Protein detection via nanoreporters

0001Related application This application was filed on October 13, 2009, US Provisional Patent Application 61 / 251,192, April 16, 2010, US Provisional Patent Application 61 / 325,224, and April 22, 2010. US Provisional Patent Application 61 / 326,787 claims the interests of the above provisional patent application, the entire contents of which are incorporated herein by reference in their entirety.

0002Field of invention The present invention generally relates to the fields of protein detection, quantification, identification, and multiplex analysis using molecular biology tools for making unique nanoreporter constructs and methods for using them. ..

0003Background of the invention With the recent completion of the analysis of the human genome, much attention is now shifting to the field of proteomics, where gene products (proteins), their variants, interacting partners, and their regulation and processing. The dynamics of the research is a priority item of research. Such studies are essential to understanding, for example, the underlying mechanisms of hereditary and environmentally-induced disorders, or the effects of drug treatment, as well as the underlying basis for further clinical and diagnostic analysis. There is a possibility that Important for these studies is the ability to qualitatively determine specific variants of the whole protein (eg, splicing variants, point mutations, post-translational modification versions, and environment / treatment-induced modifications), and their quantification. The ability to see qualitative adjustments. Moreover, it is becoming increasingly important to perform these analyzes from multiple biological liquids / extracts, not just one. The additional challenges inherent in protein samples limit the methods of multiplexed protein measurement technology.

<p num="0004"> However, the measurement of proteins in biological liquids is difficult due to their unique properties. Therefore, there is an urgent need for a rapid, sensitive, reproducible and accurate analytical approach for the analysis of proteins and their variants.</p><p num="0005"> Proteins present in various biological fluids and / or extracts can be evaluated both qualitatively and quantitatively in order to analyze the proteins of interest from and within their natural environment. An assay is needed.</p>

<p num="0006"> Abstract of the invention The present invention provides methods and compositions for protein analysis. In some embodiments, the invention provides methods and compositions for the detection and / or quantification of proteins in a sample. In some embodiments, the invention is a capture of (a) (i) at least one protein, (ii) a first protein probe specific for the first region of the at least one protein described above. A first protein probe containing a region, (iii) a second protein probe specific for a second region of at least one protein, comprising a nanoreporter containing a plurality of different detectable labels. Steps of supplying a protein probe and (iv) a matrix to which a portion of the first protein probe that can bind to a capture region (portion) is attached; (b) at least one protein, a first protein probe, a second In the step of forming at least one complex containing the protein probe and the above portion, at least one protein is bound to the first protein probe and the second protein probe, and the above portion is the first. Detecting at least a portion of a complex or complex individually by a method that is bound to a capture probe in a protein probe; and (c) individually counting the presence of one or more molecules of the nanoreporter. Provided is a method of determining the concentration of at least one protein in a sample, comprising the step, wherein the presence of one or more molecules indicates the concentration of protein in the sample. In some embodiments, the step of detecting individually further comprises detecting a digital signal.</p><p num="0007"> Moiety refers to an entity, also known as an entity. The components of the invention are operably linked to the matrix and bind to the capture region of the first protein probe. Ingredients act on the matrix by physical or chemical bonds, including, but not limited to, covalent, non-covalent, electronic, flexed, aromatic, metal, hydrogen, ionic, or van der Waals forces. It is connected as possible. Ingredients are expressed through either the physical or chemical binding described herein, a receptor-ligand interaction, a hybridization event between two oligonucleotides, or an interaction between an oligonucleotide and a polypeptide. It binds to the capture region of the first protein probe. For example, a biotin-containing capture region binds to a streptavidin-containing component to form a strong non-covalent bond, and a matrix to which streptavidin is attached binds the matrix to the capture region of the first protein probe. Allows you to (see Figure 1). All known receptor-ligand interactions are contemplated, but interactions with a dissociation constant (Kd) between 0.1 fM and 1000 nM are preferred. Hybridization events occur between oligonucleotides that have complementary sequences. However, perfect or perfect complementarity is not required. The present invention includes hybridization events between oligonucleotides having 50%, 60%, 70%, 80%, 90%, 95%, 100%, and any percentage of complementarity between them. In addition, the association of the aptamer with the first protein probe provides a non-limiting example of the preferred interaction between the oligonucleotide and the polypeptide.</p><p num="0008"> In some embodiments, the present invention is a method for determining the concentration of a plurality of target proteins by forming a plurality of complexes, wherein each complex is (i) at least one target protein. (ii) a first protein probe specific for the first region of at least one protein, the first protein probe containing a capture region, (iii) specific for the second region of at least one protein. Second protein probe, the second protein probe containing nanoreporters containing multiple different detectable labels, (iv) a component attached to the matrix, in the capture region of the first protein probe. Provided are methods that include components that can bind and each second protein probe contains a different nanoreporter region. In some embodiments, each nanoreporter in multiple complexes has a detectable signal that distinguishes it from other nanoreporters in the population. In some embodiments, the dissociation constants of the first and second protein probes are about 1.00 × 10^-10~ About 1.00 × 10^-08Is. In some embodiments, the concentration of two or more target proteins is determined. In some embodiments, 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800, 900 Concentrations of different target proteins of 1, 1000, or more than 1000 are determined. In some embodiments, the concentration of at least 972 different target proteins is determined.</p><p num="0009"> In some embodiments, the matrix is selected from the group consisting of beads and arrays. In some embodiments, the matrix is beads. In some embodiments where multiple target proteins are analyzed, the matrix is beads and each component of each complex of the multiple complexes is attached to different beads. In some embodiments, the matrix is a surface. In some embodiments where multiple target proteins are analyzed, the matrix is a surface and each component of each complex of the multiple complexes is attached to different locations on the surface.</p><p num="0010"> In some embodiments, the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids.</p><p num="0011"> In some embodiments, the nanoreporter comprises a single-stranded nucleic acid backbone, the backbones comprising a plurality of labeled attachment regions that are linearly combined and covalently linked together, and each labeled attachment region is detectable. It hybridizes to the complementary polynucleotide sequence to which it is labeled. In some embodiments, the nanoreporter is attached to a second probe through hybridization to a linker oligo. In some embodiments, the nanoreporter hybridizes to the linker oligo at a temperature of about 32 degrees Celsius (° C) to about 40 degrees Celsius. In some embodiments, the nanoreporter hybridizes to the linker oligo at a temperature of about 37 ° C. In some embodiments, the nanoreporter comprises a moiety that is complementary to the linker oligo. In some embodiments, the complementary region is about 15 to about 20 bases.</p><p num="0012"> In some embodiments, the invention is a first protein probe specific for (a) (i) at least one protein, (ii) a first region of at least one protein, the first. A first protein probe attached to a capture region or first matrix, (iii) a second protein probe specific for a second region of at least one protein, comprising a signal oligo. When the protein probe and (iv) the first probe are attached to the first capture region: the step of supplying a second matrix to which components capable of binding to the capture region of the first protein probe are attached; (b) A step of forming at least the first complex comprising at least one protein, a first protein probe, and a second protein probe, wherein the at least one protein is the first protein probe and the second. If it is attached to a protein probe and the first probe is attached to the first capture region, then the capture probe is attached to a component in the second matrix; Steps of liberation from the complex; (d) (1) Steps of forming a second complex containing at least one oligoprobe containing at least a signal oligo, (2) a signal oligo-specific region, and a region containing a nanoreporter. A second complex or method comprising counting the presence of one or more molecules of the nanoreporter individually, wherein the nanoreporter comprises a plurality of different detectable labels; At least one protein in a sample that comprises the step of individually detecting at least a portion of the second complex, wherein the presence of the second one or more molecules indicates the concentration of the protein in the sample. Provide a method for determining the concentration of protein. In some embodiments, the step of detecting individually further comprises detecting a digital signal.</p><p num="0013"> In some embodiments, the first matrix is a bead or array. Preferably, the first matrix is beads. In other embodiments, the second matrix is a bead or array.</p><p num="0014"> In some embodiments, the signal oligo is attached to the second capture region. In some embodiments, the release step of the signal oligo further comprises capturing the signal molecule directly or indirectly into a third matrix.</p><p num="0015"> In some embodiments, the nanoreporter further comprises a constant region, the constant region comprising a plurality of repeating nucleotide sequences. In some embodiments, the constant region is bound to a second component in the third matrix, and the second component may bind the constant region.</p><p num="0016"> In some embodiments, the present invention is a method for determining the concentration of a plurality of target proteins by forming a plurality of complexes, wherein each complex is (i) at least one target protein. (ii) a first protein probe specific for the first region of at least one protein, the first protein probe attached to the capture region or first matrix, (iii) at least one protein If the second protein probe specific to the second region of the protein contains a second protein probe containing a signal molecule and the first probe is attached to the first capture region, then the capture probe is the second. A method is provided in which the components in the two matrices are bound and each second protein probe in each of the multiple complexes contains a different signal oligo. In some embodiments, the concentration of two or more target proteins is determined. In some embodiments, 2, 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800 Concentrations of different target proteins are determined: 1, 900, 1000, or more than 1000. In some embodiments, the concentration of at least 972 different target proteins is determined.</p><p num="0017"> In some embodiments, the first matrix of the complex of the multiple complexes is a bead, which comprises a plurality of identical first protein probes. The term identical shall describe a protein probe that has the same sequence and either contains or is attached to the same capture region.</p><p num="0018"> In some embodiments, the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids.</p><p num="0019"> In some embodiments, the nanoreporter comprises a single-stranded nucleic acid backbone, the backbones comprising a plurality of labeled attachment regions that are linearly combined and covalently linked together, and each labeled attachment region is detectable. It hybridizes to the complementary polynucleotide sequence to which it is labeled.</p><p num="0020"> In some embodiments, the invention is a first protein probe specific for (a) (i) at least one protein, (ii) a first region of at least one protein, the first. A first protein probe attached to the oligo, and (iii) a second protein probe specific for the second region of at least one protein, which is attached to the second oligo. Steps of supplying a protein probe; (b) A step of forming a first complex comprising at least one protein, a first protein probe, and a second protein probe, wherein the at least one protein is the first. Steps bound to a protein probe and a second protein probe; (c) ligating the first and second oligos to form a signal oligo; (d) (1) first signal oligo, (2) A step of forming a second complex comprising a signal oligo-specific region and at least one oligo probe comprising a region containing a nanoreporter, wherein the nanoreporter contains a plurality of different detectable labels. Steps; as well as (e) the step of individually detecting at least a portion of the second complex or the second complex by a method comprising counting the presence of one or more molecules of the nanoreporter individually. Provided are methods for determining the concentration of at least one protein in a sample, including steps, where the presence of one or more molecules indicates the concentration of protein in the sample. In some embodiments, the step of detecting individually further comprises detecting a digital signal.</p><p num="0021"> In some embodiments, the signal oligo is released from the first complex. In some embodiments, the signal oligo comprises a capture region. In some embodiments, the step of releasing the signal oligo further comprises capturing the signal oligo directly or indirectly into the matrix.</p><p num="0022"> In some embodiments, the present invention is a method for determining the concentration of a plurality of target proteins by forming a plurality of complexes, wherein each complex is (i) at least one target protein. (ii) a first protein probe specific for the first region of at least one protein, the first protein probe attached to the first oligo, (iii) the second of at least one protein. A second protein probe specific to the region, which contains a second protein probe attached to the second oligo, and the ligation of the first oligo and the second oligo forms a signal oligo, Provided is a method in which each complex in a plurality of complexes comprises a different signal oligo. In some embodiments, the concentration of two or more target proteins is determined. In some embodiments, 2, 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800 Concentrations of different target proteins are determined: 1, 900, 1000, or more than 1000. In some embodiments, the concentration of at least 972 different target proteins is determined.</p><p num="0023"> In some embodiments, the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids.</p><p num="0024"> In some embodiments, the nanoreporter comprises a single-stranded nucleic acid backbone, the backbones comprising a plurality of labeled attachment regions that are linearly combined and covalently linked together, and each labeled attachment region is detectable. It hybridizes to the complementary polynucleotide sequence to which it is labeled.</p><p num="0025"> In some embodiments, the invention is a population of uniquely labeled protein probes, each probe containing i) a target-specific region; and ii) a nanoreporter containing a plurality of different detectable molecules. Provided is a population comprising a region comprising, each nanoreporter in each protein probe having a detectable signal that distinguishes it from other nanoreporters in the population. In some embodiments, the target-specific region is selected from the group consisting of antibodies, peptides, aptamers, and peptoids.</p><p num="0026"> In some embodiments, the nanoreporter comprises a single-stranded nucleic acid backbone, the backbones comprising a plurality of labeled attachment regions that are linearly combined and covalently linked together, and each labeled attachment region is detectable. It hybridizes to the complementary polynucleotide sequence to which it is labeled. In some embodiments, the dissociation constant of the target-specific region is about 1.00 × 10^-10~ About 1.00 × 10^-8Is.</p><p num="0027"> In some embodiments, the nanoreporter is attached to the protein probe through hybridization to a linker oligo. In some embodiments, the nanoreporter hybridizes to the linker oligo at a temperature of about 32 ° C to about 40 ° C. In some embodiments, the nanoreporter hybridizes to the linker oligo at a temperature of about 37 ° C. In some embodiments, the nanoreporter comprises a moiety that is complementary to the linker oligo. In some embodiments, the complementary region is about 15 to about 20 bases.</p><p num="0028"> Incorporation by reference All publications and patent applications referred to herein are, to the extent that each individual publication or patent application is specifically and individually indicated and incorporated by reference, herein by reference. Incorporated. For example, the present invention provides the following items. (Item 1) A method for determining the concentration of at least one protein in a sample, (a) (i) At least one protein, (ii) A first protein probe that is specific for a first region of the at least one protein, wherein the first protein probe comprises a capture region. (iii) A second protein probe specific for a second region of the at least one protein, wherein the second protein probe comprises a nanoreporter comprising a plurality of different detectable labels. Protein probe, and (iv) A matrix in which a component capable of binding to the capture region in the first protein probe is attached to the matrix. The process of supplying; (b) A step of forming at least one complex containing the at least one protein, the first protein probe, the second protein probe, and the component, wherein the at least one protein is the first. And the component is bound to the capture probe in the first protein probe, which is bound to the protein probe and the second protein probe; (c) A step of individually detecting the complex or at least a portion of the complex by a method comprising individually counting the presence of one or more molecules of the nanoreporter, the one or more. The presence of the molecule indicates the concentration of the protein in the sample, step Including methods. (Item 2) The method according to item 1, wherein the individually detecting step further comprises a step of detecting a digital signal. (Item 3) Each complex further comprises the step of determining the concentration of the plurality of target proteins by a method comprising forming the plurality of complexes. (i) At least one target protein, (ii) A first protein probe that is specific for a first region of the at least one protein, wherein the first protein probe comprises a capture region. (iii) A second protein probe specific for a second region of the at least one protein, wherein the second protein probe comprises a nanoreporter comprising a plurality of different detectable labels. Protein probe, as well (iv) A component attached to the matrix, the component capable of binding to the capture region in the first protein probe. The method of item 1, wherein each second protein probe comprises a different nanoreporter region. (Item 4) The method of item 3, wherein each nanoreporter in the plurality of complexes has a detectable signal that distinguishes it from other nanoreporters in the population. (Item 5) The dissociation constants of the first protein probe and the second protein probe are about 1.00 × 10.^-15~ About 1.00 × 10^-08The method described in item 3. (Item 6) The method of item 3, wherein the concentration of two or more target proteins is determined. (Item 7) 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000, or 1000 Item 6. The method of item 6, wherein the concentration of more than one different target protein is determined. (Item 8) The method of item 3, wherein the concentration of up to 2000 different target proteins is determined. (Item 9) The method of item 3, wherein the concentration of up to 980 different target proteins is determined. (Item 10) The method of item 1, wherein the matrix is selected from the group consisting of beads and arrays. (Item 11) The method according to item 3, wherein the matrix is beads, and each component in each complex of the plurality of complexes is attached to different beads. (Item 12) The method according to item 3, wherein the matrix is a surface, and each component in each complex of the plurality of complexes is attached to different positions on the surface. (Item 13) The method of item 1, wherein the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids. (Item 14) The nanoreporter comprises a single-stranded nucleic acid backbone, the backbone comprising a plurality of labeled attachment regions that are linearly combined and covalently attached, and each labeled attachment region hybridizes to a complementary polynucleotide sequence. The method of item 1, wherein the detectable label is attached to the complementary polynucleotide sequence. (Item 15) The method of item 1, wherein the nanoreporter is attached to the second probe through hybridization to a linker oligo. (Item 16) The method of item 15, wherein the nanoreporter hybridizes to the linker oligo at a temperature of about 32 to about 40 degrees Celsius. (Item 17) The method of item 15, wherein the nanoreporter hybridizes to the linker oligo at a temperature of about 37 degrees Celsius to about 45 degrees Celsius. (Item 18) The method of item 15, wherein the nanoreporter hybridizes to the linker oligo at a temperature of about 37 degrees Celsius. (Item 19) 15. The method of item 15, wherein the nanoreporter comprises a moiety that is complementary to the linker oligo. (Item 20) 19. The method of item 19, wherein the complementary region is from about 15 to about 20 bases. (Item 21) A method for determining the concentration of at least one protein in a sample, (a) (i) At least one protein, (ii) A first protein probe specific for the first region of the at least one protein, wherein the first protein probe is attached to a first capture region or a first matrix. First protein probe, (iii) A second protein probe specific for a second region of the at least one protein, wherein the second protein probe comprises a signal oligo and a second protein probe. (iv) When the first probe is attached to the first capture region: A component of the second matrix that can bind to the capture region in the first protein probe is attached to the second matrix. Adhering, second matrix The process of supplying; (b) A step of forming at least a first complex comprising the at least one protein, the first protein probe, and the second protein probe, wherein the at least one protein is the first protein. When the probe and the second protein probe are attached and the first probe is attached to the first capture region, the capture probe is attached to the component in the second matrix. Process; (c) Step of releasing the signal oligo from the first complex; (d) (1) At least the signal oligo, and (2) At least one oligo probe containing a signal oligo-specific region and a region containing a nanoreporter A step of forming a second complex comprising, wherein the nanoreporter comprises a plurality of different detectable labels; (e) A step of individually detecting at least a part of the second complex or the second complex by a method including individually counting the presence of one or more molecules of the nanoreporter. , The presence of the second one or more molecules indicates the concentration of the protein in the sample, step. Including methods. (Item 22) The method of item 21, wherein the signal oligo is attached to a second capture region. (Item 23) 22. The method of item 22, wherein the release step of the signal oligo further comprises a step of directly or indirectly capturing the signal molecule into a third matrix. (Item 24) 23. The method of item 23, wherein the nanoreporter further comprises a constant region, wherein the constant region comprises a plurality of repeating nucleotide sequences. (Item 25) 24. The method of item 24, further comprising a step of binding the constant region to a second component in the third matrix, wherein the second component can bind the constant region. (Item 26) 21. The method of item 21, wherein the individually detecting step further comprises a step of detecting a digital signal. (Item 27) 21. The method of item 21, wherein the first matrix is selected from the group consisting of beads and arrays. (Item 28) 21. The method of item 21, wherein the second matrix is selected from the group consisting of beads and arrays. (Item 29) Each complex further comprises the step of determining the concentration of the plurality of target proteins by a method comprising the step of forming the plurality of complexes. (i) At least one target protein, (ii) A first protein probe specific for the first region of the at least one protein, wherein the first protein probe is attached to a capture region or a first matrix. Protein probe, (iii) A second protein probe specific for a second region of the at least one protein, wherein the second protein probe contains a signal molecule. When the first probe is attached to the first capture region, the capture probe is bound to the component in the second matrix and each second in each of the plurality of complexes. 21. The method of item 21, wherein the protein probe of the protein comprises a different signal oligo. (Item 30) 29. The method of item 29, wherein the first matrix is beads, wherein the beads contain a plurality of identical first protein probes. (Item 31) 29. The method of item 29, wherein the concentration of two or more target proteins is determined. (Item 32) 2, 3, 4, 5, 10, 20, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000 , Or the method of item 29, wherein the concentration of more than 1000 different target proteins is determined. (Item 33) 29. The method of item 29, wherein the concentration of up to 2000 different target proteins is determined. (Item 34) 29. The method of item 29, wherein the concentration of up to 980 different target proteins is determined. (Item 35) 21. The method of item 21, wherein the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids. (Item 36) The nanoreporter comprises a single-stranded nucleic acid backbone, the backbone comprising a plurality of labeled attachment regions that are linearly combined and covalently attached, and each labeled attachment region hybridizes to a complementary polynucleotide sequence. The method of item 21, wherein the detectable label is attached to the complementary polynucleotide sequence. (Item 37) A method for determining the concentration of at least one protein in a sample, (a) (i) At least one protein, (ii) The first protein probe, which is a first protein probe specific to the first region of the at least one protein and is attached to the first oligo. (iii) The second protein probe that is specific to the second region of the at least one protein and is attached to the second oligo. The process of supplying; (b) A step of forming a first complex comprising the at least one protein, the first protein probe, and the second protein probe, wherein the at least one protein is the first protein probe. And binding to the second protein probe, step; (c) A step of ligating the first oligo and the second oligo to form a signal oligo; (d) (1) The first signal oligo, and (2) At least one oligo probe containing a signal oligo-specific region and a region containing a nanoreporter A step of forming a second complex comprising, wherein the nanoreporter comprises a plurality of different detectable labels; (e) A step of individually detecting at least a portion of the second complex or the second complex by a method comprising individually counting the presence of one or more molecules of the nanoreporter. , The presence of the one or more molecules indicates the concentration of the protein in the sample, step. Including methods. (Item 38) 37. The method of item 37, further comprising releasing the signal oligo from the first complex. (Item 39) 38. The method of item 38, wherein the signal oligo comprises a capture region. (Item 40) 39. The method of item 39, wherein the release step of the signal oligo further comprises the step of capturing the signal molecule directly or indirectly into a matrix. (Item 41) 37. The method of item 37, wherein the individually detected step further comprises a step of detecting a digital signal. (Item 42) Each complex further comprises the step of determining the concentration of the plurality of target proteins by a method comprising the step of forming the plurality of complexes. (i) At least one target protein, (ii) A first protein probe that is specific to the first region of the at least one protein, wherein the first protein probe is attached to a first oligo. (iii) A second protein probe that is specific for the second region of the at least one protein, wherein the second protein probe is attached to a second oligo. 37. The method of item 37, wherein the ligation of the first oligo and the second oligo forms a signal oligo, wherein each complex in the plurality of complexes comprises a different signal oligo. (Item 43) 42. The method of item 42, wherein the concentration of two or more target proteins is determined. (Item 44) 2, 3, 4, 5, 10, 20, 20, 30, 50, 100, 200, 300, 500, 600, 700, 800, 900, 1000 , Or the method of item 43, wherein the concentration of more than 1000 different target proteins is determined. (Item 45) 41. The method of item 41, wherein the concentration of at least 972 different target proteins is determined. (Item 46) 37. The method of item 37, wherein the first protein probe and the second protein probe are independently selected from the group consisting of antibodies, peptides, aptamers, and peptoids. (Item 47) The nanoreporter comprises a single-stranded nucleic acid backbone, the backbone comprising a plurality of labeled attachment regions that are linearly combined and covalently attached, and each labeled attachment region hybridizes to a complementary polynucleotide sequence. 37. The method of item 37, wherein the detectable label is attached to the complementary polynucleotide sequence. (Item 48) A population of uniquely labeled protein probes, each probe i) Target-specific regions; and ii) A region containing a nanoreporter, wherein the nanoreporter contains a plurality of different detectable molecules; Population, wherein the nanoreporter in each protein probe has a detectable signal that distinguishes it from other nanoreporters in the population. (Item 49) Item 48. The population of item 48, wherein the target-specific region is selected from the group consisting of antibodies, peptides, aptamers, and peptoids. (Item 50) The nanoreporter comprises a single-stranded nucleic acid backbone, the backbone comprising a plurality of labeled attachment regions that are linearly combined and covalently attached, and each labeled attachment region hybridizes to a complementary polynucleotide sequence. 48. The population of item 48, wherein the detectable label is attached to the complementary polynucleotide sequence. (Item 51) The dissociation constant of the target-specific region is about 1.00 × 10.^-15~ About 1.00 × 10^-08The population according to item 48. (Item 52) 48. The population of item 48, wherein the nanoreporter is attached to the protein probe through hybridization to a linker oligo. (Item 53) 52. The population of item 52, wherein the nanoreporter hybridizes to the linker oligo at a temperature of about 32 to about 40 degrees Celsius. (Item 54) 53. The population of item 53, wherein the nanoreporter hybridizes to the linker oligo at a temperature of about 37 degrees Celsius. (Item 55) 52. The population of item 52, wherein the nanoreporter comprises a moiety that is complementary to a linker oligo. (Item 56) 55. The method of item 55, wherein the complementary region is from about 15 to about 20 bases. (Item 57) 49. The population of item 49, wherein the nanoreporter is attached to the antibody through hybridization to the linker oligo, and the nanoreporter is hybridizing to the linker oligo at a temperature of about 37-45 degrees Celsius. ..</p>

