Multiplexed imaging with enzyme-mediated amplification

Abstract

To provide methods for providing an adequate signal for detection for immunological targets which are weakly expressed or which are not efficiently targeted by existing IHC reagents.SOLUTION: Methods for imaging an analyte in a sample comprises: contacting the sample with a binding agent, where the binding agent comprises a binding moiety that binds to the analyte and a first nucleotide sequence; contacting the sample with a catalytic agent, where the catalytic agent comprises a second nucleotide sequence linked to an enzyme, and where the second nucleotide sequence hybridizes to the first nucleotide sequence; contacting the sample with a localization agent, where the localization agent comprises a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate; and contacting the sample with a labeling agent, where the labeling agent comprises a fourth nucleotide sequence linked to an optical label, where the fourth nucleotide sequence hybridizes to the third nucleotide sequence.SELECTED DRAWING: Figure 2B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 908,540, filed September 30, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

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

[0003] [Patent Document 1] U.S. Patent No. 6,372,937 [Patent Document 2] U.S. Patent No. 6,828,109 [Patent Document 3] U.S. Patent Application Publication No. 2017 / 0226572 [Patent Document 4] U.S. Patent Application Publication No. 2005 / 0003462 [Patent Document 5] U.S. Patent No. 9,909,167 [Patent Document 6] U.S. Patent No. 10,370,698 [Patent Document 7] U.S. Patent Application Publication No. 16 / 902,215 [Patent Document 8] U.S. Patent No. 6,735,531 [Patent Document 9] U.S. Patent No. 7,226,788 [Patent Document 10] U.S. Patent No. 7,729,125 [Patent Document 11] U.S. Patent No. 5,391,723 [Patent Document 12] U.S. Patent No. 7,155,55 [Patent Document 13] U.S. Patent No. 7,019,777 [Patent Document 14] U.S. Patent No. 9,107,624 [Patent Document 15] PCT Patent Application Publication No. 2005 / 040769 [Non-patent literature]

[0004] [Non-Patent Document 1] Taofiq et al., Molecules 22(2): 281 (2017) [Non-patent document 2] G. Glass, J. Papin, J Mandell, J. Histochem Cytochem 2009 Oct; 57 (10): 899:905 [Non-patent document 3] Dennler et al., Antibodies 4: 197-224 (2015) [Non-patent document 4] Kozlov et al., Biopolymers 73:621 (2004) [Non-Patent Document 5] van Gijlswijk et al., Cytogenet. Cell Genet. 75: 258-262 (1996) [Non-patent document 6] Spicer et al., Chem. Rev. 118(16): 7702-7743 (2018) [Non-Patent Document 7] Winkler, Ther. Deliv. 4(7): 791-809 (2013) [Non-patent document 8] van Gijlswijk et al., Histochemie 113(3): 175-180 (2000) [Non-Patent Document 9] Wood et al., “Fluorescence Labeling of Nucleic Acids”, Encylcopedia of Biophysics (2013) [Non-Patent Document 10] Hwang, Molecules 23(1): 124 (2018) [Non-Patent Document 11] Taskova et al., Bioconjugate Chem. 30(12): 3007-3012 (2019) Summary of the Invention [Problem to be solved by the invention]

[0005] However, traditional immunohistochemistry (IHC) methods do not always provide a signal suitable for detection, especially for immunological targets that are weakly expressed or not efficiently targeted by existing IHC reagents. [Means for solving the problem]

[0006] The present disclosure features methods, compositions of matter, and kits for performing immunohistochemistry on biological samples using tyramide signal amplification, which uses chromogenic or fluorescent dyes to enhance target detection and allows the dyes to be removed after detection. Multiplexed detection can be performed at high multiplex levels through successive imaging rounds, where multiple dyes corresponding to multiple targets can be detected in each imaging round, and amplification can be used for some or all of the targets.

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

[0008] One or more dyes labeled with countersense oligonucleotide sequences are introduced, which hybridize to and detect corresponding (i.e., complementary) TSA-deposited oligonucleotide sequences, 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 corresponding 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 groups S are orthogonal, i.e., a given S i Countersense sequence of S i ' is substantially S under stringent conditions. i hybridizes only with i and 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 number of cross-links with the sequence S i Array of 'S' i This means that the amount of binding to the antibody is less than 1% of the amount of binding to the antibody.

[0010] As is typical during antibody incubations in immunohistochemistry, blocking and washing steps can be performed to minimize nonspecific binding during incubation and to remove excess antibody afterwards, which may be followed by a fixation step to more firmly bind the primary antibody to the sample and reduce the likelihood that the antibody will be removed during subsequent steps.

[0011] An enzyme, e.g., horseradish peroxidase (HRP), is introduced into the countersense sequence S i and applied to the sample, where the countersense sequence S i ' represents the corresponding sequence S linked to the primary antibody by binding at least at the first binding region i Stringent or near-stringent conditions can be used to minimize cross-hybridization with other sequences and / or other locations in the sample. The primary antibody hybridizes to a different sequence S drawn from the orthogonal group S. i Because the primary antibody is conjugated to the IgG, little or no enzyme is localized to the primary antibody that specifically binds to other target analytes.

[0012] Alternatively, the localization agent may be an oligonucleotide sequence S conjugated to a substrate for the HRP enzyme. kIn some preferred embodiments, the substrate may be a tyramine compound (i.e., a tyramine-containing compound) that is a tyramine derivative, p-hydroxycinnamic acid, or a compound containing a derivative of p-hydroxycinnamic acid. Suitable tyramine derivatives include, but are not limited to, tyramine moieties having one or more (e.g., two or more, three or more) substituents on the amine group, such as one or more alkyl, alkenyl, alkynyl, hydroxyl, halide, and / or alkoxy groups. Suitable p-hydroxycinnamic acid derivatives include, but are not limited to, those described in Taofiq et al., Molecules 22(2): 281 (2017), the entire contents of which are incorporated by reference.

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

[0014] An oligonucleotide-labeled enzyme substrate is then applied to the sample, and through tyramide signal amplification, the sequence S k This results in the deposition of oligonucleotide molecules having the following structure into the sample: A dehybridization step is performed to release the enzyme (e.g., HRP) to which the oligonucleotide is conjugated from its associated antibody, and the enzyme is removed by one or more washing steps.

