WO2024017263A1 - Composition de détection et son utilisation - Google Patents

Composition de détection et son utilisation Download PDF

Info

Publication number
WO2024017263A1
WO2024017263A1 PCT/CN2023/107980 CN2023107980W WO2024017263A1 WO 2024017263 A1 WO2024017263 A1 WO 2024017263A1 CN 2023107980 W CN2023107980 W CN 2023107980W WO 2024017263 A1 WO2024017263 A1 WO 2024017263A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
dna
oligonucleotide
hcr
antibodies
Prior art date
Application number
PCT/CN2023/107980
Other languages
English (en)
Inventor
Rui Lin
Shilin ZHONG
Minmin LUO
Original Assignee
Genans Biotechnology Co., Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genans Biotechnology Co., Ltd filed Critical Genans Biotechnology Co., Ltd
Publication of WO2024017263A1 publication Critical patent/WO2024017263A1/fr

Links

Classifications

    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

  • the present disclosure relates to the field ofmolecular biology, and particular to a composition for detecting targets and use thereof.
  • immunoassays are currently the standard and most popular methods for biomolecule detection. These methods use a primary antibody that binds selectively to a target molecule (antigen) , and this antibody-antigen interaction can be visualized via a conjugated reporter on the primary antibody. More commonly, a labeled secondary antibody that is capable of recognizing primary antibodies from a same host species are used to react with the primary antibody-epitope complex to generate signals. In classic immunoassays, fluorescence or luminescence is typically utilized to indicate the abundance and the localization of target molecules. This approach allows only low multiplexing of target molecules due to the overlapping of antibody host species and reporter spectrums. The use of photo-detection as the signal acquisition mechanism also limits the throughput of immunoassays.
  • DNA barcoding represents a promising strategy to overcome these limitations of immunoassays.
  • primary antibodies can be tagged with DNA molecules containing orthogonal barcode sequences. This allows a combination of two or more DNA-tagged primary antibodies to be used on the same sample to detect two or moretarget molecules at the same time. Each individual antibody-antigen interaction can be examined via barcode readout.
  • DNA barcoding offers much higher multiplexity far beyond the scope of conventional fluorescent or luminescent signaling molecules.
  • careful design of DNA barcode sequences also enables combined analysis of antibody DNA barcodes from parallel reactions using high through-put sequencing, which dramatically increases the detection throughput.
  • the present disclosure provides aMultiplexed and Modular Barcoding of Antibodies (MaMBA) strategy, which is a simple and cost-effective strategy to barcode antibodies with single-stranded DNAs by using nanobodies as the adaptor.
  • the MaMBA strategy is generally applicable to IgGs without the requirement of chemical modifications, allowing its rapid dissemination to a vast number of off-the-shelf antibodies.
  • the present disclosure provides a composition, which comprises an antibody linked with an oligonucleotidevia a disulfide bond, wherein the oligonucleotide is specific for the antibody.
  • the disulfide bond may be cleavable. According to some embodiments, the disulfide bond may be cut by a reducing agent. According to some embodiments, the disulfide bond may be cut by a reductive cleavage, e.g. by TCEP (Tris (2-carboxyethyl) phosphine) , DTT (dithiothreitol) , BME (Beta-mercaptoethanol) and the like, to remove the antibody specific oligonucleotide from the antibody.
  • TCEP Tris (2-carboxyethyl) phosphine
  • DTT dithiothreitol
  • BME Beta-mercaptoethanol
  • the antibody specific oligonucleotide may comprise a hybridization chain reaction (HCR) initiator.
  • HCR hybridization chain reaction
  • the antibody specific oligonucleotide may comprise a first barcode sequence which is specific for the antibody that it is associated therewith.
  • the barcode sequence may comprise5 to 15 nucleotides in length. According to some specific embodiment, the barcode sequence may comprise 5 to 10 nucleotides in length, e.g. 5, 6, 7, 8, 9, or 10 nucleotides.
  • the oligonucleotide may further comprise a unique molecular identifier (UMI) sequence.
  • UMI unique molecular identifier
  • the unique molecular identifier sequence may comprise 10 to 20 nucleotides in length.
  • the unique molecular identifiersequence may comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.
  • the oligonucleotide may further comprise a second barcode sequence.
  • the second barcode sequence may be specific forany information associated with a sample to be detected, such as the well in which the sample locates.
  • the oligonucleotide may further comprise anamplification region for performing a polymerase chain reaction (PCR) amplification reaction.
  • the oligonucleotide may further comprise a pair of primer sequences for polymerase chain reaction (PCR) , qPCR or sequencing.
  • the oligonucleotide-linkedantibody may be a secondary antibody.
  • the oligonucleotide-linkedantibody may be selected from a group consisting of an anti-IgE antibody, an anti-IgM antibody, an anti-IgG antibody, an anti-IgA antibody, and an anti-IgD antibody.
  • the oligonucleotide-linkedantibody may be a nanobody.
  • the nanobody can specifically bind to the Fc region of the anti-IgE, anti-IgM, anti-IgG, anti-IgA or anti-IgD antibody.
  • the oligonucleotide-linkedantibody may be obtained by a step of reacting an antibody with an oligonucleotide conjugated with disulfide (-S-S-) .
  • the antibody may be modified with an azide moiety.
  • the oligonucleotide may be further modified with a dibenzocyclooctyne group (DBCO) .
  • DBCO dibenzocyclooctyne group
  • the present disclosure provides a kit for detecting one or more targets, which comprises the above composition.
  • the kit may comprise one or more oligonucleotide-linkedantibodies each comprising an antibody linkedwith a specificHCR initiator, and one or more amplifiers.
  • the one or more amplifiers can trigger hybridization chain reactions.
  • each amplifier may be conjugated with a detectable label, which may include, but do not limit to, a chemical group, such as biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, Tetrazine, Alkyne and DBCO; and a fluorophoresuch as FITC, Cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluor dyes (e.g., Alexa Fluro 546, Alexa Fluor 488, and Alexa Fluor 647) .
  • a detectable label such as biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol,
  • the kit may comprise one or morepairs of primers for specifically amplify the antibody specific oligonucleotide.
  • the kit may comprise one or more oligonucleotide-linked antibodies each comprising an antibody linked with an oligonucleotide, wherein the oligonucleotide comprises a barcode sequence and a region for an amplification reaction.
  • the kit may comprise one or more pair primes for performing the amplification reaction.
  • the present disclosure provides a method for detecting one or more targets in a sample by using the above composition or the above kit.
  • the method may comprise a step of a) incubating a first primary antibody with the above composition, to form a complex having the first primary antibody associated with the composition.
  • the antibody of the composition is a secondary antibody
  • the complex is formed by the interaction between the first primary antibody and the secondary antibody.
  • the composition may comprise a secondary antibody linked with an oligonucleotidevia a disulfide bond, wherein the oligonucleotide is specific for the antibody.
  • the disulfide bond may be cut by a reducing agent.
  • the method may comprise the steps of: a) a first primary antibody with the above composition, to form a complex having the first primary antibody associated with the composition; b) contacting the sample with the complex; c) making an amplification reaction to produce detectable amplified products; and optionally, d) adding a reducing agent and incubating to cut the disulfide bond, andrepeating steps a) to c) .
  • one or more primary antibodies may be incubated with one or more secondary antibodies, respectively, toform one or more complexes, each complex may comprise a primary antibody and a secondary antibody associated a specific oligonucleotide.
