Immune signal amplification method based on hybrid chain reaction
阅读说明：本技术 基于杂交链式反应的免疫信号放大方法 (Immune signal amplification method based on hybrid chain reaction ) 是由 林睿 罗敏敏 于 2018-01-26 设计创作，主要内容包括：本发明提供了一种基于抗体的生物分析方法,特别是一种用于放大免疫信号的基于杂交链式反应的方法,被称为免疫信号HCR(isHCR),其将抗体-抗原相互作用与杂交链式反应(HCR)技术相结合来放大免疫信号。本发明还提供了一种用于执行上述isHCR的试剂盒。(The present invention provides an antibody-based bioanalytical method, in particular a hybridization chain reaction-based method for amplifying immune signals, referred to as the immune signal HCR (ishcr), which combines antibody-antigen interactions with Hybridization Chain Reaction (HCR) technology to amplify immune signals. The invention also provides a kit for carrying out the above-described isHCR.)
1. An immune signal hybrid chain reaction (isHCR) comprising coupling a Hybrid Chain Reaction (HCR) initiating chain to an analyte-specific antibody and adding a pair of HCR amplifying chains to perform the hybrid chain reaction.
2. The immune signal hybridization chain reaction of claim 1, wherein the HCR trigger chain has a region for hybridization to an HCR amplification chain and a region for coupling to an analyte-specific antibody.
3. The immune signal hybridization chain reaction according to claim 1 or 2, wherein the HCR trigger chain and/or the amplification chain are end-modified and/or internally modified with biotin, acrydite, amine, thiol, DBCO and/or a fluorescent dye, and the fluorescent dye is selected from FITC, cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluor 546, Alexa Fluor488 and Alexa Fluor 647.
4. The immune signal hybridization chain reaction of any one of claims 1-3, wherein the HCR priming chain is coupled to an antibody through an interaction selected from the group consisting of a streptavidin-biotin interaction and a covalent bond interaction.
5. The immune signal hybridization chain reaction of claim 4, wherein the HCR priming chain is a biotinylated priming chain capable of associating with a vacancy binding site of streptavidin which is capable of associating with a biotinylated antibody, thereby coupling the HCR priming chain to the antibody.
6. The immune signal hybridization chain reaction of claim 4, wherein the covalent bond interaction is through an interaction of a chemical linker selected from the group consisting of an amine-reactive linker containing a succinimide ester group and a click-on chemical linker.
7. The immune signal hybridization chain reaction of claim 6, wherein the click chemistry linker is selected from the group consisting of an NHS-azide linker, an NHS-DBCO linker, a maleimide-azide linker, and a maleimide-DBCO linker.
8. The immune signal hybridization chain reaction of any one of claims 1-7, wherein the amplification chain or pair of amplification chains is modified to enter a branched multi-round amplification for branching and growth of HCR polymers.
9. The immune signal hybridization chain reaction according to claim 8, wherein the amplification chain or pair of amplification chains is end-or internally modified with a chemical group selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, tetrazine, alkyne and/or DBCO and/or a fluorescent dye selected from FITC, cyanine dyes, Dylight fluors, Atto dyes, Janeliafluor dyes, Alexa Fluor 546, Alexa Fluor488 and Alexa Fluor 647, allowing for further rounds of amplification to be initiated.
10. The immune signal hybridization chain reaction according to claim 8 or 9, wherein a pair of fluorophore tagged amplification chains is added to the last round of the multi-round isHCR for visualization.
11. The immune signal hybridization chain reaction of any one of claims 8-10, wherein the amplification chain is modified at an internal position that is more accessible to streptavidin and serves as an anchor point for each successive branching wheel in a multi-wheeled isHCR.
12. The immune signal hybridization chain reaction according to any one of claims 1-11, wherein the antibody is a second antibody that reacts with a first antibody specific to an analyte, the second antibody is an IgG or nanobody, and the first antibody is an IgG, nanobody, or scFv.
13. The immune signal hybridization chain reaction of any one of claims 1-12, further comprising the use of Graphene Oxide (GO) to absorb unassembled HCR amplification chains.
14. The immune signaling hybridization chain reaction according to any one of claims 1-12, further comprising using Graphene Oxide (GO) to absorb unassembled HCR amplification chains and quench fluorescence, wherein the amplification chains are end-modified and/or internally modified with a fluorescent dye.
15. The immune signal hybridization chain reaction according to claim 13 or 14, wherein the particle size of GO is <500 nm.