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

[0016] Amplified deposition can be performed for multiple antibody species, and different S sequences can be detected at sample locations corresponding to multiple primary antibodies. k The number of rounds of oligodeposition amplified by TSA can be one if amplification is desired for only one target in the sample, or some number M less than the number N of primary antibodies if amplification is desired for a subset of markers, or N if amplification is desired for all markers.

[0017] Detection can be achieved by incorporating dye-labeled oligonucleotides, each of which has the sequence S k The dye molecule conjugated to each such oligonucleotide is bound to the corresponding sequence S by binding at least at the first binding region. k The dye hybridizes to an oligonucleotide sequence deposited by a TSA having the following structure: Stringent or near-stringent conditions can be imposed to minimize binding of the dye to other sites in the sample, along with a wash step to remove excess oligonucleotide-conjugated dye molecules.

[0018] The sample is then imaged under a fluorescence microscope if a fluorescent dye is used, or under a bright-field microscope if a chromogenic dye is used. This constitutes one round of detection. A dehybridization step is then performed to release the oligo-labeled dyes, which 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) are imaged in each detection round, with each dye being detected individually using a multi-channel fluorescence microscope. In certain embodiments, up to six dyes or more are imaged using spectral imaging and resolution techniques. In general, 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 images.

[0020] A counterstain such as DAPI may be imaged once or during each imaging cycle and used to register the oligonucleotide-labeled dye images obtained from successive imaging rounds to form an overall multiplexed image of the sample, where the images obtained from all rounds are spatially co-registered.

[0021] Thus, highly multiplexed images can be obtained. The overall number of targets that can be imaged, N, is based on the number of antibodies labeled with orthogonal oligonucleotide sequences and is not limited by either the number of dyes, B, that can be imaged in a single imaging round, or the number of TSA-based oligonucleotide deposition rounds, M.

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

[0023] In some embodiments, an enzyme such as HRP can be localized to the antibody site by other means, for example, by indirect labeling to deposit oligonucleotides in the sample. A 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, resulting in the enzyme being indirectly linked to the primary antibody.

[0024] As an example, a primary antibody, which for PDL-1 is the E1L3N clone, can be incubated with the sample to localize to the PDL-1 site on the sample, and a secondary antibody consisting of Leica Power Vision Poly HRP can be added to localize to the site of the primary antibody and deliver the sequence S to the sample, adjacent to the antibody location. k The deposition of oligonucleotides bearing the SEQ ID NO: 1 can be catalyzed using a stripping technique such as elution with a citrate antigen retrieval solution to remove the primary and secondary antibodies, leaving behind the deposited oligonucleotides. This can be used in conjunction with other antibodies and other selected SEQ ID NO: 1. kcan be repeated with , resulting in the deposition of multiple HRP-amplified oligonucleotides corresponding to multiple targets, which can be detected using oligo-labeled dyes as described above.

[0025] The dyes used in each labeling and imaging cycle may be the same or different, and the same or different numbers of dyes may be used and imaged in each cycle. While the goal is typically to image all of the intended targets, the specific dyes used and the manner in which they are grouped into rounds may 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 TSA-deposited oligonucleotides, while in another imaging round, a relatively short exposure time may be selected to image other targets with fewer TSA-deposited oligonucleotides. Grouping targets in a selected manner may be beneficial in terms of practical factors such as exposure time, image registration, and other factors specific to a particular workflow.

[0026] The biological sample may be selected from the group consisting of biological tissue, cultured cells, and cells harvested from an animal subject of interest. In some embodiments, the biological sample comprises material of human or murine 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 may include material derived from a tissue section, a tissue microarray (TMA), a cell pellet, a core biopsy, a needle biopsy, or cells obtained from a blood or plasma sample.

[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 comprises an 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, e.g., a protein, or another antibody or antibody fragment (e.g., indirectly linked).

[0029] In some embodiments, the oligonucleotide sequence S conjugated to the antibody i In some embodiments, S comprises a plurality of ribonucleic acids. In some embodiments, it comprises a plurality of deoxyribonucleic acids. In some embodiments, S i The oligonucleotides are 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. i The oligonucleotides are 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 length.

[0030] In some embodiments, S iOligonucleotides are 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.

[0031] In some embodiments, S i The oligonucleotide comprises one or more synthetic nucleotides. i The oligonucleotide is entirely single-stranded. i The oligonucleotide is partially double-stranded.

[0032] In some embodiments, the S i 'The first binding region of the oligonucleotide is the first oligonucleotide S i In some embodiments, S i The first 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. i The first binding region of the oligonucleotide is 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 length.

[0033] In some embodiments, S i The binding region of the oligonucleotide is 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 in length.

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

[0035] In some embodiments, S iThe 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, the second 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. In some embodiments, the second 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.

[0036] In some embodiments, S i The oligonucleotide comprises one or more synthetic nucleotides. In some embodiments, S i The oligonucleotide is entirely single-stranded. 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, such as hematin, that can mimic HRP. In some embodiments, the enzyme can be soybean peroxidase.

[0038] In some embodiments, the oligonucleotide sequence S conjugated to the substrate material k In some embodiments, S comprises a plurality of ribonucleic acids. k In some embodiments, S comprises a plurality of deoxyribonucleic acids. k The oligonucleotides are 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. k The oligonucleotides are 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 length.

[0039] In some embodiments, S k Oligonucleotides are 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.

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

[0041] In some embodiments, S k The oligonucleotide binding region is 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. k The first binding region of the oligonucleotide is 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 length.

[0042] In some embodiments, S k The binding region of the oligonucleotide is 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 in length.

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

[0044] In some embodiments, S k 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, the second 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. k 'Oligonucleotides are 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 in length.

[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 substituted variants of these species.

[0047] In some embodiments, all amplification steps are performed first, followed by one or more rounds of detection through hybridization with oligo-labeled dyes, imaging, and, optionally, removal of the oligo-labeled dyes. In some embodiments, amplification is performed on samples located elsewhere than on the microscope, which may improve utilization of the device or may allow for the use of specialized processing equipment, such as an automated stainer, for the amplification steps. In other embodiments, amplification is performed on samples on the microscope. This allows for a fully automated workflow that does not require robotics or human intervention to move samples 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 interwoven. In some embodiments, the steps described herein can be repeated.

