Lateral Flow Analyte Detection
20170370925 · 2017-12-28
Inventors
Cpc classification
G01N33/54389
PHYSICS
International classification
Abstract
Lateral flow methods and apparatuses for detecting one or more analytes are provided. Certain embodiments provide a lateral flow device, kit and method of using the same, comprising: a flow path defined by a permeable sub-assembly of the lateral flow device, a release zone comprising a plurality of peptide-tagged agents, and a detection zone comprising a plurality of anti-peptide agents present in the flow path at a ratio of at least 100:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents.
Claims
1. A method to detect a pre-determined analyte, comprising: i) exposing a sample comprising the pre-determined analyte to a plurality of peptide-tagged agents to form an analyte complex, said analyte complex comprising the pre-determined analyte and at least one of the plurality of peptide-tagged agents; and ii) transporting the analyte complex to a detection zone of a lateral flow device, said detection zone comprising a plurality of anti-peptide agents present at a ratio of at least 100:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents, said plurality of anti-peptide agents capable of immobilizing at least one of the at least one of the plurality of peptide-tagged agents; and iii) detecting the pre-determined analyte.
2. The method of claim 1, wherein the transporting comprises capillary flow action along a flow path.
3. The method of claim 2, wherein the flow path is a divergent flow path.
4. The method of claim 1, wherein the plurality of peptide-tagged agents is releasably bound to a release zone of the lateral flow device.
5. The method of claim 1, further comprising: introducing the analyte complex to the lateral flow device.
6. The method of claim 1, wherein the plurality of anti-peptide agents is present at a ratio of at least 300:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents.
7. The method of claim 1, further comprising: immobilizing the analyte complex in the detection zone of the lateral flow device.
8. The method of claim 7, wherein detecting the pre-determined analyte comprises detecting the immobilized analyte complex.
9. The method of claim 1, further comprising: exposing said sample to a plurality of detectable agents, said analyte complex further comprising at least one of said plurality of detectable agents.
10. The method of claim 9, further comprising: immobilizing the analyte complex in the detection zone; wherein detecting the pre-determined analyte comprises detecting the at least one of said plurality of detectable agents.
11. The method of claim 9, wherein the plurality of detectable agents and the plurality of peptide-tagged agents are mixed and striped onto a release zone of the lateral flow device.
12. The method of claim 9, wherein the plurality of detectable agents and the plurality of peptide-tagged agents are separately striped onto the release zone.
13. The method of claim 9, wherein said plurality of anti-peptide agents are present at a ratio of at least 8:1, on a weight:weight basis, relative to the plurality of detectable agents.
14. The method of claim 9, wherein the plurality of detectable agents are present at a ratio of at least 2:75, on a weight:weight basis, relative to the plurality of peptide-tagged agents.
15. The method of claim 1, wherein at least one of the plurality of peptide-tagged agents comprises a plurality of covalently-conjugated peptide tags.
16. The method of claim 15, wherein at least one of the plurality of anti-peptide agents comprises a specific binding partner to at least one of the plurality of covalently conjugated peptide tags.
17. The method of claim 1, wherein the sample is formed by contacting a solvent with a material present on the lateral flow device.
18. The method of claim 17, wherein the solvent is contacted with the material present on the lateral flow device.
19. A method to detect a pre-determined analyte, comprising: i) receiving a sample comprising the pre-determined analyte to a permeable release zone of a lateral flow device, said permeable release zone comprising a plurality of peptide-tagged agents and a plurality of detectable agents striped thereon; ii) forming an analyte complex comprising the pre-determined analyte bound to at least one of the plurality of peptide-tagged agents and separately bound to at least one of the plurality of detectable agents, wherein the at least one of the plurality of peptide-tagged agents: a) is a pre-determined specific binding partner with the pre-determined analyte; and b) comprises a plurality of covalently conjugated peptide tags; iii) transporting the analyte complex by capillary flow action to a detection zone of the lateral flow device, said detection zone comprising a plurality of anti-peptide agents, wherein said plurality of anti-peptide agents are present at a ratio of: a) at least 300:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents; and b) at least 8:1, on a weight:weight basis, relative to the plurality of detectable agents; iv) immobilizing the analyte complex to the detection zone by selectively binding at least one of the plurality of anti-peptide agents with at least one of the plurality of covalently conjugated peptide tags; and v) detecting the immobilized analyte complex within 8 minutes of introducing the sample.
20. (canceled)
21. The method of claim 3, wherein: i) the plurality of peptide-tagged agents is releasably bound to a central release zone of the lateral flow device; and ii) the divergent flow path is a radial flow path.
Description
DETAILED DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0095] Certain embodiments provide lateral flow apparatuses and systems to detect one or more analytes, for example: assay platforms, lateral flow devices, assemblies, single-purpose kits, multi-purpose kits, and divergent flow devices. Other types of lateral flow apparatuses are contemplated herein. Certain embodiments provide lateral flow methods to detect one or more analytes, including, for example, methods of detection, methods of making lateral flow apparatuses, and methods of making lateral flow kits. Other types of lateral flow methods are contemplated herein.
[0096] Suitable analytes may include any analyte to which a peptide-tagged agent is able to bind (for example the peptide-tagged agent may be designed or selected to bind a pre-determined analyte). In certain embodiments, the analyte may be a predetermined analyte, for example an analyte for which a specific type of peptide-tagged agent is selected. In certain embodiments, the analyte may comprise, for example, a protein, microbe or a fragment thereof, a virus or a fragment thereof, a peptide, a biomarker, an antibody (for example, an antibody to an infectious agent) or a fragment thereof, a nucleic acid, a macromolecule, a small molecule, a drug, a hormone (for example, human chorionic gonadotrophin) or a fragment thereof, a hapten or a fragment thereof, etc. In certain embodiments, the analyte may be any analyte which has heretofore been assayed using known immunoassay procedures, or known to be detectable by such procedures (see, for example, “The Immunoassay Handbook,” Fourth Ed., D. Wild ed., Elsevier Ltd. (2013)). Other types of analytes are also contemplated herein.