  • each secondary antibody may be associated with a specific oligonucleotide which can be used as abarcode.
  • Each of the complexes formed by the primary antibody and the secondary antibody can thus be conjugated with a specific barcode.
  • the specific oligonucleotide may comprise an HCR initiator.
  • the method of the present disclosure may comprise the steps of: a) a first primary antibody with the above composition, to form a complex having the first primary antibody associated with the composition; b) contacting the sample with the complex; c) performing an HCR reaction to produce detectable amplified products; and optionally, d) adding a reducing agent and incubating to cut the disulfide bond, andrepeating steps a) to c) .
  • HCR amplifiers are added in step c) .
  • the method may be used for anin situ assay or ex vivo assay.
  • the specific oligonucleotide may comprise a first barcode sequence.
  • the specific oligonucleotide may further comprise a unique molecular identifier (UMI) sequence. Both the first barcode sequence and the unique identifier sequence may be specific for the antibody that they are associated therewith.
  • the specific oligonucleotide may further comprise a second barcode sequence. The second barcode sequence may be specific for any information associated with a sample to be detected, such as the well in which the sample locates.
  • the method of the present disclosure may comprise the steps of: a) a first primary antibody with the above composition, to form a complex having the first primary antibody associated with the composition; b) contacting the sample with the complex; c) adding a reducing agent and incubating to cut the disulfide bond; d) collecting the specific oligonucleotides andperforming anamplification reactionto detect the one or more targets; and optionally, e) repeating steps a) to d) .
  • the method of the present disclosure may be used for semi-quantitatively or quantitatively measuring the one or more targets in the sample.
  • the method of the present disclosure may be used for sequencing one or more targets.
  • the method may comprise, before step b) , a step of incubating the sample with a support which is associated with a target-specific binding substance, to capture the target in the sample.
  • the target-specific binding substance may be a second primary antibody.
  • the target-specific binding substance may be an antigen, and the target to be detected may be an antibody.
  • the support may be selected from a group consisting of a microplate and magnetic beads.
  • one or more first primary antibodies may incubate with one or more compositions each linked with an oligonucleotide specific for the antibody that it is linked therewith.
  • the method of the present disclosure may be used for high-throughput sequencing.
  • a washing and/or blocking step may be requiredafter each step.
  • the method may comprise a step of blocking using a protein-free blocking buffer and sheared Salmon sperm DNA (ssDNA) .
  • ssDNA sheared Salmon sperm DNA
  • the method may comprise, before step a) , a step of contacting the sample with a plurality of target-specific binding substances to capture the plurality of targets in the sample.
  • the target-specific binding substance may be antigen and the target to be detected may be an antibody.
  • the target-specific binding substance may be a primary antibody and the target to be detected may be an antigen recognized by this primary antibody.
  • the present disclosure first combined MaMBA with a fluorescent signal amplification method (immunosignal hybridization chain reaction, isHCR) and established a highly multiplexed and sensitive immunohistochemistry for spatial protein profiling (multiplexed isHCR, misHCR) . Further a cleavable MaMBA is applied to increase the multiplexity of misHCR and developed a multi-round version of misHCR (misHCR n ) . Finally, this strategy has been demonstrated to be capable ofbeing adopted for traditional enzyme-linked immunosorbent assay (ELISA) and developed a barcode-linked immunosorbent assay (BLISA) for multiplexed and high-throughput protein detections combined with DNA sequencing.
  • ELISA enzyme-linked immunosorbent assay
  • BLISA barcode-linked immunosorbent assay
  • Fig. 1 Design of MaMBA.
  • A DNA-barcoding modules production overview. Nanobodies (Nb) targeting the Fc region of IgGs, are conjugated with an azide (N 3 ) group via OaAEP1 enzymatic reaction in which the C-terminal ‘NGL’ peptide sequences are recognized, cut, and ligated to ‘GVG-K (N 3 ) -RG’ peptide (orange dotted line shows the cut site of the enzyme) .
  • the N 3 -conjugated nanobodies are linked to DBCO-modified DNA oligonucleotide (DNA oligo) via the click reaction.
  • the final product (Nb-DNA oligo) then is ready for barcoding.
  • B MaMBA workflow.
  • Nb-DNA oligo employing a unique DNA barcode.
  • Nb-DNA oligo is bound to the Fc region of antibody to form the DNA-barcoded complex (Ab-Nb 1 to Ab-Nb 3) , following purification by filtration. Then the Ab-Nb-DNA oligos are available to be pooled and applied for assays (IHC, immunoassay, etc. ) .
  • Fig. 2 MaMBA applied for multiplexed in situ protein imaging by misHCR.
  • A Schematic of misHCR based on the HCR reaction processed by a single-strand DNA (HCR initiator) and a pair of DNA hairpins (HCR amplifiers) . One-step staining is achieved by the Nb-HCR initiator-bound antibody (Ab-HCR initiator) . Arrows indicate the 5’ -to-3’ direction.
  • HCR amplifiers are modified with fluorophores.
  • B-D Fluorescent images of mouse brain sections with or without HCR amplification.
  • Sections are stained by anti-NeuN antibody with Cy5 dye-conjugated Nb (Nb-Cy5) or misHCR using Cy5 dye-conjugated HCR amplifiers (misHCR-Cy5) , and stained by anti-Th antibody with Nb-Cy3 or misHCR-Cy3.
  • E Multiplexed misHCR imaging workflow.
  • Antibodies barcoded with unique HCR initiators are pooled and applied simultaneously to detect the antigens (Ag. 1 to Ag. n) , followed by sequential rounds of imaging via hybridization and dehybridization of orthogonal HCR amplifiers (Amp. ) .
  • F Fluorescent images of human psoriatic skin section stained for DAPI and six other epitopes with three rounds of HCR imaging. Scale bar, 300 ⁇ m. Host species of antibodies are shown in the parentheses.
  • Rb rabbit; Mus, mouse; Th, tyrosine hydroxylase; PDGFR ⁇ , platelet-derived growth factor receptor A; KRT14, keratin 14; DCT, dopachrome tautomerase; ⁇ SMA, alpha smooth muscle actin.
  • Fig. 3 Cleavable MaMBA enables increased multiplexity for in situ protein imaging with misHCR n .
  • A Cleavable DNA-barcoding module production overview. DBCO-modified DNA oligo bearing a disulfide bond is linked to N 3 -conjugated Nb via click reaction. The product (Nb-SS-DNA oligo) is ready for barcoding and the DNA oligo could be removed by reductive cleavage (TCEP treatment, etc. ) . TCEP, Tris (2-carboxyethyl) phosphine. B, misHCR n staining and imaging outline.
  • Antibodies used in the 2 nd round of misHCR could be equipped with the same set of HCR initiators as in the 1 st round of misHCR (Ab-SS-i1’ to Ab-SS-in’ ) and thus for the same set of HCR amplifiers in image rounds. Then after removing HCR initiators, another round of misHCR is available to be performed, and so on.
  • C Fluorescent images of the dorsal raphe nucleus (DRN) in mouse brain sections stained for DAPI and 12 other epitopes by two rounds of misHCR with seven image rounds in total. The imaging location was marked in the boxed region (red) of the mouse brain atlas shown in the bottom left. Host species of antibodies are shown in the parentheses.
  • nNOS neuronal nitric oxide synthase
  • GFAP glial fibrillary acidic protein
  • Th tyrosine hydroxylase
  • NF-H neurofilament-H
  • Tph2 tryptophan hydroxylase 2
  • GABA gamma-aminobutyric acid
  • TMEM119 transmembrane protein 119
  • DDC dopa decarboxylase
  • 5-HT 5-hydroxytryptamine
  • D Zoomed-in views of the dotted-box region marked in c.
  • Fig. 4 Cleavable MaMBA applied for multiplexed quantitative protein detection by BLISA.
