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WO2025054427A1 - Développement d'extincteur - Google Patents

Développement d'extincteur Download PDF

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Publication number
WO2025054427A1
WO2025054427A1 PCT/US2024/045545 US2024045545W WO2025054427A1 WO 2025054427 A1 WO2025054427 A1 WO 2025054427A1 US 2024045545 W US2024045545 W US 2024045545W WO 2025054427 A1 WO2025054427 A1 WO 2025054427A1
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Prior art keywords
nanobody
amino acid
seq
antigen
nos
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Antonius M. VAN OIJEN
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Quantum Si Inc
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Quantum Si Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • Q-body is a fluorescently labeled antibody that emits light upon binding of an antigen and is a rapid and highly sensitive agent that can be used in immunoassays.
  • Q-bodies can be costly and time consuming. There is a need for techniques for streamlining the identification and development of effective Q-bodies.
  • Q-bodies quenchbodies
  • Previous Q-bodies in the art have poor detection of proteins, particularly in complex biological samples (e.g, resulting from non-specific quenching).
  • the present disclosure provides compositions of Q-bodies that can be readily useful for antigen detection in complex biological samples (e.g., plasma) and that can be easily adapted for targeting a diverse selection of target antigens.
  • the inventors of the present disclosure have identified a novel strategy to design a nanobody scaffold that is modified to include tryptophan or phenylalanine residues in specific locations within the complementarity determining regions (CDRs) and framework regions of the nanobody.
  • Such nanobody scaffolds can then be labeled with a fluorescent molecule (e.g., labeled with a fluorescent molecule at the N-terminal end or C-terminal end of the nanobody) to generate a quenchbody (Q-body).
  • these nanobody scaffolds can be further evolved or mutated within their CDRs (while maintaining the tryptophan or phenylalanine residues introduced at specific locations) to allow for binding to any desired target antigen.
  • some aspects of the disclosure provide a nanobody labeled with a fluorescent molecule, wherein the nanobody comprises one or more amino acid substitutions in one or more complementarity determining regions (CDRs) relative to a reference nanobody, wherein the one or more amino acid substitutions introduce one or more tryptophan (W) or phenylalanine (F) residues in the one or more CDRs.
  • CDRs complementarity determining regions
  • the reference nanobody is a wild-type nanobody.
  • the reference nanobody comprises the amino acid sequence of SEQ ID NO: 4.
  • the reference nanobody comprises the amino acid sequence of SEQ ID NO: 38.
  • the reference nanobody comprises the amino acid sequence of SEQ ID NO: 42.
  • the nanobody has at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of any one of SEQ ID NOs: 1-4 or 35-42. In some embodiments, the nanobody has at least 90%, at least 95%, or at least 98% identity relative to the amino acid sequence of SEQ ID NO: 4, and wherein the one or more amino acid substitutions in a CDR introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 4.
  • W tryptophan
  • F phenylalanine
  • the nanobody has at least 90%, at least 95%, or at least 98% identity relative to the amino acid sequence of SEQ ID NO: 4, and wherein the one or more amino acid substitutions in a CDR introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 38.
  • the nanobody has at least 90%, at least 95%, or at least 98% identity relative to the amino acid sequence of SEQ ID NO: 4, and wherein the one or more amino acid substitutions in a CDR introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 42.
  • nanobody that is modified to comprise a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 4, wherein the nanobody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 4.
  • W tryptophan
  • F phenylalanine
  • nanobody that is modified to comprise a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 4, wherein the nanobody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 38.
  • W tryptophan
  • F phenylalanine
  • nanobody that is modified to comprise a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 4, wherein the nanobody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 42.
  • W tryptophan
  • F phenylalanine
  • the amino acid sequence has at least 95% or at least 98% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 38, or 42.
  • the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 4. In some embodiments, the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 38. In some embodiments, the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 42. In some embodiments, the nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 2, 36, or 40.
  • the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 4. In some embodiments, the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 38. In some embodiments, the nanobody is modified to comprise a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 42. In some embodiments, the nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 3, 37, or 41.
  • the nanobody is modified to comprise (a) a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 4; and (b) a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 4.
  • the nanobody is modified to comprise (a) a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 38; and (b) a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 38.
  • the nanobody is modified to comprise (a) a tryptophan (W) at the amino acid position corresponding to position 59 of SEQ ID NO: 42; and (b) a tryptophan (W) at the amino acid position corresponding to position 103 of SEQ ID NO: 42.
  • the nanobody comprises the amino acid sequence of any one of SEQ ID NOs: 1, 35, or 39.
  • nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 1-3.
  • the nanobody is labeled with a fluorescent molecule.
  • the nanobody specifically binds to a target antigen.
  • binding of a target antigen to the nanobody results in increased fluorescence from the fluorescent molecule, optionally at least a 1.4-fold increase.
  • the nanobody further comprises one or more residues in a framework region of the nanobody are substituted with a tryptophan residue or a phenylalanine residue.
  • the tryptophan residue or phenylalanine residue is within 10 A of an antigen binding site of the nanobody.
  • the nanobody specifically binds to a target antigen, wherein the target antigen is a protein, a peptide, a hapten, a small molecule, or a polysaccharide. In some embodiments, the nanobody specifically binds to a target antigen, wherein the target antigen is a marker for a disease.
  • the fluorescent molecule is ATTO520, ATTO655, carboxyrhodamine 110 (CR110), rhodamine 6G (R6G), or tetramethylrhodamine (TAMRA).
  • the nanobody is biotinylated.
  • kits comprising any one of the nanobodies described herein and a sample comprising a target antigen.
  • nucleic acid encoding the amino acid sequence of any one of the nanobodies described herein.
  • the nucleic acid is a plasmid or vector.
  • Further aspects of the disclosure provide a method of detecting the presence of a target antigen in a sample, the method comprising contacting the sample with any one of the nanobodies labeled with a fluorescent molecule described herein and detecting fluorescence emitted by the fluorescent molecule.
  • Further aspects of the disclosure provide a method of quantifying the amount of a target antigen in a sample, the method comprising contacting the sample with any one of the nanobodies labeled with a fluorescent molecule described herein and determining the amount of the antigen present in the sample based on intensity of fluorescence emitted by the fluorescent molecule.
  • the sample may be a blood, fecal, vaginal, plasma, or saliva sample.
  • Further aspects of the disclosure provide a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67.
  • the nanobody comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 7, 8, 16, 46, 49, 50, or 60-67.
  • the nanobody specifically binds to Interleukin-6 (IL-6).
  • the nanobody is labeled with a fluorescent molecule.
  • Some aspects provide a method of quantifying the amount of IL-6 in a sample, the method comprising contacting the sample with a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67 labeled with a fluorescent molecule and determining the amount of IL-6 present in the sample based on intensity of fluorescence emitted by the fluorescent molecule.
  • an antibody e.g., an scFv conjugated to a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide is complementary to the second oligonucleotide, wherein the first oligonucleotide is conjugated to a first position on the antibody and the second oligonucleotide is conjugated to a second position on the antibody.
  • the first oligonucleotide and/or the second oligonucleotide comprise 5-50 nucleotides, optionally 5-15 nucleotides, further optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides.
  • the first oligonucleotide is bound to the second oligonucleotide in the absence of antigen bound to the antibody. In some embodiments, the first oligonucleotide is not bound to the second oligonucleotide in the presence of antigen bound to the antibody.
  • the antibody is modified to comprise a tryptophan (W) or a phenylalanine (F) at at least one of the amino acid positions corresponding to positions 59, 103, and 114 of any one of SEQ ID NOs: 4, 38, or 42, wherein the nanobody comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 4, 38, or 42.
  • W tryptophan
  • F phenylalanine
  • the antibody comprises any one of the quenchbody (or antibody) sequences or modifications described herein.
  • the antibody comprises the amino acid sequence of any one of SEQ ID NOs: 1-3, 35-37, 39-41, 7-18, or 44-67.
  • Some aspects of the disclosure provide a method of detecting the presence of a target antigen in a sample, the method comprising:
  • Some aspects of the disclosure provide a method of quantifying the amount of a target antigen in a sample, the method comprising
  • FIGs. 1A-1G show the design and in silico modeling of nanobodies labeled with fluorescent molecules (quenchbodies).
  • FIG. 1A shows a model of a reference MBP-binding Q- body (nanobody comprises the sequence of PDB ID 5M14). The nanobody is covalently conjugated with a TAMRA label, which undergoes quenching due to complex formation with the intrinsic CDR-based tryptophan residues. Upon binding to a MBP antigen, TAMRA is sterically occluded from accessing quenching tryptophan residues (W 101, W 110, and W 115), which is associated with increased fluorescence intensity.
  • FIG. 1A shows a model of a reference MBP-binding Q- body (nanobody comprises the sequence of PDB ID 5M14). The nanobody is covalently conjugated with a TAMRA label, which undergoes quenching due to complex formation with the intrinsic CDR-based tryptophan residues. Upon binding to a
  • IB shows molecular dynamics (MD) simulations of dye-tryptophan (W) interactions in the presence and absence of MBP for W 101, WHO, and W115.
  • FIGs. 1C-1E show normalized distribution histograms illustrating the distances between TAMRA and W101 (FIG. 1C), TAMRA and WHO (FIG. ID), and TAMRA and W115 (FIG. IE), wherein the distances are derived from molecular dynamics (MD) simulations in the absence or presence of MBP.
  • FIGs. 1F-1G show normalized distribution histograms illustrating the distances between TAMRA and W103 (FIG. IF) and TAMRA and W115 (FIG. 1G), wherein the distances are derived from MD simulations in the absence or presence of lysozyme. TAMRA is considered quenched at distances ⁇ 10 A.
  • FIGs. 2A-2D show fluorescence intensity changes in TAMRA-labelled quenchbodies upon MBP binding or denaturation.
  • FIG. 2A shows the fluorescence fold-increase of Qb-MBP (quenchbodies comprising an MBP-specific nanobody) incubated in the presence of MBP.
  • FIG. 2B shows the fluorescence fold-increase of Qb-MBP incubated in the presence of MBP or in the presence of a denaturant (2% SDS/5% BME).
  • FIG. 2C shows the fluorescence intensity response of Qb-MBP to MBP.
  • FIG. 2D shows a schematic of a high-throughput 384-well plate assay for Q-body fluorescence responses. The right-most panel shows fluorescence fold-increase of the wild-type nanobody-based MBP Q-body in the presence of MBP and upon denaturation.
  • FIGs. 3A-3C show fluorescence intensity changes in TAMRA-labelled quenchbodies upon Lysozyme binding or denaturation.
  • FIG. 3A shows the fluorescence fold-increase of Qb- Lys (quenchbodies comprising an lysozyme-specific nanobody) incubated in the presence of lysozyme.
  • FIG. 3B shows the fluorescence fold-increase of Qb-Lys incubated in the presence of lysozyme or in the presence of a denaturant (2% SDS/5% BME).
  • FIG. 3C shows the fluorescence intensity response of Qb-Lys to the cognate antigen lysozyme.
  • FIGs. 4A-4E show a mechanistic model showing sites of tryptophan-mediated quenching and fluorescence intensity changes in a reference quenchbody (PDB ID 1ZVH) and associated Trp mutants.
  • FIG. 4A shows fluorescence fold-increase of wild-type, W103Y, or W115Y lysozyme quenchbody variants incubated in the absence of presence of 500 nM lysozyme to produce a maximum antigen-dependent fluorescence fold-increase.
  • FIG. 4B shows lysozyme pulldown by Qb-Lys and tryptophan substitution mutations.
  • FIG. 4C shows fluorescence foldincrease of MBP Q-body mutants in the presence of 125, 250, or 500 nM cognate antigen (MBP).