[0097] Protein analytes may include, for example, a phosphoprotein. A range of phosphoproteins are known including, for example, phosphorylated ERK, S6 p240/44, AKT pT308 or AKT pS473. In certain embodiments, the analyte may comprise, for example, a component of a cell signaling pathway, a cytokine, or a tumour suppressor. In certain embodiments, the analyte may comprise, for example, a protein that is phosphorylated, methylated, or glycosylated, etc. In certain embodiments, the analyte may comprise, for example, phospho-ERK 1/2; total ERK 1/2; phospho-Akt 1/2/3; total Akt 1/2/3; phospho-NF- Kr3 p65; total NF-K13 p65; phospho-l-kB; total-kBP; phospho-STAT3; total STAT3; phospho-STAT5 A/B; phospho-JNK 1/2/3; total JNK 1/2/3; phospho-p38 MAPK; total p38 MAPK; phospho-p53; total p53; phospho-p70S6K; total p70S6K; and GAPDH. In other embodiments, the protein may comprise, for example, an acute phase protein, for example a C-reactive protein, haptoglobin, hemopexin, alpha-1 acid glycoprotein, clusterin, alpha-2-macroglobulin, serum amyloid A (SAA) or serum amyloid P (SAP) in species including but not limited to human, mouse, rat, rabbit, cat, dog, pig, cow, chicken, and monkey. In certain embodiments, the protein may comprise, for example, a particular form or state of a protein, an endogenous protein, or a transfected protein.
[0098] In certain embodiments, the analyte may comprise, for example, a biomarker, for example a cancer biomarker (for example a prostate cancer biomarker or a bowel cancer biomarker) or a diabetic kidney disease biomarker. In certain embodiments, the analyte may comprise, for example, an analyte useful for detection in plant or contaminant diagnostics. In certain other embodiments, the analyte may comprise, for example, an analyte useful for detection in human or veterinary diagnostics. In certain embodiments, the analyte may be capable of migrating, dissolving, disconnecting, or diffusing from the sample into a solvent. Suitable solvents may comprise, for example, water, an alcohol, a buffered solution, a pH-balanced solution, an organic solvent, or a combination of two or more thereof.
[0099] In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be in a range of 0-100 ng/mL, for example, between 1-100 ng/mL, between 1-50 ng/mL, between 1-25 ng/mL, between 10-50 ng/mL, of in a range of 1-10 ng/mL. In certain embodiments, the concentration of the analyte, the one or more analytes may be below 1 ng/mL. In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be above 50 ng/mL. In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be 100 ng/mL or less, for example 50 ng/mL or less, 10 ng/mL or less, 1 ng/mL or less, 100 pg/mL or less, or 10 pg/mL or less, 1 pg/mL or less, 100 fg/mL or less, 10 fg/mL or less, or 1 fg/mL or less. In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be greater than 10 ng/mL, greater than 1 ng/mL, greater than 100 pg/mL or, greater than 10 pg/mL, greater than 1 pg/mL, greater than 100 fg/mL, greater than 10 fg/mL, or greater than 1 fg/mL. In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be in the range of 1 fg/mL to 100 ng/mL, for example 1 fg/mL to 10 ng/mL, 1 fg/mL to 1 ng/mL, 10 fg/mL to 100 ng/mL, 10 fg/mL to 10 ng/mL, 10 fg/mL to 1 ng/mL, 100 fg/mL to 100 ng/mL, 100 fg/mL to 10 ng/mL, 100 fg/mL to 1 ng/mL, 1 pg/mL to 100 ng/mL, 1 pg/mL to 10 ng/mL, or in the range of 1 pg/mL to 1 ng/mL.
[0100] In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be in a range of 0 mIU/mL (milli-International Units per milli-liter) to 100 mIU/mL, for example 0.25 mIU/mL to 0.5 mIU/mL, 0.5 mIU/mL to 1 mIU/mL, 1 mIU/mL to 2 mIU/mL, 2 mIU/mL to 5 mIU/mL, 5 mIU/mL to 10 mIU/mL, 10 mIU/mL to 15 mIU/mL, 15 mIU/mL to 25 mIU/mL, 25 mIU/mL to 50 mIU/mL, or in a range of 50 mIU/mL to 100 mIU/mL. In certain embodiments, the concentration of the analyte or any one of a plurality of analytes may be less than 100 mIU/mL, less than 50 mIU/mL, less than 25 mIU/mL, less than 15 mIU/mL, less than 10 mIU/mL, less than 5 mIU/mL, less than 2 mIU/mL, less than 1 mIU/mL, less than 0.5 mIU/mL, or less than 0.25 mIU/mL.
[0101] In certain embodiments, the analyte may have a molecular weight in a range of 10 Da to 10,000,000 kDa, for example a molecular weight in a range of 10 Da to 25 Da, 25 Da to 75 Da, 75 Da to 100 Da, 100 Da to 200 Da, 200 Da to 300 Da, 300 Da to 500 Da, 500 Da to 750 Da, 750 Da to 200 Da, 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 15 kDa, 15 kDa to 20 kDa, 20 kDa to 25 kDa, 25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125 kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDa to 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000 kDa, 5,000 kDa to 10,000 kDa, 10,000 KDa to 1,000,000 kDa, 1,000,000 kDa to 10,000,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, or a molecular weight in a range of 125 kDa to 175 kDa.
