Complex formation for parallel detection of multiple analytes in multiplexed applications

Abstract

The technology provided herein relates to multiplexing methods and kits for simultaneously detecting different analytes in a sample by using premixed macromolecular complexes, particularly complexes having a primary antibody and a secondary antibody, where the primary antibody contains the same identifier element.

Description

[Technical Field]

[0001] The technology provided herein relates to multiplexing methods and kits for simultaneously detecting different analytes in a sample by using premixed macromolecular complexes, particularly complexes having a primary antibody and a secondary antibody, where the primary antibody contains the same identifier element. [Background technology]

[0002] The analysis and detection of small amounts of analytes, such as RNA or proteins, in biological and non-biological samples has become a routine procedure in clinical and analytical environments. Numerous analytical methods have been established for this purpose. Some of them use encoding techniques that assign a specific readable code to a particular first analyte that is different from the code assigned to a particular second analyte.

[0003] One of the prior art techniques in this field is so-called "single molecule fluorescence in situ hybridization" (smFISH), which was developed essentially to detect mRNA molecules in a sample. In Non-Patent Document 1, target mRNAs are detected via specific directly labeled probe sets. After one round of hybridization and detection, a set of mRNA-specific probes is eluted from the mRNA, and the same set of probes with a different (or the same) fluorescent label is used in the next round of hybridization and imaging to generate gene-specific color-coding schemes over several rounds. This technique requires several differently tagged probe sets for each transcript, and these probe sets must be denatured after each detection round.

[0004] A further development of this technology does not use directly labeled probe sets. Instead, the oligonucleotides of the probe set provide nucleic acid sequences that act as initiators for hybridization chain reaction (HCR), a technique that allows for signal amplification. See Non-Patent Document 2.

[0005] Immunohistochemistry (IHC) refers to the process of detecting, localizing, and / or quantifying antigens, such as proteins, in biological samples using antibodies specific to those antigens. IHC offers the significant advantage of pinpointing the precise location of a particular protein within a tissue sample. IHC is also an effective method for examining tissue itself. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, including tissues (e.g., fresh-frozen, formalin-fixed, and paraffin-embedded) and cytological samples. Target recognition can be detected using a variety of labels (e.g., chromogenic, fluorescent, luminescent, and radioactive), regardless of whether the target is a nucleic acid or an antigen. Amplification of the recognition event is desirable for robust target detection, localization, and quantification in clinical settings, as the ability to confidently detect low-abundance cellular markers is becoming increasingly important for diagnostic purposes. For example, placing hundreds or thousands of labeled molecules at the site of a marker in response to a single antigen detection event improves the ability to detect that recognition event through amplification.

[0006] However, conventional immunohistochemistry (IHC) methods do not always provide adequate signals for detection, especially for immunological targets that are weakly expressed or that are not efficiently targeted by existing IHC reagents. The above-mentioned immunohistochemistry methods for labeling target analytes can be limited, for example, by the amount of probe dye that can be delivered to specifically label each target analyte in a sample. Because the probe dye is conjugated to a specific antibody, the amount of probe dye that can be applied to a specific location in a sample directly depends on the number of probe dye moieties attached directly to the antibody that binds to the analyte at that location. Therefore, certain immunohistochemistry methods are somewhat limited in their ability to amplify signals resulting from specific target analytes by selectively placing larger amounts of probe dye at locations corresponding to the specific target analytes.

[0007] One of the techniques based on the Dako ARK™ is chromogenic immunohistological staining. This system is designed for staining with mouse primary antibodies on formalin-fixed, paraffin-embedded tissues, cryostat tissues, or cell preparations from any species, including mice. The technology used in this system is based on avidin-biotin and peroxidase methodology. Mixing the primary antibody and biotinylated reagent in solution results in binding of the biotinylated secondary antibody to the primary antibody. A blocking reagent containing normal serum is then added to the mixture. Immunoglobulins present in the blocking reagent bind to any remaining biotinylated reagent not bound to the primary antibody. The biotinylated primary antibody is then applied to the specimen. The specimen is then incubated with streptavidin-peroxidase, followed by reaction with diaminobenzidine / hydrogen peroxide as the substrate-chromogen.

[0008] A drawback of this technique is that the detection system, based on the enzymatic reaction of diaminobenzidine / hydrogen peroxide as a substrate-chromogen, cannot be applied to parallel protein detection (more than two proteins per detection). Furthermore, streptavidin has a strong affinity for biotin, forming a biotin-avidin complex. As the number of parallel protein detections increases, cross-interactions between different primary antibodies and Fab-biotin complexes may occur.

[0009] Thus, the methods for detecting multiple analytes known in the art have numerous disadvantages: in particular, they are inflexible, expensive, complicated, time-consuming, and quite often produce inaccurate results.

[0010] Against this background, the objective underlying the present disclosure is to provide a method that can mitigate or even avoid the disadvantages of the prior art methods. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Lubeck et al. (2014), "Single-cell in situ RNA profiling by sequential hybridization," Nature Methods 11(4), pp. 360-361 [Non-patent document 2] Shah et al. (2016), "In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus," Neuron 92(2), pp. 342-357

[0012] The present disclosure relates to novel multiplexed methods and kits for the parallel detection of different analytes in a sample by using premixed complexes. In particular, the present disclosure relates to the parallel detection of analytes by primary decodable complexes.

[0013] In a first aspect, the present disclosure provides a method for simultaneously detecting different analytes in a sample, comprising: (A) contacting the sample with a set of at least four (4) different analyte-specific probes for detecting at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; is a complex comprising wherein the binding element (Y) and the identifier element (Z) are premixed for each set of analyte-specific probes in separate reaction tubes to form complexes between the binding element (Y) and the identifier element (Z), and then the sample is contacted with the different sets of analyte-specific probes, wherein the identifier element (Z) interacts with and binds to the same identifier element (T) of the binding element (Y); (B) detecting a signal caused by an identifier fluorophore (f) coupled to the identifier element (Z); or (C) i) contacting the sample with a set of different signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connection element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the identifier oligonucleotide (o) contained in the identifier element (Z); (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; (including (C)ii) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide is (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of an identifier oligonucleotide (o) of an identifier element (Z) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein a set of decoding oligonucleotides for an individual analyte is different from another set of decoding oligonucleotides for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connecting element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; ) and (D) detecting a signal caused by the signal element of section C)i) and / or C)ii); and optionally, (E) selectively removing the signal oligonucleotide of section C)i) or the decoding oligonucleotide and signal oligonucleotide of section C)ii) from the sample, thereby essentially maintaining specific binding of the analyte-specific probe to the encoded analyte; and optionally, (F) performing at least three (3) further cycles comprising steps C) through E) to generate an encoding scheme with one codeword per analyte (wherein, in particular, the last cycle may stop at step (D)); The present invention relates to a method, comprising:

[0014] In some embodiments, the present disclosure relates to the use of complexes, each complex comprising an antigen- (analyte-) specific primary antibody and a secondary antibody or antibody fragment, where the secondary antibody or antibody fragment is conjugated (tagged) with an oligonucleotide used to simultaneously detect the antigens (analytes) of interest. The complexes are formed in multiple reaction tubes and added to a biological specimen prior to contacting with the sample. The presence of the antigen (analyte) can be identified through various options using complementary oligonucleotides, thereby enabling simultaneous detection of the analytes.

[0015] In a second aspect, an embodiment of the present disclosure provides a method for simultaneously detecting different analytes in a sample, comprising: contacting the sample with a population of different analyte-specific probe sets that have been separately premixed, wherein the members of each analyte-specific probe set population are a) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); b) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; (including complexes having contacting the analyte-bound complex with a first population of decoding elements comprising probes having a region complementary to the identifier oligonucleotide (o) and a region having a first signal element binding domain; contacting a first population of decoding elements bound to the analyte-bound complexes with a first signal element probe comprising a first fluorophore, detecting the first fluorophore emission spectrum, removing the first population of decoding elements, contacting the analyte-bound complexes with a second population of decoding elements comprising probes having a region complementary to an identifier oligonucleotide (o) element and a region having a second signal element binding domain, contacting the identifier element-bound analyte complexes with a second signal element probe comprising a second fluorophore, detecting the second fluorophore emission spectrum, and correlating the pattern of first fluorophore emission spectra and second fluorophore emission spectra that corresponds to the order of addition of the first and second populations of decoding elements; The present invention relates to a method, comprising:

[0016] In a second aspect, an embodiment of the present disclosure provides a method for simultaneously detecting different analytes in a sample, comprising: a) contacting a biological sample with a set of at least four (4) different analyte-specific antibodies or antibody fragments for detecting at least four (4) different analytes, wherein the antibodies or antibody fragments of each set bind to a different analyte compared to the antibodies or antibody fragments of another set, and wherein the antibodies or antibody fragments in all sets comprise the same (Fc) region, and wherein the different antibodies or antibody fragments in the different sets are coupled to a secondary antibody or antibody fragments that bind to the (Fc) region of the analyte-specific antibody or antibody fragment in each set of analyte-specific probes. and antibody fragments to form complexes between the analyte-specific antibodies or antibody fragments and secondary antibodies, wherein the secondary antibodies each comprise an identifier oligonucleotide (o), and wherein the identifier oligonucleotide (o) contained in the secondary antibodies in each set is different from the identifier oligonucleotide (o) in another set; b) contacting the analyte-bound complexes with a decoding oligonucleotide; c) contacting the decoding oligonucleotides bound to the complexes with a signal oligonucleotide; and d) detecting the signal oligonucleotide bound to the decoding oligonucleotide contained in the analyte-bound complexes.

[0017] In a third aspect, an embodiment of the present disclosure provides a method for simultaneously detecting different analytes in a sample, comprising: (A) contacting the sample with a set of at least four (4) different analyte-specific probes for detecting at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) and / or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; is a complex having wherein the binding element (Y) and the identifier element (Z) are premixed in each set of analyte-specific probes to form a complex between the binding element (Y) and the identifier element (Z), and then the sample is contacted with a different set of analyte-specific probes, wherein the identifier element (Z) interacts with and binds to the identifier element (T) of the binding element (Y); (B) detecting a signal caused by an identifier fluorophore (f) coupled to the identifier element (Z); or (C) i) contacting the sample with a set of different signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connection element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the identifier oligonucleotide (o) contained in the identifier element (Z); (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; or (C)ii) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide is (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of an identifier oligonucleotide (o) of an identifier element (Z) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein a set of decoding oligonucleotides for an individual analyte is different from another set of decoding oligonucleotides for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connecting element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; ) and (D) detecting a signal caused by the signal element of section C)i) and / or C)ii); and optionally, (E) selectively removing the decoding oligonucleotides and signal oligonucleotides of section ii) from the sample, thereby essentially maintaining specific binding of the analyte-specific probes to the encoded analytes; and optionally, (F) performing at least three (3) further cycles comprising steps C)ii) through E) to optionally generate a coding scheme with one codeword per analyte (wherein, in particular, the last cycle may stop at step (D)); The present invention relates to a method, comprising:

[0018] In a third aspect, an embodiment of the present disclosure provides a mixture for simultaneously detecting different analytes in a sample, the mixture comprising at least four (4) different sets of analyte-specific probes for encoding at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe is (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; The present invention relates to a mixture comprising a complex having

[0019] In particular, this detailed description describes the use of antibodies in combination with secondary antibodies, Fabs, or proteins that specifically bind to the antibody or protein that recognizes and specifically binds to another protein. Thus, the present disclosure relates to parallel detection of analytes using primary decodable complexes. This refers to a complex of antigen-specific primary antibodies and secondary antibody or Fab-conjugated oligonucleotides used to simultaneously detect antigens of interest. The complexes between the primary and secondary antibodies are formed in multiple reaction tubes and added to a biological specimen. The presence of the antigen can be identified through various options using complementary oligonucleotides. This allows for simultaneous detection of analytes, particularly proteins.