  • FIG. 4D shows the total fraction of time dye spends with tryptophan residues in wildtype and modified nanobody-based MBP Q-bodies, in the presence or absence of MBP.
  • FIG. 4E shows the fold-quench, fold-sense, and binding characteristics of wild-type and modified nanobody -based MBP Q-bodies.
  • FIG. 5 shows the fold increase associated with modified nanobody-based lysozyme Q- bodies relative to wild- type (WT).
  • FIG. 6 shows a cartoon representation of quenchbody in the dark apo-state and bright bound-state.
  • FIGs. 7A-7F show single molecule studies of fluorescent intensity changes in mutants upon antigen binding.
  • FIG. 7A shows fluoescence in the apo-state and bound-state after addition of cognate antigen.
  • FIG. 7B shows a cartoon representation of quenchbody immobilized on coverslips. “Before” represents the 30 frames acquired before addition of cognate antigen (MBP) and sodium dodecyl sulfate (SDS); and “after” represents the 30 frames acquired after the addition of saturating levels of cognate antigen.
  • FIG. 7C shows the detection of individual nanobody-based Q-bodies using a single-molecule microscope. Detection of fluorescence in the presence or absence of MBP, and upon denaturation is shown.
  • FIG. 7A-7F show single molecule studies of fluorescent intensity changes in mutants upon antigen binding.
  • FIG. 7A shows fluoescence in the apo-state and bound-state after addition of cognate antigen.
  • FIG. 7B shows a cartoon representation of que
  • FIG. 7D shows the sub-average of images collected before and after addition of saturating (250 nm) cognate antigen (MBP).
  • FIG. 7E shows the distribution of intensity and fold-increase of single wild-type and W 115A nanobody-based MBP Q-bodies, in the presence or absence of MBP, and upon denaturation.
  • FIG. 7F shows probability density functions of fold sense value of individual nanobodies in absence of MBP (0 nM MBP), under saturating MBP (250 nM MBP) conditions, and in the presence of 1% SDS. Corresponding mean values are denoted by dashed lines and numbers.
  • FIG. 8 shows a comparison of fold-sense between in-solution and immobilized quenchbody.
  • FIGs. 9-10 show single molecule titration of MBP against wild-type nanobody-based MBP Q-body.
  • FIG. 9 shows fluorescence fold-increase of wild-type MBP Q-body incubated in the presence of 0, 2, 8, 32, 125, or 250 nM cognate antigen (MBP).
  • FIG. 10 shows the fluorescence intensity response of wild-type MBP Q-body to the cognate antigen MBP best fit to an equation describing a single site- specific binding mode to derive half-maximal sensing (Ks).
  • FIG. 11 shows the temporal change in Q-body intensity for single molecules of nanobody-based MBP Q-body, in the presence of MBP and upon SDS denaturation.
  • FIG. 12 shows an example schematic of a pixel of an integrated device.
  • FIGs. 13A-13B shows the use of Q-bodies as immunosensors.
  • FIG. 13A shows an exemplary schematic of methods of characterizing generalizable quenchbody biosensors.
  • FIG. 13B shows an exemplary schematic for a generalizable Q-body framework for protein detection.
  • FIGs. 14A-14H show reducing SDS-PAGE and fluorescence fold-increase analysis of novel IL-6 quenchbodies evolved with optimized CDR-W mutations (Trp residues introduced into CDRs).
  • FIG. 14A shows a gel stained in Coomassie blue to assess protein purity.
  • FIG. 14B shows a gel stained in in-gel fluorescence imaging to detect TAMRA.
  • FIG. 14C shows relative fluorescent intensity changes in TAMRA-labelled IL-6 quenchbodies upon antigen-binding compared to quenchbody alone.
  • FIG. 14D shows relative fluorescent intensity changes in TAMRA-labelled IL-6 quenchbodies upon denaturation (2% SDS/5% BME) compared to quenchbody alone.
  • FIG. 14E shows the fluorescence-fold increase of an exemplary IL-6 Q-body after incubation in the presence of 0, 16, 31, 63, 125, 250, 500, 1000, 2000, 4000, or 8000 nM of cognate antigen.
  • FIG. 14C shows relative fluorescent intensity changes in TAMRA-labelled IL-6 quenchbodies upon antigen-binding compared to quenchbody alone.
  • FIG. 14D shows relative fluorescent intensity changes in TAMRA-labelled IL-6 quenchbodies upon denaturation (2% SDS/5% BME) compared to quenchbody alone.
  • FIG. 14E shows the fluorescence-fold increase of an exemplary IL-6 Q-body after in
  • FIG. 14F shows the fluorescence fold-increase of another exemplary IL-6 Q- body after incubation in the presence of 0, 16, 31, 63, 125, 250, 500, 1000, 2000, 4000, or 8000 nM of cognate antigen.
  • FIG. 14G shows fluorescence intensity responses of an exemplary IL-6 Q-body to its cognate antigen best fit to an equation describing a single site- specific binding mode to derive an EC50 as a proxy measure for quenchbody binding affinity (KD).
  • FIG. 14H shows fluorescence intensity responses of another exemplary IL-6 Q-body to its cognate antigen best fit to an equation describing a single- site- specific binding mode to derive an EC50 as a proxy measure for quenchbody binding affinity (KD).
  • FIGs. 15A-15F show the performance of exemplary evolved IL-6 Q-bodies.
  • FIGs. 16A-16C show performance of three different MBP mutants using an ATTO520 fluorophore after addition of 125, 250, or 500 nM cognate antigen (MBP) or after denaturation.
  • FIG. 16A shows the performance of a wild-type MBP mutant using an ATTO520 fluorophore.
  • FIG. 16B shows the performance of a Y59W MBP mutant using an ATTO520 fluorophore.
  • FIG. 16C shows the performance of using a CGGS (SEQ ID NO: 71) linker in combination with an ATTO520 fluorophore.
  • FIG. 17 shows the performance in the MBP binder with varying linker length after addition of 125, 250, or 500 nM cognate antigen (MBP).
  • FIG. 18 shows a schematic of oligonucleotide-based biosensors.
  • FIGs. 19A-19C show the production of scFv-oligonucleotide conjugates using cell-free approaches.
  • FIG. 19A shows a schematic of an scFv gene design.
  • FIG. 19B shows a schematic of scFv protein production.
  • FIG. 19C shows SDS-PAGE analysis of functional scFv and MBP pull-down.
  • FIGs. 20A-20D show the effect of intra-oligo length on probe binding rate.
  • FIG. 20A shows a cartoon representation of a 6 bp intra-oligo for conjugated to scFv.
  • FIG. 20B shows a cartoon representation of probe-oligo hybridization in the presence of MBP.
  • FIG. 20C shows single-molecule TIRF imaging and analysis before and after the addition of MBP in the presence of probe-oligo.
  • FIG. 20D shows the proportion of bound probes in the absence or presence of excess MBP.
  • FIGs. 21A-21C show probing DNA hybridization kinetics using complementary overhangs.
  • FIG. 21A shows a schematic of DNA complex with single overhangs (+MBP mimic) or dual overhangs (Apo mimic).
  • FIG. 21B shows single molecule TIRF microscopy analysis of association-dissociation events using single or dual overhang complexes.
  • FIG. 21C shows the distribution of total binding events per trajectory using single or dual overhang complexes.
  • FIGs. 22A-22F show non-palindromic intra-oligo conjugation via unnatural amino acid (UAA) incorporation.
  • FIG. 22A shows a schematic of amber stop codon replacement.
  • FIG. 22B shows a schematic of the oxime ligation reaction.
  • FIG. 22C shows a schematic of the cys-thiol conjugation reaction.
  • FIG. 22D shows a schematic of intra-oligo and probe-oligo hybridization.
  • FIG. 22E shows binding events of single-conjugated scFv analyzed through single molecule TIRF.
  • FIG. 22F shows binding events of dual-oligo conjugated scFv analyzed through single molecule TIRF.
  • FIG. 23 shows a schematic of hybridized DNA conjugated to scFv using an 8 bp oligo design (top) or 6 bp oligo design (bottom).
  • FIG. 24 shows the trade-off between shorter intra-oligo lengths versus shorter probe oligo s.
  • FIGs. 25A-25C provide data for novel quenchbodies that bind to Interleukin-6 (IL-6).
  • FIGs. 26A-26C provide data showing the ability of scFv-oligonucleotide conjugates.
  • biomarkers are important for maintenance of human health in all aspects of modem medicine. Genomic diagnostics may require additional considerations of epigenetic control and/or post-transcriptional/translational regulation. In contrast, detection of biomarker proteins (including peptides) is advantageous because it often directly connects to physiological outcomes. Analytical mass spectrometry techniques are often employed for study of the proteome and identification of proteinaceous biomarkers. However, mass spectrometry typically requires large and expensive equipment, and involves length and complex analysis for detection. Immunoassays, which rely on the affinity of antibodies for antigenic proteins, are advantageous due to their ease of accessibility, rapid results, target selectivity, good sensitivity, and financial affordability.
  • immunoassays There are many outputs associated with analyte detection in immunoassays, including enzyme reaction monitoring, fluorescence, chemiluminescence, and chromatographic separation. Regardless, immunoassays can be generally dichotomized into heterogenous or homogenous depending on whether the antibody-bound label responsible for antigen detection requires separation from the free unbound antibody-bound label (heterogenous), or not (homogenous).
  • the enzyme-linked immunosorbent assay (ELISA) is a well-known example of a heterogenous immunoassay relying on enzyme output that is used in research and clinical settings.
  • heterogenous immunoassays typically require lengthy incubation times and extensive washes, making analyte detection laborious and time consuming.
  • homogenous “mix-and-read” immunoassays are incredibly quick and simple, provided the biological matrix where the antigen is being measured does not cause interference in the immunoassay.
  • homogenous “mix-and-read” immunoassays typically requires the use of characterized dual antibody pairs which is considered the major bottleneck in development of all sandwich-style immunoassays.
  • the present disclosure provides nanobody-based quenchbodies with superior performance against protein antigens and methods of use thereof.
  • Initial modeling revealed the importance of intrinsic tryptophan locations and high-performing tryptophan mutations informed the design of a new quenchbody sequence.
  • Evolution against interleukin-6 (IL-6) resulted in the production of a nanobody-based quenchbody which had a maximal 2.2-fold increase in fluorescence intensity (e.g., TAMRA) when detecting IL-6 antigen, representing an improvement in this class of quenchbodies.
  • This high-performance nanobody-based quenchbody forms a generalizable scaffold suitable for evolution against a variety of chosen clinical protein antigens, facilitating antigen detection via rapid homogenous fluorescence immunoassays.
  • Quenchbodies generally comprise nanobody, single-chain variable region (scFv; 32 kDa) or antigen-binding fragment (Fab; 50 kDa) antibodies which have been fluorescently labelled with a fluorescent molecule, typically at a flexible peptide linker, such as the N-terminus of the antibody.
  • This flexible linker permits the fluorophore to interact with tryptophan residues in the quenchbody. This interaction leads to a reduction in fluorescence intensity (quenching) through photoinduced electron transfer (PET), facilitated by hydrophobic/7t-7t stacking interactions, or indirectly via association to the protein, provided the fluorophore is within ⁇ 10 A to the quenching tryptophan.
  • the quenched fluorophore can increase in fluorescence intensity when it is disrupted from interacting with tryptophan. This can occur by (i) binding of the quenchbody to the cognate antigen, which sterically hinders the fluorophore from binding to tryptophan in hydrophobic pockets, or (ii) denaturing the quenchbody with chaotropic agents.
  • the positive increase in fluorescence intensity of the quenchbody can be used as a homogenous immunoassay to rapidly measure concentration of antigens, even in complex biological samples such as human plasma.
