Colocalization-by-linkage sandwich assays
11493505 · 2022-11-08
Assignee
Inventors
Cpc classification
G01N2458/10
PHYSICS
G01N2400/00
PHYSICS
G01N33/5308
PHYSICS
International classification
Abstract
There are provided methods and systems for detecting and/or quantifying an analyte. In particular, there are provided methods and systems for simultaneous detection and/or quantitation of two or more analytes in a sample. In some embodiments, there are provided colocalization-by-linkage assays on microparticles (CLAMP) comprising two sets of binders pre-assembled on a support, such that the two sets of binders are colocalized before contacting the sample.
Claims
1. A biomolecule complex for the detection or quantification of an analyte in a sample, comprising: (a) a support complex comprising: (i) a support; (ii) a capture reagent coupled with the support; (iii) an anchor oligonucleotide coupled with the support; (iv) a detection reagent coupled with a hook oligonucleotide, wherein the hook oligonucleotide is releasably coupled with the anchor oligonucleotide at a coupled region; and (b) a detectably-labeled displacer oligonucleotide configured to couple with the hook oligonucleotide at the coupled region and decouple the hook oligonucleotide from the anchor oligonucleotide; wherein the capture reagent and the detection reagent are configured to simultaneously couple with the analyte.
2. The biomolecule complex of claim 1, wherein the analyte is coupled to the capture reagent and the detection reagent and the support complex further comprises the detectably-labeled displacer oligonucleotide coupled to the hook oligonucleotide.
3. The biomolecule complex of claim 1, further wherein the analyte is not coupled to one or both of the capture reagent and detection reagent and the support complex does not comprise the detectably-labeled displacer oligonucleotide coupled to the hook oligonucleotide.
4. The biomolecule complex of claim 1, wherein the anchor oligonucleotide comprises an anchor sequence and the hook oligonucleotide comprises a linker sequence complementary to the anchor sequence.
5. The biomolecule complex of claim 4, wherein the hook oligonucleotide comprises an additional sequence adjacent to the linker sequence and the detectably-labeled displacer oligonucleotide comprises a displacer sequence complementary to the additional sequence and at least a portion of the linker sequence.
6. The biomolecule complex of claim 5, wherein the displacer sequence is complementary to the linker sequence.
7. The biomolecule complex of claim 5, wherein the additional sequence comprises at least one nucleotide.
8. The biomolecule complex of claim 5, wherein the displacer sequence has a melting temperature greater than that of the anchor sequence.
9. The biomolecule complex of claim 1, wherein the sample is a biological sample.
10. The biomolecule complex of claim 9, wherein the sample is a bodily fluid, a whole blood sample, a cell supernatant, an extract, a cell extract, a cell lysate, a tissue lysate, a solution comprising nucleic acid molecules, or a solution comprising proteins.
11. The biomolecule complex of claim 1, wherein the capture reagent is selected from the group consisting of: an antibody or an antigen-binding fragment thereof, an aptamer, a modified aptamer, a somamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, an oligonucleotide, a low molecular weight compound, and any combination thereof.
12. The biomolecule complex of claim 1, wherein the detection reagent is selected from the group consisting of: an antibody or an antigen-binding fragment thereof, an aptamer, a modified aptamer, a somamer, an affimer, an antigen, a protein, a polypeptide, a multi-protein complex, an exosome, an oligonucleotide, a low molecular weight compound, and any combination thereof.
13. The biomolecule complex of claim 1, wherein the capture reagent and the detection reagent are both antibodies or antigen-binding fragments thereof.
14. The biomolecule complex of claim 13, wherein the capture reagent and the detection reagent are a different antibody or antigen-binding fragment thereof and bind to a different epitope on the analyte.
15. The biomolecule complex of claim 1, wherein the detectably-labeled displacer oligonucleotide comprises a detectable label selected from the group consisting of a fluorescent polymer, a biotin molecule, a fluorophore, an enzyme, a nucleic acid enzyme, a riboswitch, an enzyme substrate, a specific nucleic acid sequence, and any combination thereof.
16. The biomolecule complex of claim 1, wherein the support is a microparticle, a nanoparticle, a well in a plate, an array, a microfluidic chip, a lateral flow strip, a slide, a porous polymer, or a hydrogel.
17. The biomolecule complex of claim 1, wherein the support complex comprises a plurality of the capture reagent, a plurality of the anchor oligonucleotide, and a plurality of the detection reagents coupled with the hook oligonucleotide.
18. The biomolecule complex of claim 1, wherein the detection reagent is coupled with a single hook oligonucleotide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
(2) For a better understanding of the invention and to show more clearly how it may be carried into effect, reference will now be made by way of example to the accompanying drawings, which illustrate aspects and features according to embodiments of the present invention, and in which:
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DETAILED DESCRIPTION
(29) There are provided systems and methods for detecting and/or quantifying one or more analyte using a colocalization-by-linkage assay, as described herein. In particular, there are provided systems and methods having sufficiently low background signal, sufficiently low cross-reactivity between reagents, and/or sufficiently high sensitivity to allow detection and/or quantitation of multiple biomolecules simultaneously in a sample. There are also provided multiplex sandwich assays that are rapid, sensitive, cost-effective, and/or scalable, and methods for their preparation.
(30) It should be understood that this disclosure is not limited to specific devices, systems, methods, or uses or process steps, and as such they may vary.
(31) In order to provide a clear and consistent understanding of the terms used in the present specification, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.
(32) The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one”, but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”. Similarly, the word “another” may mean at least a second or more.
(33) As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
(34) As used herein, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.
(35) As used herein, the term “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.
(36) As used herein, the term “support” refers to an immobilizing structure, surface or substrate, such as without limitation a microparticle, a nanoparticle, a well in a plate, a porous polymer, or a hydrogel. It should be understood that the support is not meant to be particularly limited, and any solid, semi-solid, gel or gel-like structure may be used. For example, a support may be an array, a bead (such as without limitation a polystyrene bead), the surface of a multi-well plate (such as a 96-well plate, a 384-well plate, etc.), the surface of a glass slide, a hydrogel matrix, a microfluidic chip, a lateral flow strip, a glass surface, a plastic surface, a silicon surface, a ceramic surface, and the like. In one embodiment, the support is a bead or microparticle or nanoparticle, typically micron-sized or nano-sized, such as without limitation a polystyrene bead, a magnetic bead, a paramagnetic bead, a plastic bead, etc. In another embodiment, the support is a planar microarray. In an embodiment, the support is a nanoparticle. In an embodiment, the support is a microparticle.
(37) As used herein, the term “analyte” refers to a targeted biomolecule or biological cell of interest which is being identified, detected, measured and/or quantified. An analyte may be any biomolecule or biological cell which can be detected using the systems and methods provided herein, such as without limitation proteins, nucleic acids (DNAs, RNAs, etc.), antibodies, antigens, proteins, cells, chemicals, biomarkers, enzymes, polypeptides, amino acids, polymers, carbohydrates, multi-protein complexes, exosomes, oligonucleotides, low molecular weight compounds, and the like. Non-limiting examples of analytes include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers, somamers, affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.
(38) As used herein, a “sample” refers to any fluid or liquid sample which is being analyzed in order to detect and/or quantify an analyte. In some embodiments, a sample is a biological sample. Examples of samples include without limitation a bodily fluid, an extract, a solution containing proteins and/or DNA, a cell extract, a cell lysate, or a tissue lysate. Non-limiting examples of bodily fluids include urine, saliva, blood, serum, plasma, cerebrospinal fluid, tears, semen, sweat, pleural effusion, liquified fecal matter, and lacrimal gland secretion.
