Multiplex imaging using enzyme-mediated amplification

Abstract

1. A method for imaging an analyte in a sample, comprising: contacting the biological sample with a binding agent, the binding agent comprising a binding moiety that binds to the analyte and a first nucleotide sequence; contacting the biological sample with a catalytic agent, the catalytic agent comprising a second nucleotide sequence linked to an enzyme, the second nucleotide sequence hybridizing to the first nucleotide sequence; contacting the biological sample with a localizing agent, the localizing agent comprising a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate; and contacting the biological sample with a labeling agent, the labeling agent comprising a fourth nucleotide sequence linked to an optical label, the fourth nucleotide sequence hybridizing to the third nucleotide sequence.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 908,540, filed on September 30, 2019, the entire content of which is incorporated herein by reference.

Background Art

[0002] Antibodies were first used in tissue section analysis in 1942 to visualize pneumococcal antigens in organ biopsies obtained from mice injected with live bacteria. Since then, immunohistochemistry has become a mainstay of clinical diagnosis and basic research.

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Summary of the Invention

Problems to be Solved by the Invention

[0005] However, conventional immunohistochemistry (IHC) methods do not necessarily provide signals suitable for detection, especially with respect to immunological targets that are weakly expressed or not efficiently targeted by existing IHC reagents.

Means for Solving the Problems

[0006] The present disclosure features a method, a substance composition, and a kit for performing immunohistochemistry on a biological sample using tyramide signal amplification, which enhances the detection of a target using a chromogenic or fluorescent dye and can remove the dye after detection. Multiplex detection can be performed at a high multiplex level through successive imaging rounds, where multiple dyes corresponding to multiple targets can be detected in each imaging round, and the amplification can be used for some or all of the targets.

[0007] In some embodiments, the method includes enzyme-mediated deposition of oligonucleotide sequences into a sample using TSA technique. The enzyme moiety linked to each antibody molecule can catalyze the deposition of multiple oligonucleotide sequence molecules into a sample, thus achieving amplification (i.e., a ratio of the amount of dye molecules to the amount of target molecules that is greater than 1:1). This process can be repeated for multiple antibodies, resulting in the deposition of multiple oligonucleotide sequences into a sample, which are amplified by TSA.

[0008] One or more dyes labeled with countersense oligonucleotide sequences are introduced which hybridize to corresponding (i.e., complementary) TSA-deposited oligonucleotide sequences and are then detected, after which the oligonucleotide-labeled dyes can be dehybridized and removed from the sample. One or more additional rounds of detection can optionally be performed.

[0009] In some embodiments, the sample is incubated with multiple primary antibodies, each of which targets a different analyte of interest and localizes to a site in the sample that corresponds to its target analyte. Each different type of antibody is assigned to a set of N sequences, S={S1, S2...S N A unique oligonucleotide sequence S derived from i where the group S is orthogonal, i.e., a given S i Countersense sequence of S i ' is substantially the same as S under stringent conditions. i hybridizes only with i and is not j j In the context of a group of sequences S, "substantially" means that the sequence S i ' and S i Array S other than j The total amount of cross-links with the sequence S i 'Array of S i This means that the binding is less than 1% of the amount of binding to the antibody.

[0010] Typically, during antibody incubation in immunohistochemistry, blocking and washing steps can be performed to minimize non-specific binding during incubation and then remove excess antibody. Subsequently, a fixation step may be performed to firmly link the primary antibody to the sample and reduce the possibility of the antibody being removed during subsequent steps.

[0011] An enzyme, such as horseradish peroxidase (HRP), is conjugated to the counter-sense sequence S i ' and applied to the sample, where the counter-sense sequence S i ' hybridizes to the corresponding sequence S i that is linked to the primary antibody by binding in at least the first binding region. Cross-hybridization with other sequences and / or other positions in the sample can be minimized using stringent or nearly stringent conditions. Since the primary antibody is conjugated to different sequences S i drawn from an orthogonal group S, there is little or no enzyme localized to the primary antibody that specifically binds to other target analytes.

[0012] Alternatively, the localizing agent is an oligonucleotide sequence S kIt includes. In some preferred embodiments, the substrate can be a tyramine compound (i.e., a tyramine-containing compound) that is a compound containing a tyramine derivative, p-hydroxy-cinnamic acid, or a derivative of p-hydroxy-cinnamic acid. Suitable derivatives of tyramine include those having one or more (e.g., two or more, three or more) substituents on the amine group, such as one or more alkyl, alkenyl, alkynl, hydroxyl, halide, and / or alkoxy groups, but are not limited thereto. Suitable derivatives of p-hydroxy-cinnamic acid include, but are not limited to, those described in Taofiq et al., Molecules 22(2): 281 (2017), the entire content of which is incorporated by reference.

[0013] In some embodiments, the oligonucleotide sequence is the same as that on the associating primary antibody (k = i). In other embodiments, it is a different sequence (k ≠ i) and is also different from the sequence conjugated to any other primary antibody.

[0014] The enzyme substrate labeled with the oligonucleotide is then applied to the sample, and through tyramide signal amplification, near the target antibody, deposition of oligonucleotide molecules having sequence S k occurs on the sample. A dehybridization step is performed to release the enzyme (e.g., HRP) conjugated with the oligonucleotide from its associating antibody, and the enzyme is removed by one or more washing steps.

[0015] The foregoing procedure corresponds to one round of amplified oligonucleotide deposition, and in the vicinity of each primary antibody, several oligonucleotide molecules of sequence S k covalently bound to the sample are generated. The average number of such molecules is the degree of amplification obtained through the TSA mechanism.

[0016] The amplified deposition can be performed on a plurality of antibody species, and at the positions of the samples corresponding to the plurality of primary antibodies, depositions of different S k types of oligonucleotide sequences occur. The number of rounds of oligonucleotide deposition amplified by TSA may be one if the amplification is sought only for one target in the sample, or may be some number M less than the number N of primary antibodies if the amplification is desired for a subset of the markers, or may be N times if the amplification is sought for all the markers.

[0017] Detection can be performed by introducing a dye labeled with one or more oligonucleotides, each oligonucleotide having the sequence S k '. The dye molecule conjugated to each such oligonucleotide hybridizes with the oligonucleotide sequence deposited by TSA having the corresponding sequence S k by binding at least in the first binding region. Stringent or nearly stringent conditions may be imposed, along with a washing step to remove dye molecules conjugated to excess oligonucleotides, to minimize the binding of dye to other sites in the sample.

[0018] The sample is then imaged with a fluorescence microscope if a fluorescent dye is used, or with a bright-field microscope if a chromogenic dye is used. This constitutes one round of detection. Thereafter, a denaturation step is performed to release the dye labeled with oligonucleotides, and they can be removed by one or more washing steps.

[0019] In some embodiments, two or more dyes (e.g., three or more, four or more, five or more, six or more, eight or more, ten or more, or even more dyes) are imaged in each detection round, and using a multi-channel fluorescence microscope, each dye is detected individually. In certain embodiments, up to six or more dyes are imaged using spectral imaging and deconvolution techniques. Generally, the number B of dyes that can be imaged in one round depends on the capabilities of the microscope and the analytical techniques used to interpret the image.

[0020] Counterstains such as DAPI may be imaged once or during each imaging cycle, which can be used to register images of dyes labeled with oligonucleotides obtained from successive imaging rounds to form an overall multiplexed image of the sample in which images obtained from all rounds are spatially registered simultaneously.

[0021] Thus, highly multiplexed images can be obtained. The total number N of targets that can be imaged is not limited to the number of antibodies labeled with orthogonal oligonucleotide sequences, the number B of dyes that can be imaged in one round, or the number M of TSA-based oligonucleotide deposition rounds.

[0022] The use of amplification through deposition of oligonucleotides catalyzed by HRP can be combined with non-amplified detection. A particular workflow can include antibodies labeled with several oligonucleotides, which hybridize with HRP labeled with an oligonucleotide and catalyze the deposition of multiple oligonucleotides onto the sample through a TSA reaction, and are detected through a dye labeled with an oligonucleotide that binds to these deposited oligonucleotides, while antibodies labeled with other oligonucleotides are detected by hybridizing with a dye labeled with an oligonucleotide. Thus, the workflow may incorporate amplification for the detection of some antibodies and not for others.

[0023] In some embodiments, an enzyme such as HRP can be localized to the antibody site by other means, e.g., by indirect labeling for depositing oligonucleotides onto the sample. The primary antibody can bind to a specific target analyte in the sample as described above, and then an enzyme conjugated to a binding entity such as a secondary antibody or nanobody that binds to the primary antibody can be introduced, such that the enzyme will be indirectly linked to the primary antibody.

[0024] As one example, a primary antibody, which is the E1L3N clone for PDL-1, can be incubated with the sample to localize it to the PDL-1 site of the sample, and a secondary antibody consisting of Leica Power Vision Poly HRP can be localized to the site of the primary antibody and catalyze the deposition of an oligonucleotide having sequence S k onto the sample adjacent to the antibody position. Stripping techniques such as elution using a citric acid antigen retrieval solution can be used to remove the primary and secondary antibodies and leave the deposited oligonucleotides there. This can be repeated with other antibodies and other selected S kRepeatedly using it can result in the deposition of multiple oligonucleotides corresponding to multiple targets amplified by HRP, which can be detected using dyes labeled with oligos as described above.

[0025] The dyes used in each labeling and imaging cycle may be the same or different, and the same number or different numbers of dyes may be used and imaged in each cycle. The goal is typically to image all the targets of interest, but the specific dyes used and the manner in which they are grouped into rounds can be selected according to the specific attributes of the specimen, sample, and workflow conditions. For example, in one imaging round, a relatively short exposure time may be selected to image some targets with a high density of oligonucleotides deposited by TSA, and in another imaging round, a relatively short exposure time may be selected to image other targets where fewer oligonucleotides deposited by TSA are present. Grouping the targets in the selected manner can be beneficial in terms of practical factors such as exposure time, image registration, and other factors specific to a particular workflow.

[0026] Biological samples can be selected from the group consisting of biological tissues, cultured cells, and cells collected from the animal subject of interest. In some embodiments, the biological sample contains materials of human or mouse origin. In some embodiments, the biological sample may be fresh, frozen, or fixed. In some embodiments, it may be a section or core obtained from a formalin-fixed paraffin-embedded (FFPE) tissue block. The sample can include materials derived from tissue sections, tissue microarrays (TMAs), cell pellets, core biopsies, needle biopsies, or cells obtained from blood or plasma samples.

[0027] In some embodiments, the biological sample is immobilized on a surface such as a slide, plate, well, or film.

[0028] In some embodiments, the primary and / or secondary antibody or antibody fragment includes IgG, IgM, monoclonal antibody, scFv, nanobody, Fab, or diabody. In some embodiments, the antibody or antibody fragment is specific for an element of the sample, such as a protein, or another antibody or antibody fragment (e.g., indirect linkage).