[0020] In a further aspect, an embodiment of the present disclosure is a kit for simultaneously detecting different analytes in a sample, the kit comprising at least four (4) different sets of analyte-specific probes for encoding the at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe is (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; The present invention relates to a kit comprising a complex having

[0021] Before describing the present disclosure in detail, it is to be understood that the present disclosure is not limited to the specific component parts of the described method steps. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Furthermore, when a range of parameters bounded by numerical values ​​is presented, it is to be understood that the range is intended to include these limits. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 shows 1) an example of an identifier element (Z) that specifically interacts with and binds to an identifier element (T) of a binding element (Y), and 2) an example of a complex comprising a binding element (Y) that specifically interacts with one of different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T) and (bb) an identifier element (Z) that specifically interacts with and binds to the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or identifier oligonucleotide (o) used to identify the identity of the identifier element (Z), and wherein the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes. [Figure 2] FIG. 1 shows a schematic diagram illustrating a method for sample analysis according to the present disclosure. [Figure 3]Figure 1 shows frozen sections of mouse kidney probed for nestin. Rabbit primary antibodies against nestin were prelabeled with Alexa Flour 647 conjugated to goat anti-rabbit IgG (upper panel) or goat anti-rabbit Fab (lower panel) at a ratio of 1:0.2 and 1:2 (molecule / molecule), respectively, conjugated to goat anti-rabbit IgG or goat anti-rabbit Fab. Prelabeling was performed for 30 minutes, and residual secondary antibodies or Fabs unbound to the primary antibodies were blocked with 0.1% (volume / volume) normal rabbit serum for 5 minutes. The antibody complexes were added to the tissue and incubated overnight. Images were taken with a ZEISS Celldiscoverer 7 using an Apochromat 5X / 0.35 objective and a 2X tube lens (exposure times: 1 second and 0.1 seconds for Alexa Flour 647 and DAPI, respectively). [Figure 4] Figure 1 shows a frozen section of a mouse kidney probed for nestin. Nestin was detected by a primary antibody (Abcam ab176571) precomplexed with an oligo-conjugated secondary antibody, followed by a complementary oligo conjugated to a fluorophore (A). Localization of the primary antibody-secondary antibody complex was confirmed by probing the tissue with a fluorophore-conjugated antibody complementary to the oligo-conjugated secondary antibody (B). DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure features a method for multiplexed labeling, identification, and quantification of target analytes in a biological sample. Using this method, multiple cycles of target analyte labeling, detection, and removal of certain agents involved in the labeling process can be performed without disrupting antibody-analyte binding in the sample. Instead, removal of agents involved in the labeling process is performed by dehybridizing the agents under relatively mild conditions, thus maintaining sample integrity and ensuring near-complete removal of agents during each labeling and detection cycle. As a result, little or no cross-species labeling occurs. Instead, each target analyte in a sample can be selectively labeled with a different labeling species, with the location of each labeling species being highly constrained to a region of the sample that specifically corresponds to the location of a particular target analyte.

[0024] The disclosed method allows for multiplexed labeling, discrimination, signal amplification, and quantification of target analytes in biological samples. Using the disclosed method, multiple cycles of target analyte labeling, detection, and removal of certain agents involved in the labeling process can be performed without disrupting antibody-analyte binding in the sample. Instead, removal of agents involved in the labeling process is performed by dehybridizing the agents under relatively mild conditions, thus maintaining sample integrity and ensuring near-complete removal of agents during each labeling and detection cycle. As a result, little or no cross-species labeling occurs. Instead, each target analyte in a sample can be selectively labeled with a different labeling species, with the placement of each labeling species highly constrained to a region of the sample that specifically corresponds to the location of a particular target analyte. The disclosure describes the use of a set of labeled and unlabeled nucleic acid sequences for specific quantitative and / or spatial detection of different analytes in parallel via specific hybridization. This technology allows for discrimination of more different analytes than the available detection signals allow. This discrimination can be achieved through sequential signal encoding of the analytes, achieved by several cycles of specific hybridization, signal detection, and selective elution of the hybridized nucleic acid sequences.

[0025] In contrast to other state-of-the-art methods, the oligonucleotide that produces a detectable signal does not directly interact with the sample-specific analyte-specific probe, but can also be mediated by a so-called "decoding oligonucleotide." This mechanism separates the dependency between the analyte-specific probe and the signal oligonucleotide. The use of decoding oligonucleotides allows for much greater flexibility, while dramatically reducing the number of different signal oligonucleotides required, thereby increasing the encoding capacity achieved in a given number of detection rounds.

[0026] The use of decoding oligonucleotides provides a sequential signal encoding technique that is more flexible, inexpensive, simple, fast, and / or accurate than other methods.

[0027] In particular, the step of premixing the binding elements (Y) and the identifier elements (Z) in multiple reaction tubes (one separate tube for each type of analyte-specific probe) to form complexes and then contacting the sample with these complexes has the following advantages:

[0028] 1) Reducing Experimental Complexity I: Typically, multiplexed protein detection requires the use of primary ABs generated in different species (goat, rabbit, chicken, etc.) (see Table 1a). Different primary ABs generated in the same species can be used if the complex between the primary antibody (primary AB) and the secondary antibody (secondary AB) or secondary antibody FAB fragment is formed before the primary AB is applied to the sample (see Table 1b below).

[0029] [Table 1]

[0030] [Table 2]

[0031] 2) Reducing Experimental Complexity II: When detection is achieved by an oligonucleotide coupled to an AB, it may be advantageous to form a complex before binding to the sample. In this case, a secondary AB (or FAB) can be used to couple to a different oligonucleotide. The advantage here is that not all primary ABs need to be coupled to the oligonucleotide, but only a limited library of secondary ABs. Secondary ABs (or FABs) can be coupled to oligos in a much larger configuration than individual primary ABs.

[0032] 3) Cost and time savings: Labeling of primary ABs is very costly and time-consuming on a small scale. On the other hand, labeling of secondary ABs (or FABs) can be done on a larger scale and more cost-effectively. Labeled secondary ABs (or FABs) can be used in a general and universal manner that is not possible with fluorophore-labeled primary ABs. Preformation of a complex of a primary AB and a fluorophore-labeled secondary AB (or FAB) can be substituted for the fluorophore-labeled primary AB.

[0033] 4) Increased sensitivity: In highly multiplexed protein detection systems, labeled primary ABs are used to avoid compatibility issues with secondary ABs. In these assays, when labeled primary ABs are used, the sensitivity is limited by the number of labels coupled to the primary ABs. If pre-formation of complexes between the primary ABs and labeled secondary ABs can be performed, multiple secondary ABs can be bound to the primary ABs, and each secondary AB can be labeled multiple times, which can increase sensitivity.

[0034] 5) Time savings: The pre-formation of complexes between the primary AB and the labeled secondary AB (or FAB) can be performed in a highly parallel manner. After pre-formation of the complexes, multiple complexes can be simultaneously incubated with the sample. Sequential detection can then be performed.

[0035] 6) Reduction of background effects I: Background effects can occur due to the primary AB and the secondary AB. When the concentration of the secondary AB is lower than that of the primary AB, the background effect due to the secondary AB can be minimized due to the pre-formation of a complex between the primary AB and the secondary AB. In this case, the secondary AB is almost completely bound to the primary AB, and no secondary AB remains.

[0036] A.Definition According to the present disclosure, an "analyte" is an entity of interest whose presence or absence in a sample is specifically detected and, if present, encoded. An analyte can be any type of entity, including a protein, polypeptide, protein, or nucleic acid molecule (e.g., RNA, PNA, or DNA) of interest. An analyte provides at least one site for specific binding with an analyte-specific probe. As used herein, the term "analyte" may be substituted for "target." An "analyte" according to the present disclosure includes an entity of interest, such as a complex of at least two individual nucleic acid, protein, or peptide molecules. In an embodiment of the present disclosure, the term "analyte" excludes chromosomes. In another embodiment of the present disclosure, the term "analyte" excludes DNA. In an advantageous embodiment, the analyte is a protein / polypeptide.

[0037] As used herein, "contacts" and "contacting" mean that an agent, chemical species, moiety, or other element is associated with a sample or another agent, chemical species, moiety, or element such that the two interact with each other. For example, when a sample containing an analyte is "contacted" with a set of different analyte-specific probes, the analyte-specific probes are in sufficient proximity to associate with the sample and interact with the analyte in the sample.

[0038] In some embodiments, the analyte may be a "coding sequence," "encoding sequence," "structural nucleotide sequence," or "structural nucleic acid molecule," which refers to a nucleotide sequence that is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of a coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding sequences can include, but are not limited to, genomic DNA, cDNA, ESTs, and recombinant nucleotide sequences.

[0039] A "sample" as referred to herein is a composition in liquid or solid form suspected of containing the analyte to be encoded. In particular, the sample is a biological sample, preferably a biological tissue, more preferably a biological sample containing biological cells and / or extracts and / or cell parts. For example, the cells are prokaryotic or eukaryotic cells, particularly mammalian cells, especially human cells. In some embodiments, the biological tissue, biological cells, extracts and / or cell parts are fixed. In particular, the analyte is fixed in a permeabilized sample, such as a sample containing cells.

[0040] As used in this disclosure, "cell," "cell line," and "cell culture" can be used interchangeably, and all such designations include progeny. Thus, the terms "transformants" or "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same functionality as screened for in the originally transformed cell are included.

[0041] A "coding scheme" may describe a set of code words associated with the analytes to be detected. Each code word refers to one of the analytes and is distinct from all other code words. In this case, a code word is a sequence of codes provided by a detection cycle of the method. A code within a code word is the presence or absence of a detectable signal. A code word need not consist of all the different signals used in the method. The number of codes within a code word is determined by the number of detection cycles.