  • A BLISA of direct antigen detection workflow. The biomolecules of samples are directly immobilized to the surface of polystyrene microplate wells, and the target antigens (Ag. 1 to Ag. 3) are detected by the pool of unique-and-cleavable DNA-barcoded antibodies (Ab-SS-DNA barcodes) . DNA barcodes are retrieved by TCEP via breaking the disulfide bonds. Released DNA barcodes are quantified by qPCR to reveal the antigen levels.
  • B Schematic of multiplexed detection for GFP, mCherry and ⁇ -tubulin by BLISA.
  • HEK293T cells were transiently co-transfected to express GFP and mCherry, which are under control of drug-inducible promoters.
  • the expression of GFP is controlled by the Tet-ON system and induced by ATc, a tetracycline derivative, which binds to rtTA transcription factor and allows it to bind DNA at the TRE promoter to trigger gene expression.
  • ATc a tetracycline derivative
  • FSK tetracycline derivative
  • the expression of mCherry is controlled by3 ⁇ CRE promoter and induced by FSK in the presence of 0.3 mM IBMX. ATc and FSK were titrated against each other to produce a series of culture conditions.
  • F Representative results of BLISA qPCR and Western blot bands for samples described in Fig. 4E.
  • Three targets (GFP, mCherry and ⁇ -tubulin) were detected by BLISA and Western blot.
  • ATc anhydrotetracycline hydrochloride
  • TRE tetracycline response element
  • CRE cAMP-response element
  • FSK forskolin
  • IBMX 3-Isobutyl-1-methylxanthine.
  • Fig. 5 Sequencing-combined BLISA for anti-SARS-CoV-2 Spike RBD IgG detection in human serum.
  • A BLISA of sandwich-based antibody detection workflow. The purified antigen is immobilized on the microplate so that the target antibody is captured during sample loading. After washing away other unbound biomolecules, the target antibody is detected by the DNA-barcoded secondary antibody (2 nd Ab-SS-DNA barcode) . DNA barcode is retrieved from the plate in solution containing TCEP and a known amount of DNA barcode for normalization (norm bc) , following amplicon generation for sequencing.
  • B Schematic of ELISA and BLISA for human IgG detection.
  • Human IgG is immobilized on the microplate and detected by anti-human IgG antibody (from Rb host species) which is DNA-barcoded for qPCR, or follows horseradish peroxidase (HRP) -conjugated anti-Rb IgG antibody for colorimetric readout of ELISA using 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrates.
  • HRP horseradish peroxidase
  • TMB TMB
  • DNA barcode contains an Ab barcode (Ab bc) and a unique molecular identifier (UMI) .
  • Released DNA barcode is further barcoded by a three-step PCR protocol for well barcode (well bc) addition, phasing spacers (PS) addition, and illumina sequencing adaptors tagging.
  • E Annotated amplicon sequence of the final product for illumina sequencing.
  • F Schematic of BLISA for human anti-SARS-CoV-2 Spike RBD IgG detection.
  • G BLISA sequencing results for large-scale anti-SARS-CoV-2 Spike RBD IgG detection in human serum samples. The scatterplot indicates averaged anti-SARS-CoV-2 Spike RBD IgG abundance normalized by normalization barcode for 505 human serum samples (unknown) listed by sub-groups of plate (S1 to S12) .
  • Each plate includes a blank and a negative control (neg. ctrl) .
  • Negative control represents serum from individuals that were neither SARS-CoV-2 infected nor vaccinated.
  • I Decreased coefficient of variation (CV) after normalization step. The histogram (left) presents relative distribution (percentage) of CV of each duplicate from raw (gray) or normalized (red) data. The boxplot (right) of the distribution of CV between raw and normalized groups is analyzed using two-sided paired t-test. ****P ⁇ 0.0001.
  • Fig. 6 2-plex sequencing-combined BLISA for hepatitis B virus (HBV) antigens detection in human serum.
  • A BLISA of sandwich-based antigen detection workflow. The capture antibody (Ab Cap ) is immobilized on the microplate. Sample and the DNA-barcoded detection antibody (Ab Det -SS-DNA barcode) are simultaneously loaded. The target antigen then is trapped between Ab Cap and Ab Det . DNA barcode is retrieved from the plate in solution containing TCEP and a known amount of normalization barcode, following amplicon generation for sequencing.
  • B Schematic of 2-plex BLISA for HBsAg and HBeAg detection.
  • C-E 2-plex antigens detection in control serum samples by BLISA.
  • qPCR results (C) of 2-plex BLISA indicates HBsAg (blue) and HBeAg (red) levels in double-negative and double-positive serum samples (neg. 1 to neg. 3, pos. 1 to pos. 4) which had been defined by commercial ELISA kits (D, E) .
  • n 2 replicates each, mean ⁇ s.e.m.
  • F Summary of the averaged antigen levels detected in negative and positive groups for HBsAg and HBeAg. mean ⁇ s.e.m. ****P ⁇ 0.0001, **P ⁇ 0.01, two-sided t-test.
  • the scatterplot indicates normalized averaged abundances of HBsAg and HBeAg for 498 human serum samples listed by sub-groups of plate (H1 to H12) .
  • n 2 replicates.
  • H Frequency distribution of the antigen abundances for HBsAg (blue) and HBeAg (red) .
  • I Correlation of the normalized abundances between duplicate for HBsAg (left) and HBeAg (right) . Pearson’s correlations were performed.
  • J Validation of the sequencing data by ELISA.
  • Fig. 7 Multiplexed detection of phospho-proteins by BLISA based on magnetic beads (MB) .
  • A Schematic of BLISA for multiplexed detection on MB. Antigens are detected by sandwich-based assay in which capture antibodies are attached to the MB, identified via different DNA barcodes (bc. 1 to bc. n) .
  • B MB-based BLISA workflow. Capture antibodies-bound MB are mixed and aliquoted into sample wells of the deep-well plate. Samples are loaded and incubated with MB. After washing steps utilizing the magnetic isolation to remove the unbound biomolecules, the captured antigens are detected by the DNA-barcoded detection antibodies (Ab Det -SS-DNA barcodes) .
  • n 3 replicates, mean ⁇ s.e.m.
  • p-p38 ⁇ phospho-p38 ⁇ (T180/Y182) ; p-ERK1/2, phospho-ERK1 (T202/Y204) /ERK2 (T185/Y187) ; p-JNK, phospho-JNK Pan Specific; p-AMPK ⁇ 1, phospho-AMPK ⁇ 1 (T183) ; p-CREB, phospho-CREB (S133) ; p-Src, phospho-Src (Y419) ; p-Akt, phospho-Akt (S473) ; NT, non-treated; FBS, fetal bovine serum; Aniso, Anisomycin.
  • Fig. 8 SDS-PAGE verification of Nb-DNA oligo production and the unaffected spatial resolution of misHCR imaging.
  • A Non-reducing SDS-PAGE gel for purified Nanobody-NGL-His 6 (Nb-NGL-His 6 ) , the OaAEP1 reaction product (Nb-NGV-N 3 ) , and the Nb-DNA oligos. The same gel was first stained for the DNA oligo by nucleic acid dye and then stained for protein by Coomassie brilliant blue.
  • B Fluorescent images of ⁇ -tubulin in HeLa cells stained by traditional method using Alexa fluor 647-conjugated secondary antibody (2 nd Ab) (left) and by misHCR using Alexa Fluor 647-conjugated HCR amplifiers (right) . Zoomed-in views were shown as marked in the boxed region. Scale bar, 10 ⁇ m.
  • C Example intensity profiles illustrated in B for straight lines drawn perpendicular to the microtubule structure.
  • D Spatial resolutions of traditional method (2 nd Ab) and misHCR by calculating the full width at half maximum (FWHM) for a series of microtubule structure intensity profiles.
  • A-B Fluorescent images of mouse brain sections before and after HCR amplifiers or initiators removal by formamide or TCEP. Scale bar, 500 ⁇ m (A) , 300 ⁇ m (B) .
  • C Fluorescent images of dorsal raphe nucleus (DRN) in mouse brain sections for NeuN, NF-H, and Th through five rounds of HCR imaging-and-washing cycle. Scale bar, 100 ⁇ m.
  • D Signal-to-noise ratios (SNRs) of each round of HCR imaging for NeuN (left) and NF-H (right) .
  • n 3 brain sections for each group, mean ⁇ s.e.m.
  • One-way ANOVA tests were performed. The images of every group were acquired using identical microscopy settings. NF-H, neurofilament-H.
  • Fig. 10misHCR imaging in healthy human skin sections Fluorescent images of healthy human skin section stained for DAPI and six other epitopes with three rounds of HCR imaging. Host species of antibodies are shown in the parentheses. Scale bar, 200 ⁇ m.
  • PDGFR ⁇ platelet-derived growth factor receptor A
  • KRT14 keratin 14
  • DCT dopachrome tautomerase
  • ⁇ SMA alpha smooth muscle actin.
  • Fig. 11 Multi-round misHCR for nine targets using three sets of HCR initiator and amplifiers repeatedly.
