisher (2019), SIMOA assay from Quanterix (Rissin DM (2010)), and SMCxPRO from MilliporeSigma (MilliporeSigma (2019)). <tm>.

[0076] In some embodiments, in step 5), qPCR detection is used, which uses the nucleic acid reporter molecule as a template for detection; in some embodiments, UNG enzyme is added before or during the qPCR detection to degrade the blocker.

[0077] In certain embodiments, an elution step is further included between step 4) and step 5) to separate the immune complex from the solid phase matrix; preferably, the elution step includes adding enzyme-free sterile water and incubating at 70°C-95°C for 10-20 minutes.

[0078] Preferably, in the elution step, water is used for high-temperature elution to break the hydrogen bonds between the double strands formed by the nucleotide sequence of the capture probe and the first proximity probe, thereby separating the immune complex from the solid matrix. The water used is enzyme-free, sterile water without other ingredients to minimize adverse effects on subsequent detection steps.

[0079] On the other hand, the disclosure also provides test kit, and the test kit includes the blocker provided by the disclosure. In some embodiments, the test kit also includes the first ortho probe, the second ortho probe and / or capture probe of the present disclosure. Alternatively, the test kit can also include the reagent required for more than one step in the detection method of the present disclosure, and such reagent can include the binding buffer, salmon sperm DNA for the junction and reaction of the ortho probe and analyte, the washing buffer for the washing step, the connection oligonucleotide, polymerase, ligase etc. for forming nucleic acid reporter molecules, and the reagent (such as qPCR primer sequence, fluorescent probe sequence) for detecting nucleic acid reporter molecules. Alternatively, the test kit can also include solid-phase matrix (such as streptavidin magnetic beads), and the test kit can also include the instructions for the use of the detection method of the present disclosure or the blocker.

[0080] In this document, the terms "comprise" or "comprising" generally mean that the features specifically stated are included, but other elements are not excluded.

[0081] The present disclosure is further described in detail below with reference to the accompanying drawings and examples. The following examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. Experimental methods not specifying specific conditions in the examples were performed according to conventional conditions known in the art or according to the conditions recommended by the manufacturer.

[0082] Example

[0083] Example 1

[0084] Experimental Materials

[0085] Sample: human IL-8 (72aa) (CXCL8), purchased from Wuhan Aimi Technology Co., Ltd.

[0086] Reagents: sulfo-SMCC, DTT, DMSO, TCEP solution (0.5 M), and EDTA disodium salt solution (0.5 M) were purchased from Sigma-Aldrich LLC. IL-8 antibody was purchased from Beijing Aibosheng Biotechnology Co., Ltd.

[0087] Binding buffer: 0.2% (BSA), 0.1% Tween 20, 2×PBS, pH 7.0.

[0088] Washing solution: 0.05% Tween 20, 1× PBS, pH 7.0.

[0089] Oligonucleotide tail sequence of PLA probe L: 5′HS-CATGCTAGTAGACTCGACGCACACTCCTTTCCTACAAGCAATCGGCTACGATCACATAATAGTCAGCAC-3′;

[0090] The oligonucleotide tail sequence of the PLA probe R is: 5'P-TCGTGACAATACGGAGGTCGGCGATACATT-3'SH.

[0091] Bio-CP sequence: 5' biotin-CCGAUUGCUUGUAGGAAAGGAGUGUGCGUCGAGUU-3'ddC.

[0092] Blocker sequence: 5′-AACUCGACGCACACUCCUUUCCUACAAGCAAUCGG-3′ddC.

[0093] CNT sequence: 5′-TTGUCACGAGUGCUGACT-3′ddC.

[0094] Upstream amplification primer sequence: 5'-CATGCTAGTAGACTCGACGCAC-3'.

[0095] Downstream amplification primer sequence: 5'-ACCTCCGTATTGTCACGA-3'.

[0096] Fluorescent probe sequence: 5'CY5-CTACAAGCAATCGGCTACG-3'BHQ2.

[0097] Bio-CP, blockers, CNTs, amplification primers, and fluorescent probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0098] Taq Pro HS U+Probe Master Mix was purchased from Novozymes Biotech Co., Ltd.

[0099] Salmon sperm DNA (10 mg / ml), Tween-20, 10× PBS buffer, and bovine serum albumin (BSA) were purchased from Solebro.