[0029] In some embodiments, the oligonucleotide sequence S conjugated to the antibody i comprises a plurality of ribonucleic acids. In some embodiments, it comprises a plurality of deoxyribonucleic acids. In some embodiments, S i oligonucleotide has a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some embodiments, S i oligonucleotide has a length of 10 - 30, 10 - 50, 10 - 70, 10 - 100, 20 - 50, 20 - 70, 20 - 100, 30 - 50, 30 - 70, 30 - 100, 40 - 70, 40 - 100, 50 - 70, 50 - 100, 60 - 70, 60 - 80, 60 - 90, or 60 - 100 nucleotides.

[0030] In some embodiments, S iThe oligonucleotide has a length of no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides.

[0031] In some embodiments, S i The oligonucleotide contains one or more synthetic nucleotides. In some embodiments, S i The oligonucleotide is single-stranded as a whole. In some embodiments, S i The oligonucleotide is partially double-stranded.

[0032] In some embodiments, the said S i The first binding region of the oligonucleotide is complementary to at least a part of the first oligonucleotide S i . In some embodiments, S i The first binding region of the oligonucleotide has a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some embodiments, S i The first binding region of the oligonucleotide has a length of 10 - 30, 10 - 50, 10 - 70, 10 - 100, 20 - 50, 20 - 70, 20 - 100, 30 - 50, 30 - 70, 30 - 100, 40 - 70, 40 - 100, 50 - 70, 50 - 100, 60 - 70, 60 - 80, 60 - 90, or 60 - 100 nucleotides.

[0033] In some embodiments, S i 'The binding region of the oligonucleotide is no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides in length.

[0034] In some embodiments, S i 'The binding region of the oligonucleotide contains one or more synthetic nucleotides. In some embodiments, S i 'The oligonucleotide contains multiple ribonucleic acids. In some embodiments, S i 'The oligonucleotide contains multiple deoxyribonucleic acids.

[0035] In some embodiments, S iThe oligonucleotide has a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some embodiments, the second oligonucleotide has a length of 10 - 30, 10 - 50, 10 - 70, 10 - 100, 20 - 50, 20 - 70, 20 - 100, 30 - 50, 30 - 70, 30 - 100, 40 - 70, 40 - 100, 50 - 70, 50 - 100, 60 - 70, 60 - 80, 60 - 90, or 60 - 100 nucleotides. In some embodiments, the second oligonucleotide has a length not exceeding 5, not exceeding 10, not exceeding 15, not exceeding 20, not exceeding 25, not exceeding 30, not exceeding 35, not exceeding 40, not exceeding 45, not exceeding 50, not exceeding 55, not exceeding 60, not exceeding 65, not exceeding 70, not exceeding 75, not exceeding 80, not exceeding 85, not exceeding 90, not exceeding 95, or not exceeding 100 nucleotides.

[0036] In some embodiments, S i The oligonucleotide contains one or more synthetic nucleotides. In some embodiments, S i The oligonucleotide is single-stranded as a whole. In some embodiments, S i The oligonucleotide is partially double-stranded.

[0037] In some embodiments, the enzyme is horseradish peroxidase (HRP). In some embodiments, the enzyme can be a hemin-containing complex that mimics HRP, for example, hemin. In some embodiments, the enzyme can be soybean peroxidase.

[0038] In some embodiments, the oligonucleotide sequence S conjugated to the substrate material k comprises a plurality of ribonucleic acids. In some embodiments, S k comprises a plurality of deoxyribonucleic acids. In some embodiments, S k The oligonucleotide has a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some embodiments, S k The oligonucleotide has a length of 10 - 30, 10 - 50, 10 - 70, 10 - 100, 20 - 50, 20 - 70, 20 - 100, 30 - 50, 30 - 70, 30 - 100, 40 - 70, 40 - 100, 50 - 70, 50 - 100, 60 - 70, 60 - 80, 60 - 90, or 60 - 100 nucleotides.

[0039] In some embodiments, S k The oligonucleotide has a length of not more than 5, not more than 10, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, not more than 45, not more than 50, not more than 55, not more than 60, not more than 65, not more than 70, not more than 75, not more than 80, not more than 85, not more than 90, not more than 95, or not more than 100 nucleotides.

[0040] In some embodiments, S k The oligonucleotide comprises one or more synthetic nucleotides. In some embodiments, S kThe oligonucleotide is single-stranded as a whole. In some embodiments, S k The oligonucleotide is partially double-stranded.

[0041] In some embodiments, S k The binding region of the 'oligonucleotide is complementary to at least a portion of the first oligonucleotide S k In some embodiments, S k The binding region of the 'oligonucleotide is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides in length. In some embodiments, S k The first binding region of the 'oligonucleotide is 10 to 30, 10 to 50, 10 to 70, 10 to 100, 20 to 50, 20 to 70, 20 to 100, 30 to 50, 30 to 70, 30 to 100, 40 to 70, 40 to 100, 50 to 70, 50 to 100, 60 to 70, 60 to 80, 60 to 90, or 60 to 100 nucleotides in length.

[0042] In some embodiments, S k The binding region of the 'oligonucleotide is no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides in length.

[0043] In some embodiments, the said Sk 'The binding region of the oligonucleotide contains one or more synthetic nucleotides. In some embodiments, S k 'The oligonucleotide contains multiple ribonucleic acids. In some embodiments, S k 'The oligonucleotide contains multiple deoxyribonucleic acids.

[0044] In some embodiments, S k 'The oligonucleotide has a length of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides. In some embodiments, the second oligonucleotide has a length of 10 - 30, 10 - 50, 10 - 70, 10 - 100, 20 - 50, 20 - 70, 20 - 100, 30 - 50, 30 - 70, 30 - 100, 40 - 70, 40 - 100, 50 - 70, 50 - 100, 60 - 70, 60 - 80, 60 - 90, or 60 - 100 nucleotides. In some embodiments, S k 'The oligonucleotide has a length of not more than 5, not more than 10, not more than 15, not more than 20, not more than 25, not more than 30, not more than 35, not more than 40, not more than 45, not more than 50, not more than 55, not more than 60, not more than 65, not more than 70, not more than 75, not more than 80, not more than 85, not more than 90, not more than 95, or not more than 100 nucleotides.

[0045] In some embodiments, one or more compounds are introduced to control or modify the TSA reaction. Examples of such compounds and TSA workflows are described, for example, in U.S. Patent Nos. 6,372,937 and 6,828,109, and U.S. Patent Application Publication Nos. 2017 / 0226572 and 2005 / 0003462, the entire contents of each of which are incorporated herein by reference.

[0046] In some embodiments, the dye conjugated to the oligonucleotide is a fluorophore, a stain, a quantum dot, or a chromogenic compound. Suitable dyes include, but are not limited to, R6G, DCC, Texas Red, FITC, Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 750, Cy3, Cy 3.5, Cy5, Cy 5.5, Cy7, coumarin, rhodamine, and fluorescent and chromogenic species that are substitution variants of these species.

[0047] In some embodiments, all amplification steps are performed first, and then one or more detection rounds are performed through hybridization with the dye labeled with the oligo, imaging, and optionally removal of the dye labeled with the oligo. In some embodiments, amplification is performed on a sample located elsewhere than on a microscope, which may improve the utilization of the device or may be for the use of specialized processing equipment such as an automated staining device for the amplification step. In other embodiments, amplification is performed on a sample on a microscope. This enables a fully automated workflow that does not require robotic or human intervention to move the sample from one device or processing station to another throughout the entire amplification and detection process.

[0048] In other embodiments, the amplification and detection steps are interleaved. In some embodiments, the steps described herein can be repeated.

[0049] In one aspect, the present disclosure is a method for imaging an analyte in a biological sample, the method comprising: contacting the biological sample with a binding agent, wherein the binding agent is characterized by a binding moiety that binds to the analyte and a first nucleotide sequence; contacting the biological sample with a catalytic agent, wherein the catalytic agent is characterized by a second nucleotide sequence linked to an enzyme, and the second nucleotide sequence hybridizes to the first nucleotide sequence; contacting the biological sample with a localization agent, wherein the localization agent is characterized by a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate; contacting the biological sample with a labeling agent, wherein the labeling agent is characterized by a fourth nucleotide sequence linked to an optical label, and the fourth nucleotide sequence hybridizes to the third nucleotide sequence; exposing the biological sample to illumination light, detecting light emitted from the biological sample, and forming an image of the biological sample in which the position of the analyte is indicated by the optical label.

[0050] In another aspect, the present disclosure is a method for imaging a plurality of analytes in a biological sample, the method comprising: (a) contacting the biological sample with a binding agent characterized by a binding moiety that selectively binds to one of the analytes and a first nucleotide sequence; (b) contacting the biological sample with a catalytic agent, the catalytic agent being characterized by a second nucleotide sequence linked to an enzyme, the second nucleotide sequence hybridizing to the first nucleotide sequence; (c) contacting the biological sample with a localization agent, the localization agent being characterized by a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate, depositing the third nucleotide sequence adjacent to one of the analytes; repeating steps (a)-(c) to deposit N different third nucleotide sequences in the biological sample such that each of the third nucleotide sequences is selectively positioned adjacent to a different one of the analytes; contacting the biological sample with M different labeling agents, each labeling agent being characterized by a fourth nucleotide sequence linked to a different optical label, the fourth nucleotide sequence hybridizing to only one of the third nucleotide sequences; and obtaining an image of the sample in which the position of one of the analytes is indicated by the position of one of the optical labels.

[0051] In a further aspect, the present disclosure is a method for imaging an analyte in a biological sample, the method comprising: linking an enzyme to the analyte such that the enzyme localizes to the position of the analyte in the biological sample; contacting the biological sample with a localization agent characterized by a substrate complementary to the enzyme and a first nucleotide sequence, depositing the first nucleotide sequence adjacent to the position of the analyte in the biological sample; contacting the biological sample with a labeling agent characterized by a second nucleotide sequence that hybridizes to the first nucleotide sequence and an optical label; and obtaining an image of the biological sample in which the position of the analyte is represented by the position of the optical label.

[0052] Any embodiment of the present method may include any of the following features.

[0053] The binding moiety may include an antibody, an antibody fragment, or an antibody analog. The antibody, antibody fragment, or antibody analog may include a member selected from the group consisting of IgG antibodies, IgM antibodies, monoclonal antibodies, single-chain variable fragments, and diabodies.

[0054] The first nucleotide sequence may include at least 5 nucleotides (e.g., at least 50 nucleotides). The first nucleotide sequence may include a DNA fragment. The first nucleotide sequence may include an RNA fragment. The first nucleotide sequence may include at least one synthetic nucleotide. The first nucleotide sequence may be a single-stranded sequence. The first nucleotide sequence may be at least partially double-stranded.

[0055] The second nucleotide sequence may be at least 80% complementary to the first nucleotide sequence. As used herein, the percentage of complementarity between two nucleotide sequences refers to the percentage of complementary bases between the binding regions of the two sequences. To achieve a reproducible binding, the second sequence may be at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%) complementary to the first sequence.