(39) As used herein, the term “encoded microparticle” refers to a microparticle that is barcoded, e.g., encoded spectrally according to either the target analyte or the specific test that is to be performed in the assay. Barcoded (or encoded) microparticles are often used in multiplexed suspension assays as they allow particles in a large mixture to be distinguished. The method of barcoding is not particularly limited. Barcoding can be performed using, for example, spectral, graphical, or chemical means. For example, spectral encoding of microparticles can be performed by labeling the microparticles with precise proportions of multicolor dyes. This approach allows simple and high throughput read-out by flow cytometry. As another example, graphically barcoded microparticles are typically engraved or otherwise patterned with a visual pattern that can be characterized via microscopy. Microparticles can also be chemically barcoded, for example using unique DNA sequences that can later be detected via DNA detection means.
(40) As used herein, the term “non-specific binding” refers to an unintended reaction between reagents and/or molecules within the sample, including but not limited to reaction between non-cognate antibodies and protein sticking through hydrophobic interactions.
(41) As used herein, the terms “affinity binder” (AB), “binder”, and “reactant” are used interchangeably to mean any molecule capable of specifically recognizing a target analyte, e.g., via a non-covalent interaction. Examples of affinity binders (ABs) include without limitation immunoglobulin-G (IgG) antibodies (e.g., whole molecules or Fab fragments), aptamers, affimers, nanobodies, ankyrins, and single-chain variable fragments (scFvs).
(42) As used herein, the term “sandwich assay” is used to mean an analyte-targeting assay wherein two ABs simultaneously bind the target analyte of interest and can be used to detect and/or quantify it.
(43) As used herein, the terms “multiplex sandwich assay”, “multiplexed sandwich assay” and “MSA” are used interchangeably to mean a sandwich assay that targets multiple (e.g., two or more) analytes from the same sample and/or assay volume at the same time, multiple AB pairs being used in the assay system at the same time.
(44) As used herein, the term “cross-reactivity” is used to mean a particular case of non-specific binding or non-specific reaction in a multiplexed sandwich assay, wherein an unintended complex is formed that includes non-cognate affinity binders, e.g., as shown in FIG.1.
(45) As used herein, the terms “capture affinity binder”, “cAB”, “capture AB”, “capture binder” and “capture reagent” are used interchangeably to refer to an AB that is attached to a support in a biomolecule complex and is not released from it. A capture AB may be attached directly to a support (e.g., via a covalent bond, a biotin-streptavidin bond, a DNA oligonucleotide linker, or a polymer linker) or indirectly (e.g., via linkage to an an anchor strand, e.g., by conjugation or through a linker such as a capture strand). Non-limiting examples of capture reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers (such as slow off-rate modified aptamers or somamers), affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.
(46) The term “capture strand” refers to a linker (e.g., an oligonucleotide, a polymer, etc.) that links a capture reagent to an anchor strand (and hence the support to which the anchor strand is attached).
(47) As used herein, the terms “detection affinity binder”, “dAB”, “detection AB”, “detection binder” and “detection reagent” are used interchangeably to refer to an AB in a biomolecule complex that is releasably attached to a support. The dAB is generally used for signal transduction and assay signalling. In some embodiments of methods and systems provided herein, for example, the fraction of dAB unbound to an analyte is released from the support such that no signal is produced in the absence of bound analyte. In some embodiments, the dAB is bound to a label or means for signal transduction and assay signalling. Non-limiting examples of detection reagents include antibodies, antibody fragments (e.g., scFv, Fab, etc.), aptamers, modified aptamers, somamers, affimers, antigens, proteins, polypeptides, multi-protein complexes, exosomes, oligonucleotides, and low molecular weight compounds.
(48) As used herein, the term “anchor strand” refers to a linker that attaches to an immobile point on a support. Non-limiting examples of anchor strands include polymers, such as polyethylene glycol (PEG), oligonucleotides (such as a single-stranded DNA oligonucleotide, a single-stranded RNA oligonucleotide, or a double-stranded DNA or RNA oligonucleotide, or a DNA-RNA hybrid), and oligosaccharides.
(49) As used herein, the term “hook strand” refers to a linker that links a detection AB to an anchor strand and hence attaches it to a support. The hook strand is typically attached releasably to the anchor strand, e.g., in such a way that the attachment can be released. Generally, when the attachment between the hook strand and the anchor strand is released, the fraction of detection AB linked to the hook strand that is not bound to a target analyte will be released from the anchor strand, and therefore also released from the support, such that no signal from the detection AB can be detected on the support in the absence of the target analyte. In this way, signal on the support is only detected when the target analyte is present and bound by the detection AB and the capture AB.
(50) In some embodiments, where a label is on the hook strand and/or the detection reagent and is only activated or detectable after the release of the hook strand and/or the detection reagent from the anchor strand, the signal is “release-dependent”, as it will only be detectable after the release of the hook strand and/or the detection reagent from the anchor strand. Similarly, in some embodiments, where the label is on a displacer agent hybridizing to the hook strand, the signal is “displacement-dependent”.
(51) As used herein, the term “displacer agent” refers to an agent that directly or indirectly causes or initiates release of the releasable linkage between the anchor strand and the hook strand, thereby releasing the hook strand (and the detection AB linked thereto) from the support. The mechanism used by the displacer agent is not particularly limited. For example, the displacer agent may directly or indirectly cause or initiate cleavage, displacement, or unbinding of the linkage between the anchor strand and the hook strand; other mechanisms are possible and are also contemplated. In some embodiments, the hook strand is displaced from the anchor strand using a DNA oligonucleotide that hybridizes to the hook strand and/or the anchor strand. Examples of displacer agents include but are not limited to a displacement DNA oligonucleotide, a source of mono- or poly-chromatic light, a restriction enzyme, and a reducing agent such as dithiothreitol (DTT). In some embodiments, where photocleavable DNA segments are used, the displacer agent may be a light which effects release via a photocleavage reaction. In some embodiments, the displacer agent is labeled, e.g., with a dye, a fluorophore, a specific DNA sequence, an enzyme, a biotin moiety, and the like. When the displacer agent is labeled, it can serve the dual-function of releasing the hook strand and labelling it simultaneously.
(52) As used herein, the term “label” refers to any molecule or a portion of a molecule that generates a signal, can be targeted with a signal-generating molecule, or is otherwise detectable. Examples of labels include but are not limited to biotin, fluorophores, enzymes, enzyme substrates, and specific DNA sequences. An “inactive” or “undetectable” label refers to a label which is not active, is masked, or is otherwise undetectable, e.g., not capable of generating a detectable signal, such as without limitation a quenched fluorescent dye.
(53) It should be understood that systems and methods provided herein can be used in virtually any type of sandwich assay wherein two sets of ABs are used. However, for simplicity, specific embodiments of the present invention are presented herein using whole-molecule Immunoglobulin-G antibodies (IgG) as ABs, which represents one of many possible embodiments. It should be understood that antibodies are not limited to whole-molecule IgG and that many different antibodies, antibody fragments, etc. can be used. Further, ABs are not limited to antibodies. Similarly, many different types of sandwich assays other than the specific ones described herein can be used.
(54) In some embodiments, there is provided a dual-AB or sandwich assay that can avoid cross-reactivity by colocalizing two ABs (a capture AB and a detection AB) on a support prior to exposure to a biological sample containing an analyte of interest. Colocalization on the support does not permit any mixing of different AB pairs prior to exposure to the analyte, and can thus reduce or eliminate cross-reactivity between reagents and/or background (such as that shown in
(55) It should be understood that “linkers” and “strands” used in methods and systems provided herein are not particularly limited. Non-limiting examples of linkers and strands include DNA oligonucleotides (also referred to as DNA oligos), polymers, polysaccharides, and the like. DNA linkages can be covalent, such as conjugation between a hook strand oligo and a detection AB, or non-covalent, such as hybridization or base-stacking between two complementary DNA sequences. To allow formation of a capture AB-antigen-detection AB tertiary complex, the hook strand is designed to have a flexible, single-stranded portion. Displacement of a DNA linkage can be performed using several methods including but not limited to a toe-hold mediated DNA displacement reaction, enzymatic cleavage, and photo-activated cleavage. Specific DNA sequences can also be used as labels, which can be either directly targeted using a complementary sequence that is fluorescently labeled, can be used as amplification triggers or primers through a hybridization-chain reaction or a polymerase-chain reaction, and can be read-out via sequencing.
