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WO2024179837A1 - Formation complexe pour la détection parallèle de multiples analytes dans de multiples applications - Google Patents

Formation complexe pour la détection parallèle de multiples analytes dans de multiples applications Download PDF

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WO2024179837A1
WO2024179837A1 PCT/EP2024/053689 EP2024053689W WO2024179837A1 WO 2024179837 A1 WO2024179837 A1 WO 2024179837A1 EP 2024053689 W EP2024053689 W EP 2024053689W WO 2024179837 A1 WO2024179837 A1 WO 2024179837A1
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identifier
analyte
specific
oligonucleotides
different
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Treewut RASSAMEGEVANON
Christian Korfhage
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Resolve Biosciences GmbH
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Resolve Biosciences GmbH
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/161—Modifications characterised by incorporating target specific and non-target specific sites
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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    • C12Q2525/205—Aptamer
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
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    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/531—Detection characterised by immobilisation to a surface characterised by the capture moiety being a protein for target oligonucleotides

Definitions

  • the ⁇ technology ⁇ provided ⁇ herein ⁇ relates ⁇ to ⁇ multiplex ⁇ methods ⁇ and ⁇ kits ⁇ for detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample simultaneously, ⁇ in ⁇ particular ⁇ by ⁇ using ⁇ premixed ⁇ macromolecule ⁇ complexes ⁇ like ⁇ complexes ⁇ having a ⁇ primary and ⁇ a ⁇ secondary ⁇ antibody, ⁇ wherein ⁇ the ⁇ primary ⁇ antibodies contain ⁇ identical ⁇ identifier ⁇ elements.
  • IHC Immunohistochemistry ⁇
  • ISH Immunohistochemistry ⁇
  • IHC ⁇ provides ⁇ the ⁇ substantial ⁇ advantage ⁇ of ⁇ identifying ⁇ exactly ⁇ where ⁇ a ⁇ particular ⁇ protein ⁇ is ⁇ located ⁇ within ⁇ the ⁇ tissue ⁇ sample.
  • One ⁇ technique ⁇ based ⁇ on ⁇ Dako ⁇ ARK TM is ⁇ a ⁇ chromogenic ⁇ immunohistological ⁇ staining.
  • the ⁇ system ⁇ is ⁇ designed ⁇ for ⁇ staining ⁇ with ⁇ mouse ⁇ primary ⁇ antibodies ⁇ on ⁇ formalin-fixed, ⁇ paraffin-embedded ⁇ tissues, ⁇ cryostat ⁇ tissues ⁇ or ⁇ cell ⁇ preparations ⁇ from ⁇ any ⁇ species, ⁇ including ⁇ mouse.
  • the ⁇ present ⁇ disclosure ⁇ pertains ⁇ to ⁇ novel ⁇ multiplex ⁇ methods ⁇ and ⁇ kits ⁇ for ⁇ detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ in ⁇ parallel ⁇ by ⁇ using ⁇ premixed ⁇ complexes.
  • ⁇ In ⁇ particular, ⁇ the ⁇ present ⁇ disclosure relates ⁇ to ⁇ a ⁇ parallel ⁇ detection ⁇ of ⁇ analytes ⁇ by ⁇ primary ⁇ decodeable ⁇ complexes.
  • each ⁇ complex comprises ⁇ an antigen(analyte)-specific ⁇ primary ⁇ antibody ⁇ and ⁇ a ⁇ secondary ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment, ⁇ wherein ⁇ the ⁇ secondary ⁇ antibody ⁇ or ⁇ the ⁇ antibody ⁇ fragment is ⁇ conjugated (tagged) ⁇ with ⁇ an oligonucleotide ⁇ that ⁇ is used ⁇ to ⁇ detect ⁇ the ⁇ antigens (analyts) of ⁇ interest ⁇ simultaneously.
  • the ⁇ presence of ⁇ antigens (analytes) can ⁇ be ⁇ identified ⁇ via ⁇ various ⁇ options ⁇ using ⁇ complementary ⁇ oligonucleotides. ⁇ This ⁇ allows ⁇ a ⁇ sim
  • ⁇ embodiments ⁇ of ⁇ the ⁇ disclosure ⁇ pertains ⁇ to ⁇ a method ⁇ for ⁇ detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ simultaneously ⁇ comprising ⁇ the ⁇ steps ⁇ of: ⁇ (A) ⁇ contacting ⁇ the ⁇ sample ⁇ with ⁇ at ⁇ least ⁇ four ⁇ (4) ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ for ⁇ detecting ⁇ of ⁇ at least ⁇ 4 ⁇ different ⁇ analytes, ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ interacting ⁇ with ⁇ a ⁇ different ⁇ analyte, ⁇ wherein ⁇ each ⁇ analyte-specific ⁇ probe ⁇ is ⁇ a ⁇ complex ⁇ having (aa) ⁇ a ⁇ binding ⁇ element ⁇ (Y) ⁇ that ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ different ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ wherein ⁇ each ⁇ binding ⁇ element ⁇ (Y) ⁇ of ⁇ each
  • ⁇ embodiments ⁇ of ⁇ the ⁇ disclosure ⁇ pertains ⁇ to ⁇ a ⁇ mixture ⁇ for ⁇ detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ simultaneously, ⁇ wherein ⁇ the ⁇ mixture ⁇ comprises ⁇ at ⁇ least ⁇ four ⁇ (4) ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ for ⁇ encoding of ⁇ at ⁇ least ⁇ 4 ⁇ different ⁇ analytes, ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ interacting ⁇ with ⁇ a ⁇ different ⁇ analyte, ⁇ wherein ⁇ each ⁇ analyte-specific ⁇ probe ⁇ comprises ⁇ complexes ⁇ having (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
  • Fig. ⁇ 2 are ⁇ schematic ⁇ diagrams ⁇ showing ⁇ methods ⁇ for ⁇ sample ⁇ analysis according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • Fig. ⁇ 3 ⁇ shows ⁇ a ⁇ cryosection ⁇ of ⁇ murine ⁇ kidney ⁇ was ⁇ probed ⁇ for ⁇ nestin.