0029The novel features of the present invention are shown in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description showing exemplary embodiments in which the principles of the present invention are utilized, and the accompanying drawings:<figref num="1">FIG. 1 is a schematic diagram showing an embodiment of the present invention in which two antibodies specific for a target protein bind to the target protein in solution. The first antibody is attached to an affinity tag such as biotin (indicated by the circled "B"), while the second antibody is attached to a partially double-stranded nucleic acid probe. .. Binding of the first and second antibodies to the target protein forms a complex, which is isolated from the solution by the affinity tag of the first antibody. One of the strands of a partially double-stranded nucleic acid probe can be eluted to yield a signal oligo, which signal oligo can then be analyzed by any of the methods described herein. it can.</figref><figref num="2">FIG. 2 is a graph showing the results of detection assays using IL-2 target proteins at different concentrations. Specifically, IL- in a blocker-free solution, 0.03% milk-containing solution, 0.1% bovine serum albumin (BSA) -containing solution, or 98 ng / ml salmon sperm (SS) -containing solution. Detection of 2 was measured as the total number detected as a function of increasing IL-2 target protein molar concentration ([IL2 target, M]).</figref><figref num="3">FIG. 3 is a graph showing the efficiency of IL-2 detection in the assay used in FIG. The total number detected was standardized for 600 molecules per field of view (FOV) and expressed as a function of the increasing concentration of IL2 target protein molecules ([IL2 target, molecule]). The efficiency of detection is the slope of a line shown in this graph.</figref><figref num="4">FIG. 4 is a schematic diagram showing two selectable embodiments of the present invention for solution tripartite binding. According to this method, in solution, two antibodies specific for the target protein bind to the target protein. The first antibody is attached to an affinity tag such as biotin and contains a constant region, the constant region containing, for example, an F repeat. The second antibody is attached to the nanoreporter probe and the second constant region, which contains, for example, the G repeats. Binding of the first and second antibodies to the target protein forms a complex, which can be isolated from solution by the affinity tag of the first antibody. The "standard" elution of the complex is achieved by melting the G and F beads. The "alternative" elution of the complex is achieved by digestion. Nanoreporter labeled monomers (drawn as circles) emit individually signals of spatially distinct qualitatively different wavelengths, from left to right, red (R), yellow (drawn). Y), green (G), blue (B), red (R), and purple (V).</figref><figref num="5">FIG. 5 is a graph showing the dissociation constant (Kd) and probe concentration for one of the embodiments of the present invention, represented as the ratio of bound targets to Kd for nanoreporter probes and protein probes.</figref><figref num="6">FIG. 6 is a schematic diagram showing an embodiment of the present invention in which a capture antibody specific for a target protein binds to the target protein to form a complex in solution. The complex can then be isolated from the solution. The complex is then contacted with a second antibody, which is attached to a partially double-stranded nucleic acid probe. One of the strands of a partially double-stranded nucleic acid probe can be eluted to yield a signal oligo, which signal oligo can be analyzed by any of the methods described herein.</figref><figref num="7">7A and 7B are schematics showing embodiments of the invention in which two antibodies specific for the target protein bind to the target protein in solution. The first antibody is a captive antibody, while the second antibody is attached to a partially double-stranded nucleic acid probe, and one of the strands in the probe is attached to an affinity tag such as biotin. ing. Binding of the first and second antibodies to the target protein forms a complex, which can be isolated from solution by the capture antibody. One of the strands of a partially double-stranded nucleic acid probe can be eluted to yield a signal oligo containing an affinity tag. The signal oligo can then hybridize to the nanoreporter to form a nanoreporter-signal oligo complex, which can be isolated and / or immobilized on a solid surface. Nanoreporter-signal oligo complexes can be analyzed by any of the methods described herein. Nanoreporter labeled monomers (drawn as circles) emit individually signals of spatially distinct qualitatively different wavelengths, from left to right, red (R), yellow (drawn). Y), green (G), blue (B), red (R), and purple (V).</figref><figref num="8A">FIG. 8A is a schematic diagram showing a particular embodiment of the present invention using proximity ligation. The first and second oligos are attached to the first and second antibodies, respectively, and both antibodies are specific for the target protein. The first and second antibodies bind to the target protein, bringing the first and second oligos into close proximity. Crosslinked oligos and ligases are added to the solution and the first and second oligos are ligated to yield a signal oligo. The signal oligo can then be analyzed by any of the methods described herein.</figref><figref num="8B">8B-D are schematics showing how the signal oligos shown in FIG. 8A can be liberated and purified.</figref><figref num="8C">8B-D are schematics showing how the signal oligos shown in FIG. 8A can be liberated and purified.</figref><figref num="8D">8B-D are schematics showing how the signal oligos shown in FIG. 8A can be liberated and purified.</figref><figref num="9">FIG. 9 is a schematic diagram showing a particular embodiment of the present invention using proximity ligation. Nanoreporter labeled monomers (drawn as circles) emit individually signals of spatially distinct qualitatively different wavelengths, from left to right, red (R), yellow (drawn). Y), green (G), blue (B), red (R), and purple (V).</figref><figref num="10">FIG. 10 is a graph showing the results of multiplexed protein detection of TNF alpha, IL1 alpha, IL6, and VEGF, measured as the total number detected as a function of increasing protein target concentration ([protein target]). In this example, a sandwich detection assay in solution was used. A quadruple measurement is shown.</figref><figref num="11">FIG. 11 is a graph showing the analyzed data in FIG. 10 plotted by lanes instead of concentration. Specifically, this figure shows that two target proteins were titrated-effective, while two other proteins were titrated-ineffective.</figref><figref num="12">FIG. 12 is a graph showing the results of a limit of detection (LOD) experiment using two protein targets, IL1 alpha and IL6. The total number detected was plotted as a function of the increasing molar concentration of the target protein ([target] molar concentration). Experiments have shown that detection limits are 1.4 x 10 for IL1 alpha (IL1α) and IL6, respectively.^-12M and 1.9 × 10^-12There were 26 picograms and 38 picograms (pg / ml) per milliliter, corresponding to M. The detection limit was two standard deviations above the background detection level. Six negative controls were performed, resulting in an average number of positive or -1 standard deviations, namely 3196 ± 265 and 6703 ± 585, respectively.</figref><figref num="13">FIG. 13 shows the various components of the antibody reporter complex after purification and rinsing steps with either water or SSPE buffers of various multiple concentrations (0.01 ×, 0.03 ×, or 0.1 ×) (PROX01, It is a graph which shows the total number held by PROX03, PROX04, PROX05, and PROX06). At 0.03 × SSPE, the oligo representing the ligated product, PROX05, was retained.</figref><figref num="14">FIG. 14 shows each antibody probe (S17, S8, S22, S14, S23, S6, S13, S7, S18, S9, S10, S11, S12, S15, S16) on a bound, stretched and immobilized reporter. , S19, S20, and S21) are graphs showing the number per field of view (FOV). The numbers are shown only for reporters with antibody probes bound to the surface.</figref>