  • Q-bodies As described herein, the inventors have developed techniques for optimizing the amino acid sequences of Q-bodies (antibodies labeled with a fluorescent molecule near the antigenbinding site).
  • the interaction of the dye with amino acids in the antibody results quenches the fluorescence of the dye. Binding of the antigen to the antibody causes de-quenching, leading to an increase in fluorescence.
  • Q-bodies can detect a range of target molecules quickly by simply mixing with a sample (WO 2011/061944, WO 2013/065314, R. Abe et al., J. Am. Chem. Soc., 2011, 133(43), 17386-17394).
  • an antibody is an immunoglobulin molecule capable of specific binding to a target antigen through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • the term “antibody” encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies (e.g., IgG), but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a Q-body of the disclosure comprises an IgG, sc-Fv,
  • a Q-body of the disclosure is a nanobody based Q-body .
  • the terms “nanobody”, “VHH”, “VHH antibody fragment” and “single-domain antibody” are used without distinction and denote the variable domain of the single heavy chain of antibodies of the type of those found in camelids, which are naturally devoid of light chains.
  • the nanobodies In the absence of a light chain, the nanobodies each have three complementarity determining regions (CDRs), denoted CDR1, CDR2 and CDR3 respectively.
  • a nanobody generally has an amino acid sequence with the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in which FR1-FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively.
  • Nanobodies may be camel, dromedary, llama or alpaca nanobodies.
  • a Q-body may comprise a sequence selected from Table 1.
  • a nanobody (e.g., for use in a quenchbody) may comprise one or more amino acid substitutions in one or more complementarity determining regions (CDRs) or framework regions relative to a reference nanobody.
  • the one or more amino acid substitutions introduce one or more amino acid residues that are capable to quenching the fluorescence of a fluorescent molecule (e.g., a tryptophan (W) or phenylalanine (F) amino acid residue).
  • the one or more amino acid substitutions to introduce a tryptophan (W) or phenylalanine (F) amino acid residue are substitutions at amino acid positions corresponding to a reference nanobody e.g., a wild-type nanobody).
  • the one or more amino acid substitutions to introduce a tryptophan (W) or phenylalanine (F) amino acid residue are substitutions at amino acid positions corresponding to an amino acid position of SEQ ID NO: 4.
  • the one or more amino acid substitutions to introduce a tryptophan (W) or phenylalanine (F) amino acid residue are substitutions at amino acid positions corresponding to an amino acid position of SEQ ID NO: 38. In some embodiments, the one or more amino acid substitutions to introduce a tryptophan (W) or phenylalanine (F) amino acid residue are substitutions at amino acid positions corresponding to an amino acid position of SEQ ID NO: 42.
  • SEQ ID NO: 4 provides a sequence of a core nanobody scaffold that is composed of the following amino acid architecture: [N-terminal end]-[first framework region] -[complementarity determining region 1 (CDRl)]-[second framework region] -[CDR2]- [third framework region]- [CDR3]- [fourth framework region] -[C-terminal end].
  • the framework regions of SEQ ID NO: 4 are composed of fixed amino acid residue sequences while the majority of the amino acid positions within the CDRs of SEQ ID NO: 4 can be any amino acid (specific identity of residues depends on the desired target antigen).
  • Positions 101, 110, and 115 of SEQ ID NO: 4 are tryptophan residues within or near CDR3.
  • SEQ ID NO: 38 provides a sequence of a core nanobody scaffold that is composed of the following amino acid architecture: [N-terminal end]-[first framework region] -[complementarity determining region 1 (CDRl)]-[second framework region] -[CDR2]- [third framework region]- [CDR3]- [fourth framework region] -[C-terminal end].
  • the framework regions of SEQ ID NO: 38 are composed of fixed amino acid residue sequences while the majority of the amino acid positions within the CDRs of SEQ ID NO: 38 can be any amino acid (specific identity of residues depends on the desired target antigen).
  • Positions 101, 110, and 115 of SEQ ID NO: 38 are tryptophan residues within or near CDR3.
  • SEQ ID NO: 42 provides a sequence of a core nanobody scaffold that is composed of the following amino acid architecture: [N-terminal end]-[first framework region] -[complementarity determining region 1 (CDRl)]-[second framework region] -[CDR2]- [third framework region]- [CDR3]- [fourth framework region] -[C-terminal end].
  • the framework regions of SEQ ID NO: 42 are composed of fixed amino acid residue sequences while the majority of the amino acid positions within the CDRs of SEQ ID NO: 42 can be any amino acid (specific identity of residues depends on the desired target antigen).
  • Positions 101, 110, and 115 of SEQ ID NO: 42 are tryptophan residues within or near CDR3.
  • the one or more amino acid substitutions introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 4.
  • a nanobody comprises a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 4.
  • the one or more amino acid substitutions introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 38.
  • a nanobody comprises a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 38.
  • the one or more amino acid substitutions introduce a tryptophan (W) residue or a phenylalanine (F) residue at position 59 and/or position 103 of SEQ ID NO: 42.
  • a nanobody comprises a tryptophan (W) or a phenylalanine (F) at one or both of the amino acid positions corresponding to positions 59 and 103 of SEQ ID NO: 42.
  • a nanobody comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 3. [0081] In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 37.
  • a nanobody comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a nanobody comprises the amino acid sequence of SEQ ID NO: 41.
  • a nanobody that specifically binds to Interieukin-6 comprises the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67.
  • a nanobody that specifically binds to Interieukin-6 comprises the amino acid sequence of any one of SEQ ID NOs: 7, 8, 16, 46, 49, 50, or 60-67.
  • a nanobody e.g., a nanobody that specifically binds to Interleukin-6 (IL-6)
  • comprises one or two affinity peptides e.g., affinity tags.
  • An affinity peptide may be present at the N-terminal end of the nanobody and/or at the C-terminal end of the nanobody.
  • an N-terminal affinity peptide comprises or consists of the amino acid sequence of SEQ ID NO: 68.
  • a C-terminal affinity peptide comprises or consists of the amino acid sequence of SEQ ID NO: 69.
  • a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67 further comprises one or two affinity peptides (e.g., affinity tags).
  • a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67 further comprises an N-terminal affinity peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 68.
  • a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67 further comprises a C-terminal affinity peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 69.
  • a nanobody comprising the amino acid sequence of any one of SEQ ID NOs: 7-18 or 44-67 further comprises an N-terminal affinity peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 68 and a C-terminal affinity peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 69.
  • the disclosure provides a nanobody based Q-body, i.e., a nanobody labeled with a fluorescent molecule and targeting an antigen and wherein the binding of the antigen leads to an increase in fluorescence, wherein the nanobody is engineered to substitute one or more residues in a CDR with a tryptophan residue or a phenylalanine residue.
  • the CDR is CDR1.
  • the CDR is CDR2.
  • the CDR is CDR3.
  • one or more residues in a framework region of the nanobody are substituted with a tryptophan residues or a phenylalanine residue.
  • the framework region is FR1.
  • the framework region is FR2. In some embodiments, the framework region is FR3. In some embodiments, the framework region is FR4. In some embodiments, one or more tyrosine residues of the nanobody are substituted with a tryptophan residue or a phenylalanine residue. In some embodiments, the tyrosine residue is in CDR1. In some embodiments, the tyrosine residue is in CDR2. In some embodiments, the tyrosine residue is in CDR3. In some embodiments, the tyrosine residue is in FR1. In some embodiments, the tyrosine residue is in FR2. In some embodiments, the tyrosine residue is in FR3. In some embodiments, the tyrosine residue is in FR4.
  • one or more aromatic residues of the nanobody that do not contact the antigen are substituted to a non-aromatic residue.
  • the aromatic residue is in a framework region.
  • the aromatic residue is in FR1.
  • the aromatic residue is in FR2.
  • the aromatic residue is in FR3.
  • Non-limiting examples of an amino acid comprising a nonpolar, aromatic side chain include phenylalanine, tyrosine, and tryptophan.
  • a substituted residue is within 10 A of the antigen binding site. In some embodiments, the substituted residue is within 15 A of the antigen binding site. In some embodiments, the substituted residue is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 A of the antigen binding site.
  • a substituted residue is within 10 A of the fluorescent molecule. In some embodiments, the substituted residue is within 15 A of the fluorescent molecule. In some embodiments, the substituted residue is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 A of the fluorescent molecule.
  • At least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 residues of the nanobody are substituted.
  • a Q-body of the disclosure shows at least a 1.4-fold increase upon antigen binding relative to the fluorescence of the dye in the absence of the antigen.
  • a Q-body of the disclosure shows at least a 1.5-fold increase, at least a 1.6-fold increase, at least 1.7-fold increase, at least a 1.8-fold increase, at least a 1.9-fold increase, at least a 2.0-fold increase, at least a 2.5-fold increase, at least a 3-fold increase, at least a 3.5-fold increase, at least a 4-fold increase, at least a 4.5-fold increase, at least a 5-fold increase, at least a 5.5-fold increase, or at least a 6-fold increase in fluorescence upon antigen binding relative to the fluorescence of the Q-body in the absence of the antigen.
  • Methods of measuring fluorescence are known in the art.
  • fluorescence is measured by a fluorescence plate reader.
  • fluorescence fluorescence is measured by a fluorescence plate reader.
  • a Q-body may be labeled with one or more fluorescent molecules.
  • a Q-body is labeled at the N-terminus near the antigen-binding site.
  • a Q-body is labeled with a single fluorescent molecule.
  • a Q-body is labeled with two or more fluorescent molecules.
  • a Q-body is labeled with a two fluorescent molecules that form a FRET pair.
  • the fluorescent molecule is a rhodamine-based molecule. In some embodiments, the fluorescent molecule is a cyanine-based molecule. Typically, the fluorescent molecule comprises an aromatic or hetero aromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole, benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline, ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate, coumarin, 21etection21n, rhodamine or other like compound.
  • the fluorescent molecule comprises an aromatic or hetero aromatic compound and can be a pyrene, anthracene, naphthalene, acridine, stilbene, indole, benzindole, oxazole
  • fluoresecent dyes include xanthene dyes, such as fluorescein or rhodamine dyes, including 5- carboxyfluorescein (FAM), 2’7’-dimethoxy-4’5’-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N’,N’-tetramethyl-6- carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).
  • fluoresecent dyes include xanthene dyes, such as fluorescein or rhodamine dyes, including 5- carboxyfluorescein (FAM), 2’7’-dimethoxy-4’5’-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,
  • naphthylamino compounds include l-dimethylaminonaphthyl-5-sulfonate, l-anilino-8- naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate, 5-(2’- aminoethyl)aminonaphthalene-l- sulfonic acid (EDANS).
  • dyes include coumarins, such as 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such as indodicarbocyanine 3 (Cy®3), (2Z)-2-[I-3-[3-(5-carboxypentyl)-l,l-dimethyl-6,8- disulfobenzo[e]indol-3-ium-2-yl]prop-2-enylidene]-3-ethyl-l,l-dimethyl-8- (trioxidanylsulfanyl)benzo [e] indole-6-sulfonate (Cy ® 3.5), 2- ⁇ 2- [(2,5-dioxopyrrolidin- 1 -yl)oxy]- 2-oxoethyl]-16,
  • the fluorescent molecule is a dye selected from Table 2.
  • the dyes listed in Table 2 are non-limiting, and the luminescent labels or luminescent molecules of the application may include dyes not listed in Table 2.
  • an antibody is produced with an N-terminal Cys tag. In some embodiments, an antibody is labeled based on a cysteine-maleimide reaction. In some embodiments, an antibody is labeled using transamination at the N-terminus. Methods of labeling Q-bodies with fluorescent molecules are known in the art. See, e.g., Sensors (Basel) 21(4): 1223 (2021).