  • A Fluorescent images of mouse brain sections stained for DAPI and 9 other targets by three rounds of misHCR with one image round of each. The imaging location was marked in the boxed region of the mouse brain atlas shown in the bottom right. Scale bar, 300 ⁇ m.
  • B Zoomed-in views of the dotted-box region marked in A. Different target signals were not co-localized in misHCR imaging (marked with arrows) . Scale bar, 25 ⁇ m.
  • MAP2 microtubule-associated protein 2
  • DDC dopa decarboxylase
  • nNOS neuronal nitric oxide synthase
  • Iba1 ionized calcium binding adaptor molecule 1
  • GFAP glial fibrillary acidic protein
  • Th tyrosine hydroxylase
  • NF-H neurofilament-H
  • Tph2 tryptophan hydroxylase 2.
  • Fig. 12Co-localization of antibody staining patterns for traditional method and misHCR Mouse brain sections were stained with antibodies against different epitopes and then incubated with equal amounts of Cy3-conjugated secondary antibodies (2 nd Ab-Cy3) and Nb-HCR initiators following HCR using Alexa Fluor 647-conjugated amplifiers (misHCR-647) . Scale bar, 100 ⁇ m.
  • Fig. 13 Endogenous proteins detection in HEK293T cells by BLISA and Western blot, and sequencing-combined BLISA for multiplexed detection of GFP, mCherry and ⁇ -tubulin.
  • A Endogenous GFP detection.
  • HEK293T cells were transiently transfected to express GFP, which are under control of the Tet-ON system and induced by ATc, a tetracycline derivative, which binds to rtTA transcription factor and allows it to bind DNA at the TRE promoter to trigger gene expression.
  • C Endogenous mCherry detection. HEK293T cells were transiently transfected to express mCherry, which are under control of the3 ⁇ CRE promoter and induced by FSK in the presence of 0.3 mM IBMX.
  • D Representative results of BLISA qPCR and Western blot bands (left panel) , and their corresponding correlation of relative expression levels of mCherry (normalized to ⁇ -tubulin) (right panel) .
  • DNA barcode design and amplicon generation for BLISA sequencing contains an Ab barcode (Ab bc) and a unique molecular identifier (UMI) . Released DNA barcode is further barcoded by a two-step PCR protocol for well barcode (well bc) addition and illumina sequencing adaptors tagging.
  • F UMI counts for the three targets (GFP, mCherry, and ⁇ -tubulin) in the samples as designed in samples containing purified GFP and mCherry loaded against each other with 1 ⁇ g/mL cell lysates ( ⁇ -tubulin) .
  • Fig. 14 Reproductivity and plate-to-plate variability of sequencing-combined BLISA.
  • A Standard curves of purified phospho-proteins (p-p38 ⁇ , p-ERK1/2, p-JNK, p-AMPK ⁇ 1, p-CREB, p-Src, and p-Akt) in BLISA and ELISA.
  • B R 2 of the standard curves.
  • nanobodies which are single-domain antibodies derived from camelid heavy-chain antibodies, increasingly become popular as powerful tools in cell biology, structural biology, and therapy, taking advantage of their excellent functionality (small size, easy production, flexible manipulation, etc) .
  • secondary nanobodies nanobodies against antibodies
  • different antibodies even sharing the same host species can be simply pre-incubated with the secondary nanobodies and then applied to sample together, which is infeasible for bivalent secondary antibodies.
  • MaMBA an efficient modular barcoding strategy that is generally applicable to antibodies via nanobodies for simple and site-specific DNA barcoding.
  • misHCR a multiplexed in situ protein imaging method, which combined HCR technology with immunohistochemistry using orthogonal HCR initiator-barcoded antibodies.
  • a cleavable MaMBA by employing a disulfide linker between nanobody and DNA, and further applied it to the multi-round version of misHCR (misHCR n ) .
  • misHCR n the multi-round version of misHCR
  • Fluorescent immunostaining has become a routine approach in both biological and clinical laboratories.
  • traditional methods offer limited multiplexing targets.
  • strategies like sequential antibody staining strips the bound antibodies and starts a new round of staining
  • enhanced multiplexing it requires multiple rounds of antibodies incubation (hours or even days per cycle) and takes weeks to image tens of targets.
  • DNA barcoding approaches enable simultaneous antibodies binding with orthogonal DNA barcodes, followed by sequential docking-strand readout to achieve faster multiplexed imaging.
  • DNA conjugation should be applied to antibodies, which makes sequential docking-strand imaging weaker signal than traditional immunostaining because it loses the signal enhancement from using secondary antibodies.
  • DNA-based signal amplification methods were applied such as rolling circle amplification (RCA) , signal amplification by exchange reaction (SEBAR) , and HCR.
  • RCA rolling circle amplification
  • SEBAR signal amplification by exchange reaction
  • HCR utilizes a pair of HCR hairpins that iteratively opened, triggered by a complementary HCR initiator.
  • Signal amplification by HCR happens in situ and could be tuned by changing the concentration of HCR amplifiers.
  • misHCR not only achieved the multiplexing relied on the power of DNA barcodes, but also obtained amplified signal levels via the HCR reaction.
  • misHCR is not limited to fluorescent imaging.
  • methods based on Raman microscopy were developed for multiplexed protein imaging, which utilize orthogonal Raman dyes that possess much narrower vibrational peaks than fluorescence.
  • Raman dye imaging shows the advantage of multiplexing in one shot, which could greatly accelerate the experimental procedure.
  • Immunoassays based on DNA-barcoded antibodies have shown the advantage that dramatically enhances the sensitivity of conventional immunoassays. For example, taking advantage of DNA amplification techniques, immuno-PCR and its related methods showed excellent performance in ultrasensitive detection for low abundance proteins, typically leading to 10-10, 000-fold increase in sensitivity compared with analogous enzyme-amplified immunoassay. Therefore, combined with the sequencing technique, a variety of immunoassays utilizing DNA-barcoded antibodies were developed and demonstrated for highly multiplexed and high-throughput proteins detection (e.g. ID-seq) , further single-cell level readout (e.g. CITE-seq) , and even transcriptome-combined expression profiling (e.g.
  • ID-seq highly multiplexed and high-throughput proteins detection
  • CITE-seq further single-cell level readout
  • transcriptome-combined expression profiling e.g.
  • BLISA adopts the MaMBA strategy for non-covalent and site-specific barcoding of antibodies using secondary nanobodies as modular mediators.
  • Pipeline-synthesized nanobody-DNA conjugates can be directly mixed with antibodies, and each purified DNA-barcoded antibody then is ready for the multiplexed assembly.
  • This straightforward DNA barcoding workflow is designed to be suitable for large-scale pipeline production, which enables wider applications of DNA-barcoded-antibodies-based techniques with higher efficiency and lower costs.
  • a recombinant AAV virion includes a plurality of such virions and reference to “microglia” includes reference to one or more microglia cells and equivalents thereof known to those skilled in the art, and so forth.
  • hybridization chain reaction or “HCR” used herein is a technique, based on a chain reaction of recognition and hybridization events between two sets of DNA hairpin molecules, offers an enzyme-free alternative for the rapid detection of specific DNA sequences.
  • HCR uses a pair of complementary, kineticallytrapped hairpin oligomers to propagate a chain reaction of hybridization events.
  • hybridization chain reaction initiator or “HCR initiator” used herein refers to a nucleic acid region that can trigger the polymerization of two metastable HCR hairpin monomer species to form an HCR amplification polymer.
  • An exposed HCR initiator is functional and triggers polymerization of the metastable HCR hairpin monomers under polymerizing conditions.
  • a sequestered HCR initiator is non-functional and does not trigger polymerization of the metastable HCR hairpins monomers under polymerizing conditions.
  • a sequestered HCR initiator (hence, initially non-functional) can be exposed (hence, becoming functional) upon binding of another molecule to the sequestering molecule.
  • nanobody as defined herein includes, but not limited to a VHH sequence.
