Patent Application
20190360025
Quantitative detection of protein biomarkers in biological fluids is essential for diagnosis, monitoring and personalized treatment of disease. Despite considerable progress in recent years the clinical use of validated proteomic biomarkers remains limited (1). The benchmark for affinity-based protein measurements is defined by the enzyme-linked immunosorbent assay (ELISA) where affinity ligands (e.g. antibodies) are used in a sandwich format to detect and quantify the protein of interest (2, 3). ELISA generally involves several steps starting with sample incubation where the target analyte is captured on a surface precoated with primary antibodies, followed by washing steps and recognition with a secondary antibody, which facilitates detection with a colorimetric, fluorescent or luminescent label. ELISA offers reasonable sensitivity but requires a large sample volume, has limited dynamic range and frequently suffers from false positives due to nonspecific binding (4, 5). These limitations interfere with the discovery and validation of novel biomarker candidates that have the potential to enable early diagnosis and regular molecular monitoring of disease.
Immunoassays combined with nucleic acid-based amplification and detection have facilitated new approaches and have extended the analytical sensitivity beyond that achievable with ELISA (6-9). One particularly promising approach is the proximity ligation assay (PLA) (10, 11). In PLA, pairs of affinity probes are individually conjugated to short, single-stranded DNA molecules to form proximity-probes that carry either a phosphorylated 5′-end or a 3′-hydroxyl group. When the probe pairs subsequently bind to their cognate target analyte in solution, the associated DNA strands are brought into close proximity and aligned by hybridization to a third bridging oligonucleotide. The free DNA ends are ligated, forming a new DNA sequence that is amplified and quantified using quantitative PCR (qPCR). It has previously been demonstrated that PLA provides femtomolar sensitivity and a wide dynamic range over 5 orders of magnitude, consuming as little as 1 μL of sample (12). One key benefit of PLA is that it addresses the widespread problem of cross-reactivity in antibody-based protein detection. Potential signals from cross-reactive antibodies are eliminated by tailoring the DNA sequences such that ligation only takes place when cognate proximity-probes are bound to the target analyte. This technology has been applied to detect more than 20 different biomarkers in clinical plasma samples using panels of seven multiplex PLA reactions (13, 14). Others have expanded the multiplexing capability even further (15).
One of the limitations of affinity-based immunoassays, including PLA, is the so called hook effect in which the signal decreases at high antigen concentrations resulting in incorrectly low signals or even false negatives (16). The hook effect becomes predominant when the analyte concentration exceeds the concentration of proximity-probes (>>1 nM) and it effectively determines the upper quantification limit (17). The proximity-probe concentration can potentially be increased, but must be carefully balanced against a deteriorating signal-to-noise ratio from random background ligation events when probes and the bridge oligo come together by chance. The hook effect can ordinarily be avoided by sample dilution; however, dilution alters binding equilibrium. This does not present a problem for high affinity interactions but it may prevent low affinity antibodies to bind to their target analytes, thereby limiting the effectiveness of PLA to high-affinity capture agents (18). While incompatibility with low-affinity antibodies is a major drawback for PLA, it is not restricted to this assay alone. The availability of high affinity antibodies or other capture reagents is a general limitation for all sandwich immunoassays because the sensitivity is ultimately determined by the quality of the reagents used (19).
Numerous approaches have been developed to improve PLA performance including concentration of minute amounts of analyte on a solid-phase prior to ligation (10, 18, 20), use of multivalent proximity-probes (21), addition of multiple affinity probes (22, 23), special design of asymmetric bridge oligos (17), inclusion of protecting oligonucleotides prehybridized to proximity-probes in order to reduce background ligation events (22, 24), and the use of novel amplification schemes (25-28). Some of the amplification schemes entail enzymatic manipulations where the ligation products are released from antibodies and converted into circles used for isothermal rolling circle amplification (26, 29-31). Many of these approaches have improved assay reproducibility, although precision still remains a challenge for adaptation into clinical diagnostics.
As such, there is a need for new assays for detecting analytes in a sample, particularly assays that can use lower affinity binding reagents.
Provided herein, among other things, is a circular proximity ligation assay in which proximity-probes are employed as bridges to covalently join two free oligonucleotides via a dual ligation event, resulting in the formation of a circle. The circles are then quantified by, e.g., qPCR. In some embodiments, the method may comprise: incubating a sample comprising a target analyte with: (i) a first conjugate comprising a binding agent and first splint oligonucleotide, and (ii) a second conjugate comprising a binding agent and a second splint oligonucleotide, under conditions suitable for binding of the binding agents of the first and second conjugates to the target analyte, to produce a product, incubating at least some of the product with: (i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides; and (ii) a ligase, to produce a reaction mix comprising covalently closed circular molecules, treating at least some of the reaction mix with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and, after the exonuclease treatment, quantifying the amount of covalently closed circular molecules produced in the ligation step. A kit for performing the method is also provided.
Relative to other methods, the addition of an extra oligonucleotide is believed to enhance specificity by decreasing the probability of random background ligation events. In addition, circle formation has selective advantages, as uncircularized DNA can be removed by a simple exonuclease treatment and it has streamlined the workflow by eliminating preamplification prior to qPCR. As a result, this assay is believed to be much more straightforward than not only prior assays, but most existing protein detection methods, and can be performed in a single reaction tube with a tiny sample volume (e.g., 2 uL). The assay format can utilize the same proximity-probes as used in traditional proximity ligation assays, which enables a direct performance comparison between the two methods. Moreover, it has been demonstrated that the enhanced specificity in this assay can be used to increase proximity-probe concentration while maintaining a low probability of background ligation events. This results in a better signal-to-noise ratio, improved assay performance and provides a path for compatibility with low affinity reagents without the need for pre-concentration on solid-phase.
Quantitative detection of protein biomarkers over a wide concentration range from minute amounts of blood is essential for clinical diagnostics. Proximity ligation assay combines antibody-oligo conjugates, enzymatic ligation and PCR amplification into a sensitive method for quantitative protein detection from small volumes. The method herein describes a streamlined and more stringent assay format that takes advantage of DNA circle formation to remove unwanted DNA molecules. Kinetic analysis of antibody-antigen interactions demonstrates that variation in assay performance between various biomarkers is an effect of antibody quality. It has been shown that this new assay format enables compatibility with low affinity reagents, a major limitation for most protein quantitation methods, while improving sensitivity and reproducibility.
These and other features of the present teachings are set forth herein.
The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Unless defined otherwise herein, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.
All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
Unless defined 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. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.
The term “sample” refers to a sample of organic material from a biological source and, as such, may comprise protein, nucleic acid, carbohydrates, small molecules, etc. A sample may be from an animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may include materials taken from a patient including, but not limited to cultures, blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, semen, needle aspirates, and the like. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention.
The term “specific binding” refers to the ability of a binding reagent to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).
The term “affinity” refers to the strength of binding between two entities. The affinity between two proteins (e.g., a capture agent and an analyte) when they are specifically bound together in a capture agent/analyte complex may be characterized by a KD (equilibrium dissociation constant) of less than 10−6M, less than 10−7 M, less than 10−8 M, less than 10−9 M, less than 10−10 M, less than 10−11 M, or less than about 10−12 M.