[0042] As used herein, "oligonucleotide" refers to a short nucleic acid molecule such as DNA, PNA, LNA, or RNA. The length of an oligonucleotide is 4 nucleotides (nt) to 200 nt, preferably 6 nt to 80 nt, more preferably 8 nt to 60 nt, more preferably 10 nt to 50 nt, and more preferably 12 nt to 35 nt, depending on the number of consecutive sequence elements. A nucleic acid molecule can be completely or partially single-stranded. An oligonucleotide can be linear or contain a hairpin or loop structure. An oligonucleotide can contain modifications such as biotin, a labeling moiety, a blocking moiety, or other modifications.

[0043] An "analyte-specific probe" is a complex consisting of at least two elements, namely, so-called binding elements (Y) that specifically interact with one of the analytes, where each binding element (Y) of each set of analyte-specific probes contains the same identifier element (T), and a so-called identifier element (Z) that specifically interacts with and binds to the same identifier element (T) of the binding element (Y) and contains an identifier fluorophore (f) or an identifier oligonucleotide (o). The binding element (Y) may be a nucleic acid such as a hybridization sequence or an aptamer, or a peptide structure such as an antibody.

[0044] In particular, the binding element (Y) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts with and binds to the analyte, wherein in particular the antibody fragment is selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandab.

[0045] In particular, the identifier element (Z) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts and binds to the analyte, wherein in particular the antibody fragment is selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandab.

[0046] In advantageous embodiments of the present disclosure, binding element (Y) is an antibody or an antibody fragment such as a Fab, scFv, single domain, bis-scFv, Fab2, Fab3 fragment, and identifier element (Z) is also an antibody or an antibody fragment such as a Fab, scFv, single domain, bis-scFv, Fab2, Fab3 fragment. In advantageous embodiments of the present disclosure, binding element (Y) is an antibody and identifier element (Z) is a secondary antibody.

[0047] In an advantageous embodiment of the present disclosure, the analyte is a protein / polypeptide contained in a biological sample, the binding element (Y) is an antibody or antibody fragment comprising an (Fc) region, and the identifier element (Z) is a secondary antibody, Fab, Fab2 and / or Fab3 fragment.

[0048] In particular, in some embodiments, the binding element (Y) comprises a portion or whole affinity substance that is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptide allergen, a recombinant allergen, an allergen-idiotypic antibody, an autoimmune inducing structure, a tissue rejection inducing structure, an immunoglobulin constant region, and derivatives, mutants, or combinations thereof. In further advantageous embodiments, the antibody fragment is a Fab, scFv, single domain or fragment thereof, bis-scFv, Fab2, Fab3, minibody, maxibody, diabody, triabody, tetrabody, or tandbab, in particular a single chain variable fragment (scFv).

[0049] The "identical identifier element (T)" contained in a binding element (Y) of an analyte-specific probe has the same sequence as other identical identifier elements (T) contained in other binding elements (Y) of other analyte-specific probes. In other words, the identical identifier elements (T) contained in all binding elements (Y) in a set of all analyte-specific probes are identical. In other words, there is only one type of identical identifier element (T) sequence for all binding elements (Y) used in one approach. The identical identifier element may be a sequence element of a binding element (Y) attached directly or via a linker, covalent bond, or high-affinity binding method such as antibody-antigen interaction, streptavidin-biotin interaction, etc.

[0050] The term "analyte-specific probe" is understood to include multiple complexes in which each probe binds to the same analyte but the binding elements (Y) may be different such that they may bind to different portions of the analyte, e.g., different (e.g., adjacent) or overlapping sections of a polypeptide sequence comprising the protein to be detected. However, each of the multiple probes contains the same identical identifier element (T).

[0051] A "decoding oligonucleotide" or "adapter" or " / adapter segment" consists of at least two sequence elements: one sequence element (called the "identifier connection element" (t) or "first connection element" (t)) that can specifically bind to a unique identifier sequence, and a second sequence element (called the "translator element" (c)) that specifically binds to a signal oligonucleotide. The length of the sequence elements ranges from 8 nt to 60 nt, preferably 12 nt to 40 nt, and more preferably 14 nt to 20 nt, depending on the number of analytes to be encoded in parallel, the required stability of the interaction, and the number of different signal oligonucleotides used. The lengths of the two sequence elements may or may not be the same.

[0052] In some embodiments, the decoding oligonucleotide in the kits and / or methods of the present disclosure may be a "multi-decoder." A "multi-decoder" is a decoding oligonucleotide consisting of at least three sequence elements. One sequence element (the identifier connection element (t)) can specifically bind to the identifier oligonucleotide (o) of the identifier element (Z) of the corresponding analyte-specific probe set, and at least two other sequence elements (the translator elements (c)) specifically bind to different signal oligonucleotides (each of these sequence elements specifically binds to a signal oligonucleotide that is different from all other signal oligonucleotides recruited by other elements of the multi-decoder). The length of the sequence elements ranges from 8 nt to 60 nt, preferably 12 nt to 40 nt, and more preferably 14 nt to 20 nt, depending on the number of analytes to be detected in parallel, the required stability, and the number of different signal oligonucleotides used. The lengths of the sequence elements may or may not be the same.

[0053] Thus, in some advantageous embodiments, the decoding oligonucleotide comprises: - an identifier connecting element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of an identifier oligonucleotide (o) of an identifier element (Z) of a corresponding analyte-specific probe set; - at least two translator elements (c), each containing a nucleotide sequence that allows specific hybridization of a different signal oligonucleotide; A multi-decoder including:

[0054] Thus, the first translator element binds to a different signal oligonucleotide than the second translator element, in particular the signal oligonucleotides differ in the type of signal element, e.g., fluorophore, contained therein.

[0055] As used herein, a "signal oligonucleotide" or "reporter" comprises at least two elements: a so-called "translator connection element" (C) or "second connection element" (C) that has a nucleotide sequence capable of specifically hybridizing to at least a section of the nucleotide sequence of the identifier oligonucleotide (o) contained in the identifier element (Z) and / or translator element (c) of the decoding oligonucleotide, and a "signal element" that provides a detectable signal and / or an additional sequence that can be detected by hybridizing to one or multiple labeled complementary oligonucleotides. This element can either actively generate a detectable signal or provide such a signal via manipulation, e.g., fluorescence excitation. Typical signal elements are, for example, enzymes, fluorophores, radioactive elements, or dyes that catalyze a detectable reaction.

[0056] A "set" refers to a plurality of moieties or entities, e.g., analyte-specific probes, signal oligonucleotides, or decoding oligonucleotides, whether the individual members of the plurality are identical or different from one another. In all analyte-specific probe sets, the identity identifier elements (T) of the binding elements (Y) of the analyte-specific probes are identical.

[0057] Thus, in an advantageous embodiment, each analyte-specific probe set comprises premixed complexes in which the complexes of one analyte-specific probe set contain a different binding element (Y) that specifically interacts with one of the different encoded analytes compared to the complexes of the other analyte-specific probe set, but contain the same identifier element (T) that interacts with an identifier element (Z) that contains a different identifier fluorophore (f) or identifier oligonucleotide (o) compared to the identifier element (Z) of another analyte-specific probe set.

[0058] "Selective denaturation" can be a process that maximizes the removal of bound decoding and signal oligonucleotides while simultaneously maintaining the target-specific probe hybridized with maximum efficiency. The combined efficiency of these two events can be at least 0.22 for 2 detection cycles, 0.37 for 3 detection cycles, 0.47 for 4 detection cycles, 0.55 for 5 detection cycles, 0.61 for 6 detection cycles, 0.65 for 7 detection cycles, 0.69 for 8 detection cycles, 0.72 for 9 detection cycles, 0.74 for 10 detection cycles, 0.76 for 11 detection cycles, or 0.78 for 12 detection cycles.

[0059] In one embodiment of the present disclosure, a single set refers to a plurality of oligonucleotides.

[0060] An "analyte-specific probe set" refers to multiple moieties or entities, e.g., analyte-specific probes that are different from each other and bind to separate regions of an analyte. Single analyte-specific probe sets are further characterized by the same identifier element (Z).

[0061] A "decoding oligonucleotide set" refers to multiple decoding oligonucleotides specific to a particular unique identifier that are required to achieve length-independent encoding of a codeword. All decoding oligonucleotides in a "decoding oligonucleotide set" bind to the same identifier oligonucleotide (o) of an analyte-specific probe.

[0062] In certain embodiments, this pattern of binding or hybridization of the decoding oligonucleotides can be converted into a "code word." For example, the code word could be "101" and "110" for one analyte, where a value of 1 represents binding and a value of 0 represents no binding. In other embodiments, the code word may have a longer length (see Figure 13). The code word may be directly associated with the particular unique identifier sequence of the analyte-specific probe. Thus, different analyte-specific probes may match a particular code word, which can then be used to distinguish different analytes of the analyte-specific probe based on the binding pattern of the decoding oligonucleotides. However, if no binding is apparent, the code word in this example would be "000."

[0063] In some embodiments, the values ​​in each codeword can be assigned differently. For example, a value of 0 can represent binding, while a value of 1 can represent no binding. Similarly, a value of 1 can represent binding of the secondary nucleic acid probe to one type of signaling entity, while a value of 0 can represent binding of the secondary nucleic acid probe to another type of distinguishable signaling entity. These signaling entities can be distinguished, for example, via different fluorescent colors. In some cases, the values ​​in the codeword need not be limited to 0 and 1. Values ​​can also be drawn from a larger alphabet, such as a ternary system (e.g., 0, 1, and 2) or a quaternary system (e.g., 0, 1, 2, and 3). Each different value can be represented by a different distinguishable signaling entity, including, for example, one value that can (in some cases) be represented by the absence of a signal.

[0064] The codeword for each analyte may be assigned sequentially or randomly. For example, a first analyte may be assigned 101, while a second nucleic acid target may be assigned 110. Furthermore, in some embodiments, the codeword may be assigned using an error detection or correction scheme, such as a Hamming scheme, Golay code, or extended Hamming scheme (or SECDED scheme, i.e., single error correction, double error detection). Generally speaking, such schemes can be used to identify where an error occurred and, in some cases, can also be used to correct the error and determine what the correct codeword should be. For example, a codeword such as 001 may be detected as invalid and corrected to 101 using such a scheme if 001 has not previously been assigned to a different target sequence. Various types of error correction codes can be used, many of which have previously been developed for use in the computer industry, but such error correction schemes have not typically been used in biological systems. Additional examples of such error correction codes are discussed in more detail below.

[0065] "Essentially complementary" means, with respect to two nucleotide sequences, that both sequences are capable of specifically hybridizing to each other under stringent conditions, thereby forming a hybrid nucleic acid molecule in which the sense and antisense strands are connected to each other via hydrogen bonds (Watson-Crick base pairing). "Essentially complementary" encompasses not only perfect base pairing along the entire strand, i.e., perfect complementary sequences, but also non-perfect complementary sequences that are still capable of hybridizing to each other under stringent conditions. Experts generally agree that "essentially complementary" sequences have at least 88% sequence identity to a completely or perfectly complementary sequence.