[0056] The second nucleotide sequence may include at least 5 nucleotides. The first and second nucleotide sequences may include different numbers of nucleotides.

[0057] The enzyme may include horseradish peroxidase or a derivative thereof. The enzyme may include a compound that mimics horseradish peroxidase. The compound may include a hemin-containing complex. The compound may include hemin. The enzyme may include soybean peroxidase.

[0058] The third nucleotide sequence may be the same as the first nucleotide sequence. The third nucleotide sequence may be different from the first nucleotide sequence. The third nucleotide sequence may contain at least 5 nucleotides (e.g., at least 50 nucleotides). The third nucleotide sequence may contain a DNA fragment. The third nucleotide sequence may contain an RNA fragment. The third nucleotide sequence may contain at least one synthetic nucleotide. The third nucleotide sequence may be a single-stranded sequence. The third nucleotide sequence may be at least partially double-stranded.

[0059] The fourth nucleotide sequence may be at least 80% complementary to the third nucleotide sequence. The fourth nucleotide sequence may contain at least 5 nucleotides. The third and fourth nucleotide sequences may contain different numbers of nucleotides.

[0060] The optical label may contain a fluorescent species. The optical label may contain a chromogenic stain.

[0061] The biological sample may be a tissue sample. The tissue sample may be a fresh tissue sample, a frozen tissue sample, or a formalin-fixed paraffin-embedded (FFPE) tissue sample.

[0062] The specimen may contain a protein. The specimen may contain a peptide or a peptide fragment.

[0063] The method may include a step of contacting the biological sample with a contrast stain before exposing the biological sample to illumination light, and a step of exposing the biological sample to illumination light, detecting the light emitted from the biological sample, and forming a second image of the sample indicating the position of the contrast stain in the biological sample. The contrast stain may contain DAPI.

[0064] The ratio of the amount of the fourth nucleotide sequence to the amount of the first nucleotide sequence in the biological sample may be greater than 1 (e.g., greater than 5, greater than 50).

[0065] The binder can be a first binder, the catalytic agent can be a first catalytic agent, the localizing agent can be a first localizing agent, the labeling agent can be a first labeling agent, the analyte can be a first analyte, the binding moiety can be a first binding moiety, and the method can include a step of contacting a biological sample with a second binder, wherein the second binder is characterized by a second binding moiety that binds to a second analyte in the biological sample and a fifth nucleotide sequence. The second analyte can be different from the first analyte. The second analyte can include a protein, a peptide, or a peptide fragment. The method can include a step of contacting the biological sample with the first and second binders simultaneously. The method can include a step of contacting the biological sample with the first and second binders sequentially.

[0066] The catalytic agent can be a first catalytic agent, the enzyme can be a first enzyme, the localizing agent can be a first localizing agent, the substrate can be a first substrate, the labeling agent can be a first labeling agent, the optical label can be a first optical label, and the method can include a step of contacting a biological sample with a second catalytic agent, wherein the second catalytic agent is characterized by a sixth nucleotide sequence linked to a second enzyme, and the sixth nucleotide sequence hybridizes to the fifth nucleotide sequence, a step of contacting the biological sample with a second binder, wherein the second binder is characterized by a second substrate complementary to the second enzyme and a seventh nucleotide sequence linked to the second substrate, and a step of contacting the biological sample with a second labeling agent, wherein the second labeling agent is characterized by an eighth nucleotide sequence linked to a second optical label, and the eighth nucleotide sequence hybridizes to the seventh nucleotide sequence. The first and fifth nucleotide sequences may be the same. The first and fifth nucleotide sequences may be different. The second and sixth nucleotide sequences may be the same or different. The third and seventh nucleotide sequences may be different. The fourth and eighth nucleotide sequences may be different.

[0067] The first and second optical labels may be different. The second binding moiety may include an antibody, an antibody fragment, or an antibody analog. The antibody, antibody fragment, or antibody analog may include a member selected from the group consisting of IgG antibodies, IgM antibodies, monoclonal antibodies, single-chain variable fragments, and diabodies. The fifth nucleotide sequence may include at least 5 nucleotides. The fifth nucleotide sequence may include at least one member selected from the group consisting of DNA fragments and RNA fragments. The fifth nucleotide sequence may include at least one synthetic nucleotide. The fifth nucleotide sequence may be a single-stranded sequence. The fifth nucleotide sequence may be at least partially double-stranded.

[0068] The sixth nucleotide sequence may be at least 80% complementary to the fifth nucleotide sequence. The sixth nucleotide sequence may include at least 5 nucleotides. The fifth and sixth nucleotide sequences may include different numbers of nucleotides.

[0069] The first and second enzymes may be different. The second enzyme may include horseradish peroxidase, a derivative of horseradish peroxidase, a compound mimicking horseradish peroxidase, a hemin-containing complex, and hematin. The second enzyme may include soybean peroxidase. The seventh nucleotide sequence may be the same as the fifth nucleotide sequence. The seventh nucleotide sequence may be different from the fifth nucleotide sequence. The seventh nucleotide sequence may include at least 5 nucleotides. The seventh nucleotide sequence may include at least one member selected from the group consisting of DNA fragments and RNA fragments. The seventh nucleotide sequence may include at least one synthetic nucleotide. The seventh nucleotide sequence may be a single-stranded sequence. The seventh nucleotide sequence may be at least partially double-stranded.

[0070] The eighth nucleotide sequence can be at least 80% complementary to the seventh nucleotide sequence. The eighth nucleotide sequence can include at least 5 nucleotides. The seventh and eighth nucleotide sequences can include different numbers of nucleotides.

[0071] The second optical label can include a fluorescent species. The second optical label can include a chromogenic dye.

[0072] The ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample can be greater than 1 (e.g., can be greater than 50). The ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample can be different from the ratio of the amount of the fourth nucleotide sequence to the amount of the first nucleotide sequence in the biological sample. The ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample may not be greater than 1. The ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample can be greater than 1, and the ratio of the amount of the fourth nucleotide sequence to the amount of the first nucleotide sequence in the biological sample can be greater than 1.

[0073] The method can include a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second labeling agent. The method can include a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second localization agent. The method can include a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second catalytic agent. The method can include a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second binding agent. The method can include a step of removing the first labeling agent from the biological sample by dehybridizing the fourth nucleotide sequence from the third nucleotide sequence.

[0074] The image of the biological sample can be the first image, and the method can include exposing the biological sample to illumination light, detecting the light emitted from the biological sample, and forming a second image of the biological sample in which the position of the second analyte is indicated by a second optical label. The first optical label can be present in the biological sample when exposing the biological sample to illumination light to form a second image of the biological sample.

[0075] The method can include removing a first optical label from the biological sample before exposing the biological sample to illumination light to form a second image of the biological sample. The step of removing the first optical label from the biological sample can include dehybridizing a fourth nucleotide sequence from a third nucleotide sequence.

[0076] The method is a step of contacting the biological sample with a second catalyst agent after contacting the biological sample with a localizing agent and before contacting the biological sample with a labeling agent, wherein the second catalyst agent is characterized by a fifth nucleotide sequence linked to a second enzyme, and the fifth nucleotide sequence hybridizes to the third nucleotide sequence, and a step of contacting the biological sample with a second localizing agent, wherein the second localizing agent is characterized by a second substrate complementary to the second enzyme and a sixth nucleotide sequence linked to the second substrate, and the fourth nucleotide sequence of the labeling agent hybridizes to the sixth nucleotide sequence. The fifth nucleotide sequence and the first nucleotide sequence may be the same. The sixth nucleotide sequence and the fourth nucleotide sequence may be the same. The second enzyme can be selected from the group consisting of horseradish peroxidase and its derivatives, hemin-containing complexes, hemin, and soybean peroxidase.

[0077] Embodiments of the method can also include any of the other features described herein, including combinations of features described in connection with different embodiments, unless explicitly stated otherwise.

[0078] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0079]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

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Figure 2C

Figure 2D

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DETAILED DESCRIPTION OF THE INVENTION

[0080] Similar elements within the figures are labeled with common reference numerals.

[0081] Overview Immunofluorescence techniques can be used to observe multiple antigen targets in a single sample, for example, to visualize or measure the expression of several proteins, peptides, or other amino acid-containing targets in a given cell or tissue section. This so-called multiplex immunofluorescence method can be performed in several ways. For example, one technique involves contacting the sample with several directly labeled primary antibodies, where each primary antibody can target the antigen of interest and can be conjugated to a different fluorescent dye. In such a method, the antibodies can be applied in a single step, however, the dyes are distinguishable from each other during imaging, and thus, the number of antigen targets, i.e., the degree of multiplexing, is limited by the number of dyes that can be resolved. Also, since there is no amplification mechanism, the number of dye molecules per antigen is set by the dye-antibody conjugation and can be relatively low.

[0082] Indirect labeling can be used to obtain brighter signals. In that technique, secondary antibodies of different species can bind to various primary antibodies, and a fluorescent dye can bind to the secondary antibody. This can result in the possibility of amplification through secondary binding at multiple sites, which can be a more complex approach than direct labeling as it may require that each primary antibody is raised in a different species or alternatively that the secondary antibody can recognize different antibodies derived from the same species.

[0083] A continuous staining technique has been developed that allows a sample to be contacted with a single primary antibody targeting a first antigen. A secondary antibody conjugated to horseradish peroxidase (HRP) is introduced, which can localize to the primary antibody site. Tyramide signal amplification (TSA) can be used to deposit dye molecules near these sites through a reaction catalyzed by HRP. The TSA reaction can result in relatively high amplification. After dye deposition, the primary and secondary antibodies can be stripped or denatured, but most of the dye remains bound to the sample. This process can be repeated multiple times, with different primary antibodies targeting different antigens to deposit different dyes being used in each round. Once the dyes are deposited, the sample is imaged.

[0084] Since this is a sequential approach where only one primary antibody is targeted at a time, there are no cross-reactivity issues. This is an excellent practical advantage as antibodies can be selected without issues related to the animal species in which they were produced. However, because the dyes remain bound to the sample for extended periods, the degree of multiplexing is limited by the number of dyes that can be reliably distinguished from each other in a single imaging round.

[0085] In some embodiments, multiple primary antibodies can be conjugated to oligomers. For example, several antibodies each targeting a desired antigen can be used, where each can be conjugated to a different oligomer. The oligomer sequences can be selected or engineered such that cross-hybridization between different oligomers is low. A sample can be contacted with the primary antibodies, which can localize to antigen sites depending on their type. A fluorescent dye or other detection moiety, such as a quantum dot, can be conjugated to an oligomer sequence tailored for use with the various primary antibodies. One or more of these can be contacted with the sample under conditions that can facilitate hybridization of the oligomer linked to the detection moiety to its conjugate linked to the antibody. In this way, the detection moiety can localize to the antigen sites of the associating antibodies, and the sample can be imaged. As used herein, this is referred to as non-amplified oligomer-mediated detection. Aspects of such methods are described, for example, in U.S. Patent Nos. 9,909,167 and 10,370,698, the entire contents of which are incorporated by reference.