16. A kit for immune signal hybridization chain reaction (isHCR), the kit comprising: (1) an analyte-specific antibody; (a) HCR initiating chain; and (3) a pair of HCR amplifier chains, wherein the HCR trigger chains have a region for hybridization to the HCR amplifier chains and a region for coupling to the antibody.
17. The kit of claim 16, wherein said HCR trigger strand and/or said amplification strand are end-modified and/or internally modified with biotin, acrydite, amine, thiol, DBCO and/or a fluorescent dye, and said fluorescent dye is selected from FITC, cyanine dyes, Dylight fluorochromes, Atto dyes, Janelia Fluor dyes, Alexa Fluor 546, Alexa Fluor488, and Alexa Fluor 647.
18. The kit of claim 16 or claim 17, wherein the HCR priming chains are coupled to antibodies by an interaction selected from the group consisting of streptavidin-biotin interactions and covalent bond interactions.
19. The kit of claim 18, wherein said HCR priming chain is a biotinylated priming chain capable of associating with a vacancy binding site of streptavidin, which is capable of associating with a biotinylated antibody, thereby coupling said HCR priming chain to said antibody.
20. The kit of claim 18, wherein the covalent bond interaction is through an interaction of a chemical linker selected from the group consisting of an amine-reactive linker containing a succinimide ester group and a click chemical linker.
21. The kit of claim 20, wherein the click chemistry linker is selected from the group consisting of a NHS-azide linker, a NHS-DBCO linker, a maleimide-azide linker, and a maleimide-DBCO linker.
22. The kit of any one of claims 16-21, wherein the amplification strand or pair of amplification strands is modified to enter a branching multi-round amplification for branching and growth of HCR polymers.
23. The kit of claim 22, wherein said amplification strand or said amplification strand is terminally or internally modified with a chemical group selected from biotin, digoxigenin, acrydite, amine, succinimidyl ester, thiol, azide, TCO, tetrazine, alkyne and/or DBCO and/or a fluorescent dye selected from FITC, cyanine dyes, Dylight fluors, Atto dyes, Janelia Fluor dyes, Alexa Fluor 546, Alexa Fluor488 and Alexa Fluor 647, which allows for the initiation of further rounds of amplification.
24. The kit of claim 22 or claim 23, wherein a pair of fluorophore tagged amplification strands are added to the last round of the multi-round isHCR for visualization.
25. The kit of any one of claims 22-24, wherein the amplification strand is modified at an internal position that is more accessible to streptavidin and serves as an anchor point for each successive branching wheel in a multi-wheeled isHCR.
26. The kit of any one of claims 16-25, wherein the antibody is a second antibody that reacts with a first antibody specific for an analyte, the second antibody is an IgG or nanobody, and the first antibody is an IgG, nanobody, or scFv.
27. The kit of any one of claims 16-26, further comprising Graphene Oxide (GO) for absorbing unassembled HCR-amplified chains.
28. The kit of any one of claims 16-26, further comprising Graphene Oxide (GO) for absorbing unassembled HCR amplified chains and quenching fluorescence.
29. Use of the immune signal hybridization chain reaction of any one of claims 1-15 or the kit of any one of claims 16-28 for the robust amplification of an immune signal, for the analysis of an analyte in a biological sample, or in laboratory, clinical, or diagnostic applications.
30. The use of claim 29, wherein the application is selected from Western blotting and ELISA.
31. The use of claim 29 or claim 30, wherein the biological sample is selected from the group consisting of cultured cells, dispersed cells, tissue sections, body fluids, and whole organs.
32. The use of claim 31, wherein the clinical or diagnostic application is selected from biological studies, forensic examinations, clinical tests or diagnostics.
Background
Because of their ease of use, speed, and cost-effectiveness, antibody-based immunoassays have been the most popular methods for detecting and identifying the location of proteins and other biomolecules in biological samples. These methods use a primary antibody that binds selectively to the target molecule (antigen), and this antibody-antigen interaction can be visualized by a conjugated reporter or labeled secondary antibody that can recognize and react with the primary antibody-epitope complex (Han, k.n., Li, c.a. & Seong, g.h.annu.rev.anal.chem.6, 119-141 (2013)). A major limitation when using immunoassays is that the low abundance of a given target molecule in a sample often requires signal amplification before detection is possible. Amplification can be achieved using coupled enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase, etc., which catalyze the deposition of chromogenic substrates on the target complex (Bobrow, m.n., Harris, t.d., shaughanesy, K.J. & Litt, g.j.j.immunol. methods 125, 279- & 285 (1989)). Fluorogenic substrates, especially those based on HRP-tyramine reaction chemistry, have been developed to support high resolution fluorescence microscopy (Stack, e.c., Wang, c., Roman, K.A. & Hoyt, c.c. methods70, 46-58 (2014)). Although current amplification methods are very useful and widely adopted, they have several disadvantages: they often produce high background, they can reduce spatial resolution due to dye diffusion, they are difficult to use for simultaneous detection of multiple amplified signals (Carvajal-Hausdorf, d.e., Schalper, k.a., Neumeister, V.M. & Rimm, d.l.lab.invest.95, 385-.