0030Detailed description of the invention Particularly preferred embodiments of the present invention will be referred to in detail. Examples of preferred embodiments are shown in the following Examples section.

0031Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. All patents and publications referred to herein are incorporated herein by reference in their entirety.

0032The present invention provides compositions and methods for the detection and quantification of individual target molecules in biomolecular samples. In particular, the present invention provides protein probes capable of binding individual target molecules. The present invention also provides the use of nanoreporters. Binding of the protein probe to the target molecule through the nanoreporter labeling code results in the identification of the target molecule. Methods for making and using such protein probes and / or nanoreporters are also provided. The methods and compositions described herein can be used in a wide variety of applications such as diagnostic, prognostic, quality control, and screening applications.

0033One particular aspect of the invention relates to the detection of a large number of target molecules. Multiplexing is the measurement of more than one target molecule in a sample without the need to separate the sample. The methods described herein provide potential benefits in the areas of multiplexing, quantification, and sensitivity. For example, in some embodiments, the target molecule is a protein. Measuring protein concentration is a challenge. Proteins are sticky and tend to aggregate. In addition, proteins are unstable and tend to unfold more easily than RNA or DNA. Extreme pH, temperature, solute concentration, and the presence of denaturants are conditions that can interfere with protein stability and complicate measurements. In some embodiments, the present invention provides methods and compositions for sensitive and reliable multiplexing protein measurements.

0034Multiplexing within a liquid sample is an important advantage of this approach. Multiplexing within a single sample saves significant labor, reduces sample requirements in proportion to the number of measurements, and increases accuracy with separate sample manipulation and measurement steps. Improve. In some embodiments, the methods described herein allow different samples to be pooled together during treatment and analyzed at once. This gives a throughput advantage and can accelerate the analysis of different samples, for example up to 8 times.

0035In some embodiments, the invention provides a protein probe for analysis of a target molecule. In some embodiments, the invention provides a population of protein probes for use in a multiplexing assay. Each protein probe in the population is specific for the target molecule. The binding of the target molecule to the protein probe is then detected using a nanoreporter. Each nanoreporter contains a unique labeling code that can be associated with a particular target molecule.

0036In some embodiments, the nanoreporter is attached directly or indirectly to the protein probe. The unique nanoreporter labeling code is then assigned to a particular protein probe so that the labeling code for each nanoreporter can associate with the target molecule bound to the protein probe.

0037In other embodiments, the protein probe is attached directly or indirectly to the signal oligo. Each protein probe is attached to a unique signal oligo. Nanoreporters used in the analysis of signal oligos contain moieties that are complementary to the signal oligos. The unique nanoreporter labeling code is assigned to a particular signal oligo sequence so that the labeling code of each nanoreporter can associate with the target molecule via the signal oligo sequence.

0038In another aspect of the invention, the invention provides a method for detecting a target molecule by digitally measuring the signal. Current technology uses analog fluorescent signals to quantify the presence of target molecules. Quantification using fluorescence is error-prone for a variety of reasons. For example, fluorophores can be photobleached. There can be spectral changes in the presence of proteins or due to the local environment, eg pH, salts. In addition, the light source can vary in intensity over time. For example, an arc lamp, which is a commonly used light source, exhibits a phenomenon called arc wonder that can cause significantly different illumination levels over time. In embodiments of the invention, the target molecule is digitally detected. Fluorescence may be used to read the nanoreporter's labeling code, but the signal is high and the spots are either present or absent, and thus digital detection. Digital detection of target molecules results in more accurate quantification.

0039Protein probe A protein probe is a molecule or assembly designed to bind at least one target protein, at least one target protein surrogate, or both; under appropriate conditions, a molecular complex comprising the protein probe and the target protein. Can form a body. The terms "protein", "polypeptide", "peptide", and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids or synthetic amino acids. The term also includes amino acid polymers that have been modified by any other operation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeled components. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including both glycine and D- or L-type optical isomers, as well as amino acid analogs. Examples include, but are not limited to, bodies and peptide mimetics.

0040The methods of the invention also include protein probes designed to bind targets other than proteins. Examples of targets other than proteins include, but are not limited to, nucleic acids, lipids, sugars, ions, small molecules, organic monomers, and drugs. For convenience only, most of the embodiments described herein have been described in the context of protein probes that bind to the target protein. However, these embodiments can also be applied to other target molecules.

0041A protein probe is typically part of at least one probe set that includes at least one first probe and at least one second probe. However, in certain embodiments, at least one probe set can include only the first probe or only the second probe, but not both the first and second probes. .. Probes typically sequence them into at least one target protein, at least one portion of at least one target protein, at least one target protein surrogate, at least a portion of the target protein surrogate, or a combination thereof. It is possible to bind or interact with specific modes, confirmation-specific modes, or both, eg, but not limited to, antigen-antibody binding, aptamer-target binding, and the like. Includes at least one reaction part.

0042In certain embodiments, the protein probe comprises an identity part or at least a portion of the identity part, such as a signal oligo, a nanoreporter, and / or a linker oligo. In certain embodiments, the protein probe comprises a capture region. In some embodiments, the capture region is used for isolation of the protein probe and / or immobilization of the protein probe on the surface. The capture area can be an affinity tag, bead, slide, or array as described below.

0043In some embodiments, the protein probe is an antibody. As used herein, the term antibody (s) is used in a broad sense to include intact antibody molecules such as, but not limited to, immunoglobulin A, immunoglobulin G, and immunoglobulin M. It also includes any immunoreactive component (s) of an antibody molecule that immunospecifically binds to at least one epitope. Such immunoreactive components include Fab fragments, Fab'fragments, F (ab').₂Fragments, single chain antibody fragments (scFv), mini-antibodies, bispecific antibodies, cross-linked antibody fragments, Affibody^TM, Cyclotides, molecules, etc., but not limited to them. Immunoreactive products derived using antibody or protein engineering techniques are also clearly within the meaning of the term antibody. A detailed description of antibody engineering and / or protein engineering, including related protocols, is described, among other things, by J. Maynard and G. Georgiou, Ann. Rev. Biomed. Eng. 2: 339 76 (2000); Antibody Engineering, R. Kontermann. And S. Dubel, Springer Lab Manual, Springer Verlag (2001); US Pat. No. 5,831,012; and S. Paul, Antibody Engineering Protocols, Humana Press (1995).

0044Antibodies can be obtained from a variety of sources, including, but not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, recombinant expression antibodies, humanized antibodies, plant antibodies, etc. Those skilled in the art will recognize that they can be obtained from a variety of animal species, including humans, horses, cows, guinea pigs, chickens, sheep, donkeys, humans and the like. A wide variety of antibodies are commercially available, and custom-made antibodies are available from several contract laboratories. A detailed description of antibodies, including relevant protocols, is, among other things, Current Protocols in Immunology, edited by Colligan et al., John Wiley &amp; Sons (including updates 1999, August 2003); The Electronic Notebook; Basic Methods in Antibody Production. and TEXT, edited by G. Howard and D. Bethel, CRC Press (2000); J. Goding, Monoclonal Antibodies: Principles and Practice, 3rd Edition, Academic Press (1996); E. Harlow and D. Lane, Using Antibodies, Cold Spring Harbor Lab Press (1999); P. Shepherd and C. Dean, Monoclonal Antibodies: A Practical Approach , Oxford University Press (2000); A. Johnstone and M. Turner, Immunochemistry 1 and 2, Oxford University Press (1997); C. Borrebaeck, Antibody Engineering, 2nd Edition, Oxford university Press (1995); A. Johnstone and R.Thorpe, Immunochemistry in Practice, Blackwell Science, Ltd. (1996); H.Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives (Basics: From Background to Bench), Springer Verlag (2000); and S. Hockfield et al., Selected Methods for Antibody and Nucleic Acid Probes, Cold Spring Harbor Lab Press (1993) Can be found in. In addition, a vast number of commercially available antibodies, including labeled or unlabeled, polyclonal antibodies, monoclonal antibodies, and monospecific antibodies, as well as their immunoreactive components; custom antibody suppliers, etc., World Antibody Search page on the wide web, especially on biocompare.com, Antibody Resource on antibodyresource.com It can be found on Page and on the Antibody Explorer page at sigmaaldrich.com.

0045In some embodiments, the antibodies described herein are attached to nucleic acids such as signal oligos, linker oligos, and / or nanoreporters. Methods for attaching nucleic acids to antibodies are known in the art. Any suitable method for attaching nucleic acid to an antibody is included in the methods of the invention. The antibodies described herein are described in Gullberg et al., PNAS 101 (22): 228420-8424 (2004); and Boozer et al., Analytical Chemistry, 76 (23): 6967-6972 (2004). It can be attached to nucleic acids by method and both references are incorporated herein by reference. The antibodies described herein can attach to nucleic acids by random amine attachment. In some embodiments, the antibodies described herein can attach to nucleic acids by random amine attachment using a 10: 1 nucleic acid to antibody ratio. The antibodies described herein are Kozlov et al., Biopolymers, which are incorporated herein by reference. It can be attached to nucleic acids by the method described in 5:73 (5): 621-630 (2004). The antibodies described herein can attach to nucleic acids by hydrazine chemistry. Antibodies described herein attach to nucleic acids using "tadpoles" as described in Nolan, Nature Methods 2, 11-12 (2005) incorporated herein by reference. can do. The antibodies described herein can be attached to nucleic acids by any suitable method known in the art for making engineered antibodies, including those described herein.

0046In some embodiments, the protein probe is an aptamer. Aptamers include nucleic acid aptamers (ie, single-stranded DNA molecules or single-stranded RNA molecules) and peptide aptamers. Aptamers bind target molecules in a highly specific, higher-order structure-dependent manner, typically with very high affinities, but aptamers with lower binding affinities can be selected as needed. it can. Aptamers have been shown to distinguish between targets based on very small structural differences, such as the presence or absence of methyl or hydroxyl groups, and certain aptamers refer to D-enantiomers and L-enantiomers. Can be distinguished. Small molecule targets, including drugs, metal ions, and organic dyes, peptides, biotin, and aptamers that bind to proteins, including, but not limited to, streptavidin, VEGF, and viral proteins have been obtained. Aptamers have been shown to retain functional activity after biotinogenesis, after fluorescein labeling, and when attached to glass surfaces and microspheres.

0047Nucleic acid aptamers, including speigelmers, are identified by an in vitro selection process known as In vitro Evolution (SELEX). In the SELEX process, a very large combinatorial library of oligonucleotides, eg, often as large as 60-100 nucleotides in length, 10¹⁴~10¹⁵Individual sequences are routinely screened by a repetitive process of in vitro selection and amplification. Most targets are affinity enriched within 8-15 cycles and the process is automated, allowing faster aptamer isolation. Peptide aptamers are typically identified by several different protein engineering techniques known in the art, such as phage display, ribosome display, mRNA display, selective infectious phage technology (SIP), etc. However, it is not limited to them. Those skilled in the art will appreciate that nucleic acid aptamers and peptide aptamers can be obtained according to conventional procedures and without undue experimentation. A detailed description of the aptamer, including the relevant protocol, is given, among other things, L. Gold, J. Biol. Chem., 270 (23): 13581 84 (1995); S. Jayasena, Clin. Chem., 45: 1628-50. (1999); V. Sieber et al., Nat Biotechnol.16 (10): 955-60 (1998); D. Wilson and J. Szostak, Ann. Rev. Biochem. 68: 611-47 (1999); L. Jermutus et al., Eur. Biophys. J., 31 : 179-84 (2002); S S. Spada et al., Biol. Chem., 378: 445-56 (1997); B. Wlotzka et al., Proc. Natl. Acad. Sci., 99: 8898-8902 (2002) Can be found in.

0048In some embodiments, the aptamer is ligated or hybridized to a signal oligo, a linker oligo, and / or a nanoreporter. In some embodiments, ligation of the aptamer to the nanoreporter is performed prior to annealing the labeled segment to the nanoreporter. Hybridization or ligation of aptamers can be performed by any suitable method known in the art. For example, the ligation may be at least one DNA ligase or at least one RNA ligase, such as, but not limited to, T4 DNA ligase, T4 RNA ligase, Thermophilus (Tth) ligase, Thermus aquaticus (Taq) DNA ligase, or Pyrococcus. It can be performed enzymatically with furiosus (Pfu) ligase. Ligation is also chemical with activators and reducing agents such as carbodiimide, cyanogen bromide (BrCN), imidazole, 1-methylimidazole / carbodiimide / cystamine, N-cyanoimidazole, dithiothreitol (DTT), and UV light. It can be carried out by ligation.

0049In some embodiments, the protein probe is a peptoid. Peptoids are short sequences of synthetic peptides of N-substituted glycine that bind proteins. In some embodiments, small size peptoids improve the diffusion and kinetics of the methods described herein. Any suitable method known in the art for making peptoids is included in the methods described herein. See Simon et al., PNAS 15; 89 (20): 9376-9371 (1992), incorporated herein by reference.