[0086] The oligonucleotide linked to the detection moiety can create conditions favorable for dehybridization and can be removed by performing a washing step. Many primary antibodies each having a different oligonucleotide sequence can be used in a single experiment, and the oligomers linked to the corresponding detection moieties are hybridized, imaged, and removed in groups. In some workflows, the imaging step can only distinguish some of the dyes present within any one group, but overall measurements can achieve a high multiplexing level through repetition.

[0087] Labeling and Imaging of Samples The present disclosure features a method for labeling and imaging a sample, involving enzyme-mediated amplification of a signal corresponding to a specific target analyte in the sample. In some embodiments, the method comprises contacting a biological sample with an antibody or antibody fragment conjugated to a first oligonucleotide S i and contacting the first oligonucleotide with a binding region of a second oligonucleotide S i ', wherein the binding region of the second oligonucleotide S i ' is complementary to at least a portion of the first oligonucleotide S i , and the second oligonucleotide S i ' is conjugated to an enzyme, such that the enzyme mediates deposition of a substance onto the biological sample through a TSA reaction. This substance is itself complementary to at least a portion of a fourth oligonucleotide S k ' conjugated to a dye that is imaged using a microscope or similar device, and is a third oligonucleotide S k .

[0088] In this context, the subscript i in S i represents its use in relation to the first oligonucleotide sequence and a selected target in the imaged sample. The notation S i ' indicates a second oligonucleotide sequence that is complementary to S i and can selectively bind to S i over at least a portion of its length. S i is drawn from an orthogonal set of sequences, i.e., S i ' does not hybridize to any other sequence S j (j ≠ i) in the set under stringent conditions.

[0089] S k The subscript k in represents its use in relation to a particular oligonucleotide sequence and a selected target in the imaged sample.k The notation 'indicates an oligonucleotide sequence that is complementary to S k and can selectively bind to S over at least a portion of its length. S k is drawn from an orthogonal set of sequences, i.e., S k ' will not hybridize to any other sequence S k in the set under stringent conditions (where k ≠ m). m (k≠m) does not hybridize.

[0090] When describing the amplification detection of multiple targets according to the present disclosure, the same subscript and prime notations are used in relation to each target, but the actual sequences referred to by S i and S k are different for each target. Thus, in a multiplex experiment using M different targets for which amplification is used, each target has an antibody conjugated to an oligonucleotide represented by S i , but the actual sequence referred to by S i will be different for each antibody. Similarly, an oligonucleotide sequence represented by S k is deposited by the TSA reaction associated with its target, but the actual sequence referred to by S k will be different for each target. Similarly, the oligo-labeled HRP used in relation to its target is represented herein as having a sequence of S i ', but the actual sequence referred to by S i ' is different for each target, and the oligo-labeled dye has a sequence of S k ', but the actual sequence referred to by S k ' will be different for each target.

[0091] Techniques exist for designing or selecting a set of sequences {S1, S2... S N} that are orthogonal and have a low probability of selectively binding to naturally occurring oligonucleotide sequences. See, for example, U.S. Patent No. 10,370,698, which lists an exemplary set of complementary sequences that meet these conditions.

[0092] Biological samples can be contacted with two or more (e.g., three or more, four or more, five or more, six or more, eight or more, ten or more, twelve or more, fifteen or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, eighty or more, or even more) antibodies, antibody fragments, or combinations thereof. The sample may be contacted with a cocktail of all the antibodies or antibody fragments or combinations of plural subsets of the total number of antibodies. In addition to the time savings and simplification obtained by using a single such incubation, this can result in improved detection of co-localized targets in the sample. Without wishing to be bound by theory, incubation with a single cocktail is seen to reduce or eliminate systematic interference between antibodies that bind to adjacent or overlapping targets in the sample.

[0093] The steps of antigen activation, blocking, and washing associated with antibody incubation can be used according to the conventions of conventional immunohistochemistry. The specific steps, compounds used, times, temperatures, and order of operations can be optimized based on the target being imaged to obtain good sensitivity, localization, selectivity, or other criteria of interest.

[0094] Each of one or more antibodies, antibody fragments, or combinations thereof is conjugated to a first oligonucleotide S i that is unique to that antibody or antibody fragment. After contacting the antibody or antibody fragment with the sample, the first oligonucleotide is i' can be brought into contact with. For example, the first oligonucleotide can hybridize to the second oligonucleotide by complementary base pairing or the like. Each first oligonucleotide S i can correspond to a unique second oligonucleotide S i '. This can be achieved by a barcoding system that includes an orthogonal set of sequences {S1, S2…S N} as described above.

[0095] In some cases, the first oligonucleotide S i is indirectly linked to the antibody or fragment, for example, via an additional linker oligonucleotide. As another method of indirect linkage, the first oligonucleotide S i conjugated to a secondary antibody, nanobody, or other entity that specifically targets the primary antibody, such that the primary antibody is indirectly linked to the first oligonucleotide.

[0096] Each second oligonucleotide S i ' is conjugated to an enzyme that can mediate the deposition of a detectable substance onto the biological sample. In some preferred embodiments, the second oligonucleotide S i ' can be conjugated to a horseradish peroxidase (HRP) enzyme. In other embodiments, the second oligonucleotide Si' is conjugated to a polymer containing several HRP molecules. In some embodiments, the enzyme can be a hemin-containing complex that can mimic HRP, for example, hemin. In some embodiments, the enzyme can be soybean peroxidase. In some cases, the second oligonucleotide is indirectly linked to the enzyme, for example, via an additional linker oligonucleotide or a click chemistry reaction system.

[0097] The intended S on the target antibody iTo ensure that little or no binding occurs at sites other than the barcode, stringent or near-stringent conditions may be used. To further reduce the possibility of non-specific binding of the enzyme to the sample, other oligonucleotide sequences that do not associate with the enzyme and do not selectively bind to the intended binding target S i may similarly be applied.

[0098] Excess catalyst agent molecules corresponding to the enzyme labeled with the oligonucleotide can be removed from the sample by washing after the enzyme is localized only or mainly in the vicinity of the primary antibody with which it associates.

[0099] Use the TSA reaction to deposit oligonucleotide S k on the sample. Oligo sequence S k can be conjugated to a tyramine compound or a substrate such as p-hydroxycinnamic acid that binds to the sample according to the TSA mechanism, or another substrate catalyzed by HRP. The linkage between oligo sequence S k and the substrate material may be indirect, for example, via an additional linker oligonucleotide or by another mechanism.

[0100] A localizing agent containing an enzyme substrate labeled with an oligonucleotide having sequence S k is introduced into the sample, while an enzyme linked to an oligonucleotide having sequence S i ' binds through hybridization to an antibody linked to an oligonucleotide having sequence S i . Thereby, deposition of oligo sequence S k occurs only nominally at the position corresponding to this antibody.

[0101] As described above, the deposition of the localizing agent results in amplification of the imaging signal that associates with the specific target analyte. The amplification factor is represented by α in this consideration, and is the oligonucleotide sequence S deposited on the sample per antibody molecule localized at the target site of the sample.k The number of molecules having the deposited sequence S k Each of the oligonucleotides may be labeled to provide an imaging signal (e.g., a fluorescent signal or a signal corresponding to the absorption, transmission, or reflection of light), so that such a sequence S for each target analyte molecule can be k The number of oligonucleotides corresponds to the amplification factor or degree of amplification α.

[0102] Each target analyte molecule has the sequence S i Since each sequence is labeled with a primary antibody linked to a single oligonucleotide molecule, the amplification factor α is effectively the amplification factor for the sequence S in the sample. i of oligonucleotides in a sample versus the amount or concentration of sequence S k The ratio corresponds to the ratio of the amount or concentration of oligonucleotides. The ratio of the amounts or concentrations (i.e., the amplification factor) may be 1.1 or greater (e.g., 1.5 or greater, 2.0 or greater, 3.0 or greater, 4.0 or greater, 5.0 or greater, 7.0 or greater, 10.0 or greater, 20.0 or greater, 30.0 or greater, 40.0 or greater, 50.0 or greater, 60.0 or greater, 70.0 or greater, 80.0 or greater, 90.0 or greater, 100.0 or greater, 200.0 or greater, 500.0 or greater, 1000 or greater, 5000 or greater, 10000 or greater, or even higher). The amplification factor α can be adjusted to balance the level of signal between multiple antibodies, to achieve a desired staining pattern, or for other purposes based on the assay being performed.

[0103] The enzyme-mediated amplification process can be controlled by adjusting the concentration of the enzyme substrate labeled with oligonucleotides in the localizing agent, the reaction time, the reaction temperature, and the replacement or replenishment of the enzyme substrate conjugated to the oligonucleotide. This can also be modified by the addition of compounds, such as inorganic salts, or organic enhancing compounds, such as those described in U.S. Patent No. 6,372,937, the entire content of which is incorporated by reference. The degree of amplification can be adjusted separately for each target analyte by separately depositing the localizing agent that associates with each enzyme-mediated target analyte.

[0104] Under some conditions, the TSA reaction results in dimerization of the enzyme substrate molecules rather than deposition onto the sample. This can occur when the density of the enzyme molecules or the enzyme substrate molecules labeled with oligonucleotides is too high. By reducing one or both of these factors, the effect of dimerization can be reduced, resulting in a higher level of deposition. For example, the concentration of the enzyme substrate labeled with oligonucleotides can be reduced, or a non-polymeric HRP enzyme can be used instead of the polymeric HRP enzyme.

[0105] The deposition cycle is for the oligonucleotide sequence S i linked to the antibody corresponding to the oligonucleotide of, the oligonucleotide sequence S i ' having an enzyme (e.g., HRP) linked to the oligonucleotide, and can be performed sequentially for each antibody, and the oligonucleotide sequence S k is deposited on the sample. This is repeated for each antibody for which amplified detection is desired, where each type of antibody is linked to a different oligonucleotide having a different sequence S i .

[0106] In some embodiments, the oligonucleotide having the sequence S k deposited for a given antibody is conjugated to the sequence S iIt is the same. In such an embodiment, the result of the deposition step is to bind a plurality of oligonucleotide molecules having the same sequence as the oligonucleotide sequence conjugated to the antibody, adjacent to the antibody, to the sample.

[0107] In certain embodiments, the sequence S deposited on a given antibody k is different from the sequence S conjugated to that antibody i In such an embodiment, the effect of the deposition step is to bind a plurality of oligonucleotide molecules having a sequence different from the oligonucleotide sequence conjugated to the antibody, adjacent to the antibody, to the sample.

[0108] The oligonucleotide sequence S deposited on the sample k hybridizes selectively with S k over at least a portion of its length, and can be detected using a dye molecule conjugated to an oligonucleotide sequence S k '.