In the present invention, we have found that an enzyme-free amplification approach can overcome many of these limitations. In particular, the Hybrid Chain Reaction (HCR) technique is suitable for amplifying immune signals. HCR, based on recognition and hybridization events occurring between sets of DNA hairpin oligomers self-assembled as polymers, has so far been used mainly for amplification of mRNA signals from in situ hybridized samples (Choi, H.M.T., Beck, V.A. & Pierce, N.A.ACS Nano 8, 4284-. In a typical use case, a nucleic acid probe complementary to a target mRNA molecule is used as a 'priming strand' oligonucleotide. Starting with a priming strand oligonucleotide, a fluorophore-labeled nucleic acid 'amplification strand' oligonucleotide is added to the target mRNA-priming strand complex using a series of polymerization reactions; the fluorophore is then visualized.
Disclosure of Invention
The present invention provides an antibody-based bioanalytical method, in particular a method called immune signal HCR (ishcr), which combines antibody-antigen interactions with Hybrid Chain Reaction (HCR) technology. The invention also provides a kit for carrying out the above-described isHCR. The invention further provides an antibody-based bioanalytical method, in particular a method called multi-wheeled immune signal HCR (isHCR) (multi-wheeled isHCR) which combines antibody-antigen interactions with Hybrid Chain Reaction (HCR) in which multiple rounds of amplification are used to branch and grow HCR polymers. Accordingly, the invention also provides a kit for performing a multi-wheeled isHCR.
In a first aspect, the present invention provides an isHCR method comprising coupling HCR priming chains with analyte-specific antibodies (including but not limited to traditional IgG and nanobodies) and adding a pair of amplification chains to perform a hybridization chain reaction.
In a second aspect, the present invention provides a kit for performing the isHCR method, the kit comprising (1) an analyte-specific antibody; (a) HCR initiation chain (HCR initiator); and (3) a pair of HCR amplifier chains (hcrampliifiers), wherein the HCR trigger chains have a region for hybridization with the HCR amplifier chains and a region for coupling to the antibody.
In a third aspect, the invention provides an HCR priming chain having a region for hybridisation to an HCR amplification chain and a region for coupling to an analyte-specific antibody to be analysed.
In a fourth aspect, the invention provides an antibody specific for the analyte to be analysed, which antibody is coupled directly or indirectly to the HCR priming chain.
The HCR priming strand can hybridize to any of several types of self-assembling DNA HCR amplification strands, including fluorophore-labeled amplification strand oligonucleotides that can be used to visualize the original target signal.
The amplification strand is a fluorescence-tagged amplification strand or a biotinylated HCR amplification strand.
The HCR priming chains are coupled to the antibody using a number of interactions, such as streptavidin-biotin interactions, covalent bond interactions (chemical linkers, e.g., amine-reactive linkers, thiol-reactive linkers, or click-chemical linkers), and the like. The amine reactive linker may be a linker containing a succinimide ester group. The thiol-reactive linker may be a maleimide group-containing linker. The click chemistry linker may be a linker containing a click chemistry functional group, such as a NHS-azide linker, NHS-DBCO linker, maleimide-azide linker, or maleimide-DBCO linker.
Preferably, the HCR priming chain is a biotinylated priming chain capable of associating with a vacancy binding site of streptavidin which is capable of associating with a biotinylated antibody, thereby coupling the HCR priming chain to the antibody.
Preferably, the HCR initiating and amplifying strands (H1 and H2) used in the present isHCR method may be end-or internally modified to improve signal intensity or as an interface into other chemical reactions. The HCR initiating and amplifying strands (H1 and H2) used in the present invention isHCR methods can be end-modified or internally modified with chemical groups and/or fluorescent dyes. For example, the HCR priming and amplification strands (H1 and H2) used in the present isHCR methods can be terminally and/or internally modified with biotin, acrydite, amines, thiols, DBCO, and/or fluorescent dyes. The fluorescent dye may be FITC, cyanine dyes, Alexa fluorochromes, Dylight fluorochromes, Atto dyes, or Janelia Fluor dyes.