[0102] Certain embodiments may provide an assay platform for detecting at least one type of an analyte in a sample, optionally in the presence of one or more type of non-analyte antibody. In certain embodiments, for example, the assay platform may comprise, for example, at least one type of peptide-tagged agent and at least one type of anti-peptide tag. In certain embodiments, the affinity, or rate constant, for binding one of the at least one type of anti-peptide agent to one of the at least one type of peptide-tagged agent may be approximately the same as the affinity, or rate constant, for binding the one of the at least one type of anti-peptide agent to any of the one or more type of non-analyte antibody. In certain embodiments, the affinity, or rate constant, for binding one of the at least one type of anti-peptide agent to one of the at least one type of peptide-tagged agent may be at least 2 times larger than the affinity, or rate constant, for binding the one of the at least one type of anti-peptide agent to any of the one or more type of non-analyte antibody, for example at least 5, 10, 25, 50, 75, or at least 100 times larger. In certain embodiments, the affinity, or rate constant, for binding one of the at least one type of anti-peptide agent to one of the at least one type of peptide-tagged agent may be in the range of 2-1000 times larger than the affinity, or rate constant, for binding the one of the at least one type of anti-peptide agent to any of the one or more type of non-analyte antibody, for example in the range of 2-500, 2-100, 2-10, 10-250, 20-100, or in the range of 5-50 times larger.
[0103] In certain embodiments, the dissociation constant between one of the at least one type of anti-peptide agent and one of the at least one type of peptide-tagged agent may be approximately the same as the dissociation constant between one of the at least one type of anti-peptide agent and any of the one or more type of non-analyte antibody. In certain embodiments, the dissociation constant between one of the at least one type of anti-peptide agent and one of the at least one type of peptide-tagged agent may be at least 2 times larger than the dissociation constant between one of the at least one type of anti-peptide agent and any of the one or more type of non-analyte antibody, for example at least 5, 10, 25, 50, 75, or at least 100 times larger. In certain embodiments, the dissociation constant between one of the at least one type of anti-peptide agent and one of the at least one type of peptide-tagged agent may be in the range of 2-1000 times larger than the dissociation constant between one of the at least one type of anti-peptide agent and any of the one or more type of non-analyte antibody, for example in the range of 2-500, 2-100, 2-10, 10-250, 20-100, or in the range of 5-50 times larger.
[0104] In certain embodiments, one of the at least one type of peptide-tagged agent may form a first specific binding pair with one of one or more pre-determined type of analyte, wherein said specific binding pair may have a dissociation constant (Kd) of greater than 10.sup.−6M. In further embodiments, said first specific binding pair may have a Kd of greater than 10.sup.−7M, 10.sup.−8M or 10.sup.−9M. In certain embodiments, said first specific binding pair may have a dissociation constant (Kd) in the range from 10.sup.−8M to 10.sup.−12M. In certain embodiments, the one of the at least one type of peptide-tagged agent may form a second specific binding pair with one of the at least one type of anti-peptide agent, wherein said second specific binding pair may have a dissociation constant (Kd) of greater than 10.sup.−6M. In further embodiments, said second second specific binding pair may have a Kd of greater than 10.sup.−7M, 10.sup.−8M or 10.sup.−9M. In certain embodiments, said second specific binding pair may have a dissociation constant (Kd) in the range from 10.sup.−8M to 10.sup.−12M. In certain embodiments, the first specific binding pair may have a Kd in the range of 10.sup.−8M to 10.sup.−12M and the second specific binding pair may have a Kd in the range of greater than 10.sup.−6M. In certain embodiments, the second specific binding pair may have a Kd in the range of 10.sup.−8M to 10.sup.−12M and the first specific binding pair may have a Kd in the range of greater than 10.sup.−6M.
[0105] In certain embodiments, one of the at least one type of peptide-tagged agent may be present in the assay platform in an amount in the range of between 1 ng and 50 ng, for example in the range of between 3 ng and 30 ng, 4 ng and 20 ng, 4 ng and 15 ng, 5 ng and 10 ng, or in the range of between 5 ng and 8 ng, for example 6.5 ng. In certain embodiments, one of the least one type of peptide-tagged agent may be present in the assay platform in an amount of less than 10 ng, for example less than 9 ng, 8 ng, 7.5 ng, 7 ng, 6.5 ng, 6 ng, 5.5 ng, 5 ng, 4.5 ng, 4 ng, 3.5 ng, 3 ng, 2.5 ng, or in an amout of less than 2 ng.
[0106] In certain embodiments, one of the at least one type of peptide-tagged agent may be present in the assay platform in an amount in the range of between 10 ng/cm.sup.2 and 500 ng/cm.sup.2 of test strip area striped by the one of the at least one type of peptide-tagged agent, for example in the range of between 30 ng/cm.sup.2 and 300 ng/cm.sup.2, 40 ng/cm.sup.2 and 200 ng/cm.sup.2, 40 ng/cm.sup.2 and 150 ng/cm.sup.2, 50 ng/cm.sup.2 and 100 ng/cm.sup.2, or in the range of between 50 ng/cm.sup.2 and 80 ng/cm.sup.2, for example 65 ng/cm.sup.2 of test strip area striped by the one of the at least one type of peptide-tagged agent. In certain embodiments, one of the at least one type of peptide-tagged agent may be present in the assay platform in an amount of less than 100 ng/cm.sup.2 of test strip area striped by the one of the at least one type of peptide-tagged agent, for example less than 90 ng/cm.sup.2, 80 ng/cm.sup.2, 75 ng/cm.sup.2, 70 ng/cm.sup.2, 65 ng/cm.sup.2, 60 ng/cm.sup.2, 55 ng/cm.sup.2, 50 ng/cm.sup.2, 45 ng/cm.sup.2, 40 ng/cm.sup.2, 35 ng/cm.sup.2, 30 ng/cm.sup.2, 25 ng/cm.sup.2, or in an amout of less than 20 ng/cm.sup.2 of test strip area striped by the one of the at least one type of peptide-tagged agent. In certain further embodiments, the test strip area striped by the one of the at least one type of peptide-tagged agent may have a surface area in the range of 0.01- 5 cm.sup.2, for example 0.1-2 cm.sup.2, 0.15-2 cm.sup.2, or a surface area in the range of 0.25-1 cm.sup.2.