[0109] Detection involves introducing a labeling agent comprising a dye molecule labeled with an oligonucleotide, providing stringent or near-stringent hybridization conditions under which the oligonucleotide sequence S k ' conjugated to the dye molecule hybridizes selectively with an oligonucleotide having the sequence S k deposited on the sample or conjugated to the antibody, removing the dye molecules labeled with oligonucleotides that did not hybridize, optionally applying a counterstain, such as DAPI, imaging the sample using a microscope to form an image of the dye molecules, and optionally removing the dye molecules labeled with oligonucleotides by a dehybridization and washing step.

[0110] In summary, the steps of binding a dye molecule labeled with an oligonucleotide to a site of a sample in the presence of a complementary localizing agent, applying a contrast stain, imaging the sample, and removing the dye molecule form one detection cycle.

[0111] As described above, an important aspect of certain methods described herein is that amplified detection of a target analyte can be performed, followed by removal of the labeling agent from the sample, thereby generating a signal observed during imaging of the sample. As a result, multiple cycles consisting of labeling, imaging, and optionally removal of the label of the sample can be performed continuously. The dyes used in each cycle can be removed by denaturation and washing steps, so they do not interfere with labeled imaging in subsequent cycles. A globally high degree of multiplexing for detecting N target analytes can be achieved through successive cycles of labeling and detection, where a smaller number B of dyes, or even a single dye (B = 1), is used in each cycle.

[0112] Generally, the number N of target analytes that can be detected using the methods described herein is one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, or even more).

[0113] The number B of dyes detected in a single imaging process can be one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, twenty or more, thirty or more, forty or more, fifty or more, seventy or more, or even more).

[0114] The number M of different types of labeling agents that can be deposited on the sample to detect the target analyte can be one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, twelve or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty-five or more, forty or more, forty-five or more, fifty or more, or even more). In some embodiments, M is less than or equal to N.

[0115] In some embodiments, the amplified deposition of the localization agent comprising the oligonucleotide is performed for all of the targets being imaged. In certain embodiments, the amplified deposition of the localization agent comprising the oligonucleotide is performed for only a single target or for a subset of the N targets being imaged.

[0116] When amplification is performed, detection of the target analyte can be performed at any time after deposition of the localization agent for that target. It is possible to perform the deposition step and the detection step alternately, and in some embodiments, one or more deposition steps can be performed to introduce one or more different types of localization agents that associate with different target analytes, and one or more detection steps can be performed to label and detect the localization agent, and further deposition and detection cycles may follow.

[0117] In some embodiments, the localization agent may be deposited on the sample for all of the target analytes of interest before performing any detection step (e.g., introduction of the labeling agent and imaging of the sample). The localization agent can be deposited using equipment constructed for a specialized purpose, such as an automated staining device, a microfluidic system having a staining chamber, and other systems. Such systems automatically dispense reagents and manage temperature, treatment time, and flow rate without user intervention. One advantage of such systems is that the sample is not repeatedly circulated between the staining device and the imaging station. Suitable systems for applying the reagents and methods of this specification are described, for example, in U.S. Patent Application No. 16 / 902,215, and U.S. Patents Nos. 6,735,531 and 7,226,788, the entire contents of each of which are incorporated herein by reference.

[0118] Detection may be performed on one analyte at a time, but may be performed on two, three, four, five, six, or more analytes at a time, together with one or more contrast staining agents (e.g., one or more tissue contrast staining agents, one or more nuclear contrast staining agents) that bind non-specifically to regions in the sample. Different labeling agents having dye molecules each labeled with an oligonucleotide are introduced and hybridized in one step, and the nucleotide sequence S k ' is orthogonal as described above, so that each dye molecule localizes to the corresponding antibody that binds to only one of the target analytes (or equivalently, its associated sequence S in the sample)k (Deposition by TSA of a localization agent having an oligonucleotide having). The number of dyes, the dyes selected, and the imaging process are chosen to ensure that the signals associated with each dye, and thus each target analyte, can be distinguished from one another in the resulting image.

[0119] It is beneficial to compare the methods described herein with other sequential multiplexing methods. The method called Sequential ImmunoPeroxidase Labeling and Erasing (SIMPLE, G. Glass, J. Papin, J Mandell, J. Histochem Cytochem 2009 Oct, 57 (10): 899:905) involves successive rounds of IHC using 3-amino-9-ethylcarbazole (AEC), which staining is imaged and then dissolved using alcohol. Each IHC round yields a picture of a single marker and requires blocking, primary antibody incubation, washing, secondary antibody incubation, further washing, and staining, then imaging the sample and washing in water, three dilutions of ethanol, water, potassium permanganate, and water. These steps take a total of approximately 3 hours per marker, so a 12-plex assay requires 36 hours of sample processing excluding the time taken to image the sample 12 times.

[0120] The multiplex imaging method is described in U.S. Patent No. 7,729,125, where normal prostate samples were imaged using 11 immune markers and DAPI. It included two rounds of two-channel indirect IHC, followed by seven rounds of direct-labeled IHC, bleaching of the dyes using NaOH solution between each staining round, and subsequent washing in PBS. Each indirect IHC round took approximately two hours, each direct IHC round took approximately one hour, and the bleaching steps took over 15 minutes each. Four markers had signal amplification through the secondary antibody, and there was no amplification in the seven rounds. Overall, excluding the time taken to image the sample nine times, a total of 14 hours of sample processing was required.

[0121] The method described herein can be performed in a single incubation step for all primary antibodies. Amplification of TSA deposition can be performed individually for each target analyte for which amplification is desired and takes approximately 30 minutes per target analyte, including hybridization, washing, TSA deposition, washing, and dehybridization. Detection can be performed, for example, for four or more species per cycle using four dyes (e.g., dyes Opal 520, Opal 570, Opal 620, Opal 690, available from Akoya Biosciences, Inc., Menlo Park, CA) together with a DAPI counterstain, and imaging can be performed using a Vectra Polaris instrument (available from Akoya Biosciences, Inc.). The time required for each detection round is approximately 30 minutes, including approximately 10 minutes for imaging. Excluding the primary incubation, the overall sample processing time for this example is three and a half hours if four species are amplified or seven and a half hours if twelve species are amplified.

[0122] This comparison illustrates some of the beneficial aspects of the methods described herein, including the option of using a single incubation with one cocktail of all primary antibodies, the ability to image multiple N targets using amplification, the option of using amplification for some targets and not for others, the ability to obtain high-level amplification of markers when desired, the ability to label and image multiple target Bs in a single detection round, and being fast overall.

[0123] In embodiments using fluorescent dye molecules, imaging can be performed using a fluorescence microscope. This can be done using a wide-field epifluorescence method, or it can use techniques such as confocal imaging, super-resolution imaging, multispectral imaging, two-photon microscopy, or total internal reflection fluorescence microscopy. The imaging system used to acquire the images can include an upright and / or inverted microscope, a digital slide scanner, or a custom device.

[0124] In embodiments using chromogenic dye molecules, imaging can be performed using a bright-field microscope. This can be done using a white light source and transmitted light optics, or it can use techniques such as laser scanning, narrow-band imaging, or multispectral imaging.

[0125] In certain embodiments, successive amplification cycles can be performed on each of one or more target analytes. For example, a primary antibody conjugated to an oligonucleotide sequence S i is contacted with an enzyme labeled with an oligonucleotide, e.g., HRP conjugated to S i '. An enzyme substrate labeled with an oligonucleotide having the sequence S k is introduced into the sample, and oligonucleotide molecules having the sequence S k are deposited onto the sample with an amplification factor α1 through a TSA mechanism catalyzed by the enzyme that associates with the primary antibody.

[0126] The sequence Si An enzyme labeled with an oligonucleotide having ', is optionally removed from the sample through dehybridization, and the sample is the sequence S k Contacted with an enzyme labeled with an oligonucleotide (e.g., HRP) conjugated to an oligonucleotide of '. Next, the sequence S k An enzyme substrate labeled with an oligonucleotide having is applied to the sample, and the sequence S k Hybridized to an oligonucleotide of ', and the deposited oligonucleotide molecules of the sequence S k Are deposited on the sample through the TSA mechanism with an amplification factor α2 (the amplification factor α2 is the sequence S k Of the amount or concentration of oligonucleotide molecules of'corresponding to the amount or concentration of oligonucleotide molecules of the sequence S k ). The deposition is by the enzyme conjugated to the oligonucleotide of the sequence S k And, if not removed from the sample, by the enzyme conjugated to the oligonucleotide of the sequence S i '. Overall, an amplification of (α1 × α2) is achieved by two deposition steps.

[0127] Using successive amplified deposition rounds, a higher overall amplification than would be practical in a single deposition step can be achieved and / or the degree of amplification can be finely controlled. The foregoing examples illustrate two deposition steps for the amplification of a signal that associates with a single target analyte, but more generally, any number of deposition steps (e.g., one or more, two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, or even more) can be performed, each step involving the introduction of a catalytic agent having an enzyme conjugated to an oligonucleotide and a localization agent having a complementary oligonucleotide. Specifically, for weakly expressed target analytes, multiple deposition steps can be advantageous for detection. Further, the number of deposition steps can be selected independently for each target analyte, and any two target analytes can be detected after the same or different numbers of deposition steps for amplification.

[0128] In some embodiments, one or more amplification cycles or steps are performed on a given target analyte and detection is performed on that target analyte. Optionally, a determination is made as to whether further amplification is desired (e.g., based on a measured imaging signal corresponding to the target analyte). In at least some embodiments, one or more further deposition cycles or steps for amplification are then performed on this target analyte and detection is performed again. Thereby, two or more images of samples having different levels of amplification are obtained. As described above, the number of images of a particular target analyte can be one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, or even more).

[0129] In some embodiments, the differential amplification method described above is used to image samples with widely varying expression, and the optimal amplification for it is not known prior to imaging. In other embodiments, this method is used to image strong expression regions in a sample in a first image and weaker expression regions of the same sample in a second image. In certain embodiments, evaluation of the second amplification factor α2 is obtained by comparing the signal levels in the two images. Optionally, one image is assembled from the first and second images with a high dynamic range in the target expression.

[0130] The biological sample may be fresh, frozen, or fixed. The biological sample may be of animal origin, such as from human, mouse, rat, bovine, porcine, ovine, monkey, or rabbit.

[0131] The biological sample can be immobilized on a surface. In some embodiments, the surface can be a slide, plate, well, membrane, or film. The biological sample may be fixed using an aldehyde, alcohol, oxidizing agent, mercury, picric acid, or HOPE fixative. The biological sample may alternatively be fixed using heat fixation. Fixation may be achieved by immersion or perfusion. The biological sample may be fresh or frozen. In some preferred embodiments, the sample includes formalin-fixed paraffin-embedded (FFPE) tissue.

[0132] In some embodiments, when contacting the biological sample, the antibody or antibody fragment may be bound to an element of the biological sample. The antibody or antibody fragment can bind reversibly or irreversibly to an element of the biological sample.