[0066] "Percent sequence identity" or "percent identity" similarly refers to aligning the sequence to be compared ("comparison sequence") with the sequence set forth in the detailed description or claims ("reference sequence"), followed by comparing the sequence to the sequence set forth in the claims or detailed description, where percent identity is determined according to the following formula: percent identity = 100[1 - (C / R)], where C is the number of differences between the reference and comparison sequences over the length of the alignment between the reference and comparison sequences, and where (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the comparison sequence; and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence that differs from an aligned base or amino acid in the comparison sequence constitutes a difference, and (iiii) the alignment must start at position 1 of the aligned sequence, and R is the number of bases or amino acids in the reference sequence over the entire length of the alignment with the comparison sequence, where any gaps that occur in the reference sequence are also counted as bases or amino acids.

[0067] If there is an alignment between the comparison sequence and the reference sequence where the percent identity calculated above is about equal to or greater than the specified minimum percent identity, then the comparison sequence has the specified minimum percent identity relative to the reference sequence, even though there may be an alignment where the percent identity calculated above is less than the specified percent identity herein.

[0068] In an "incubation" step as understood herein, the respective moieties or entities, such as probes or oligonucleotides, are brought into contact with each other under conditions well known to those skilled in the art that allow a specific binding or hybridization reaction, such as pH, temperature, salt conditions, etc. Such a step can therefore preferably be carried out in a liquid environment, such as a buffer system well known in the art.

[0069] The "removal" step according to the present disclosure may involve washing away the moiety or entity to be removed, such as a probe or oligonucleotide, under certain conditions known in the art, such as pH, temperature, salt conditions, etc.

[0070] It is understood that in embodiments of the method according to the present disclosure, multiple analytes can be simultaneously encoded. This requires the use of different sets of analyte-specific probes in step (A). The analyte-specific probes of a particular set are different from the analyte-specific probes of another set. This means that the analyte-specific probes of set 1 bind to analyte 1, the analyte-specific probes of set 2 bind to analyte 2, and the analyte-specific probes of set 3 bind to analyte 3. However, all the analyte-specific probes contain the same identical identifier element (T).

[0071] A "kit" is a combination of individual elements useful for carrying out the uses and / or methods of the present disclosure, where the elements are optimized for use together in the method. The kit may also include additional reagents, chemicals, buffers, reaction vials, etc. that may be useful for carrying out the methods of the present disclosure. Such kits integrate all essential elements needed to carry out the methods of the present disclosure, thereby minimizing the risk of error. Thus, such kits allow even semi-skilled laboratory personnel to carry out the methods of the present disclosure.

[0072] The present method is particularly suitable for encoding, identifying, detecting, counting, or quantifying analytes or single analyte molecules in biological samples, i.e., samples that contain nucleic acids or proteins as said analytes. It is understood that the biological sample may be in its natural form (i.e., liquid, semi-liquid, solid, etc.) in its natural environment, or may be processed, for example, as a dry coating on the surface of a device that can be re-liquefied before performing the present method.

[0073] In another embodiment of the present disclosure, the biological tissue and / or biological cells are fixed prior to step (A). For example, in some embodiments, cells and / or tissues are fixed prior to the introduction of probes to preserve the location of analytes, such as proteins or nucleic acids, within the cells. Techniques for fixing cells are known to those skilled in the art. As non-limiting examples, cells can be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, etc. In one embodiment, cells can be fixed using Hepes-glutamate buffer-mediated organic solvent (HOPE).

[0074] This measure has the advantage that the analyte to be encoded, e.g., a nucleic acid or a protein, is immobilized and cannot escape, thus preparing the analyte to be better detected or encoded by the method according to the present disclosure.

[0075] In still further embodiments, within the set of analyte-specific probes, each analyte-specific probe comprises a binding element (S1, S2, S3, S4, S5) that specifically interacts with a different substructure of one of the encoded analytes.

[0076] This measure increases the signal strength obtained at the end of the method or at the end of a cycle, respectively, making the method even more robust and reliable. It is understood that the individual probes of a set bind to the same analyte, but their binding positions or binding sites in or on the analyte are different. Thus, the binding elements S1, S2, S3, S4, S5, etc. of the first, second, third, fourth, fifth, etc. analyte-specific probes bind to different positions or at different positions, which may or may not overlap.

[0077] In some advantageous embodiments according to the present disclosure, at least 20, particularly at least 25, particularly at least 30 different analytes are detected and / or quantified in parallel in a sample. For example, there may be at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 300, at least 1000, at least 3000, at least 10000, or at least 30000 distinguishable analyte-specific probes, which are applied to the sample, e.g., simultaneously or sequentially.

[0078] In some advantageous embodiments for multiplexing, a set of twenty (20) or more different analyte-specific probes is required to encode at least 20 or more, particularly more than 50, 100, or 200 different analytes. In particular, in the multiplexing methods of the present disclosure, at least 20 different groups of analytes (e.g., mRNA molecules, polypeptides / proteins), i.e., tags, are targeted.

[0079] In some advantageous embodiments, at least four rounds are performed to collect information for analyte identification, where multiple readings increase the accuracy of identification and avoid false positives. Unique tags can be identified directly or indirectly by various techniques, including, for example, hybridization with labeled probes, or by sequencing (by synthesis, ligation). In particular, the identity of a tag can be coded using one signal (binary code), two or more signals, where the signal can be a fluorescent label (e.g., attached to an oligonucleotide).

[0080] In some advantageous embodiments according to the present disclosure, the kit does not include a set of analyte-specific probes as defined in item A).

[0081] Preferably, when the analytes in the kit or method according to the present disclosure are nucleic acids, each set of analyte-specific probes comprises at least five (10) analyte-specific probes, particularly at least fifteen (15) analyte-specific probes, particularly at least twenty (20) analyte-specific probes that specifically interact with different substructures of the same analyte. Nucleic acid analytes include specific DNA molecules, such as genomic DNA, nuclear DNA, mitochondrial DNA, viral DNA, bacterial DNA, extracellular DNA or intracellular DNA, and specific mRNA molecules, such as hnRNA, miRNA, viral RNA, bacterial RNA, extracellular RNA or intracellular RNA.

[0082] Preferably, when the analyte in the kit or method according to the present disclosure is a peptide, polypeptide, or protein, each set of analyte-specific probes comprises at least two (2) analyte-specific probes, particularly at least three (3) analyte-specific probes, particularly at least four (4) analyte-specific probes that specifically interact with different substructures of the same analyte.

[0083] In some advantageous embodiments according to the present disclosure, the kit comprises at least two different sets of signal oligonucleotides, where the signal oligonucleotides in each set comprise different signal elements and comprise different connecting elements (C).

[0084] In particular, the kit may comprise at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets comprise the same identifier connection element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets per analyte have different translator elements (c) that comprise nucleotide sequences that allow specific hybridization of the signal oligonucleotides.

[0085] In some embodiments, the kit comprises at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets comprise the same identifier connection element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets for at least one analyte differ in their translator elements (c), which comprise a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0086] In some advantageous embodiments, the number of different sets of decoding oligonucleotides containing different translator elements (c) per analyte corresponds to the number of different sets of signal oligonucleotides containing different connector elements (C). However, the decoding oligonucleotides in a particular set of decoding oligonucleotides may interact with the same identifier oligonucleotide (o) of an identifier element (Z) unique to a particular analyte. In particular, all sets of decoding oligonucleotides for different analytes may contain the same type of translator element(s) (c).

[0087] In another aspect, the present disclosure generally relates to a method including exposing a sample to a plurality of analyte-specific probes; determining, for each analyte-specific probe, the binding of the analyte-specific probe in the sample; creating a code word based on the binding of the analyte-specific probe, a decoding oligonucleotide, and a signal oligonucleotide; and, for at least some of the code words, matching the code word with a valid code word. In certain embodiments, this pattern of binding or hybridization of the analyte-specific probe, the decoding oligonucleotide, and the signal oligonucleotide can be converted into a "code word." For example, the code words for the first analyte and the second analyte can be "101" and "110," respectively, where a value of 1 represents binding, and a value of 0 represents no binding of the decoding oligonucleotide and / or binding of the signal oligonucleotide that does not contain a signal element and / or contains a quenched signal element. Thus, the analyte in the detection round / cycle cannot be detected during imaging.

[0088] To create such zeros (0) in the codeword for each analyte, the kit may include (D) at least a set of signal-free decoding oligonucleotides that bind to specific identifier elements (T) of the analyte-specific probe, wherein the decoding oligonucleotides in the same set of signal-free decoding oligonucleotides interact with the same different identifier elements (T), and wherein each signal-free decoding oligonucleotide includes an identifier connection element (t) that includes a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence, and does not include a translator element (c) that includes a nucleotide sequence that allows specific hybridization of the signal oligonucleotide.

[0089] In some advantageous embodiments, all steps are automated, particularly by using a robotic system and / or an optical multiplexing system according to the present disclosure. In some instances, the steps may be performed in a fluidic system.

[0090] As described above, the method according to the present disclosure creates an encoding scheme with one code word per analyte. Thus, each analyte may be associated with a specific code word, where the code word includes several positions, and each position corresponds to one cycle, resulting in multiple distinct encoding schemes with multiple code words. In particular, the encoding schemes can be predetermined and assigned to the analytes to be encoded.

[0091] As mentioned above, the encoded analyte may be a nucleic acid, preferably DNA, PNA, RNA, in particular mRNA, a peptide, a polypeptide, a protein, or a combination thereof. Accordingly, the binding element (Y) may comprise an amino acid sequence that allows specific binding to the encoded analyte. Examples of binding elements (Y) include an affinity moiety derived from an affinity element selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptide allergens, recombinant allergens, allergen-idiotypic antibodies, autoimmune-inducing structures, tissue rejection-inducing structures, immunoglobulin constant regions, and combinations thereof, or the entire affinity element. In particular, the binding element (Y) is an antibody or an antibody fragment selected from the group consisting of Fab, scFv, single domains or fragments thereof, bis-scFv, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and tandabs.

[0092] By this measure, the method is further developed to the extent that the encoded analyte can be detected by any means adapted to visualize the signal element. Examples of detectable physical characteristics include, for example, light, chemical reaction, molecular weight, radioactivity, etc.

[0093] In some advantageous embodiments, the signal generated by the signal element, and thus in particular the signal oligonucleotide bound to the decoding oligonucleotide and interacting with the corresponding analyte probe bound to the respective analyte, is (a) imaging at least a portion of the sample; and / or (b) the use of optical imaging techniques; and / or (c) the use of fluorescent imaging techniques; and / or (d) multicolor fluorescence imaging techniques, and / or (e) Super-resolution fluorescence imaging technology, is determined by.