0050Target protein A target protein is a protein that is detected or measured by the binding of a protein probe to it and is recognized by the target-specific region (s) of the protein probe. However, the present invention includes the detection of other targets than proteins such as nucleic acids, lipids, sugars, small molecules, organic monomers, or drugs. Nucleic acids that can be analyzed by the methods herein include double-stranded DNA, single-stranded DNA, single-stranded DNA hairpins, DNA / RNA hybrids, RNA (eg, mRNA or miRNA), and RNA hairpins. Be done. For convenience only, the methods described herein have been described mostly in connection with the analysis of proteins. However, the embodiments described herein can also be used to detect non-protein targets.

0051The target protein can be part of a biomolecular sample containing other components, or it can be the only or major component of the sample. The target protein can be a whole cell or tissue, a cell extract or tissue extract, a fractionated solubilized solution thereof, or a component of a substantially purified molecule. The target protein can be attached in solution or to a solid phase containing a solid surface such as a chip, microarray, or bead. Also, the target molecule can have either a known or unknown structure or sequence.

0052The compositions, methods, and kits disclosed herein can also be used in a wide variety of applications that determine the presence of a target protein in a sample. For example, but not limited to compositions, methods, and kits, pharmacokinetic studies, including, but not limited to, drug metabolism, ADME profiling, and toxicity studies; targeted validation for drug discovery; protein expression profiling; proteome analysis; Metabolomics studies; post-translational modification studies including, but not limited to, glycosylation, phosphorylation, acetylation, and amino acid modifications such as glutamate formation to form gamma-carboxyglutamic acid, and hydroxylation of proline to form hydroxylation; It is useful for analysis of serum or mucosal antibody levels; evaluation of non-nucleic acid diagnostic indicators; detection of foreign antigens, etc.

0053In certain embodiments, at least one first protein probe, at least one second protein probe, or both the first protein probe and the second protein probe in at least one probe set are at least one. Includes at least one antibody, aptamer, or peptoid that specifically reacts with a target protein or at least one target protein surrogate. In certain embodiments, at least one first protein probe, at least one second protein probe, or both the first protein probe and the second protein probe in at least one probe set are at least one. Includes binding proteins that specifically interact with the target protein or at least one target protein surrogate.

0054For antibody probes, the reaction moiety typically contains the antigen binding site and associated residues of the antibody molecule; and the target sequence comprises a portion of the analyte that contains an epitope, such sequence is linear. Those skilled in the art understand that it does not matter whether it is a higher-order structure, a higher-order structure, or a combination thereof. The molecular complexes described herein and at least a portion of the molecular complexes are anchored to a substrate, depending on the nature of the particular molecular complex or cleaving component, and the SMD technology and detector used, among others. Those skilled in the art will recognize that they can be detected individually, either while they are or adhere to them, or even in solution.

0055Protein isolation techniques are also well known in the art, and kits using at least some of these techniques are commercially available. Protein isolation techniques typically use one or more of the following: salting out and cell lysis, including physical, chemical, and enzymatic methods; centrifugation; size exclusion chromatography and Separation by molecular weight, such as preparative electrophoresis; selective precipitation, eg, salting and salting out procedures; various chromatographic methods, etc. A detailed description of protein purification techniques and related protocols are available, among others, Marchak et al., Strategies for Protein Purification and characterization: A Laboratory Course Manual, Cold Spring Harbor Press (1996); Essentials from Cells: A Laboratory Manual, D. .Spector and R. Goldman, Cold Spring Harbor It can be found in Press (2003); R. Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Press (2003); and D. Liebler, Introduction to Proteomics, Humana Press (2002). Commercially available kits are also available, including Proteo Extract available from CALBIOCHEM.RTM., La Jolla, Calif.^TMPartial Proteome Extraction Kits (P-PEK), and Proteo Extract^TMComplete Proteome Extraction Kits (C-PEK), but not limited to them. Those skilled in the art recognize that non-nucleic acid analysts used with the compositions, methods, and kits of the invention can be readily obtained using such purification techniques and commercially available kits without undue experimentation. There will be.

0056Method The present invention provides methods for the detection and quantification of individual target proteins in biomolecular samples. In particular, the present invention provides protein probes capable of binding individual target proteins. The present invention also provides the use of nanoreporters. Binding of the protein probe to the target molecule through the nanoreporter labeling code results in the identification of the target molecule. Methods for making and using such protein probes and / or nanoreporters are also provided.

0057In some embodiments, the invention provides a method for the detection and / or quantification of a target protein by binding the protein probe to the target protein. A protein probe is an antigen, for example, but not limited to, in a sequence-specific mode, a higher-order structure-specific mode, or both, with the probe being a target protein, a target protein surrogate, or a combination thereof. -Contains at least one reaction moiety that allows it to bind or interact with them in a manner such as antibody binding, aptamer-target binding.

0058A protein probe is typically part of at least one probe set that includes at least one first probe and at least one second probe. Thus, in some embodiments, the present invention is a method for the detection and / or quantification of a target protein by binding the protein probe set to the target protein, wherein the protein probe set is the first. A method comprising a protein probe and a second protein probe is provided. The first protein probe and the second protein probe bind to different regions of the target protein, or target protein surrogate, or a combination thereof, for example, in a sequence-specific manner, a higher-order structure-specific manner, or both. Includes at least one reaction moiety that allows it to interact with, or interact with them.

0059In some embodiments, the methods described herein further comprise a protein probe comprising an identity part or at least a portion of the identity part, such as a signal oligo, a nanoreporter, and / or a linker oligo. The identity part allows identification of the presence or absence of a protein probe (s) bound to a target protein during the detection steps of the methods described herein. Thus, in some embodiments, the present invention is a method for the detection and / or quantification of a target protein by binding the protein probe or protein probe set to the target protein, the protein probe or protein probe. Provided is a method in which at least one of the protein probes in the set comprises an identity part (eg, a signal oligo, a nanoreporter, and / or a linker oligo).

0060In some embodiments, the identity part is a signal oligo. The signal oligo comprises a polynucleotide sequence. Each protein probe or protein probe set has a specific and / or unique signal oligo so that the signal oligo can associate with the target protein in the assay. In certain embodiments, the signal oligos are approximately 4, 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 Contains, 70, or more nucleotide bases. In one embodiment, the signal oligo comprises between 40 and 120 bases, or between 80 and 100 bases. In some embodiments, the signal oligo is biotinylated and is used with capture probes and nanoreporters as described below. Signal oligos can attach directly or indirectly to protein probes. Methods for attaching nucleic acids to protein probes are known in the art, including those described herein. The signal oligo can be a designed synthetic nucleic acid sequence or a natural sequence from a natural source, such as a sequence from a viral genome, bacteriophage, or animal genome.

0061In some embodiments, the signal oligo is indirectly attached to the protein probe through hybridization with the linker oligo attached to the protein probe. The linker oligo comprises a polynucleotide sequence. In embodiments where linker oligos are used, each linker oligo is specific and / or specific for a protein probe or protein probe set in the assay so that complementary signal oligos can associate with the target protein. The signal oligo contains a moiety that is complementary to the linker oligo attached to the protein probe. In some embodiments, the complementary moieties of the signal oligos are 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 , 70, or more nucleotide bases. In some embodiments, the complementary portion of the signal oligo is 10-25 bases. In some embodiments, the complementary portion of the signal oligo is in the range of 15-20 bases. In some embodiments, the complementary portion of the signal oligo is 40 bases. In some embodiments, the complementary portion of the signal oligo is 30 bases. In some embodiments, the complementary portion of the signal oligo is 20 bases. The linker oligo can be a designed synthetic nucleic acid sequence or a natural sequence from a natural source, such as a sequence from a viral genome, bacteriophage, or animal genome.

0062FIG. 1 shows one schematic of an embodiment of the invention in which signal oligos are used to detect target proteins. The embodiment shown in FIG. 1 is set to separate the binding of the target protein from hybridization of the nanoreporter. FIG. 1 in step 1) shows a first protein probe containing a signal oligo attached to the probe via hybridization with a linker oligo; and a second protein attached to an affinity tag. In the embodiment shown in FIG. 1, the protein probe is an antibody and the affinity tag is biotin. However, the embodiments shown in this figure can be utilized with any of the protein probes and affinity tags described herein. Both the first protein probe and the second protein probe contain a target-specific region that can bind one or more parts of the target. In steps 2) and 3), the target protein is mixed with the first protein probe and the second protein probe. In step 4), the complex of the target protein and the protein probe is purified. In the example shown in FIG. 1, the target protein-protein probe complex is purified using streptavidin-bound magnetic beads such as Dynabeads® (Invitrogen). However, in any of the other embodiments described herein, including this, the complex of the target protein and the protein probe (s) can be, but not limited to, HPLC, FPLC, size exclusion (gel). Chromatography including (filtration) chromatography, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, immunoaffinity chromatography, and reverse phase chromatography; biotin-avidin, maltose-maltose binding protein (MBP), calcium -Ligands such as calcium-binding peptides-receptor binding; aptomer-target binding;

0063In step 5) of FIG. 1, the signal oligo is eluted from the complex of the target protein and the protein probe and analyzed using a nanoreporter as described below. Methods for elution of signal oligos are known in the art and include those shown in FIG. 1 and those described herein. In some embodiments, the method shown in FIG. 1 is used to detect and / or quantify multiple target proteins. Each target protein is detected by a probe set containing a first probe and a second probe as shown in FIG. Each probe set has a specific and / or unique signal oligo, which can then associate with the target protein of each probe set.

0064In some embodiments, the protein probe comprises a capture region. In some embodiments, the capture region is used for isolation of the protein probe and / or immobilization of the protein probe on the surface. The capture area can be an affinity tag such as the one below, or a solid surface such as a bead, slide, or array.

0065FIG. 6 shows one schematic view of an embodiment of the present invention. In this embodiment, the protein probe is attached to a capture area, eg, a magnetic bead. Figure 6 shows the use of antibodies. However, the embodiments shown in this figure can be utilized with either the protein probe and the capture region described herein. Protein probes (eg, antibodies) can be attached to the capture region by any suitable method known in the art, including the methods described herein. The target protein is mixed with a protein probe containing the capture region. The target protein-protein probe complex is then contacted with a second protein probe attached to the signal oligo via a linker oligo. Purify the complex of target protein and protein probe. In this example, the target protein-antibody complex is purified using the magnetic beads in the captured antibody. However, in any of the other embodiments described herein, including this, the complex of the target protein and protein probe can be obtained by any suitable method known in the art, such as the methods described above. Can be purified. If the capture region is a slide or array, the target protein-protein probe complex can be purified by flushing out the overbound sample and protein probe. The isolated target protein / protein probe complex is then washed to elute the signal oligo. Signal oligos are analyzed using nanoreporters such as: Methods for elution of signal oligos are known in the art, including the methods described herein. In this embodiment, the protein and the nanoreporter are well separated, which eliminates concerns about protein stickiness. In some embodiments, the method shown in FIG. 6 is used to detect and / or quantify multiple target proteins. Each target protein is described in Figure 6. Detected by a probe set containing a first probe and a second probe. Each probe set has a specific and / or unique signal oligo, which can then associate with the target protein of each probe set.

0066In some embodiments, the signal oligo is attached to an affinity tag. Affinity tags in signal oligos can be used to isolate and / or immobilize signal oligos. Signal oligos can be attached to affinity tags in any of the methods described herein that utilize signal oligos.

0067FIG. 7 shows one schematic view of an embodiment of the present invention. This embodiment can be used with any of the methods described herein. The figure in FIG. 7 shows an antibody as a protein probe, but this example can be used with any of the protein probes described herein. FIG. 7 shows an antibody attached directly or indirectly (eg, by hybridization through an oligo) to a capture region (eg, magnetic beads), and a second antibody attached to a biotinylated signal oligo. However, the embodiments shown in this figure can be utilized with either the capture region and the affinity tag described herein. Mix the target protein with the protein probe. The complex of target protein and antibody is purified using the magnetic beads in the captured antibody. However, in any of the other embodiments described herein, including this, the complex of the target protein and protein probe can be obtained by any suitable method known in the art, such as the methods described above. Can be purified. If the capture region is a slide or array, the target protein-protein probe complex can be purified by flushing out the overbound sample and protein probe. The isolated target protein / antibody complex is then washed and the signal oligos are eluted by any suitable method known in the art, including those described herein. In the embodiment of FIG. 7, signal oligos are purified using oligonucleotide-bound beads such as Dynabeads®. However, the signal oligo can be purified by any suitable method according to the affinity tag attached to it. Signal oligos are analyzed using nanoreporters such as: In some embodiments, the method shown in FIG. 7 is used to detect and / or quantify multiple target proteins. Each target protein is a number as described in Figure 7. Detected by a probe set containing one probe and a second probe. Each probe set has a specific and / or unique signal oligo, which can then associate with the target protein of each probe set. The embodiment described in FIG. 7 offers the advantage that it requires only two bead purifications. In addition, in this embodiment, the protein and the nanoreporter are well separated, which eliminates concerns about protein stickiness.

0068In some embodiments, signal oligos are made by ligating two oligos in close proximity, eg, close ligation. A diagram of proximity ligation is shown in FIG. In step 1) of FIG. 8, the oligo-containing probe is designed to bind to the target protein as a pair and to form a signal oligo by ligation when the probes are in close proximity. FIG. 8 shows an embodiment in which an antibody is used as a protein probe. However, the method described in FIG. 8 can be used with any of the protein probes described herein. The oligo-containing probe can be prepared and purified by any method known in the art, such as that described by Gullberg et al., PNAS 101 (22), p8420-24 (2004). In step 2) of FIG. 8, the target protein is then mixed with a probe containing oligos and crosslinked oligos.

0069The crosslinked oligo contains a polynucleotide sequence. The oligo attached to the protein probe contains moieties that are complementary to the crosslinked oligo. In some embodiments, the complementary moieties of the oligo are 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 14, 15, 16, 17 pcs, 18 pcs, 19 pcs, 20 pcs, 21 pcs, 22 pcs, 23 pcs, 24 pcs, 25 pcs, 26 pcs, 27 pcs, 28 pcs, 29 pcs, 30 pcs, 40 pcs, 50 pcs, 60 pcs , 70, or more nucleotide bases. In some embodiments, the complementary portion of the crosslinked oligo with each of the oligos attached to the protein probe is 6-15 bases and the total length of the crosslinked oligo is 12-30 bases. In some embodiments, the complementary portion of the oligo is 40 bases. In some embodiments, the complementary portion of the oligo is 30 bases. In some embodiments, the complementary portion of the oligo is 20 bases.