  • the ⁇ prelabelling ⁇ was ⁇ performed ⁇ for ⁇ 30 ⁇ minutes ⁇ and ⁇ residual ⁇ secondary ⁇ antibody ⁇ or ⁇ Fab ⁇ not ⁇ bound ⁇ to ⁇ primary ⁇ antibody ⁇ was ⁇ blocked ⁇ with ⁇ 0.1
  • Fig. ⁇ 4 shows ⁇ a ⁇ cyrosection ⁇ of ⁇ murine ⁇ kidney ⁇ probed ⁇ for ⁇ Nestin.
  • Detection ⁇ of ⁇ Nestin was ⁇ done ⁇ by ⁇ precomplexed ⁇ primary ⁇ antibody ⁇ (Abcam ⁇ ab176571) ⁇ with ⁇ oligos ⁇ conjugated ⁇ secondary ⁇ antibody ⁇ followed ⁇ by ⁇ complementary ⁇ oligos ⁇ conjugated ⁇ to ⁇ fluorophores ⁇ (A).
  • the ⁇ localization ⁇ of ⁇ the ⁇ primary-secondary ⁇ antibody ⁇ complexes ⁇ was ⁇ confirmed ⁇ by ⁇ probing ⁇ the tissue ⁇ with ⁇ fluorophores ⁇ conjugated ⁇ antibody ⁇ complemented ⁇ to ⁇ the ⁇ oligo ⁇ conjugated ⁇ secondary ⁇ antibody ⁇ (B).
  • ⁇ DETAILED ⁇ DESCRIPTION ⁇ OF ⁇ THE DISCLOSURE RES-PA13-PCT The ⁇ present disclosure ⁇ features ⁇ methods ⁇ for ⁇ performing ⁇ multiplexed ⁇ labeling, ⁇ identification and ⁇ quantification ⁇ of ⁇ target ⁇ analy
  • the methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ multiplexed ⁇ labeling, ⁇ identification, ⁇ signal ⁇ amplification, ⁇ and ⁇ quantification ⁇ of ⁇ target ⁇ analytes ⁇ in ⁇ a ⁇ biological ⁇ sample may ⁇ be ⁇ performed.
  • the ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ can ⁇ be ⁇ used ⁇ to ⁇ perform ⁇ multiple ⁇ cycles ⁇ of ⁇ target ⁇ analyte ⁇ labeling, ⁇ detection, ⁇ and ⁇ removal ⁇ of ⁇ certain ⁇ agents ⁇ involved ⁇ in ⁇ the ⁇ labeling ⁇ process, ⁇ without ⁇ disrupting ⁇ antibody-analyte ⁇ binding ⁇ in ⁇ the ⁇ sample.
  • ⁇ removal ⁇ of ⁇ agents ⁇ involved ⁇ in ⁇ the ⁇ labeling ⁇ process ⁇ is ⁇ performed ⁇ by ⁇ dehybridizing ⁇ the ⁇ agents ⁇ under ⁇ relatively ⁇ mild ⁇ conditions, ⁇ preserving ⁇ sample ⁇ integrity ⁇ and ⁇ ensuring ⁇ that ⁇ removal ⁇ of ⁇ the ⁇ agents ⁇ during ⁇ each ⁇ labeling ⁇ and ⁇ detection ⁇ cycle ⁇ is ⁇ nearly ⁇ complete. ⁇ As ⁇ a ⁇ resul
  • ⁇ the ⁇ analyte ⁇ is ⁇ a ⁇ protein/polypeptide.
  • ⁇ the ⁇ terms“contacts” ⁇ and“contacting” ⁇ mean ⁇ that ⁇ an ⁇ agent, ⁇ species, ⁇ moiety, ⁇ or ⁇ other ⁇ element ⁇ is ⁇ brought ⁇ into ⁇ association ⁇ with ⁇ a ⁇ sample, ⁇ or ⁇ another ⁇ agent, ⁇ species, ⁇ moiety, ⁇ or ⁇ element, ⁇ such ⁇ that ⁇ the ⁇ two ⁇ interact ⁇ with ⁇ one ⁇ another.
  • ⁇ the ⁇ sample ⁇ ⁇ comprising ⁇ the ⁇ analytes ⁇ is “contacted” ⁇ with ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes
  • ⁇ the ⁇ analyte-specific ⁇ probes ⁇ are ⁇ brought ⁇ into ⁇ close ⁇ enough ⁇ association ⁇ with ⁇ the ⁇ sample ⁇ that ⁇ they ⁇ interact ⁇ with ⁇ the ⁇ anayltes ⁇ in ⁇ the ⁇ sample.
  • an ⁇ "oligonucleotide ⁇ as ⁇ used ⁇ herein, ⁇ refers ⁇ to ⁇ s ⁇ short ⁇ nucleic ⁇ acid ⁇ molecule, ⁇ such ⁇ as ⁇ DNA, ⁇ PNA, ⁇ LNA ⁇ or ⁇ RNA.
  • the ⁇ length ⁇ of ⁇ the ⁇ oligonucleotides ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 4-200 ⁇ nucleotides ⁇ (nt), ⁇ preferably ⁇ 6-80 ⁇ nt, ⁇ more ⁇ preferably ⁇ 8-60 ⁇ nt, ⁇ more ⁇ preferably ⁇ 10-50 ⁇ nt, ⁇ more ⁇ preferably ⁇ 12 ⁇ to ⁇ 35 ⁇ depending ⁇ on ⁇ the ⁇ number ⁇ of ⁇ consecutive ⁇ sequence ⁇ elements.