[0049] In certain aspects, the disclosure features a method for imaging an analyte in a biological sample, the method including: contacting the biological sample with a binding agent, where 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, where the catalytic agent is characterized by a second nucleotide sequence linked to an enzyme, where the second nucleotide sequence hybridizes to the first nucleotide sequence; contacting the biological sample with a localizing agent, where the localizing 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, where the labeling agent is characterized by a fourth nucleotide sequence linked to an optical label, where the fourth nucleotide sequence hybridizes to the third nucleotide sequence; and 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 location of the analyte is indicated by the optical label.

[0050] In another aspect, the disclosure provides a method for imaging multiple analytes in a biological sample, comprising the steps of: (a) contacting the biological sample with a binding agent characterized by a first nucleotide sequence and a binding moiety that selectively binds to one of the analytes; (b) contacting the biological sample with a catalytic agent characterized by a second nucleotide sequence linked to an enzyme, the second nucleotide sequence hybridizing to the first nucleotide sequence; and (c) contacting the biological sample with a localizing agent characterized by a substrate complementary to the enzyme and a third nucleotide sequence linked to the substrate, the third nucleotide sequence hybridizing to the first nucleotide sequence. repeating steps (a)-(c) to deposit N different third nucleotide sequences in the biological sample such that each one 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 featuring a fourth nucleotide sequence linked to a different optical label, wherein the fourth nucleotide sequence hybridizes to only one of the third nucleotide sequences; and acquiring an image of the sample, in which the location of one of the analytes is indicated by the location of one of the optical labels.

[0051] In a further aspect, the disclosure features a method for imaging an analyte in a biological sample, the method including: linking an enzyme to the analyte such that the enzyme localizes to the location of the analyte in the biological sample; contacting the biological sample with a substrate complementary to the enzyme and a localization agent characterized by a first nucleotide sequence to deposit the first nucleotide sequence adjacent to the location 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 acquiring an image of the biological sample, in which the location of the analyte is represented by the location of the optical label.

[0052] Embodiments of any of the methods may include any of the following features.

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

[0054] The first nucleotide sequence may comprise at least 5 nucleotides (e.g., at least 50 nucleotides). The first nucleotide sequence may comprise a DNA fragment. The first nucleotide sequence may comprise an RNA fragment. The first nucleotide sequence may comprise 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 can 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 reproducible binding, the second sequence can be at least 70% (for example, 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 comprise at least 5 nucleotides. The first and second nucleotide sequences may comprise a different number 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 hematin. 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 comprise at least 5 nucleotides (e.g., at least 50 nucleotides). The third nucleotide sequence may comprise a DNA fragment. The third nucleotide sequence may comprise an RNA fragment. The third nucleotide sequence may comprise 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 comprise at least 5 nucleotides. The third and fourth nucleotide sequences may comprise different numbers of nucleotides.

[0060] Optical labels may include fluorescent species. Optical labels may include chromogenic stains.

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

[0062] The analyte may comprise a protein. The analyte may comprise a peptide or peptide fragment.

[0063] The method may include contacting the biological sample with a counterstain before exposing the biological sample to the illuminating light, and exposing the biological sample to the illuminating light and detecting light emitted from the biological sample to form a second image of the sample indicating the location of the counterstain in the biological sample. The counterstain may include DAPI.

[0064] 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 (eg, greater than 5, greater than 50).

[0065] The binding agent may be a first binding agent, the catalytic agent may be a first catalytic agent, the localizing agent may be a first localizing agent, the labeling agent may be a first labeling agent, the analyte may be a first analyte, and the binding moiety may be a first binding moiety. The method may include contacting the biological sample with a second binding agent, wherein the second binding agent is characterized by a second binding moiety that binds to the second analyte in the biological sample and a fifth nucleotide sequence. The second analyte may be different from the first analyte. The second analyte may comprise a protein, peptide, or peptide fragment. The method may include contacting the biological sample with the first and second binding agents simultaneously. The method may include contacting the biological sample with the first and second binding agents 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, and the optical label can be a first optical label, and the method includes contacting the biological sample with a second catalytic agent, wherein the second catalytic agent is characterized by a sixth nucleotide sequence linked to the second enzyme, the sixth nucleotide sequence hybridizing to the fifth nucleotide sequence. The method may include contacting the biological sample with a second binding agent, wherein the second binding agent is characterized by a second substrate complementary to the second enzyme and a seventh nucleotide sequence linked to the second substrate, and 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, wherein 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 comprise an antibody, antibody fragment, or antibody analog. The antibody, antibody fragment, or antibody analog may comprise a member selected from the group consisting of an IgG antibody, an IgM antibody, a monoclonal antibody, a single-chain variable fragment, and a diabody. The fifth nucleotide sequence may comprise at least five nucleotides. The fifth nucleotide sequence may comprise at least one member selected from the group consisting of a DNA fragment and an RNA fragment. The fifth nucleotide sequence may comprise 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 comprise at least 5 nucleotides. The fifth and sixth nucleotide sequences may comprise different numbers of nucleotides.

[0069] The first and second enzymes may be different. The second enzyme may include horseradish peroxidase, a horseradish peroxidase derivative, a horseradish peroxidase mimic, 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 five nucleotides. The seventh nucleotide sequence may include at least one member selected from the group consisting of a DNA fragment and an RNA fragment. 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 may be at least 80% complementary to the seventh nucleotide sequence. The eighth nucleotide sequence may comprise at least 5 nucleotides. The seventh and eighth nucleotide sequences may comprise different numbers of nucleotides.

[0071] The second optical label may comprise a fluorescent species. The second optical label may comprise a chromogenic stain.

[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., 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 does not have to 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 may include removing the first labeling agent from the biological sample before contacting the biological sample with a second labeling agent. The method may include removing the first labeling agent from the biological sample before contacting the biological sample with a second localizing agent. The method may include removing the first labeling agent from the biological sample before contacting the biological sample with a second catalytic agent. The method may include removing the first labeling agent from the biological sample before contacting the biological sample with a second binding agent. The method may include 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 may be a first image, and the method may include exposing the biological sample to illumination light, detecting light emitted from the biological sample, and forming a second image of the biological sample in which the location of a second analyte is indicated by a second optical label. The first optical label may be present in the biological sample when the biological sample is exposed to the illumination light to form the second image of the biological sample.