(56) It should be understood that “oligonucleotides” (also referred to as “oligos”) used in methods and systems provided herein are not particularly limited. For example, oligos can be modified using fluorescent dyes on 5′ or 3′ termini, modified with a photocleavable phosphodiester back-bone, conjugated to a protein, a biotin, or an enzyme, etc.
(57) These embodiments may also be referred to herein as a “Colocalization-by-Linkage Assay” or “CLA”. In some embodiments of CLA, the detection AB is labeled (i.e., attached to a label). In some embodiments of CLA, the hook strand linking the detection AB to the anchor strand is labeled (i.e., attached to a label). Generally the label attached to the detection AB or the hook strand is inactive or undetectable, such that the label can be detected after release of the detection AB from the support (i.e., after the hook strand is released from the anchor strand). Signal detection from the label is thus release-dependent (also referred to, in some embodiments, as “displacement-dependent”). In this way, only detection ABs bound to the analyte in a tertiary complex with a capture AB and released from the anchor strand will be detected, as unbound detection AB will be released from the support (and can be removed e.g., by washing). Background signal may also be reduced since the label is inactive or undetectable prior to release, or if a given hook strand is not released (i.e., due to the release-dependent or displacement-dependent nature of the signal). In some embodiments, therefore, methods and systems provided herein may be referred to as “release-dependent transduction” (or “RDT”) or “displacement-dependent detection”, to reflect the release-dependent (or displacement-dependent) signal transduction.
(58) Conventional sandwich assays generally rely on the presence of detection ABs to transduce a signal and detect the presence of an analyte. Similarly, in certain embodiments of systems and methods presented herein, the detection AB and/or the hook strand can act as signal transducers. However, in contrast to conventional assays, in systems and methods provided herein the detection AB and/or the hook strand optionally linked thereto can remain on the support only when a tertiary complex is formed with the analyte and the capture AB. It will be appreciated that, if the detection reagent and/or the hook strand is not successfully or completely released from the anchor strand, then it can remain on the support even in the absence of the analyte. In this case, if the detection AB and/or the hook strand is attached to a label that is active or detectable even when attached to the anchor strand, then any non-released, labeled detection AB and/or hook strand would transduce a signal. In other words, in that case, any labeled and non-released detection reagent and/or hook strand could result in a signal independent of the presence of the analyte, contributing to non-specific background signal, and reducing assay performance and/or sensitivity. It will be appreciated that the background signal in that case will be proportional to the fraction of non-released detection reagents and/or hook strands. It should also be appreciated that near-complete release of complexes from supports may be difficult to achieve due to steric hindrance, sticking, and/or incomplete washing. However, release-dependent transduction (RDT) can minimize or eliminate these problems, as no signal transduction occurs if the release of the detection reagent and/or the hook strand from the anchor strand is not complete, as demonstrated in
(59) In some embodiments, therefore, systems and methods provided herein include an additional level of redundancy to reduce background signal and/or increase sensitivity by the use of release-dependent transduction (RDT). In RDT, signal transduction occurs only if both of the following conditions are satisfied: (i) formation of a tertiary capture AB-analyte-detection AB complex, and (ii) release of the corresponding detection AB and/or hook strand from the anchor strand. In such cases, a non-released detection AB and/or hook strand will not contribute to the background signal. This signal transduction mechanism, which we herein refer to as “release-dependent transduction (RDT)”, can be achieved through various means. For example, some embodiments can include a label on the hook strand, wherein the label is inactive or undetectable until after the release from the anchor strand, such that a non-released (e.g., non-displaced) hook strand and/or detection AB) will not contribute to or transduce the signal.
(60) In some embodiments of RDT, a hook strand is labeled with a fluorescent dye quenched by a quencher on the anchor or another proximal strand, such that release results in unquenching or activation of the fluorescent dye.
(61) In some embodiments of RDT, the detection reagent and the hook strand are not labeled, and instead the displacer agent is labeled. In this case, the displacer agent hybridizes to the hook strand, displacing it from the anchor strand, and simultaneously labeling it. If the detection AB is not bound to analyte and capture AB in a tertiary complex, then the hook strand, the displacer agent, and the label are washed off the support. Since the label is attached to the displacer agent, the label is only present on the support when both conditions are met: (i) release or displacement from the anchor strand has occurred, and (ii) analyte has bound to both capture and detection ABs (shown in
(62) It will be appreciated that other embodiments of RDT are possible, and the mechanism of RDT is not meant to be particularly limited.
(63) In some embodiments of RDT, the detection AB or the anchor strand is attached to a label. In some embodiments, the hook strand linking the detection AB to the anchor strand is labeled (i.e., attached to a label). Generally the label attached to the detection AB, the anchor strand, or the hook strand is inactive or undetectable, such that the label can only be detected after release of the detection AB from the support (i.e., after the hook strand is released from the anchor strand, as shown for example in
(64) In other embodiments of RDT, the hook strand contains a label that remains inactive or undetectable until the hook strand is released from the anchor strand. For example, this can be achieved when the hook strand and the anchor strand are DNA oligonucleotides bound together via hybridization, wherein the hook strand contains a DNA sequence label normally hybridized to the anchor strand and hence unavailable for binding, or undetectable. Release of the hook strand oligo from the anchor strand oligo reveals a detectable label on the hook strand. Such release can be achieved e.g., via enzymatic cleavage, DNA displacement, or photocleavage using light.
(65) In some such embodiments, there is provided a release or displacer agent which is an oligonucleotide that displaces the anchor strand-hook strand hybrid by binding to the anchor strand oligo via a toe-hold displacement reaction. In an embodiment, the hook strand and the detection AB are both not labeled, and a labeled displacer agent (e.g., a fluorescently-labeled oligonucleotide) performs RDT through the dual-function of release (displacement) and labeling. In this way, through only labeling the displaced hook strands, a detectable signal/signal transduction only occurs on a support when two conditions are met (displacement of the hook strand and presence of the analyte), akin to an “AND” logical gate (shown in
(66) In some embodiments of assays and systems provided herein, one or more set of capture and detection ABs is attached to a support, each set being specific for an analyte of interest. In this way, the capture and the detection AB are pre-assembled and colocalized on the support, prior to exposure to a biological sample containing the analyte of interest. As described above, the detection AB is attached to the support releasably. In some embodiments, the detection AB is attached to the support by a releasable linker (a hook strand) which is linked to an anchor strand attached to the support. The hook strand is generally flexible and allows the detection AB to diffuse freely within the bounds allowed by the lengths of the hook strand and/or the anchor strand. The hook strand and the releasable link are not particularly limited and may vary in size, flexibility, structure, etc., as long as they allow simultaneous binding of the analyte by the detection AB and the capture AB. The capture AB and the detection AB generally bind separate regions of the analyte, although they may bind overlapping sites, as long as they are capable of binding the analyte simultaneously.
(67) In some embodiments, the detection AB is linked to the support using a hook strand which is a DNA oligonucleotide that can bind specifically to the anchor strand attached to the support. After contacting and incubating with the biological sample (i.e., target recognition step), the detection AB is separated from the anchor strand by breaking the linkage between the hook strand and the anchor strand on the surface. This release the fraction of detection AB that has not formed a tertiary capture AB-analyte-detection AB complex. It should be understood that the linkage between the hook strand and the anchor strand may be released or broken in several ways, such as without limitation DNA strand displacement, enzymatic cleavage, photo-activated cleavage, and the like.