[0094] The kits and methods according to the present disclosure may ideally be used in in vitro methods of diagnosing diseases selected from the group including cancer, neurological diseases, cardiovascular diseases, inflammatory diseases, autoimmune diseases, diseases caused by viral or bacterial infections, skin diseases, musculoskeletal diseases, dental diseases, and prenatal diseases.

[0095] Furthermore, the kits and methods according to the present disclosure may also ideally be used in in vitro methods of diagnosing diseases in plants selected from the group comprising diseases caused by biotic stress, preferably of infection and / or parasite origin, or diseases caused by abiotic stress, preferably of nutrient deficiency and / or adverse environment.

[0096] Additionally, kits and methods according to the present disclosure are ideally in vitro methods for screening, identifying, and / or testing substances and / or drugs, comprising: (a) contacting a test sample containing a sample with a substance and / or drug; (b) detecting different analytes in a sample by sequential signal encoding of said analytes using a method according to the present disclosure; The method may also be used in a method including:

[0097] An optical multiplexing system suitable for the method according to the present disclosure, comprising at least: - a reaction vessel containing a kit or part of a kit according to the present disclosure, a detection unit comprising a microscope, in particular a fluorescence microscope; - camera, - liquid handling devices, An optical multiplexing system comprising:

[0098] In some embodiments, the optical multiplexing system may further comprise a heating and cooling device and / or a robotic system.

[0099] In some advantageous embodiments, the methods described herein are used to specifically detect many different analytes in parallel. This technique allows for the differentiation of a greater number of analytes than the number of different signals available. The process involves at least four successive rounds of specific binding, signal detection, and selective denaturation (if a subsequent round is required), ultimately producing a signal code. To separate the dependency between the specific binding of the analyte and the oligonucleotide that produces the detectable signal, a so-called "decoding" oligonucleotide is introduced. The decoding oligonucleotide transfers the information of the analyte-specific probe set to the signal oligonucleotide.

[0100] The present disclosure will now be further described by embodiments that provide additional features, characteristics, and advantages of the present disclosure. The embodiments are purely exemplary in nature and do not limit the scope or scope of the disclosure. Features mentioned in specific embodiments are general features of the present disclosure, which are not only applicable in specific embodiments but also applicable alone in the context of any embodiment of the present disclosure.

[0101] The method disclosed herein is used to specifically detect many different analytes in parallel. This technique allows for the differentiation of a greater number of analytes than the number of different signals available. The process preferably involves at least two successive rounds of specific binding, signal detection, and selective denaturation (if a subsequent round is required), ultimately producing a signal code. To separate the dependency between analyte-specific binding and the oligonucleotide that produces a detectable signal, a so-called "decoding" oligonucleotide is introduced. The decoding oligonucleotide transcribes the information of the analyte-specific probe set into a signal oligonucleotide.

[0102] The present disclosure further provides a method for detecting an analyte, comprising: - attaching a plurality of pre-mixed analyte-specific probes to the analyte, wherein the analyte-specific probes are independently attached to the analyte and the analyte-specific probes in all sets share the same identifier element (T) and include an identifier element (Z) that specifically interacts and binds to the identifier element (T) of the binding element (Y), the identifier element (Z) being coupled to an identifier fluorophore (f) or identifier oligonucleotide (o) used to identify the identity of the identifier element (Z), and wherein the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; - detecting a signal caused by an identifier fluorophore (f) coupled to the identifier element (Z), or - annealing a signal oligonucleotide to the identifier oligonucleotide (o) of the identifier element (Z) contained in the analyte-specific probe complex and hybridizing one or a number of complementary oligonucleotides to the labeled signal oligonucleotide; - detecting a signal caused by the signal element of the signal oligonucleotide or a complementary oligonucleotide, or - annealing a plurality of first decoding oligonucleotides to the analyte-specific probes; - contacting the sample with a set of at least one signal oligonucleotide; - detecting the signal oligonucleotide; - removing a plurality of first decoding oligonucleotides; - annealing a plurality of second decoding oligonucleotides to the analyte-specific probes; - annealing a second signal oligonucleotide to at least one of the plurality of second decoding oligonucleotides; The present invention relates to a method, comprising:

[0103] In summary, the present disclosure relates, inter alia, to the combined use of a primary antibody together with a secondary antibody, FAB, or protein that specifically binds to the primary antibody, or a protein that recognizes and specifically binds to another protein.

[0104] The analyte is specifically a protein, nucleic acid, or another molecule that is specifically recognized and bound by the binding element (Y). The analyte may be part of a eukaryotic, prokaryotic, animal, fungal, or plant cell, an organelle, or an extracellular region within an organism. The analyte may be part of an intact or disintegrated organism or cell. Disintegration of an organism is defined as any type of surgery, extraction, lysis, purification, or other method that disrupts an organism or cell.

[0105] A binding element (Y) is generally defined as a protein or nucleic acid that specifically recognizes and binds to another molecule, X. Y can be an antibody, a fragment of an antibody, such as FAB or F(ab)2, any other protein, an aptamer, or a nucleic acid that specifically interacts with and binds to X.

[0106] Identifier element (Z) is generally defined as a protein or nucleic acid that specifically recognizes and binds to Y. Z can be an antibody, an antibody fragment such as FAB or F(ab)2, any other protein, aptamer, or nucleic acid that specifically interacts with and binds to Y. Furthermore, Z is coupled to an identifier fluorophore (f) or identifier (o) oligonucleotide that is used to identify the identity of Z. The same type of Z can be coupled to different oligonucleotide identifiers (T) to obtain a library of Z coupled to different oligonucleotides. The library can have a size of at least 5, 10, 20, 40, 80, or more than 80 Z coupled to different identifier oligonucleotides.

[0107] The process is divided into several steps:

[0108] 1) preforming at least four different conjugates of each Y and each Z, where Z is coupled to a specific oligonucleotide; a) Y1-Z (identifier oligo T1) b) Y2-Z (identifier oligo T2) c) Y3-Z (identifier oligo T3) d) Y4-Z (identifier oligo T4) forming four different combinations, such as (Time, temperature, concentration, and other conditions may vary for various Y and Z).

[0109] 2) Binding of the complex to the analyte (time, temperature, concentration, and other conditions may vary depending on the application), after which a washing step may be required to reduce the number of non-specific binding events.

[0110] 3) Detecting the analyte-bound YZ complex by hybridizing it to an oligonucleotide coupled to Z. A washing step may then be required to reduce the number of non-specific binding events. Various options are possible for the identification of the identifier: a) The identifier may hybridize to a labeled oligonucleotide that is complementary to the sequence of the identifier. The label may be a fluorophore, an enzyme, or another label that can be detected. b) The identifier may hybridize to an unlabeled oligonucleotide that is complementary to the sequence of the identifier, the oligonucleotide having an additional sequence that can be detected by hybridizing to one or multiple labeled complementary oligonucleotides. c) Identifying an identifier may require multiple rounds of codeword construction.

[0111] This process may require additional steps to complex formation, such as filtration, purification, absorption, size selection, etc., to purify the appropriate complex from the individual components or by-products.

[0112] In particular, the methods of the present disclosure can be used to identify regions of interest, particularly in organs, tissues, or single cells.

[0113] In some advantageous embodiments, the method of the present disclosure can be used for image analysis. In this context, the method of the present disclosure can be used to develop cell segmentation algorithms using immunofluorescence images. The method of the present disclosure can be used to assist in the segmentation of cells in a sample, particularly in tissues. Cell segmentation is an important process because errors in identifying cells and their boundaries directly affect the ability to correctly quantify the expression levels of proteins in these cells. For example, information about the levels of proteins in single cells can be used to answer questions about the set of cell types in a sample, the spatial distribution of these types, and the interactions between different cells and cell types in a tissue.

[0114] A unique advantage of spatial proteomics is that, unlike traditional methods, specific markers do not need to be pre-selected. Because dozens of proteins are profiled in these studies, markers can be selected post-experimentally. Furthermore, there is no need to select a single marker. Even if a single marker does not work for all cells, a combination of such markers may be useful for segmenting all cells.

[0115] Furthermore, the disclosed methods provide spatial information regarding the location of the profiled cells, thereby enabling analysis of cell-cell signaling and cell type composition.

[0116] The method of the present disclosure for detecting analytes such as biomolecules in fixed biological samples can be used to detect up to three analytes, such as proteins on cell surfaces, thereby visualizing cell boundaries. The analytes may be bound by antibodies, antibody fragments, aptamers, or nucleic acids that specifically interact and bind to the analytes, particularly antibody fragments selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibodies, diabodies, triplebodies, tetrabodies, and tandabs.

[0117] In particular, the analytes detected may be sodium-potassium ATPase, plasma membrane calcium ATPase (PMCA), cadherin, CD98, caveolae, HER2, β-catenin, CAH9, E-cadherin, c-MYC, vimentin, among others. The choice of binding element, such as an antibody, will depend on the cell type to be detected. Each tissue has a different cell type composition. Visualizing cell boundaries allows the user to segment cells visually or through computational tools.

[0118] In some further advantageous embodiments, the disclosed method can be used to establish an AI-based image analysis algorithm using immunofluorescence images to identify lesions. Furthermore, advantageous embodiments of the disclosed method can be used for feature extraction, such as nuclear morphology, signal uniformity, signal intensity, and / or centroid. For example, cell morphology often reflects the specific function or state of a tissue. Cells undergo morphological changes and become motile in response to various stimuli, either as part of normal physiological function and development or due to pathological disorders. In some cancers, epithelial cells lose their intercellular connections and adopt prominent exploratory morphologies as they transition from a static to a migratory phenotype. Recognizing such abnormal cell shapes can help appropriately identify malignant tumors (as distinct from benign tumors).

[0119] A further application for the methods of the present disclosure is to determine the expression of proteins and genes of interest via semi-quantitative approaches, for example, semi-quantitative immunohistochemistry (IHC) is a powerful method for investigating protein expression and localization within tissues.

[0120] In some further advantageous embodiments, the methods of the present disclosure can be used for diagnostic and prognostic applications. In particular, the methods of the present disclosure can be used to determine the distribution of target proteins in healthy and diseased organs, tissues, and / or cells. Furthermore, the methods of the present disclosure can be used to predict disease prognosis by using disease-related prognostic and / or predictive biomarkers. A patient's clinical outcome is influenced by various factors, including the intrinsic characteristics of the patient, the disease, or the condition, and the effectiveness of any treatment the patient receives. Some intrinsic characteristics are reflected in prognostic biomarkers, i.e., biomarkers used to identify the likelihood of a clinical event, disease recurrence, or progression in patients suffering from the disease or condition of interest, while other intrinsic characteristics are reflected in predictive biomarkers, i.e., biomarkers used to identify individuals who are more likely to experience favorable or unfavorable effects from exposure to a pharmaceutical or environmental factor than similar individuals without the biomarker.