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

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

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

[0078] All publications, patents, and patent applications mentioned in this specification are herein 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 explanation of the drawings]

[0079] [Figure 1A] FIG. 1A is a schematic diagram showing binding of a binding agent comprising a primary antibody conjugated to a first oligonucleotide to a sample. [Figure 1B] FIG. 1B is a schematic diagram showing hybridization of a catalytic agent to the binder of FIG. 1A. [Figure 1C] FIG. 1C is a schematic diagram showing enzyme-mediated deposition of a localization agent comprising a tyramide-conjugated oligonucleotide in a sample in proximity to the primary antibody of FIG. 1A. [Figure 1D] FIG. 1D is a schematic diagram showing a population of localization agents positioned in proximity to the primary antibody of FIG. 1A in a sample. [Figure 2A] FIG. 2A is a schematic diagram showing a localization agent molecule deposited in a sample in proximity to a primary antibody bound to an analyte of interest. [Figure 2B] FIG. 2B is a schematic diagram showing hybridization of a labeling agent molecule to a localization agent molecule in this system. [Figure 2C] FIG. 2C is a schematic diagram showing imaging of the labeled sample in FIG. 2B. [Figure 2D] FIG. 2D is a schematic diagram showing the sample of FIG. 2B after removal of the labeling agent through dehybridization. [Figure 3] FIG. 3 is a flow chart showing an exemplary set of steps for labeling and imaging a sample to detect multiple different analytes. [Figure 4] FIG. 4 is an image of a tissue section stained with a CD20 antibody linked to an oligonucleotide and labeled with a labeling agent that localizes in the vicinity of the CD20 antibody. [Figure 5] FIG. 5 is an image of the tissue section of FIG. 4 after removal of the labeling agent. 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 multiplexed immunofluorescence method can be performed in several ways. For example, one technique involves contacting a sample with several directly labeled primary antibodies, where each primary antibody can target an antigen of interest and can be conjugated to a different fluorescent dye. In such methods, the antibodies can be applied in a single step; however, the dyes are distinguishable from each other during imaging, so the number of antigen targets, i.e., the degree of multiplexing, is limited by the number of dyes that can be resolved. Also, because there is no amplification mechanism, the number of dye molecules per antigen can be relatively low, set by the dye-antibody conjugation.

[0082] Brighter signals can be obtained using indirect labeling, a technique in which secondary antibodies of different species may be bound to various primary antibodies and fluorescent dyes may be bound to the secondary antibodies. This may offer the possibility of amplification through secondary binding at multiple sites, but it is a more complex approach than direct labeling because it may require that each primary antibody be raised in a different species, or alternatively, that the secondary antibodies be able to recognize different antibodies from the same species.

[0083] Sequential staining techniques have been developed in which a sample can be contacted with a single primary antibody targeting a first antigen. A secondary antibody conjugated to horseradish peroxidase (HRP) can be introduced and localized 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 the dye remains largely bound to the sample. This process can be repeated multiple times, each time using a different primary antibody targeting a different antigen to deposit a different dye. Once the dye is deposited, the sample is imaged.

[0084] Because this is a sequential approach, where only one primary antibody is targeted at a time, there are no issues with cross-species reactivity. This is a significant practical advantage, as antibodies can be selected without concerns about the animal species in which they were raised. 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 an oligomer. For example, several antibodies can be used, each targeting a desired antigen, where each can be conjugated to a different oligomer. The oligomer sequence can be selected or engineered to reduce cross-hybridization between different oligomers. The sample can be contacted with the primary antibodies, which can localize to the antigenic site depending on their type. Fluorescent dyes or other detection moieties, such as quantum dots, can be conjugated to oligomer sequences tailored for use with various primary antibodies. One or more of these can be contacted with the sample under conditions that promote hybridization of the oligomer linked to the detection moiety to its antibody-linked counterpart. In this way, the detection moiety can localize to the antigenic site of the associated antibody, and the sample can be imaged. This is referred to herein as non-amplified oligo-mediated detection. Aspects of such methods are described, for example, in US Pat. Nos. 9,909,167 and 10,370,698, the entire contents of which are incorporated by reference.

[0086] The oligos linked to the detection moieties can be removed by creating conditions favorable for dehybridization and performing a washing step. Multiple primary antibodies, each with a different oligo sequence, can be used in a single experiment, with the corresponding detection moiety-linked oligos hybridized in groups, imaged, and removed. In some workflows, the imaging step may only distinguish between several dyes present in any one group, but the overall measurement can achieve a high level of multiplexing through repetition.

[0087] Sample labeling and imaging The present disclosure features methods 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 methods include labeling a biological sample with a first oligonucleotide S i and contacting the first oligonucleotide with an antibody or antibody fragment conjugated to a second oligonucleotide S i and contacting the second oligonucleotide S with the binding region of i The binding region of the first oligonucleotide S i and a second oligonucleotide S i The fourth oligonucleotide S' is conjugated to an enzyme, which then mediates the deposition of a substance into the biological sample through a TSA reaction. This substance is itself conjugated to a dye that can be imaged using a microscope or similar device. k a third oligonucleotide S that is complementary to at least a portion of k is.

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

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

[0090] In describing the amplification and detection of multiple targets according to the present disclosure, the same subscript and prime notation is used in association with each target, but with S i and S k The actual sequence that S refers to will be different for each target. Thus, in a multiplex experiment with M different targets for which amplification is used, each target will have a sequence of S i and an antibody conjugated to an oligonucleotide represented by S i The actual sequence that S refers to will be different for each antibody. Similarly, the TSA reaction associated with its target will determine the S k The oligonucleotide sequence represented by S is deposited. k The actual sequence referred to will be different for each target. Similarly, the oligo-labeled HRP used in connection with that target is referred to herein as S. i ', but S i The actual sequence indicated by ' is different for each target, and the oligo-labeled dye is k ', but S k The actual sequence that ' refers to will be different for each target.

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

[0092] A biological sample 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 also be contacted with a cocktail of all antibodies or antibody fragments, or a combination of multiple subsets of the total number of antibodies. In addition to the time savings and simplification afforded 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 appears to reduce or eliminate systematic interference between antibodies binding to adjacent or overlapping targets in the sample.

[0093] Antibody incubation and associated antigen retrieval, blocking, and washing steps can be used according to standard immunohistochemistry practices. The specific steps, compounds used, times, temperatures, and order of actions can be optimized based on the target being imaged to obtain good sensitivity, localization, selectivity, or other criteria of interest.