0070In step 4) of FIG. 8, the components required for probe ligation are added. Oligos in protein probes can be ligated by any suitable method known in the art. Ligation according to the invention involves any enzymatic or chemical process, in which nucleotide-to-nucleotide linkages are formed between the opposite ends of a nucleic acid sequence that hybridizes adjacent to a crosslinked oligo. Examples of enzymes that can be used for ligation include T4 DNA ligase, T4 RNA ligase, Thermophilus (Tth) ligase, Thermus aquaticus (Taq) DNA ligase, or Pyrococcus. DNA ligases such as furiosus (Pfu) ligase and RNA ligases include, but are not limited to. Chemical ligation uses activators and reducing agents such as carbodiimide, cyanogen bromide (BrCN), imidazole, 1-methylimidazole / carbodiimide / cystamine, N-cyanoimidazole, dithiothreitol (DTT), and UV light. Can be carried out. Ligation techniques such as, but not limited to, gap-filling ligation, including gap-filling OLA and LCR, cross-linked oligonucleotide ligation, and correction ligation are also within the scope of the invention. Descriptions of these techniques can be found, among other things, in US Pat. Nos. 5,185,243, European Patent Application Publications EP 320308 and EP 439182, and International Patents WO 90/01069 and WO 01/57268. it can.

0071In step 5) of FIG. 8, after ligation, the signal oligo is released by disulfide reduction, uracil removal, restriction digestion, proteinase K, or any other suitable method known in the art. In addition, signal oligos can be released by the methods shown in Figures 8B-8D. FIG. 8B shows an embodiment in which the signal oligo has an affinity tag such as biotin or sequence. Affinity tags can be used to isolate and / or immobilize signal oligos as described herein. FIG. 8C shows an embodiment in which the crosslinked oligo has an affinity tag such as biotin or sequence. Affinity tags can be used to isolate and / or immobilize signal oligos as described herein. Only ligated oligos have sufficient duplication to remain hybridized to signal oligos during the isolation and / or immobilization process. FIG. 8D shows an embodiment in which the embodiments of FIGS. 8B and 8C are combined. Signal oligos are analyzed using nanoreporters such as: In some embodiments, the method shown in FIG. 8 is used to detect and / or quantify multiple target proteins. Each target protein is detected by a probe set containing a first probe and a second probe as shown in FIG. Each probe set has a specific and / or unique signal oligo, which can then associate with the target protein of each probe set. The embodiments described in FIG. 8 have several benefits centered on sensitivity, cross-reactivity minimization, and multiplexing. Proximity ligation exhibits high sensitivity and has the effect of reducing apparent Kd by essentially reducing off-speed.

0072In some embodiments that utilize proximity ligation, one of the oligos is attached to the nanoreporter. FIG. 9 shows one such embodiment.

0073In step 1) of FIG. 9, the oligo-containing probe is designed to bind to the target protein as a pair. One of the oligos in one of the protein probes is attached to the nanoreporter. FIG. 9 shows an embodiment in which an antibody is used as a protein probe. However, the method described in FIG. 9 can be used with any of the protein probes described herein. The oligo-containing probe can be prepared and purified as described above. In steps 2) and 3) of FIG. 9, the target protein is then mixed with a probe containing oligos and crosslinked oligos. The cross-linked oligo binds to the oligo in the first protein probe and the portion of the nanoreporter attached to the second protein probe.

0074The oligos and nanoreporters attached to the first protein probe contain moieties that are complementary to the crosslinked oligos. In some embodiments, the complementary moieties are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, and 17. , 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70 One or more nucleotide bases. In some embodiments, the complementary moiety is 40 bases. In some embodiments, the complementary moiety is 30 bases. In some embodiments, the complementary moiety is 20 bases. In some embodiments, the complementary portion of the crosslinked oligo with each of the oligos attached to the protein probe and the nanoreporter is 6 to 15 bases, and the total length of the crosslinked oligo is 12 to 30 bases. In step 4) of FIG. 9, the components required for probe ligation are then added. The oligo and nanoreporter in the first protein probe can be ligated by any suitable method known in the art as described above. In step 5) of FIG. 9, after ligation, the signal oligo can optionally be released by disulfide reduction, uracil removal, restriction digestion, proteinase K, or any other suitable method known in the art. ..

0075In addition, signal oligos can be released by the methods shown in Figures 8B-8D. For example, using the approach described in FIG. 8C, the purification step is performed to separate the ligated oligo from the unligated oligo after release of the signal oligo, eg, antibody. This purification step can be performed using any other method known in the art for the physical separation of magnetic beads or proteins. Importantly, if the amount of antibody used is higher than the amount of reporter used, the resulting over-ligated oligo can block the hybridization of the reporter to the oligo. is there. As described in Example 7, the purification step further comprises a rinsing step with a buffer solution. FIG. 13 shows how different components of the antibody reporter complex are purified and rinsed under different buffer conditions. The preferred rinsing buffer is SSPE; however, other buffers with similar ability to retain the number of receptor complexes or their components and all concentrations are included in these methods.

0076Signal oligos are analyzed using nanoreporters such as: In some embodiments, the method shown in FIG. 9 is used to detect and / or quantify multiple target proteins. Each target protein is detected by a probe set containing a first probe and a second probe as shown in FIG. Each probe set has a specific and / or unique signal oligo, which can then associate with the target protein of each probe set. The embodiment described in FIG. 9 takes advantage of the reduction in Koff due to proximity ligation. Lower Koff means the ability to act with lower Kd and lower concentrations of protein probes. This reduction in Kd makes it easier to act at the required concentration for the reporter and, accordingly, to design a direct detection approach for multiplex analysis and reduce reagent costs. These embodiments do not require a hybridization step to the reporter within the assay. Therefore, these assays will be faster and will respond in a shorter time.

0077In some embodiments, signal oligos are analyzed / detected using nanoreporters (s) as described in the section below. In these embodiments, the nanoreporter (s) comprises a moiety that is complementary to the signal oligo. In some embodiments, the complementary moieties are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, and 17. , 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70 One or more nucleotide bases. In some embodiments, the complementary moiety is 40 bases. In some embodiments, the complementary moiety is 30 bases. In some embodiments, the complementary moiety is 20 bases. In some embodiments, the complementary moiety is 15-20 bases.

0078In some embodiments, the methods described herein further comprise a protein probe comprising a nanoreporter. Thus, in some embodiments, the present invention is a method for the detection and / or quantification of a target protein by binding the protein probe or protein probe set to the target protein, the protein probe or probe set. Provided is a method in which at least one of the protein probes in the invention comprises a nanoreporter.

0079FIG. 4 shows a schematic view of one of the embodiments of the present invention. In this embodiment, the nanoreporter is attached to one of the antibodies. The method described in FIG. 4 can be utilized with any of the protein probes described herein. In some embodiments, the nanoreporter can attach directly to the protein probe. In other embodiments, the nanoreporter can attach to the protein probe by hybridization through a linker oligo. Therefore, the nanoreporter contains a portion of the protein probe that is complementary to the linker oligo. In some embodiments, the complementary moieties are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 15, 16, and 17. , 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70 One or more nucleotide bases. In some embodiments, the linker oligo is 15-20 bases. In some embodiments, the complementary moiety is 40 bases. In some embodiments, the complementary moiety is 30 bases. In some embodiments, the complementary moiety is 20 bases. In some embodiments, the complementary moiety is 15 bases.

0080Hybridization of nanoreporters to linker oligos can occur at different temperatures depending on the length of the complementary moiety. In some embodiments, the nanoreporter can hybridize to a linker oligo attached to a protein probe at temperatures in the range of 32 ° C to 40 ° C. In some embodiments, the nanoreporter can hybridize to a linker oligo attached to a protein probe at a temperature of 35 ° C. In some embodiments, the nanoreporter can hybridize to a linker oligo attached to a protein probe at a temperature of 37 ° C. In some embodiments, the nanoreporter can hybridize to a linker oligo attached to a protein probe at a temperature of 45 ° C. In some embodiments, the nanoreporter can hybridize to the linker oligo attached to the protein probe at a temperature of 52 ° C to 57 ° C. In some embodiments, the nanoreporter hybridizes to the linker oligo attached to the protein probe at a temperature 15 ° C to 20 ° C below the melting temperature (Tm) of the complementary portion of the nanoreporter with the linker oligo. can do. Those skilled in the art will appreciate that the length of the complementary portion of the nanoreporter with the linker oligo and their hybridization temperature will depend on the type of protein probe used. In some embodiments, the protein probe is an antibody and the length of the complementary portion of the nanoreporter with the linker oligo is 15-20 bases, which is about 57 ° C, ie 37 ° C. Give Tm 15 ° C to 20 ° C above the ideal antibody temperature. Thus, in some embodiments, the protein probe is an antibody, the length of the complementary portion of the nanoreporter with the linker oligo is 15-20 bases, and the hybridization temperature is 37 ° C.

0081FIG. 4 shows that one of the antibodies is bound to biotin and the other antibody forms a complex of target protein and antibody to which the nanoreporter is attached. The method described in FIG. 4 can use any affinity tag described herein other than biotin. Purification of the target protein-antibody complex can be performed by any suitable method known in the art, including those described herein. Elution of the nanoreporter can be achieved by melting the G and F beads, by digestion, or by any other suitable method known in the art. In embodiments where the protein-antibody complex comprises an affinity tag, the complex is coated with, for example, streptavidin, a cover glass (Optichem®, Accelr8 Technology). Can be combined with Corporation). The nanoreporter is analyzed as follows. In some embodiments, the method shown in FIG. 4 is used to detect and / or quantify multiple target proteins. Each target protein is detected by a probe set containing a first probe and a second probe as shown in FIG. Each probe set has a specific and / or unique nanoreporter, which can then associate with the target protein of each probe set.

0082Although not intended to be limited to any theory or any particular embodiment, embodiments of the invention utilizing signal oligos provide several advantages: (1) These embodiments are targeted. Separate proteins and protein probes from nanoreporters. Separation of the protein from the reporter eliminates the potential problems of solubility and stickiness associated with using the nanoreporter to measure the protein. Separation of the target protein from the nanoreporter avoids the challenge of Kd mismatch between DNA and protein, allowing the use of ideal concentrations for both maximum signal and minimum noise, if desired. Allows the use of low Kd antibodies; (2) The indirect signal oligo approach can be performed as an upstream process of the nanoreporter assay described below, thereby utilizing an optimized nanoreporter assay. (3) Protein probe sets (eg, antibody pairs) are used, if desired, in their usual arrangements, eg, as capture antibodies on the surface (eg, on magnetic beads) and as detection antibodies in solution. be able to. Some antibodies work best in this arrangement; (4) for these embodiments, the protein probe is off target (Koff rate), eg, only the antibody is targeted during binding and purification on the beads. Problems related to having to stay bound to are minimized. This allows the use of a wide range of antibodies, including antibodies with lower binding affinities; and (5) proteins can be read in the same lane as nucleic acids, eg RNA or DNA. Divide the sample first: Some are subjected to the protein detection embodiments described herein (cells are lysed with a detergent, then bound and purified as described herein) and some are Cut off and treat as nucleic acid sample (cells are lysed with GITC). The samples are then recombined and analyzed using a nanoreporter such as the one below, but may be done in the same lane. Measurement of both nucleic acid (eg, RNA) and protein in the same lane minimizes measurement differences, makes protein and nucleic acid expression data more comparable, and multiple measurement methods to obtain the required data. Eliminate the need for.

0083In some embodiments, the methods described herein provide a measurement of a nucleic acid, such as RNA or DNA, in combination with a measurement of a protein.

0084Any of the embodiments described herein can be used to detect multiple target proteins. In some embodiments, the invention provides a method comprising a protein probe for analysis of a target protein. In some embodiments, the invention provides a population of protein probes for use in a multiplexing assay. Each protein probe in the population is specific for the target molecule. The binding of the target protein to the protein probe is then detected using a nanoreporter. Each nanoreporter contains a unique labeling code that can be associated with a particular target molecule, as described below.

0085In some embodiments, the detection of nanoreporters, such as the ones below, is inherently digital in that one molecule is counted at a time. Fluorescence is used to read the code, but the signal is high, spots are either present or absent, and are therefore digital detection. Using digital detection rather than the analog fluorescent signal used to quantify the signal results in more accurate quantification. Therefore, the methods described herein allow multiplexing at levels above currently possible levels, more accurate quantification, and optionally higher sensitivity.

0086Nano reporter A nanoreporter that provides a code for a signal that associates with a specific target (nanoreporter labeling code). In some embodiments, upon binding of the nanoreporter to a signal oligo or linker oligo associated with the protein probe, the nanoreporter code identifies the signal oligo or protein probe to which the nanoreporter is bound. Thus, in some embodiments, the nanoreporter of the invention comprises two main parts: (i) a sequence specific for a signal oligo or linker oligo associated with a protein probe; and (ii) labeling. Nano reporter. In some embodiments, the nanoreporter is attached directly to the protein probe.

0087The nanoreporter has a modular structure. In some embodiments, the nanoreporter comprises a plurality of different detectable molecules. In some embodiments, labeled nanoreporters are molecular components that include certain basic elements: (i) multiple labeled attachment regions that are linearly combined and attached, and (ii) backbone. Complementary polynucleotide sequence attached to the labeled attachment region. In some embodiments, labeled nanoreporters are linearly combined and attached 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Includes a unique labeled attachment region of the backbone and a complementary polynucleotide sequence attached to the labeled attachment region of the backbone. In some embodiments, the labeled nanoreporter comprises three or more labeled attachment regions that are linearly combined and attached, and a complementary polynucleotide sequence that is attached to the labeled attachment region of the backbone. The term labeled attachment region includes a region of a defined polynucleotide sequence within a given backbone that can serve as an individual attachment point for a detectable molecule.

0088Multiple labeled attachment regions attached in a linear combination may include a uniquely designed sequence. In addition, multiple labeled attachment regions that are linearly combined and attached within the nanoreporter include at least one template, eg, but not limited to, cloning cassettes, derivatives including polylinkers, etc. Linear or linear viral genomes such as hepatitis virus, herpesvirus, rotavirus genomes, or at least one nucleic acid sequence such as at least a portion of a bacteriophage such as a lambda, M13, φX-174, T-series bacteriophage. Includes plasmids such as pBR322 and pUC series plasmids, including derivatives including cloning cassettes, polylinkers, etc .; synthetic templates; templates containing artificial sequences, etc. Virtually any piece of nucleic acid can be combined with at least one other nucleic acid sequence if it is large enough to contain at least two labeled attachment regions, and the combined sequence is Those skilled in the art will appreciate that it can serve as a template for making nanoreporters if it is large enough to contain at least two labeled attachment regions.

0089In some embodiments, the labeled nanoreporter also comprises a backbone that includes a constant region. The constant region can attach directly or indirectly to the nanoreporter. Thus, the constant region can be covalently attached to the nanoreporter, or the constant region can be attached to the nanoreporter later in the assay. The term constant region comprises a column repeat of about 10 to about 25 nucleotides. The constant region can be attached in either the 5'region or the 3'region of the nanoreporter, and the nano for imaging or detection, such as by attaching a sequence complementary to the constant region to a solid substrate. It may be used for capture and immobilization of reporters.