[0100] SA magnetic beads: Streptavidin magnetic microspheres, 1 mg / mL, particle size 0.6 μm-3 μm, purchased from Weidu Biotechnology Co., Ltd.

[0101] Experimental procedures

[0102] a) Preparation of PLA probes: (i) Weigh 0.5 mg of sulfo-SMCC and dissolve it in 20 μL of DMSO; (ii) Replace the storage solution of the IL-8 antibody with PBS using an ultrafiltration tube; (iii) Add 20 times the molar amount of sulfo-SMCC to the IL-8 antibody. After activation at room temperature for 2 hours, purify the activated antibody using a 50 kDa MWCO ultrafiltration tube; (iv) Mix 20 μL of 100 μM oligonucleotide tail, 5 μL of 0.5 M TCEP solution, 1 μL of 0.5 M EDTA disodium salt solution, and 14 μL of water. Incubate at 37°C for 1 hour. Remove excess TCEP using a MicroSpin G50 desalting column. (v) Mix the activated antibody and TCEP-treated oligonucleotide tail, incubate overnight at room temperature, and remove unbound components using a 50 kDa MWCO ultrafiltration tube again. Recover the liquid from the filter cartridge and determine the antibody concentration. (vi) Calculate the antibody molar concentration based on the antibody mass concentration and dilute to 20 fmol / μL to obtain the PLA probe.

[0103] b) Binding of PLA probe L to SA magnetic beads: Add 17 μL of PLA probe L, 3.4 μL of 1 μM Bio-CP, 17 μL of salmon sperm DNA, and 34 μL of SA magnetic beads to a 2 mL protein low-adsorption centrifuge tube. Add binding buffer to make up to 1700 μL. Vortex to mix thoroughly, then aliquot into new 2 mL protein low-adsorption centrifuge tubes, 100 μL per tube (tubes are numbered 1-16). Incubate at 37°C with shaking for 10 minutes. After incubation, place the centrifuge tube on a magnetic rack. After the solution has clarified, discard the solution in the tube, then add 150 μL of wash buffer, vortex to mix thoroughly, centrifuge briefly, return to the magnetic rack, let stand for 1 minute, and discard the wash buffer. Wash three times in total.

[0104] c) Binding of PLA Probe L to Antigen: IL-8 antigen was serially diluted using binding buffer to obtain 1, 10, and 100 pg / mL IL-8 dilutions. The diluted antigen and blocking agent according to Table 1 were added to the corresponding numbered centrifuge tubes from step b), mixed by vortexing, and incubated at 37°C for 30 minutes. After the reaction, the centrifuge tubes were placed on a magnetic rack. After the solution clarified, the solution in the tubes was discarded, and 150 μL of wash buffer was added. The tubes were mixed by vortexing, centrifuged briefly, and returned to the magnetic rack. The tubes were allowed to stand for 1 minute, and the wash buffer was discarded. Washing was performed three times in total.

[0105] Table 1: Antigens and blocking agents

[0106] d) Binding with PLA probe R: Add 17 μL of PLA probe R and 17 μL of salmon sperm DNA to a 2 mL protein low-adsorption centrifuge tube, add binding buffer to make up to 1700 μL, vortex to mix, and add 100 μL to each centrifuge tube that has completed step c), and incubate at 37°C with shaking for 30 minutes; after the reaction is completed, place the centrifuge tube on a magnetic rack, wait for the solution to clarify, discard the solution in the tube, then add 150 μL of wash solution, vortex to mix, centrifuge briefly, return to the magnetic rack, let stand for 1 minute, and discard the wash solution; wash three times in total.

[0107] e) Add 120 μL of wash buffer to the centrifuge tube from step d), vortex to mix, centrifuge briefly, and transfer the entire solution to a 0.2 mL 8-tube strip. Place the strip on a magnetic rack and allow the solution to clear. Discard the solution.

[0108] f) Ligation Reaction: Mix 6.8 μL of 5 U / μL T4 DNA Ligase, 34 μL of 10× T4 Ligase Buffer, 34 μL of 1 μM CNT, and 34 μL of salmon sperm DNA. Add water to a total volume of 340 μL. Vortex to mix thoroughly, then aliquot into the corresponding 8-tube strips in step d), 20 μL per tube. Vortex to mix thoroughly, centrifuge briefly, and place in a PCR instrument. Incubate at 37°C for 10 minutes.