The term “epitope” as used herein is defined as a site in an antigen molecule that is bound by an antibody or non-antibody scaffold. An antigen can have one or more epitopes. In some cases, an epitope can be a linear sequence of at least five amino acids. In some cases, an antibody will only bind to an antigen molecule if the epitope has a specific three-dimensional structure.
A “subject” of diagnosis or treatment is a plant or animal, including a human. Non-human animals subject to diagnosis or treatment include, for example, livestock and pets.
As used herein, the term “incubating” refers to maintaining a sample and binding agent under conditions that are suitable for specific binding of the binding agent to molecules in the sample. Such conditions typically include a period of time, a temperature, and an appropriate binding buffer (e.g., PBS or the like). Such conditions are well known for antibodies, aptamers, and other binding agents.
The term “nucleotide” is intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, are functionalized as ethers, amines, or the likes.
The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotides, ribonucleotides or a combination thereof, and may be produced enzymatically or synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) and which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. A locked nucleic acid (LNA), often referred to as an inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose in the 3′-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid”, or “UNA”, is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G′ residue and a C′ residue, where these residues correspond to non-naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA.
The term “oligonucleotide” as used herein denotes a single-stranded multimer of nucleotides of from about 2 to 200 nucleotides, up to 500 nucleotides in length. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 30 to 150 nucleotides in length. Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides) or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides in length, for example. Oligonucleotides can have nucleotide analogs and, in some embodiments, at least some of the linkages between the nucleotides do not need to be phosphate. In some embodiments, an oligonucleotide can have a phosporothioate linkage, particularly at the 3′ end and/or 5′ end of the oligonucleotide.
The term “primer” as used herein refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be single-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. The primers herein are selected to be substantially complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
The term “hybridization” or “hybridizes” refers to a process in which a nucleic acid strand anneals to and forms a stable duplex, either a homoduplex or a heteroduplex, under normal hybridization conditions with a second complementary nucleic acid strand, and does not form a stable duplex with unrelated nucleic acid molecules under the same normal hybridization conditions. The formation of a duplex is accomplished by annealing two complementary nucleic acid strands in a hybridization reaction. The hybridization reaction can be made to be highly specific by adjustment of the hybridization conditions (often referred to as hybridization stringency) under which the hybridization reaction takes place, such that hybridization between two nucleic acid strands will not form a stable duplex, e.g., a duplex that retains a region of double-strandedness under normal stringency conditions, unless the two nucleic acid strands contain a certain number of nucleotides in specific sequences which are substantially or completely complementary. “Normal hybridization or normal stringency conditions” are readily determined for any given hybridization reaction. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. As used herein, the term “hybridizing” or “hybridization” refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The term “binding agents” refers to any member of a pair of molecules that specifically bind to each other. For example, an antibody (a type of capture agent) binds to an antigen. In this example, both the antibody and the antigen can be a binding agent. The complex that contains a pair of binding agents contains a capture agent (e.g., an antibody or a non-antibody scaffold) and a moiety that is not a capture agent, e.g., a protein, a metabolite, small molecule, carbohydrate, drug, etc.
The term “capture agent” refers to proteins that have a domain that specifically binds to other moieties. For example, antibodies and non-antibody binding scaffolds that have the ability to specifically bind to another moiety. Non-antibody binding proteins include aptamers and non-antibody proteins such as those described in Binz et al. (Curr Opin Biotechnol. 2005 16:459-69), Binz et al. (Nat. Biotechnol. 2005 23:1257-68), Forrer et al. (Chembiochem. 2004 5:183-9), Gronwall et al. (J. Biotechnol. 2009 140:254-69), Hosse et al. (Protein Sci. 2006 15:14-27) and Skerra et al. (Curr. Opin. Biotechnol. 2007 18:295-304), which are incorporated by reference herein.
The terms “antibody” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, polyclonal antibodies (which may or may not be affinity purified), monoclonal antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. This term encompasses by antibody fragments (e.g., Fab′, Fv, F(ab′)2 etc.) that retain specific binding to antigen. An antibody may be monovalent or bivalent.
A nucleic acid is considered to be “selectively hybridizable” to a nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). One example of high stringency conditions includes hybridization at about 42C in 50% formamide, 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1 ×SSC and 0.5% SDS at 42° C.
The term “duplex,” or “duplexed,” as used herein, describes two complementary polynucleotides that are base-paired, i.e., hybridized together.
The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent.
The term “ligating”, as used herein, refers to the enzymatically catalyzed joining of the terminal nucleotide at the 5′ end of a first nucleic acid molecule to the terminal nucleotide at the 3′ end of a second nucleic acid molecule.
The terms “plurality”, “set” and “population” are used interchangeably to refer to something that contains at least 2 members. In certain cases, a plurality may have at least 10, at least 100, at least 100, at least 10,000, at least 100,000, at least 106, at least 107, at least 108 or at least 109 or more members.
The term “strand” as used herein refers to a nucleic acid made up of nucleotides covalently linked together by covalent bonds, e.g., phosphodiester bonds.
The term “covalently linking” refers to the production of a covalent linkage between two separate molecules. Ligation produces a covalent linkage.
As used herein, the term “ligatably adjacent” in the context of two oligonucleotide sequences that are ligatably adjacent to one another, means that there are no intervening nucleotides between two oligonucleotides and they can be ligated to one another.
As used herein, the term “splint oligonucleotide,” as used herein, refers to an oligonucleotide that, when hybridized to two or more other polynucleotides, acts as a “splint” to position the 5′ and 3′ ends of two other polynucleotides next to one another so that they can be ligated together, as illustrated in
The terms “ligatable circle” and “ligatable circular complex” refer to a circular complex in which the various oligonucleotides are ligatably adjacent to one another in a circle, held together by splint oligonucleotides, as illustrated in
As used herein, the term “a set of probes that produces a ligatable circle only when the probes are hybridized to first and second splint oligonucleotides” comprises a pair of probes that (a) contains ends that hybridize to different splint oligonucleotides (i.e., a first splint oligonucleotide and a second splint oligonucleotide) and (b) hybridize to the first and second splint oligonucleotides to form a ligatable circle. In a complex comprising a first probe molecule and a second probe molecule, the 5′ end of the first probe molecule is ligatably adjacent to the 3′ end of the second probe molecule and the 3′ end of the first probe molecule is ligatably adjacent to the 5′ end of the second probe molecule. In this example, two ligation events are required for circularization. The term “only” in this phrase is intended to mean that the probes do not produce a ligatable circle if they are hybridized to only one of the first and second splint oligonucleotides (i.e., only one but not the other). A set of “probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides” is illustrated in
As used herein, the term “covalently closed circular molecule” refers to a strand that is in the form of a closed circle that has no free 3′ or 5′ ends.
The term “corresponds to” and grammatical equivalents, e.g., “corresponding”, as used herein refers to a specific relationship between the elements to which the term refers. For example, an RCA product corresponds to an analyte if the RCA product can be used to quantify that analyte in a sample.
As used herein, the term “rolling circle amplification” or “RCA” for short refers to an isothermal amplification that generates linear concatemerized copies of a circular nucleic acid template using a strand-displacing polymerase. RCA is well known in the molecular biology arts and is described in a variety of publications including, but not limited to Lizardi et al. (Nat. Genet. 1998 19:225-232), Schweitzer et al. (Proc. Natl. Acad. Sci. 2000 97:10113-10119), Wiltshire et al. (Clin. Chem. 2000 46:1990-1993) and Schweitzer et al. (Curr. Opin. Biotech 2001 12:21-27), which are incorporated by reference herein. This term includes linear RCA as well as exponential RCA (which can use random primers in some cases).