  • the ⁇ oligonucleotides may ⁇ be ⁇ linear ⁇ or ⁇ may ⁇ comprise ⁇ hairpin ⁇ or ⁇ loop ⁇ structures.
  • the ⁇ oligonucleotides ⁇ may ⁇ comprise ⁇ modifications
  • the ⁇ identifier ⁇ element ⁇ (Z) ⁇ is ⁇ an ⁇ antibody, ⁇ an ⁇ antibody ⁇ fragment, ⁇ an ⁇ aptamer ⁇ or ⁇ a ⁇ nucleic ⁇ acid ⁇ that ⁇ specifically ⁇ interacts ⁇ and ⁇ binds ⁇ to ⁇ the ⁇ analyte, wherein ⁇ in ⁇ particular ⁇ the ⁇ antibody ⁇ fragment ⁇ is ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ a ⁇ Fab, ⁇ scFv, ⁇ single ⁇ domain, ⁇ bis ⁇ scFv, ⁇ Fab 2 , ⁇ Fab 3 , ⁇ minibody, ⁇ diabody, ⁇ triplebody, ⁇ tetrabody ⁇ and ⁇ tandab.
  • the ⁇ analyte ⁇ is ⁇ a ⁇ protein/polypeptide comprised ⁇ in ⁇ a ⁇ biological ⁇ sample, ⁇ the ⁇ binding ⁇ element ⁇ (Y) ⁇ is ⁇ an ⁇ antibody ⁇ or ⁇ an ⁇ antibody ⁇ fragment ⁇ comprising ⁇ a ⁇ (Fc) ⁇ region ⁇ and ⁇ the ⁇ identifier ⁇ element ⁇ (Z) ⁇ is ⁇ a ⁇ secondary ⁇ antibody, ⁇ a ⁇ Fab, ⁇ Fab2 and/or ⁇ a ⁇ Fab 3 ⁇ fragment.
  • the ⁇ binding ⁇ element ⁇ (Y) ⁇ comprises ⁇ moieties ⁇ which ⁇ are ⁇ affinity ⁇ moieties ⁇ from ⁇ affinity ⁇ substances ⁇ or ⁇ affinity ⁇ substances ⁇ in ⁇ their ⁇ entirety ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ consisting ⁇ of ⁇ antibodies, ⁇ antibody ⁇ fragments, ⁇ receptor ⁇ ligands, ⁇ enzyme ⁇ substrates, ⁇ lectin
  • the ⁇ "identical ⁇ identifier ⁇ element ⁇ (T)" ⁇ as ⁇ comprised ⁇ in ⁇ a ⁇ binding ⁇ element ⁇ (Y) ⁇ of ⁇ an analyte-specific ⁇ probe is ⁇ identical in ⁇ its ⁇ sequence ⁇ compared ⁇ to ⁇ other ⁇ identical ⁇ identifier ⁇ elements (T) in ⁇ other ⁇ binding ⁇ element ⁇ (Y) ⁇ of another analyte-specific ⁇ probe. ⁇ In ⁇ other ⁇ words, ⁇ the ⁇ identical identifier ⁇ elements (T) comprised ⁇ in ⁇ all ⁇ binding ⁇ elements ⁇ (Y) ⁇ in ⁇ all ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ are ⁇ identical.
  • a ⁇ "decoding ⁇ oligonucleotide” ⁇ or ⁇ an ⁇ “adapter” ⁇ or ⁇ a ⁇ /adapter ⁇ segment” ⁇ consists ⁇ of ⁇ at ⁇ least ⁇ two ⁇ sequence ⁇ elements.
  • the ⁇ length ⁇ of ⁇ the ⁇ sequence ⁇ elements ⁇ is ⁇ within ⁇ the ⁇ range ⁇ 8-60 ⁇ nt, ⁇ preferably ⁇ 12-40 ⁇ nt, ⁇ more ⁇ preferably ⁇ 14-20 ⁇ nt, ⁇ de-pending ⁇ on ⁇ the ⁇ number ⁇ of ⁇ analytes ⁇ to ⁇ be ⁇ encoded ⁇ in ⁇ parallel, ⁇
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ in ⁇ the ⁇ kits ⁇ and/or ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ a ⁇ “multi-decoder”.
  • ⁇ the ⁇ decoding ⁇ oligonucleotide ⁇ is ⁇ a ⁇ multi-decoder ⁇ comprising ⁇ - an ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ is ⁇ essentially ⁇ complementary ⁇ to ⁇ at ⁇ least ⁇ a ⁇ section ⁇ of ⁇ the ⁇ identifier ⁇ oligonucleotide ⁇ (o) ⁇ of ⁇ an ⁇ identifier ⁇ element ⁇ (Z) ⁇ of ⁇ the ⁇ corresponding ⁇ analyte-specific ⁇ probe ⁇ set, ⁇ and - at ⁇ least ⁇ two ⁇ translator ⁇ elements ⁇ (c) ⁇ comprising ⁇ each ⁇ a ⁇ nucleotide ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ hybridization ⁇ of ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide.
  • the ⁇ first ⁇ ⁇ translator ⁇ element ⁇ binds ⁇ a ⁇ different ⁇ signal ⁇ oligonucleotide ⁇ as ⁇ the ⁇ second ⁇ translator ⁇ element.
  • the ⁇ signal ⁇ oligonucleotides ⁇ differ ⁇ in ⁇ the ⁇ signal ⁇ element ⁇ comprised ⁇ in ⁇ the ⁇ signal ⁇ oligonucleotide, ⁇ e.g. ⁇ in ⁇ the ⁇ kind ⁇ of ⁇ the ⁇ fluorophore.