  • the antibody may be further conjugated to another moiety. In some embodiments, the antibody may be further conjugated to a moiety that facilitates immobilization. In some embodiments, the antibody may be biotinylated.
  • the Q-bodies of the disclosure can target a wide range of antigens.
  • the antigen may be a protein, a peptide, a carbohydrate, a lipid, a glycolipid, a polynucleotide, or a small molecule.
  • the antigen is a protein or a peptide with a post-translational modification.
  • Non-limiting examples of post-translational modifications include acetylation, ADP-ribosylation, caspase cleavage, citrullination, formylation, N-linked glycosylation, O-linked, hydroxylation, methylation, myristoylation, neddylation, nitration, chlorination, oxidation/reduction, carbonylation, palmitoylation, phosphorylation, prenylation, S-nitrosylation, sulfation, glycation, sumoylation, and ubiquitination.
  • the antigen is a marker for a disease.
  • the antigen is associated with a pathogen (e.g., viral, bacterial, fungal).
  • a Q-body of the disclosure specifically binds to its target antigen.
  • “Specifically binds” a is a term well understood in the art.
  • a molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and/or with greater affinity with a particular target antigen than it does with alternative targets.
  • a Q-body “specifically binds” to a target antigen if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • a Q-body that specifically (or preferentially) binds to an antigen is a Q-body that binds this target antigen with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens. It is also understood with this definition that, for example, a Q-body that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. In some examples, a Q-body that “specifically binds” to a target antigen may not bind to other antigens or other epitopes in the same antigen.
  • a Q-body that specifically binds to an antigen will bind with lesser affinity (if at all) to other antigens.
  • Lesser affinity may include at least 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less, or 95% less.
  • a Q-body as described herein has a suitable binding affinity for the target antigen.
  • binding affinity refers to the apparent association constant or KA.
  • the KA is the reciprocal of the dissociation constant (KD).
  • the nanobodies described herein may have a binding affinity (KD) of at least 10' 9 , IO 0 M, 10 41 , 10 2 , or lower for the target antigen. An increased binding affinity corresponds to a decreased KD.
  • Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, biolayer interferometry or spectroscopy (e.g., using a fluorescence assay).
  • Antibodies can be produced by conventional polypeptide production techniques. For example, they can be synthesized using the well-known solid-phase synthesis method (Merrifield (1962) Proc. Soc. Ex. Boil. 21: 412; Merrifield (1963) J. Am. Chem. Soc. 85: 2149; Tam et al. (1983) J. Am. Chem. Soc. 105: 6442), preferably using a commercially available peptide synthesis instrument (such as the one made by Applied Biosystems, Foster City, Calif.) and by following the manufacturer’s instructions.
  • a commercially available peptide synthesis instrument such as the one made by Applied Biosystems, Foster City, Calif.
  • antibodies e.g., nanobodies
  • antibodies can be synthesized by recombinant DNA techniques well known to those skilled in the art (Maniatis et al. (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, NY, 51-54 and 412-430).
  • they can be obtained as DNA expression products after incorporation of the DNA sequences encoding the polypeptide of interest into expression vectors and introduction of these vectors into the appropriate prokaryotic or eukaryotic hosts that will express the polypeptide of interest, from which they can then be isolated using techniques well known to those skilled in the art.
  • polypeptide of interest is linked to a tag that facilitates its purification.
  • tags are well known to those skilled in the art and include, but are not limited to, for example, hexahistidine (6His) (SEQ ID NO: 81), glutathione S-transferase (GST), FLAG tag, the myc tag or influenza virus hemagglutinin (HA).
  • hexahistidine 6His
  • GST glutathione S-transferase
  • FLAG tag FLAG tag
  • HA influenza virus hemagglutinin
  • a protein when a protein is linked to a tag that facilitates its purification, such a protein comprises, between the native sequence and this tag, a sequence which allows enzymatic cleavage between the protein and this tag.
  • nucleic acid comprising a nucleic sequence (or a set of nucleic acids) encoding an antibody (e.g., a nanobody) according to the present disclosure.
  • said nucleic acid is a DNA or RNA molecule, which can be included in any appropriate vector, such as a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector.
  • vector cloning vector” and “expression vector” mean the carrier by which the DNA or RNA sequence can be introduced into the host cell, in such a way as to transform the host and to promote the expression (e.g., transcription and translation) of the sequence introduced.
  • vector comprising a nucleic acid according to the invention.
  • Such vectors can comprise regulatory elements, such as a promoter, an activator, a terminator, etc., for causing or directing the expression of the polypeptide.
  • regulatory elements such as a promoter, an activator, a terminator, etc.
  • promoters and activators used in expression vectors for animal cells include the SV40 early promoter and activator, the Moloney mouse leukemia virus LTR promoter and activator, the immunoglobulin chain promoter and activator, etc.
  • Any expression vector for animal cells can be used.
  • vectors include replicating plasmids comprising an origin of replication, or integrating plasmids, such as for example pUC, pcDNA, pBR, etc.
  • vectors include viral vectors such as adenoviral, retroviral, herpes virus and AAV vectors.
  • viral vectors such as adenoviral, retroviral, herpes virus and AAV vectors.
  • Such recombinant viruses can be produced by techniques well known to those skilled in the art, such as by transfection of packaging cells or by transient transfection with helper plasmids or viruses.
  • virus packaging cells include PA317 cells, PsiCRIP cells, Gpenv+ cells, 293 cells, etc.
  • Another aspect of the present disclosure relates to a cell that has been transfected, transduced or transformed with a nucleic acid and/or a vector according to the invention.
  • the nucleic acids according to the invention can be used to produce an antibody (e.g., nanobody) according to the invention in an appropriate expression system.
  • expression system means a host cell and a vector compatible under appropriate conditions, e.g., for the expression of a protein encoded by the foreign DNA carried by the vector and introduced into the host cell.
  • Conventional expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and their vectors.
  • Other examples of host cells include prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • Escherichia coli Kluyveromyces or Saccharomyces yeasts
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.
  • primary or established mammalian cell cultures e.g., produced from lymphoblasts, fibroblasts, epithelial cells, nerve cells, adipocytes, etc.
  • mice SP2/0-Agl4 cells ATCC CRL1581
  • mouse P3X63- Ag8.653 cells ATCC CRL1580
  • CHO cells in which a dihydrofolate reductase gene is defective rat YB2/3HL.P2.G11.16Ag.2O cells (ATCC CRL1662), etc.
  • the disclosure also relates to a method for producing an antibody (e.g., nanobody according to the invention, said method comprising the steps of: (i) culturing a cell comprising a nucleic acid or vector of the disclosure under conditions suitable for allowing the expression of said antibody (e.g., nanobody), and (ii) recovering the nanobody expressed.
  • the antibodies (e.g., nanobodies) according to the invention can be suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as for example protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, affinity dialysis or chromatography.
  • the Q-bodies of the disclosure may be used to detect a target analyte (an antigen).
  • the Q-bodies of the disclosure are useful for a wide range of applications that involve detecting an analyte (e.g., diagnosing and/or monitoring a disease, drug detection, environmental monitoring, etc.).
  • the disclosure provides a method of detecting the presence of an antigen in a sample, comprising contacting the sample with a Q-body of the disclosure and detecting the fluorescence emitted by the fluorescent molecule.
  • the disclosure provides a method of quantifying the amount of an antigen present in a sample, comprising contact the sample with a Q-body of the disclosure and detecting the fluorescence emitted by the fluorescent molecule.
  • the disclosure provides a method of detecting the presence of an antigen in a sample in a single molecule detection assay, comprising contacting the sample with a Q-body of the disclosure and detecting the fluorescence emitted by the fluorescent molecule.
  • the disclosure provides a method of quantifying the presence of an antigen in a sample in a single molecule detection assay, comprising contacting the sample with a Q- body of the disclosure and detecting the fluorescence emitted by the fluorescent molecule.
  • single molecule detection refers to the detection and/or measurement of a single molecule of an analyte in a test sample.
  • the analyte is at very low levels of concentration (such as pg/mL or femtogram/mL levels).
  • a number of different single molecule analyzers or devices are known in the art and include nanopore and nano well devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016/161402. Examples of nanowell device are described in International Patent Publication No. WO 2016/161400. Additional single molecule devices are described herein.
  • the method comprises determining the presence or absence of an antigen associated with a disease or condition in a subject and determining whether the subject has the disease or condition.
  • the method comprises measuring the presence or absence of an antigen associated with a disease or condition in a sample isolated from a subject at a first time point and a second time point and determining the progression of the disease or condition to an advanced stage based on changes in the presence or absence of the antigen at the first and second time points, wherein i) when the antigen is present at a higher level in the isolated sample from the second time point, the disease or condition has progressed to an advanced stage, and ii) when the antigen is present at a lower level in the isolated sample from the second time point, the disease or condition has regressed to a less advanced stage.
  • the levels of antigen may be compared with either a reference or threshold amount or with amounts found in other samples. For instance, if the presence or levels of antigens are measured in a diseased tissue or a corresponding normal tissue, those can be compared to provide a relative assessment of the progression of the disease. Alternatively, the levels may be compared with an amount that is known (or is shown) to be an amount of the antigen that is not associated with the disease. The value that is used in the comparison is referred to as the reference or threshold level.
  • threshold values may vary for different diseases or conditions or under different circumstances, such as the conditions of the assay to determine expression. However, the skilled artisan would be able to identify the correct threshold values based on the circumstances. For example, threshold values could easily be generated using normal tissue under similar circumstances.
  • the reference sample can be any of a variety of biological samples against which a diagnostic assessment may be made.
  • Examples of reference samples include biological samples from control populations or control samples.
  • Reference samples may be generated through manufacture to be supplied for testing in parallel with the test samples, e.g., reference sample may be supplied in diagnostic kits. Appropriate reference samples will be apparent to the skilled artisan.
  • the sample can be any sample that may contain a target analyte and is suitable for use in an assay that detects fluorescence.
  • the sample is a liquid sample.
  • the sample is a non-liquid sample.
  • a non-liquid sample may be liquefied e.g., dissolved in or suspended in) prior to use.
  • the sample is a biological sample.
  • the biological sample may be obtained from a subject.
  • the term “biological sample” is used to generally refer to any biological material obtained from a subject.
  • the biological sample typically is a fluid sample. Solid tissues may be made into fluid samples using routine methods in the art.
  • the biological sample is tissue, feces, or a cell obtained from a subject.
  • the biological sample comprises a bodily fluid from a subject.
  • the bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, intraorgan system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid or combinations thereof.
  • reaction conditions for contacting a Q-body with its antigen are known in the art. See, e.g., reaction conditions described in International Publication No. WO 2013/065314.
  • the methods of disclosure comprise irradiating the sample with excitation light for the fluorescent molecule (or for the donor molecule if there is FRET pair), and detecting the resulting fluorescence.
  • the wavelength of the excitation light to be used can be selected based on the fluorescent molecule being used.
  • An appropriate light source for irradiating the sample with the excitation light can be selected. Examples of the light source can include mercury lamps, xenon lamps, LED, and laser beam. Excitation light with a particular wavelength can be obtained using an appropriate filter.
  • a device usually used in fluorescence observation can be used as a fluorescence measurement apparatus, , a microscope equipped with a fluorescence image capturing system, flow cytometry, or the like.
  • Fluorescence intensity has a positive correlation with the concentration of the antigen.
  • the fluorescence intensity can be measured using a sample containing the antigen having a known concentration to prepare a standard curve that indicates the relationship between the antigen concentration and the fluorescence intensity.
  • the concentration of the antigen having an unknown concentration can be calculated based on the standard curve.