Preferably, the isHCR method uses a biotin-streptavidin interaction, where the DNA-biotin HCR priming strand links to a biotinylated antibody and in turn triggers self-assembly of labeled HCR amplification strands into polymers. For example, the labeled HCR amplification strand is a fluorophore-labeled HCR amplification strand.
The isHCR method uses unlabeled streptavidin, which allows the synthetic 5' -biotinylated DNAHCR priming strand to phase with the vacancy binding site of streptavidin, which in turn is linked to a biotinylated antibody.
The biotinylated antibody may be a biotinylated secondary antibody that reacts with a primary antibody specific for the target antigen.
Preferably, the present invention provides a kit comprising: (1) an analyte-specific biotinylated antibody; (2) streptavidin; (3) a biotinylated priming strand; and (4) a pair of HCR amplification chains.
The present isHCR methods or kits can be used to amplify immune signals strongly at different subcellular locations (e.g., cell and vesicle membranes, cytosol, mitochondria, and nucleus) and in various types of samples (e.g., blots, cultured cells, tissue sections, and whole organs). Thus, the isHCR method can be used to analyze analytes in biological samples. In particular, the present isHCR methods are useful in laboratory, clinical or diagnostic applications, especially in field applications, such as clinical site care.
The isHCR method is particularly useful in applications requiring sensitive detection of immune signals. We demonstrate here, among other applications, its use in amplifying immune signals with monoclonal antibodies, and in detecting translocation bacterial effector signals that are in very low abundance. The actual amplification performance of the isHCR depends on the particular application and abundance of a given target signal: for signal amplification in Western blot and tissue section samples, the isHCR performed up to two orders of magnitude beyond the standard IHC.
In a fifth aspect, the invention provides a multi-wheeled isHCR method comprising coupling HCR priming chains with analyte-specific antibodies (including but not limited to traditional IgG and nanobodies) and adding a pair of amplification chains to perform a hybridization chain reaction, wherein the amplification chain or pair of amplification chains is modified to enter a branched multi-round amplification for branching and growth of HCR polymers.
In a sixth aspect, the invention provides a kit for performing a multi-wheeled isHCR, the kit comprising (1) an analyte-specific antibody; (a) HCR initiating chain; and (3) a pair of HCR amplifier chains, wherein the HCR trigger chain has a region for hybridization with the HCR amplifier chain and a region for coupling to the antibody, and the amplifier chain or pair of amplifier chains is modified to enter a branching multi-round amplification for branching and growth of the HCR polymer.
In a seventh aspect, the invention provides a pair of amplification strands, wherein the amplification strand or pair of amplification strands is modified to enter a branching multi-round amplification for branching and growth of the HCR polymer.
The HCR priming strand can hybridize to any of several types of self-assembling DNA HCR amplification strands, including fluorophore-labeled amplification strand oligonucleotides that can be used to visualize the original target signal.
The HCR priming chain is coupled to the antibody using a number of interactions, such as streptavidin-biotin interactions, covalent bond interactions (chemical linkers, e.g., amine-reactive linkers or click-chemistry linkers), and the like. The amine reactive linker may be a linker containing a succinimide ester group. The click chemistry linker may be a linker containing a click chemistry functional group, such as a NHS-azide linker, NHS-DBCO linker, maleimide-azide linker, or maleimide-DBCO linker.
Preferably, the HCR priming chain is a biotinylated priming chain capable of associating with a vacancy binding site of streptavidin which is capable of associating with a biotinylated antibody, thereby coupling the HCR priming chain to the antibody.
The HCR initiating and/or amplifying chains (H1 and H2) used in the present multi-round HCR can be end-or internally modified to improve signal intensity or as an interface into other chemical reactions.