  • each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes comprises ⁇ premixed ⁇ complexes, ⁇ wherein ⁇ the ⁇ complexes ⁇ of ⁇ one ⁇ analyte-specific ⁇ probe set ⁇ compared ⁇ to ⁇ the ⁇ complexes ⁇ of ⁇ another ⁇ analyte-specific ⁇ probe set ⁇ comprises ⁇ a ⁇ different ⁇ binding ⁇ element ⁇ (Y) ⁇ that ⁇ specifically ⁇ interacts ⁇ with ⁇ one ⁇ of ⁇ the ⁇ different ⁇ analytes ⁇ to ⁇ be ⁇ encoded but ⁇ comprises ⁇ identical ⁇ identifier ⁇ elements (T) that ⁇ interact ⁇ with ⁇ identifier ⁇ elements (Z) comprising ⁇ different identifier ⁇ fluorophores (f) ⁇ or ⁇ identifier ⁇ oligonucleotides (o) compared ⁇ to ⁇ the ⁇ the ⁇ identifier ⁇ elements (Z) of ⁇ another ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes.
  • “Selective ⁇ denaturation” ⁇ may ⁇ be ⁇ the ⁇ process ⁇ of ⁇ eliminating ⁇ bound ⁇ decoding ⁇ oligonucleotides ⁇ and ⁇ signal ⁇ oligonucleotides ⁇ with ⁇ highest ⁇ efficiency ⁇ while ⁇ at ⁇ the ⁇ same ⁇ time ⁇ the ⁇ target ⁇ specific ⁇ probes ⁇ have ⁇ to ⁇ stay ⁇ hybridized ⁇ with ⁇ the ⁇ highest ⁇ efficiency.
  • the ⁇ total ⁇ efficiency ⁇ of ⁇ these ⁇ two ⁇ combined ⁇ events may ⁇ to ⁇ be ⁇ at ⁇ least ⁇ 0.22 ⁇ for ⁇ two ⁇ detection ⁇ cycles, ⁇ 0.37 ⁇ for ⁇ three ⁇ detection ⁇ cycles, ⁇ 0.47 ⁇ for ⁇ four ⁇ detection ⁇ cycles, ⁇ 0.55 ⁇ for ⁇ five ⁇ detection ⁇ cycles, ⁇ 0.61 ⁇ for ⁇ six ⁇ detection ⁇ cycles, ⁇ 0.65 ⁇ for ⁇ seven ⁇ detection ⁇ cycles, ⁇ 0.69 ⁇ for ⁇ eight ⁇ detection ⁇ cycles, ⁇ 0.72 ⁇ for ⁇ nine ⁇ det
  • a ⁇ single ⁇ set refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ oligonucleotides.
  • An ⁇ "analyte ⁇ specific ⁇ probe ⁇ set” refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ moieties ⁇ or ⁇ subjects, ⁇ e.g. ⁇ analyte-specific ⁇ probes ⁇ that ⁇ are ⁇ different ⁇ from ⁇ each ⁇ other ⁇ and ⁇ bind ⁇ to ⁇ independent regions ⁇ of ⁇ the ⁇ analyte. ⁇ A ⁇ single ⁇ analyte ⁇ specific ⁇ probe ⁇ set ⁇ is ⁇ further ⁇ characterized ⁇ by ⁇ the ⁇ same ⁇ identifier ⁇ element ⁇ (Z).
  • a ⁇ "decoding ⁇ oligonucleotide ⁇ set refers ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ specific ⁇ for ⁇ a ⁇ certain ⁇ unique ⁇ identifier ⁇ needed ⁇ to ⁇ realize ⁇ the ⁇ encoding ⁇ independent ⁇ of ⁇ the ⁇ length ⁇ of ⁇ the ⁇ code ⁇ word.
  • Each ⁇ and ⁇ all ⁇ of ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ included ⁇ in ⁇ a ⁇ "decoding ⁇ oligonucleotide ⁇ set” bind ⁇ to ⁇ the ⁇ same ⁇ identifier ⁇ oligonucleotide ⁇ (o) ⁇ of ⁇ the ⁇ analyte-specific ⁇ probe.
  • the ⁇ values ⁇ in ⁇ each ⁇ code ⁇ word can ⁇ also ⁇ be ⁇ assigned ⁇ in ⁇ different ⁇ fashions ⁇ in ⁇ some ⁇ embodiments. ⁇ For ⁇ example, ⁇ a ⁇ value ⁇ of ⁇ 0 ⁇ could ⁇ represent ⁇ binding ⁇ while ⁇ a ⁇ value ⁇ of ⁇ 1 ⁇ represents ⁇ no ⁇ binding. ⁇ Similarly, ⁇ a ⁇ value ⁇ of ⁇ 1 ⁇ could ⁇ represent ⁇ binding ⁇ of ⁇ a ⁇ secondary ⁇ nucleic ⁇ acid ⁇ probe ⁇ with ⁇ one ⁇ type ⁇ of ⁇ signaling ⁇ entity ⁇ while ⁇ a ⁇ value ⁇ of ⁇ 0 ⁇ could ⁇ represent ⁇ binding ⁇ of ⁇ a ⁇ secondary ⁇ nucleic ⁇ acid ⁇ probe ⁇ with ⁇ another ⁇ type ⁇ of ⁇ distinguishable ⁇ signaling ⁇ entity. ⁇ These ⁇ signaling ⁇ entities ⁇ could ⁇ be ⁇ distinguished, ⁇ for ⁇ example, ⁇ via ⁇ different ⁇ colors ⁇ of ⁇ fluorescence. ⁇ In ⁇ some cases, ⁇ values ⁇ in ⁇ code ⁇ words need ⁇ not ⁇ be ⁇ confined ⁇
  • RES-PA13-PCT "Essentially ⁇ complementary” ⁇ means, ⁇ when ⁇ referring ⁇ to ⁇ two ⁇ nucleotide ⁇ sequences, ⁇ that ⁇ both ⁇ sequences ⁇ can ⁇ specifically ⁇ hybridize ⁇ to ⁇ each ⁇ other ⁇ under ⁇ stringent ⁇ conditions, ⁇ thereby ⁇ forming ⁇ a ⁇ hybrid ⁇ nucleic ⁇ acid ⁇ molecule ⁇ with ⁇ a ⁇ sense ⁇ and ⁇ an ⁇ antisense ⁇ strand ⁇ connected ⁇ to ⁇ each ⁇ other ⁇ via ⁇ hydrogen ⁇ bonds ⁇ (Watson-and-Crick ⁇ base ⁇ pairs).