As used herein, the term “rolling circle amplification products” refers to the concatamerized products of a rolling circle amplification reaction.
As used herein, the term “counting” refers to determining the number of individual objects in a greater collection. “Counting” requires detecting separate signals from individual objects in a plurality (not a collective signal from the plurality of objects) and then determining how many objects there are in the plurality by counting the individual signals. In the context of the present method, “counting” is done by determining the number of individual signals in an array of signals.
Other definitions of terms may appear throughout the specification.
Provided herein is a method for sample analysis. In some embodiments, the method may comprise: (a) incubating a sample comprising a target analyte with: (i) a first conjugate comprising a binding agent and first splint oligonucleotide, and (ii) a second conjugate comprising a binding agent and a second splint oligonucleotide, under conditions suitable for binding of the binding agents of the first and second conjugates to the target analyte, to produce a product, (b) incubating at least some of the product with: (i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides; and (ii) a ligase, to produce a reaction mix comprising covalently closed circular molecules, (c) treating at least some of the reaction mix with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and, (d) after step (c), quantifying the amount of covalently closed circular molecules produced in the ligation step, i.e., the covalently closed circular molecules comprising the probes of step (b).
In some embodiments, the binding agents of the first and second conjugates are capture agents. In these embodiments, the method for sample analysis may comprise: (a) incubating a sample comprising an target analyte with: (i) a first conjugate comprising a capture agent and a first splint oligonucleotide, and (ii) a second conjugate comprising a capture agent and a second splint oligonucleotide, under conditions suitable for binding of the capture agents of the first and second conjugates to the target analyte, to produce a product,(b) incubating at least some of the product of step (a) with: (i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides; and (ii) a ligase, to produce a reaction mix comprising covalently closed circular molecules,(c) treating at least some of the reaction mix of step (b) with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and (d) after step (c), quantifying the amount of covalently closed circular molecules produced in step (b).
For example, in some embodiments, the proteins of the first and second conjugates may be polyclonal antibodies, e.g., affinity-selected polyclonal antibodies, i.e., antibodies that have been obtained from an animal that has been immunized with the target analyte, or portion thereof. In these embodiments, two portions of a single batch of polyclonal antibody may be linked to different splint oligonucleotides, as described below. In other embodiments, the binding agents of the first and second conjugates may be matched monoclonal antibodies, where matched monoclonal antibodies bind to different sites in the antigen. Alternative non-antibody capture agents include, but are not limited to, aptamers and non-antibody proteins such as those described in Binz et al. (Curr Opin Biotechnol. 2005 16:459-69), Binz et al. (Nat. Biotechnol. 2005 23:1257-68), Forrer et al. (Chembiochem. 2004 5:183-9), Gronwall et al. (J. Biotechnol. 2009 140:254-69), Hosse et al. (Protein Sci. 2006 15:14-27) and Skerra et al. (Curr. Opin. Biotechnol. 2007 18:295-304), which are incorporated by reference herein.
In some embodiments, the binding agents of the first and second conjugates are not capture agents. In these embodiments, the target analyte may be an antibody (which, in its native form, contains two binding sites and, as such, can bind to two other molecules). In these embodiments, the method for sample analysis may comprise:(a) incubating a sample comprising an antibody with: (i) a first conjugate comprising a non-capture agent moiety, e.g., a protein, and a first splint oligonucleotide, and (ii) a second conjugate comprising a non-capture agent moiety, e.g., a protein and a second splint oligonucleotide, under conditions suitable for binding of the non-capture agent moieties of the first and second conjugates to the antibody, to produce a product,(b) incubating at least some of the product of step (a) with: (i) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides; and (ii) a ligase, to produce a reaction mix comprising covalently closed circular molecules;(c) treating at least some of the reaction mix of step (b) with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and (d) after step (c), quantifying the amount of covalently closed circular molecules produced in step (b). In these embodiments, two portions of a single batch of an isolated non-capture agent moiety, e.g., a protein, may be linked to different splint oligonucleotides, and used in the method.
In some embodiments, the binding agent may have a low affinity for the target analyte and some embodiments may have a KD in the range of 10−5 M to 10−9 M, 10−5 M to 10−8 M or 10−5 M to 10−7 M. In these embodiments, the binding agent may be a polyclonal antibody.
If the binding agent in a conjugate is an antibody, the antibody may be a “natural” antibody in which the heavy and light chains have been naturally selected by the immune system of a multi-cellular organism. Such antibodies have a stereotypical “Y”-shaped molecule that consists of four polypeptide chains; two identical heavy chains and two identical light chains connected by disulfide bonds. In other cases, an antibody may be an antibody fragment, a single chain antibody, or a phage display antibody, for example. An antibody may be a monoclonal antibody or a polyclonal antibody. If the antibodies used in the method are monoclonal, the antibodies bind to different epitopes. If polyclonal antibodies are used, an animal may be immunized with a single analyte (e.g., a protein) or portion thereof and a polyclonal population of antibodies may be affinity purified from the animal using the analyte or portion thereof. The affinity purified antibodies can be split and a first portion of the antibody population can be conjugated to the first splint oligonucleotide and a second portion of the antibody population can be conjugated to the second splint oligonucleotide.
If the binding agent in a conjugate is a non-capture agent polypeptide, the polypeptide may be prepared, a first portion (e.g., an aliquot) of the polypeptide may be linked to the first splint oligonucleotide, a second portion (e.g., an aliquot) of the polypeptide may be linked to the second splint oligonucleotide, and the products may be used to quantify the amount of an antibody that binds to the protein. Antibodies, in their natural form, contain two binding sites and, as such, the method can be readily implemented using a single polypeptide.
In a conjugate, the binding agent and splint oligonucleotide may be linked non-covalently (e.g., via a streptavidin/biotin interaction) or covalently (e.g., via a cycloaddition reaction or alternative chemistry). The splint oligonucleotide and the binding agent can be linked together via a number of different methods, including those that use a maleimide or halogen-containing group, which are cysteine-reactive. Oligonucleotides may be linked to agents, e.g., antibodies, using any convenient method (see, e.g., Gong et al., Bioconjugate Chem. 2016 27: 217-225 and Kazane et al., Proc Natl Acad Sci 2012 109: 3731-3736). A variety of linkage methods are available. For example, the splint oligonucleotides may be linked to the capture agents directly using any suitable chemical moiety on the capture agent (e.g., a cysteine residue or via an engineered site). In other embodiments, the splint oligonucleotides may be linked to the capture agents directly or indirectly via a non-covalent interaction, e.g., via a biotin/streptavidin or an equivalent thereof, via an aptamer or secondary antibody, or via a protein-protein interaction such as a leucine-zipper tag interaction or the like. The binding agent and the oligonucleotide may be linked at a site that is proximal to or at the 5′ end of the oligonucleotide, proximal to or at the 3′ end of the oligonucleotide, or anywhere in-between.
In some embodiments, each end of the probes is perfectly complementary to at least 6 contiguous nucleotides (e.g., 6 to 50 or 8 to 30 contiguous nucleotides, e.g., 6, 7, 8 or more nucleotides) of the splint oligonucleotides, although the method is expected to work if there are a few mismatches that do not significantly interfere with hybridization or ligation. In some embodiments, the oligonucleotides may be linked to the agents by a linker that spaces the oligonucleotide from the agents, if needed.