  • Methods in accordance with the disclosure may involve immobilizing a Q-body on a surface of a substrate.
  • the substrate is a solid support, such as a biosensor, a microarray, a chip, or an integrated device as described herein.
  • a plurality of Q-bodies are attached to a plurality of sites (e.g., with each site having one Q-body of the plurality attached thereto) on an array.
  • a Q- body may be immobilized on a surface of a sample well (e.g., on a bottom surface of the sample well) on a substrate comprising an array of sample wells.
  • a Q-body is immobilized (e.g., attached to the surface) directly or indirectly (e.g., through a linker or through a moiety that is attached to the surface).
  • the immobilized Q-body can be attached using any suitable covalent or non-covalent linker or linkage group, for example, as described in this disclosure.
  • a Q-body is attached to a surface through a covalent linkage group, which may be formed using techniques (e.g., click chemistry) known in the art.
  • a Q-body is attached to a surface through a non-covalent linkage group.
  • the non-covalent linkage group comprises an avidin protein.
  • Avidin proteins are biotin-binding proteins, generally having a biotin binding site at each of four subunits of the avidin protein.
  • Avidin proteins include, for example, avidin, streptavidin, traptavidin, tamavidin, bradavidin, xenavidin, and homologs and variants thereof.
  • the monomeric, dimeric, or tetrameric form of the avidin protein can be used.
  • the avidin protein of an avidin protein complex is streptavidin in a tetrameric form (e.g., a homotetramer).
  • the biotin binding sites of an avidin protein provide attachment points for a biotinylated surface, a biotinylated Q-body, and/or a biotinylated analyte.
  • the disclosure provides an apparatus comprising a substrate having an array of single-molecule confinement sites.
  • a plurality of the singlemolecule confinement sites each comprise a single molecule comprising a Q-body as described herein.
  • the Q-body is immobilized to a surface of the single-molecule confinement site.
  • the apparatus comprises a receptacle or other means for keeping reagents (e.g., a sample, one or more antigen molecules, or any one or more of the compositions described herein) in contact with the substrate.
  • the substrate comprises a plurality of different Q-bodies in contact with one or more antigens.
  • the substrate is an integrated device.
  • the plurality of the single-molecule confinement sites comprise a plurality of sample wells.
  • the system may include an integrated device and an instrument configured to interface with the integrated device.
  • the integrated device may include an array of pixels, where individual pixels include a reaction chamber and at least one photodetector.
  • the reaction chambers of the integrated device may be formed on or through a surface of the integrated device and be configured to receive a sample placed on the surface of the integrated device. Collectively, the reaction chambers may be considered as an array of reaction chambers.
  • the plurality of reaction chambers may have a suitable size and shape such that at least a portion of the reaction chambers receive a single sample (e.g., a single molecule, such as a target analyte).
  • the number of samples within a reaction chamber may be distributed among the reaction chambers of the integrated device such that some reaction chambers contain one sample while others contain zero, two or more samples.
  • Excitation light is provided to the integrated device from one or more light sources external to the integrated device.
  • Optical components of the integrated device may receive the excitation light from the light source and direct the light towards the array of reaction chambers of the integrated device and illuminate an illumination region within the reaction chamber.
  • a reaction chamber may have a configuration that allows for the sample to be retained in proximity to a surface of the reaction chamber, which may ease delivery of excitation light to the sample and detection of emission light from the sample.
  • a sample positioned within the illumination region may emit emission light in response to being illuminated by the excitation light.
  • the sample may be labeled with a fluorescent label, which emits light in response to achieving an excited state through the illumination of excitation light.
  • Emission light emitted by a sample may then be detected by one or more photodetectors within a pixel corresponding to the reaction chamber with the sample being analyzed.
  • one or more photodetectors When performed across the array of reaction chambers, which may range in number between approximately 10,000 pixels to 1,000,000 pixels according to some embodiments, multiple samples can be analyzed in parallel.
  • the integrated device may include an optical system for receiving excitation light and directing the excitation light among the reaction chamber array.
  • the optical system may include one or more grating couplers configured to couple excitation light to other optical components of the integrated device and direct the excitation light to the other optical components.
  • the optical system may include optical components that direct the excitation light from the grating coupler(s) towards the reaction chamber array.
  • Such optical components may include optical splitters, optical combiners, and waveguides.
  • one or more optical splitters may couple excitation light from a grating coupler and deliver excitation light to at least one of the waveguides.
  • the optical splitter may have a configuration that allows for delivery of excitation light to be substantially uniform across all the waveguides such that each of the waveguides receives a substantially similar amount of excitation light.
  • Such embodiments may improve performance of the integrated device by improving the uniformity of excitation light received by reaction chambers of the integrated device.
  • suitable components e.g., for coupling excitation light to a reaction chamber and/or directing emission light to a photodetector, to include in an integrated device are described in U.S. Patent Application No. 14/821,688, filed August 7, 2015, titled “INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING
  • Additional photonic structures may be positioned between the reaction chambers and the photodetectors and configured to reduce or prevent excitation light from reaching the photodetectors, which may otherwise contribute to signal noise in detecting emission light.
  • metal layers which may act as a circuitry for the integrated device, may also act as a spatial filter. Examples of suitable photonic structures may include spectral filters, a polarization filters, and spatial filters and are described in U.S. Patent Application No. 16/042,968, filed July 23, 2018, titled “OPTICAL REJECTION PHOTONIC STRUCTURES,” and U.S. Provisional Patent Application No.
  • Components located off of the integrated device may be used to position and align an excitation source to the integrated device.
  • Such components may include optical components including lenses, mirrors, prisms, windows, apertures, attenuators, and/or optical fibers.
  • Additional mechanical components may be included in the instrument to allow for control of one or more alignment components.
  • Such mechanical components may include actuators, stepper motors, and/or knobs.
  • suitable excitation sources and alignment mechanisms are described in U.S. Patent Application No. 15/161,088, filed May 20, 2016, titled “PULSED LASER AND SYSTEM,” which is incorporated by reference in its entirety.
  • Another example of a beam-steering module is described in U.S. Patent Application No. 15/842,720, filed December, 14, 2017, titled “COMPACT BEAM SHAPING AND STEERING ASSEMBLY,” which is incorporated herein by reference.
  • Additional examples of suitable excitation sources are described in U.S. Patent Application No. 14/821,688, filed August 7, 2015, titled “INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZING
  • the photodetector(s) positioned with individual pixels of the integrated device may be configured and positioned to detect emission light from the pixel’s corresponding reaction chamber.
  • suitable photodetectors are described in U.S. Patent Application No. 14/821,656, filed August 7, 2015, titled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” which is incorporated by reference in its entirety.
  • a reaction chamber and its respective photodetector(s) may be aligned along a common axis. In this manner, the photodetector(s) may overlap with the reaction chamber within the pixel.
  • Characteristics of the detected emission light may provide an indication for identifying the label associated with the emission light. Such characteristics may include any suitable type of characteristic, including an arrival time of photons detected by a photodetector, an amount of photons accumulated over time by a photodetector, and/or a distribution of photons across two or more photodetectors. In some embodiments, such characteristics can be any one or a combination of two or more of luminescence lifetime, luminescence intensity, brightness, absorption spectra, emission spectra, luminescence quantum yield, wavelength (e.g., peak wavelength), and signal characteristics (e.g., pulse duration, interpulse durations, change in signal magnitude).
  • wavelength e.g., peak wavelength
  • signal characteristics e.g., pulse duration, interpulse durations, change in signal magnitude
  • a photodetector may have a configuration that allows for the detection of one or more timing characteristics associated with a sample’s emission light (e.g., luminescence lifetime).
  • the photodetector may detect a distribution of photon arrival times after a pulse of excitation light propagates through the integrated device, and the distribution of arrival times may provide an indication of a timing characteristic of the sample’s emission light (e.g., a proxy for luminescence lifetime).
  • the one or more photodetectors provide an indication of the probability of emission light emitted by the label (e.g., luminescence intensity).
  • a plurality of photodetectors may be sized and arranged to capture a spatial distribution of the emission light.
  • Output signals from the one or more photodetectors may then be used to distinguish a label from among a plurality of labels, where the plurality of labels may be used to identify a sample within the sample.
  • a sample may be excited by multiple excitation energies, and emission light and/or timing characteristics of the emission light emitted by the sample in response to the multiple excitation energies may distinguish a label from a plurality of labels.
  • parallel analyses of samples within the reaction chambers are carried out by exciting some or all of the samples within the chambers using excitation light and detecting signals from sample emission with the photodetectors.
  • Emission light from a sample may be detected by a corresponding photodetector and converted to at least one electrical signal.
  • the electrical signals may be transmitted along conducting lines in the circuitry of the integrated device, which may be connected to an instrument interfaced with the integrated device.
  • the electrical signals may be subsequently processed and/or analyzed. Processing or analyzing of electrical signals may occur on a suitable computing device either located on or off the instrument.
  • the instrument may include a user interface for controlling operation of the instrument and/or the integrated device.
  • the user interface may be configured to allow a user to input information into the instrument, such as commands and/or settings used to control the functioning of the instrument.
  • the user interface may include buttons, switches, dials, and a microphone for voice commands.
  • the user interface may allow a user to receive feedback on the performance of the instrument and/or integrated device, such as proper alignment and/or information obtained by readout signals from the photodetectors on the integrated device.
  • the user interface may provide feedback using a speaker to provide audible feedback.
  • the user interface may include indicator lights and/or a display screen for providing visual feedback to a user.
  • the instrument may include a computer interface configured to connect with a computing device.
  • the computer interface may be a USB interface, a FireWire interface, or any other suitable computer interface.
  • a computing device may be any general purpose computer, such as a laptop or desktop computer.
  • a computing device may be a server (e.g., cloud-based server) accessible over a wireless network via a suitable computer interface.
  • the computer interface may facilitate communication of information between the instrument and the computing device.
  • Input information for controlling and/or configuring the instrument may be provided to the computing device and transmitted to the instrument via the computer interface.
  • Output information generated by the instrument may be received by the computing device via the computer interface.
  • Output information may include feedback about performance of the instrument, performance of the integrated device, and/or data generated from the readout signals of the photodetector.
  • the instrument may include a processing device configured to analyze data received from one or more photodetectors of the integrated device and/or transmit control signals to the excitation source(s).
  • the processing device may comprise a general purpose processor, a specially-adapted processor e.g., a central processing unit (CPU) such as one or more microprocessor or microcontroller cores, a field-programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a custom integrated circuit, a digital signal processor (DSP), or a combination thereof).
  • the processing of data from one or more photodetectors may be performed by both a processing device of the instrument and an external computing device. In other embodiments, an external computing device may be omitted and processing of data from one or more photodetectors may be performed solely by a processing device of the integrated device.
  • the instrument that is configured to analyze samples based on luminescence emission characteristics may detect differences in luminescence lifetimes and/or intensities between different luminescent molecules, and/or differences between lifetimes and/or intensities of the same luminescent molecules in different environments.
  • the inventors have recognized and appreciated that differences in luminescence emission lifetimes can be used to discern between the presence or absence of different luminescent molecules and/or to discern between different environments or conditions to which a luminescent molecule is subjected.
  • discerning luminescent molecules based on lifetime can simplify aspects of the system.
  • wavelengthdiscriminating optics such as wavelength filters, dedicated detectors for each wavelength, dedicated pulsed optical sources at different wavelengths, and/or diffractive optics
  • wavelengthdiscriminating optics may be reduced in number or eliminated when discerning luminescent molecules based on lifetime.
  • a single pulsed optical source operating at a single characteristic wavelength may be used to excite different luminescent molecules that emit within a same wavelength region of the optical spectrum but have measurably different lifetimes.