[0109] g) Washing: Place the 8-tube strip on a magnetic rack. Let stand for 2 minutes, then discard the solution in the tube. Then add 120 μL of wash buffer, vortex to mix, centrifuge briefly, and return to the magnetic rack. Wash twice in total.

[0110] h) Elution: Add 20 μL of water to each tube strip, vortex to mix, centrifuge briefly, and place in a PCR instrument. Incubate at 70°C for 10 minutes. Vortex to mix, centrifuge briefly, and place on a magnetic rack. Let stand for 2 minutes. Transfer the supernatant to a new tube strip to use as a template for real-time PCR.

[0111] i) Real-time fluorescence PCR: Mix 5.1 μL of 10 μM upstream amplification primer, 5.1 μL of 10 μM downstream amplification primer, 10.2 μL of 10 μM fluorescent probe, and 170 μL of 2× Taq Pro HS U+Probe Master Mix. Add water to a total volume of 340 μL. Vortex to mix thoroughly, centrifuge briefly, and aliquot into 8 new tube strips, 20 μL per tube. Then, add 10 μL of the template obtained in step h) to each tube. Vortex to mix thoroughly, centrifuge briefly, and perform assays on a 7500 qPCR instrument. The reaction protocol is shown in Table 2. After de-incubation, data were processed using 7500 software V2.3.

[0112] Table 2: Real-time fluorescence PCR detection procedures

[0113] 3) Experimental results

[0114] The detection data and analysis of IL-8 antigen are shown in Table 3 , Figures 2 , and 3 .

[0115] Table 3: Detection data and analysis of IL-8 antigen

[0116] From the above results, it can be seen that after adding the blocking agent to the reaction system, the detection Ct value (background Ct value) of the 0 pg / mL IL-8 antigen sample increased by 2.53, which means that the background signal was reduced by about 5.8 times (2 2.53 ), indicating that the use of blockers has a very significant effect on reducing background signals; in addition, after adding blockers, the ΔCt value (background Ct value - sample Ct value) of the 1 pg / mL IL-8 antigen sample increased by 2.13, further indicating that the use of blockers has a significant effect on improving detection sensitivity.

[0117] Example 2

[0118] 1) Experimental materials: The sequence of Bio-CP is 'biotin-AGGAGUGUGCGUCGA-3'ddC; the sequence of the blocker is 5'-UCGACGCACACUCCU-3'ddC.

[0119] The remaining reagents are the same as those in Example 1.

[0120] 2) Experimental procedures: The same as those in Example 1.

[0121] 3) Experimental results: as shown in Table 4.

[0122] Table 4: Experimental results and analysis of Example 2

[0123] The above results indicate that good detection effects can still be achieved when the nucleotide length of the capture probe Bio-CP and the blocker is 15 nucleotides.

[0124] Example 3

[0125] 1) Experimental Materials: Blocker 1 is the blocker used in Example 2, and its sequence is completely complementary to Bio-CP. Blocker 2 is 12 nucleotides long and has the sequence: 5'-GACGCACACUCC-3'ddC. The remaining reagents are the same as in Example 2.

[0126] 2) Experimental procedures: No blocker was used for samples 1-8, Blocker 1 was used for samples 9-16, and Blocker 2 was used for samples 17-24. The remaining experimental procedures were the same as those in Example 2.

[0127] 3) Experimental results: as shown in Table 5.

[0128] Table 5: Experimental results and analysis of Example 3

[0129] The above results show that Blocker 2 has a very poor blocking effect, and the detection results are very similar to those without the addition of blocker, indicating that Blocker 2 has almost no blocking effect. This indicates that when the blocker length is less than 15 nucleotides, the binding ability of the blocker and the capture probe is significantly reduced, and the blocking ability is also reduced accordingly.

[0130] Example 4

[0131] 1) Experimental Materials: Blocker 1 is the blocker used in Example 1. Blocker 2 has a sequence of 5'-GACGCACACUCCUUUCCUACAAGCAAUC-3'ddC and is 28 nucleotides long. Compared to Blocker 1, it lacks 5 nucleotides at the 5' end and 2 nucleotides at the 3' end. All other reagents are the same as in Example 1.