[0133] An antibody or antibody fragment may include IgG, IgM, polyclonal antibodies, monoclonal antibodies, scFv, nanobodies, Fab, or diabodies. An antibody or antibody fragment may have specificity for an element of a sample such as a protein. Selecting a particular antibody from among available candidates is done according to the needs of a given experiment and may be based on several factors, such as cost, the antibodies available for its target, the specificity of each candidate antibody for its target, the amount of background (non-specific) binding, and other factors used in immunohistochemistry design. These may be selected preferentially one clone over another, or monoclonal over polyclonal (or vice versa).

[0134] For some clones, these properties change when the antibody is conjugated to oligonucleotide S i Accordingly, the antibody is tested by conventional IHC techniques to form an initial assessment of its behavior and then investigated after conjugation with an oligonucleotide sequence for use in the present disclosure to ensure that its performance remains acceptable after conjugation.

[0135] Exemplary workflow The methods described herein can be implemented in a wide variety of different workflows. Figures 1A - 1D and Figures 2A - 2D are schematic diagrams illustrating one exemplary implementation of the method. In Figure 1A, sample 102 is contacted with a binder that includes a binding portion 104 that binds to analyte 101 in sample 102. Binding portion 104 is linked to a first oligonucleotide having sequence S i Suitable binding portions 104 include any of the binding portions described herein that specifically bind to a target analyte of interest in sample 102, such as antibodies and antibody fragments.

[0136] In Figure 1B, sample 102 is contacted with a complementary sequence S linked to enzyme 110 iContact with a catalytic agent comprising a second oligonucleotide 108 having '. When considered herein, suitable enzymes include HRP, soybean peroxidase, and other species that mimic the functional catalytic properties of these peroxidases. The second oligonucleotide 108 selectively hybridizes to the first oligonucleotide 106 such that the catalytic agent is selectively localized to the position of the target analyte 101 in the sample 102.

[0137] In FIG. 1C, the sample 102 is contacted with a localizing agent comprising a substrate 112 complementary to the enzyme 110. The substrate 112 is linked to a third oligonucleotide having the sequence S k Suitable substrates include, but are not limited to, tyramine and tyramine derivatives, p-hydroxycinnamic acid, and their derivatives, as described herein. Due to the catalytic reaction between the enzyme 110 and the substrate 112, multiple localizing agent molecules are deposited at positions in the sample proximal to the analyte 101.

[0138] In FIG. 1D, the catalytic agent is removed from the sample 102 through denaturation and washing. Since the deposited localizing agent molecules are covalently bound to the sample, they remain in the sample as shown.

[0139] FIG. 2A shows a view of the same sample 102 as in FIG. 1D. In FIG. 2B, a labeling agent comprising a fourth oligonucleotide 116 having the sequence S k ' linked to an optical label 118 is contacted with the sample. The sequences of the third and fourth oligonucleotides 114 and 116 are complementary, and the fourth oligonucleotide 116 hybridizes to the third oligonucleotide 114, localizing the labeling agent in the sample 102 in proximity to the analyte 101. Since multiple localizing agent molecules were deposited in the sample for each analyte molecule 101, the measured signal associated with the analyte 101 is amplified.

[0140] In FIG. 2C, illumination light 120 is incident on sample 102, and optical label 118 generates light that is emitted (e.g., by emitting fluorescence or by absorbing a portion of the illumination light). The emitted light is detected to form an image of sample 102 in which the light emitted from optical label 118 indicates the location of analyte 101.

[0141] In FIG. 2D, the labeling agent molecules are optionally removed from sample 102 by denaturation and washing. As described above, additional deposition and / or detection cycles can be performed to identify additional target analytes in sample 102.

[0142] FIG. 3 is a flowchart showing a series of exemplary steps corresponding to one implementation of the method described herein for detecting analytes in a sample. In a first step 302, the sample is incubated with a plurality of different types of binding agents. Each different type of binding agent includes a binding moiety specific for a particular target analyte and a first nucleotide having a sequence that associates with the binding moiety.

[0143] Next, in step 304a, the sample is contacted with an agent having a catalyst including an enzyme linked to a second oligonucleotide. The sequence of the second oligonucleotide is complementary to only one of the different first oligonucleotides from step 302, selectively hybridizes to that first oligonucleotide, and localizes the agent having a catalyst in the sample. Then, in step 306a, the sample is contacted with a localizing agent including an enzyme substrate linked to a third oligonucleotide. A catalytic reaction between the enzyme and the substrate deposits molecules of the localizing agent in the vicinity of a particular target analyte in the sample.

[0144] Steps 304a and 306a are repeated N times (as steps 304n and 306n, where n = b…N) for each of the N different target analytes in the sample. Each different type of catalyst agent introduced in step 304n selectively hybridizes to a different one of the binding agents specific to different target analytes in the sample. Each different type of localizing agent introduced in step 306n is deposited in proximity to the target analyte to which the catalyst agent selectively associates in step 304n and has a third oligonucleotide sequence that is unique among the sequences of the different types of localizing agents.

[0145] Next, in step 308a, a group of one or more different types of labeling agents, each having a fourth oligonucleotide having a sequence that is complementary to only one of the plurality of types of localizing agents, is contacted with the sample. Each different type of labeling agent hybridizes to its complementary localizing agent and has an optical label. In step 310a, an image of the sample is acquired that shows the contribution from each of the different optical labels of the labeling agents. Due to the localization of the labeling agents utilizing hybridization to the complementary localizing agents, the optical labels of each type of labeling agent indicate the presence of a different one of the target analytes in the sample.

[0146] Steps 308a and 310a are repeated P times (as steps 308p and 310p, where p = a…P) for P different groups of labeling agents. In some embodiments, for example, each group P of labeling agents includes only a single labeling agent. In a particular embodiment, each group P of labeling agents includes more than one (e.g., 2, 3, 4, 5, 6, 8, 10, or even more than 10) labeling agents, each of which is detected in the image acquired in step 310p. Note that the number of labeling agents detected in each step 310p may be the same or different.

[0147] After all labeling agents have been introduced into the samples and all sample images have been acquired, the procedure shown in FIG. 3 ends.

[0148] Oligonucleotide An oligonucleotide is a molecule that includes a plurality of nucleotides (for example, at least some of which may be connected to form a chain). The oligonucleotides described herein may include ribonucleic acids. The oligonucleotides described herein may include deoxyribonucleic acids. In some embodiments, the oligonucleotide can be of any sequence, including the sequence specified by the user.

[0149] Often, oligonucleotides can be composed of bases that can base pair with high reliability with G, A, T, and C, or complementary nucleotides. 7-Deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza-guanosine, 2-thio-adenine, 2-thio-7-deaza-adenine, isoguanine, 7-deaza-guanine, 5,6-dihydro-uridine, 5,6-dihydro-thymine, xanthine, 7-deaza-xanthine, hypoxanthine, 7-deaza-xanthine, 2,6-diamino-7-deazapurine, 5-methyl-cytosine, 5-propynyl-uridine, 5-propynyl-cytidine, 2-thio-thymine, or 2-thio-uridine are examples of such bases, but many others are known. The oligonucleotide may be, for example, an LNA, PNA, UNA, or morpholino oligomer. The oligonucleotides used herein may contain natural or non-natural nucleotides or linkages.

[0150] As used herein, an antibody, antibody fragment, or other analyte-targeting moiety can be conjugated to a first oligonucleotide to form a binding agent such that at least a portion of the antibody, antibody fragment, or other analyte-targeting moiety can contact an analyte of a biological sample. The first oligonucleotide can then hybridize to a binding region of a second oligonucleotide, which is conjugated to an enzyme that can mediate the deposition of another molecule onto the biological sample.

[0151] In some embodiments, the first oligonucleotide comprises a plurality of ribonucleic acids. In some embodiments, the first oligonucleotide comprises a plurality of deoxyribonucleic acids. In some embodiments, the first oligonucleotide can comprise one or more synthetic nucleotides. Examples of synthetic nucleotides can include RNA analogs or DNA analogs. Some synthetic nucleotides can include artificial nucleic acids, which can include peptide nucleic acids, morpholinos, and locked nucleic acids, glycolic acid, or threose nucleic acids.

[0152] The first oligonucleotide can have a given length suitable for a particular workflow. In some embodiments, longer oligonucleotides can be selected. In some embodiments, shorter oligonucleotides can be selected. Factors that can affect the selection of the oligonucleotide length can include, for example, melting temperature, secondary structure, affinity, specificity, selectivity, cost, and / or the number of possible sequence combinations.

[0153] In some embodiments, the first oligonucleotide can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides in length.

[0154] In some embodiments, the first oligonucleotide can be 5 to 30, 5 to 25, 5 to 20, 10 to 20, 10 to 30, 10 to 50, 10 to 70, 10 to 100, 20 to 50, 20 to 70, 20 to 100, 30 to 50, 30 to 70, 30 to 100, 40 to 70, 40 to 100, 50 to 70, 50 to 100, 60 to 70, 60 to 80, 60 to 90, or 60 to 100 nucleotides in length.

[0155] In some embodiments, the first oligonucleotide can be no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides in length.

[0156] In some embodiments, the first oligonucleotide can be single-stranded overall. In some embodiments, the first oligonucleotide can be partially double-stranded. In some embodiments, the partially double-stranded region can be at the 3' end of the nucleotide, at the 5' end of the nucleotide, or between the 5' end and the 3' end of the nucleotide. In some embodiments, more than one double-stranded region may be present. Some first oligonucleotides can have a secondary structure. Some first oligonucleotides may have a secondary structure, and as a result, one or more double-stranded regions containing single strands can be brought about by single-strand folding and / or complementarity to itself.

[0157] The second oligonucleotide conjugated to an enzyme to form a catalytic agent can hybridize to the first oligonucleotide in the binding region of the second oligonucleotide. This interaction can occur through base pairing.

[0158] The binding region of the second oligonucleotide can be at least partially complementary to at least a portion of the first oligonucleotide. In some embodiments, the binding region can be complementary to the 3' end of the first oligonucleotide. In some embodiments, the first binding region can be complementary to the 5' end of the first oligonucleotide. In some embodiments, the first binding region can be complementary to a region between the 3' end and the 5' end of the first oligonucleotide. In some embodiments, the binding region can be complementary to the entire first oligonucleotide. In some embodiments, the binding region can be complementary to less than 100% of the first oligonucleotide, as described above.

[0159] In some embodiments, the second oligonucleotide can be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides in length.

[0160] In some embodiments, the second oligonucleotide can be 5 to 30, 5 to 25, 5 to 20, 10 to 20, 10 to 30, 10 to 50, 10 to 70, 10 to 100, 20 to 50, 20 to 70, 20 to 100, 30 to 50, 30 to 70, 30 to 100, 40 to 70, 40 to 100, 50 to 70, 50 to 100, 60 to 70, 60 to 80, 60 to 90, or 60 to 100 nucleotides in length.

[0161] In some embodiments, the second oligonucleotide can be no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, or no more than 100 nucleotides in length.