(68) As encompassed herein, many ABs targeting many different analytes can be mixed in the same assay volume (i.e., multiplexing); interaction between different ABs on different supports (or between different ABs on different locations/positions on the same support) are limited by the linkages to the support(s), so that interaction between ABs from different supports/locations is avoided. This is in contrast to conventional multiplexing technologies that can not limit interactions between ABs when all ABs are mixed in solution. Further, with methods and systems described herein, different microparticle populations can be fabricated separately in large batches, each containing a different AB capture-detection pair needed to detect a specific antigen, ensuring that cross-reactivity does not occur during manufacturing.
(69) In some embodiments, multiplexed CLA methods and systems can thus avoid the cross-reactivity scenarios shown in FIG.1. For example, as will be appreciated by those skilled in the art, the colocalization of cognate capture and detection ABs on their respective supports (e.g., microparticles) will eliminate unwanted interactions such as, for example, binding between non-cognate detection and capture ABs. In addition to those scenarios shown in FIG.1, those skilled in the art will recognize that, as opposed to conventional multiplexed sandwich assays, analytes that indiscriminately bind, or stick, to off-target supports cannot be detected by their cognate detection AB in methods and systems provided herein, and hence do not contribute to increase the background signal.
(70) In some embodiments, on each support, the local concentrations of the capture and detection ABs can be high, which can serve to concentrate the analytes and increase the sensitivity. On the other hand, the total concentration of each capture and detection AB in the entire assay volume is only dependent on the concentration of target-specific supports (e.g., microparticles, microarray spots) and can be designed to yield low bulk-concentrations of detection ABs upon release. For example, while the local-concentrations can be in the micromolar range, the use of a low number of target-specific microparticles can yield bulk detection AB concentrations too low (<pM) to yield any off-target binding, as shown for example in
(71) In some embodiments, simultaneous binding of two colocalized binders (capture AB, detection AB) to two different epitopes of the same analyte (that is, increased binding avidity) can result in a much lower effective off-rate (koff) in comparison to conventional sandwich assays where capture and detection ABs are added sequentially. After sample introduction and incubation, the supports in methods and systems provided herein can be stringently washed, since the analytes are bound with high avidity. Hence, in some embodiments of methods and systems provided herein, stringent washing can be used to reduce assay background and/or improve sensitivity and/or specificity. In some embodiments, it may be desirable to rapidly execute the assay steps following the release of a hook strand from an anchor strand and up until read-out of the assay signal, since off-binding of analytes can result in a reduced signal which can contribute to reduced sensitivity, although such effects are generally reduced in CLA.
(72) In one embodiment of methods and systems provided herein, a support is an encoded micron-sized microparticle, and capture reagent and the detection reagent are both antibodies, wherein the capture reagent and its cognate detection reagent are colocalized on the surface of the same support using DNA linkages (in other words, the hook strand and the anchor strands are single-stranded DNA oligonucleotides, linked together via a double-stranded DNA hybrid). In some such embodiments, the detection reagent linked to the hook strand and the anchor strand are homogeneously mixed and attached to the surface of the microparticle, wherein the anchor strand is linked to the hook strand through partial hybridization, the hook strand being conjugated to the detection reagent, the hook strand being a flexible and releasable DNA linker. The hybrid between the anchor and hook strands is generally stable during conditions of sample incubation. In some embodiments, the capture reagent is also linked to the microparticle via a DNA linker as well. In some such embodiments, release of the hook strand from the anchor strand can be performed via a toe-hold mediated DNA displacement reaction. In such embodiments, a displacer agent is an oligonucleotide designed to bind to a toe-hold sequence on the hook strand to drive the displacement reaction forward. In some such embodiments, release of the hook strand from the anchor strand can be performed without a displacer agent, e.g., by raising the temperature so that the DNA hybrid “melts” or is unbound.
(73) In an embodiment, a detection AB and/or a hook strand is labeled, e.g., with a dye, a biotin moiety that can be detected using a fluorescently-labeled streptavidin in a subsequent step etc. In certain embodiments, a detection AB can be detected after binding an analyte with a labeled-binder, for example, an IgG can be targeted using a labeled species-specific secondary-IgG. In some embodiments, the detection AB and the hook strand are not labeled, and instead a displacer agent used to release the hook strand from the anchor strand is labeled. In such embodiments, the labeled displacer agent attaches to the hook strand and/or the detection AB after the release of the hooks strand from the anchor strand.
(74) In some embodiments, the label is a specific DNA sequence that can be detected or targeted in a subsequent step(s). For example, a specific DNA sequence can be targeted with a subsequent DNA hybridization step that labels it with a dye. In an embodiment, the specific DNA sequence is detected and amplified through Polymerase Chain Reaction (PCR) or other enzymatic DNA amplification means. Specific DNA sequences can also be cleaved and detected by other means such as sequencing. Embodiments using DNA sequence as a label are not limited and may include the sequence being part of the hook strand (and hence, initially inactive/undetectable), or present on the displacer agent (shown for example in
(75) In some embodiments, there is provided a detection AB linked to a hook strand and attached to a microparticle indirectly via a releasable link to an anchor strand attached thereto. The hook strand is partially complementary to the anchor strand attached to the microparticle. The anchor strand may be attached to the microparticle via for example a streptavidin/biotin interaction or a chemical bond. The detection AB is thus attached to the microparticle. In this embodiment there is further provided a capture AB which is attached to the microparticle surface, and wherein the detection AB recognizes the same antigen as the capture AB and both ABs can bind the antigen simultaneously. In addition, there is provided a displacement oligonucleotide (the displacer agent) that has a sequence that is complementary to the hook strand, overlapping with the sequence of the anchor strand, so that the detection AB is released from the anchor strand and thus released from the microparticle, if no antigen is bound (i.e., if there is no tertiary complex between capture AB-antigen-detection AB). In a further embodiment, there is also provided a fluorescently-labeled secondary antibody that binds to the detection AB remaining on the microparticle after the displacement reaction.
(76) It should be noted that, in embodiments where capture and detection ABs are pre-assembled on a support, and detection ABs are labeled with a detectable label, any non-released hook strand-detection AB complexes will result in an analyte-independent signal, which could contribute to the background noise (as shown in
(77) In one embodiment, a labeled displacer agent (e.g., oligonucleotide) can perform the dual-function of release (displacement) and labeling. In this way, through only labeling the displaced hook strands, a detectable signal/signal transduction necessitates two conditions, akin to an “AND” logical gate (
(78) In some embodiments, an additional level of redundancy can be achieved by using a hook strand with an inactive or undetectable label which is only activated or detectable upon displacement from the anchor strand. For example, in one embodiment a hook strand is labeled with a dye that is quenched by a dye quencher that can be conjugated to the anchor strand. In another embodiment, displacement can be similarly achieved using a restriction enzyme, followed by signal generation using a labeled-oligo that targets the previously-hybridized (and hence unavailable for binding) portion of the hook strand, thereby only hybridizing to and labeling already displaced hook strands.
(79) In certain embodiments, there is provided a detection AB linked to a microparticle via a hook strand, the hook strand being an oligo, which is linked to the detection AB. The hook strand oligo is partially complementary to an anchor strand, which is also an oligo, linked to the microparticle via e.g., a streptavidin/biotin interaction or a chemical bond, thus attaching the detection AB to the microparticle. There is further provided a capture AB which is linked to the microparticle surface and wherein the detection AB recognizes the same antigen as the capture AB and both ABs can bind the antigen simultaneously. In addition, there may be provided a displacement agent which is an oligonucleotide containing a fluorescent label or a DNA barcode sequence and has a sequence complementary to the hook strand oligonucleotide, overlapping with the sequence of the anchor strand oligonucleotide so that the detection Ab is released from the anchor strand oligo and thus may be released from the microparticle.