[0132] 2) Experimental procedures: No blocker was used for samples 1-8, Blocker 1 was used for samples 9-16, and Blocker 2 was used for samples 17-24. The remaining experimental procedures were the same as those in Example 1.

[0133] 3) Experimental results: as shown in Table 6.

[0134] Table 6: Experimental results and analysis of Example 4

[0135] The above results show that the effects of blocker 2 and blocker 1 are similar, which indicates that even if the binding region of the blocker to the capture probe does not completely cover the entire length of the capture probe (a total of 7 nucleotides are exposed at the 5' and 3' ends of the capture probe), there is still a good blocking effect.

[0136] Example 5

[0137] 1) Experimental Materials: Blocker 1 is the blocker used in Example 1. Blocker 2 has a sequence of 5'-GCACACUCCUUUCCUACAAGC-3'ddC and is 21 nucleotides long. Compared to Blocker 1, it lacks 8 nucleotides at the 5' end and 6 nucleotides at the 3' end. Other reagents are the same as in Example 1.

[0138] 2) Experimental procedures: No blocker was used for samples 1-8, Blocker 1 was used for samples 9-16, and Blocker 2 was used for samples 17-24. The remaining experimental procedures were the same as those in Example 1.

[0139] 3) Experimental results: as shown in Table 7.

[0140] Table 7: Experimental results and analysis of Example 5

[0141] The above results show that the blocking effect of blocker 1 is normal, but the blocking effect of blocker 2 is very poor, indicating that when the length of the blocker is too short relative to the length of the capture probe and the number of bases exposed by the capture probe is large (a total of 14 nucleotides are exposed at the 5' and 3' ends of the capture probe), the capture probe will continue to non-specifically bind to other oligonucleotides, and the blocking effect will deteriorate.

[0142] Example 6

[0143] 1) Experimental Materials: Blocker 1 is the blocker used in Example 1. Blocker 2 has the same sequence as Blocker 1, but without ddC modification at the 3' end. The remaining reagents are the same as in Example 1.

[0144] 2) Experimental procedures: No blocker was used for samples 1-8, Blocker 1 was used for samples 9-16, and Blocker 2 was used for samples 17-24. The remaining experimental procedures were the same as those in Example 1.

[0145] 3) Experimental results: as shown in Table 8.

[0146] Table 8: Experimental results and analysis of Example 6

[0147] The experimental results showed that both Blocker 1 and Blocker 2 had good blocking effects, but Blocker 1 had a lower background signal and a larger ΔCt value compared to Blocker 2. This result suggests that phosphorylation at the 3' end of the blocker prevents nonspecific extension of the blocker itself, reducing its impact on the qPCR detection stage, thereby improving the blocking effect of the blocker.

[0148] Example 7

[0149] 1) Experimental Materials: Blocker 1 is the blocker used in Example 1, and the sequence of Blocker 2 is 5'-AACTCGACGCACACTCCTTTCCTACAAGCAATCGG-3'ddC, ie, U in Blocker 1 is replaced by T.

[0150] 2) Experimental procedures: Samples numbered 1 to 8 were treated with Blocker 1, and samples numbered 9 to 16 were treated with Blocker 2. The remaining experimental procedures were the same as those in Example 1.

[0151] 3) Experimental results: as shown in Table 9.

[0152] Table 9: Experimental results and analysis of Example 7

[0153] The experimental results showed that both Blocker 1 and Blocker 2 had good blocking effects, but Blocker 1 had a lower background signal and a larger ΔCt value than Blocker 2. This result indicates that U-containing blockers can be effectively degraded by UNG enzyme during the qPCR detection stage, thus preventing their impact on amplification.

[0154] Example 8

[0155] 1) Experimental Materials:

[0156] Blocker 1: The sequence is 5'-CGCACACUCCUUUCCUACAAGCAAUCGG-3'ddC. Compared with the blocker in Example 1, the 5' end lacks 7 nucleotides.

[0157] Blocker 2: The sequence is 5'-GACGCACACUCCUUUCCUACAAGCAAUC-3'ddC, which is the same as the blocker 2 in Example 4, that is, compared with the blocker in Example 1, it lacks 5 nucleotides at the 5' end and 2 nucleotides at the 3' end.