[0094] Each of the one or more antibodies, antibody fragments, or combinations thereof comprises a first oligonucleotide S that is unique to that antibody or antibody fragment. i After contacting the antibody or antibody fragment with the sample, the first oligonucleotide is conjugated to a second oligonucleotide S iFor example, the first oligonucleotide can hybridize to the second oligonucleotide by complementary base pairing, etc. Each first oligonucleotide S i is a unique second oligonucleotide S i ', which corresponds to the orthogonal set of sequences {S1, S2...S N This can be achieved by a bar coding system including

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

[0096] Each second oligonucleotide S i In some preferred embodiments, the second oligonucleotide S' is conjugated to an enzyme capable of mediating the deposition of a detectable substance in a biological sample. i The second oligonucleotide Si' may 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 may be a hemin-containing complex, such as hematin, that can mimic HRP. In some embodiments, the enzyme may 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] Intended S on target antibody iStringent or near-stringent conditions can be used to ensure that little or no binding occurs at sites other than the barcode. To further reduce the possibility of non-specific binding of the enzyme to the sample, the enzyme is not associated with the intended binding target S. i Other oligonucleotide sequences that do not selectively bind to the nucleotide sequence may be similarly applied.

[0098] Excess catalytic agent molecules corresponding to the oligonucleotide-labeled enzyme can be removed from the sample by washing after the enzyme has been localized only or primarily in the vicinity of the associated primary antibody.

[0099] Add oligonucleotide S to the sample using a TSA reaction k The oligo sequence S k is conjugated to a substrate, which may be a tyramine compound or p-hydroxycinnamic acid, or another substrate catalyzed by HRP, so as to bind to the sample according to the TSA mechanism. k The link between the and the substrate material may be indirect, for example, via an additional linker oligonucleotide or by another mechanism.

[0100] Array S k A localization agent containing an enzyme substrate labeled with an oligonucleotide having the sequence S i The enzyme linked to the oligonucleotide having the sequence S' binds to the i An oligonucleotide having the oligo sequence S is bound to the linked antibody, nominally only at the position corresponding to this antibody. k Deposition occurs.

[0101] As mentioned above, deposition of a localization agent results in an amplification of the imaging signal associated with a particular target analyte. The amplification factor, represented in this discussion by α, is the ratio of the number of oligonucleotide sequences S deposited in the sample per antibody molecule localized to the target site in the sample.k The number of molecules having the deposited sequence S k Each of the oligonucleotides in 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 each of the sequences S for each target analyte molecule 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 Because the sequence is labeled with a primary antibody linked to a single oligonucleotide molecule, the amplification factor α is effectively i of the sequence S in the sample relative to the amount or concentration of the oligonucleotide k corresponds to the ratio of the amount or concentration of oligonucleotides. The ratio of amounts or concentrations (i.e., amplification factor) may be 1.1 or greater (e.g., may be 1.5 or greater, may be 2.0 or greater, may be 3.0 or greater, may be 4.0 or greater, may be 5.0 or greater, may be 7.0 or greater, may be 10.0 or greater, may be 20.0 or greater, may be 30.0 or greater, may be 40.0 or greater, may be 50.0 or greater, may be 60.0 or greater, may be 70.0 or greater, may be 80.0 or greater, may be 90.0 or greater, may be 100.0 or greater, may be 200.0 or greater, may be 500.0 or greater, may be 1000 or greater, may be 5000 or greater, may be 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 oligonucleotide-labeled enzyme substrate in the localization agent, reaction time, reaction temperature, and replacement or supplementation of the enzyme substrate conjugated to the oligonucleotide. It can also be modified by adding compounds such as inorganic salts or organic enhancing compounds, such as those described in U.S. Patent No. 6,372,937, the entire contents of which are incorporated by reference. The degree of amplification can be adjusted separately for each target analyte by separately depositing the enzyme-mediated localization agent associated with each target analyte.

[0104] Under some conditions, the TSA reaction results in the dimerization of enzyme substrate molecules rather than their deposition in the sample. This can occur when the density of enzyme molecules or oligonucleotide-labeled enzyme substrate molecules is too high. Reducing one or both of these factors can reduce the effect of dimerization and result in higher levels of deposition. For example, the concentration of oligonucleotide-labeled enzyme substrate can be reduced, or non-polymeric HRP enzyme can be used instead of polymeric HRP enzyme.

[0105] The deposition cycle consists of the sequence S i the oligonucleotide sequence S corresponding to the antibody linked to the oligonucleotide i This can be done for each antibody in turn using an enzyme (e.g., HRP) linked to an oligonucleotide having the sequence S k This is repeated for each antibody for which amplified detection is desired, where each type of antibody has a different sequence S i is linked to a different oligonucleotide having

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

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

[0108] Oligonucleotide sequence S deposited on the sample k is S over at least a portion of its length k an oligonucleotide sequence S that selectively hybridizes with k The detection can be performed using dye molecules conjugated to the

[0109] Detection is achieved by introducing a labeling agent containing a dye molecule labeled with an oligonucleotide, an oligonucleotide sequence S conjugated to the dye molecule, k ' is a sequence S deposited in a sample or conjugated to an antibody k providing stringent or near-stringent hybridization conditions that selectively hybridize to oligonucleotides having the formula: (I) (II) (III) (III), removing dye molecules labeled with non-hybridized oligonucleotides, optionally applying a counterstain, e.g., DAPI, imaging the sample using a microscope to form an image of the dye molecules, and optionally removing the oligo-labeled dye molecules by dehybridization and washing steps.

[0110] In summary, the steps of binding an oligonucleotide-labeled dye molecule to a site in the sample where a complementary localization agent is present, applying a counterstain, imaging the sample, and removing the dye molecule form one detection cycle.

[0111] As mentioned above, an important aspect of certain methods described herein is that amplified detection of target analytes can be performed, followed by removal of the labeling agent from the sample, thereby generating a signal that is observed during imaging of the sample. As a result, multiple cycles consisting of sample labeling, imaging, and optionally label removal can be performed consecutively. The dyes used in each cycle can be removed by dehybridization and washing steps so that they do not interfere with labeling imaging in subsequent cycles. An overall high degree of multiplexing to detect 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 step can be 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, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 70 or more, or even more).

[0114] The number M of different types of labeling agents that can be deposited in a sample to detect target analytes can be 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). In some embodiments, M is less than or equal to N.