(80) It should be understood that, in methods and systems provided herein, the use of colocalization and linkages may necessitate rational topological design to optimize the availability of both ABs (capture AB and detection AB) across a support. In some embodiments, with stochastically distributed capture ABs and/or detection ABs attached to the support, appropriate binding of an analyte may require optimization of two important design parameters: (i) the relative density of the capture and detection ABs, and (ii) the length of the hook strand. These two parameters serve to control the time-averaged distance between capture and detection ABs by considering the gyration radius of the detection AB. In some cases, the distance between capture and detection ABs, and ultimately the effective-affinity at the single-molecule level, may be stochastic and difficult to control. Therefore, in some embodiments it may be desirable to optimize the aforementioned two parameters for optimal assay performance.
(81) In another embodiment, the capture AB and the detection AB are both linked to the anchor strand, allowing concomitant control over capture and detection AB densities whilst maintaining colocalization at the nano-scale, potentially allowing more accurate control of assay performance (such as shown in
(82) In some such embodiments where the effective affinity can be tuned by changing the length of the hook strand or the anchor strand (i.e., the linker length), or by tuning the surface densities of capture and detection ABs, multiplexed arrays (such as multiplexed microparticles) can be fabricated that are designed with different effective affinities. This can be useful to extend the dynamic range of a particular assay for a particular analyte. For example, those skilled in the art will appreciate that some proteins are present in blood in concentrations ranging >5 orders of magnitude; for such targets, several assays can be designed, with different barcodes, to be able to quantify such proteins over a larger dynamic range.
(83) In an embodiment, a capture AB is conjugated to a capture oligonucleotide which hybridizes to one sequence domain of the support-linked anchor strand. Another sequence domain of the anchor strand may be hybridized to the hook strand which is linked to the detection AB. All the aforementioned strategies for signal transduction and generation can also be utilized in this embodiment.
(84) In an embodiment, two or more sets of distinguishable (i.e., multiplexed) complexes detecting the same target can be designed to increase the dynamic range of a multiplexed assay, wherein the lengths of the hook strand oligos for the two or more sets, and hence the stringency of the binding, can be controlled. For example, two or more sets of microparticles with different barcodes but targeting the same analyte can be fabricated, wherein the first microparticle set includes a shorter hook strand oligo to reduce flexibility and increase stringency of binding, and wherein the second microparticle set includes a longer hook strand oligo to increase flexibility and reduce stringency of binding, and so on. In this way, the first microparticle set can be designed to quantitate the analyte when it is present at higher concentrations.
(85) Those skilled in the art will recognize that another challenge of multiplexed assays is interference and matrix effects, which can be difficult to control at the analyte-level. One of the advantages of methods and systems provided herein, in some embodiments, is the ability to contact the same biological sample with multitudes of assay configurations within the same assay volume. This flexibility may provide the ability to individually control for matrix effects on specific ABs and assay reagents. For example, certain samples could contain endogenous antibodies and other molecules which could positively or negatively impact the intensity of the assay signal for specific analytes.
(86) In another embodiment, there are provided distinct supports or biomolecule complexes, with every analyte-specific support lacking either one of the capture or detection ABs and acting as an analyte-specific internal standard that controls for matrix effects and other potential modes of failures of the assay. The assay signal of the fully-formed biomolecule complex on the support can then be compared to these single-AB controls. These internal controls can be used as flags for potential false positives.
(87) Those skilled in the art will recognize that another challenge of assays, particularly when using binders with non-zero or fast off-rate (k-off), is the unbinding of analytes, and hence drop in the assay signal, that can occur in the time between the washing of the biological sample to the read-out of the assay signal. This unbinding is especially problematic for low concentration analytes, and read-out methods that cannot measure the different assays in multiplex (e.g. cytometry). This problem may also be present in the CLA sensor procedure, whereby post-release (e.g., post-displacement), unbinding of the analyte to either the capture AB or the detection AB may result in signal loss. In yet another embodiment, therefore, the CLA methods and systems provided herein can be modified to mitigate this problem of unbinding and time-dependent signal by transducing the assay signal from a reversible reaction (e.g., an AB-analyte) into a stable oligo hybrid to stop further unbinding and is linked to the support enabling storage and read-out at a later time (such as shown in
(88) In some such embodiments, there is provided a displacer agent which is an oligo that displaces the anchor strand-hook strand hybrid by binding to the anchor strand oligo via a toe-hold displacement reaction, followed by washing of released and unbound hook strand oligo-detection AB complexes, followed by addition of a replacement oligo that enables re-binding of the hook strand oligo to the anchor strand oligo by hybridizing to both oligos.
(89) Several applications will benefit significantly from the methods and systems provided herein, which serve in some embodiments to address several sources of background noise and false-positives in multiplexed sandwich assays. In particular, in some embodiments multiplexing of protein analyses will be significantly enabled by the methods and systems provided herein. For example, profiling of proteins such as cytokines and other soluble factors has been limited in conventional multiplexing due to reagent-cross reactivity. In some embodiments, methods and systems provided herein can significantly improve multiplexed serological analyses. For example, multiplexed autoantibody assays that are used to detect many specific autoantibodies have been severely hindered by specificity. Autoantibodies are typically captured by specific recombinant or native antigens on a solid-support, and are then detected by a species-specific detection antibody (e.g., anti-human Fc IgG). As a result, any non-specific binding of autoantibodies present in sera will be detected and often leads to a false-positive, making this type of assay a single-binder assay (in other words, limited to single-plex form). In contrast, methods and systems provided herein can be utilized to perform a dual-binder assay; that is, one where the analyte (here an autoantibody) is recognized and detected by two specific ABs (here, the specific antigen). In such embodiments, recombinant or native antigens can be divided into two fractions, representing capture AB and detection AB, that are conjugated to a capture strand and a hook strand, respectively, wherein the capture strand and the hook strand are both linked to the same anchor strand, wherein the anchor strand is attached to the support (as in
(90) In some embodiments, methods and systems provided herein can address a major challenge in the multiplexed analyses of protein-protein interactions using ABs. For purposes thereof, AB pairs can be pre-assembled, each AB pair targeting one protein of interest, allowing for the CLA to detect interactions between the pair in question, as shown in
(91) In some embodiments, methods and systems provided herein can address another major challenge in the multiplexed analyses of post-translational modifications (PTM) using ABs. For example, accurate protein phosphorylation analysis can be used to reveal cellular signaling events not evident from protein expression levels. Current methods and workflows for quantifying the fraction of PTM of a specific protein are severely limited in multiplexing because PTM-specific ABs possess inadequate specificity for the protein itself (that is, a phosphor-specific AB is highly susceptible to the problem of reagent-driven cross-reactivity). As a result, conventional PTM panels are not multiplexed. The multiplexed CLA assay methods and systems provided herein can address this problem by confining the anti-PTM binder to an analyte-specific support (as in
(92) In some embodiments, a hook strand is a flexible and releasable linker and is an oligonucleotide, which allows for the formation of a capture AB-analyte-detection AB tertiary complex, such that upon release of one of the unbound hook strand oligos from the support, a signal is generated only in response to recognition of a sandwich capture AB-analyte-detection AB.