[0121] In some further advantageous embodiments, the methods of the present disclosure can be used to provide a diagnosis of diseases, such as tumors of unknown primary origin, neurodegenerative disorders, muscle disorders, brain trauma, and infectious diseases. In particular, the methods of the present disclosure are used to predict treatment response. A further application of the methods of the present disclosure is disease subclassification. Disease classification is crucial for compiling statistics on causes of illness (morbidity) and causes of death (mortality).

[0122] Additionally, the methods of the present disclosure can be used to examine gene expression and study gene regulation in normal and pathological tissues.

[0123] In some further advantageous embodiments, the methods of the present disclosure can be used for morphological studies. In particular, the methods of the present disclosure are used to recognize tissue structures and / or classify cell types. In this context, cell type is a classification used to identify cells that share morphological or phenotypic characteristics. Multicellular organisms can contain cells of a wide variety of specialized cell types, such as muscle cells and skin cells, that differ in appearance and function but have the same genome sequence. Cells may have the same genotype but belong to different cell types due to different regulation of the genes they contain. Classification of specific cell types is often performed using microscopy (such as classification from differentiation cluster families, which are commonly used for this purpose in immunology).

[0124] In some further advantageous embodiments, the disclosed methods can be used for multi-omics research. Multi-omics is a biological analysis approach that uses multiple "omes" (such as genomes, proteomes, transcriptomes, epigenomes, metabolomes, and microbiomes) as datasets. By combining these "omes," scientists can analyze complex biological big data to discover novel associations between biological entities, identify associated biomarkers, and construct sophisticated markers of disease and physiological function. In this way, multi-omics integrates diverse omics data to discover consistent and concordant gene-phenotype-environment relationships or associations. In particular, the disclosed methods can be used to study quantitative correlations and spatial colocalization between ISH and ICH. As mentioned above, immunohistochemistry (IHC) is an invaluable tool for detecting, localizing, and quantifying antigens in preserved tissues for research and diagnostic purposes. In situ hybridization (ISH) is a unique molecular analysis method because it provides precise microscopic localization of analytes such as proteins, DNA, mRNA, and nucleic acids such as microRNAs in metaphase spreads and cell and tissue specimens. In particular, the disclosed methods are used for cell type classification based on such multi-omics data. In further embodiments, the disclosed methods are used to study epigenetic modifications and chromatin structure. In particular, the disclosed methods are used to identify spatial distribution and cell-specific ISH signals (cell segmentation).

[0125] In some further advantageous embodiments, the methods of the present disclosure can be used to determine the correlation of genetic abnormalities and / or copy number variations (CNVs) to corresponding proteins, where CNVs are phenomena in which sections of the genome are repeated and the number of repeats within the genome varies between individuals.

[0126] Accordingly, the present disclosure relates to the following:

[0127] Item 1. A method for simultaneously detecting different analytes in a sample, comprising: (A) contacting the sample with a set of at least four (4) different analyte-specific probes for detecting at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; is a complex comprising wherein the binding element (Y) and the identifier element (Z) are premixed for each set of analyte-specific probes in separate reaction tubes to form complexes between the binding element (Y) and the identifier element (Z), and then the sample is contacted with the different sets of analyte-specific probes, wherein the identifier element (Z) interacts with and binds to the same identifier element (T) of the binding element (Y); (B) detecting a signal caused by an identifier fluorophore (f) coupled to the identifier element (Z); or (C) i) contacting the sample with a set of different signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connection element (C) comprising a nucleotide sequence that is essentially complementary to at least a section of the nucleotide sequence of the identifier oligonucleotide (o) contained in the identifier element (Z); (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; (including (C)ii) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide is (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of an identifier oligonucleotide (o) of an identifier element (Z) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein a set of decoding oligonucleotides for an individual analyte is different from another set of decoding oligonucleotides for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connecting element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; ) and (D) detecting a signal caused by the signal element of section C)i) and / or C)ii); and optionally, (E) selectively removing the signal oligonucleotide of section C)i) or the decoding oligonucleotide and signal oligonucleotide of section C)ii) from the sample, thereby essentially maintaining specific binding of the analyte-specific probe to the encoded analyte; and optionally, (F) performing at least three (3) further cycles comprising steps C) through E) to generate an encoding scheme with one codeword per analyte (wherein, in particular, the last cycle may stop at step (D)); A method comprising:

[0128] Item 2. The method according to Item 1, wherein the sample is a biological sample, preferably a biological tissue from a human, animal, or plant, more preferably a biological sample comprising biological cells and / or extracts and / or cell parts.

[0129] Item 3. The method according to any one of Items 1 to 2, wherein the cell is a prokaryotic or eukaryotic cell, an animal cell, a plant cell, in particular a cell from a vertebrate animal, in particular a mammalian cell, in particular a human cell.

[0130] Item 4. The method according to Item 2, wherein the biological tissue, biological cell, extract and / or cell portion is fixed.

[0131] Item 5. The method according to any one of Items 1 to 4, wherein the analyte is fixed in a permeabilized sample, such as a sample containing cells.

[0132] Item 6. The method according to any one of Items 1 to 5, wherein the analyte is a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA.

[0133] Item 7. The method according to any one of Items 1 to 5, wherein the analyte is a peptide, polypeptide, or protein.

[0134] Item 8. The method according to any one of Items 1 to 7, wherein the binding element (Y) is a protein and / or a nucleic acid that specifically recognizes and binds to the analyte.

[0135] Item 9. The method according to any one of items 1 to 8, wherein the binding element (Y) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts with and binds to the analyte, wherein the antibody fragment is selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandab.

[0136] Item 10. The method according to any one of Items 1 to 9, wherein the identifier element (Z) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts with and binds to the identifier element (Z), and wherein the antibody fragment is selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandab.

[0137] Item 11. The method of any one of items 1 to 10, wherein the identifier element (Z) is an antibody fragment selected from the group consisting of Fab, scFv, single domain, bis-scFv, Fab2, Fab3, minibody, diabody, triplebody, tetrabody, and tandab, wherein the antibody fragment is particularly Fab.

[0138] Item 12. The method according to any one of Items 1 to 11, wherein the analyte is a protein contained in a biological sample, the binding element (Y) is an antibody or antibody fragment comprising an (Fc) region, and the identifier element (Z) is a secondary antibody, Fab, Fab2 and / or Fab3 fragment.

[0139] Item 13. The method according to any one of Items 1 to 12, wherein after step A) and before step B), the identifier element (Z) is blocked to prevent cross-reactivity by mixing with a binding element that is specifically specific for the identifier element (Z).

[0140] Item 14. The method according to any one of Items 1 to 13, wherein after step A) and before step B), unbound analyte-specific probes are removed, in particular by washing.

[0141] Item 15. The method according to any one of Items 1 to 14, wherein all steps are automated, particularly by using a robot system.

[0142] Item 16. The method according to any one of Items 1 to 15, wherein all steps are carried out in a fluid system.

[0143] Item 17. The method of any one of items 1 to 16, wherein each analyte is associated with a specific code word, where the code word includes several positions and each position corresponds to one cycle, resulting in multiple distinguishable encoding schemes with multiple code words.

[0144] Item 18. The method according to any one of Items 1 to 17, wherein the encoding scheme is determined in advance and assigned to the analyte to be encoded.

[0145] Item 19. The method according to any one of items 1 to 18, wherein the code word obtained for each analyte in the performed cycle includes a detected signal and at least one element corresponding to an undetected signal.

[0146] Item 20. The method of any one of claims 1 to 19, wherein no signal is detected for at least one analyte within at least one cycle.

[0147] Item 21. The method of any one of Items 1 to 20, wherein for at least one individual analyte, the position of the code word is zero (0).

[0148] Item 22. Codeword zero (0 [CK1]22. The method according to any one of items 1 to 21, wherein the analyte-specific probes for each individual analyte are generated without using a decoding oligonucleotide having an identifier connecting element (t) that comprises a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for each individual analyte.

[0149] Item 23. The method according to any one of Items 1 to 21, wherein if for at least one individual analyte, the position of the code word is zero (0) in this cycle, a corresponding decoding oligonucleotide having an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte is not used.

[0150] Item 24. The method according to any one of Items 1 to 23, wherein the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set contain different signal elements and different connecting elements (C).

[0151] Item 25. The method according to any one of Items 1 to 24, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets contain the same identifier connection element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides in the different sets per analyte have different translator elements (c) comprising a nucleotide sequence that enables specific hybridization of the signal oligonucleotide.

[0152] Item 26. The method according to any one of Items 1 to 25, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets contain the same identifier connection element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides in the different sets per analyte differ in their translator elements (c) comprising a nucleotide sequence that enables specific hybridization of the signal oligonucleotide, wherein only one set of decoding oligonucleotides per analyte is used per cycle, and / or in different cycles, different sets of decoding oligonucleotides are used in combination with the corresponding set of signal oligonucleotides in the same cycle.

[0153] Item 27. The method according to any one of Items 1 to 26, wherein the number of sets of different decoding oligonucleotides containing different translator elements (c) per analyte corresponds to the number of sets of different signal oligonucleotides containing different connection elements (C).

[0154] Item 28. The method according to any one of Items 1 to 27, wherein all sets of decoding oligonucleotides for different analytes contain the same type of translator element(s) (c).

[0155] Item 29. The method according to any one of Items 1 to 28, wherein the set of different decoding oligonucleotides may be included in a premix of the set of different decoding oligonucleotides or may be present separately.

[0156] Item 30. The method of any one of Items 1 to 29, wherein the sets of different analyte-specific probes may be included in a premix of the sets of different analyte-specific probes or may be present separately.

[0157] Item 31. The method of any one of Items 1 to 30, wherein the set of different signal oligonucleotides may be included in a premix of the set of different signal oligonucleotides or may be present separately.

[0158] Item 32. The method according to any one of Items 1 to 31, wherein the sample is a biological sample, preferably a biological tissue, more preferably a biological sample containing biological cells and / or extracts and / or cell parts.

[0159] Item 33. The method according to Item 32, wherein the cell is a prokaryotic or eukaryotic cell, particularly a mammalian cell, particularly a human cell.

[0160] Item 34. The method according to Item 33, wherein the biological tissue, biological cell, extract and / or cell portion is fixed.

[0161] Item 35. The method according to any one of Items 1 to 34, wherein the analyte is fixed in a permeabilized sample, such as a sample containing cells.

[0162] Item 36. The method according to any one of Items 1 to 33, wherein unbound identifier elements (Z) in the set of premixed analyte-specific probes are removed, particularly by washing.

[0163] Item 37. The method according to any one of Items 1 to 35, wherein after step A) and before step B), unbound analyte-specific probes are removed, in particular by washing.

[0164] Item 38. The method according to any one of Items 1 to 37, wherein after step C) and before step D), unbound signal oligonucleotides and / or unbound decoding oligonucleotides are removed, particularly by washing.