[0107] Certain embodiments may provide a lateral flow apparatus or system to detect an analyte. The lateral flow apparatus or system may comprise, for example, a plurality of peptide-tagged agents. In certain embodiments, the plurality of peptide-tagged agents may be capable of forming an analyte complex that may comprise at least one of the plurality of peptide-tagged agents and the analyte. In certain embodiments, the lateral flow apparatus or system may further comprise, for example, a plurality of anti-peptide agents present, for example, at a ratio of at least 100:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents. In certain embodiments, the lateral flow apparatus or system may further comprise, for example, a plurality of detectable agents, and, optionally, the analyte complex may further comprise, for example, at least one of the plurality of detectable agents.
[0108] In certain embodiments, the sample may be any mixture, composition, or solution that may or may not contain the analyte. In certain assays, it may be useful, for example, to determine that an analyte may not be present in a sample. In certain embodiments, the sample may comprise, for example, a laboratory sample, a medical sample, a biological sample, a water sample, a food sample, an agricultural sample, a serum sample, a serum containing sample, a cell lysate, or a combination of two or more thereof. The sample may comprise, for example, urine, blood, or another bodily fluid of a human or animal.
[0109] In certain embodiments, the sample may be a liquid, for example an aqueous solution, a suspension, or a liquid mixture; a solid; a gel; or congealed matter. In certain embodiments, the sample may be dissolved, mixed, passively mixed, or diffused in a solvent. In certain embodiments, the sample may be pretreated, for example the sample may be precleared, concentrated, diluted, or processed to remove one or more components or impurities from the sample.
[0110] A peptide-tagged agent may comprise, for example, an agent that may be capable of binding with the analyte, for example an antibody, inclusive, for example, of a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment; an aptamer; an affimer; a protein; a protein receptor; a protein ligand; or a fusion protein, comprising, for example, an immunoglobulin fusion partner, a fusion partner that stabilizes a receptor or a ligand, or a fusion partner that provides a target for binding.
[0111] In certain embodiments, the peptide-tagged agent may have a molecular weight in a range of 100 Da to 10,000 kDa, for example a molecular weight in a range of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125 kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDa to 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000 kDa, 5,000 kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, or a molecular weight in a range of 125 kDa to 175 kDa.
[0112] In certain embodiments, the peptide-tagged agent may be capable of binding to an epitope of the analyte, for example the peptide-tagged agent may comprise a complementary determinant region, or a portion thereof, that may be capable of binding with one or more epitopes of the analyte, for example a phospho-epitope. In certain embodiments, the analyte may be a target for the peptide-tagged agent. In certain embodiments, the peptide-tagged agent may be non-covalently bound to the analyte, for example releasably bound, partially bonded, hydrogen-bonded, ionically bonded, or a combination of two or more thereof.
[0113] In certain embodiments, the peptide-tagged agent may further comprise, for example, a plurality of peptide tags. In certain embodiments, the peptide-tagged agent may further comprise, for example, a plurality of a single type of peptide tag. In certain embodiments, the peptide-tagged agent may further comprise, for example, a plurality of two, three, or four types of peptide tags. In certain embodiments, the plurality of peptide tags may be attached, conjugated, and/or covalently bound to the peptide-tagged agent. In certain embodiments, the plurality of peptide tags may be bound to the peptide-tagged agent via cross-linking to the peptide-tagged agent. In certain further embodiments, the cross-linking may comprise, for example, covalent conjugation by primary amines. In certain embodiments, Recombinant DNA technology may be used to form a peptide-tagged agent comprising the plurality of peptide tags.
[0114] In certain embodiments, the plurality of peptide tags may comprise, for example, in a range of 1-50 or 2-50 peptide tags, such as 5-8, 3-7, 2-5, 10-15, 15-20, or in the range of 25-50 peptide tags. In certain embodiments, the plurality of peptide tags may comprise, for example, no more than 50 peptide tags, such as no more than 12, no more than 10, no more than 8, no more than 7, no more than 6, no more than 5, or no more than 3 peptide tags. In certain embodiments, the plurality of peptide tags may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 peptide tags.