  • An analytic system that uses a single pulsed optical source, rather than multiple sources operating at different wavelengths, to excite and discern different luminescent molecules emitting in a same wavelength region can be less complex to operate and maintain, more compact, and may be manufactured at lower cost.
  • analytic systems based on luminescence lifetime analysis may have certain benefits, the amount of information obtained by an analytic system and/or detection accuracy may be increased by allowing for additional detection techniques.
  • some embodiments of the systems may additionally be configured to discern one or more properties of a sample based on luminescence wavelength and/or luminescence intensity.
  • luminescence intensity may be used additionally or alternatively to distinguish between different luminescent labels.
  • some luminescent labels may emit at significantly different intensities or have a significant difference in their probabilities of excitation (e.g., at least a difference of about 35%) even though their decay rates may be similar. By referencing binned signals to measured excitation light, it may be possible to distinguish different luminescent labels based on intensity levels.
  • different luminescence lifetimes may be distinguished with a photodetector that is configured to time-bin luminescence emission events following excitation of a luminescent label.
  • the time binning may occur during a single chargeaccumulation cycle for the photodetector.
  • a charge-accumulation cycle is an interval between read-out events during which photo-generated carriers are accumulated in bins of the timebinning photodetector. Examples of a time-binning photodetector are described in U.S. Patent Application No. 14/821,656, filed August 7, 2015, titled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” which is incorporated herein by reference.
  • a time-binning photodetector may generate charge carriers in a photon absorption/carrier generation region and directly transfer charge carriers to a charge carrier storage bin in a charge carrier storage region.
  • the time-binning photodetector may not include a carrier travel/capture region.
  • Such a time-binning photodetector may be referred to as a “direct binning pixel.” Examples of time-binning photodetectors, including direct binning pixels, are described in U.S. Patent Application No. 15/852,571, filed December, 22, 2017, titled “INTEGRATED PHOTODETECTOR WITH DIRECT BINNING PIXEL,” which is incorporated herein by reference.
  • different numbers of fluorophores of the same type may be linked to different reagents in a sample, so that each reagent may be identified based on luminescence intensity.
  • two fluorophores may be linked to a first labeled recognition molecule and four or more fluorophores may be linked to a second labeled recognition molecule.
  • optical excitation may be performed with a single-wavelength source (e.g., a source producing one characteristic wavelength rather than multiple sources or a source operating at multiple different characteristic wavelengths).
  • a single-wavelength source e.g., a source producing one characteristic wavelength rather than multiple sources or a source operating at multiple different characteristic wavelengths.
  • wavelength discriminating optics and filters may not be needed in the detection system.
  • a single photodetector may be used for each reaction chamber to detect emission from different fluorophores.
  • characteristic wavelength or “wavelength” is used to refer to a central or predominant wavelength within a limited bandwidth of radiation (e.g., a central or peak wavelength within a 20 nm bandwidth output by a pulsed optical source). In some cases, “characteristic wavelength” or “wavelength” may be used to refer to a peak wavelength within a total bandwidth of radiation output by a source.
  • an exemplary integrated device may be configured to perform single-molecule analysis in combination with an instrument as described above. It should be appreciated that the exemplary integrated device described herein is intended to be illustrative and that other integrated device configurations may be configured to perform any or all techniques described herein.
  • FIG. 12 illustrates a cross-sectional view of a pixel 1-112 of an integrated device 1-102.
  • Pixel 1-112 includes a photodetection region, which may be a pinned photodiode (PPD), and a charge storage region, which may be a storage diode (SD0).
  • a photodetection region and charge storage regions may be formed in semiconductor material of a pixel by doping regions of the semiconductor material.
  • the photodetection region and charge storage regions can be formed using a same conductivity type (e.g., n-type doping or p-type doping).
  • excitation light may illuminate sample well 1-108 causing incident photons, including fluorescence emissions from a sample, to flow along the optical axis to photodetection region PPD.
  • pixel 1-112 may include a waveguide 1-220 configured to optically couple light from the sample well 1-108 toward photodetection region PPD.
  • pixel 1-112 may also include photonic structure 1-230, which may include one or more optical rejection structures such as a spectral filter, a polarization filter, and/or a spatial filter.
  • pixel 1-112 may include one or more metal layers 1-240, which may be configured as a filter and/or may carry control signals from a control circuit configured to control transfer gates, as described further herein.
  • pixel 1-112 may include one or more transfer gates configured to control operation of pixel 1-112 by applying an electrical bias to one or more semiconductor regions of pixel 1-112 in response to one or more control signals.
  • transfer gate STO induces a first electrical bias at the semiconductor region between photodetection region PPD and storage region SDO
  • a transfer path e.g., charge transfer channel
  • Charge carriers e.g., photo-electrons
  • the first electrical bias may be applied during a collection period during which charge carriers from the sample are selectively directed to storage region SDO.
  • drain gate REJ may provide a channel to drain D to draw noise charge carriers generated in photodetection region PPD by the excitation light away from photodetection region PPD and storage region SDO, such as during a rejection period before fluorescent emission photons from the sample reach photodetection region PPD.
  • transfer gate STO may provide the second electrical bias and transfer gate TXO may provide an electrical bias to cause charge carriers stored in storage region SDO to flow to the readout region, which may be a floating diffusion (FD) region, for processing.
  • FD floating diffusion
  • transfer gates described herein may include semiconductor material(s) and/or metal, and may include a gate of a field effect transistor (FET), a base of a bipolar junction transistor (BJT), and/or the like.
  • FET field effect transistor
  • BJT bipolar junction transistor
  • operation of pixel 1-112 may include one or more collection sequences, each collection sequence including one or more rejection (e.g., drain) periods and one or more collection periods.
  • a collection sequence performed in accordance with one or more pulses of an excitation light source may begin with a rejection period, such as to discard charge carriers generated in pixel 1-112 (e.g., in photodetection region PD) responsive to excitation photons from the light source.
  • the excitation photons may arrive at pixel 1-112 prior to the arrival of fluorescence emission photons from the sample well.
  • Transfer gates for the charge storage regions may be biased to have low conductivity in the charge transfer channels coupling the charge storage regions to the photodetection region, blocking transfer and accumulation of charge carriers in the charge storage regions.
  • a drain gate for the drain region may be biased to have high conductivity in a drain channel between the photodetection region and the drain region, facilitating draining of charge carriers from the photodetection region to the drain region.
  • Transfer gates for any charge storage regions coupled to the photodetection region may be biased to have low conductivity between the photodetection region and the charge storage regions, such that charge carriers are not transferred to or accumulated in the charge storage regions during the rejection period.
  • a collection period may occur in which charge carriers generated responsive to the incident photons are transferred to one or more charge storage regions.
  • the incident photons may include fluorescent emission photons, resulting in accumulation of fluorescent emission charge carriers in the charge storage region(s).
  • a transfer gate for one of the charge storage regions may be biased to have high conductivity between the photodetection region and the charge storage region, facilitating accumulation of charge carriers in the charge storage region.
  • Any drain gates coupled to the photodetection region may be biased to have low conductivity between the photodetection region and the drain region such that charge carriers are not discarded during the collection period.
  • Some embodiments may include multiple rejection and/or collection periods in a collection sequence, such as a second rejection period and second collection period following a first rejection period and a collection period, where each pair of rejection and collection periods is conducted in response to a pulse of excitation light.
  • charge carriers generated in the photodetection region during each collection period of a collection sequence may be aggregated in a single charge storage region.
  • charge carriers aggregated in the charge storage region may be read out for processing prior to the next collection sequence.
  • charge carriers aggregated in a first charge storage region during a first collection sequence may be transferred to a second charge storage region sequentially coupled to the first charge storage region and read out simultaneously with the next collection sequence.
  • a processing circuit configured to read out charge carriers from one or more pixels may be configured to determine one or more of luminescence intensity information, luminescence lifetime information, luminescence spectral information, and/or any other mode of luminescence information associated with performing techniques described herein.
  • a first collection sequence may include transferring, to a charge storage region at a first time following each excitation pulse, charge carriers generated in the photodetection response in response to the excitation pulse
  • a second collection sequence may include transferring, to the charge storage region at a second time following each excitation pulse, charge carriers generated in the photodetection response in response to the excitation pulse.
  • the number of charge carriers aggregated after the first and second times may indicate luminance lifetime information of the received light.
  • pixels of an integrated device may be controlled to perform one or more collection sequences using one or more control signals from a control circuit of the integrated circuit, such as by providing the control signal(s) to drain and/or transfer gates of the pixel(s) of the integrated circuit.
  • charge carriers may be read out from the FD region of each pixel during a readout pixel associated with each pixel and/or a row or column of pixels for processing.
  • FD regions of the pixels may be read out using correlated double sampling (CDS) techniques.
  • CDS correlated double sampling
  • kits comprising a Q-body of the disclosure.
  • the kit may further comprise assay diluents, standards, and/or controls.
  • the assay diluents, standards and/or controls may be optimized for a particular sample.
  • reagents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.
  • kits may include one or more containers housing the components of the disclosure and instructions for use.
  • Instructions typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
  • kits may include one or more agents described herein, along with instructions describing the intended therapeutic application and the proper administration of these agents.
  • agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • the kit may be designed to facilitate use of the methods described herein by physicians and can take many forms.
  • Each of the compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • the kit may contain any one or more of the components described herein in one or more containers.
  • the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kit may include a container housing agents described herein.
  • the agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely.
  • the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • a nanobody labeled with a fluorescent molecule and targeting an antigen wherein the nanobody is engineered to substitute one or more residues in a complementarity determining region (CDR) with a tryptophan residue or a phenylalanine residue, and, wherein the binding of the antigen leads to an increase in fluorescence.
  • CDR complementarity determining region
  • a library comprising a plurality of nanobodies according to any of paragraphs 1-12.
  • a method of detecting the presence of an antigen in a sample comprising contacting the sample with the nanobody of any one of paragraphs 1-12 and detecting fluorescence emitted by the fluorescent molecule.
  • a method of quantifying the amount of an antigen present in a sample comprising contacting the sample with the nanobody of any of paragraphs 1-12 and calculating the amount of the antigen contained in the sample based on intensity of fluorescence emitted by the fluorescent molecule.
  • a method of detecting and/or quantifying an antigen molecule in a sample comprising contacting the sample with the nanobody of any one of paragraphs 1-12 and detecting fluorescence emitted by the fluorescent molecule, wherein the method is performed using a single molecule detection assay.
  • a kit for detecting and/or quantifying an antigen comprising the nanobody of any one of paragraphs 1-12.
  • a method of detecting and/or quantifying an antigen molecule in a sample in a single molecule detection assay comprising contacting the sample with an antibody or fragment thereof that targets the antigen, wherein the antibody is labeled with a fluorescent molecule, and detecting the increase in fluorescence emitted by the fluorescent molecule when the antigen binds to the antibody or fragment thereof.
  • MBP-binding (PDB ID: 5M14) and lysozyme-binding (PDB ID: 1ZVH) nanobodies were chosen as model starting scaffolds due to the availability of high-quality ( ⁇ 2 A) crystal structures and antigen accessibility.
  • the conjugated fluorophore e.g., TAMRA
  • TAMRA conjugated fluorophore
  • W 101, W110, and W115 the conjugated fluorophore
  • CDRs complementary determining regions
  • TAMRA is sterically occluded from the tryptophans and dequenching occurs, leading to an increase in fluorescence intensity of the fluorophore (FIG. 1A).
  • Nanobody-coding sequences were grafted into an in vitro transcription and translation (IVTT) expression scaffold containing a Cys-tag to enable conjugation of a malemide-TAMRA fluorophore at the N-terminus, which has previously been used as a tryptophan-sensitive dye in quenchbodies.