In some embodiments, the initial steps of the method may comprise combining a volume of a liquid sample with the conjugates in a suitable binding buffer, incubating the mix for a period of time, and then adding the probes and ligase to the mix. This initial step may be done by adding a relatively small volume of sample (e.g., 1-5 uL) directly to larger volume of the first and second conjugates in a binding buffer (e.g., 10 uL to 50 uL of buffer). In these embodiments, the sample may be diluted by at least 2×, at least 3×, at least 4×, at least 5×, or at least 10×. In the ligation step, the ligase used can be a ligase that has a substrate preference for sealing nicks in double-stranded DNA molecules, rather than for ligating single stranded DNA molecules together, thereby lowering background ligations. After the covalently closed circular molecules have been produced, the method may comprise treating at least some of the reaction mix with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule. In some cases, the exonuclease may comprise both exonuclease I and exonuclease III, although other one or more other exonucleases, e.g., exonuclease T, exonuclease V, exonuclease VII, T5 exonuclease or T7 exonuclease could be used instead in some cases. In some embodiments, the exonuclease treatment step may be implemented by directly adding the one or more exonucleases to the ligation. The exonuclease treatment step should terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule. This step cannot be incorporated into other methods in which the binding agents are linked to oligonucleotides that are used primers because, as would be apparent, the exonuclease would destroy the primers. In the present method, the first and second splint oligonucleotides are solely used as splints for ligating the probes together. The method does not comprise using the splint oligonucleotides as primers to amplify the covalently closed circular molecules. After exonuclease treatment, the exonucleases can be inactivated by any convenient method. In some embodiments, the exonucleases can be inactivated by heat treatment, e.g., by heating the reaction to a temperature of at least 60° C. for an extended period of time, e.g., at least 10 minutes. The conditions used for inactivating the exonuclease may depend on the exonucleases used.
After the covalently closed circular molecules are made, the method comprises quantifying the amount of covalently closed circular molecules. This may be done by a variety of different ways, with or without a preamplification step, e.g., by quantitative PCR or endpoint PCR (e.g., digital PCR, which can be implemented on plates or in droplets), using microarrays, or by sequencing. In sequencing embodiment, one or more of the probes may be indexed, which allows the molecules to be counted after sequencing.
In some embodiments, the covalently closed circular molecules may be quantified by quantitative PCR (qPCR), which assay may employ a double-stranded DNA-specific dye or a hydrolysis probe (e.g., a TaqMan assay or the like). Such quantitative PCR assays typically use a first primer that hybridizes to the covalently closed circular molecule and a second primer that hybridizes to the complement of the covalently closed circular molecule, thereby allowing part of the sequence of the covalently closed circular molecule to be amplified by PCR. In some embodiments, the first and second primers used for quantitative PCR may target the ligation junctions in the covalently closed circular molecules, thereby making the qPCR more specific. In these embodiments, the first primer hybridizes to a sequence that encompasses one of the ligation junctions, and the second primer hybridizes to the complement of a sequence that encompasses the other of the ligation junctions. In these embodiments, the 3′ end of the first and second primers may extend one, two, three or, in some cases, four or more nucleotides past the ligation junction. In some embodiments, no pre-amplification of the covalently closed circular molecule is required prior to performing qPCR. Such pre-amplification methods typically used primers that bind to sites that are outside of the sites bound by the qPCR primers (such that the pre-amplification primers and the qPCR primers have a nested relationship). As such, the qPCR may be done without a pre-amplification step. Alternatively, the covalently closed circular molecules may be quantified by amplifying the covalently closed circular molecules by rolling circle amplification (RCA) to produce RCA products, and counting the RCA products. Such methods may be adapted from Larsson et al. (Nature Methods 2004 1: 227-232) among others. Several other methods for quantifying circular nucleic acid molecules are known or would be apparent to one of ordinary skill in the art.
In some embodiments, the initial steps of the method (up until the quantification step) can be implemented in a “single tube” format, in which reagent (e.g., enzyme, probes, etc.) are added directly to the prior reaction (after the reaction has been completed). In these embodiments, the binding, probe hybridization/ligation and exonuclease reactions (i.e., steps (a)-(c)) may be done in the same vessel. Specifically, the probe hybridization/ligation step (step (b)) may comprise adding reagents (the probes and ligase) to the vessel comprising the product of the binding step (step (a)), and the exonuclease treatment (step (c)) may comprises adding reagents (one or more exonucleases) to the vessel comprising the ligation product (of step (b)).
The method can be multiplexed using different sets of first and second conjugates for each target analyte. In these embodiments, the splint oligonucleotides of the conjugates that bind to a first analyte as well as the probes that hybridize to those splint oligonucleotides may be different from the splint oligonucleotides of the conjugates that bind to a second analyte as well as the probes that hybridize to those splint oligonucleotides. Since the different probe sets have different sequences, they can be assayed independently from one another by qPCR. As such, in some embodiments the sample may comprise a plurality of analytes, step (a) of the method may comprise incubating the sample with multiple pairs of said first and second conjugates, wherein each pair of conjugates binds to a different analyte; and step (d) comprises quantifying the number of covalently closed circular molecules corresponding to each analyte. In some embodiments, the analytes may be quantified in the same reaction, e.g., using multiplex qPCR. In other embodiments, the analytes may be quantified in different assays. In some embodiments, the analytes may be quantified in different assays, where each assay is, itself, a multiplex assay that contains an internal control (which can be used to, e.g., normalize the amount of the target analyte across samples). At least 2, at least 5 or at least 10 or more analytes may be quantified in a multiplexed manner using the present method.
The target analyte detected by the method may be any type of biological molecule to which antibodies can bind to different sites. Proteins are one type of target analyte. In some embodiments, both conjugates may bind to a single protein. In other embodiments, the analyte may be a complex of proteins. In these embodiments, the first and second binding agents may bind to different proteins in the complex.
In some embodiments, sample is a bodily fluid or a processed form thereof. Bodily fluids of interest include plasma, saliva and urine, although several other bodily fluids may be used in the present method. Bodily fluids include but are not limited to, amniotic fluid, aqueous humour, vitreous humour, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, and urine. In some embodiments, a sample may be obtained from a subject, e.g., a human, and it may be processed prior to use in the subject assay. For example, prior to analysis, the protein may be extracted from a tissue sample prior to initiating the present method. In particular embodiments, the sample may be a clinical sample, e.g., a sample collected from a patient.
The present method may have a sensitivity of at least 5 fM, 10 fM, 50 fM, 100 fM, 0.5 pM, 1pM, 5 pM, 10 pM, 50 pM, 100 pM, 0.5 nM, 1 nM, 5 nM, 10 nM, 50 nM or 100 nM depending on the target analyte.