[0115] Suitable peptide tags may include a FLAG-tag, for example DYKDDDDK (SEQ ID NO: 1); a peptide tag having the amino acid sequence KRITVEEALAHPYLEQYYDPTDE (SEQ ID NO: 2); a peptide tag having the amino acid sequence HHHHHH (SEQ ID NO: 3); a peptide tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 4); a peptide tag having the amino acid sequence YPYDVPDYA (SEQ ID NO: 5); a peptide tag having the amino acid sequence YTDIEMNRLGK (SEQ ID NO: 6); a peptide tag having the amino acid sequence QPELAPEDPED (SEQ ID NO: 7); a peptide tag having the amino acid sequence CDYKDDDDK (SEQ ID NO: 8) a FLAG octapeptide; a polypeptide protein tag; or a polypeptide sequence that does not include a plurality of consecutive amino acids with the same charge. In certain further embodiments, the suitable peptide tags may also be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or myc-tag. In certain embodiments, at least a portion of the peptide tags may not be naturally occurring. In certain embodiments, the suitable peptide tags do not denature or inactivate the peptide-tagged agent to which they are attached. In certain embodiments, at least one of the suitable peptide tags is more hydrophilic than the FLAG-tag. In certain embodiments, at least a portion of the suitable peptide tags may be removed from the peptide-tagged agent to which they are attached by treatment with the specific proteinase, enterokinase (enteropeptidase). In certain embodiments, at least a portion of the suitable peptide tags may not be naturally occurring in an organism from which a sample is taken. In certain embodiments, suitable peptide tags may include amino acid sequences having no more than 100 amino acids, for example, no more than 50, no more than 30, no more than 20, no more than 15, no more than 12, no more than 10, no more than 9, or no more than 8 amino acids. In certain embodiments, suitable peptide tags may include amino acid sequences having in the range of 4-100 amino acids, for example in the range of 4-50, 4-30, 4-20, 6-15, 6-12, 8-20, 8-15, or in the range of 8-12 amino acids.
[0116] In certain embodiments, an anti-peptide agent may comprise, for example, an agent capable of binding with at least one of the plurality of peptide tags, for example at least 10%, 25%, 50%, 90%, or 95% of the plurality of peptide tags, such as for example, each of the plurality of peptide tags. In certain embodiments, a suitable anti-peptide agent may comprise, for example, an antibody, inclusive, for example, of a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment; an aptamer; an affimer; a protein; a protein receptor; a protein ligand; or a fusion protein, comprising, for example, an immunoglobulin fusion partner, a fusion partner that stabilizes a receptor or a ligand, or a fusion partner that provides a target for binding.
[0117] In certain embodiments, the anti-peptide agent may have a molecular weight in a range of 100 Da to 10,000 kDa, for example a molecular weight in a range of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125 kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDa to 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000 kDa, 5,000 kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, or a molecular weight in a range of 125 kDa to 175 kDa.
[0118] In certain embodiments, at least one of the plurality of peptide tags may be a target for the anti-peptide agent. In certain embodiments, at least one of the plurality of peptide tags and the anti-peptide agent may be specific binding partners. In certain embodiments, the anti-peptide agent may comprise, for example, a binding region highly selective for at least one of the peptide tags, or a portion of at least one of the peptide tags, such as, for example for a peptide tag having the amino acid sequence DYKDDDDK (SEQ ID NO: 1); a peptide tag having the amino acid sequence CDYKDDDDK (SEQ ID NO: 8); a peptide tag having the amino acid sequence KRITVEEALAHPYLEQYYDPTDE (SEQ ID NO: 2; a peptide tag having the amino acid sequence HHHHHH (SEQ ID NO: 3); a peptide tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 4); a peptide tag having the amino acid sequence YPYDVPDYA (SEQ ID NO: 5); YTDIEMNRLGK (SEQ ID NO: 6); or a peptide tag having the amino acid sequence QPELAPEDPED (SEQ ID NO: 7). For example, the binding region of the anti-peptide agent may be highly selective for at least 60% of amino acid sequence of at least one of the peptide tags, such as 70%, 80%, 90%, 95%, or 100%, of the amino acid sequence of at least one of the peptide tags. Commercially available anti-peptide antibodies antibodies that are highly selective for a peptide tag having the amino acid sequence DYKDDDDK (SEQ ID NO: 1) include Sigma-Aldrich product codes F7425, F3040, F1804, F3165, F4042, F2555 and SAB4200071. In certain embodiments, the anti-peptide agent may be selective to a peptide tag only when the peptide tag occupies a certain type of position on a peptide-tagged agent, for example a peptide attached at the N-terminal position. In other embodiments, the anti-peptide agent may be insensitive to how a peptide tag is positioned on an anti-peptide agent.
[0119] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of at least 100:1, 125:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 750:1, or 1000:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of in a range of 100:1-1000:1, 100:1-500:1, 100:1-300:1, 250:1-300:1, 300:1-1000:1, 300:1-500:1, 300:1-400:1, 300:1-350:1, 325:1-375:1, 300:1-310:1, 310:1-320:1, 320:1-330:1, 330:1-340:1, 340:1-350:1, or 350:1-360:1, on a weight:weight basis, relative to the plurality of peptide-tagged agents.
[0120] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of at least 100:1, 125:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 750:1, or 1000:1, on a mole:mole basis, relative to the plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of in a range of 100:1-1000:1, 100:1-500:1, 100:1-300:1, 250:1-300:1, 300:1-1000:1, 300:1-500:1, 300:1-400:1, 300:1-350:1, 325:1-375:1, 300:1-310:1, 310:1-320:1, 320:1-330:1, 330:1-340:1, 340:1-350:1, or 350:1-360:1, on a mole:mole basis, relative to the plurality of peptide-tagged agents.
[0121] A detectable agent may comprise, for example, an agent that may be capable of binding with the analyte, for example an antibody, inclusive, for example, of a monoclonal antibody, a polyclonal antibody, a multivalent antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment; an aptamer; an affimer; a protein; a protein receptor; a protein ligand; or a fusion protein, comprising, for example, an immunoglobulin fusion partner, a fusion partner that stabilizes a receptor or a ligand, or a fusion partner that provides a target for binding, or a fusion partner that provides a detectable signal. In certain embodiments, the detectable agent may be capable of binding to an epitope of the analyte, for example the detectable agent may comprise a complementary determinant region, or a portion thereof, that may be capable of binding with one or more epitopes of the analyte, for example a phospho-epitope.