[0162] In some embodiments, the second oligonucleotide can be single-stranded overall. In some embodiments, the first oligonucleotide can be partially double-stranded. In some embodiments, the partially double-stranded region can be at the 3' end of the nucleotide, at the 5' end of the nucleotide, or between the 5' and 3' ends of the nucleotide. In some embodiments, more than one double-stranded region may be present. Some second oligonucleotides can have a secondary structure. Some second oligonucleotides may have a secondary structure, and as a result, one or more double-stranded regions containing single strands can be brought about by single-strand folding and / or complementarity to itself. In some embodiments, the second oligonucleotide can include more than one oligonucleotide.

[0163] Sequence S k The third oligonucleotide having [sequence S] is conjugated to an enzyme substrate to form a localization agent suitable for TSA amplification, such as the aforementioned tyramide compound, p-hydroxycinnamic acid, or derivatives thereof. The conjugation can be indirect, for example, via an additional linker oligonucleotide, via a secondary antibody or nanobody, or by any of the other mechanisms described above.

[0164] Sequence S k The fourth oligonucleotide having '[sequence S]' is conjugated to an optical label to form a labeling agent. The conjugation can be indirect, for example, via an additional linker oligonucleotide or by other mechanisms. Typically, the optical label is a dye molecule, a fluorescent moiety, a chromogenic moiety, or more generally, any other type of moiety that generates a detectable signal when exposed to illumination light.

[0165] Optical labels can be fluorescent dye molecules or moieties, such as Alexa 488, Alexa 514, Alexa 568, Alexa 547, Alexa 750, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, fluorescein, rhodamine, tetramethylrhodamine, Texas red, coumarin, DyLight dyes, Atto dyes, or others. In some embodiments, the dye molecule or moiety may include one or more quantum dots. In certain embodiments, the dye molecule or moiety may include one or more chromogenic species.

[0166] The fourth oligonucleotide can hybridize to the third oligonucleotide in the binding region of the fourth oligonucleotide. This interaction can occur through base pairing.

[0167] The binding region of the fourth oligonucleotide can be complementary to at least a portion of the third oligonucleotide. In some embodiments, the binding region can be complementary to the 3' end of the third oligonucleotide. In some embodiments, the binding region can be complementary to the 5' end of the third oligonucleotide. In some embodiments, the binding region can be complementary to a region between the 3' end and the 5' end of the third oligonucleotide. In some embodiments, the binding region can be complementary to the entire third oligonucleotide. In some embodiments, the binding region can be complementary to less than 100% of the third oligonucleotide.

[0168] In the methods described herein, a sample is array S iContact the sample with one or more binding agents that include a binding moiety conjugated to an oligonucleotide having a first oligonucleotide having . As described above, suitable binding moieties include antibodies, antibody fragments, and other moieties that selectively bind to proteins, markers, peptides, peptide fragments, and other amino acid-containing species that are target analytes in the sample. Methods for preparing suitable binding agents by conjugating a binding moiety to an oligonucleotide are described, for example, in U.S. Patent No. 5,391,723, as well as in Dennler et al., Antibodies 4: 197-224 (2015) and Kozlov et al., Biopolymers 73:621 (2004), the entire contents of each of which are incorporated herein by reference.

[0169] Further contact the sample with one or more catalytic agents that include an enzyme linked to a second oligonucleotide having sequence S i '. Methods for preparing suitable catalytic agents by linking an enzyme to an oligonucleotide are described, for example, in van Gijlswijk et al., Cytogenet. Cell Genet. 75: 258-262 (1996), the entire contents of which are incorporated herein by reference.

[0170] Further contact the sample with one or more localization agents that include an enzyme substrate linked to a third oligonucleotide having sequence S k '. Methods for preparing suitable localization agents by linking an enzyme substrate to an oligonucleotide are described, for example, in Spicer et al., Chem. Rev. 118(16): 7702-7743 (2018), Winkler, Ther. Deliv. 4(7): 791-809 (2013), and van Gijlswijk et al., Histochemie 113(3): 175-180 (2000), the entire contents of each of which are incorporated herein by reference.

[0171] Further contact the sample with one or more localization agents that include an enzyme substrate linked to a third oligonucleotide having sequence S kContact with one or more localization agents that include an optical label (e.g., a fluorescent or chromogenic moiety) linked to an oligonucleotide of '. Suitable methods for preparing suitable labeling agents are described, for example, in Wood et al., "Fluorescence Labeling of Nucleic Acids", Encylcopedia of Biophysics (2013), Hwang, Molecules 23(1): 124 (2018), and Taskova et al., Bioconjugate Chem. 30(12): 3007-3012 (2019), the entire contents of each of which are incorporated herein by reference.

[0172] Reagents and Conditions of the Process An antibody, antibody fragment, or other analyte targeting moiety (binding agent) conjugated to a first oligonucleotide can be delivered to the sample in a first buffer. The first buffer can include PBS, PBS-T, TBS, TBS-T water, saline, or Krebs buffer and can include a blocking material. In some embodiments, the blocking material can include BSA, casein, sheared salmon sperm DNA, other oligonucleotide components, rat IgG, and / or mouse IgG.

[0173] An enzyme molecule (catalytic agent) conjugated to an oligonucleotide can be delivered to the sample in a second buffer. In some embodiments, the second buffer can include PBS, PBS-T, TBS, TBS-T water, saline, or Krebs buffer.

[0174] An enzyme substrate (localization agent) conjugated to an oligonucleotide and a dye molecule (labeling agent) conjugated to an oligonucleotide can be delivered to the sample in the first or second buffer.

[0175] In some embodiments, the first buffer may be essentially the same as the second buffer.

[0176] In some embodiments, the antibody conjugated to the first oligonucleotide may be in the same buffer as the second oligonucleotide, which may be a first alternative buffer. In some embodiments, the first alternative buffer may include PBS, PBS-T, TBS, TBS-T water, saline, or Krebs buffer.

[0177] In some embodiments, the sample is subjected to a buffer that may include a DNA component, a protein component, a chaotropic reagent at a concentration of 5%, 10%, 15%, or 20%, and a surfactant solution, which promote hybridization.

[0178] In some embodiments, the sample is subjected to a buffer that may include a chaotropic reagent at a concentration of 60%, 70%, 80%, or 90%, such as DMSO and formamide, which promote dehybridization.

[0179] The enzyme molecule (e.g., a catalytic agent) linked to the oligonucleotide can be removed from the sample, for example, through dehybridization between oligonucleotides directly or indirectly conjugated to the enzyme and antibody components. In some embodiments, this dehybridization can be performed using a chaotropic reagent, such as DMSO or formamide. The dehybridization step can thus remove the directed enzyme activity from the sample surface, thereby enabling subsequent rounds of hybridization and enzyme-catalyzed oligonucleotide deposition without signal contamination from non-targeted portions.

[0180] Dye molecules (e.g., labeling agents) conjugated to oligonucleotides can be removed from the sample surface, for example, through the dehybridization between oligonucleotides directly or indirectly conjugated to the oligonucleotide components deposited by the dye and TSA. In some embodiments, this dehybridization can be performed using chaotropic reagents such as DMSO or formamide. The dehybridization step can thus remove the dye from the sample surface, thereby enabling subsequent detection rounds without signal contamination from this round of dye.

[0181] In some preferred embodiments of this type, at least some of the primary antibodies can be used to identify the cell type or identity, and at least some of the primary antibodies indicate the cell activity, expression, or signaling state.

[0182] Compositions and Kits Compositions can be formed by combining any of the reagents, molecules, and other substances described herein and delivering them to a biological sample for the purpose of performing one or more steps of the various methods described. Reagents, molecules, and other substances described in any of the documents incorporated by reference may also be present in the composition.

[0183] Kits containing any of the compositions may also include instructions for performing any of the method steps described herein. Such kits may include a housing or packaging formed from one or more materials such as paper, metal, and plastic. The housing can be implemented in various forms including one or more tube-like containers such as vials, blister packs, and other sealed containers. The instructions may be placed within, affixed to, or associated with the housing of the kit.

[0184] Imaging Systems and Methods A wide variety of different imaging systems can be used to acquire the images described herein. One particular commercially available system, for example, the Vectra Polaris system (available from Akoya Biosciences, Inc.), can be used. Aspects of imaging systems that can be used are described, for example, in U.S. Patent Nos. 7,155,55, 7,019,777, 9,107,624, and PCT Patent Application Publication No. WO 2005 / 040769, the entire contents of each of which are incorporated herein by reference.

[0185] To acquire an image of a sample described herein, the sample is exposed to illumination light from a light source of an imaging system. An image of the sample is acquired by using a detector of the imaging system (e.g., an imaging detector, e.g., a CCD array) to detect the light emitted from the sample in response to the illumination light. The emitted light can be fluorescence emission, illumination light transmitted through the sample, illumination light reflected from the sample, or any combination thereof. Individual elements of the detector measure the emitted light and form a two-dimensional image of the biological sample. Since the emitted light corresponds to the location in the sample where the labeling agent is located, the location of the specimen is indicated by the optical label of the labeling agent.

[0186] Using a similar method, an image of a contrast stain applied to a sample described herein is acquired. Since the contrast stain binds non-specifically to the structure of the sample, the image of the contrast stain typically does not indicate the location of a particular specimen, but rather provides more general information regarding the structure, features, and morphology of the sample.

Examples

[0187] To evaluate the above method, tissue sections with a thickness of 5 microns were cut from formalin-fixed paraffin-embedded human tonsil blocks. The sections were deparaffinized, hydrated, and subjected to antigen retrieval with citrate buffer. The samples were then stained with a CD20 (L20) antibody obtained from Akoya Biosciences, Inc. (Menlo Park, CA) according to the staining instructions attached to the antibody. The CD20 antibody used for staining was pre-conjugated to an oligonucleotide (sequence BX015, obtained from Akoya Biosciences, Inc.) according to the manufacturer's instructions.

[0188] After staining, the tissue sections were fixed with paraformaldehyde, ice-cold methanol, and CODEX® fixation reagent (obtained from Akoya Biosciences, Inc.), and then washed. The tissue sections were equilibrated for 10 minutes with 20% dimethyl sulfoxide (DMSO) in 1× CODEX® assay buffer (obtained from Akoya Biosciences, Inc.).

[0189] After equilibration, the tissue sections were hybridized with 5 μL of a 20 μM solution of an oligonucleotide conjugated to horseradish peroxidase (HRP). The oligonucleotide conjugated to HRP had a nucleotide sequence complementary to the BX015 sequence. After hybridization, the tissue sections were washed three times with CODEX® assay buffer.

[0190] A reagent consisting of a DNA oligonucleotide with sequence BX006 (obtained from Akoya Biosciences, Inc.) was conjugated to a tyramine moiety at the C10 carboxyl linker at the 5'-end of the sequence. The tissue samples were then contacted with the reagent in 1× CODEX® buffer and reacted for 10 minutes.

[0191] After the reaction, the oligonucleotide conjugated to HRP was removed from the tissue sections according to the CODEX® Clear Tissue protocol described in the CODEX® user manual available from Akoya Biosciences, Inc., for example, at the Internet address www.akoyabio.com / support / reagents / .