0090The elements of the nanoreporter can be found as a single molecular component (single nanoreporter) or two separate molecular components (double nanoreporter). Each molecular component may be composed of one molecule or more than one molecule attached to each other by covalent or non-covalent means. In some embodiments, each component of the dual nanoreporter has a signal oligo-specific sequence that binds to different sites on the same signal oligo molecule. When using a dual nanoreporter system, one of the nanoreporter probes may be unlabeled. In some embodiments, the unlabeled nanoreporter probe may include a capture region. In some embodiments, the unlabeled nanoreporter probe may include a signal oligo-specific region and a backbone that can be single-stranded. In some embodiments, the unlabeled nanoreporter probe may include a signal oligo-specific region and a backbone that can be double-stranded.

0091Complementary polynucleotide sequences attached to the nanoreporter backbone serve to attach detectable molecules or labeled monomers to the nanoreporter backbone. Complementary polynucleotide sequences may be labeled directly, for example, by covalent incorporation of one or more detectable molecules into the complementary polynucleotide sequence. Alternatively, the complementary polynucleotide sequence may be indirectly labeled, such as by incorporating biotin or another molecule capable of specific ligand interaction into the complementary polynucleotide sequence. In such an example, the ligand (eg, streptavidin in the case of biotin incorporation into a complementary polynucleotide sequence) may be covalently attached to a detectable molecule. If the detectable molecule attached to the labeled attachment region is not directly incorporated into the complementary polynucleotide sequence, this sequence acts as a bridge between the detectable molecule and the labeled attachment region. , A cross-linked molecule, for example, sometimes referred to as a cross-linked nucleic acid.

0092In some embodiments, the invention is incorporated herein by reference in its entirety, U.S. Pat. No. 7,473,767; U.S. Patent Application No. 10 / 542,458; No. 12 / 324,357; No. 11 / 645,270, And the nanoreporter described in 12 / 541,131 is used.

0093The nucleic acid-based nanoreporter, nanoreporter-signal oligo complex, or nanoreporter-protein probe complex of the present invention is affinity purified or purified using nucleic acids such as oligonucleotides that are complementary to the constant region of the nanoreporter. Contains nucleic acids that can be immobilized. As mentioned above, in some embodiments, the nanoreporter can serve as an affinity tag for purification and / or immobilization (eg, on a solid surface) at least one constant region. including. The constant region typically comprises two or more tandem repeat regions of repeating nucleotides, such as a series of 15-base repeats. In such an exemplary embodiment, the nanoreporter is coated with a 15-base oligonucleotide that is the inverse complement of the repeating unit, whether or not it is complexed with a signal oligo, the target molecule. It can be purified or immobilized with an affinity reagent.

0094The nanoreporter, nanoreporter-signal oligo complex, or nanoreporter-protein probe complex can be purified in two or more affinity selection steps. For example, in embodiments where the nanoreporter is attached to a protein probe, the nanoreporter may include an affinity tag. In other embodiments where signal oligos and dual nanoreporters are used, one nanoreporter probe may contain a first affinity tag and the other nanoreporter probe will carry a second (different) affinity tag. Can include. The nanoreporter probe is mixed with the signal oligo, and the complex containing the two probes of the dual nanoreporter is not bound by affinity purification to one or both individual affinity tags (eg, signal oligo, or nano). Separated from the reporter's individual probes). In the first step, the mixture can be attached to an affinity reagent for the first affinity tag, thereby purifying only the probe containing the first affinity tag and the desired complex. The bound material is released from the first affinity reagent and optionally attached to the affinity reagent for the second affinity tag, allowing separation of the complex from the nanoreporter probe containing the first affinity tag. At this point, only the complete complex will be bound. The complex is finally released from the affinity reagent for the second affinity tag, then preferably stretched and imaged. The affinity reagent can be any solid surface coated with an affinity tag binding partner, such as a column, beads (eg, latex or magnetic beads), or slides coated with a binding partner. Immobilization and stretching of nanoreporters with affinity reagents are incorporated herein by reference in their entirety, Sean M. et al.

0095The sequence of signals supplied by the labeled monomers associated with various labeled attachment regions of a given nanoreporter backbone allows for the unique identification of the nanoreporter. For example, when using a fluorescent label, a nanoreporter with a unique identity or unique spectral signature is associated with a signal oligo-specific sequence or protein probe that recognizes a particular target molecule or portion thereof. Detection of nanoreporter signals, such as the spectral code of fluorescently labeled nanoreporters, associated with the nanoreporter enables detection of the presence of target molecules in the mixture (qualitative analysis). Counting all labeled monomers associated with a given spectral code or signature allows counting of all molecules in a signal oligo-specific sequence or in a mixture associated with a protein probe bound to a nanoreporter (quantitative). analysis). In embodiments where the signal oligo is used, the signal oligo can then correlate with the target molecule through binding of the target molecule to a protein probe associated with the signal oligo. Therefore, nanoreporters are useful in diagnosing or prognosis of different biological conditions (eg, disease vs. health) by quantitative analysis of known biological markers.

0096In addition, due to the elaborate sensitivity of signal molecule detection and quantification provided by the nanoreporters of the present invention, fluctuations between different biological states can be combined with specific biological states using traditional molecular methods. Allows the identification of new diagnostic and prognostic markers, including those that are too minor to detect correlations. The sensitivity of molecular detection based on nanoreporters allows detailed pharmacokinetic analysis of therapeutic and diagnostic agents in small biological samples.

0097The synthesis of nanoreporters can be carried out by any suitable method known in the art. Examples of nanoreporter synthesis are incorporated herein by reference in their entirety, U.S. Pat. No. 7,473,767; U.S. Patent Application No. 10 / 542,458; No. 12 / 324,357; No. 11 / 645,270, and No. 12. It is described in / 541,131.

0098In one embodiment, the invention provides a nanoreporter that further comprises an affinity tag attached to the nanoreporter backbone, whereby attachment of the affinity tag to the support results in backbone stretch and different labeled attachment regions on the backbone. Allows the degradation of the signal provided by the corresponding labeled monomer. Nanoreporter stretches can include any stretch means known in the art, including but not limited to means including physical, hydraulic, or electrical means. The affinity tag may include a constant region.

0099The uniqueness of each nanoreporter probe in the population of probes allows multiplex analysis of multiple target molecules. For example, in some embodiments, each nanoreporter probe may contain 6 labeled attachment regions, and each labeled attachment region on each backbone is different from other labeled attachment regions on that same backbone. The label recognition area is to be labeled with one of four colors, and there are 24 possible unique sequences for the label attachment area, and each label attachment area is assigned a specific color. If so, each labeled attachment region on each backbone would consist of one of four sequences. In this example there would be 4096 possible nanoreporters. The number of possible nanoreporters can be increased, for example, by increasing the number of colors, increasing the number of unique sequences for labeled attachment regions, and / or increasing the number of labeled attachment regions per backbone. Can be made to. Similarly, the number of possible nanoreporters can be increased by reducing the number of colors, the number of unique sequences for the labeled attachment region, and / or the number of labeled attachment regions per backbone. Can be reduced.

0100In certain embodiments, the detection method is performed in a multiplex assay where multiple target molecules are detected in the same assay (single reaction mixture). In a preferred embodiment, the assay is a hybridization assay in which multiple target molecules are detected simultaneously. In certain embodiments, the plurality of target molecules detected in the same assay are at least 2, at least 5 different target molecules, at least 10 different target molecules, at least 20 different target molecules, and at least 50. Different target molecules, at least 75 different target molecules, at least 100 different target molecules, at least 200 different target molecules, at least 500 different target molecules, or at least 750 different target molecules, or at least 1000 Are different target molecules. In other embodiments, the plurality of target molecules detected in the same assay are up to 50 different target molecules, up to 100 different target molecules, up to 150 different target molecules, up to 200 different target molecules, Up to 300 different target molecules, up to 500 different target molecules, up to 750 different target molecules, up to 1000 different target molecules, up to 2000 target molecules, or up to 5000 target molecules. In yet another embodiment, the plurality of target molecules detected may be any range between the aforementioned numbers of different target molecules, eg, 20 to 50 different target molecules, 50 to 50. Up to 200 different target molecules, 100 to 1000 different target molecules, 500 to 5000 different target molecules, and much more.

0101In addition to the qualitative analysis capabilities provided by the nanoreporters of the present invention, and analytical techniques based on them, the nanoreporters of the present invention are uniquely suitable for performing quantitative analysis. Target molecules present in a biomolecular sample by providing a one-to-one bond between the nanoreporters of the invention (whether single nanoreporters or double nanoreporters) and their target molecules. All or representative parts of can be identified and counted. Individual counts of this various molecular species provide an accurate and direct method for determining the absolute or relative concentration of a target molecule in a biomolecular sample. In addition, the ability to individually address each molecule in the mixture provides high sensitivity, minimal sample requirements, high kinetics provided by liquid phase kinetics in small volumes, and ultimately very low reagent costs. The benefits of miniaturization, including, are being utilized individually.

0102Detectable molecule or labeled monomer The nanoreporters of the invention can be radioactive isotopes, fluorescent dyes, dyes, enzymes, nanoparticles, chemical luminescent markers, biotin, or directly (eg, by luminescence) or indirectly (eg, by binding of fluorescently labeled antibodies). It can be labeled with any of a variety of labeled monomers, such as other monomers known in the art that can be detected. Generally, one or more of the labeled attachment regions in the nanoreporter are labeled with one or more labeled monomers, and the signal provided by the labeled monomer attached to the labeled attachment regions of the nanoreporter is the nanoreporter. Consists of a detectable code that identifies the target to which the target-specific region of the In certain embodiments, the lack of a given signal from the labeled attachment region (eg, dark spots) can also constitute a portion of the nanoreporter code.

0103Examples of labeled monomers that can be used with the nanoreporters described herein, and examples of methods for incorporating labeled monomers into nanoreporters, are incorporated herein by reference in their entirety, U.S. Pat. No. 7,473,767. No.; US Patent Application No. 10 / 542,458; No. 12 / 324,357; No. 11 / 645,270, and No. 12 / 541,131.

0104Affinity tag Various affinity tags known in the art can be used, for example, to purify and / or immobilize nanoreporters. In some embodiments, a biotin anchor is attached to the nanoreporter, allowing immobilization of the nanoreporter on a streptavidin-coated slide.

0105In some embodiments, the labeled nanoreporter comprises an affinity tag, A1 and A2, at each end. Labeled nanoreporters can be immobilized on the surface through binding of A1 to an immobilized affinity partner. In the absence of an affinity binding partner for A2, the A2 end of the nanoreporter remains in solution, but in the presence of the affinity binding partner (A2'), the A2 end of the nanoreporter is also immobilized. In some embodiments, the labeled nanoreporter comprises a single affinity tag, A1. Another affinity tag, A2, can attach to the nanoreporter by direct binding of the nanoreporter to a molecule containing A2 (eg, if the nanoreporter is or contains nucleic acid, it is , Can hybridize directly with another nucleic acid to which A2 is attached). Alternatively, either affinity tag can be attached to the labeled nanoreporter via a cross-linked molecule such as a cross-linked nucleic acid. In some embodiments, upon immobilization of A1, the nanoreporter stretches or "stretches", for example, by electrostretching, for the separation of labeled attachment regions in a manner that allows detection of the nanoreporter code. can do. Optionally, at the same time that the nanoreporter is in an elongated state, A2 is introduced and descends to the surface to bind to the end of the nanoreporter, which is complementary to A2.

0106In some embodiments, the affinity tag is attached to the protein probe, for example, to purify and / or immobilize the protein probe.

0107Affinity tags can be used for attachment to beads or other matrices for a variety of useful applications, including but not limited to purification.

0108Examples of affinity tags, as well as examples of how to make them and / or attach them to the nanoreporters described herein, are incorporated herein by reference in their entirety, US Pat. No. 7,473,767. It is described in US Patent Application Nos. 10 / 542,458; 12 / 324,357; 11 / 645,270, and 12 / 541,131.

0109Biomolecular sample The protein probe and nanoreporter system of the present invention can be used to detect a target protein in any biomolecular sample. As recognized by those skilled in the art, the sample may contain any number of substances, examples include, but are not limited to: cells (including both primary and cultured cell lines). , Cell solubilized or cell extracts, and biological samples such as tissues and tissue extracts; body fluids (blood, urine, serum, lymph, bile, cerebrospinal fluid, interstitial fluid, atrioventricular fluid or vitreous fluid, primary milk , Sputum, sheep water, saliva, anal and vaginal fluids, sweat and semen, leaks, exudates (eg, abscesses, or fluids obtained from any other site of infection or inflammation) or virtually any organ Fluids obtained from joints (eg, normal joints or joints affected by diseases such as rheumatoid arthritis, osteoarthritis, gout, or purulent arthritis), but mammalian samples are preferred, human samples are preferred. Particularly preferred; environmental samples (including, but not limited to, air, agricultural, water, and soil samples); biological warfare samples; extracellular fluids from cell cultures, extracellular supernatants, inclusions in bacteria. , Cell compartments, cell periplasm, mitochondrial compartments, etc.

0110The biomolecular sample can be indirectly withdrawn from the biological sample. For example, if the target protein of interest is a kinase, the biomolecular sample of the invention can be a sample containing an isolated protein derived from a cell solubilized product. In another example, the biomolecular sample of the invention is made by subjecting a biological sample to a fraction, eg, a size fraction or a membrane fraction.

0111The biomolecular sample of the present invention may be natural (eg, unengineered or untreated) or processed (exposure to candidate agents, including drugs, genetically engineered (eg, genes). Can include any number of processes, including additions or deletions of).

0112Biomolecular samples can also include environmental samples such as those containing bacteria, or other organisms such as diatoms, whirlpool algae, and algae, especially in samples based on certain oceans or the earth.

0113Nanoreporter detection Nanoreporters are detected by any means available in the art capable of detecting a particular signal on a given nanoreporter. If the nanoreporter is fluorescently labeled, appropriate considerations for a suitable source of excitation may be examined. Possible sources include, but are not limited to, arc lamps, xenon lamps, lasers, light emitting diodes, or some combination thereof. A suitable source of excitation is used with a suitable optical detection system, such as an inverted fluorescence microscope, an epi-fluorescence microscope, or a confocal microscope. Preferably, a microscope is used that can allow detection with sufficient spatial resolution to determine the sequence of spots on the nanoreporter. For example, in one embodiment, an image of a dual nanoreporter hybridized to a target molecule can be obtained. For example, the nanoreporter has three different colors, Alexa 488, Cy3, and Alexa (named 1, 2, and 3, respectively). When labeled with 647, colors 1, 2, and 3 are acquired in different channels, respectively, and the first and second registers, which can be seen as a sequence of spots, may indicate each register individually. It is shifted up by a few pixels so that it can be done.

0114Examples of methods for detecting nanoreporters that can be used in the methods of the present invention are U.S. Pat. No. 7,473,767, "Methods for detection and quantification," entitled "Methods for detection and quantification of analytes in complex laminate." U.S. Patent Publication No. 2007/0166708, entitled "compositions comprising oriented," U.S. Patent Application No. 11 / 645,270 with the title of the invention "imposed macromolecules and methods for their preparation", PCT Application No. US06 / 049274 with the name of the invention "Nanoreporters and methods of manufacturing and use thereof", and "Stable nanoreporter" The title of the invention is described in US Provisional Application No. 60 / 08,988, all of which are incorporated herein by reference in their entirety.