[0122] In certain embodiments, the detectable agent may have a molecular weight in a range of 100 Da to 10,000 kDa, for example a molecular weight in a range of 100 Da to 1 kDa, 1 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 50 kDa, 50 kDa to 75 kDa, 75 kDa to 100 kDa, 100 kDa to 125 kDa, 125 kDa to 150 kDa, 150 kDa to 175 kDa, 175 kDa to 200 kDa, 200 kDa to 250 kDa, 250 kDa to 500 kDa, 500 kDa to 1,000 kDa, 1,000 kDa to 5,000 kDa, 5,000 kDa to 10,000 kDa, 75 kDa to 400 kDa, 100 kDa to 200 kDa, or a molecular weight in a range of 125 kDa to 175 kDa.
[0123] In certain embodiments, the analyte may be a target for the detectable agent. In certain embodiments, the detectable agent may be non-covalently bound to the analyte, for example releasably bound, partially bonded, hydrogen-bonded, ionically bonded, or a combination of two or more thereof.
[0124] In certain embodiments, one or more of the detectable agents may comprise, for example, a detectable tag that may be capable of producing a detectable signal. In certain embodiments, the magnitude, or another characteristic, of the signal may be related to the quantity or concentration of an analyte in a sample. In certain embodiments, the detectable tag may be applied to or bound to another portion of the detectable agent to form the complete detectable agent. In certain embodiments, the detectable tag may be integral to the rest of the detectable agent. For example, the detectable agent may include the detectable tag as a fusion partner, a labelled amino acid, or a labelled nucleotide.
[0125] Suitable detectable tags may include, for example, antigens; enzymes; fluorophores; quenchers; radioactive isotopes; luminescent labels; chemiluminescent labels; one or more lanthanide ions, for example Eu.sup.3+, Sm.sup.3+, Tb.sup.3+, and/or Dy.sup.3+, nucleic acids that may be capable of PCR amplification; coloured particles, inclusive, for example, of coloured latex beads, metal sols (for example, collidal gold), gold labels, or dye labels; tags that may be capable of interaction by fluorescence resonance energy transfer; and tags that may be capable of chemical transfer proximity interaction.
[0126] In certain embodiments, the detectable signal may be produced directly by the detectable agent or detectable tag, if present, or indirectly by a further molecule that can produce a detectable signal, such as, for example, a further molecule that may comprise an enzyme. In certain embodiments, detectable signals include, but are not limited to, light signals, inclusive of, for example, signals having a color or no color that may be detectable by human vision; stains; and signals that may be detected with a detector, inclusive of, for example, a spectrophotometer, a fluorometer, for example a fluorometer capable of measuring time-resolved fluorescence, a luminometer, a radioactivity-based counter, or an electrochemical detection apparatus. In certain embodiments, the detector may be a hand-held detector, a detector in a laboratory, and/or a mobile detector. In certain embodiments, detectable signals may include the lack of any of the foregoing types of signals, for example a quenched signal, when measured by a detector capable of detecting a signal if one were present.
[0127] Metal sols and other types of colored particles useful as detectable tags in immunoassay procedures are known. See, for example, U.S. Pat. No. 4,313,734; Horisberger, Evaluation of Colloidal Gold as a Cytochromic Marker for Transmission and Scanning Electron Microscopy, Biol. Cellulaire, 36, 253 258 (1979); Leuvering et al., Sol Particle Immunoassay, J. Immunoassay, 1 (1): 77 91 (1980), and Frens, Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions, Nature, Physical Science, 241: 20 22 (1973).
[0128] Use of metal sols, for example colloidal gold, may produce a visibly detectable signal. In certain embodiments, detectable tags may comprise, for example, colloidal gold providing a red detectable signal. In certain embodiments, detectable tags may comprise, for example, colloidal gold having a mean particle size in a range of 50-100 nm, such as, for example, colloidal gold having a mean particle size in a range of 55-90, 55-85, 40-47, 60-80, 60-75, or 65-75 nm. In certain embodiments, detectable tags may comprise, for example, colloidal gold having a mean particle size greater than 50 nm, greater than 52 nm, greater than 55 nm, greater than 57 nm, greater than 60 nm, or greater than 100 nm in size.
[0129] In certain embodiments, detectable tags may comprise, for example, particles, at least a portion of which may be substantially or predominantly spherical in shape. In certain embodiments, at least a portion of the detectable tags may include particles having substantially or predominantly non-spherical shape or shapes. In certain embodiments, detectable tags may comprise, for example, particles that may be substantially or predominantly monodisperse or have a narrow particle size distribution, for example a monodispersity greater than 50%, 75% or 95% as determined by a Coulter N4 Particle Analyzer (Beckman Coulter, Fullerton, Calif.).
[0130] Antigens that may be used as detectable tags may include, for example, any antigenic component of a detectable agent that may be targeted by a secondary deteclable agent. In certain embodiments, an antibody may be used as a secondary detectable agent to detect an antigen detectable tag. In certain further embodiments, the antibody secondary detectable agent may be fluorescently or enzymatically labelled. In embodiments where the portion of the detectable agent that may bind with the analyte is part of an antibody, the antibody secondary detectable agent may have a binding affinity to an antigen on the detectable agent.
[0131] Enzymes that may be used as detectable tags include, for example, enzymes that result in the conversion of a portion of the detectable agent into a detectable product. In certain embodiments, the conversion may result in a change in colour or fluorescence or generation of an electrochemical signal by the detectable agent. Such enzymes may include, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), p-galactosidase, acetylcholinesterase, luciferase, or catalase.