Without wishing to be bound to any particular use, the present method has particular utility in analyzing blood plasma. Blood plasma can be obtained non-invasively and it contains a variety of different, low abundance proteins that are diagnostic, prognostic or theranostic (see, generally, Anderson et al., Molecular & Cellular Proteomics 2002 1: 845-867 and Anderson et al., Clinical Chemistry 2010 56: 177-185). As such, in some embodiments, the present method may be used to quantify any one or combination (e.g., 2, 3, 4, 5 or more) of the following proteins in plasma: acid phosphatase, IgG, alanine aminotransferase (ALT or SGPT), IgM, albumin, inhibin-A, aldolase, insulin, alkaline phosphatase (ALP), insulinlike growth factor-I (IGF-I), α-1-acid glycoprotein (orosomucoid), insulinlike growth factor-II (IGF-II), α-1-antitrypsin, IGFBP-1, α-2-antiplasmin, IGFBP-3, α-2-HS-glycoprotein, interleukin-2 receptor (IL-2R), α-2-macroglobulin, isocitric dehydrogenase, α-fetoprotein (tumor marker), K light chains, amylase, lactate dehydrogenase heart fraction (LDH-1), amylase, lactate dehydrogenase liver fraction (LLDH), ACE, lactoferrin, antithrombin III (ATIII), A light chains, apolipoprotein A1, lipase, apolipoprotein B, Lp(a), aspartate aminotransferase (AST or SGOT), lipoprotein-associated phospholipase A2 (LP-PLA2), 3-2 microglobulin, LH, 3-thromboglobulin, lysozyme, biotinidase, macrophage migration inhibitory factor (MIF) myeloperoxidase (MPO), cancer antigen 125 (CA 125), myoglobin, cancer antigen 15-3 (CA 15-3), osteocalcin, cancer antigen, human epididymis protein (HE4), parathyroid hormone, carcinoembryonic antigen (CEA), phosphohexose isomerase, ceruloplasmin, plasminogen, cholinesterase, plasminogen activator inhibitor (PAI), complement C1, prealbumin, complement C1 Inhibitor, NTproBNP, complement C1Q, procalcitonin (PCT), complement C3, prolactin, complement C4, properdin factor B, complement C5, prostatic acid phosphatase (PAP), CRP, prostatic specific antigen (PSA), creatine kinase-BB (CKBB), protein C, creatine kinase-MM (CKMM), protein S, cystatin C, pseudocholinesterase, erythropoietin, pyruvate kinase, factor IX antigen, renin, factor X, retinol binding protein (RBP), factor XIII, sex hormone-binding globulin, ferritin, soluble mesothelin-related peptide, fibrinogen, sorbital dehydrogenase (SDH), fibronectin, thyroglobulin, FSH, TSH, GGT, thyroxine binding globulin (TBG), haptoglobin, tissue plasminogen activator (T-PA), human chorionic gonadotropin (hCG), transferrin, hemopexin, transferrin receptor (TFR), her-2/neu protein, troponin T (TnT), human growth hormone (HGH), TnI (cardiac), human placental lactogen (HPL), trypsin, IgA, urokinase, IgD, Von Willebrand factor, IgE, nucleotidase, IgG subclass 4, ADAMTS13 activity and inhibitor, inhibin B (infertility), adenosine deaminase, IGFBP-2, adiponectin, intercellular adhesion molecule 1, a subunit of pituitary glycoprotein hormones, interferon-γ, α-galactosidase, interferon-α, EIA, α-N-acetylglucosaminidase, interleukin-1 receptor antagonist, amyloid 13-protein, interleukin-1 soluble receptor type II, angiotensinogen, interleukin-1α, anti-Mullerian hormone (AMH), interleukin-113, 3-glucuronidase, interleukin-2, 3-N-acetylglucosaminidase, interleukin-3, calprotectin, interleukin-4, cancer antigen 72-4, interleukin-5 cholecystokinin, interleukin-6, complement C2, interleukin-7, complement C4 binding protein, interleukin-8, complement C6, interleukin-9, complement C7 level, interleukin-10, complement C8 level, interleukin-11, complement C9 level, interleukin-12, corticosteroid binding globulin (transcortin), interleukin-13, CYFRA 21-1 (soluble cytokeratin fragment), interleukin-14, dopa decarboxylase, interleukin-15, elastase, interleukin-16, eosinophil cationic protein, interleukin-17, epidermal growth factor, interleukin-18, epidermal growth factor receptor (EGFR), kallikrein, factor II, leptin, factor V, leucine aminopeptidase, factor VII, mannose-binding lectin, factor VIII, neuron-specific enolase (NSE), factor XI, neurophysin, factor XII, pancreastatin, fibroblast growth factor (FGF2), pepsinogen I, gastric inhibitory polypeptide (GIP), pepsinogen II, Glial cell-derived neurotrophic factor (GDNF), glutathione peroxidase, proteasome activity, plasma-based Leumeta, granulocyte colony-stimulating factor, S-100B protein, granulocyte-macrophage colony-stimulating factor, soluble CD30, growth hormone binding protein, squamous cell carcinoma antigen, hemoglobin, thyrotropin releasing hormone (TRH), heparin cofactor II, transforming growth factor-131, hexosaminidase A and total hexosaminidase, tumor necrosis factor receptor 1, high molecular weight kininogen, tumor necrosis factor receptor 2, human growth hormone-releasing hormone (HGH-RH), tumor necrosis factor-α, IgG subclass 1, tumor necrosis factor-13, IgG subclass 2, vascular endothelial growth factor (VEGF), IgG subclass 3, and vitamin D-binding protein.
As would be apparent, the method may also be employed to identify a microbial (e.g., bacterial or viral) pathogen in a clinical sample, e.g., a cell surface protein or secreted protein. In these embodiments, the capture agents may target proteins or other moieties from a pathogen. If circles are detected, then the subject may be diagnosed as being infected by that pathogen. Microbes that might be identified using the present methods, compositions and kits include but are not limited to: viruses, yeast, Gram (+) bacteria, Gram (−) bacteria, bacteria in the family Enterobacteriaceae, bacteria in the genus Enterococcus, bacteria in the genus Staphylococcus, and bacteria in the genus Campylobacter, Escherichia coli (E. coli), E. coli of various strains such as, K12-MG1655, CFT073, O157:H7 EDL933, O157:H7 VT2-Sakai, etc., Streptococcus pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, coagulase-negative staphylococci, a plurality of Candida species including C. albicans, C. tropicalis, C. dubliniensis, C. viswanathii, C. parapsilosis, Klebsiella pneumoniae, a plurality of Mycobacterium species such as M. tuberculosis, M. bovis, M. bovis BCG, M. scrofulaceum, M. kansasii, M. chelonae, M. gordonae, M. ulcerans, M. genavense, M. xenoi, M. simiae, M. fortuitum, M. malmoense, M. celatum, M. haemophilum and M. africanum, Listeria species, Chlamydia species, Mycoplasma species, Salmonella species, Brucella species, Yersinia species, etc. Thus, the subject method enables identification of microbes to the level of the genus, species, sub-species, strain or variant of the microbe.
In some embodiments, the results of the method may be diagnostic (e.g., may provide a diagnosis of a disease or condition or the type or stage of a disease or condition, etc.), prognostic (e.g., indicating a clinical outcome, e.g., survival or death within a time frame) or theranostic (e.g., indicating which treatment would be the most effective). In some embodiments, the method may be used to analyze a group of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more analytes that are independently either present at a higher concentration or lower concentration relative to a control (e.g., an internal control), where collectively the identity of the analytes and their abundance correlate with a phenotype.
The method may be used to analyze a patient sample. In this embodiment, the method may comprise: (a) quantifying, using the above-described method, one or more analytes in a sample and (b) providing a report indicating a correlation with phenotype. This embodiment may further comprise making a diagnosis, prognosis or theranosis based on the report. The report may indicate the normal range of the analyte.