  • the nanobody of the present disclosure specifically binds to Fc domain of the primary antibody.
  • UMI unique molecular identifier
  • DNA oligos were synthesized by Thermo Fisher Scientific, Genewiz and Sangon Biotech. Detailed sequences and modifications of DNA oligos are listed in Table 1. All oligos were dissolved in nuclease-free water (Thermo Fisher Scientific, AM9932) and stored at -20°C.
  • SSC sodium chloride citrate
  • AM9763 sheared Salmon sperm DNA
  • FNN0011 cell extraction buffer
  • PFBB protein-free blocking buffer
  • DNA fragments were amplified by PCR using primers (Genewiz) with 17-20 bp overlaps for Gibson assembly, and plasmid sequences were verified using Sanger sequencing.
  • pET28a-His 6 -Ubiquitin-OaAEP1 C247A was constructed as described 1 , and DNA sequences encoding TP897-NGL-His 6 and TP1107-NGL-His 6 were synthesized (Genewiz) and cloned into pET28a vector.
  • EGFP and mCherry sequences were cloned into the pET28a vector fused with C-terminal His 6 -tag sequences.
  • EGFP and rtTA sequences were cloned into a pLJM1 vector (Addgene, 91980) 2 under control of a TREpromoter and the PGK promoter (by replacing the original puromycin N-acetyltransferase gene) respectively, and sequences encoding mCherry were cloned into another pLJM1 vector following a 3 ⁇ CRE promoter.
  • Recombinant OaAEP1 (C247A) was expressed and obtained. Briefly, pET28a-His 6 -Ubiquitin-OaAEP1 (C247A) was transformed into E. coli SHuffle and the protein expression was induced overnight with 1 mM IPTG at 18°C. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole) , and lysed by sonication. Cell debris was cleared via 1 hr of centrifugation at 39, 000 g at 4 °C.
  • lysis buffer 50 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl, 10 mM imidazole
  • the supernatant was bound to Ni-NTA resin, washed with AEP wash buffer (50 mM NaH 2 PO 4 , pH 8.0, 0.3 M NaCl, 20 mM imidazole) , and eluted with elution buffer (50 mM NaH 2 PO 4 , pH 8.0, 0.3 M NaCl, 250 mM imidazole) .
  • AEP wash buffer 50 mM NaH 2 PO 4 , pH 8.0, 0.3 M NaCl, 20 mM imidazole
  • elution buffer 50 mM NaH 2 PO 4 , pH 8.0, 0.3 M NaCl, 250 mM imidazole
  • Fractions containing mature AEP were pooled and concentrated using 10 K MWCO concentrator (Sartorius) .
  • the final product was analyzed by SDS-PAGE, quantified via A 280 reading ( N60, IMPLEN) according to the protein molecular weight and extinctions coefficient, and then stored in aliquots at -80°C until use.
  • Nanobodies against Fc domain of IgG were expressed and purified. Briefly, pET21a-TP897-NGL-His 6 and pET21a-TP1107-NGL-His 6 were transformed into E. coli Shuffle. Expression was induced for 16-18 h with 1 mM IPTG at 28°C. After harvested by centrifugation, cells were resuspended in Nb lysis buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM Imidazole, 10%Glycerol) and lysed by sonication. Cells debris was removed by centrifugation and the supernatant was applied to Co-NTA resin.
  • Nb lysis buffer 50 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM Imidazole, 10%Glycerol
  • the resin was washed with Nb wash buffer (20 mM HEPES, pH 7.5, 300 mM NaCl. 10 mM Imidazole, 10%Glycerol) and the His-tagged nanobodies were eluted with Nb elution buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 500 mM Imidazole, 10%Glycerol) .
  • the nanobodies were further purified and buffer exchanged by size exclusion column (SEC) in Nb exchange buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 10%Glycerol) . Fractions containing nanobodies were pooled and concentrated using Amicon 3 K MWCO concentrator (Millipore) .
  • Protein concentrations were determined by A 280 reading as described above. Nanobodies were stored in aliquots at -80°C until use. The amino acids sequences of TP897-NGL-His 6 and TP1107-NGL-His 6 are listed in Table 2.
  • pET28a-GFP and pET28a-mCherry were transformed into E. coli BL21 (DE3) . Expression was induced for 16-18 h with 0.5 mM IPTG at 16°C. GFP and mCherry were purified using Co-NTA resin as described above, and were further purified by SEC in PBS. Proteins were concentrated using Amicon 10 K MWCO concentrator and quantified by A 280 reading.
  • NGL-tagged nanobodies were labeled C-terminally using GVG-K (N 3 ) -RG by OaAEP1 reaction. This was carried out by reacting 50 ⁇ M nanobody, 1 mM GVG-K (N 3 ) -RG, and 750 nM OaAEP1 (C247A) in reaction buffer (100 mM sodium phosphate buffer, pH 6.5, supplemented with 2 mM DTT) overnight with gentle shaking at RT. The reaction mixture was then 1: 1 diluted in Nb lysis buffer and bound to Ni-NTA resin for removing GL-His 6 and unreacted His-tagged nanobody. Resin was washed with Nb lysis buffer until A 280 reached baseline.
  • Flowthrough and wash fractions containing Azide-labeled nanobodies were pooled and remaining GVG-K (N 3 ) -RG was removed using 3,000 MWCO concentrator. Azide-labeled nanobodies were also quantified by A 280 reading and analyzed by SDS-PAGE.
  • Nanobody-DNA oligonucleotide conjugation
  • Nb-SS-HCR initiator and Nb-SS-DNA barcode we used DBCO-PEG 3 -SS-NHS (10 mM in anhydrous DMSO; CP-2089, Conju-Probe, LLC) as the linker. HCR initiators and DNA barcodes modified with a 5’ NH 2 -C6 modification were reacted with DBCO-PEG 3 -SS-NHS (20-fold molar excess) in 0.091 M NaB (pH 8.5) at RT for 2 h with gentle shaking.
  • oligonucleotides were precipitated by mixing with one-tenth volume of 3 M sodium acetate at pH 5.2 and 2 volumes of cold absolute ethanol following incubation for 1 h at -20°C. After a 30-min spin at 20, 000g at 4°C, supernatant was removed and pellet was carefully rinsed twice by cold 70%ethanol. Pellet was air-dried and re-dissolved in TE buffer. Excess cross-linkers were further removed by Zeba spin desalting column (7K MWCO) .
  • HCR initiators modified with a 5’ DBCO moiety (2-fold molar excess) or the DBCO-PEG 3 -SS-conjugated oligos (2-fold molar excess) were mixed with 25 ⁇ M azide-labeled nanobodies, and reacted at 4°C with shaking for 16 h.
  • the DNA oligo-conjugated nanobodies were analyzed by SDS-PAGE.
  • Antibodies were premixed with 3-fold molar excess of DNA oligo-conjugated nanobodies (Nb-HCR initiator, Nb-SS-HCR initiator, or Nb-SS-DNA barcode) respectively in 50 ⁇ L premixing buffer (0.1%BSA, 0.05%Triton X-100, 5 mM EDTA in PBS) at RT with shaking for 3-5 h. The mixtures were then added to Amicon Ultra-0.5 100 K centrifugal filters and washed ten times with 400 ⁇ L PBS containing 0.05%Tween-20 and 5 mM EDTA. Then the purified DNA-barcoded antibodies were stored at 4°Cwith 0.03%NaN 3 or stored at -20°C with 50%glycerol until assaying.
  • mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (China) , and adult (8-12 weeks old) mice of either sex were used. Mice were maintained with a 12-h light/dark photoperiod (light on at 8 AM) and were provided food and water ad libitum.
  • mice were anesthetized with an overdose of pentobarbital and perfused intracardially with PBS followed by 4%paraformaldehyde (PFA; 4% (w/v) in PBS) .
  • Brains were dissected out and postfixed in 4%PFA for 4 h at RT. Samples were cryoprotected in 30%sucrose until they sank. Coronal sections (35 ⁇ m) were prepared on a Cryostat microtome (Leica CM1950) .
  • Human skin samples were obtained surgically, from 28-year-old female arm skin with psoriasis and 23-year-old male health arm skin. Samples were embedded in O.C.T compound, frozen at -80°C, and sectioned into 30- ⁇ m-thick specimens. Skin sections were fixed in 4%PFA for 15 min and washed three times with PBS.
  • Nb-HCR initiators or Nb-SS-HCR initiators
  • PBST PBS
  • blocking buffer 2%BSA, 5 mM EDTA, 0.3%Triton X-100 in PBS
  • Ab-HCR initiators or Ab-SS-HCR initiators
  • MaMBA strategy All prepared Ab-HCR initiators (or Ab-SS-HCR initiators) by MaMBA strategy were pooled into incubation buffer (1%BSA, 0.1%Triton X-100, 5 mM EDTA, 0.5 mg/mL sheared Salmon sperm DNA, 1%dextran sulfate, 150 mM NaCl, 0.05%NaN 3 ) supplemented with excess Nb-NGL-His 6 according to the nanobody type used above. Sections were then incubated with the Ab-HCR initiators (or Ab-SS-HCR initiators) pool at 4°C for 12-16 h, and then washed three times with washing buffer (2%BSA, 0.1%Triton X-100 in PBS) for 10 min.
  • washing buffer 20%BSA, 0.1%Triton X-100 in PBS