0115Application of protein detection by nanoreporter technology The compositions and methods of the invention can be used for diagnostic, prognosis, treatment, patient stratification, drug development, treatment selection, and screening. The present invention provides the advantage that many different target proteins can be analyzed from a single biomolecular sample at one time using the methods of the present invention. This allows, for example, several diagnostic tests to be performed on a single sample.

0116The compositions and methods of the present invention can be used in proteomics. The methods described herein typically provide an answer quickly, which is highly desirable for this application. The methods and compositions described herein can be used in the process of finding biomarkers that can be used as indicators of diagnosis or prognosis, as well as health and disease. The methods and compositions described herein can be used to screen for drugs, eg, to identify targets for drug development, treatment selection, treatment efficacy determination, and / or drug development. it can. The ability to test protein expression in drug-related screening assays is very important because the protein is the final gene product in the body. In some embodiments, the methods and compositions described herein simultaneously measure both protein and gene expression, which provides the most information regarding the particular screening to be performed. It will be.

0117The method is obtained from a patient to determine whether the affected cell type is present in the sample, the stage of the disease, the prognosis of the patient, the ability of the patient to respond to a particular treatment, or the best treatment for the patient. It can be applied to the analysis of biomolecular samples derived from or derived from patients. The method can also be applied to biomarkers identified for a particular disease.

0118In some embodiments, the methods described herein are used to diagnose the condition. As used herein, the term "diagnosing" or "diagnosing" a condition means predicting or diagnosing the condition, determining the predisposition to the condition, monitoring the treatment of the condition, of the disease. Includes diagnosing a therapeutic response, as well as the prognosis of the condition, progression of the condition, and response to specific treatments of the condition. For example, blood samples are described herein for determining the presence and / or amount of disease markers or malignant cell types in a sample, thereby diagnosing or staging a disease or cancer. The assay can be performed according to any of the above methods.

0119In some embodiments, the methods and compositions described herein are used for diagnosis and prognosis of the condition.

0120A large number of immunological, proliferative, and malignant diseases and disorders are particularly suitable for the methods described herein. Immunological diseases and disorders include allergic diseases and disorders, impaired immune function, and autoimmune diseases and conditions. Allergic diseases and disorders include, but are not limited to, allergic rhinitis, allergic conjunctivitis, allergic asthma, atopic eczema, atopic dermatitis, and food allergies. Immunodeficiency includes severe combined immunodeficiency (SCID), eutropenia syndrome, chronic granulomatous disease, leukocyte adhesion deficiency I and II, hyperimmunoglobulin syndrome, Chediak-Higashi syndrome, and neutropenia. , Neutropenia, dysplasia, agammaglobulinemia, hyperimmunoglobulin syndrome, DiGeorg / palatal cardio-facial syndrome, and interferon gamma-TH1 pathway defects, but not limited to them. Autoimmune and immunodysregulation disorders include rheumatoid arthritis, diabetes, systemic lupus erythematosus, Graves' disease, Graves' eye disease, Crohn's disease, multiple sclerosus, psoriasis, systemic sclerosus, goiter and lymphomatous goiter ( Hashimoto thyroiditis, lymph node-like goiter), circular alopecia, autoimmune myocarditis, sclerosing lichen, autoimmune Graves' disease, Addison's disease, atrophic gastric inflammation, severe myasthenia, idiopathic thrombocytopenia Tissue destruction from purpura, hemolytic anemia, primary biliary cirrhosis, Wegener's granulomatosis, nodular polyarteritis, and inflammatory bowel disease, allogeneic transplant rejection and allergic reactions to infectious microorganisms or environmental antigens However, it is not limited to them.

0121Proliferative disorders and disorders that can be assessed by the methods of the invention include hemanomasis in neonates; secondary progressive multiple sclerosis; chronic progressive myelopathy; neurofibrosis; ganglion neuromatosis; Keroid formation; Bone Paget's disease; Fibrotic cystic disease (eg, breast or uterus); Sarcoidosis; Peylony and Dupuytran fibrosis, cirrhosis, atherosclerosis, and vascular restenosis, among others Not limited.

0122Malignant diseases and disorders that can be evaluated by the methods of the invention include both hematological malignancies and solid tumors.

0123Blood malignancies are particularly suitable for the methods of the invention, as such malignancies are involved in changes in blood-derived cells when the sample is a blood sample. Such malignant tumors include non-Hodgkin's lymphoma, Hodgkin's lymphoma, non-B-cell lymphoma, and other lymphomas, acute or chronic leukemia, erythrocytosis, thromboemia, multiple myeloma, myeloproliferative disorders, myeloproliferative disorders. These include sexual disorders, myeloma fibrosis, atypical immune lymphocyte proliferation, and plasma cell disorders.

0124Plasma cell disorders that can be assessed by the methods of the invention include multiple myeloma, amyloidosis, and Waldenström macroglobulinemia.

0125Examples of solid tumors include colon cancer, breast cancer, lung cancer, prostate cancer, brain tumor, central nervous system tumor, bladder tumor, melanoma, liver cancer, osteosarcoma and other bone cancers, testicular and ovarian cancers, head and neck tumors, and cervical neoplasms. Examples include, but are not limited to, organisms.

0126The methods described herein are used to diagnose pathogenic infections, such as those by intracellular bacteria and viruses, by determining the presence and / or amount of each marker in the sample, bacteria or virus. be able to.

0127A wide variety of infectious diseases can be detected by the processes of the invention. Typically, they are caused by infectious agents of bacteria, viruses, parasites, and fungi. Resistance to drugs of various infectious agents can also be determined using the present invention.

0128Bacterial infectious agents that can be detected by the present invention include Escherichia coli, Streptococcus salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium aviumintracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis. Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus Included are mirabilis, Helicobacter pylori, Treponema pallidum, Borrelia burgdorferi, Borrelia recurrentis, Rickettsial pathogens, Nocardia, and Acitnomycetes.

0129Fungal infectious agents that can be detected by the present invention include Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candida albicans, Aspergillus fumigautus, Phycomycetes, Phycomycetes (Rhizopus), Sporoth. Be done.

0130Infectious agents of viruses that can be detected by the present invention include human immunodeficiency virus, human T lymphocyte tropic virus, hepatitis virus (eg, hepatitis B virus and hepatitis C virus), Epstein-Burvirus, site. Examples include megalovirus, human papillomavirus, orthomixovirus, paramixovirus, adenovirus, coronavirus, rabdovirus, poliovirus, togavirus, buniavirus, arenavirus, eczema virus, and leovirus.

0131Parasite pathogens that can be detected by the present invention include Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmadium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia sp. Spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.

0132The present invention is also useful for detecting drug resistance by infectious agents. For example, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multidrug-resistant Mycobacterium tuberculosis, and AZT-resistant human immunodeficiency virus can all be identified in the present invention.

0133Thus, the target molecule detected using the compositions and methods of the invention can be either a patient marker (such as a cancer marker) or a marker of infection with a foreign pathogen such as a bacterial or viral marker.

0134Due to the quantitative nature of nanoreporters, the compositions and methods of the invention include target proteins whose amounts indicate biological or disease states, such as blood markers whose amount is upregulated or downregulated as a result of the condition. It can be used for quantification.

0135In some embodiments, the compositions and methods of the invention can be used for cytokine detection. The low sensitivity of the methods described herein will be useful for early detection of cytokines, for example, as biomarkers of the condition, as a diagnosis or prognosis of diseases such as cancer, and as identification of asymptomatic conditions. ..

0136kit The present invention further provides a kit comprising one or more components of the present invention. The kit may include, for example, one or more protein probe sets and / or one or more nanoreporters. The kit can be used for any purpose apparent to those skilled in the art, including those described above.

0137In certain embodiments, the invention also provides a kit useful for stretching and selective immobilization of nanoreporters. The kit may include a substrate for immobilization and one or more binding partners to facilitate the extension or immobilization of the nanoreporter. The binding partner may, in certain embodiments, contain components useful for the extension of the nanoreporter with appropriate force. In certain embodiments, the binding partner can facilitate immobilization or selective immobilization of the nanoreporter on the surface. In a further embodiment, the kit may include a nanoreporter for extension and immobilization. In a further embodiment, the kit may include a device capable of extending the nanoreporter.

0138The kit may include a population of protein probes and / or nanoreporters as described herein.

0139The kit may include a pre-labeled nanoreporter, or an unlabeled nanoreporter with one or more components for labeling the nanoreporter. In addition, the nanoreporters provided in the kit may or may not have pre-attached target-specific sequences. In one embodiment, the target sequence is provided within the kit without attaching to the nanoreporter backbone.

0140The kit may include other reagents such as signal oligos, linker oligos, and crosslinked oligos. In some embodiments, the kit can separate protein probe pairs into different premixes.

0141The kit may also include other reagents, such as buffers, linkers, restriction endonucleases, and DNA I ligases to carry out hybridization reactions.

0142The kit also includes instructions for using the components of the kit and / or for making and / or using labeled nanoreporters.

0143Preferred embodiments of the present invention will be presented and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will come to mind for those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims define the scope of the invention and are intended to cover methods and structures within the scope of these claims and their equivalents.