(93) In some embodiments, there is provided a detection AB which is an antibody attached to a support, such as a microparticle, via a hook strand which is an oligonucleotide linked to the detection AB. The hook strand oligonucleotide is partially complementary to an anchor strand oligonucleotide attached to the support (e.g., microparticle) via a streptavidin/biotin interaction for example or a chemical bond, thus attaching the detection AB to the support. There is further provided a capture AB which is an antibody attached to the support and wherein the detection AB recognizes the same antigen but not the same epitope as the capture AB. In some embodiments, there is provided a displacer agent which is an oligonucleotide which contains a fluorescent label or a DNA barcode sequence and has a sequence complementary to the hook strand oligonucleotide, overlapping with the sequence of the anchor strand oligonucleotide so that the detection AB is released from the anchor strand oligo and thus may be released from the support in the absence of the target analyte. It should be understood that once the capture AB and the detection AB bind to the analyte, a tertiary capture AB-analyte-detection AB complex is formed on the support (e.g., on the microparticle). After formation of the tertiary complex, unbound detection AB is removed from the support by washing, while the tertiary complexes are retained on the support. The presence of the tertiary complexes on the support afterwards can be detected and/or quantified.
(94) In some embodiments, methods and systems provided herein may be referred to as “colocalization-by-linkages assay on microparticles” or “CLAMP”. CLAMP methods and systems described herein may be highly accessible and advantageous for users. For example, by providing microparticles that have pre-assembled AB pairs (pairs of capture and detection ABs), users can rapidly mix-and-match panels at will, perform multiplexed assays rapidly, and read-out the assay results using e.g. any multicolour flow cytometer. CLAMP assays provided herein can thus fit within existing experimental workflows in biology, and in some embodiments can be read out using any multicolor flow cytometer.
(95) It will be appreciated that CLAMP embodiments are uniquely amenable for large, industrial-scale fabrication of multiplexed panels that avoid cross-reactivity. As opposed to planar arrays, CLAMPs can be fabricated separately in large batches, optionally stored, and then mixed prior to the assay. This fabrication method allows CLAMPs to be manufactured independently without interaction between non-cognate ABs, and hence without cross-reactivity during the manufacturing step, a key advantage over other CLA embodiments.
(96) In some embodiments, to fabricate multiplexed CLAMPs, AB pairs are attached on sets of microparticles, wherein each target-specific AB pair is attached on its respective set of microparticles in a separate vessel. The microparticles can be barcoded prior to the AB attachment, or can be barcoded during this process as well. This reaction can be performed in large batches, and the fabricated CLAMPs can be stored. To conduct an assay, fractions of beads for each barcode/target are mixed together before contacting with the biological sample. Microparticles can be barcoded using any means, for example spectrally, graphically, or chemically.
(97) In some embodiments, where the support is a microparticle (MP), certain advantages may be obtained. For example, in some embodiments the ability to rapidly read out a large number of MPs by flow cytometry can afford increased precision and sample throughput In addition, MPs may be functionalized in large batches and then stored, used, and read-out while in solution, which can reduce lot-to-lot variability and enable quantitative analysis (Tighe, P. J., et al., Proteomics-Clinical Applications 9, 406-422, 2015; Jani, I. V., et al., The Lancet 2, 243-250, 2002; Krishhan, V. V., Khan, I. H. & Luciw, P. a. Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts; Tighe, P., et al., Utility, reliability and reproducibility of immunoassay multiplex kits. Methods (San Diego, Calif.) 1-7 2013; Fu, Q., et al., Clinical applications 4, 271-84, 2010).
(98) In some embodiments, methods and systems provided herein can reduce or eliminate reagent cross-reactivity. As shown in
(99)
(100) In some embodiments, CLAMP panels can have lower development costs than traditional immunoassays; not only is costly re-optimization of panels avoided as new target analytes are added, but CLAMP can also use significantly lower quantities of antibodies per assay.
(101) In some embodiments, in addition to overcoming reagent cross-reactivity, the pair of surface-tethered antibodies in CLAMP can result in a binding avidity effect, giving CLAMP further advantages over conventional sandwich immunoassays. CLAMP can exhibit a higher affinity for targets, as the off-rate (koff) of targets from antibody sandwich complexes in CLAMP can be much lower than in assays using sequential antibody addition. In some embodiments, CLAMP assays can be stringently washed after incubation, reducing assay background and improving specificity. In addition, in some embodiments CLAMP may have a reduced liability for false positives: mis-binding events in CLAMP do not form complete sandwich complexes, and hence they do not lead to false positive signals.
EXAMPLES
(102) The present invention will be more readily understood by referring to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope thereof in any manner.
(103) Unless defined otherwise or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention.
Example 1
One-Pot Bead Barcoding and CLAMP Manufacturing
(104) In some embodiments, the multiplexed assay system was implemented on spectrally-encoded beads, wherein a one-pot bead barcoding strategy and automated decoding method can be used in methods and systems provided herein. Examples of such barcoding/decoding methods are described in U.S. patent application Ser. No. 16/153,071 and in Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018, the contents of each of which are incorporated by reference herein in their entirety. Such methods use accurate models of fluorophore spectral overlap and multicolor Forster-resonance energy transfer (FRET). For example, such strategies may have a capacity for more than 580 barcodes using two lasers for barcoding and a third laser for assay readout (shown in
(105) The same manufacturing workflows were used to build a version of a colocalized antibody assay as described herein. Namely, in a first step, streptavidin beads were co-coupled with biotinylated capture antibodies and biotinylated anchor or capture oligos modified with different dyes to yield a distinguishable barcode. Each barcode, and target-specific antibody, were fabricated in separate tubes. In a second step, the detection antibodies (monoclonal) conjugated to a hook oligo were added to the corresponding functionalized beads from the first step. The hook strand oligo was complementary to the anchor strand oligo and hybridized to it, resulting in the assembly and colocalization of matched antibody pairs. The beads can be separately stored for use at a later time.
(106) In some embodiments, a low oligo:antibody conjugation ratio or valency and/or two-step purification can be used to optimize (i.e., lower) background signal. For example, low valency antibody-oligo conjugates were shown to maximize CLAMP strand displacement efficiency and minimize background signal (
(107) In one embodiment, a CLAMP system as described herein comprises the following components: 1) microparticles holding all the other components in place; 2) a type of capture antibodies (cAb) covalently coupled to the microparticle; 3) a type of detection antibodies (dAb) covalently linked to a hook oligonucleotide wherein the detection antibody recognizes the same antigen but not the same epitope as the cAb; 4) an anchor oligonucleotide (AO) linked to the microparticles via a streptavidin/biotin interaction, for example; 5) a stem oligonucleotide (SO) that is fully or partially complementary to the AO and thus renders it at least partially double-stranded; 6) a hook oligonucleotide (HO) covalently-linked to the cAb and partially complementary to the anchor oligonucleotide thus attaching the cAb to the microparticle; and 7) a displacement oligonucleotide (DO) having 2 functions: a) containing a fluorescent label and b) having a sequence complementary to the HO and overlapping with the sequence of the AO so that the dAb is released from the AO and thus released from the microparticle.
(108) A 5-plex CLAMP using antibodies was assembled wherein the antibodies were highly cross-reactive in a conventional sandwich immunoassay, and confirmed that CLAMP completely avoided cross-reactivity (
(109) CLAMP was used to profile human serum. Conjugated antibodies and barcoded beads were independently stored for >1 month, and CLAMP yielded good spike-in recovery of PSA in serum (data not shown).
(110) In an embodiment, the CLAMP system described herein is a 10-plex cytokine panel. Cytokines encompassed herein are for example, but not limited to IL1 to IL17, MCP1/3, TNF, EGF/R, and/or VEGF/R. In another embodiment, the CLAMP system encompassed herein is a 10-plex panel focused on breast cancer metastasis, targeting for example, but not limited to, HER2, CEA, p53 and/or CA15-3.