  • IVTT in vitro transcription and translation
  • a C-terminal Avi-tag, a 3xFLAG, and a lOxHis tag were also added to facilitate biotinylation and purification procedures.
  • quenchbodies were subjected to on-column TAMRA labelling, biotinylation, and finally FLAG purification.
  • Purified Qb-MBP and purified Qb-Lys were analyzed by reducing SDS-PAGE and subjected to in-gel fluorescence imaging to detect TAMRA or stained with Coomassie blue to assess protein purity.
  • Both the Qb-MBP and Qb-Lys appeared as a single band by reducing SDS-PAGE analysis , indicating high purity (data not shown).
  • Purified quenchbodies also showed a single fluorescent band upon TAMRA excitation of reducing SDS-PAGE with a lack of unbound fluorescent molecule, indicating successful labeling of quenchbodies. TAMRA-labeling efficiency was close to 100% by spectrophotometric analysis, suggesting conjugation stoichiometries were approximately 1:1 quenchbody:TAMRA.
  • Quenchbody functionality was further probed by reducing SDS-PAGE analysis of pulldown assays, in which each of the quenchbodies were immobilized to FLAG beads and incubated with 1 pM cognate antigen prior to extensive washing and FLAG elution. Under the same conditions, FLAG beads exhibited zero pulldown of lysozyme or MBP in the absence of either of the quenchbodies.
  • the model of 1ZVH shows that W 103 is sterically occluded by lysozyme, W115 is freely accessible regardless of antigen binding, and W36 is buried within the beta-sheet barrel and unable to participate in quenching at all. This illustrates that CDR-W tryptophans that directly interface with antigens are likely to be key in the mechanism for successful quenchbody performance.
  • the nanobodies were also exposed to denaturing conditions in order to determine the fold-quench, which can function as a positive control to demonstrate the upper limit of fluorescence that can be achieved when the nanobody is unable to quench the fluorophore (FIG. 4A).
  • MBP Q-body The wild-type MBP Q-body (5M 14- wild- type, also referred to herein as wild-type MBP Q-body) has three quenching tryptophans at positions 101, 110, and 115. W101 and WHO are in the CDRs while W115 is in a framework region. Mutational analysis was carried out by knocking out existing tryptophan residues, e.g., by substituting a non-tryptophan residue for a tryptophan, or by introducing additional tryptophan residues, e.g., by substituting a non-tryptophan residue for a tryptophan. Five positions for introducing additional tryptophans were selected - G32, Y33, Y54, Y59, and Y114.
  • a Y114W mutant was first produced, which improved the quenchbody fold-sense to 1.6 (compared to 1.4 in the wild-type), while retaining its binding affinity (Table 8). Therefore, the Y114 mutant was maintained in further Y to W substitutions. Unfortunately, all subsequent mutants had binding affected when compared with the wild-type, confounding the ability to draw conclusions about the favorability of these CDR-tryptophan positions in general across future quenchbody scaffolds. Despite this, the Y59W/Y114W mutant (which had the strongest pulldown of MBP relative to all other mutants) had an improved fold-sense of 1.9, suggesting a cumulative improvement due to the addition of favorable tryptophans had still been achieved (FIG. 4D).
  • ⁇ old-sense is the fluorescence fold-increase of the quenchbody in the presence of antigen, relative to the fluorescence of the quenchbody alone.
  • Fold-quench is the fluorescence fold-increase of the quenchbody in the presence of denaturant, relative to the fluorescence of the quench body alone.
  • 3 Mutation provides a description of the mutation and highlights whether the amino acid binds directly to the antigen (antigen) or not (non-antigen).
  • ⁇ Binding is a semi-quantitative measure reporting the effect the mutation had on the binding affinity of the quenchbody, as assessed by a reducing SDS-PAGE pulldown assay with comparison to a wild-type control.
  • the fold-quench, fold-sense, and Ks (half-maximal sensing) values of the MBP Q-bodies are shown in FIG. 4E.
  • the decreased fold-quench values of the tryptophan knockout mutants indicate that CDR tryptophans are key for quenching.
  • Lysozyme Q-body Improved version of Qb-Lys were produced based on the information learned from mutational modelling of 1ZVH, whereby CDR-based tryptophans were important for producing increased fluorescence upon antigen binding. Three tyrosine residues were selected in the CDRs for introducing tryptophan residues - Y27W, Y104W, and Y110W. FoldX predicted Y27W, Y104W, and Y HOW would be suitable for maintaining protein stability and antigen binding (Table 8), while MD simulations showed that Y27W, Y104W, and Y110W could each contribute to the quenching of TAMRA in the apo-state but not the antigen-bound state. In particular, TAMRA was quenched approximately 50% of the time in the apo-state and approximately 5% in the antigen-bound state for each of the individual mutants (Table 9).
  • Q-bodies are a novel antibody derivative with a fluorescent tag close to the complementarity determining regions. This fluorescent molecule exists in a quenched state when the antibody is unbound (apo-state) due to photoinduced electron transfer with intrinsic tryptophan residues (FIG. 6). When bound to cognate antigen quenching is disrupted, resulting in an increase in fluorescence (FIG. 7A). This signal can be used to measure analyte concentration in solution.
  • Q-bodies offer several advantages over traditional antibody based fluorometric assays by combining capture and detection events, in particular the removal of necessary wash steps.
  • Q-bodies developed herein have been shown to have a robust response to their cognate antigen in bulk.
  • Bulk assays are the standard by which analyte concentration measurements are done but they require relatively large amount of sample.
  • the possibility of using total internal reflection microscopy and single molecule techniques to determine analyte concentration in solution using surface tethered Q-bodies was investigated.
  • the wild-type MBP Q-body showed an average fold-sense of 1.4-fold, while the W115A MBP Q-body did not show a change in intensity in response to MBP.
  • TIRF total internal reflection
  • Fold- sense and fold-quench values were also determined on a molecule-by-molecule basis. The ratio of the mean intensity before and after was measurqued for each particle to determine an individual fold-sense for each particle. This was done across multiple MBP concentrations. Fold-sense values were plotted as a histogram and, after a clear two population distribution was observed in the saturating MBP conditions, fit with a two-component Gaussian- mixture model. In the absence of MBP (0 nM), there is a clear single population distribution with a mean of ⁇ 1. As MBP concentration is increased, a greater proportion of particles falls under the second ‘responding’ population. This response confirms that a considerable proportion of quenchbody molecules are non-functional, in line with bulk data observations showing a discrepancy in fold-sense and fold-quench.
  • Dye sensitized photo-oxidation is possibly one cause for the existence of this non-functional subpopulation when observed via single molecule techniques, given the fold-sense of the quenchbody was more pronounced at lower laser intensities.
  • the laser intensities encountered in plate reader experiments are unlikely to result in this effect due to their pulsed nature and lower wattage. Therefore, the major reason for the partially non-functional population observed by bulk and single molecule techniques remains unknown, but reducing the discrepancy in fold-sense and fold-quench could lead to improved version of the quenchbody.
  • Single-molecule titration of MBP against wild-type MBP Q-body was also studied.
  • the sandwich ELISA is a heterogenous immunoassay currently employed as the golden standard in clinical and research settings for specific and sensitive detection of biomolecules. Compared to the sandwich ELISA which involves lengthy washing and incubation steps (taking as long as 48 hours to complete in some cases), a rapid “mix-and-read” style homogenous immunoassay using Q-bodies provides results in under 1 hour.
  • nanobodies instead of IgG-style antibodies immunosensing scaffold, the time and financial requirements involved in the antibody production process, which is one of the major limitations in developing conventional sandwich ELISAs, have been greatly simplified.
  • the nanobody sequence has optimally placed tryptophans which quench the covalently-attached dye (e.g., TAMRA).
  • FIG. 13A An exemplary schematic for a generalizable approach is illustrated in FIG. 13B.
  • Example 7 Evolution of an improved quenchbody scaffold for IL-6 detection
  • Naive libraries were assembled and sequenced on an Illumina MiSeq instrument. 7.23 x 10 5 reads were generated, of which 63.6% correctly encoded a Q-body sequence. Assessment of the naive library diversity revealated 97% of 4.5 x 10 5 reads corresponded to a unique CDR combination, indicating high library diversity. Analysis of amino-acid distributions in randomized positions also closely matched the expected distributions, indicating the library matched the quenchbody designs.
  • the synthetic Q-body libraries were evolved against recombinant human interleukin-6 (IL-6) using SNAP-display.
  • Naive libraries were assembled with a C-terminal SNAP tag, T7-RNA polymerase promoter, 5’ ribosome binding site, and 3’ T7 transcription terminator signal sequence.
  • Eight benzylguanine molecules per gene were then attached using dendrimer-like DNA.
  • Libraries were subjected to five rounds of selection against IL-6 coated magnetic beads, with a parallel control of blank beads to remove nanobodies binding to the matrix. After the first found, the library was split and selected IL-6 for a further 4 rounds (two replicates) and blank beads. A model selection was done at each round to control for IVTT activity.
  • a qPCR on the SNAP gene was used to track the active fraction of libraries during selection rounds 2-5, with an increase in recovered library of between 5- and 35-fold. All five rounds were sequenced on an Illumina MiSeq instrument, producing on average 5.4 x 10 5 reads per library per round. An average of 63% of reads per round passed quality filtering and encoded a nanobody sequence. This increased by approximately 10% from round 1 to 5, from 59% in round 1 to an average of 71% ( ⁇ 2.6% standard deviation), indicating a purifying selection on correctly assembled nanobody genes. Linally, the top twelve hits from these screens based on their abundance in round 5 (in either selection 1 or 2) were extracted, and sequences that were enriched on blank beads were removed.
  • Nanobody sequences corresponding to the top twelve hits were then grafted into the quenchbody scaffold. Following IVTT expression, quenchbodies were subjected to on-column TAMRA labelling, biotinylation, and finally FLAG purification. Reducing SDS-PAGE indicated that most IL-6 quenchbodies were of excellent purity (FIG. 14A). In addition, some quenchbodies migrated as two bands of similar size instead of one, which may have indicated proteolytic cleavage of the products. Spectrophotometry quantification indicated a TAMRA labelling efficiency of approximately 100%.
  • IL-6 quenchbodies were prescreened with IL-6 in fluorescence plate assays. Quenchbodies selected against IL-6 (QB-IL6-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) were incubated for 60 minutes at 25°C in the presence of 500, 125, or 62 nM of IL-6 or in the presence of a denaturant (2% SDS/5% BME).
  • This disclosure therefore demonstrates a generalizable strategy for the rapid production of nanobody-based quenchbody biosensors against proteins of interest with high fluorescence performance.
  • high-performing quenchbodies against IL-6 a master mediator of human inflammation, were successfully evolved, these tryptophan-optimized quenchbodies hold great promise for clinical, diagnostic, or therapeutic applications, such as the detection of IL-6 and potentially other biomarkers in biological fluids.
  • This disclosure is also the first in silico guided study to understand the quenching mechanism and identify the key quenching tryptophans of nanobody-based quenchbodies.
  • This disclosure supports a working mechanism for nanobody-based quenchbodies, whereby CDR-based tryptophans that directly interface with antigens are the most important tryptophans for quenching TAMRA and subsequently becoming dequenched upon antigen binding, resulting in the measured fluorescence increase in the antigenbound state.
  • CDR-based tryptophans that directly interface with antigens are the most important tryptophans for quenching TAMRA and subsequently becoming dequenched upon antigen binding, resulting in the measured fluorescence increase in the antigenbound state.
  • the same general principle applies to scFv and Fab-based quenchbodies, whereby tryptophan residues in the variable region of the antibody (Fv; VH+VL) quench the flexible N-terminal fluorescent molecule of the quenchbody by PET in the apo-state but not in the antigen-bound state.