[0132] Radioactive isotopes that may be used as detectable tags include, for example, .sup.3H, .sup.14C.sub., .sup.32.sub.P, .sup.35S, or .sup.131I. In certain embodiments, the radioisotope may be conjugated to a detectable agent or incorporated into a detectable agent by translation of mRNA encoding the detectable agent in the presence of radiolabelled amino acids. Radioisotopes and methods for conjugating radioactive isotopes to molecules such as proteins are known in the art and include methods discussed by Slater (Radioisotopes in Biology: A Practical Approach, Oxford University Press, 2002). Radioisotopes may be detected using gamma, beta or scintillation counters.
[0133] Fluorophores that may be used as detectable tags include, for example, resorufin; fluorescein, inclusive of, for example, fluorescein isothiocyanate, FITC; rhodamine, inclusive of, for example, tetramethyl rhodamine isothiocyanate, TRITC; green fluorescent protein, GFP; and phycobiliproteins, inclusive of, for example, allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin, or derivatives of any of the foregoing.
[0134] In certain embodiments, fluorophores may be subjected to applied stimulation, for example light of a suitable excitation wavelength to promote fluorescence. Alternatively, in certain embodiments stimulation may be provided by a fluorescence resonance energy transfer (FRET) partner, for example a donor molecule. When the fluorophore comes into close vicinity to the FRET partner, for example during formation of the analyte complex, the fluorophore may become excited by the FRET partner and fluoresce. FRET donors may include luminescent and/or fluorescent agents.
[0135] In certain embodiments, a detectable tag may comprise, for example, a quencher. Quenchers may be able to absorb excitation energy from fluorophores and may be used to suppress the fluorophore's emission when in close proximity. In this regard, the reaction is similar to a FRET reaction, except that the readout is a loss of fluorescence.
[0136] Luminescent compounds that may be used as detectable tags include, for example, chemiluminescent and bioluminescent compounds. In certain embodiments, these compounds may be used to label the detectable agent. In certain embodiments, the presence of the chemiluminescent-tag may be determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of useful chemiluminescent labelling compounds include, but are not limited to, luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In certain embodiments, the presence of a bioluminescent antibody is determined by detecting the presence of luminescence. Examples of bioluminescent compounds include, but are not limited to luciferin, luciferase and aequorin.
[0137] Nucleic acids that may be used as detectable tags include a suitable nucleic acid that may be capable of PCR amplification and/or hybridisation to a probe. The nucleic acid may be of sufficient length to allow binding of a forward and/or a reverse primer. In certain embodiments, the nucleic acid tag may be included in an aptamer or bound to a protein. In certain embodiments, nucleic acid detectable tags may be detected by performing a PCR reaction, whereby the nucleic acid tag is amplified and measured, or using a labelled nucleic acid probe with a complementary sequence to at least a portion of the nucleic acid tag. Methods of preparing and binding detectable nucleic acid tags to detectable agents are known in the art and include methods described in US 2009/0053701.
[0138] Detectable tags that may be capable of detection derived from chemical transfer proximity interaction may comprise, for example, a photoactive indicator precursor, for example a photoactive indicator precursor bound to a particle (such as a bead), which may be activated (for example, activated by singlet oxygen) to form a photoactive indicator. In certain embodiments, the photoactive indicator may be stimulated (for example, by irradiation) to produce a measurable light signal. Suitable photoactive indicator precursors may include, for example, rubrene, europium, europium chelate, samarium, or terbium. In embodiments where a photoactive indicator precursor is bound to a bead, suitable beads may include, for example, latex beads and magnetic beads.
[0139] In certain embodiments, a photoactive indicator may be capable of producing a light signal when irradiated by light. In certain further embodiments, the irradiating light may have a wavelength in a range of 250-1100 nm, for example, light at a wavelength in a range of 300-1000 nm, between 450-950 nm, between 360-441 nm, between 620-700 nm, between 600-630 nm, between 620-650 nm, between 640-700 nm, between 650-700 nm, between 670-690 nm, between 680-700 nm, or between 660-680 nm, such as by light at a wavelength of 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, or at a wavelength of 700 nm.
[0140] In certain embodiments, the photoactive indicator, when irradiated, may fluoresce (i.e., emit a fluorescence) at a wavelength in a range of 500-625 nm, such as at a wavelength in a range of 525-575 nm, between 525-550 nm, between 540-560 nm, between 540-550 nm, 590-620 nm, between 600-625 nm, or in the range of 610-620 nm, such as at a wavelength of 520 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, or at a wavelength of 625 nm.
[0141] In certain embodiments, detection of an analyte may comprise detecting a loss or gain of a light emission that may be derived from an interaction between at least a portion of the plurality of detectable tags and at least a portion of the permeable detection members. In certain embodiments, for example in certain embodiments utilizing a quencher, a FRET fluorophore, or photoactive indicator precursor as at least one of the detectable tags, an interacting component may be provided on, or integrated with, at least a portion of the permeable detection member. For example, in certain embodiments where at least a portion of the detectable tags comprise a fluorophore, the at least a portion of the permeable detection member may comprise a FRET partner (either donor or acceptor) or a quencher. In certain embodiments, in other embodiments where at least a portion of the plurality of detectable tags comprises a quencher, the at least a portion of the permeable detection member may comprise a suitable fluorophore. In certain embodiments, at least a portion of the plurality of detectable tags may comprise a chemical transfer interaction acceptor, for example a photoactive indicator precursor and, optionally, at least a portion of the permeable detection members may comprise a chemical transfer interaction donor, for example a donor capable of providing singlet oxygen upon stimulation by an appropriate light source. In certain embodiments, at least a portion of the plurality of permeable detection members may comprise a chemical transfer interaction acceptor, for example a photoactive indicator precursor and, optionally, at least a portion of the detectable tags may comprise a chemical transfer interaction donor, for example a donor capable of providing singlet oxygen upon stimulation by an appropriate light source.