Example 2
CLAMP Assay Architecture
(111) We prepared and tested a colocalization-by-linkage assay on microparticles (MPs) called “CLAMP”, in accordance with one embodiment. CLAMP is a multiplexed assay designed to eliminate reagent-driven cross reactivity (“rCR”) by colocalizing and confining each antibody pair onto a set of barcoded MPs, thereby avoiding interaction between non-cognate antibodies (
(112) The architecture and operative principle of one embodiment, referred to herein as a CLAMP assay, are illustrated schematically in panels d and e of
(113) To mitigate this effect in CLAMP assays, we designed a detection scheme to exclusively label ‘successfully’ released conjugates through the use of a fluorescently-labeled displacer oligo (DO) that binds to a toe-hold domain on HO, displacing and labeling it simultaneously (
Example 3
CLAMP Assay Optimization
(114) We first optimized the efficiency of the toe-hold mediated displacement reaction by displacing unconjugated, Cy5-labeled HOs (
(115) Next, we studied the impact of antibody-oligonucleotide conjugates on assay background by measuring the residual signal on the MPs following a labeled-displacement step in buffer (see Methods below). We first conjugated HOs to immunoglobulin-G (IgGs) using a commercial kit (Solulink) leading to approx. 90% antibody conjugation yield and an average of 2 HOs per IgG (i.e., λ˜2). Using these conjugates, the assay background was an order of magnitude greater than the assay background of unconjugated HOs (
(116) To optimize assay performance, we modulated the dAb-HO density. In CLAMP, adequate local dAb concentrations are key for sensitive and high capacity sandwich binding which, for a set HO length, is chiefly dependent on the surface densities of dAb-HOs and, through hybridization capture, COs. CLAMPs against urokinase plasminogen activator (anti-uPA CLAMP) with varying CO densities were prepared using low valency dAb-HO conjugates with fewer than 8% multivalent conjugates (
Example 4
Multiplexed CLAMP Assay
(117) To test CLAMP's efficacy in eliminating reagent-driven cross reactivity (“rCR”), we screened the assay specificity of a multiplexed CLAMP in accordance with one embodiment. In addition, to challenge the CLAMP assay we selected antibody pairs that have been shown to exhibit different types of rCR when used together in a conventional multiplexed sandwich assay (“MSA”). To this end, antibody pairs against six targets (EpCAM, PSA, E-Cadherin, EGF, uPA and MCP) were shortlisted from a 35-protein panel that we previously characterized for specific and non-specific binding in a conventional MSA (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018). For conventional MSAs, the specificity screen consisted of incubating each individual antigen with a pool of cAb-coated barcoded MPs, followed by addition of mixed dAb cocktail and secondary-antibody (“sAb”) for detection and labeling, respectively (
(118) In summary, we successfully demonstrated use of CLAMP, a homogeneous MSA that uses oligonucleotides to precolocalized antibody pairs on MPs. By confining each antibody pair to their respective MPs during sample incubation, CLAMP can be multiplexed while maintaining single-plex assay environments on each MP and, in doing so, eliminates reagent-driven CR. Notably, the pre-colocalization of antibodies in CLAMP represents a departure from conventional sandwich immunoassays, where matched antibodies are separate at the beginning of the assay. To detect correct sandwich binding, we have shown that a labeled displacer oligo can be used to simultaneously release and label dAb-oligo complexes. We studied and demonstrated the importance of using monovalent antibody-oligo conjugates to avoid labeling unreleased complexes and increasing background signals. We have experimentally validated the assay, both in single-plex and multiplex, and screened the specificity of the assay in multiplex using five antibody pairs pre-selected for CR, demonstrating that CLAMP eliminates all rCR experienced in a conventional MSA.
(119) CLAMP can provide several distinct advantages over currently available MSAs. First, CLAMP can be easily deployable as it does not necessitate dedicated equipment for readout or introduce new workflows. Second, CLAMP can be a rapid assay as it can be completed in little over three hours. Finally, by eliminating the need to incubate detection antibodies in solution (which is typically done at high concentrations), CLAMP can provide significant reductions in reagent consumption. Owing to its highly scalable and highly efficient nature, CLAMP can be used to provide a truly-scalable multiplexed ELISA platform that meets the increasing demands in biomarker discovery and drug development.
Example 6
Low Antibody Concentration Minimizes Cross-Reactivity in a CLAMP Assay
(120) Conventional multiplexed sandwich immunoassays are commonly conducted with a mixture of reagents in the solution phase. In particular, the detection antibodies (dAbs) against different targets are mixed and applied to the reaction together. The application of such dAb cocktail leads to spurious binding and generates false-positive signals from non-specific binding events (between a cAb or dAb and a non-targeted analyte) that are difficult to discriminate from the real target protein-binding signal. The risk of reagent-driven CR scales as ˜4N.sup.2 with the number of target analyte N.
(121) In contrast, in embodiments of CLAMP, reagent (e.g., antibody) pairs can be pre-assembled and colocalized on barcoded microparticles to avoid the reagents mixing. The detection antibodies (dAbs) will only be released in solution after the displacement reaction, as described herein. In some embodiments, to avoid re-binding on off-target beads after the displacement reaction, the dAbs released into solution should optimally remain at sufficiently low concentrations.
(122) To ensure that off-binding is avoided after release, the amount of antibodies per target should ideally be kept <10 pM. In a volume of 100 uL, the amount of antibodies is <1 fmoles. In a CLAMP assay, in some embodiments, the amount of released Ab from 1000 microparticles was estimated to be 0.1-1 fmoles (
Example 7
Displacement-Dependent Signal Transduction Minimizes Background Signal in a CLAMP Assay
(123) In an embodiment of a CLAMP colocalized assay where both antibodies are pre-colocalized on the support, signal transduction could be performed by detecting all dABs remaining on the surface after release and washing. However, any non-released hook oligo-dAb complexes could result in an analyte-independent signal, significantly contributing to the background noise. Hence, it will be appreciated that to avoid increasing the background signal, in some embodiments a near-complete anchor-hook displacement and washing of hook oligo-dAB complexes are required.
(124) In some embodiments, the problem of increased background signal due to inefficient release can be addressed through a displacement-dependent signal transduction mechanism. Such a mechanism would ensure that only displaced hook-anchor strands are detectable, and as such, non-displaced strands, which might occur due to inefficient displacement, do not yield a background signal. In such embodiments, signal transduction at the molecular level only occurs if both of the following conditions are satisfied: (i) formation of a tertiary complex, and (ii) displacement of the hook-anchor strands.
(125) In some embodiments, therefore, the detection Ab and the hook strand are not labeled, and displacement occurs using a labeled (e.g., fluorescently-labeled) dispacer oligo. In this embodiment, the displacer oligo can bind to the hook strand preferentially which (i) releases it from the anchor strand and (ii) labels it. On the other hand, a non-displaced hook oligo is not labeled and does not contribute to the signal. This mechanism is equivalent to an AND gate where the signal (output) is dependent on both displacement (input 1) and analyte presence (input 2), as shown in
(126) To demonstrate the effectiveness of the displacement-dependent signal transduction, we performed calibration assays for IL-7, IFN-gamma, and MMP-9. In a first test, the displacer oligos were not labeled, and the mouse-dAbs were targeted using anti-mouse BV421 secondary antibody. The BV421-labelled secondary antibody was targeting at the dAb independent of whether it was released and hence the labelling occurred regardless of the displacement. In a second test, the displacement oligo was labeled using Cy5, which tested the displacement-dependent signal transduction. As shown in the logic gate representation chart (
Example 8
Low Valency Antibody-Oligo Minimizes Background Signal in a CLAMP Assay
(127) In some embodiments, the hook and anchor strands are DNA oligonucleotides. Antibody-DNA conjugation can be performed, for example, by targeting the lysine groups on an IgG molecule. Heterobifunctional linkers such as sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) can be used to therefore link a thiol-terminated DNA to an IgG molecule. This reaction, however, results a heterogenous conjugates, wherein the number of oligos per antibody is dependent on the stoichiometry of DNA:antibody during the reaction. Multivalent conjugates (more than one oligo per antibody) can reduce displacement efficiency and hence increase the background signal.