  • a ⁇ "kit” ⁇ is ⁇ a ⁇ combination ⁇ of ⁇ individual ⁇ elements ⁇ useful ⁇ for ⁇ carrying ⁇ out ⁇ the ⁇ use ⁇ and/or ⁇ method ⁇ of ⁇ the ⁇ disclosure, ⁇ wherein ⁇ the ⁇ elements ⁇ are ⁇ optimized ⁇ for ⁇ use ⁇ together ⁇ in ⁇ the ⁇ methods.
  • the ⁇ kits ⁇ may ⁇ also ⁇ contain ⁇ additional ⁇ reagents, ⁇ chemicals, ⁇ buffers, ⁇ reaction ⁇ vials ⁇ etc. ⁇ which ⁇ may ⁇ be ⁇ useful ⁇ for ⁇ carrying ⁇ out ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure.
  • Such ⁇ kits unify ⁇ all ⁇ essential ⁇ elements ⁇ required ⁇ to ⁇ work ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure, ⁇ thus ⁇ minimizing ⁇ the ⁇ risk ⁇ of ⁇ errors. ⁇ Therefore, ⁇ such ⁇ kits ⁇ also ⁇ allow ⁇ semi-skilled ⁇ laboratory ⁇ staff ⁇ to ⁇ perform ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • ⁇ a ⁇ cell ⁇ may ⁇ be ⁇ fixed ⁇ using ⁇ chemicals ⁇ such ⁇ as ⁇ formaldehyde, ⁇ paraformaldehyde, ⁇ glutaraldehyde, ⁇ ethanol, ⁇ methanol, ⁇ acetone, acetic ⁇ acid, ⁇ or ⁇ the ⁇ like.
  • This ⁇ measure ⁇ has ⁇ the ⁇ advantage ⁇ that ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded, ⁇ e.g. ⁇ the ⁇ nuclei ⁇ acids ⁇ or ⁇ proteins, ⁇ are ⁇ immobilized ⁇ and ⁇ cannot ⁇ escape. ⁇ In ⁇ doing ⁇ so, ⁇ the ⁇ analytes ⁇ then ⁇ prepared ⁇ for ⁇ a ⁇ better ⁇ detection ⁇ or ⁇ encoding ⁇ by ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ disclosure. ⁇ In ⁇ yet ⁇ a ⁇ further ⁇ embodiment ⁇ within ⁇ the ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ the ⁇ individual ⁇ analyte-specific ⁇ probes ⁇ comprise ⁇ binding ⁇ elements ⁇ (S1, ⁇ S2, ⁇ S3, ⁇ S4, ⁇ S5) ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ one ⁇ of ⁇ the ⁇ analytes ⁇ to ⁇ be ⁇ encoded.
  • ⁇ 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 ⁇ 1,000, ⁇ at ⁇ least ⁇ 3,000, ⁇ at ⁇ least ⁇ 10,000, ⁇ or ⁇ at ⁇ least ⁇ 30,000 ⁇ distinguishable ⁇ analyte-specific ⁇ probes ⁇ that ⁇ are ⁇ applied ⁇ to ⁇ a ⁇ sample, ⁇ e.g., ⁇ simultaneously ⁇ or ⁇ sequentially.
  • the ⁇ unique ⁇ tag ⁇ can ⁇ be ⁇ identified ⁇ by ⁇ various ⁇ techniques, ⁇ including ⁇ hybridization, ⁇ e.g. ⁇ with ⁇ labeled ⁇ probes, ⁇ directly ⁇ or ⁇ indirectly ⁇ or ⁇ by ⁇ sequencing ⁇ (by ⁇ synthesis, ⁇ ligation).
  • the ⁇ kit ⁇ does ⁇ not ⁇ comprise ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ as ⁇ defined ⁇ under ⁇ item ⁇ A).
  • ⁇ if ⁇ the ⁇ analyte ⁇ in ⁇ the ⁇ kits ⁇ or ⁇ methods ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure ⁇ is ⁇ a ⁇ nucleic ⁇ acid ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ comprises ⁇ at ⁇ least ⁇ five ⁇ (10) ⁇ analyte-specific ⁇ probes, ⁇ in ⁇ particular ⁇ at ⁇ least ⁇ fifteen ⁇ (15) ⁇ analyte-specific ⁇ probes, ⁇ in ⁇ particular ⁇ at ⁇ least ⁇ twenty ⁇ (20) ⁇ analyte- specific ⁇ probes ⁇ which ⁇ specifically ⁇ interact ⁇ with ⁇ different ⁇ sub-structures ⁇ of ⁇ the ⁇ same ⁇ analyte.