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

[0116] If amplification is performed, detection of the target analyte can occur at any time after deposition of the localization agent for that target. Deposition and detection steps can be alternated, 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, one or more detection steps can be performed to label and detect the localization agents, and further cycles of deposition and detection can follow.

[0117] In some embodiments, localization agents may be deposited on a sample for all target analytes of interest prior to any detection steps (e.g., introduction of a labeling agent and imaging the sample). Localization agents can be deposited using purpose-built equipment, such as automated stainers, microfluidic systems with staining chambers, and other systems. Such systems automatically dispense reagents and manage temperatures, processing times, and flow rates without user intervention. One advantage of such systems is that the sample is not repeatedly cycled between the stainer and the imaging station. Suitable systems for applying the reagents and methods herein are described, for example, in U.S. Patent Application No. 16 / 902,215 and U.S. Patent 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 specimen at a time, or on two, three, four, five, six, or even more specimens at a time, along with one or more counterstains that nonspecifically bind to regions in the sample (e.g., one or more tissue counterstains, one or more nuclear counterstains). Different labeling agents, each carrying an oligonucleotide-labeled dye molecule, are introduced and hybridized at a time, and the nucleotide sequence S of the labeling agent is detected. k Since the 's' are orthogonal as described above, each dye molecule localizes to a 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 containing the dye.) The number of dyes, the dyes selected, and the imaging process are chosen to ensure that the signals associated with each dye, and therefore each target analyte, can be distinguished from one another in the resulting image.

[0119] It is instructive to compare the method described herein with other sequential multiplexing methods. A 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 is imaged and then dissolved using alcohol. Each IHC round yields a single marker and requires blocking, primary antibody incubation, washing, secondary antibody incubation, further washing, and staining. The sample is then imaged and washed in water, three dilutions of ethanol, water, potassium permanganate, and water. These steps take a total of approximately three hours per marker; therefore, a 12-plex assay requires 36 hours of sample processing, excluding the time it takes to image the samples 12 times.

[0120] A multiplexed imaging method was described in U.S. Patent No. 7,729,125, in which normal prostate samples were imaged using 11 immunomarkers and DAPI. It involved two rounds of two-channel indirect IHC, followed by seven rounds of direct-labeled IHC, with dye quenching using NaOH solution between each staining round, followed by washing in PBS. Each indirect IHC round took approximately two hours, each direct IHC round took approximately one hour, and each quenching step took more than 15 minutes. Four markers had signal amplification through secondary antibodies, and seven rounds had no amplification. Overall, a total of 14 hours of sample processing was required, excluding the time it took to image the samples nine times.

[0121] The methods described herein can be performed with a single incubation step for all primary antibodies. Amplification of TSA deposition can be performed separately for each target sample for which amplification is desired, and takes approximately 30 minutes per target sample, including hybridization, washing, TSA deposition, washing, and dehybridization. Detection can be performed for four or more species per cycle using, for example, four dyes (e.g., Opal 520, Opal 570, Opal 620, and Opal 690, available from Akoya Biosciences, Inc., Menlo Park, CA) with a DAPI counterstain, and imaging can be performed using a Vectra Polaris instrument (available from Akoya Biosciences, Inc.). Each detection round requires approximately 30 minutes, including approximately 10 minutes for imaging. Excluding the primary incubation, the overall sample processing time for this example is 3.5 hours if 4 species are amplified, or 7.5 hours if 12 species are amplified.

[0122] This comparison illustrates several beneficial aspects of the methods described herein, including the option to use a single incubation with one cocktail of all primary antibodies, the ability to image many N targets with amplification, the option to use amplification for some targets and not others, the ability to obtain high amplification of markers when desired, the ability to label and image multiple targets B in a single detection round, as well as the overall speed.

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

[0124] In embodiments using chromogenic dye molecules, imaging can be performed using bright-field microscopy, which 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 may be performed for each of one or more target analytes. For example, the oligonucleotide sequence S i The primary antibody conjugated to an oligonucleotide-labeled enzyme, e.g., S i The antibody is contacted with HRP conjugated to sequence S'. k An enzyme substrate labeled with an oligonucleotide having the sequence S k An oligonucleotide molecule having the formula: is deposited on the sample with an amplification factor α1 through a TSA mechanism catalyzed by an enzyme associated with the primary antibody.

[0126] Array Si The enzyme labeled oligonucleotide having the sequence S' is optionally removed from the sample through dehybridization, and the sample is k The oligonucleotide is then contacted with an enzyme (e.g., HRP) conjugated to an oligonucleotide of sequence S'. k An enzyme substrate labeled with an oligonucleotide having the sequence S is applied to the sample. k ' and hybridized with the oligonucleotide of sequence S k The deposited oligonucleotide molecules are amplified by the TSA mechanism with an amplification factor α2 (where α2 is the amplification factor for the sequence S k ' the amount or concentration of oligonucleotide molecules in sequence S k The deposition is carried out in a sample (corresponding to the amount or concentration of oligonucleotide molecules of sequence S k If not removed from the sample by the enzyme conjugated to the oligonucleotide of sequence S, i The amplification is catalyzed by an enzyme conjugated to the oligonucleotide α1. Overall, the two deposition steps achieve an amplification of (α1 × α2).

[0127] Successive rounds of amplified deposition can be used to achieve a higher overall amplification than would be practical in a single deposition step, and / or to finely control the degree of amplification. While the preceding example describes two deposition steps for amplifying a signal associated with a single target analyte, 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 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. Furthermore, 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 amplification deposition steps.

[0128] In some embodiments, one or more amplification cycles or steps are performed on a given target analyte, and detection is performed on the target analyte. Optionally, a determination is made whether further amplification is desired (e.g., based on the measured imaging signal corresponding to the target analyte). In at least some embodiments, one or more further deposition cycles or steps of amplification are then performed on the target analyte, and detection is again performed. This results in two or more images of the sample with different levels of amplification. As noted 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 technique described above is used to image samples with widely varying expression, for which optimal amplification is not known prior to imaging. In other embodiments, the technique is used to image strongly expressing regions of a sample in a first image and weakly expressing regions of the same sample in a second image. In certain embodiments, a comparison of the signal levels in the two images provides an estimate of a second amplification factor, α2. Optionally, a single image is assembled from the first and second images, with a high dynamic range in target expression.