  • tissue sections with the anchored Ab-SS-HCR initiators were incubated with 50 mM TCEP in PBS (to remove HCR initiators via reductive cleavage) for 15 min at RT followed by three washes with PBST. Samples were blocked in blocking buffer for 1 h at RT, and a new round of staining was performed as above.
  • hybridized amplifiers were first removed with 50%formamide in 0.1 ⁇ PBS at 37°C for 5 min twice. Samples were then washed three times with PBST and blocked again in amplification buffer for 1 h at RT followed by the basic HCR amplification process.
  • Hybridized amplifiers should be removed before the HCR initiators removal for a new round of staining.
  • Images were processed using Zeiss ZEN, Leica LAS X and FIJI, and colored for display using FIJI and Photoshop.
  • a basic alignment and registration was done by descriptor-based registration plugin based on DAPI channel of each image using FIJI.
  • HEK293T cells ATCC CRL-3216 and HeLa cells (ATCC CCL-2) were incubated according to standard procedures in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 10%(v/v) fetal bovine serum (FBS, Gibco) at 37°C with 5%CO 2 .
  • DMEM Dulbecco modified Eagle medium
  • FBS fetal bovine serum
  • Cells were seeded in a 24-well plate and grown to 70-90%viable.
  • cover slips pretreated with poly-L-lysine solution (P8920, Sigma-Aldrich) .
  • Cell medium was replaced with fresh complete medium 2 h before transfection.
  • plasmid and 0.5 ⁇ L DNA transfection reagent were mixed in 50 ⁇ L serum-free medium and incubated for 15-20 min at RT. The mixture was added to the cell culture medium, and cells were incubated for 18-24 h before assaying for transgene expression.
  • Tet system approved FBS Biological Industries
  • Drug used in this study were anhydrotetracycline hydrochloride (ATc; 541642, J&K) , 3-Isobutyl-1-methylxanthine (IBMX; I5879, Sigma-Aldrich) and Forskolin (FSK; F3917, Sigma-Aldrich) .
  • Cells transfected with TRE-EGFP-PGK-rtTA were induced with increasing dose of ATc as described.
  • Cells transfected with 3 ⁇ CRE-mCherry-hPEST were induced with 0.3 mM IBMX and increasing dose of FSK as described.
  • Cells co-transfected with TRE-EGFP-PGK-rtTA and 3 ⁇ CRE-mCherry-hPEST were treated with 0.3 mM IBMX, increasing dose of ATc and decreasing dose of FSK as described.
  • cells were lysed in cell extraction buffer supplemented with 1 mM PMSF and protease inhibitor cocktail following manufacturer’s instructions, quantified using BCA protein assay (23225, Thermo Fisher Scientific) and stored in aliquots at -80°C until use.
  • BCA protein assay 23225, Thermo Fisher Scientific
  • the DNA retrieved solutions were measured immediately or stored at -20°C until analysis. Volume of 1 ⁇ L was used as template for qPCR or sequencing library preparation.
  • samples were incubated with antibody (diluted in PFBB) for 1 h at 37°C and washed three times with plate wash buffer. Samples were then incubated with HRP-conjugated secondary antibody diluted in PFBB for 1 h at 37°C and washed five times with plate wash buffer.
  • the HRP-driven colorimetric readout was done by adding 50 ⁇ L TMB solution and incubating for 10 min at RT. Then 50 ⁇ L 1.8 N H 2 SO 4 was added to stop the reaction, and samples were immediately measured absorbance at 450 nm with results subtracted by the value of blank well.
  • sandwich immunoassay was adopted by coating SARS-CoV-2 Spike RBD-His recombinant proteins (40592-V08B, Sino Biological) (1 ⁇ g/mL diluted in PBS) on the microplate at RT overnight and washing for three times with plate wash buffer by microplate washer (Wellwash TM Versa Microplate Washer, Thermo Fisher Scientific) . After incubation in blocking buffer for 1 h at RT following three times of washing, 1 ⁇ L serum and 49 ⁇ L PBS were added to the coated microplate and incubated for 1 h at 37°C. After five times of washing, captured anti-SARS-CoV-2 Spike RBD IgG was detected by Ab-SS-DNA barcode (antibody against human IgG) diluted in incubation buffer following washing and DNA retrieval as described above.
  • Ab-SS-DNA barcode antibody against human IgG
  • Ab-SS-DNA barcodes pool detection antibodies against HBsAg and HBeAg were added to the microplate coated with the capture antibodies, following washing and DNA retrieval.
  • each vial of capture antibody was coupled to 18 mg Epoxy magnetic beads (Beijing Yunci Technology) .
  • Beads were mixed with 0.1 M sodium phosphate (pH 7.4) , capture antibody and 1 M ammonium sulfate. The mixtures were incubated for 16-24 h at 37°C with rotation (5 rpm) . Then beads were washed three times with PBS containing 1%Triton X-100, followed by blocking in PBS with 1%BSA and 0.05%Tween-20 for 2 h at RT.
  • beads were maintained in PBS with 0.1%BSA, 0.05%Tween-20 and 0.03%NaN 3 at a concentration of 10 mg/mL, and stored at 4°C.
  • the antibody-coupled beads for all targets were mixed before use.
  • beads wash buffer (0.1%Triton X-100 in PBS)
  • beads were resuspended in wash buffer and distributed into each well of a 96-well deep well plate (501102, NEST) .
  • beads were mixed with 100 ⁇ L cell lysates samples or standard proteins for 30 min at RT in a plate mixer (800 rpm) .
  • the beads were isolated using a 96-well magnetic stand following 3 ⁇ wash steps with beads wash buffer.
  • the antigen-captured beads were detected by Ab-SS-DNA barcodes pooled in beads incubation buffer (0.5%BSA, 0.05%Triton X-100, 0.1%dextran sulfate, 0.4 mg/mL sheared Salmon sperm DNA, 5 ⁇ M TP1107-NGL-His 6 , 2 ⁇ M TP897-NGL-His 6 in PBS) for 30 min at RT in a plate mixer (800 rpm) , followed by 5 ⁇ wash steps with beads wash buffer.
  • beads incubation buffer 0.5%BSA, 0.05%Triton X-100, 0.1%dextran sulfate, 0.4 mg/mL sheared Salmon sperm DNA, 5 ⁇ M TP1107-NGL-His 6 , 2 ⁇ M TP897-NGL-His 6 in PBS
  • the DNA barcode sequences (62 bp) were designed to include a 6-bp antibody-dedicated barcode (Ab bc) and a 15-bp unique molecular identifier (UMI) .
  • Ab bc 6-bp antibody-dedicated barcode
  • UMI 15-bp unique molecular identifier
  • DNA barcodes are designed with different both-end sequences for specific amplification using different pairs of primers. The sequences of all DNA barcodes were listed in Table 1.
  • a 25 ⁇ L PCR was performed per sample containing 1 ⁇ L released DNA barcodes, 0.1 mM dNTPs, 0.25 ⁇ L Q5 high-fidelity DNA polymerase (NEB) , 1 ⁇ Q5 reaction buffer, 0.4 ⁇ M well-specific forward primer (6-bp well barcode) and 0.4 ⁇ M reverse primer (Tables 4-6) .
  • PCR thermocycling conditions were performed as followed (PCR condition 1) : (1) 30 s at 98°C; (2) 10 s at 98°C; (3) 30 s at 50°C; (4) 10 s at 72°C; (5) repeat step 2-4 nine times; (6) 2 min at 72°C; and (7) ⁇ 12°C.
  • 1 ⁇ L of each well-barcoded DNA from one plate were pooled and purified using 1.8 ⁇ AMPure XP beads (Beckman Coulter) following the manufacturer’s protocol. The well-barcoded DNA pool was eluted with 30 ⁇ L nuclease-free water.
  • PCR was prepared with 29 ⁇ L eluted DNA, 0.2 mM dNTPs, 0.5 ⁇ L Q5 polymerase, 1 ⁇ Q5 reaction buffer, 0.5 ⁇ M adaptor forward primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG-3’ , SEQ ID NO: 63) and 0.5 ⁇ M adaptor reverse primer containing i7 index (5’-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACG-3’ , SEQ ID NO: 64) .
  • PCR condition 1 was performed and the products were purified by 1.8 ⁇ AMPure XP beads.
  • the final sequencing library was eluted in 30 ⁇ L nuclease-free water.
  • PCR condition 1 was performed. 0.73 ⁇ L of each well-barcoded DNA from the same plate were pooled and purified by 1.8 ⁇ AMPure XP beads with elution in 85 ⁇ L nuclease-free water.
  • PCR thermocycling conditions were performed as followed: (1) 30 s at 98°C; (2) 10 s at 98°C; (3) 30 s at 55°C; (4) 10 s at 72°C; (5) repeat step 2-4 nine times; (6) 2 min at 72°C; and (7) ⁇ 12°C.
  • each phasing spacer-tagged DNA product 8.75 ⁇ L of each phasing spacer-tagged DNA product were pooled (70 ⁇ L) and purified by 1.8 ⁇ AMPure XP beads with elution in 30 ⁇ L nuclease-free water. Then 50 ⁇ L PCR was prepared with 5 ⁇ L eluted DNA, 0.2 mM dNTPs, 0.5 ⁇ L Q5 polymerase, 1 ⁇ Q5 reaction buffer, 0.5 ⁇ M forward primer 151AR containing i5 index and 0.5 ⁇ M reverse primer 117JvA containing i7 index (plate index) .
  • PCR thermocycling conditions were performed as followed: (1) 30 s at 98°C; (2) 10 s at 98°C; (3) 30 s at 60°C; (4) 10 s at 72°C; (5) repeat step 2-4 nine times; (6) 2 min at 72°C; and (7) ⁇ 12°C.