(128) As shown in
Example 9
Cross-Reactivity Characterization at 40-Plex
(129) To assess cross-reactivity in multiplexed assays with higher multiplexing, a panel of 40 targets was tested, wherein mixtures of CLAMPS against 40 targets (as shown in
(130) Methods
(131) Materials and Reagents. HPLC-purified oligonucleotides were purchased from IDT (Coralville, Iowa, USA); the sequences and modifications are shown in
(132) Synthesis of CLAMPs. CLAMPs were assembled on streptavidin-coated magnetic MPs with a 2.7 μm diameter (M270-Streptavidin) in two steps. The first step consisted of the immobilization of a biotinylated mixture of antibodies and oligos to functionalize the MPs and simultaneously encode them as described in detail elsewhere (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018). Briefly, 90 pmols biotinylated oligos (COs, and SOs) and a total of 90 pmols of LOs (LO0-LO2) were mixed together in 25 μL of PBS+0.05% Tween20+300 mM NaCl (PBST0.05+NaCl300). Whereas the proportions of each LO0:LO1:LO2 is designed to generate a unique ensemble fluorescence to define the barcode, the proportion of CO: SO allows tuning of the surface density of pulled dAb-HOs. The mixture is annealed by heating to 80 $C and cooling back to room temperature by removing the mixture from the heat source. Next, 5 μg biotinylated cAb in 17 μL of PBST0.05+NaCl300 were added to and mixed with the annealed oligonucleotide mixture. The biotinylated reagents are thereafter coimmobilized on the MPs in a single step by adding 3.25M MPs in 10 μL PBST0.05+NaCl300 and immediately mixing by pipetting. The mixture was incubated for 90 min with end-over-end mixing at room temperature, followed by 3x washing by magnetic aggregation in 150 μL PBST0.1. The barcoded and functionalized MPs were stored at 4 $C until needed. In a second step, 100,000 of the prepared MPs were mixed with the HO-containing solution (e.g. dAb-HOs) diluted in PBST0.05+NaCl300 for 30 minutes. After pull-down of HOs, the fully-assembled CLAMPs were washed 3× in PBST0.01, and were stored until the time of the assay for up to a week at 4° C.
(133) Characterization of CLAMPs. To characterize CLAMPs, the immobilization of antibodies and oligos was confirmed by labeling using an anti-goat IgG conjugated with Alexa-Fluor 647 (AF647), or hybridization of a Cy5-labeled oligo (LO) targeting the HOs. The density of COs was estimated by fitting the ensemble fluorescence response of multicolour MPs using a multicolour fluorescence model, as described elsewhere (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018). To determine the expected assay background signal for a particular set of CLAMPs, the MPs were incubated with 1 μM Cy5-labeled DOs in PBST0.05+NaCl300 for one hour, followed by 3× magnetic washing in PBST0.05, and the residual signal was determined by cytometry.
(134) Antibody oligo conjugation, purification, and characterization. Anti-uPA monoclonal antibodies were conjugated to amine-modified HOs using a hydrazone chemistry (Solulink) followed by purification according to the manufacturer's protocol. Alternatively, monoclonal antibodies were conjugated to thiol-terminated HOs using a heterobifunctional amine/thiol-reactive crosslinker. 40 μL of 30 μM thiol-modified HOs were first reduced in 200 mM dithiothreitol (DTT) in PBST at 37° C. for one hour. The reduced oligos were (i) buffer exchanged into PBS pH 7.0 using a Zeba desalting spin-colum (7K MWCO, Thermo), (ii) activated for 10 min using 8 μL of 9 mM sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) dissolved in 80% PBS pH7.0 and 20% anhydrous dimethyl solfoxide, (iii) buffer exchanged again into PBS pH 7.0 to remove excess sulfo-SMCC, and (iv) a 1-10 μL fraction (depending on the desired) reacted with 10 μpL of 1 mg/mL antibodies. The reaction was left at room temperature for 1 hr and incubated overnight at 4 degrees C. thereafter. The conjugates were purified thereafter in two purification steps, an antibody and a DNA purification step, respectively.
(135) Antibody oligo conjugation, purification, and characterization. Anti-uPA monoclonal anti bodies were conjugated to amine-modified HOs using a hydrazone chemistry (Solulink) followed by purification according to the manufacturer's protocol. Alternatively, monoclonal antibodies were conjugated to thiol-terminated HOs using a heterobifunctional amine/thiol-reactive crosslinker. 40 μL of 30 μM thiol-modified HOs are first reduced in 200 mM DTT in PBST at 37° C. for one hour. The reduced oligos were (i) buffer exchanged into PBS pH 7.0 using a Zeba desalting spin-colum (7K MWCO, Thermo), (ii) activated for 10 min using 8 μL of 9 mM sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) dissolved in 80% PBS pH7.0 and 20% anhydrous dimethyl solfoxide, (iii) buffer exchanged again into PBS pH 7.0 to remove excess sulfo-SMCC, and (iv) a 1-10 μL fraction (depending on the desired valency) reacted with 10 μL of 1 mg/mL antibodies. The reaction was left at room temperature for 1 hr and incubated overnight at 4° C. thereafter. The conjugates were purified in two purification steps.
(136) Single-plex and multiplex CLAMP assay. Incubations were performed in a conical bottom 96-well plate at room temperature with horizontal shaking at 950 rpm. CLAMPS were mixed at roughly 80 MPs per barcode per μL and blocked with PBST0.05+NaCl150+0.5% BSA (PBST0.05+NaCl150+BSA0.5) for 30 min. A 25 μL aliquot of the blocked, multiplexed CLAMP mixture was added into each well and incubated with 25 μL containing the specified antigen(s) at 2× the specified concentrations in PBST0.05+NaCl150+BSA0.25, the incubation was performed for 3 hr at 950 rpm shaking. Magnetic aggregation and washing with 150 μL of PBST0.1 was repeated 4× in over a total of 30 min. Finally, detection-by-displacement is performed through the addition of 1 μM DO-Cy5 in PBST0.05+NaCl300+BSA0.25 and incubation for 1 hr with shaking, followed by 3× washing in PBST0.1.
(137) Conventional MSA. To screen the specificity and non-specific binding in conventional MSA format, MPs were barcoded and coupled with their respective biotinylated cAbs during synthesis as described above. MP mixtures were combined to a final concentration of 2,000 MPs per barcode per assay. Incubations were performed in a conical bottom 96-well plate at room temperature with horizontal shaking at 950 rpm. Prior to incubation with assay reagents, MPs were first blocked for one hour with 1% bovine serum albumin in 0.05% Tween-20 in PBS (PBST0.05). Incubation with antigens was conducted for 120 min at the specified concentrations. BMPs were incubated with the dAb cocktail for 60 min at 2 μg/mL, followed by incubation with sAbs for 45 mins at 4 μg/mL. SNRAg was calculated by subtracting the cAb-specific mean assay background (n=6) from the MFI signals and normalizing to the global standard-deviation (i.e. across all barcodes, n=210) of the assay background.
(138) Read-out and data analysis. MPs were read out using the FACS CANTO II cytometer by BD with blue (488 nm), red (633 nm), and violet (405 nm) lasers. In blue-laser flow cell, 530/30 and 585/42 band-pass filters were used for FAM and Cy3, respectively. In the red-laser flow cell, 660/20 band-pass filter was used for Cy5/AF647, respectively. The MPs were decoded using an automated algorithm implemented on MATLAB (Dagher, M. et al., Nature Nanotechnology, vol. 13, pp. 925-932, 2018). All data analysis was performed in MATLAB. Single-beads were distinguished from bead aggregates and other particulates by using forward and side-scatter intensities and gating was automated.
(139) While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations, including such departures from the present disclosure as come within known or customary practice within the art to and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
(140) The contents of all documents and references cited herein are hereby incorporated by reference in their entirety.