  • Short-range electrostatics were calculated together with long-range electrostatics particle mesh Ewale (PME) with a cut-off of 9.0 A and a PME grid size of 1.0 A.
  • energy minimization 10,000 steps
  • 125 ps equilibration were performed first with positional restraints placed on all the protein-heavy atoms (with a force constant of 1.0 kcal/mol/A on the backbone atoms and 0.5 kcal/mol/A on the side chain atoms) and TAMRA heavy atoms (with a force constant of 0.5 kcal/mol/A ). This was followed by 12 and 6 ps production runs for the apo- and antigen-bound quenchbodies, respectively.
  • DNA and protein sequence design of quenchbodies Protein coding sequences from either the MBP-binding nanobody or the lysozyme-binding nanobody were designed with (i) an N- terminal Cys-tag as a target for fluorophore labelling, (ii) a C-terminal Avi-tag to enable biotinylation, and (iii) a 2x LLAG-tag and lOxHis-tag to facilitate purification procedures. The entire protein coding element was then converted to DNA and codon optimized using IDT’ s Escherichia coli B strain optimizer.
  • This protein-coding DNA element was then combined into a gene expression cassette and ordered as a Geneblock from IDT, featuring a flanking T7 promoter and terminator for cell-free in vitro transcription and translation (IVTT) of the protein product.
  • the Geneblock also featured flanking DNA elements suitable for polymerase chain reaction (PCR) replication of the Geneblock, specifically DNA compatible with forward primer 5’- ACCCGGCATGACAGGAG-3’ (SEQ ID NO: 73) and reverse primer 5’- GTGGCGGCCGCTCTA-3’ (SEQ ID NO: 74).
  • PCR replication of Geneblocks was conducted using Q5® Hot Start High-Fidelity Master Mix (as per the manufacturer’s instructions) at a scale of 100 pL, using 50 ng of Geneblock as template and 0.5 pM of forward and reverse primer in a PCR Mastercycler (Eppendorf), with initial heating at 98°C for 30s, followed by 32 cycles of denaturation (98°C, 10 s), annealing (60°C, 30 s), and extension (72°C, 15 s).
  • Quenchbody PCR products were purified using Wizard Clean up kits (Promega; as per the manufacturer’s instructions) and were quantified by of A260 absorbance on a Nanodrop 2000C spectrophotometer.
  • Quenchbody PCR products were further analyzed by agarose gel electrophoresis to ensure PCR products were of the expected size and purity before they were used for IVTT expression. All mutant quenchbodies were generated by in silico sequence modification of 5M14 and 1ZVH quenchbody constructs and re-ordered as a Geneblock from IDT.
  • Expressed quenchbodies were then purified from the crude IVTT mixture (100 pL) by combining with 12.5 pL of Pierce Anti-DYKD4K (FLAG) Magnetic Agarose beads (equivalent to 50 pL of original resuspension) prewashed in PBS using a MagJET separation magnet (used for all subsequent wash steps).
  • the crude IVTT-FLAG bead mixture was then mixed at room temperature for 30 minutes at 1200 rpm to allow binding of the FLAG- tagged quenchbody to the beads, and subsequently washed with 0.2 mL PBS.
  • the beads were subjected to an additional wash with 0.2 mL of 0.5 M maltose dissolved in PBS to remove endogenous MBP (present in the endogenous E. coli cell-free lysate as a contaminant), as maltose competes with the nanobody for MBP-binding.
  • the beads were then washed with 100 uL of 1 mM TCEP in PBS for 10 minutes at 16°C with shaking at 1200 rpm, followed by washing with 0.2 mL of degassed PBS.
  • Beads were immediately combined with 100 pL of 250 pM 5(6)-carboxytetramethylrhodamine maleimide with C6-linker dissolved in degassed PBS at a final concentration of 1% (v/v) DMSO, and incubated for 3 hours (room temperature, 1200 rpm). Beads were then subjected to 9 x 0.2 mL washes with PBS to remove unconjugated dye. Finally, quenchbodies were eluted from the beads using 50 pL of 1.5 mg/mL PierceTM 3x DYKDDDDK Peptide (ThermoFisher).
  • Pulldown binding assays To assess their antigen binding, quenchbodies were expressed as above and were purified from the crude IVTT mixture (100 pL) by combining with 12.5 pL beads/50 uL suspension of Pierce Anti-DYKD4K (FLAG) Magnetic Agarose (30 minutes, 1200 rpm).
  • the crude IVTT-FLAG bead mixture was then mixed at room temperature for 30 minutes, 1200 rpm, to allow binding of the FLAG-tagged quenchbody to the beads, and subsequently combined with 100 pL of (i) 1 pM MBP for 5M 14-based quenchbodies, or (ii) 10 pM lysozyme for 1ZVH quenchbodies, and incubated for 30 minutes (room temperature, 1200 rpm). Beads were then subjected to 6 x 0.2 mL washes with PBS to remove any unconjugated or non- specifically bound antigen, and finally eluted with 50 pL of 1.5 mg/mL PierceTM 3x DYKDDDDK Peptide.
  • the eluted quenchbody-antigen complex obtained by FLAG-pulldown was subjected to reducing SDS-PAGE using and the effect of the mutation on antigen binding was semi-quantitatively analyzed by comparison to the binding of the wild-type control.
  • Pierce Anti-DYKD4K (FLAG) Magnetic Agarose exhibited no pulldown of any of the antigens tested when subjected to identical mock pulldown procedures, featuring all IVTT components except with the absence of quenchbody DNA.
  • Fluorescence spectrophotometry plate assays Quenchbodies were diluted to 20 nM in PBS/0.05% (v/v) Tween-20 (PBST) in the absence or presence of 8000, 4000, 2000, 1000, 500, 250, 125, 64, 32, 16, 8, 4, 2, or 1 nM, cognate antigens (lysozyme, MBP, or IL-6), or 2% (w/v) SDS with 5% (v/v) BME as denaturant, and incubated for 1 h at room temperature.
  • PBST PBS/0.05%
  • 8000, 4000, 2000, 1000, 500, 250, 125, 64, 32, 16, 8, 4, 2, or 1 nM, cognate antigens (lysozyme, MBP, or IL-6), or 2% (w/v) SDS with 5% (v/v) BME as denaturant and incubated for 1 h at room temperature.
  • TIRF total internal reflection fluorescence
  • Samples were illuminated using a 532-nm laser (Coherent, Sapphire 532 CW) at 8.3 W/cm -2 . Fluorescence was captured with an EMCCD camera (Andor iXonLife 897) through a band pass emission filter (ET600/50 M, Chroma). For all measurements samples were visualised with an exposure time of 100 ms and an approximately equivalent frame-rate (excluding read time), unless otherwise specified.
  • Example 10 Development of scFv-oligonucleotide conjugates for biomolecular binder detection at the single-molecule level.
  • Oligonucleotide-based biosensors have diverse applications, especially in in vitro protein diagnostics.
  • This Example provides a DNA-based biosensor to detect antibody-antigen interaction at the single molecule level.
  • a single-chain variable fragment (scFv) antibody against a model antigen MBP an “oligobody” was created by attaching complementary oligonucleotides to the scFv.
  • intra-oligo hybridization occurs, while the presence of antigen blocks this process. This allows the detection of antigen binding by introducing a replenishable DNA probe that binds to the free oligo ‘arm’.
  • This Example describes a versatile design scaffold for detecting a diverse library of biomolecular targets using a high-throughput single-molecule detection platform (FIG. 18).
  • ScFv oligonucleotide conjugates were prepared using cell-free approaches.
  • scFv genes were designed with heavy (Vn) and light (VL) chains, liked by a 15-residue amino acid linker and C-terminal tags for biotinylation (avi-tag) and protein purification (FLAG-tag and His-tag) as previously described (FIG. 19A).
  • avi-tag biotinylation
  • FLAG-tag and His-tag protein purification
  • Single molecule TIRF allows visualization of biotinylated DNA complexes or scFv- oligo conjugates attached to streptavidin-coated surfaces. Acquisition post- flow inside allows time-gated fluorescent intensity measurements (FIG. 18). Post-processing involves background fluorescence and drift correction, followed by localization of particles. Intensity integration as a function of time reveal bursts of spikes indicated an association-dissociation event. Dwell-time analysis can reveal dynamics of DNA hybridization to complementary targets. Using this technique, palindromic DNA oligonucleotides with intra-oligo length of 6 bp were evaluated (FIG. 20A).
  • Oligonucleotide-conjugated scFv were immobilized on glass coverslips and A647- conjugated DNA probe-oligo (10 nM) complementary to the scFv-overhangs (8 bp) were flown in and imaged under single molecule TIRF (FIG. 20B). Images before and after addition of MBP (200 nM) in the presence of probe-oligo were analysed. Dwell-time analysis showed only a minor increase in probe binding in the presence of excess MBP (200 nM), albeit consistently (FIGs. 20C-20D).
  • FIG. 22A A non-palindromic 5bp intra-oligo conjugation via unnatural amino acid (UAA) incorporation was also evaluated (FIG. 22A). Amber- stop codon replacement was found to enable UAA p-acetyl-phenylalanine incorporation for dual, site-specific oligo conjugation (FIGs. 22B-24D). Single-conjugated scFv analyzed through single molecule TIRF showed frequent probe binding events (FIG. 22E). The presence of dual intra-oligos abolished this, with no impact in the absence or presence of MBP (FIG. 22F).
  • oligonucleotide-conjugated scFv oligobodies
  • the MBP scaffold e.g., comprising an anti-MBP scFv
  • FIG. 26A It was found that the tested oligonucleotide-conjugated scFv (oligobodies) based on the MBP scaffold (e.g., comprising an anti-MBP scFv) can be imaged for over two hours, demonstrating that these oligobodies are stable over a lengthy time course (FIG. 26A).
  • oligobodies can detect low antigen concentrations.
  • an oligobody based on the MBP scaffold e.g., comprising an anti-MBP scFv
  • concentrations of MBP as low as 7 picomolar (pM) (FIG. 26B) and 7 femtomolar (fM) (FIG. 26C).
  • pM picomolar
  • fM femtomolar
  • top 24 hits were clustered according to their CDR3 sequences, with 15 of those top 24 corresponding to parent Qb-IL6-1, 7 corresponding to parent Qb-IL6-2, and 2 corresponding to parent Qb-IL6-10. These top 24 were further screened in a fluorescence plate assay. It was observed that 11 out of the 24 quenchbodies provided fold-sense between 2.4-1.5 (FIG. 25A) and EC50 values between 436-20 nM (higher than the parent Qb-IL6-1, Qb-IL6-2 and Qb-IL6- 10 quenchbodies, which had EC50 values between 1,113-185 nM) (FIG. 25C).
  • each of the IL-6 quenchbodies (Qb-IL6-1 to Qb-IL6-36) were incubated (60 min, 25°C) in the presence of denaturant (2% SDS/5% B -mercaptoethanol) and analysed in a CLARIOstar fluorescence plate assay to determine fold-quench (FIG. 25B).
  • denaturant 2% SDS/5% B -mercaptoethanol
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.
  • the invention, or aspects of the invention is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

La présente divulgation concerne des anticorps marqués par fluorescence (par exemple, des nanocorps), des procédés de fabrication et des procédés d'utilisation de ceux-ci. Les anticorps marqués peuvent être utilisés pour détecter la présence d'un analyte dans un échantillon.
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WO2022026475A2 (fr) * 2020-07-27 2022-02-03 Igm Biosciences, Inc. Molécules de liaison à un coronavirus multimériques et leurs utilisations
WO2022067091A1 (fr) * 2020-09-25 2022-03-31 DNARx Systèmes et méthodes pour exprimer des biomolécules chez un sujet
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