In some embodiments, the method may involve creating a report as described above (an electronic form of which may have been forwarded from a remote location) and forwarding the report to a doctor or other medical professional to determine whether a patient has a phenotype (e.g., cancer, etc.) or to identify a suitable therapy for the patient. The report may be used as a diagnostic to determine whether the subject has a disease or condition, e.g., a cancer. In certain embodiments, the method may be used to determine the stage or type of cancer, to identify metastasized cells, or to monitor a patient's response to a treatment, for example.
In any embodiment, report can be forwarded to a “remote location”, where “remote location,” means a location other than the location at which the image is examined. For example, a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one item is indicated as being “remote” from another, what is meant is that the two items can be in the same room but separated, or at least in different rooms or different buildings, and can be at least one mile, ten miles, or at least one hundred miles apart. “Communicating” information refers to transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network). “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. Examples of communicating media include radio or infra-red transmission channels as well as a network connection to another computer or networked device, and the internet or email transmissions and information recorded on websites and the like. In certain embodiments, the report may be analyzed by an MD or other qualified medical professional, and a report based on the results of the analysis of the image may be forwarded to the patient from which the sample was obtained.
Also provided by this disclosure are kits that contain reagents for practicing the subject methods, as described above. The subject kits contain one or more of any of the components described above. In some embodiments a kit may comprise: (i) a first splint oligonucleotide, (ii) a second splint oligonucleotide, (iii) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides, and (iv) one or more exonucleases. In these embodiments, the splint oligonucleotides may have reactive ends that can react with/bind to a binding agent, as discussed above. In some embodiments, the kit may also contain a binding agent and, in some embodiments, the splint oligonucleotides are conjugated to the binding agent in the kit. In some embodiments, a kit may further comprise a ligase and/or primers for performing quantitative PCR analysis, as described above. In some embodiments, the primers target the ligation junctions in the ligatable circles produced by hybridizing the splint oligonucleotides to the probes. In these embodiments, one primer may hybridize to a sequence that encompasses one of the ligation junctions, and the other primer hybridizes to the complement of a sequence that encompasses the other of the ligation junctions.
The various components of the kit may be present in separate containers or certain compatible components may be precombined into a single container, as desired.
In addition to the above-mentioned components, the subject kits may further include instructions for using the components of the kit to practice the subject methods, i.e., instructions for sample analysis. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.
Embodiment 1. A method for sample analysis comprising:
(a) incubating a sample comprising a target analyte with:
(b) incubating at least some of the product of step (a) with:
(c) treating at least some of the reaction mix of step (b) with an exonuclease to terminate the ligation and degrade any nucleic acid that is not a covalently closed circular molecule; and
(d) after step (c), quantifying the amount of covalently closed circular molecules produced in step (b).
Embodiment 2. The method of embodiment 1, wherein the binding agents of the first and second conjugates are capture agents.
Embodiment 3. The method of any prior embodiment, e.g., embodiment 1, wherein the binding agents of the first and second conjugates are affinity-selected polyclonal antibodies.
Embodiment 4. The method of any prior embodiment, e.g., embodiment 1, wherein the binding agents of the first and second conjugates are matched monoclonal antibodies.
Embodiment 5. The method of embodiment 1, wherein the binding agent of the first and second conjugates are not capture agents.
Embodiment 6. The method of any prior embodiment, wherein the first and second conjugates bind to the target analyte with a low affinity.
Embodiment 7. The method of any prior embodiment, wherein the quantifying step (d) is done by quantitative PCR, digital PCR, by hybridization to a microarray or by sequencing.
Embodiment 8. The method of embodiment 7, wherein the primers used for the quantitative PCR target the ligation junctions in the covalently closed circular molecules of (d).
Embodiment 9. The method of any prior embodiment, e.g., any of embodiments 1-2, wherein step (d) comprises amplifying the covalently closed circular molecules by rolling circle amplification (RCA) to produce RCA products.
Embodiment 10. The method of embodiment 9, wherein the method comprises counting the RCA products.
Embodiment 11. The method of any prior embodiment, wherein the reactions of steps (a)-(c) are done in the same vessel.
Embodiment 12. The method of embodiment 11, wherein step (b) comprises adding the set of probes and ligase to the vessel comprising the product of step (a), and step (c) comprises adding one or more exonucleases to the vessel comprising the ligation product of step (b).
Embodiment 13. The method of any of prior embodiment, wherein:
(i) the sample comprises a plurality of target analytes,
(ii) step (a) comprises incubating the sample with multiple pairs of said first and second conjugates, wherein each pair of conjugates binds to a different target analyte; and
(iii) step (d) comprises quantifying the number of covalently closed circular molecules corresponding to each target analyte.
Embodiment 14. The method of any prior embodiment, wherein the sample is from a bodily fluid.
Embodiment 15. The method of embodiment 11, wherein the bodily fluid is blood plasma, saliva or urine.
Embodiment 16. The method of any prior embodiment, wherein the target analyte is a protein.
Embodiment 17. A kit comprising:
(i) a first splint oligonucleotide, and
(ii) a second splint oligonucleotide;
(iii) a set of probes that produces a ligatable circle only when the probes are hybridized to the first and second splint oligonucleotides;
(iv) one or more exonucleases.
Embodiment 18. The kit of embodiment 17, further comprising a ligase.
Embodiment 19. The kit of embodiment 17 or 18, further comprising a binding agent to which the first and second splint oligonucleotides can be conjugated.
Embodiment 20. The kit of any of embodiments 17-19, further comprising primers for performing quantitative PCR (qPCR) analysis.
Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
Presented herein is a highly specific protein detection method, referred to as the circular proximity ligation assay (c-PLA), that outperforms traditional PLA in stringency, ease of use, reproducibility and compatibility with low affinity reagents. In c-PLA, two proximity-probes bind to an analyte providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity-probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity since the uncircularized DNA can be removed enzymatically. This method has been compared to traditional PLA on several biomarkers and demonstrate that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit-of-detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analysis using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit-of-detection is due to the variation in antibody affinity, and that c-PLA outperforms traditional PLA for low affinity antibodies. The lower background signal can be used to increase proximity-probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low affinity reagents in a homogeneous assay format. It is anticipated that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high affinity reagents are lacking.
c-PLA a highly specific, sensitive, and convenient assay for quantitative protein detection. The method builds on the existing PLA, but also benefits from the formation of circular DNA molecules. Proximity ligation assays combined with circle formation have traditionally been used for rolling circle amplification and have shown impressive results for in-situ detection and digital quantification. It has been demonstrated that the circle formation increases reproducibility by minimizing noise across the linear dynamic range. This is facilitated by the inclusion of exonuclease treatment, which hydrolyzes non-circular DNA. This design also simplifies the workflow as it eliminates the need for preamplification prior to qPCR quantification without any loss in assay performance. Both methods were compared using six commonly used biomarkers and demonstrate equal or better performance for circular PLA.
The kinetic analysis of the required antibody-antigen interactions, an essential precaution rarely taken during assay development for protein detection, demonstrates a direct correlation between kinetic constants and assay performance. The value of kinetic information to screen for suitable antibodies prior to proximity-probe conjugation and to optimize proximity-probe concentrations as well as assay procedures has established. This work thus indicates that the regular use of kinetic analysis will be highly beneficial in improving assay performance across a wide variety of analytes.