[0192] In preparation for imaging, the tissue sections were incubated in CODEX® hybridization buffer with the CODEX® reporter reagent Cy5-RX006 (available from Akoya Biosciences, Inc.). The tissue sections were imaged using a Keyence microscope.

[0193] Figure 4 shows an image of a tissue section. Within the tissue section, the bright regions correspond to the oligonucleotide sequence conjugated to tyramine where CD20 is localized (i.e., the BX006 sequence).

[0194] After imaging, the reporter reagent was removed from the tissue sections using the CODEX® Clear Tissue protocol. Another image of the tissue section with the reporter reagent removed is shown in Figure 5. This image exemplifies the absence of signal intensity corresponding to the oligonucleotide sequence conjugated to tyramine where CD20 is localized, suggesting near-complete removal of the reporter reagent during the dehybridization.

[0195] Other Embodiments While certain embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and alternative forms will be apparent to those skilled in the art. It should be understood that various alternative forms to the embodiments specifically described herein are within the scope of the present disclosure.

Description of Reference Numerals

[0196] 101 Specimen 102 Sample 104 Binding portion 106 First oligonucleotide 108 Second oligonucleotide 110 Enzyme 112 Substrate 114 Third oligonucleotide 116 Fourth oligonucleotide 118 Optical label 120 Illumination light

Claims

1. A method for imaging an analyte in a biological sample, comprising contacting the biological sample with a binder, wherein the binder comprises a binding moiety that binds to the analyte and a first nucleotide sequence; contacting the biological sample with a catalytic agent, wherein the catalytic agent comprises a second nucleotide sequence linked to an enzyme, and the second nucleotide sequence hybridizes to the first nucleotide sequence; contacting the biological sample with a localizing agent, wherein the localizing agent comprises a substrate specific for the enzyme and a third nucleotide sequence linked to the substrate; contacting the biological sample with a labeling agent, wherein the labeling agent comprises a fourth nucleotide sequence linked to an optical label, and the fourth nucleotide sequence hybridizes to the third nucleotide sequence; exposing the biological sample to illumination light, detecting light emitted from the biological sample, and forming an image of the biological sample in which the position of the analyte is indicated by the optical label; removing the catalytic agent from the biological sample by dehybridizing the first and second nucleotide sequences; and a method comprising the steps of:

2. The method according to claim 1, wherein the binding moiety comprises an antibody, an antibody fragment, or an antibody analog.

3. The method according to claim 2, wherein the antibody, antibody fragment, or antibody analog comprises a member selected from the group consisting of IgG antibodies, IgM antibodies, monoclonal antibodies, single-chain variable fragments, and diabodies.

4. The method according to claim 1, wherein the first nucleotide sequence comprises at least one synthetic nucleotide.

5. The method according to claim 1, wherein the first nucleotide sequence is a single-stranded sequence.

6. The method according to claim 1, wherein the first nucleotide sequence is at least partially double-stranded.

7. The method according to claim 1, wherein the first and second nucleotide sequences contain different numbers of nucleotides.

8. The method according to claim 1, wherein the enzyme comprises horseradish peroxidase or a derivative thereof.

9. The method according to claim 1, wherein the enzyme comprises a compound that mimics horseradish peroxidase.

10. The method according to claim 9, wherein the compound comprises a hemin-containing complex.

11. The method according to claim 9, wherein the compound comprises hemin.

12. The method according to claim 1, wherein the enzyme comprises soybean peroxidase.

13. The method according to claim 1, wherein the optical label comprises a fluorescent species.

14. The method according to claim 1, wherein the optical label comprises a chromogenic stain.

15. The method according to claim 1, wherein the sample comprises at least one member selected from the group consisting of proteins, peptides, and peptide fragments.

16. Before exposing the biological sample to illumination light, contacting the biological sample with a contrast stain; and exposing the biological sample to illumination light, detecting the light emitted from the biological sample, and forming a second image of the sample indicating the position of the contrast stain in the biological sample. The method according to claim 1, further comprising.

17. The method according to claim 16, wherein the contrast dye comprises DAPI.

18. The method according to claim 1, wherein the ratio of the amount of the fourth nucleotide sequence to the amount of the first nucleotide sequence in the biological sample is greater than 1.

19. The binding agent is a first binding agent, the catalytic agent is a first catalytic agent, the localization agent is a first localization agent, the labeling agent is a first labeling agent, the specimen is a first specimen, the binding moiety is a first binding moiety, and the method further comprises a step of contacting the biological sample with a second binding agent, wherein the second binding agent comprises a second binding moiety that binds to a second specimen in the biological sample and a fifth nucleotide sequence. The method according to claim 1.

20. The method according to claim 19, wherein the second specimen is different from the first specimen.

21. The method according to claim 20, wherein the second specimen comprises a protein, a peptide, or a peptide fragment.

22. The method according to claim 19, comprising a step of contacting the biological sample with the first and second binding agents simultaneously.

23. The method according to claim 19, comprising a step of sequentially contacting the biological sample with the first and second binding agents.

24. The catalytic agent is a first catalytic agent, the enzyme is a first enzyme, the localization agent is a first localization agent, the substrate is a first substrate, the labeling agent is a first labeling agent, the optical label is a first optical label, and the method is The step of contacting the biological sample with a second catalyst, wherein the second catalyst comprises a sixth nucleotide sequence linked to a second enzyme, and the sixth nucleotide sequence hybridizes to the fifth nucleotide sequence; The step of contacting the biological sample with a second binder, wherein the second binder comprises a second substrate specific for the second enzyme and a seventh nucleotide sequence linked to the second substrate; The step of contacting the biological sample with a second labeling agent, wherein the second labeling agent comprises an eighth nucleotide sequence linked to a second optical label, and the eighth nucleotide sequence hybridizes to the seventh nucleotide sequence; The method according to claim 19, further comprising. **Claim 25** The method according to claim 19, wherein the fifth nucleotide sequence is a single-stranded sequence. **Claim 26** The method according to claim 19, wherein the fifth nucleotide sequence is at least partially double-stranded. **Claim 27** The method according to claim 24, wherein the fifth and sixth nucleotide sequences contain different numbers of nucleotides. **Claim 28** The method according to claim 24, wherein the first and second enzymes are different. **Claim 29** The method according to claim 24, wherein the second enzyme comprises horseradish peroxidase, a derivative of horseradish peroxidase, a compound mimicking horseradish peroxidase, a hemin-containing complex, and hemin. **Claim 30** The method according to claim 24, wherein the second enzyme comprises soybean peroxidase. **Claim 31** The method according to claim 24, wherein the seventh nucleotide sequence is a single-stranded sequence. **Claim 32** The method according to claim 24, wherein the seventh nucleotide sequence is at least partially double-stranded.

33. The method according to claim 24, wherein the seventh and eighth nucleotide sequences contain different numbers of nucleotides.

34. The method according to claim 24, wherein the second optical label contains a fluorescent species.

35. The method according to claim 24, wherein the second optical label contains a chromogenic dye.

36. The method according to claim 24, wherein the ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample is greater than 1.

37. The method according to claim 24, wherein the ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample is different from the ratio of the amount of the fourth nucleotide sequence to the amount of the first nucleotide sequence in the biological sample.

38. The method according to claim 37, wherein the ratio of the amount of the eighth nucleotide sequence to the amount of the fifth nucleotide sequence in the biological sample is not greater than 1.

39. The method according to claim 24, further comprising a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second labeling agent.

40. The method according to claim 39, comprising a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second localizing agent.

41. The method according to claim 39, comprising a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second catalytic agent.

42. The method according to claim 39, further comprising a step of removing the first labeling agent from the biological sample before the step of contacting the biological sample with the second binding agent.

43. The method according to claim 39, further comprising a step of removing the first labeling agent from the biological sample by dehybridizing the fourth nucleotide sequence from the third nucleotide sequence.

44. The method according to claim 24, wherein the image of the biological sample is a first image, the method further comprising a step of exposing the biological sample to illumination light, detecting light emitted from the biological sample, and forming a second image of the biological sample, wherein the position of the second analyte is indicated by the second optical label.

45. The method according to claim 44, wherein the first optical label is present in the biological sample when the biological sample is exposed to the illumination light to form the second image of the biological sample.

46. The method according to claim 44, further comprising a step of removing the first optical label from the biological sample before the step of exposing the biological sample to the illumination light to form the second image of the biological sample.

47. After the biological sample is contacted with the localizing agent and before the biological sample is contacted with the labeling agent, a step of contacting the biological sample with a second catalytic agent, wherein the second catalytic agent comprises a fifth nucleotide sequence linked to a second enzyme, and the fifth nucleotide sequence hybridizes to the third nucleotide sequence; a step of contacting the biological sample with a second localizing agent, wherein the second localizing agent comprises a second substrate specific for the second enzyme and a sixth nucleotide sequence linked to the second substrate; further comprising The fourth nucleotide sequence of the labeling agent hybridizes to the sixth nucleotide sequence. The method according to claim 1.

48. The method according to claim 47, wherein the second enzyme is selected from the group consisting of horseradish peroxidase and its derivatives, hemin-containing complexes, hemin, and soybean peroxidase.

49. A method for imaging a plurality of specimens in a biological sample, comprising: (a) contacting the biological sample with a binding agent comprising a binding moiety that selectively binds to one of the specimens and a first nucleotide sequence; (b) contacting the biological sample with an agent that catalyzes, the agent comprising a second nucleotide sequence linked to an enzyme, the second nucleotide sequence hybridizing to the first nucleotide sequence; (c) contacting the biological sample with a localization agent, the localization agent comprising a substrate specific for the enzyme and a third nucleotide sequence linked to the substrate, depositing the third nucleotide sequence adjacent to one of the specimens; repeating steps (a) to (c) to deposit N different third nucleotide sequences in the biological sample such that each of the third nucleotide sequences is selectively arranged adjacent to a different one of the specimens; contacting the biological sample with M different labeling agents, each labeling agent comprising a fourth nucleotide sequence linked to a different optical label, the fourth nucleotide sequence hybridizing to only one of the third nucleotide sequences; obtaining an image of the sample, wherein the position of one of the specimens is indicated by the position of one of the optical labels. After at least one repetition of steps (a) to (c) and before the next repetition of steps (a) to (c), removing the catalytic agent from the biological sample by dehybridizing the first and second nucleotide sequences A method comprising **Claim 50** A method for imaging an analyte in a biological sample, comprising: Linking an enzyme to the analyte such that the enzyme is localized at the position of the analyte in the biological sample; Contacting the biological sample with a localization agent comprising a substrate specific for the enzyme and a first nucleotide sequence, and depositing the first nucleotide sequence adjacent to the position of the analyte in the biological sample; Contacting the biological sample with a labeling agent comprising a second nucleotide sequence that hybridizes to the first nucleotide sequence and an optical label; Obtaining an image of the biological sample, wherein the position of the analyte is represented by the position of the optical label; Removing the enzyme from the biological sample by dehybridizing the first nucleotide sequence linked to the analyte and the second nucleotide sequence linked to the enzyme; A method comprising

Images