[0130] The biological sample may be fresh, frozen, or fixed. The biological sample may be of animal origin, such as from a human, mouse, rat, cow, pig, sheep, 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 can be fixed using aldehydes, alcohols, oxidizing agents, mercury, picric acid, or HOPE fixatives. The biological sample can alternatively be fixed using heat fixation. Fixation can be achieved by immersion or perfusion. The biological sample can be fresh or frozen. In some preferred embodiments, the sample comprises formalin-fixed, paraffin-embedded (FFPE) tissue.

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

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

[0134] For some clones, these properties were confirmed by combining the antibodies with oligonucleotide S i Therefore, antibodies should be tested by traditional IHC techniques to form an initial assessment of their behavior, and then investigated after conjugation with oligonucleotide sequences for use in the present disclosure to ensure that their performance remains acceptable after conjugation.

[0135] Example Workflow The methods described herein can be implemented in a wide variety of different workflows. Figures 1A-1D and 2A-2D are schematic diagrams illustrating one exemplary implementation of the method. In Figure 1A, a sample 102 is contacted with a binding agent that includes a binding moiety 104 that binds to an analyte 101 in the sample 102. The binding moiety 104 has the sequence S i Suitable binding moieties 104 include any of the binding moieties described herein that specifically bind to a target analyte of interest in sample 102, such as antibodies and antibody fragments.

[0136] In FIG. 1B, the sample 102 is ligated to a complementary sequence S iThe first oligonucleotide 106 is contacted with a catalytic agent comprising a second oligonucleotide 108 having the sequence '. As discussed 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 location of the target analyte 101 in the sample 102.

[0137] 1C, the sample 102 is contacted with a localization agent that includes a substrate 112 that is complementary to the enzyme 110. The substrate 112 has the sequence S k and a third oligonucleotide having the formula: ##STR00002## Suitable substrates include, but are not limited to, tyramine and tyramine derivatives, p-hydroxycinnamic acid, and derivatives thereof, as described herein. A catalytic reaction between the enzyme 110 and the substrate 112 deposits a plurality of localization agent molecules in the sample in proximity to the analyte 101.

[0138] In Figure ID, the catalytic agent has been removed from the sample 102 through dehybridization and washing. The deposited localization agent molecules remain in the sample as shown, since they are covalently bound to the sample.

[0139] 2A shows the same view of sample 102 as in FIG. 1D. In FIG. 2B, the sequence S k A labeling agent comprising a fourth oligonucleotide 116 having the sequence ' 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 proximity to the analyte 101 in the sample 102. Because multiple localization agent molecules have been deposited in the sample for each analyte molecule 101, the measured signal associated with the analyte 101 is amplified.

[0140] 2C, illumination light 120 is incident on the sample 102, and optical labels 118 produce emitted light (e.g., by emitting fluorescence or by absorbing a portion of the illumination light). The emitted light is detected and an image of the sample 102 is formed, where the light emitted from the optical labels 118 indicates the location of the analyte 101.

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

[0142] 3 is a flow chart showing a series of exemplary steps corresponding to one implementation of the methods described herein for detecting an analyte 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 a catalytic agent comprising 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 hybridizing to that first oligonucleotide and localizing the catalytic agent in the sample. Then, in step 306a, the sample is contacted with a localization agent comprising an enzyme substrate linked to a third oligonucleotide. A catalytic reaction between the enzyme and the substrate deposits molecules of the localization agent in the sample near specific target analytes.

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

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

[0146] Steps 308a and 310a are repeated P times for P different groups of labeling agents (as steps 308p and 310p, where p = a...P). In some embodiments, for example, each group of labeling agents P includes only a single labeling agent. In certain embodiments, each group of labeling agents P 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 to the sample and all sample images have been acquired, the procedure shown in Figure 3 is complete.

[0148] Oligonucleotides An oligonucleotide is a molecule comprising multiple nucleotides (e.g., at least some of which may be joined to form a chain). The oligonucleotides described herein may comprise ribonucleic acid. The oligonucleotides described herein may comprise deoxyribonucleic acid. In some embodiments, the oligonucleotides may be of any sequence, including user-specified sequences.

[0149] Often, oligonucleotides can be composed of G, A, T, and C, or bases that can base pair with a high degree of fidelity to complementary nucleotides. Examples of such bases include 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-dihydrouridine, 5,6-dihydrothymine, 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, and 2-thio-uridine, although many others are known. The oligonucleotide may be, for example, an LNA, a PNA, a UNA, or a morpholino oligomer. As used herein, oligonucleotides 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 binder 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 the binding region of a second oligonucleotide, the second oligonucleotide being conjugated to an enzyme capable of mediating the deposition of another molecule on 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 may comprise one or more synthetic nucleotides. Examples of synthetic nucleotides include RNA analogs or DNA analogs. Some synthetic nucleotides may include artificial nucleic acids, which may include peptide nucleic acids, morpholinos and locked nucleic acids, glycolic acid, or threose nucleic acids.

[0152] The first oligonucleotide may have a predetermined length suitable for a particular workflow. In some embodiments, a longer oligonucleotide may be selected. In some embodiments, a shorter oligonucleotide may be selected. Factors that influence the selection of the length of the oligonucleotide may 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 may be entirely single-stranded. In some embodiments, the first oligonucleotide may be partially double-stranded. In some embodiments, the partially double-stranded region may 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, there may be more than one double-stranded region. Some first oligonucleotides may have secondary structure. Some first oligonucleotides may have secondary structure, such that folding of the single strand and / or complementarity to itself may result in one or more double-stranded regions comprising a single strand.

[0157] A second oligonucleotide, which is conjugated to an enzyme to form a catalytic agent, can hybridize to the first oligonucleotide at the binding region of the second oligonucleotide. This interaction can occur through base pairing.

[0158] The binding region of the second oligonucleotide may be at least partially complementary to at least a portion of the first oligonucleotide. In some embodiments, the binding region may be complementary to the 3' end of the first oligonucleotide. In some embodiments, the first binding region may be complementary to the 5' end of the first oligonucleotide. In some embodiments, the first binding region may be complementary to a region between the 3' end and the 5' end of the first oligonucleotide. In some embodiments, the binding region may be complementary to the entire first oligonucleotide. In some embodiments, the binding region may 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 may be entirely single-stranded. In some embodiments, the first oligonucleotide may be partially double-stranded. In some embodiments, the partially double-stranded region may 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, there may be more than one double-stranded region. Some second oligonucleotides may have secondary structure. Some second oligonucleotides may have secondary structure, such that folding of a single strand and / or complementarity to itself may result in one or more double-stranded regions, including a single strand. In some embodiments, the second oligonucleotide may comprise more than one oligonucleotide.