The advantages of c-PLA over t-PLA for low affinity interactions are aided by suppressed dissociation provided by longer DNA duplexes. It has been shown that the additional proofreading step facilitated by the extra oligo in circular PLA can be exploited to increase proximity-probe concentration without a concomitant increase in background ligation. This improves the signal-to-noise ratio and limit-of-detection compared to t-PLA and enables the use of low affinity reagents. This is a major benefit as low-affinity antibodies and variation in antibody specificity often contribute to systematic errors in research and clinical diagnostics (51). It has been also demonstrated that the benefits of c-PLA over t-PLA persist in complex media such as human plasma. This will facilitate new applications when high affinity reagents are not available, allowing not only new analytes to be detected, but also greater sensitivity for protein detection. These advantages are strengthened by the ease of application of the assay as well as its amenability to automation and a variety of quantification methods (digital, fluorescent, electrical, and more). The formation of circular DNA also supports detection using rolling circle amplification and other isothermal amplification methods that can be accomplished on low-cost point-of-care devices (52, 53). It is anticipated that an assay of this versatility and performance will help pave the way for much broader adoption of protein quantification into clinical and even resource-poor settings.
Circular proximity ligation assay workflow. The workflows for both the traditional proximity ligation assay (t-PLA) and the circular proximity ligation assay (c-PLA) are shown in
Assay characteristics. c-PLA was designed from existing traditional PLA probes that have been optimized for minimization of heteroduplex formation (12). This allowed a comparison of the two methods using the same set of proximity-probes. For c-PLA, the ligation conditions were relaxed to account for the requirement of two ligation events and to accommodate the efficiency of Ampligase at higher temperature. Ligation was consequently performed for 30 min at 45° C. instead of 15 min at 30° C. A comparable modification of t-PLA did not yield any improvements.
A direct comparison between traditional and circular PLA for six biomarkers are shown in
c-PLA was compared to traditional PLA for five additional biomarkers, glial cell line derived neurotrophic factor (GDNF), interleukin 6 (IL-6), macrophage migration inhibitory factor (MIF), tumor necrosis factor alpha (TNF-α), and insulin-like growth factor II (IGF-II) (
A notable observation is that the limit-of-detection for the analytes varies more than 5 orders of magnitude from low femtomolar to nanomolar concentration and that the dynamic range is confined by the limit-of-detection on the lower end and the proximity-probe concentration on the upper end where the hook effect starts to interfere. Consequently IGF-II, which exhibits the worst limit-of-detection (1 nM) among the analytes that were tested, also displays a restricted dynamic range of about 2 orders of magnitude. The assay performance for the same biomarkers in t-PLA generally follows the same trend as found for c-PLA, albeit with a slightly higher limit-of-detection. It is noted that previous proximity ligation studies of the same analytes have reported limit-of-detection using various definitions (12, 15). Here the limit-of-detection is strictly defined as the mean signal corresponding to a blank sample plus three standard deviations (40). Discrepancies in reported limit-of-detection can be affected by the use of alternative definitions, differences in protocol, or by batch-to-batch variation in proximity-probe conjugation, as affinity probes without conjugated oligonucleotides may bind to target analytes and interfere with the ability to facilitate ligation. One benefit of using the same batch of proximity-probes for both traditional and circular proximity ligation is that such effects are eliminated, making comparison between the two methods more straightforward. Furthermore, keeping the oligonucleotide sequences constant for all analytes eliminates any variation that may arise from sequence specificity, albeit this limits the possibility of multiplexing for the purpose of this investigation. The similarities in performance between the two methods indicate that the variation in limit-of-detection between analytes is inherently an effect of the quality of the proximity-probes and their ability to enable antibody-antigen interactions.
Kinetic analysis of affinity reagents. Knowledge about the kinetics of binding between antibodies and antigens is important for all immunoassays (3, 19). The relationship between kinetics and assay performance was explored. A previous report showed good correlation in traditional PLA between limit-of-detection and theoretical affinity, but did not determine equilibrium dissociation constants (KD) for the antibodies used (11). Furthermore, no analyses have been reported on either association rate constants, ka (on-rates), or dissociation rate constants, kd (off-rates), and their significance in proximity ligation assays. To address this, surface plasmon resonance (SPR) (41) was used to determine KD values as well as on- and off-rates for the antibody-antigen interactions investigated. Kinetic parameters are listed in Table 2 and SPR sensorgrams for individual analytes fitted to a 1:1 binding model are provided in
Because some of the off-rates determined by SPR were found to be below the sensitivity range of the Biacore instrument complementary analysis was performed using biolayer interferometry (BLI) (43). These measurements corroborated the SPR results; off-rates for GDNF and IL-6 were also beyond the sensitivity of the Fortebio Octet RED instrument. Kinetic data for BLI measurements, binding curves for individual analytes fitted to a 1:1 binding model, and an isoaffinity chart for all analytes are provided in
All proximity-probes described in this work originate from affinity-purified polyclonal antibodies. These antibodies are mixtures derived from different cell lineages producing antibodies that recognize distinct epitopes of the antigen each with their own individual kinetic characteristics. Due to this heterogeneity, kinetic properties of polyclonal antibody are inherently difficult to characterize with precision and the derived kinetic constants and affinities are considered an average for the different subpopulations existing within a batch of antisera. One prospective effect is that extended incubation time between sample and proximity-probes will allow continued exchanges that progress towards higher affinity interactions.
Using kinetics data to improve c-PLA performance. The isoaffinity graph in
The detailed information obtained by kinetic analysis is beneficial for assay development as it suggests modifications that may result in improved signals. The kinetic constants were used to predict the number of complexes formed at equilibrium during sample incubation, and the new equilibrium that is established when the volume is increased by addition of the ligation mixture. Details are provided in the section named “Modeling of antibody-antigen-antibody complex formation at equilibrium” and
The large volume addition associated with ligation was originally introduced to minimize background ligation in t-PLA (12). Background ligation events in t-PLA occur when affinity probes and bridge oligo are randomly brought into close proximity. For c-PLA, the likelihood of background ligation is lower as this complex requires an additional connector oligo and two ligation events before producing a detectable signal. This results in less background and a better signal-to-noise ratio. Additional modeling studies provided in the
Stringency of c-PLA improves signal-to-background ratio and limit-of-detection. Based on simulations described above the impact of increased proximity-probe concentration for both traditional and circular PLA was tested. The TNF-α assay was chosen as it displayed the highest KD and kd, providing a good example of low affinity reagents with fast off-rates. Both assay formats were performed with the standard affinity probe concentration (1×=0.25 nM) as well as a ten-fold increase. The results are shown in
c-PLA effectively detects proteins in human plasma. The results described thus far were all performed in buffer solutions as the goal was investigate the relationship between affinity and assay performance. Affinity analysis using SPR is not amenable to complex mixtures like plasma and although biolayer interferometry is less sensitive to matrix effects (43), both methods require pure ligands for accurate determination of kinetic constants. The correlation between kinetic constants and assay performance is established in
It has been previously shown that multiplexed t-PLA enables quantitation of biomarkers in plasma samples (12-14). To determine the performance of c-PLA in a complex matrix, tested two analytes were tested: VEGF, as an example of a high affinity interaction (low KD and slow off-rates), and TNF-α, as an example of a low affinity interaction (high KD and fast off-rates). VEGF was effectively detected in human plasma by c-PLA down to physiological single-digit pM concentrations (12, 49) (
The advantages in assay performance of c-PLA over t-PLA for TNF-α (a low affinity interaction) also persist in human plasma (
Materials. Affinity-purified polyclonal antibodies and antigen targets were from R&D Systems. Product numbers are provided in the section named “Antibodies and Antigens”. All oligonucleotides were from Integrated DNA Technologies. Sequences are listed in the section named “DNA sequences” and were kept the same for all analytes to allow for an unbiased comparison. Oligonucleotides used for proximity-probe conjugation were HPLC purified and designed to minimize probe-probe heteroduplex formation. All other reagents were from Sigma-Aldrich unless otherwise indicated.