  • Nucleic ⁇ acid ⁇ analyte ⁇ includes
  • the ⁇ kit ⁇ may ⁇ comprise ⁇ at ⁇ least ⁇ two ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ per ⁇ analyte, wherein ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ comprised ⁇ in ⁇ these ⁇ different ⁇ sets ⁇ comprise ⁇ the ⁇ same ⁇ identifier ⁇ connector ⁇ element ⁇ (t) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ which ⁇ 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 ⁇ wherein ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ of ⁇ the ⁇ different ⁇ sets ⁇ per ⁇ analyte ⁇ differ ⁇ in ⁇ the ⁇ translator ⁇ element ⁇ (c) ⁇ comprising ⁇ a ⁇ nucleotide ⁇ sequence ⁇ allowing ⁇
  • ⁇ the ⁇ present ⁇ disclosure ⁇ is generally ⁇ directed ⁇ to ⁇ a ⁇ methods ⁇ including ⁇ acts ⁇ of ⁇ exposing ⁇ a ⁇ sample ⁇ to ⁇ a ⁇ plurality ⁇ of ⁇ analyte-specific ⁇ probes; ⁇ for ⁇ each ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ determining ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes ⁇ within ⁇ the ⁇ sample; ⁇ creating ⁇ code ⁇ words based ⁇ on ⁇ the ⁇ binding ⁇ of ⁇ the ⁇ analyte-specific ⁇ probes, ⁇ the ⁇ decoding ⁇ oligonucleotides and ⁇ the ⁇ signal ⁇ oligonucleotides; ⁇ and ⁇ for ⁇ at ⁇ least ⁇ some ⁇ of ⁇ the ⁇ code ⁇ words, ⁇ matching ⁇ the ⁇ code ⁇ word to ⁇ a ⁇ valid ⁇ code ⁇ RES-PA13-PCT word.
  • this ⁇ pattern ⁇ of ⁇ binding ⁇ or ⁇ hybridization ⁇ of the ⁇ is generally ⁇ directed ⁇ to ⁇ a ⁇ methods
  • To ⁇ create ⁇ such ⁇ a ⁇ zero ⁇ (0) ⁇ in ⁇ a ⁇ code ⁇ word for ⁇ an ⁇ individual ⁇ analyte ⁇ the ⁇ kit ⁇ may ⁇ comprise: ⁇ (D) at ⁇ least ⁇ a ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ for ⁇ binding ⁇ to ⁇ a ⁇ particular ⁇ identifier ⁇ element ⁇ (T) ⁇ of ⁇ analyte-specific ⁇ probes, ⁇ wherein ⁇ the ⁇ decoding ⁇ oligonucleotides ⁇ in ⁇ the ⁇ same ⁇ set ⁇ of ⁇ non-signal ⁇ decoding ⁇ oligonucleotides ⁇ interacting ⁇ with ⁇ the ⁇ same ⁇ different ⁇ identifier ⁇ element ⁇ (T), wherein ⁇ each ⁇ non-signal ⁇ decoding ⁇ oligonucleotide ⁇ comprises ⁇ an ⁇ identifier ⁇ connector ⁇ element ⁇ (t
  • said ⁇ encoding ⁇ scheme ⁇ may ⁇ be predetermined ⁇ and ⁇ allocated ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded ⁇ may ⁇ be a ⁇ nucleic ⁇ acid, ⁇ preferably ⁇ DNA, ⁇ PNA, ⁇ RNA, ⁇ in ⁇ particular ⁇ mRNA, ⁇ a ⁇ peptide, ⁇ polypeptide, ⁇ a ⁇ protein or ⁇ combinations ⁇ thereof. Therefore, ⁇ the ⁇ binding ⁇ element ⁇ (Y) ⁇ may ⁇ comprise ⁇ an ⁇ amino ⁇ acid ⁇ sequence ⁇ allowing ⁇ a ⁇ specific ⁇ binding ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded.
  • ⁇ the ⁇ signal ⁇ caused ⁇ by ⁇ the ⁇ signal ⁇ element therefore ⁇ in ⁇ particular ⁇ the ⁇ binding ⁇ of ⁇ the ⁇ signal ⁇ oligonucleotides ⁇ to ⁇ the ⁇ decoding ⁇ oligonucleotides, ⁇ interacting ⁇ with ⁇ the ⁇ corresponding ⁇ analyte ⁇ probes, ⁇ bound ⁇ to ⁇ the ⁇ respective ⁇ analyte ⁇ is ⁇ determined ⁇ by: (a) Imaging ⁇ at ⁇ least ⁇ a ⁇ portion ⁇ of ⁇ the ⁇ sample; ⁇ and/or (b) Using ⁇ an ⁇ optical ⁇ imaging ⁇ technique; ⁇ and/or (c) Using ⁇ a ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or (d) Multi-color ⁇ fluorescence ⁇ imaging ⁇ technique; ⁇ and/or ⁇ (e) Super-resolution ⁇ fluorescence ⁇ imaging ⁇ technique.
  • the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ ideally ⁇ for ⁇ in ⁇ vitromethods ⁇ for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising ⁇ cancer, ⁇ neuronal ⁇ diseases, ⁇ cardiovascular ⁇ diseases, ⁇ inflammatory ⁇ diseases, ⁇ autoimmune ⁇ diseases, ⁇ diseases ⁇ due ⁇ to ⁇ a ⁇ viral ⁇ or ⁇ bacterial ⁇ infection, ⁇ skin ⁇ diseases, ⁇ skeletal ⁇ muscle ⁇ diseases, ⁇ dental ⁇ diseases ⁇ and ⁇ prenatal ⁇ diseases.
  • ⁇ the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ also ⁇ ideally ⁇ for ⁇ in ⁇ vitro methods ⁇ for ⁇ diagnosis ⁇ of ⁇ a ⁇ disease ⁇ in ⁇ plants ⁇ selected ⁇ from ⁇ the ⁇ group ⁇ comprising: ⁇ diseases ⁇ caused ⁇ by ⁇ biotic ⁇ stress, ⁇ preferably ⁇ by ⁇ infectious ⁇ and/or ⁇ parasitic ⁇ origin, ⁇ or ⁇ diseases ⁇ caused ⁇ by ⁇ abiotic ⁇ stress, ⁇ preferably ⁇ caused ⁇ by ⁇ nutritional ⁇ deficiencies ⁇ and/or ⁇ unfavorable ⁇ environment.