The HCR amplification chains (H1 and H2) used in the present multi-wheeled isHCR can be end-or internally modified with chemical groups and/or fluorescent dyes, allowing for more rounds of amplification to be initiated. In this case, the amplification strands (H1 and H2) may be terminally or internally modified with biotin, digoxigenin, acrydite, amines, succinimidyl esters, thiols, azides, TCO, tetrazine, alkynes, and/or DBCO. Fluorescent dyes, e.g. FITC, cyanine dyes, Alexa fluorochromes, DyLightfluorochromes, Atto dyes or JaneliaFluor dyes, wherein Alexa Fluors may be, for example, Alexa Fluro 546, Alexa Fluor488 and/or Alexa Fluor 647, may also be labeled on the amplification strand with biotin, digoxigenin, acrydite, amines, succinimidyl esters, thiols, azides, TCO, tetrazines, alkynes and/or DBCO. For example, the amplification strand may be labeled with a biotin group. Once these DNA-biotin amplified strands self-assemble and link to the growing isHCR polymer, their biotin can react with newly added streptavidin (and thus can react with more HCR priming strands, etc.), thereby initiating more rounds of polymer production. A pair of signal molecule modified amplification strands (e.g., a pair of fluorophore tagged amplification strands) can be added to an ishCRnFor visualization.
Preferably, the amplification strand is modified at an internal position which is more accessible to a binding partner, e.g. streptavidin, acting as a multi-wheeled isHCR (isHCR)n) Of each successive branch wheel.
The HCR priming strand used in the present multi-round isHCR method can be end-modified or internally modified to improve signal strength. The HCR initiating strand used in the present multi-wheeled isHCR method can be end-modified or internally modified with chemical groups and/or fluorescent dyes. For example, the HCR priming strand used in the present multi-round HCR can be terminally and/or internally modified with biotin, acrydite, amine, thiol, DBCO, and/or a fluorescent dye. The fluorescent dye may be FITC, cyanine dyes, AlexaFluors, Dylight Fluors, Atto dyes, or Janelia Fluor dyes, wherein Alexa Fluors may be, for example,
Preferably, the multi-wheeled isHCR uses a biotin-streptavidin interaction, where the DNA-biotin HCR priming strand links to a biotinylated antibody and in turn triggers self-assembly of labeled HCR amplification strands into a polymer. For example, the labeled HCR amplification strand is a fluorophore-labeled HCR amplification strand.
The multi-wheeled isHCR uses unlabeled streptavidin, which allows the synthetic 5' -biotinylated DNAHCR priming strand to phase with the vacancy binding site of streptavidin, which in turn is linked to a biotinylated antibody.
The biotinylated antibody may be a biotinylated secondary antibody that reacts with a primary antibody specific for the target antigen.
The multi-wheeled isHCR can be used to amplify immune signals strongly at different subcellular locations (e.g., cell and vesicle membranes, cytosol, mitochondria, and nucleus) and in various types of samples (e.g., blots, cultured cells, tissue sections, and whole organs). Thus, the isHCR method can be used to analyze analytes in biological samples. In particular, the present isHCR method may be used in laboratory, clinical or diagnostic applications, especially in field applications, such as clinical site care.
The multi-wheeled isHCR is particularly useful in applications where sensitive detection of immune signals is required. The isHCR method can be used to amplify immune signals with monoclonal antibodies, to detect signals with very low abundance of translocating bacterial effectors, and to enhance diluted immune signals in ExM, among other applications. The actual amplification performance of the isHCR depends on the particular application and abundance of a given target signal: for signal amplification in Western blot and tissue section samples, isHCR performs up to two orders of magnitude above standard IHC; an improvement of 3 orders of magnitude was achieved with the ExM sample. Testing of western blots based on HA-tagged scFv showed an additional two rounds of amplification compared to non-multiwheeled isHCR (isHCR)3) Resulting in a tenfold improvement in protein detection sensitivity. When we tested isHCRnEach round of isHCR amplification increased the intensity of the immunopositive signal for immunostaining performance against TH.
In an eighth aspect, the present invention provides an improved isHCR method or an improved isHCR for amplifying immunofluorescence with low backgroundnA method of eliminating the above-mentioned isHCR or isHCRnIn addition, the use of Graphene Oxide (GO) to absorb unassembled HCR amplification chains is also included. Graphene Oxide (GO) may also quench fluorescence if the amplification chain is end-modified and/or internally modified with a fluorescent dye.
In a ninth aspect, the present invention provides a kit for performing the improved isHCR method, the kit comprising (1) an antibody specific for an analyte; (a) HCR initiating chain; (3) a pair of HCR amplifier chains, wherein the HCR trigger chains have a region for hybridization to the HCR amplifier chains and a region for coupling to the antibody; and (4) Graphene Oxide (GO).