Proximity-probe conjugation. Antibody-oligonucleotide conjugation was performed using hydrazone chemistry (Solulink) followed by purification according to the manufacturer's protocol (
Proximity-probe target incubation. 2 μL of sample (diluted in either 1× PBS with 0.1% BSA or neat plasma from chicken or human (Sigma)) was added to 2 μL of proximity-probe mix and incubated for 2 hours at 37° C. to establish complex formation between the target analyte and antibodies in proximity-probes. The combined 4 μL incubation mixture contained a final concentration of 250 pM for each proximity-probe and was supplemented with 0.35 mg/mL polyadenylic acid potassium salt; 1% BSA; 0.1% Triton X-100; 0.05% IgG; 0.01% aprotinin; 1 mM phenylmethanesulfonyl fluoride; and 4 mM EDTA (Thermo Fisher Scientific) in 0.375× PBS.
Ligation step for circular PLA. 120 μL of ligation mixture was added to each 4 μL sample and incubated for 30 min at 45° C. The ligation mixture contained 100 nM each of the two circle-forming connector oligos; 0.025 U/μL Ampligase (Epicentre); 0.5 mM NAD; 1 mM DTT (Sigma-Aldrich); 0.01% Triton X-100; and 0.01% BSA in 20 mM Tris pH 8.4, 10 mM MgCl2, and 50 mM KCl, (Thermo Fisher Scientific). Ligation was terminated by adding 10 μL of exonuclease mixture and incubated for 30 min at 37° C. followed by heat inactivation of exonuclease enzymes for 20 min at 80° C. Exonuclease mixture contained 2 U/μL exonuclease I (New England Biolabs); and 2 U/μL exonuclease III (New England Biolabs) in 1× NEBuffer 1 containing 10 mM Bis-Tris-Propane-HCl pH 7.0; 10 mM MgCl2, and 1 mM DTT (New England Biolabs).
Ligation step for traditional PLA. 120 μL of ligation mixture was added to each 4 μL sample and incubated for 15 min at 30° C. The ligation mixture contained 100 nM t-PLA bridge oligo; 0.025 U/μL Ampligase (Epicentre); 0.25 mM NAD; 10 mM DTT; 0.02% Triton X-100; and 0.01% BSA in 20 mM Tris pH 8.4, 1.5 mM MgCl2, and 50 mM KCl, (Thermo Fisher Scientific). Ligation was terminated by adding 2 μL of stop ligation mixture followed by a 5 min incubation at room temperature. The stop ligation mixture contained 0.125 U/μL Uracil-DNA Excision Mix (Epicentre) in 20 mM Tris pH 8.4, 50 mM KCl (Thermo Fisher Scientific).
Pre-amplification step for traditional PLA. 25 μL of the terminated ligation mixture was added to 25 μL preamplification mixture and cycled with the following conditions: 95° C. for 3 min (1 cycle); 95° C. for 30 s and 60° C. for 4 min (13 cycles) and a final hold at 4° C. The preamplification mixture contained 20 nM pre-amplification primers; 0.06 U/μL Platinum Taq DNA Polymerase (Thermo Fisher Scientific); and 1.6 mM dNTP Mix (Agilent Technologies) in 20 mM Tris pH 8.4, 6 mM MgCl2, and 50 mM KCl, (Thermo Fisher Scientific). Following pre-amplification the product was diluted 10-fold in TE buffer and stored at 4° C. until qPCR quantification.
qPCR quantification and data analysis. 2 μL of either exonuclease-treated ligation product (for circular PLA) or diluted preamplification product (for traditional PLA) was added to qPCR master mix to a final volume of 10 μL containing 400 nM primers and 1× Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Samples were quantified using real time qPCR (ABI 7900 HT) with the following thermal cycling conditions: 95° C. for 10 min (1 cycle); 95° C. for 15 s and 60° C. for 60 s (40 cycles). Ct values were converted to an estimated number of ligated molecules using the formula 10−0.301×Ct+11.439 as previously described (13). Experiments were performed with three biological replicates that were quantified in triplicate qPCR experiments generating nine data points per concentration analyzed. Limit-of-detection was derived relative to the blank sample where the dose-response curve and the dashed line (mean signal of a blank sample+3 standard deviations) intersects (40). Statistical analysis was performed using bootstrapping (random sampling with replacement on the independent measurements of biological replicates 1000 times).
Kinetic analysis using SPR. SPR analysis was performed at 25° C. using a Biacore T200 instrument and a Biotin CAPture Kit (GE Healthcare,
Kinetic analysis using BLI. BLI analysis was performed on a Fortebio Octet RED instrument using protein G biosensors (Fortebio,
Antibodies and Antigens. Affinity reagents used were from R&D Systems and had the following product numbers for antibodies & antigens: GDNF: AF-212-NA & 212-GD-010; IL-6 AF-206-NA & 206-IL-010; VEGF: AF-293-NA & 293-VE-010; TNF-α: AF-210-NA & 210-TA-005; IGF-II: AF-292-NA & 292-G2-050; MIF: AF-289-PB & 289-MF-002.
DNA Sequences.
Modeling of Antibody-Antigen-Antibody Complex Formation at Equilibrium.
Using parameters derived from kinetic analysis the formation of antibody-antigen-antibody (Ab-Ag-Ab) complexes was modeled at various conditions used in proximity ligation assay. This information is valuable to gain a better understanding of the assay and to support assay development for equilibrium and pre-equilibrium conditions, which often exist in cases of low analyte concentration and high affinity interactions. Using the equations described in
It is noted that these calculations only consider the formation of complexes and do not take into account whether it will result in a ligation event. For example ligation events will not be produced by complexes formed between analogous proximity-probes or when one of the antibodies in the complex lacks a conjugated oligo. Free oligonucleotides that may contribute to background ligation or stabilize formed complexes are also not considered in the calculations.
The following tables provide supporting data for graphs shown in the figures.
It will also be recognized by those skilled in the art that, while the invention has been described above in terms of preferred embodiments, it is not limited thereto. Various features and aspects of the above described invention may be used individually or jointly. Further, although the invention has been described in the context of its implementation in a particular environment, and for particular applications those skilled in the art will recognize that its usefulness is not limited thereto and that the present invention can be beneficially utilized in any number of environments and implementations where it is desirable to examine analytes. Accordingly, the claims set forth below should be construed in view of the full breadth and spirit of the invention as disclosed herein.
This application claims the benefit of U.S. provisional application Ser. No. 62/465,320, filed on Mar. 1, 2017, which application is incorporated by reference herein.
This invention was made with Government support under contract HG000205 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/18859 | 2/20/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62465320 | Mar 2017 | US |