  • the adaptor-tailed DNA libraries were purified by 1.8 ⁇ AMPure XP beads and eluted with 20 ⁇ L nuclease-free water. Each plate library was analyzed and quantified to pool together for sequencing.
  • the “UMI-tools whitelist” utility was used to generate a whitelist of barcode and UMI combinations from the position 1-6, 21-26, 27-41 of the fastq reads. The whitelist was then washed with respect to a ground truth barcode list to reduce false-positive rates of the read quantification. Then the “UMI-tools extract” utility was used to extract barcode and UMI, while base pair corrections were performed on barcode-UMI pairs 2 Hamming Distance from the whitelist barcode-UMI pairs. Finally, unix commands were used to demultiplex the well barcode, antibody-specific barcode, and UMI to acquire the final count table that stores the barcode-UMI quantification.
  • nanobodies targeting the fragment crystallizable (Fc) region of IgGs are recombinantly expressed, fused with Asn-Gly-Leu (NGL) tripeptide recognition motif and 6 ⁇ His tag (for protein purification) at the C-termini.
  • NGL Asn-Gly-Leu
  • 6 ⁇ His tag for protein purification
  • OaAEP1 cuts the NGL motif, ligates the GV-based azide-modified peptide, GVGK (N 3 ) RG, and yields nanobody-NGVGK (N 3 ) RG products which are poorly recognized by OaAEP1, making it efficient for the production. Then the azide-functionalized nanobodies are conjugated with DBCO-modified (azide-reactive) DNA oligonucleotides (DNA oligos) via the click reaction (Fig. 8A and Fig. 1A) . The DNA oligo-conjugated nanobodies (Nb-DNA oligos) are ready for barcoding antibodies to execute multiplexed detection.
  • each antibody is mixed with an antibody-specific Nb-DNA oligo (specific DNA sequence) .
  • DNA oligos are conjugated to the Fc domain of antibodies through the site-specific binding of nanobodies to form the DNA-barcoded complex (Ab-Nb1 to Ab-Nbn) , and the excess Nb-DNA oligos are removed by filtration. Then each DNA oligo-conjugated antibody could be directly pooled and applied to different assay such as immunostaining, quantitative protein detection and the like (Fig. 1B) .
  • Example 2 MaMBA enables multiplexed in situ protein imaging by misHCR.
  • HCR a DNA-based method for amplification of immunofluorescence signals, which utilizes iterative DNA hairpins opening by a pair of fluorophore-modified DNA hairpins (HCR amplifiers) triggered by an initiating DNA strand (HCR initiator) bound to the epitope (the antigen target of antibody) through multiple-step binding.
  • HCR amplifiers fluorophore-modified DNA hairpins
  • HCR initiator an initiating DNA strand bound to the epitope
  • the redundant protocol makes it laborious and time-consuming to anchor the HCR initiator to the epitope, and the secondary antibodies staining strategy limits the multiplexity.
  • Abs-HCR initiators we could easily minimize HCR initiators anchoring procedure to only one step, and expand the multiplexity utilizing the power of DNA (Fig. 2A) .
  • HCR initiator-conjugated nanobodies Compared with fluorophore-conjugated nanobodies (Nb-Cy5/Cy3) , HCR initiator-conjugated nanobodies following the signal amplification (misHCR-Cy5/Cy3) showed dramatical signal increase, and the target specificity was confirmed by the same pattern displayed in the higher contrast images of Nb-Cy5/Cy3 staining (Figs. 2B-2D) .
  • misHCR did not affect the spatial resolution in diffraction-limited confocal imaging as performed by the traditional method (Figs. 8B-8D) .
  • PDGFR ⁇ platelet-derived growth factor receptor A
  • KRT14 keratin 14
  • DCT dopachrome tautomerase
  • CD31 a marker for endothelial cells
  • ⁇ SMA alpha smooth muscle actin
  • CD45 a marker for hematopoietic cells except erythrocytes and platelets, that consists of four rabbit (Rb) IgG antibodies and two mouse (Mus) IgG1 antibodies (Fig.
  • Example 3 Cleavable MaMBA enables increased multiplexity with multi-round misHCR (misHCR n ) .
  • HCR initiators could be released via reductive cleavage and washed away. Then the next round of misHCR could be performed and re-employed the same set of HCR initiators (Fig. 3B) .
  • Fig. 9B We compared the pre-and post-washing images between misHCR rounds, and signals were efficiently removed by removing the HCR initiators (Fig. 9B) .
  • misHCR n multi-round misHCR
  • GFAP glial fibrillary acidic protein
  • NF-H neurofilament-H
  • Tph2 tryptophan hydroxylase 2
  • Iba1 ionized calcium binding adaptor molecule 1
  • Example 4 Expanding cleavable MaMBA to multiplexed quantitative protein detection by BLISA.
  • DNA-barcoded antibodies are then loaded to detect their targets on the plate. After washing away the unbound antibodies, the DNA barcodes are retrieved by reductant (e.g. TCEP) and quantified by qPCR (Fig. 4A) .
  • reductant e.g. TCEP
  • the DNA barcode was designed to contain a 6-bp antibody-dedicated barcode (Ab-bc) and a 15-bp unique molecular identifier (UMI) . Retrieved DNA barcodes are added by a well-specific barcode (well-bc) through PCR amplification. Then each well-specific DNA barcodes are pooled to prepare indexed library for sequencing (Fig. 13E) . Each target proteins of each well are identified by their antibody barcode and well barcode, and quantified by UMI counts.
  • Example 5 Sequencing-combinedBLISA for anti-SARS-CoV-2 Spike RBD IgG detection in human serum.
  • SARS-CoV-2 anti-severe acute respiratory syndrome coronavirus 2
  • RBD Spike receptor-binding-domain
  • sandwich-based immunoassay in which the analytes (the anti-SARS-CoV-2 Spike RBD IgGs in serum) are captured by the immobilized antigens (the purified SARS-CoV-2 Spike RBD proteins) and detected by the DNA-barcoded secondary (2 nd ) antibodies (the anti-human IgG antibodies) .
  • DNA barcodes are retrieved by TCEP with a known amount of normalization barcodes (norm bc) , following library preparation for sequencing (Figs. 5A and 5F) .
  • BLISA human IgG detection curves between BLISA and ELISA.
  • BLISA known amounts of human IgGs are immobilized on microplate and detected by DNA-barcoded anti-human IgG antibodies following qPCR readouts.
  • ELISA immobilized human IgGs are detected by the anti-human IgG antibodies (from Rb host species) which are detected by horseradish peroxidase (HRP) -conjugated anti-Rb IgG antibodies following colorimetric readouts with 3, 3', 5, 5'-Tetramethylbenzidine (TMB) substrates (Fig. 5B) .
  • HRP horseradish peroxidase
  • TMB horseradish peroxidase
  • the retrieved DNA barcodes and normalization barcodes were further barcoded by three-step PCR amplifications that sequentially adding well bc, phasing spacers and sequencing adaptors (Figs. 5D and 5E) .
  • well-barcoded DNA were pooled and separated to 8 PCR reactions for adding various numbers of bases as spacers to both ends with 7 bases in total (0 base and 7 bases, 1 base and 6 bases, 2 bases and 5 bases, etc. ) , which allows higher input for the amplicon sequencing by enriching the base diversity.
  • HBV hepatitis B virus
  • HBsAg hepatitis B surface antigen
  • HBeAg hepatitis B e antigen
  • anti-SARS-CoV-2 Spike RBD IgG detection used an IgG-detected sandwich strategy
  • HBsAg and HBeAg detection employed an antigen-detected sandwich strategy using a pair of antibodies, namely, capture antibodies (Ab Cap ) and detection antibodies (Ab Det ) (Figs. 6A and 6B) .
  • HBsAg or HBeAg are captured by the immobilized Ab Cap and detected by the DNA-barcoded Ab Det , which are distinguished by the antibody-dedicated barcodes.
  • Example 7 Multiplexed phospho-proteins detection in cell lysates by BLISA based on magnetic beads.
  • MB conjugated with capture antibodies for each target proteins are mixed and aliquoted into each wells of the deep-well plate. Samples were loaded, and the target proteins were captured, followed by washing steps to remove the unbound biomolecules. Then, the target proteins were detected by the Ab Det -SS-DNA barcodes mixtures.
  • DNA barcodes were retrieved by TCEP and quantified (Fig. 7B) .
  • Fig. 7B We confirmed the standard curves of seven phospho-proteins in BLISA, which indicated higher sensitivity of BLISA compared with ELISA (Fig. 15) .
  • Fig. 15 To verified the multiplexing of MB-based BLISA, we simultaneously detected the seven purified phospho-proteins (p-p38 ⁇ , p-ERK1/2, p-JNK, p-AMPK ⁇ 1, p-CREB, p-Src, and p-Akt) , which were loaded within seven sample wells in different concentration gradient frames (Fig. 7C) .