In a tenth aspect, the invention provides a kit for performing the improved multi-wheeled isHCR, the kit comprising (1) an analyte-specific antibody; (a) HCR initiating chain; (3) a pair of HCR amplifier chains, wherein the HCR trigger chains have a region for hybridisation to the HCR amplifier chains and a region for coupling to the antibody, and the amplifier chain or pair of amplifier chains is modified to enter a branching multi-round amplification for branching and growth of the HCR polymer; and (4) Graphene Oxide (GO).
The particle size of the graphene oxide in the present invention is <500 nm. It is critical that, in addition to eliminating the fluorescence of HCR amplifier chains, the addition of HCR trigger chains with HCR amplifier chains and GO results in significant recovery of fluorescence, most likely because the trigger chains trigger the HCR amplifier chains to form double-stranded gapped polymers, protecting them from the adsorbing activity of GO. That is, GO can be used to suppress background levels, further improving performance of the isHCR.
The addition of GO reduced the background but did not attenuate the signal strength, resulting in an improved signal-to-noise ratio compared to isHCR amplification without GO. Surprisingly, further analysis using antibody serial dilution experiments showed that isHCR with GO significantly increased signal intensity compared to standard IHC staining methods, reaching greater than 80 x magnification when the first antibody was highly diluted.
The present invention includes all combinations of the specific embodiments recited herein.
Drawings
FIG. 1 the ISHCR method can greatly amplify immunofluorescence signals in various types of biological samples.
FIG. 2 determination of dynamic Range of Western blots using ISHCR. The purified scFv-GCN4-HA-GB1 protein was serially diluted.
FIG. 3 ISHCR amplification maintains spatial resolution of membrane-bound GFP (mGFP) immune signals in cultured cells.
Figure 4 TH immunostaining using isHCR revealed much more abundant catecholaminergic innervation in the brain.
FIG. 5 illustrates ISHCR optimization for noise floor reduction.
Figure 6.isHCR greatly increased IHC signal in mouse brain sections.
FIG. 7 ISHCR specifically amplifies Vglut3 immune signals in mouse brain sections.
Figure 8.isHCR effectively amplifies various immunofluorescent signals.
Figure 9.isHCR is able to detect biomolecules challenging to standard IHC methods.
Figure 10.isHCR amplification enables detection of low abundance proteins.
Figure 11.HCR priming chains can be coupled directly to antibodies using chemical linkers.
FIG. 12 use of ISHCRnAnd carrying out multiple rounds of amplification.
Detailed Description
In a first embodiment, the inventors analyzed purified HA-tagged proteins using isHCR using classical western blotting. The analyte proteins were purified single-chain variable fragments (scFv) (Tanenbaum, m.e., Gilbert, l.a., Qi, l.s., Weissman, j.s., and Vale, r.d. cell 159, 635-. The ISHCR amplified fluorescence signal for 1ng scFv was comparable in intensity to that of 100ng scFv measured by the traditional Alexa Fluor 546-coupled streptavidin method (SA-546), indicating that ISHCR can increase the protein detection sensitivity by 100-fold (FIG. 1 b). Compared to popular commercially available enzyme-based chemiluminescence detection methods, isHCR exhibits similar detection sensitivity but a broader dynamic range (fig. 2).
Then, in the second embodiment, the inventors next examined its performance in cells. Cultured HEK293T cells expressing membrane-bound enhanced GFP (mgfp) were immunostained for GFP. The immune signal is amplified by an isHCR using the DNA fluorophore HCR amplification strand. The fluorescence signal amplified by the isHCR of the target is greatly enhanced compared to the signal measured with the typical fluorophore-conjugated streptavidin method (fig. 1 c). To confirm the specificity of the isHCR amplification, additional control experiments and analyses with cells expressing mGFP were performed. An antibody mixture containing equal amounts of Alexa Fluor 488-conjugated secondary antibody and biotinylated secondary antibody was used to detect the anti-GFP primary antibody and the signal of the biotinylated secondary antibody was amplified using isHCR-546. Confocal microscopy showed that the unamplified and amplified signals were co-localized, indicating that the isHCR amplifies the true mGFP signal (fig. 3 a). Furthermore, these experiments demonstrate that isHCR does not affect the spatial resolution of diffraction limited confocal imaging at the sub-cellular level: the mean width of the cell membrane calculated from the unmagnified gfp signal data was not different from the mean width calculated from the isfcr-amplified gfp signal data (fig. 3b, c).