  • ⁇ the ⁇ kits ⁇ and ⁇ method ⁇ according to ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ also ⁇ ideally ⁇ for ⁇ in ⁇ vitro methods ⁇ for ⁇ screening, ⁇ identifying ⁇ and/or ⁇ testing ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ comprising: (a) contacting ⁇ a ⁇ test ⁇ sample ⁇ comprising ⁇ a ⁇ sample ⁇ with ⁇ a ⁇ substance ⁇ and/or ⁇ drug ⁇ (b) detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ by ⁇ sequential ⁇ signal-encoding ⁇ of ⁇ said ⁇ analytes ⁇ with ⁇ a ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure.
  • optical ⁇ multiplexing ⁇ system ⁇ suitable ⁇ for ⁇ the ⁇ method ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure comprising ⁇ at ⁇ least: - a reaction ⁇ vessel ⁇ for ⁇ containing ⁇ the ⁇ kits ⁇ or ⁇ part ⁇ of ⁇ the ⁇ kits ⁇ according ⁇ to ⁇ the ⁇ present ⁇ disclosure; - a ⁇ detection ⁇ unit ⁇ comprising ⁇ a ⁇ microscope, ⁇ in ⁇ particular ⁇ a ⁇ fluorescence ⁇ microscope; - a ⁇ camera; - a ⁇ liquid ⁇ handling ⁇ device.
  • optical ⁇ multiplexing ⁇ system ⁇ may ⁇ comprises ⁇ further ⁇ a ⁇ heat ⁇ and ⁇ cooling ⁇ device and/or ⁇ a ⁇ robotic ⁇ system.
  • the ⁇ technology ⁇ allows ⁇ distinguishing ⁇ a ⁇ higher ⁇ number ⁇ of ⁇ analytes ⁇ than ⁇ different ⁇ signals ⁇ are ⁇ available.
  • the ⁇ process ⁇ preferably ⁇ includes ⁇ at ⁇ least ⁇ two ⁇ consecutive ⁇ rounds ⁇ of ⁇ specific ⁇ binding, ⁇ signal ⁇ detection ⁇ and ⁇ selective ⁇ denaturation ⁇ (if ⁇ a ⁇ next ⁇ round ⁇ is ⁇ required), ⁇ eventually ⁇ producing ⁇ a ⁇ signal ⁇ code.
  • the ⁇ decoding ⁇ oligonucleotide ⁇ transcribes ⁇ the ⁇ information ⁇ of ⁇ the ⁇
  • the ⁇ analyte ⁇ is ⁇ in ⁇ particular a ⁇ protein, ⁇ a ⁇ nucleic ⁇ acid, ⁇ or ⁇ another ⁇ molecule ⁇ that ⁇ specifically ⁇ is ⁇ recognized ⁇ and ⁇ bound ⁇ by ⁇ the ⁇ binding ⁇ element ⁇ (Y).
  • An ⁇ analyte may ⁇ be ⁇ part ⁇ of ⁇ a ⁇ eucaryotic, ⁇ prokaryotic, ⁇ animal, ⁇ fungi, ⁇ plant ⁇ cell, ⁇ an ⁇ organelle, ⁇ or ⁇ an ⁇ extracellular ⁇ area ⁇ within ⁇ an ⁇ organism.
  • It ⁇ can ⁇ be ⁇ part ⁇ of ⁇ an ⁇ intact ⁇ or ⁇ disintegrated ⁇ organisms ⁇ or ⁇ cell.
  • Disintegration ⁇ of ⁇ an ⁇ organism: ⁇ is ⁇ defined ⁇ as ⁇ any ⁇ type ⁇ of ⁇ surgery, ⁇ extraction, ⁇ lysis, ⁇ purification ⁇ or ⁇ other ⁇ methods ⁇ that ⁇ disintegrate ⁇ the ⁇ organism ⁇ or ⁇ cell.
  • the ⁇ binding ⁇ element ⁇ (Y) is ⁇ defined ⁇ in ⁇ general ⁇ as ⁇ a ⁇ protein ⁇ or ⁇ nucleic ⁇ acid ⁇ that ⁇ specifically ⁇ recognizes ⁇ and ⁇ binds ⁇ another ⁇ molecule ⁇ X.
  • the ⁇ identifier ⁇ element ⁇ (Z) ⁇ is ⁇ defined ⁇ in ⁇ general ⁇ as ⁇ a ⁇ protein ⁇ or ⁇ nucleic ⁇ acid ⁇ that ⁇ specifically ⁇ recognizes ⁇ and ⁇ binds ⁇ to ⁇ Y. ⁇ Z ⁇ can ⁇ be ⁇ an ⁇ antibody, ⁇ a ⁇ fragment ⁇ of ⁇ an ⁇ antibody, ⁇ e.g., ⁇ FAB ⁇ or ⁇ F(ab)2, ⁇ any ⁇ other ⁇ proteins, ⁇
  • Identifier ⁇ may ⁇ hybridize ⁇ to ⁇ a ⁇ non-labeled ⁇ oligonucleotide ⁇ that ⁇ is ⁇ complementary ⁇ to ⁇ the ⁇ sequence ⁇ of ⁇ the ⁇ identifier.
  • This ⁇ oligonucleotide has ⁇ an ⁇ addition ⁇ sequence ⁇ that ⁇ can ⁇ be ⁇ detected ⁇ by ⁇ hybridizing ⁇ to ⁇ one ⁇ or ⁇ multiple ⁇ complementary ⁇ oligonucleotides ⁇ that ⁇ are ⁇ labeled.