Abstract

L'invention concerne une composition comprenant un anticorps lié à un oligonucléotide par l'intermédiaire d'une liaison disulfure, l'oligonucléotide étant spécifique pour l'anticorps. L'invention concerne en outre un kit comprenant celle-ci et le procédé de détection d'une ou de plusieurs cibles dans un échantillon au moyen de la composition.
PCT/CN2023/107980 2022-07-18 2023-07-18 Composition de détection et son utilisation WO2024017263A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2022106344 2022-07-18
CNPCT/CN2022/106344 2022-07-18

Publications (1)

Publication Number Publication Date
WO2024017263A1 true WO2024017263A1 (fr) 2024-01-25

Family

ID=89617167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/107980 WO2024017263A1 (fr) 2022-07-18 2023-07-18 Composition de détection et son utilisation

Country Status (1)

Country Link
WO (1) WO2024017263A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007092552A2 (fr) * 2006-02-07 2007-08-16 Antara Biosciences Inc. Dispositif et procédés de détection et de quantification d'un ou plusieurs agents cibles
CN101988920A (zh) * 2009-07-30 2011-03-23 韩国科学技术研究院 抗原检测试剂盒和方法
US20180088112A1 (en) * 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
CN110462058A (zh) * 2017-03-24 2019-11-15 新加坡国立大学 用于多重检测分子的方法
CN111655865A (zh) * 2018-01-26 2020-09-11 北京生命科学研究所 使用基于杂交链式反应的方法的多重免疫信号放大
US20200377926A1 (en) * 2018-01-26 2020-12-03 National Institute Of Biological Sciences, Beijing Hybridization chain reaction-based method for amplifying immunosignals

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007092552A2 (fr) * 2006-02-07 2007-08-16 Antara Biosciences Inc. Dispositif et procédés de détection et de quantification d'un ou plusieurs agents cibles
CN101988920A (zh) * 2009-07-30 2011-03-23 韩国科学技术研究院 抗原检测试剂盒和方法
US20180088112A1 (en) * 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
CN110462058A (zh) * 2017-03-24 2019-11-15 新加坡国立大学 用于多重检测分子的方法
CN111655865A (zh) * 2018-01-26 2020-09-11 北京生命科学研究所 使用基于杂交链式反应的方法的多重免疫信号放大
US20200377926A1 (en) * 2018-01-26 2020-12-03 National Institute Of Biological Sciences, Beijing Hybridization chain reaction-based method for amplifying immunosignals

Similar Documents

Publication Publication Date Title
Lin et al. A hybridization-chain-reaction-based method for amplifying immunosignals
US20220026433A1 (en) Cleavable fluorescent tyramide for sensitive and multiplexed analysis of biological samples
CA3163623A1 (fr) Analyse d'arn in situ a l'aide d'une ligature de paire de sondes
CN111819288B (zh) 基于杂交链式反应的免疫信号放大方法
WO2006049289A1 (fr) Procede de detection de substances cibles
US10233442B2 (en) Method for affinity purification
JP2023505195A (ja) 核酸結合免疫サンドイッチアッセイ（ｎｕｌｉｓａ）
CN114127282A (zh) 适体的筛选方法和使用适体的免疫分析方法
WO2015159122A1 (fr) Capteur à base d'acide nucléique et procédés associés
EP2189539B2 (fr) Complexes de conjugués pour une détection d'analytes
Ma et al. A multiple-target simultaneous detection method for immunosorbent assay and immunospot assay
Pang et al. Bioscreening specific peptide-expressing phage and its application in sensitive dual-mode immunoassay of SARS-CoV-2 spike antigen
US20230220445A1 (en) Multiplexed immunosignal amplification using hybridization chain reaction-based method
WO2024017263A1 (fr) Composition de détection et son utilisation
US20050003360A1 (en) Array systems and methods
US11814677B2 (en) Methods and systems for sensitive and multiplexed analysis of biological samples using cleavable fluorescent streptavidin and anti-hapten antibodies
CN110468182A (zh) 一种检测血小板衍生生长因子bb的均相生物分析方法及其应用
WO2013095137A2 (fr) Procédés, moyens et coffrets pour détecter des analytes peu abondants
CN111491667A (zh) 肽核酸缀合物
CN113125747B (zh) 一种isPLA-Seq的高通量检测蛋白质相互作用的方法和试剂盒及其应用
US20210381026A1 (en) NOVEL IMMUNO-PCR METHOD USING cDNA DISPLAY
Feng et al. High-affinity aptamers enable the rapid optical detection and differentiation of three SARS-CoV-2 VOCs
Zurla et al. A novel method to quantify RNA–protein interactions in situ using FMTRIP and proximity ligation
US20230088664A1 (en) Method of Detecting Analytes in a Sample
US20230332207A1 (en) Epoxides for multimodal detection of biomolecules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23842326

Country of ref document: EP

Kind code of ref document: A1