[0165] Item 39. The method according to any one of Items 1 to 38, wherein the analyte-specific probe is incubated with the sample to allow specific binding between the analyte-specific probe and the encoded analyte.

[0166] Item 40. The method according to any one of items 1 to 39, wherein the decoding oligonucleotide is incubated with the sample to allow specific hybridization of the decoding oligonucleotide with the identifier element (T) of each analyte-specific probe.

[0167] Item 41. The method according to any one of items 1 to 40, wherein the binding element (Y) comprises an amino acid sequence that allows specific binding to the encoded analyte, in particular, the analyte is a protein.

[0168] Item 42. The method according to any one of Items 1 to 41, wherein the binding element (Y) comprises a portion or the whole affinity substance that is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, an anticalin protein, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptide allergen, a recombinant allergen, an allergen-idiotype antibody, an autoimmune-inducing structure, a tissue rejection-inducing structure, an immunoglobulin constant region, and combinations thereof.

[0169] Item 43. The method according to any one of Items 1 to 42, wherein the binding element (S) is an antibody or an antibody fragment selected from the group consisting of Fab, scFv, single domain or fragment thereof, bis-scFv, Fab2, Fab3, minibody, diabody, triabody, tetrabody, and tandab.

[0170] 44. The signal caused by the identifier fluorophore (f) or signal element is (a) imaging at least a portion of the sample; and / or (b) the use of optical imaging techniques; and / or (c) the use of fluorescent imaging techniques; and / or (d) multicolor fluorescence imaging techniques, and / or (e) Super-resolution fluorescence imaging technology, 44. The method according to any one of items 1 to 43, wherein the detection is performed by

[0171] Item 45. The method of any one of Items 1 to 44, wherein the analyte is a protein, the identifier element (Y) is an antibody comprising a fragment crystallizable region (Fc region) as the identifier element (T), and the identifier element (Z) is a secondary antibody, Fab, Fab2, and / or Fab3 fragment comprising an identifier oligonucleotide (o).

[0172] Item 46. The analyte is a protein, the identifier element (Y) is an antibody comprising a fragment crystallizable region (Fc region) as the identifier element (T), and the identifier element (Z) is a secondary antibody, Fab, Fab2, and / or Fab3 fragment comprising an identifier oligonucleotide (o), wherein the sample is contacted with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide is (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of an identifier oligonucleotide (o) of an identifier element (Z) of a corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein a set of decoding oligonucleotides for an individual analyte is different from another set of decoding oligonucleotides for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connecting element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; 46. ​​The method according to any one of items 1 to 45, comprising:

[0173] Item 47. A premixing step is carried out before step (A) of carrying out at least four different complexes of each Y and each Z, wherein Z is coupled to a specific identifier oligonucleotide (o), - Y1-Z (identifier oligo 1) - Y2-Z (identifier oligo 2) - Y3-Z (identifier oligo 3) - Y4-Z (identifier oligo 4) 47. The method according to any one of items 1 to 46, wherein four different combinations of

[0174] Item 48. The method according to any one of Items 1 to 47, wherein a set of analyte-specific probes comprising a complex between a binding element (Y) and an identifier element (Z) is stored, and then the sample is contacted with a different set of analyte-specific probes.

[0175] Item 49. The method according to any one of Items 1 to 48, wherein the complex between the binding element (Y) and the identifier element (Z) is purified and / or filtered, and then the sample is contacted with the complex contained in the set of different analyte-specific probes.

[0176] Item 50. The method according to any one of Items 1 to 49, wherein the complexes between the binding element (Y) and the identifier element (Z) are premixed in multiple separate tubes.

[0177] Item 51. A kit for simultaneously detecting different analytes in a sample, the kit comprising at least four (4) different sets of analyte-specific probes for encoding at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe is (aa) binding elements (Y) that specifically interact with one of the different encoded analytes, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; A kit comprising a complex having

[0178] Item 52. The kit of Item 51, wherein the sets of different analyte-specific probes are contained in separate multiple tubes.

[0179] Item 53. Identifying regions of interest, image analysis, developing cell segmentation algorithms using immunofluorescence images, assisting in the segmentation of cells in samples, establishing AI-based image analysis algorithms using immunofluorescence images to identify disorders, feature extraction such as nuclear morphology, signal uniformity, signal intensity, and / or centroid, determining the expression of proteins and genes of interest via semi-quantitative approaches, diagnostic and prognostic applications, determining the distribution of target proteins in healthy and diseased organs, tissues, and / or cells, predicting disease prognosis by using disease-related prognostic and / or predictive biomarkers, disease, e.g., primary 51. The method according to items 1 to 50 for use in an application selected from the group consisting of providing a diagnosis of unknown tumors, neurodegenerative disorders, muscle disorders, brain trauma, infectious diseases, predicting treatment response, disease subclassification, investigating gene expression and studying gene regulation in normal and pathological tissues, morphological studies, tissue structure recognition and / or cell type classification, multi-omics studies, studying quantitative correlation and spatial co-localization between ISH and ICH, studying epigenetic modifications and chromatin structure, identifying spatial distribution and cell-specific ISH signals (cell segmentation), and determining correlation of genetic abnormalities and / or copy number variations (CNV) to corresponding proteins.

[0180] Experimental data Frozen sections of mouse kidneys were probed for nestin. The rabbit primary antibody against nestin was prelabeled with Alexa Flour 647 conjugated to goat anti-rabbit IgG (upper panel) or goat anti-rabbit Fab (lower panel) at a ratio of 1:0.2 and 1:2 (molecule / molecule), respectively. Prelabeling was performed for 30 minutes, and residual secondary antibody or Fab unbound to the primary antibody was blocked with 0.1% (volume / volume) normal rabbit serum for 5 minutes. The antibody complex was added to the tissue and incubated overnight. Images were taken using a ZEISS Celldiscoverer 7 with an Apochromat 5X / 0.35 objective and a 2X tube lens (exposure times: 1 second and 0.1 seconds for Alexa Flour 647 and DAPI, respectively). The results are shown in Figure 3.

[0181] Frozen sections of mouse kidneys were probed for nestin. Nestin was detected using a primary antibody (Abcam ab176571) precomplexed with an oligo-conjugated secondary antibody, followed by a complementary oligo conjugated to a fluorophore (A). Localization of the primary antibody-secondary antibody complex was confirmed by probing the tissue with a fluorophore-conjugated antibody complementary to the oligo-conjugated secondary antibody (B). The results are shown in Figure 4.

Claims

1. 1. A method for simultaneously detecting different analytes in a sample, comprising: (A) contacting the sample with a set of at least four (4) different analyte-specific probes for detecting at least four different analytes, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe: (aa) a binding element (Y) that specifically interacts with one of the different analytes to be encoded, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; is a complex comprising wherein the binding element (Y) and the identifier element (Z) are premixed in separate reaction tubes for each set of analyte-specific probes to form complexes between the binding element (Y) and the identifier element (Z), and then the sample is contacted with the different sets of analyte-specific probes, wherein the identifier element (Z) interacts with and binds to the identical identifier element (T) of the binding element (Y); (B) detecting a signal caused by the identifier fluorophore (f) coupled to the identifier element (Z); or (C) i) contacting the sample with a set of different signal oligonucleotides, wherein each signal oligonucleotide comprises: (aa) a translator connection element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the identifier oligonucleotide (o) contained in the identifier element (Z); (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; including), or (C) ii) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide comprises: (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of the identifier oligonucleotide (o) of the identifier element (Z) of the corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein the decoding oligonucleotides of a set for an individual analyte are different from the decoding oligonucleotides of another set for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connection element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; ) and (D) detecting a signal caused by said signal element of sections C)i) and / or C)ii); and optionally, (E) selectively removing the signal oligonucleotide of section C)i) or the decoding oligonucleotide and the signal oligonucleotide of section C)ii) from the sample, thereby essentially maintaining specific binding of the analyte-specific probe to the encoded analyte; and optionally, (F) performing at least three (3) additional cycles comprising steps C) through E) to generate an encoding scheme with one codeword per analyte (wherein, in particular, the last cycle may stop at step (D)); A method comprising:

2. 2. The method of claim 1, wherein the sample is a biological sample, preferably comprising biological tissue from a human, animal or plant, more preferably comprising biological cells and / or extracts and / or cell parts.

3. The method according to any one of claims 1 to 2, wherein the cell is a prokaryotic or eukaryotic cell, an animal cell, a plant cell, in particular a cell from a vertebrate animal, in particular a mammalian cell, in particular a human cell.

4. The method of claim 2 , wherein the biological tissue, biological cell, extract and / or cell part is fixed.

5. The method of any one of claims 1 to 4, wherein the analyte is fixed in a permeabilized sample, such as a sample containing cells.

6. The method according to any one of claims 1 to 5, wherein the analyte is a nucleic acid, preferably DNA, PNA or RNA, in particular mRNA.

7. The method of any one of claims 1 to 5, wherein the analyte is a peptide, polypeptide, or protein.

8. The method according to any one of claims 1 to 7, wherein the binding element (Y) is a protein and / or a nucleic acid that specifically recognizes and binds to the analyte.

9. The binding element (Y) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts with and binds to the analyte, where in particular the antibody fragment is a Fab, scFv, single domain, bis-scFv, Fab ２ , Fab ３ 9. The method of any one of claims 1 to 8, wherein the antibody is selected from the group consisting of: a minibody, a diabody, a triplebody, a tetrabody, and a tandab.

10. The identifier element (Z) is an antibody, antibody fragment, aptamer, or nucleic acid that specifically interacts and binds to the identifier element (Z), where in particular the antibody fragment is a Fab, scFv, single domain, bis-scFv, Fab ２ , Fab ３ 10. The method of any one of claims 1 to 9, wherein the antibody is selected from the group consisting of: a minibody, a diabody, a triple body, a tetrabody, and a tandab.

11. The identifier element (Z) can be a Fab, scFv, single domain, bis-scFv, Fab ２ , Fab ３ 11. The method according to any one of claims 1 to 10, wherein the antibody fragment is an antibody fragment selected from the group consisting of: a minibody, a diabody, a triple body, a tetrabody and a tandabody, wherein in particular the antibody fragment is a Fab.

12. The analyte is a protein contained in a biological sample, the binding element (Y) is an antibody or antibody fragment containing an (Fc) region, and the identifier element (Z) is a secondary antibody, Fab, Fab ２ and / or Fab ３ The method according to any one of claims 1 to 11, wherein the polypeptide is a fragment.

13. 13. The method according to any one of claims 1 to 12, wherein after step A) and before step B), the identifier element (Z) is blocked to prevent cross-reactivity, in particular by mixing with the binding element specific for the identifier element (Z).

14. The method according to any one of claims 1 to 3, wherein after step A) and before step B), unbound analyte-specific probes are removed, in particular by washing.

15. The method according to any one of claims 1 to 14, wherein all steps are automated, in particular by using a robotic system.