In a third embodiment, the isHCR is applied to enhance Immunohistochemical (IHC) staining signals in tissues. Mouse brain sections were immunostained for Tyrosine Hydroxylase (TH), a key enzyme in catecholamine biosynthesis. Our isHCR analysis revealed a more widespread distribution of TH positive signals throughout the brain neuronal processes compared to the traditional fluorophore-conjugated streptavidin method performed in parallel. In addition to the strong signals seen by both methods, isHCR amplification also revealed numerous discrete TH positive neuronal processes in cortical and subcortical structures; in conventional immunostaining assays, the TH-positive signals of these protrusions were very weak or negligible (FIG. 1d and FIG. 4 a). The non-specific label generated in the brain by the primary antibody against TH was omitted (fig. 4 b). In a traditional IHC assay, an additional control TH immunostaining experiment on two brain regions showed strong TH signals: dorsal Striatum (DS) and parabrain nuclei (PVN) (fig. 1e and fig. 4 c). For both brain regions, unamplified and isHCR amplified signals were co-localized and no significant difference was observed in the mean width of TH positive neuronal processes (fig. 1e and 4c), further confirming the specificity of isHCR amplification and showing that isHCR amplification did not affect the spatial resolution of diffraction-limited confocal imaging at the cellular and sub-cellular level.
Given the large number of commercially available biotinylated antibodies (secondary and monoclonal primary), the compatibility of the isHCR with streptavidin-biotin interactions would allow rapid adoption of the isHCR as an optional "additional" amplification step for most existing immunoassays. The streptavidin-biotin version of isHCR may also be used in conjunction with other protein-streptavidin technologies (e.g., Strep tags and in vivo biotinylation using biotin ligase).
These direct coupling strategies will reduce the size of the isHCR amplification complex, potentially facilitating the use of the isHCR in large volume samples and high resolution imaging. All reagents required for the isHCR are commercially available and very inexpensive. In view of the wide range of biotechnology that may be incorporated into the isHCR (e.g. protein tagging, DNA modification, reaction chemistry, etc.), it is envisaged that a wide range of biosensors and antibody-based methods in life science research could be improved or extended by the inventive application of these methods.
Although the performance of isHCR on immune signal amplification is impressive in immunoblotting, cultured cell and tissue section applications, the inventors noted that in brain sections, isHCR produced higher background than traditional fluorescent IHC (fig. 5a and fig. 4a, b). Incubation of brain sections with DNA-fluorophore HCR amplified strands alone resulted in high background levels, even after extensive washing (fig. 5a), suggesting that unassembled HCR amplified strands in tissue sections contribute to the high background levels we observed. Considering that reducing background intensity would be expected to improve signal-to-noise ratio, a way to modify the isHCR process to suppress background fluorescence was explored.
In a fourth embodiment, the HCR priming strand is reacted with a DNA-biotin amplification strand. Once these DNA-biotin amplified strands self-assemble and link to the growing isHCR polymer, their biotin can react with newly added streptavidin (thus reacting with more HCR priming strands, etc.), thus initiating more rounds of polymer production (fig. 12 a). Last round of isHCRnAn amplification strand (e.g., a DNA-fluorophore amplification strand) should be added for visualization. Testing of western blots based on HA-tagged scFv showed that two additional rounds were performedAmplification (ISHCR)3) Resulting in a tenfold improvement in protein detection sensitivity (figure 12 b).
In a fifth embodiment, we tested an isHCRnFor the performance of immunostaining against TH, each round of isHCR amplification increased the immunopositive signal intensity (fig. 12 c).
In addition, in this embodiment, we have found that the biotinylation position is critical in determining the amplification efficiency. Compared to amplification chains with biotin at the end, amplification chains with biotin groups at internal positions are more accessible to streptavidin, which acts as an ishCRnOf each successive branch wheel (fig. 12 c).
Control experiments confirmed that multiple rounds of ishCR were performednThe magnification, specificity and optical resolution remain (fig. 12d, e). Further tests showed that the immune signal of neuropeptide substance P was progressively amplified by three rounds of ishCR, according to which3Substance-rich expression in neuronal processes of the striatum and basal forebrain was revealed (fig. 12f, g).