<p num="0144"> Example 1. Protein detection using indirect measurements-sandwich assay in solution FIG. 1 shows a diagram of the protocol for this embodiment. In this example, the assay is configured to separate protein target binding from reporter hybridization to eliminate problems with the mismatch between the low binding affinity of the protein probe and the concentration of action of the reporter.</p><p num="0145"> Labeling and purification of detection antibody with oligo Linker oligos were attached to the antibodies by random amine attachment using a 10: 1 linker oligo to antibody ratio. Briefly, the bifunctional crosslinker sulfosuccinimidyl 4- [p-maleimidephenyl] butyrate (SMPB) (Thermo Fisher, Inc., Waltham, MA) was bound to anti-IL2 antibody A and then at room temperature. Then, the antibody: oligo ratio of 1: 3 was reacted with the thiolated oligo and cross-linked. The mixture is passed through a Zeba column 2X, 1000G (Thermo Fisher, Inc., Waltham, MA) to purify antibody A linked to SMPB and determine yield.</p><p num="0146"> To bind the oligo to SMPB-linked IL-2 antibody A, the oligo was added to the purified SMPB-linked antibody at 4 ° C.</p><p num="0147"> Antibody-SMPB-oligo and PBE, which are oligos linked to IL-2 antibody A, are separated into Pall Nanosep® centrifuges (MWCO 100kDa, Sigma-Aldrich, Inc., St. Louis, MO) with Omega Membrane. ), Washed and centrifuged.</p><p num="0148"> Hybridization of signal oligos to detection antibodies Oligo-linked Il2 antibody A and signal oligo were added in a 3: 2 signal oligo: antibody ratio to pre-anneal the signal oligo to the oligo linked to IL-2 antibody A. Other ratios are planned.</p><p num="0149"> Formation of target-antibody complex IL-2 antibody A annealed to signal oligo, about 1 × 10^-15~1×10^-8Biotinylated antibody B in M (BAF202, R &amp; D systems, Inc., Minneapolis, MN), and blocker (salmon sperm) were mixed, leaving room for the target solution to be added. Desirable dilution of target protein IL-2 (&lt;1 × 10)^-8Added up to M). Antibodies, 10^0~15~10^0~8The estimated Kd of M was set to 10 × concentration. The mixture was incubated.</p><p num="0150"> The target protein-antibody complex was purified according to the protocol using streptavidin-bound Dynabeads® (Invitrogen).</p><p num="0151"> Signal oligo elution The isolated target protein / antibody complex was washed and the signal oligos were eluted with 0.1 × SSPE at temperatures above 45 ° C for 10-15 minutes. Shorter and longer times are planned.</p><p num="0152"> Detection of signal oligos Detection of signal oligos in each sample was performed using a dual nanoreporter system with both labeled and unlabeled nanoreporter probes. Signal oligos from each sample were hybridized to final concentrations of hybridization reagents such as: unlabeled biotinylated probe, labeled reporter probe, 5 x SSPE (pH 7.5), 5 x Denhardt reagent (Sigma). , Shredded salmon sperm DNA (Sigma), and surfactants. The reagents were mixed and incubated in a thermocycler block with a heating lid for 16 hours.</p><p num="0153"> Purification after hybridization To remove non-hybridized reporters, the reactants were purified for magnetic beads (InvitrogenTM) bound to oligonucleotides complementary to the 3'-repetition contained in each biotinylated probe. The reaction was first diluted with SSPE mixture / TE in 0.1% detergent and attached to the beads at temperatures above 20 ° C. with continuous rotation. The beads were washed 3 times in SSPE and detergent and the hybridized complex was eluted in a 0.1 x SSPE / 0.1% detergent mixture at 45 ° C for 15 minutes. After elution, the sample was purified a second time to remove excess biotinylated probe by binding to magnetic beads bound to oligonucleotides complementary to the 5'-repetition sequence contained in each reporter probe. .. Elution from anti-3'-repeated beads was provided at a final concentration of 1 x SSPE and bound at 22.5 ° C for 15 minutes with rotation. The beads were washed as described above and eluted in a 0.1 x SSPE / 0.1% surfactant mixture at a temperature above 40 ° C. The doubly purified sample was then prepared for capture as described below.</p><p num="0154"> NanoString Reporter Capture, Stretching, and Imaging A solution of a custom formulation of Tetraspeck Fluorescent Microspheres (InvitrogenTM) was added to each sample. Samples were charged into the NanoString fluid system, processed and imaged.</p><p num="0155"> result: The result is shown in figure 2. The results of this experiment showed that IL-2 was detected by the assay described herein (Fig. 2). From this experiment, about 110^-11~110^-10The sensitivity of M was shown. The efficiency of detection is the slope shown in the plot in FIG. The observed efficiency was probably due to the binding affinity of the antibody to which the signal oligo was attached. This antibody is about 1.3 x 10^-7Seems to have Kd. Efficiency is about 10 for this antibody^-9It is expected that it can be increased by 100 × by exchanging with an antibody having Kd.</p><p num="0156"> With improved efficiency and moderate background improvement, the sensitivity is 1x10.^-13It should reach the level of, but further improvement is expected to be possible with a continuous decline in background.</p><p num="0157"> This technology has the potential to enable multiplexing at levels beyond what is currently possible, allowing for more accurate quantification, and there will only be further development, but perhaps higher sensitivity is the last point. You will understand.</p><p num="0158"> Example 2. Protein detection using direct measurement-Solution tripartite-Protein probe attached to reporter FIG. 4 shows a diagram of the protocol for this embodiment. In this example, the nanoreporter is attached to one of the antibodies. Preparation of the antibody and binding to the sample occur similar to the protocol described in Example 1. In the assay described in FIG. 4, a complex of target protein and antibody is formed, with one of the antibodies bound to biotin and the other antibody attaching the nanoreporter.</p><p num="0159"> This approach will work best by using medium to low Kd, ie antibodies with strong binding affinity. Although not limited to any theory, the Kd (dissociation constant) of an antibody is usually much higher than the concentration of action of the nanoreporter. In order to work with the antibody attached to the nanoreporter, Kd needs to be one tenth of the reporter's concentration to ensure&gt; 90% binding to the target probe. Figure 5 shows the ideal Kd calculation for the probe required for this assay. Figure 5 shows the ratio of bound targets to the nanoreporter probe and protein probe Kd. From Fig. 5, the ideal Kd for the protein probe is about 1.0 × 10.^-15~1.0×10^-10It is shown that it will be. This Kd is 1x10^-7Allows the concentration of multiplexed reporters below the entanglement threshold of M reporters.</p><p num="0160"> Adhesion of nanoreporter to antibody One approach is to attach the antibody to the reporter prior to binding the antibody to the target protein. This approach is 1x10^-7A very strong binding affinity (Kd) antibody is required to allow concentrations of the multiplexed reporter significantly below the M reporter entanglement threshold.</p><p num="0161"> Linker oligos are added to the antibody as described in Example 1. The nanoreporter is attached to the antibody by hybridization to the linker oligo at temperatures between 37 ° C and 45 ° C. The antibody: reporter ratio was 1: 1. Labeled antibodies and reporters were hybridized overnight at 0.05 nM, 37 ° C, 1 × SSPE.</p><p num="0162"> The second direct approach is to first bind the antibody to the target in solution to purify the complex as described in Example 1. After purification, the nanoreporter is hybridized to the antibody (after target binding) using the protocol described above. In this approach, the problem of mismatch between hybridization and protein binding is avoided.</p><p num="0163"> For both of these approaches, strong binding affinity is also required to remain bound during purification and imaging. Basically, the Koff rate (antibody dissociation from the target protein) should be longer than the time for purification and assay reading.</p><p num="0164"> Purification of the target protein-antibody complex can be performed as described in Example 1. Elution can be achieved by melting the G and F beads or by digestion. However, those skilled in the art understand that melting the complex may require optimization of the conditions to allow the affinity tag to be released, although the antibody remains bound. There will be.</p><p num="0165"> As described in Example 1, the protein-antibody complex is bound to a streptavidin-coated cover glass (Optichem®, Accelr8 Technology Corporation), stretched and imaged. Can be done.</p><p num="0166"> Stretching of the reporter hybridized to the antibody to ensure that the presence of the antibody does not cause non-specific binding or stickiness to the surface that interferes with normal binding, stretching, and imaging processes. Tested (data not shown).</p><p num="0167"> Example 3. Protein Detection Using Indirect Measurements-Surface Sandwich Assay FIG. 6 shows a diagram of the protocol for this embodiment. In this example, the captured antibody is attached to a surface, eg, a magnetic bead, and the second antibody is attached to a signal oligo. Antibodies can be prepared by any method known in the art, including the method described in Example 1. In this example, the protein and the nanoreporter are far enough apart, which eliminates concerns about protein stickiness. In this assay, local antibody concentrations on the surface can be high.</p><p num="0168"> The target protein is mixed with the captured antibody on magnetic beads (2 hours to overnight, 1 x PBS, and room temperature). Rinse off unbound protein samples. A complex of labeled antibody signal oligos is added to the beads with a blocker (1 x PBS and room temperature). After a period of binding, the excess labeled antibody signal oligo complex is washed away. The isolated target protein / antibody complex is then washed, the signal oligos are eluted and analyzed as described in Example 1.</p><p num="0169"> Example 6. Protein Detection Using Indirect Measurements-Biotinylated Signal Oligo FIG. 7 shows a diagram of the protocol for this embodiment. In this example, the captured antibody is attached to a surface, eg, a magnetic bead, and the second antibody is either in solution or attached to a biotinylated signal oligo. This assay offers the advantage that it requires only two bead purifications. In addition, in this assay, as in Example 2, the protein and nanoreporter are well separated, which eliminates concerns about protein stickiness. As described in Example 3, the target protein is mixed with a captive antibody, a complex of labeled antibody signal oligos, and a blocker. As described in Example 3, the target protein-antibody complex is purified using the magnetic beads in the captured antibody.</p><p num="0170"> The isolated target protein / antibody complex is then washed and the signal oligos are eluted as described in Example 1. The signal oligo can then be purified using streptavidin-bound Dynabeads® (Invitrogen) according to the manufacturer's protocol. The signal oligos are then analyzed as described in Example 1.</p><p num="0171"> Example 7. Detection of Protein Using Proximity Ligase-Indirect Measurement FIG. 8 shows a diagram of the protocol for this embodiment. In this assay, two physically close oligos are ligated. Oligo-containing probes are designed to bind to the target protein as a pair and to form a signal oligo by ligation when the probes are in close proximity.</p><p num="0172"> This approach has several benefits centered around sensitivity, cross-reactivity minimization, and multiplexing. Proximity ligation exhibits high sensitivity and has the effect of reducing apparent Kd by essentially reducing off-speed.</p><p num="0173"> Probes containing oligos are prepared and purified as described in Gullberg et al., PNAS 101 (22), p8420-24 (2004). The target protein is then mixed with the oligo-containing probe and cross-linked oligo by incubating the sample for 1 hour. Then add the components needed for probe ligation, as described by Gullberg et al. After 5 minutes of ligation at room temperature, the signal oligos are released by disulfide reduction, uracil removal, restriction digestion, proteinase K, or any other suitable method known in the art. In addition, signal oligos can be released by the methods shown in Figures 8B-8D.</p><p num="0174"> Signal oligos are analyzed as described in Example 1.</p><p num="0175"> Alternatively, the assay can be performed as shown in FIG. In this approach, one of the oligos is attached to the nanoreporter.</p><p num="0176"> This approach takes advantage of the reduced Koff due to proximity ligation. Lower Koff means the ability to act with lower Kd and lower concentrations of protein probes. This reduction in Kd makes it easier to act at the required concentration for the reporter and, accordingly, to design a direct detection approach for multiplex analysis and reduce reagent costs. This approach does not require a hybridization step to the reporter within the assay, as some of the other methods proposed herein. Therefore, these assays will be faster and will respond in less time.</p><p num="0177"> The approach described in Figure 8C is used to optimize purification conditions so that after liberation from the antibody, the ligated oligo is removed from the unligated oligo. For example, this purification step can be performed using magnetic beads. Importantly, if the amount of antibody used is higher than the amount of reporter used, the resulting over-ligated oligo can block the hybridization of the reporter to the oligo. is there.</p><p num="0178"> The antibody pair was oligo-labeled using the method described in Example 8. These oligos were designed to contain 9 base overlaps and cross-linked oligos with a melting temperature of 37 ° C in 1 × PBS. The crosslinked oligo had 18 bases and a biotin tag for purification purposes. The ligated oligo has biotinylated crosslinks and 18 base overlaps, so that the ligated oligo is more stable than the unligated oligo bound to the antibody. The biotinylated oligo is isolated from the solution on magnetic beads coated with streptavidin. It was determined that only the ligated oligos had a melting temperature high enough to remain attached to the biotinylated oligos after the rinsing step with stringent buffer conditions.</p><p num="0179"> Figure 13 shows how different components are purified under different buffer conditions. The component was present in solution at the concentration used for the assay. Prior to purification, the solution was digested with protease to release the oligo from the antibody, as performed in the assay. As predicted by the melting temperature estimates, 0.03 × SSPE provided the most efficient buffer. PROX05 represents the ligated product retained after this rinsing process.</p><p num="0180"> Example 8. Protein Detection Using Indirect Measurements-Multiplex Assay in Solution This example is similar to Example 1, but with multiple detections, utilizing different binding chemistries between the antibody and the oligo.</p><p num="0181"> Bioinformatics Signal oligos were designed to have minimal cross-reactivity at a range of temperatures, typically from about 4 ° C to about 37 ° C, and at 1 × PBS. The unique overlap between the signal oligo and the labeled oligo had a melting temperature of 51-56 at 1 × PBS, allowing the signal oligo to hybridize to the labeled oligo at 37 ° C. The duplication of these 15-17 bases had a melting temperature of 41 ° C to 45 ° C at 0.1 × SSPE, allowing elution after purification of the magnetic beads.</p><p num="0182"> Binding of oligos to antibodies Oligos were attached to the antibody using aldehyde-hydrazine chemistry. All antibodies and targets were purchased from R &amp; D systems, Inc. (Minneapolis, MN). Each antibody A (see Table 1) was desalted using a size exclusion spin column (0.5 ml Zeba Spin Column, Fisher Scientific, Pittsburgh, PA). Succinimidyl 6-hydrazinonicotinate acetone hydrazone (Solulink, San Diego, CA) was reacted with each antibody A. Each antibody was purified again using a size exclusion spin column.</p><p num="0183"> Table 1. Antibodies</p><p num="0184"><tables num="1"><img id="000002" he="38" wi="83" file="JP2016136162A_D0001.tif" img-format="tif" img-content="drawing" /></tables> Amine oligos were desalted using a membrane spin column (5K MWCO VivaSpin, Fisher Scientific, Pittsburgh, PA). 20 molar equivalents of succinimidyl-4-formylbenzoate (Solulink, San Diego, CA) were reacted with each oligo. The oligo was purified again using a membrane spin column.</p><p num="0185"> Each corresponding modified oligo was reacted with the corresponding modified antibody in a molar ratio of 3: 1. This was purified on a spin column (2 ml Zeba Spin Column, Fisher Scientific, Pittsburgh, PA). Table 2 shows that between 1-2 oligos were attached to each antibody at the end of the counting process (oligos: Ab).</p><p num="0186"> Table 2. Molar substitution rate indicating quality control and quantification ability of antibody-oligo binding process</p><p num="0187"><tables num="2"><img id="000003" he="47" wi="142" file="JP2016136162A_D0001.tif" img-format="tif" img-content="drawing" /></tables> Hybridization of signal oligos to detection antibodies Each unique signal oligo was pre-annealed separately by adding oligo-linked antibody A and the corresponding signal oligo to the unique oligo linked to each antibody A in a 4: 1 ratio, signal oligo: antibody ratio. Other ratios are planned.</p><p num="0188"> Formation of target-antibody complex Approximately 1 x 10 with a blocker (salmon sperm)^-15~1×10^-8A 2x master mix containing each antibody A and biotinylated antibody B (4 pairs) annealed to signal oligos in a single tube with M was made. Desirable dilution for aliquots of this master mix (&lt;1 x 10)^-8The target protein was added up to M). Antibody is 10^-15~10^-8It was 10 × concentration for the estimated Kd of M. The mixture was incubated.</p><p num="0189"> The target protein-antibody complex was purified according to the protocol using streptavidin-bound Dynabeads® (Invitrogen).</p><p num="0190"> Signal oligo elution The isolated target protein / antibody complex was washed and the signal oligos were eluted with 0.1 × SSPE at temperatures above 45 ° C for 10-15 minutes. Shorter and longer times are planned.</p><p num="0191"> Detection of signal oligos Detection of signal oligos in each sample was performed using a dual nanoreporter system with both labeled and unlabeled nanoreporter probes. Signal oligos from each sample were hybridized to final concentrations of hybridization reagents such as: unlabeled biotinylated probe, labeled reporter probe, 5 x SSPE (pH 7.5), 5 x Denhardt reagent (Sigma). , Shredded salmon sperm DNA (Sigma), and surfactants. The reagents were mixed and incubated in a thermocycler block with a heating lid for 16 hours.</p><p num="0192"> Purification after hybridization To remove non-hybridized reporters, the reactants were purified for magnetic beads (InvitrogenTM) bound to oligonucleotides complementary to the 3'-repetition contained in each biotinylated probe. The reaction was first diluted with SSPE mixture / TE in 0.1% detergent and attached to the beads at temperatures above 20 ° C. with continuous rotation. The beads were washed 3 times in SSPE and detergent and the hybridized complex was eluted in a 0.1 x SSPE / 0.1% detergent mixture at 45 ° C for 15 minutes. After elution, the sample was purified a second time to remove excess biotinylated probe by binding to magnetic beads bound to oligonucleotides complementary to the 5'-repetition sequence contained in each reporter probe. .. Elution from anti-3'-repeated beads was provided at a final concentration of 1 x SSPE and bound at 22.5 ° C for 15 minutes with rotation. The beads were washed as described above and eluted in a 0.1 x SSPE / 0.1% surfactant mixture at a temperature above 40 ° C. The doubly purified sample was then prepared for capture as described below.</p><p num="0193"> NanoString Reporter Capture, Stretching, and Imaging A solution of a custom formulation of Tetraspeck fluorescent microspheres (InvitrogenTM) was added to each sample. Samples were charged into the NanoString fluid system, processed and imaged.</p><p num="0194"> result The results are shown in Fig. 10. The results of this experiment showed that four proteins were detected simultaneously by the assays described herein (Fig. 10). The proteins detected were TNFα, IL1α, IL6, and VEGF. From this experiment, about 1x10^-12The sensitivity of M was shown. FIG. 11 shows the same data plotted against a liquid sample. In the following experiment (Figure 12), the detection limits for two of these proteins were 26 pg / ml and 38 pg / ml (1.4 × 10 for IL1α and IL6, respectively).^-12M and 1.9 × 10^-12It was shown to be M).</p><p num="0195"> Improvements in the background, i.e. reducing background detection or increasing the ratio of background to target detection, allow for an increase in sensitivity of about two orders of magnitude, and accordingly the sensitivity is 1x10.^-14It will reach M, or significantly &lt;1 pg / ml.</p><p num="0196"> Example 9. Anti-streptavidin probe reporter Anti-streptavidin antibodies (Affinity Bioreagents, Rockford, IL) were labeled with oligonucleotides (oligos) as described in Example 1.</p><p num="0197"> In a particular example of this embodiment, antibody-labeled oligos were hybridized to a reporter overnight at a concentration of 0.05 nM at a temperature of 45 ° C. in 1 × SSPE buffer. There was a 25 base overlap between the reporter and the oligo bound to the antibody. This duplication may be shortened, if desired. In certain embodiments, the duplication is optionally one, five, ten, fifteen, twenty, twenty-five bases, or any length between them. The shorter overlap of bases between the reporter and the oligo on the antibody allows increased efficiency of hybridization of the antibody-oligo to the reporter at temperatures that result in antibody stability. A reporter with an anti-streptavidin antibody probe was introduced into the flow chamber of the cartridge (at a concentration of 0.025 nM in 0.25 x SSPE buffer) and the first end of the reporter was bound to the streptavidin surface for 10 minutes. The chamber was then washed with TAE buffer. The reporter is stretched by first using a 200 volt (V) / centimeter (cm) electric field and then introducing a biotinylated oligo to attach the second end of the reporter to the surface, and then the surface. Fixed to. Samples are washed again with TAE and SlowFade to stabilize the dye^TMWas introduced. The sample was then imaged.</p><p num="0198"> FIG. 14 shows that only the reporter (S16) with the anti-streptavidin probe was detected.</p><p num="0199"> Other embodiments Preferred embodiments of the present invention will be presented and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will come to mind for those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein can be used to practice the invention. The following claims define the scope of the invention and are intended to cover methods and structures within the scope of these claims and their equivalents.</p>