[0158] Blocker 3: 5′-AACUCGACGCACACUCCUUUCCUACAAG-3′ddC, which lacks 7 nucleotides at the 3′ end compared to the blocker in Example 1.

[0159] The remaining reagents are the same as those in Example 1.

[0160] 2) Experimental procedures: No blocker was used for samples 1-8, blocker 1 was used for samples 9-16, blocker 2 was used for samples 17-24, and blocker 3 was used for samples 25-32. The remaining experimental procedures were the same as those in Example 1.

[0161] 3) Experimental results: as shown in Table 10.

[0162] Table 10: Experimental results and analysis of Example 8

[0163] The experimental results showed that compared with the test group without added blockers, blockers 1-3 all had a good blocking effect, which indicates that when the blocker nucleotide length is shorter than the capture probe, when the capture probe exposes the same number of nucleotides, the blocker can obtain a good blocking effect by binding to any position of the capture probe; further analysis of the blocking effects of blockers 1-3 showed that the blocking effect of blocker 2 was more significant than that of blockers 1 and 3, which indicates that when the blocker nucleotide length is shorter than the capture probe, when the capture probe exposes the same number of nucleotides, the complementary pairing of the blocker sequence and the middle region of the capture probe can prevent the appearance of long continuous exposed bases at both ends of the capture probe, thereby further improving the blocking effect.

[0164] All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated herein by reference in their entirety.

[0165] Although the present disclosure has been illustrated and described with reference to certain preferred embodiments thereof, it should be understood by those skilled in the art that the above description is provided to further illustrate the present disclosure in conjunction with specific embodiments thereof, and that the present disclosure is not limited to these descriptions. Those skilled in the art may make various changes in form and detail, including simple deductions or substitutions, without departing from the spirit and scope of the present disclosure.< / tm>

Claims

1. A detection method, the method comprising the following steps in sequence: 1) contacting a first proximity probe and a capture probe with a solid phase matrix in a solution, wherein the capture probe binds to the first proximity probe and the solid phase matrix, thereby capturing the first proximity probe on the solid phase matrix, thereby obtaining a first binding product, wherein the first proximity probe comprises a first nucleotide portion and a first binding portion for binding to an analyte in a sample to be tested; 2) adding a blocking agent and a sample to be tested containing an analyte, wherein the blocking agent is used to block the capture probe, and the first proximity probe in the first binding product binds to the analyte in the sample to be tested, thereby obtaining a second binding product; 3) adding a second proximity probe so that the second proximity probe binds to the second binding product, wherein the second proximity probe comprises a second nucleotide portion and a second binding moiety for binding to an analyte in the sample to be tested, and the second binding moiety and the first binding moiety bind to the same analyte to form an immune complex; 4) allowing the first nucleotide portion of the first proximity probe and the second nucleotide portion of the second proximity probe to interact to produce a nucleic acid reporter molecule; 5) Detecting the nucleic acid reporter molecule.

2. The method according to claim 1, wherein: The blocker comprises an oligonucleotide sequence, which comprises or consists of at least 15 consecutive nucleotides that complementarily hybridize with the capture probe; preferably, after the blocker binds to the capture probe, the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the capture probe is less than 7, preferably, less than 5.

3. The method according to claim 2, wherein: The blocker is an oligonucleotide sequence, which comprises or consists of 15-35 consecutive nucleotides that complementarily hybridize with the capture probe.

4. The method according to claim 2 or 3, wherein: The oligonucleotide sequence comprises adenine deoxyribonucleotide A, guanine deoxyribonucleotide G, cytosine deoxyribonucleotide C and thymine deoxyribonucleotide T; preferably, one or more thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced by uracil deoxyribonucleotides U.

5. The method according to any one of claims 2 to 4, wherein: The 3' end of the oligonucleotide sequence has a modification that blocks the extension of the oligonucleotide sequence; preferably, the modification is dideoxycytidine ddC modification.

6. The method according to any one of claims 1 to 5, wherein: The concentration ratio of the blocker to the capture probe is 1:1 to 5:

1.