In a sixth embodiment, unassembled HCR amplification chains are adsorbed using GO, quenching their fluorescence (fig. 5 b). In the wells of the microplate, GO completely abolished the fluorescence of the HCR amplification strand (fig. 5 c). It is critical that the addition of HCR initiating chains together with HCR amplifying chains and GO results in a significant recovery of fluorescence, probably because the initiating chains trigger the HCR amplifying chains to form double-stranded gapped polymers, protecting them from the adsorbing activity of GO (fig. 5 c). In brain sections, the mixture of GO and DNA-fluorophore HCR amplified strands produced significantly lower background fluorescence levels than DNA-fluorophore HCR amplified strands alone (fig. 5 a). We immunostained brain sections against NeuN to quantitatively assess the effect of GO on isHCR amplification in tissue samples. Addition of GO reduced the background but did not attenuate the signal strength, resulting in improved signal-to-noise ratio compared to isHCR amplification without GO (fig. 5 d). Further analysis using antibody serial dilution experiments showed that isHCR with GO significantly increased signal intensity compared to standard IHC staining method, reaching greater than 80 x magnification when the primary antibody was highly diluted (fig. 6).
In a seventh embodiment, immunostaining for membrane proteins, peptide neurotransmitters and two enzymes was performed to check the versatility of isHCR with GO in brain sections. It has been challenging to localize the expression of vesicular glutamate transporter 3(Vglut 3; vesicular membrane protein) using standard IHC methods (FIG. 5e and FIG. 7 a). isHCR performed without GO addition produced strong labeling signals in the brainstem raphe nucleus, but also showed high background noise. Encouraging, the isHCR with GO added reduced background and showed clear dotted labeled vgut 3 positive synaptic ends (fig. 5e and fig. 7a, c). No specific markers were observed in brain sections without primary antibody or in mutant mice lacking the functional gene encoding vgout 3 (fig. 7 b). Additional control experiments confirmed that the isHCR faithfully amplifies the true vgut 3 signal and maintained the spatial resolution of confocal imaging (fig. 7d, e). Next, the present inventors examined the expression patterns of Vasoactive Intestinal Peptide (VIP), neuronal nitric oxide synthase (nNOS), and aromatic L-Amino Acid Decarboxylase (AADC) in brain sections using HCR. In all cases, isHCR with GO produced strong immunopositive signals with low background, consistently revealing that the expression pattern of each of these proteins is much more abundant than that observed with the traditional IHC method (fig. 8).
Our success in using GO to reduce background signal intensity prompted us to explore the use of isHCR with monoclonal antibodies. Although monoclonal antibodies are often more specific than polyclonal antibodies, they often do not provide a sufficiently strong signal for detection by traditional IHC staining. In a sixth embodiment, the inventors used two strategies to test the HCR of monoclonal antibodies in mouse brain sections: biotinylated primary monoclonal antibody against GAD67, and a combination of monoclonal primary antibody against c-Fos and biotinylated secondary antibody (fig. 9a, b and fig. 10 a). In both cases, much stronger signals were obtained in the isHCR amplified samples than in the non-isHCR samples.
More importantly, isHCR using GO is able to detect immune signals that are too weak for traditional IHC. Bacterial pathogens deliver protein effectors into host cells to manipulate their physiology; however, the presence and exact distribution of some metathesis effectors is still unclear, because their concentration is extremely low. In a seventh embodiment, the inventors tagged Salmonella enterica serovar typhimurium (Salmonella serovarum typhimurium) effectors SteA and
HCR is an abbreviation for Hybridization Chain Reaction (Hybridization Chain Reaction). When a single-stranded DNA priming strand is added to the reaction, it opens one kind of hairpin (H1 amplified strand), exposing a new single-stranded region that opens another kind of hairpin (H2 amplified strand). This process in turn exposes a single stranded region identical to the original priming strand. The chain reaction thus produced results in the formation of a nicked double helix that grows until the hairpin supply is depleted.
Click chemistry is a class of biocompatible reactions primarily intended to link a selected substrate to a specific biomolecule. Click chemistry reactions are not single specific reactions, but describe a way to follow examples in nature to generate products, which also generate substances by connecting small modular units. In general, click reactions typically link a biomolecule and a reporter. Click chemistry is not limited to biological conditions: the concept of "click" reactions has been used in pharmacology and various biomimetic applications. However, they are particularly useful in the detection, localization and characterization of biomolecules.
Typical click chemistry reactions include: (1) cycloaddition, such as
Graphite oxide is a compound of carbon, oxygen and hydrogen in variable proportions obtained by treating graphite with a strong oxidizing agent. The bulk product that is maximally oxidized is a yellow solid with a C to O ratio between 1.3 and 2.9, which retains the layer structure of graphite, but with much larger and irregular spacing.
Antibodies of the invention include, but are not limited to, traditional IgG and nanobodies.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：自组装诊断阵列平台