16. The method of any one of claims 1 to 15, wherein all steps are carried out in a fluid system.

17. 17. A method according to any one of claims 1 to 16, wherein each analyte is associated with a particular codeword, wherein the codeword comprises several positions and each position corresponds to one cycle, resulting in a plurality of distinguishable encoding schemes with multiple codewords.

18. The method of any one of claims 1 to 17, wherein said encoding scheme is predetermined and assigned to said analytes to be encoded.

19. 19. The method of any one of claims 1 to 18, wherein the code word obtained for the individual analyte in the cycle performed comprises the signal detected and further at least one element corresponding to a signal not detected.

20. The method of any one of claims 1 to 19, wherein no signal is detected for at least one analyte within at least one cycle.

21. The method of any one of claims 1 to 20, wherein for at least one individual analyte, the position of the code word is zero (0).

22. 22. The method of any one of claims 1 to 21, wherein the codeword zero (0) is generated by not using a decoding oligonucleotide having an identifier connecting element (t) that comprises a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for each analyte.

23. 23. The method of any one of claims 1 to 22, wherein if for at least one individual analyte, the position of the code word is zero (0) in this cycle, then a corresponding decoding oligonucleotide having an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe for the individual analyte is not used.

24. 24. The method of any one of claims 1 to 23, wherein the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connecting element (C).

25. 25. The method of any one of claims 12 to 24, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets contain the same identifier connecting element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides in the different sets per analyte have different translator elements (c) that comprise a nucleotide sequence that allows specific hybridization of a signal oligonucleotide.

26. 26. The method of any one of claims 1 to 25, wherein the sample is contacted with at least two different sets of decoding oligonucleotides per analyte, wherein the decoding oligonucleotides in these different sets comprise the same identifier connection element (t) comprising a nucleotide sequence that is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and the decoding oligonucleotides of the different sets per analyte differ in the translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide, wherein only one set of decoding oligonucleotides per analyte is used per cycle and / or different sets of decoding oligonucleotides are used in different cycles in combination with corresponding sets of signal oligonucleotides in the same cycle.

27. 27. The method of any one of claims 1 to 26, wherein the number of sets of different decoding oligonucleotides comprising different translator elements (c) per analyte corresponds to the number of sets of different signal oligonucleotides comprising different connection elements (C).

28. The method according to any one of claims 1 to 27, wherein all sets of decoding oligonucleotides for different analytes contain the same type of translator element(s) (c).

29. 29. The method of any one of claims 1 to 28, wherein the set of different decoding oligonucleotides may be included in a pre-mixture of the set of different decoding oligonucleotides or may be present separately.

30. 30. The method of any one of claims 1 to 29, wherein the sets of different analyte-specific probes may be included in a pre-mixture of sets of different analyte-specific probes or may be present separately.

31. 31. The method of any one of claims 1 to 30, wherein the set of different signal oligonucleotides may be included in a pre-mixture of the set of different signal oligonucleotides or may be present separately.

32. 32. The method according to any one of claims 1 to 31, wherein the sample is a biological sample, preferably comprising a biological tissue, more preferably comprising biological cells and / or extracts and / or parts of cells.

33. 33. The method of claim 32, wherein the cell is a prokaryotic or eukaryotic cell, in particular a mammalian cell, in particular a human cell.

34. 33. The method of claim 32, wherein the biological tissue, biological cell, extract and / or cell portion is fixed.

35. The method of any one of claims 1 to 34, wherein the analyte is fixed in a permeabilized sample, such as a sample containing cells.

36. The method according to any one of claims 1 to 35, wherein unbound identifier elements (Z) in the set of pre-mixed analyte-specific probes are removed, in particular by washing.

37. The method according to any one of claims 1 to 36, wherein after step A) and before step B), the unbound analyte-specific probes are removed, in particular by washing.

38. 38. The method according to any one of claims 1 to 37, wherein after step C) and before step D) unbound signal oligonucleotides and / or unbound decoding oligonucleotides are removed, in particular by washing.

39. 39. The method of any one of claims 1 to 38, wherein the analyte-specific probe is incubated with the sample to allow specific binding of the analyte-specific probe to the encoded analyte.

40. 40. The method of any one of claims 1 to 39, wherein the decoding oligonucleotides are incubated with the sample to allow specific hybridization of the decoding oligonucleotides with the identifier element (T) of each analyte-specific probe.

41. 41. The method according to any one of claims 1 to 40, wherein the binding element (Y) comprises an amino acid sequence that allows specific binding to the encoded analyte, in particular the analyte is a protein.

42. 42. The method of any one of claims 1 to 41, wherein the binding element (Y) comprises a portion or whole affinity substance which is an affinity moiety derived from an affinity substance selected from the group consisting of an antibody, an antibody fragment, an anticalin protein, a receptor ligand, an enzyme substrate, a lectin, a cytokine, a lymphokine, an interleukin, an angiogenic or virulence factor, an allergen, a peptide allergen, a recombinant allergen, an allergen-idiotype antibody, an autoimmune inducing structure, a tissue rejection inducing structure, an immunoglobulin constant region, and combinations thereof.

43. The binding element (S) may be an antibody or a Fab, scFv, single domain, or fragment thereof, a bis-scFv, a Fab ２ , Fab ３ 43. The method of any one of claims 1 to 42, wherein the antibody fragment is an antibody fragment selected from the group consisting of: a minibody, a diabody, a triabody, a tetrabody, and a tandab.

44. The signal caused by the identifier fluorophore (f) or the signaling element is (a) imaging at least a portion of the sample; and / or (b) the use of optical imaging techniques; and / or (c) the use of fluorescent imaging techniques; and / or (d) multicolor fluorescence imaging techniques, and / or (e) super-resolution fluorescence imaging technology; The method according to any one of claims 1 to 43, wherein the detection is performed by

45. The analyte is a protein, the identifier element (Y) is an antibody comprising a fragment crystallizable region (Fc region) as the identifier element (T), and the identifier element (Z) is a secondary antibody, Fab, Fab, or a combination thereof comprising an identifier oligonucleotide (o). ２ , and / or Fab ３ The method of any one of claims 1 to 44, wherein the polypeptide is a fragment.

46. The analyte is a protein, the identifier element (Y) is an antibody comprising a fragment crystallizable region (Fc region) as the identifier element (T), and the identifier element (Z) is a secondary antibody, Fab, Fab, comprising an identifier oligonucleotide (o). ２ , and / or Fab ３ fragments, wherein the sample is contacted with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for each analyte, each decoding oligonucleotide comprises: (aa) an identifier connecting element (t) comprising a nucleotide sequence essentially complementary to at least a section of the identifier oligonucleotide (o) of the identifier element (Z) of the corresponding analyte-specific probe set; (bb) a translator element (c) comprising a nucleotide sequence that allows specific hybridization of a signal oligonucleotide; Including, wherein the decoding oligonucleotides of a set for an individual analyte are different from the decoding oligonucleotides of another set for a different analyte in the first connecting element (t), and contacting the sample with at least one set of signal oligonucleotides, wherein each signal oligonucleotide is (aa) a translator connection element (C) comprising a nucleotide sequence essentially complementary to at least a section of the nucleotide sequence of the translator element (c) contained in the decoding nucleotide; (bb) a signal element and / or an additional sequence that can be detected by hybridizing with one or more labeled complementary oligonucleotides; 46. ​​The method of any one of claims 1 to 45.

47. The premixing step is carried out before step (A) of performing at least four different complexes of each Y and each Z, wherein Z is coupled to a specific identifier oligonucleotide (o), - Y1-Z (identifier oligo 1) - Y2-Z (identifier oligo 2) - Y3-Z (identifier oligo 3) - Y4-Z (identifier oligo 4) 47. The method of any one of claims 1 to 46, wherein four different combinations are formed, such as

48. 48. The method of any one of claims 1 to 47, wherein the set of analyte-specific probes comprising the complex between the binding element (Y) and the identifier element (Z) is stored before contacting the sample with the different set of analyte-specific probes.

49. 49. The method of any one of claims 1 to 48, wherein the complexes between the binding element (Y) and the identifier element (Z) are purified and / or filtered before contacting the sample with the complexes comprised in the different sets of analyte-specific probes.

50. 50. The method of any one of claims 1 to 49, wherein the complexes between the binding elements (Y) and the identifier elements (Z) are premixed in a number of separate tubes.

51. 51. The method of any one of claims 1-50, wherein the sample is contacted with at least four (4) additional sets of analyte-specific probes for detecting at least four additional analytes, wherein each additional set of analyte-specific probes comprises an identical identifier element (T) that is not identical to the identical identifier element (T) comprised in the complex of the first set of four analyte-specific probes.

52. 1. A kit for simultaneously detecting different analytes in a sample, said kit comprising at least four (4) different sets of analyte-specific probes for encoding at least four different analytes, separately, wherein each set of analyte-specific probes interacts with a different analyte, and wherein each analyte-specific probe is (aa) a binding element (Y) that specifically interacts with one of the different analytes to be encoded, wherein each binding element (Y) of each set of analyte-specific probes comprises the same identifier element (T); (bb) an identifier element (Z) that specifically interacts and binds with the identifier element (T) of the binding element (Y), wherein the identifier element (Z) is coupled to an identifier fluorophore (f) or an identifier oligonucleotide (o) that is used to identify the identity of the identifier element (Z), and the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in a particular set of analyte-specific probes is different from the identifier fluorophore (f) or the identifier oligonucleotide (o) of the identifier element (Z) in another set of analyte-specific probes; A kit comprising a complex having

53. 53. The kit of claim 52, wherein the sets of different analyte-specific probes are contained in multiple separate tubes.

54. Identifying regions of interest, image analysis, developing cell segmentation algorithms using immunofluorescence images, assisting in the segmentation of cells in a sample, establishing AI-based image analysis algorithms using immunofluorescence images to identify lesions, feature extraction such as nuclear morphology, signal uniformity, signal intensity, and / or centroid, determining the expression of proteins and genes of interest via semi-quantitative approaches, diagnostic and prognostic applications, determining the distribution of target proteins in healthy and diseased organs, tissues, and / or cells, predicting disease prognosis by using disease-related prognostic and / or predictive biomarkers, disease, e.g., tumors of unknown primary origin, neurological 52. The method of any one of claims 1 to 51 for use in an application selected from the group consisting of providing a diagnosis of degenerative disorders, muscular disorders, brain trauma, infectious diseases, predicting treatment response, disease subclassification, investigating gene expression and studying gene regulation in normal and pathological tissues, morphological studies, tissue structure recognition and / or cell type classification, multi-omics studies, studying quantitative correlation and spatial co-localization between ISH and ICH, studying epigenetic modifications and chromatin structure, identifying spatial distribution and cell-specific ISH signals (cell segmentation), and determining the correlation of genetic abnormalities and / or copy number variations (CNV) to the corresponding proteins.

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