  • the ⁇ identification ⁇ of ⁇ the ⁇ identifier ⁇ may ⁇ require ⁇ several ⁇ rounds ⁇ to ⁇ build ⁇ up ⁇ a ⁇ code ⁇ word.
  • the ⁇ process ⁇ may ⁇ require ⁇ additional ⁇ steps ⁇ for ⁇ complex ⁇ formation ⁇ such ⁇ as ⁇ filtration, ⁇ purification, ⁇ absorption, ⁇ size ⁇ selection ⁇ etc. ⁇ to ⁇ purify ⁇ proper ⁇ complexes ⁇ from ⁇ individual ⁇ components ⁇ or ⁇ side ⁇ products.
  • In ⁇ particular, ⁇ the ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ to ⁇ identify ⁇ regions ⁇ of ⁇ interest, ⁇ in ⁇ particular ⁇ in ⁇ an ⁇ organ, ⁇ tissue ⁇ or ⁇ single ⁇ cell.
  • the ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ provide ⁇ spatial ⁇ information ⁇ about ⁇ the ⁇ location ⁇ of ⁇ the ⁇ cells ⁇ being ⁇ profiled ⁇ enabling ⁇ the ⁇ analysis ⁇ of ⁇ cell-cell ⁇ signaling ⁇ and ⁇ cell ⁇ type ⁇ organization.
  • ⁇ the ⁇ methods ⁇ of ⁇ the ⁇ present ⁇ disclosure ⁇ may ⁇ be ⁇ used ⁇ for ⁇ morphological ⁇ studies.
  • ⁇ a ⁇ cell ⁇ type ⁇ is ⁇ a ⁇ classification ⁇ used ⁇ to ⁇ identify ⁇ cells ⁇ that ⁇ share ⁇ morphological ⁇ or ⁇ phenotypical ⁇ features.
  • the ⁇ method ⁇ according ⁇ to ⁇ any ⁇ one ⁇ of ⁇ items ⁇ 1 ⁇ to ⁇ 12, ⁇ wherein ⁇ after ⁇ step ⁇ A) ⁇ and ⁇ before ⁇ step ⁇ B) ⁇ the ⁇ identifier ⁇ elements ⁇ (Z) ⁇ are ⁇ 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 ⁇ items ⁇ 1 ⁇ to ⁇ 17, ⁇ wherein ⁇ said ⁇ encoding ⁇ scheme ⁇ is ⁇ predetermined ⁇ and ⁇ allocated ⁇ to ⁇ the ⁇ analyte ⁇ to ⁇ be ⁇ encoded. 19.
  • the ⁇ method ⁇ according ⁇ to ⁇ any ⁇ one ⁇ of ⁇ items ⁇ 1 ⁇ to ⁇ 18, ⁇ wherein ⁇ the ⁇ code ⁇ words ⁇ obtained ⁇ for ⁇ the ⁇ individual ⁇ analytes ⁇ in ⁇ the ⁇ performed ⁇ cycles ⁇ comprise ⁇ the ⁇ detected ⁇ signals ⁇ and ⁇ additionally ⁇ at ⁇ least ⁇ one ⁇ element ⁇ corresponding ⁇ to ⁇ no ⁇ detected ⁇ signal. 20.
  • the ⁇ method ⁇ according ⁇ to ⁇ any ⁇ one ⁇ of ⁇ items ⁇ 1 ⁇ to ⁇ 28, ⁇ wherein ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ decoding ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately. ⁇ 30.
  • the ⁇ method ⁇ according ⁇ to ⁇ any ⁇ one ⁇ of ⁇ items ⁇ 1 ⁇ to ⁇ 30, ⁇ wherein ⁇ the ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ may ⁇ be ⁇ comprised ⁇ in ⁇ a ⁇ pre-mixture ⁇ of ⁇ different ⁇ sets ⁇ of ⁇ signal ⁇ oligonucleotides ⁇ or ⁇ exist ⁇ separately.
  • RES-PA13-PCT 37 37.
  • the ⁇ method ⁇ according ⁇ to ⁇ any ⁇ one ⁇ of ⁇ items ⁇ 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.
  • 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 ⁇ separate ⁇ multiple ⁇ tubes. 51.
  • a ⁇ kit ⁇ for ⁇ detecting ⁇ different ⁇ analytes ⁇ in ⁇ a ⁇ sample ⁇ simultaneously comprises ⁇ separately ⁇ at ⁇ least ⁇ four ⁇ (4) ⁇ different ⁇ sets ⁇ of ⁇ analyte-specific ⁇ probes ⁇ for ⁇ encoding ⁇ RES-PA13-PCT of ⁇ at ⁇ least ⁇ 4 ⁇ different ⁇ analytes, ⁇ each ⁇ set ⁇ of ⁇ analyte-specific ⁇ probes ⁇ interacting ⁇ with ⁇ a ⁇ different ⁇ analyte, ⁇ wherein ⁇ each ⁇ analyte-specific ⁇ probe ⁇ comprises ⁇ complexes ⁇ having (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 ⁇ an ⁇ identical ⁇ identifier ⁇ e

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Abstract

La technologie de l'invention concerne des procédés multiplex et des kits pour détecter différents analytes dans un échantillon simultanément, en particulier en utilisant des complexes de macromolécules prémélangées comme complexes ayant un anticorps primaire et un anticorps secondaire, les anticorps primaires contenant des éléments d'identification identiques.
PCT/EP2024/053689 2023-03-01 2024-02-14 Formation complexe pour la détection parallèle de multiples analytes dans de multiples applications Pending WO2024179837A1 (fr)

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