7. The method according to any one of claims 1 to 6, wherein: A washing step is further included between step 1) and step 2), between step 2) and step 3), between step 3) and step 4), and / or between step 4) and step 5); preferably, the washing step is performed 2-4 times; preferably, the washing buffer used in the washing step is: 0.05% Tween 20, 1×PBS, pH 7.

0.

8. The method according to any one of claims 1 to 7, wherein: The analyte comprises a protein.

9. The method according to any one of claims 1 to 8, wherein: The solid phase matrix has a modification group a that binds to the capture probe; preferably, the solid phase matrix is ​​a magnetic bead; preferably, the modification group a is oligo-dT or streptavidin.

10. The method according to any one of claims 1 to 9, wherein: The capture probe is one or more single-stranded DNAs, the capture probe has a modification group b bound to the modification group a, and the capture probe is complementary hybridized with the first nucleotide portion of the first proximity probe; preferably, the modification group b is poly A or biotin; preferably, the capture probe is a single-stranded DNA, and the single-stranded DNA consists of 15-35 nucleotides.

11. The method according to any one of claims 1 to 10, wherein: The first binding part and the second binding part are each a nucleic acid aptamer, an antibody or an antigen-binding fragment of an antibody, the first nucleotide part and the second nucleotide part are each one or more single-stranded DNAs, and the one or more single-stranded DNAs of the first nucleotide part contain a nucleotide sequence that hybridizes complementarily with the capture probe; preferably, the length of the one or more single-stranded DNAs is 30-100 nucleotides; preferably, one end of each of the one or more single-stranded DNAs has a modification group c selected from a thiol, an amino group, and an azide group, and the modification group c reacts with a coupling agent to couple the one or more single-stranded DNAs of the first nucleotide part to the first binding part, and to couple the one or more single-stranded DNAs of the second nucleotide part to the second binding part; preferably, the coupling agent is selected from one or more of EMCS, sulfo-SMCC, Sulfo-LC-SPDP or SMPB; preferably, the first binding part and the second binding part respectively bind to different epitopes of the same analyte.

12. The method according to any one of claims 1 to 11, wherein: The immune complex consists of the first proximity probe, the second proximity probe and the analyte.

13. The method according to any one of claims 1 to 12, wherein: In the step 4), a connecting oligonucleotide and a ligase are added to connect the free end of the first nucleotide part with the free end of the second nucleotide part to form the nucleic acid reporter molecule; preferably, the ligase is selected from T4 DNA ligase, T7 DNA ligase, SplintR ligase, and Taq ligase.

14. The method according to any one of claims 1 to 13, wherein: The detection is performed by qPCR detection; preferably, UNG enzyme is added before or during the qPCR detection to degrade the blocker.

15. The method according to any one of claims 1 to 14, wherein: An elution step is also included between step 4) and step 5) to separate the immune complex from the solid phase matrix; preferably, the elution step includes adding enzyme-free sterile water and incubating at 70°C-95°C for 10-20 minutes.

16. A blocking agent for use in the method of any one of claims 1 to 15, wherein The blocking agent is used to block the capture probe.

17. The blocking agent according to claim 16, wherein The blocker comprises an oligonucleotide sequence, which comprises or consists of at least 15 consecutive nucleotides that complementarily hybridize with the capture probe; preferably, after the blocker binds to the capture probe, the number of consecutive nucleotides exposed at the 3' end and / or 5' end of the capture probe is less than 7, preferably, less than 5.

18. The blocking agent according to claim 17, wherein The blocker is an oligonucleotide sequence, which comprises or consists of 15-35 consecutive nucleotides that complementarily hybridize with the capture probe.

19. The blocking agent according to claim 17 or 18, wherein The oligonucleotide sequence comprises adenine deoxyribonucleotide A, guanine deoxyribonucleotide G, cytosine deoxyribonucleotide C and thymine deoxyribonucleotide T; preferably, one or more thymine deoxyribonucleotides T in the oligonucleotide sequence are replaced by uracil deoxyribonucleotides U.

20. The blocking agent according to any one of claims 17 to 19, wherein The 3' end of the oligonucleotide sequence has a modification that blocks the extension of the oligonucleotide sequence; preferably, the modification is dideoxycytidine ddC modification.

21. A kit comprising the blocking agent according to any one of claims 16 to 20.

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