[0163] Array S k is conjugated to an enzyme substrate to form a localization agent suitable for TSA amplification, such as the aforementioned tyramide compounds, p-hydroxycinnamic acid, or derivatives thereof. Conjugation may be indirect, for example, via an additional linker oligonucleotide, via a second antibody or nanobody, or by any of the other mechanisms described above.

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

[0165] The optical label can be a fluorescent dye molecule or moiety, 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 can comprise one or more quantum dots. In certain embodiments, the dye molecule or moiety can comprise one or more chromogenic species.

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

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

[0168] In the methods described herein, a sample is prepared by subjecting a sample to a sequence S iThe oligonucleotide is contacted with one or more binding agents comprising a binding moiety conjugated to the oligonucleotide, the first oligonucleotide having the following structure: (I) a ...

[0169] The sample is further divided into arrays S i and contacting the oligonucleotide with one or more catalytic agents comprising an enzyme linked to a second oligonucleotide having the sequence: '. 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] The sample is further divided into arrays S k The oligonucleotide is contacted with one or more localization agents comprising an enzyme substrate linked to a third oligonucleotide having the following structure: 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] The sample is further divided into arrays S kThe oligonucleotide is contacted with one or more localization agents comprising an optical label (e.g., a fluorescent or chromogenic moiety) linked to the oligonucleotide. 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] Process Reagents and Conditions The antibody, antibody fragment, or other analyte targeting moiety (binder) conjugated to the first oligonucleotide can be delivered to the sample in a first buffer solution. The first buffer solution can include PBS, PBS-T, TBS, TBS-T water, saline, or Kreb's 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] The enzyme molecule (catalytic agent) conjugated to the oligonucleotide can be delivered to the sample in a second buffer solution, which in some embodiments can include PBS, PBS-T, TBS, TBS-T water, saline, or Kreb's buffer.

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

[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, which may comprise PBS, PBS-T, TBS, TBS-T water, saline, or Kreb's buffer.

[0177] In some embodiments, the sample is subjected to a buffer solution that may contain DNA components, protein components, chaotropic agents at concentrations of 5%, 10%, 15%, or 20%, and detergent solutions to promote hybridization.

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

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

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

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

[0182] Compositions and Kits Any of the reagents, molecules, and other substances described herein can be combined to form a composition that is delivered to a biological sample for purposes 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 may be implemented in a variety of forms, including one or more tubular containers, such as vials, blister packages, and other sealed containers. The instructions may be located within, affixed to, or associated with the housing of the kit.

[0184] Imaging system and method A wide variety of different imaging systems can be used to obtain the images described herein. Certain commercially available systems can be used, such as the Vectra Polaris system (available from Akoya Biosciences, Inc.). Embodiments of imaging systems that can be used are described, for example, in U.S. Pat. Nos. 7,155,555, 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 obtain an image of a sample as described herein, the sample is exposed to illumination light from a light source of an imaging system. A detector (e.g., an imaging detector, e.g., a CCD array) of the imaging system is used to detect light emitted from the sample in response to the illumination light, thereby obtaining an image of the sample. The emitted light can be fluorescent 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 to form a two-dimensional image of the biological sample. The emitted light corresponds to the location in the sample where the labeling agent is located, and therefore the location of the analyte is indicated by the optical label of the labeling agent.

[0186] Similar methods are used to obtain images of counterstains applied to samples described herein. Because counterstains bind non-specifically to sample structures, images of the counterstains typically do not reveal the location of specific analytes, but instead provide more general information about the structure, characteristics, and morphology of the sample. [Example]

[0187] To evaluate the above-described method, 5-micron-thick tissue sections 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 CD20 (L20) antibody obtained from Akoya Biosciences, Inc. (Menlo Park, CA) according to the staining instructions provided with 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, tissue sections were fixed in paraformaldehyde, ice-cold methanol, and CODEX® Fixation Reagent (obtained from Akoya Biosciences, Inc.), then washed. Tissue sections were equilibrated with 20% dimethyl sulfoxide (DMSO) in 1× CODEX® Assay Buffer (obtained from Akoya Biosciences, Inc.) for 10 minutes.

[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 HRP-conjugated oligonucleotide 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 having the sequence BX006 (obtained from Akoya Biosciences, Inc.) was conjugated to a tyramine moiety at the 5' end of the sequence via a C10 carboxy linker. The tissue sample was then contacted with the reagent in 1x CODEX® buffer and allowed to react for 10 minutes.

[0191] After the reaction, the HRP-conjugated oligonucleotides were dehybridized 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, tissue sections were incubated with CODEX® reporter reagent Cy5-RX006 (available from Akoya Biosciences, Inc.) in CODEX® hybridization buffer. Tissue sections were imaged using a Keyence microscope.

[0193] Figure 4 shows an image of a tissue section, in which the bright areas correspond to the tyramine-conjugated oligonucleotide sequence (i.e., the BX006 sequence) that localized CD20.

[0194] After imaging, the reporter reagent was dehybridized from the tissue sections using the CODEX® Clear Tissue protocol. Another image of the tissue section from which the reporter reagent had been removed is shown in Figure 5. This image illustrates the absence of signal intensity corresponding to the CD20-localized tyramine-conjugated oligonucleotide sequence, suggesting near-complete removal of the reporter reagent during 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 alternatives will be apparent to those skilled in the art. It will be understood that various alternatives to the embodiments specifically described herein are included within the scope of the present disclosure. [Explanation of symbols]

[0196] 101 specimens 102 samples 104 Joining part 106 First Oligonucleotide 108 Second Oligonucleotide 110 Enzymes 112 Substrate 114 Third Oligonucleotide 116 Fourth Oligonucleotide 118 Optical beacon 120 Illumination

Claims

[Claim 1] 1. A method for imaging an analyte in a biological 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 localization agent, the localization agent comprising 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 comprises a fourth nucleotide sequence linked to an optical label, wherein the fourth nucleotide sequence hybridizes to the third nucleotide sequence; exposing the biological sample to illumination light and detecting light emitted from the biological sample to form an image of the biological sample, with the location of the analyte indicated by the optical label; A method comprising:

Images