[0142] In certain embodiments, detecting the presence of the immobilised analyte complex on at least a portion of the permeable detection membrane may utilise time-resolved fluorescence (TRF) and (fluorescence resonance energy transfer) FRET technologies, inclusive of, for example, the TRF-FRET technologies as described in EP 569,496, U.S. Pat. No. 5,527,684 or U.S. Pat. No. 6,861,264.
[0143] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1, 50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relative to a plurality of detectable agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of in a range of 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1, 2:1-100:1, 2:1-50:1, 2:1-25:1, 2:1-15:1, 5:1-15:1, 5:1-15:1, 5:1-10:1, 5:1-8:1, or 8:1-10:1, on a weight:weight basis, relative to the plurality of detectable agents.
[0144] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1, 50:1, 100:1, 300:1, or 500:1, on a mole:mole basis, relative to a plurality of detectable agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio of in a range of 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1, 2:1-100:1, 2:1-50:1, 2:1-25:1, 2:1-15:1, 5:1-15:1, 5:1-15:1, 5:1-10:1, 5:1-8:1, or 8:1-10:1, on a mole:mole basis, relative to the plurality of detectable agents.
[0145] In certain embodiments, a plurality of detectable agents may be present at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1, 50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents. In certain further embodiments, the plurality of detectable agents may be present at a ratio of in a range of 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1, 2:1-100:1, 2:1-50:1, 10:1-50:1, 20:1-50:1, 25:1-45:1, 30:1-45:1, 30:1-40:1, 35:1-40:1, or 37:1-40:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents .
[0146] In certain embodiments, a plurality of detectable agents may be present at a ratio of at least 0.1:1, 1:1, 2:1, 5:1, 8:1, 10:1, 15:1, 25:1, 50:1, 100:1, 300:1, or 500:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents. In certain further embodiments, the plurality of detectable agents may be present at a ratio of in a range of 0.1:1-500:1, 0.1:1-300:1, 0.1:1-300:1, 0.1:1-500:1, 2:1-500:1, 2:1-100:1, 2:1-50:1, 10:1-50:1, 20:1-50:1, 25:1-45:1, 30:1-45:1, 30:1-40:1, 35:1-40:1, or 37:1-40:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents.
[0147] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-20:1, on a weight:weight basis, relative to the plurality of detectable agents, and the plurality of anti-peptide agents may be present at a ratio of in a range of 100:1-500:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-10:1, on a weight:weight basis, relative to the plurality of detectable agents and the plurality of anti-peptide agents may be present at a ratio of in a range of 150:1-400:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-10:1, on a weight:weight basis, relative to the plurality of detectable agents and the plurality of anti-peptide agents may be present at a ratio of in a range of 200:1-500:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 6:1-10:1, on a weight:weight basis, relative to the plurality of detectable agents and the plurality of anti-peptide agents may be present at a ratio of in a range of 250:1-400:1, on a weight:weight basis, relative to a plurality of peptide-tagged agents.
[0148] In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-20:1, on a mole:mole basis, relative to the plurality of detectable agents, and the plurality of anti-peptide agents may be present at a ratio of in a range of 100:1-500:1, on a mole:mole basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-10:1, on a mole:mole basis, relative to the plurality of detectable agents, and the plurality of anti-peptide agents may be present at a ratio of in a range of 150:1-400:1, on a mole:mole basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 5:1-10:1, on a mole:mole basis, relative to the plurality of detectable agents, and the plurality of anti-peptide agents may be present at a ratio of in a range of 200:1-500:1, on a mole:mole basis, relative to a plurality of peptide-tagged agents. In certain embodiments, a plurality of anti-peptide agents may be present at a ratio in a range of 6:1 -1 0:1 , on a mole:mole basis, relative to the plurality of detectable agents, and the plurality of anti-peptide agents may be present at a ratio of in a range of 250:1-400:1, on a mole:mole basis, relative to a plurality of peptide-tagged agents.
[0149] In certain embodiments, the analyte complex may comprise, for example, the analyte bound to one peptide-tagged agent. In certain embodiments, the analyte complex may comprise, for example, the analyte bound to a plurality of peptide-tagged agents, for example the analyte bound to 2, 3, less than 5, less than 10, or bound to in the range of 10-20 peptide-tagged agents. In certain embodiments, the analyte complex may further comprise, for example, the analyte bound to one detectable agent. In other embodiments, the analyte complex may comprise, for example, the analyte bound to a plurality of detectable agents, for example the analyte bound to 2, 3, less than 5, less than 10, or bound to in the range of 10-20 detectable agents. For example, the analyte complex may comprise the analyte bound to 1 peptide-tagged agent and 1 detectable agent, or 2 peptide-tagged agents and 1 detectable agent. In certain embodiments, the analyte complex may comprise, for example, a peptide-tagged agent bound to 2 or more analytes, for example the peptide-tagged agent bound to 2, 3, less than 5, less than 10, or bound to in the range of 10-20 analytes. In certain embodiments, the analyte complex may comprise, for example, a detectable agent bound to 2 or more analytes, for example the detectable agent bound to 2, 3, less than 5, less than 10, or bound to in the range of 10-20 analytes. For example, the analyte complex may comprise 3 analytes bound to 2 peptide-tagged agents and 1, 2, or 3 detectable agents; or, for example, the analyte complex may comprise 2 analytes bound to 3 peptide-tagged agents and 6 detectable agent.
[0150] In certain embodiments, at least one peptide-tagged agent may be capable of binding to a first epitope of the analyte and at least one detectable agents may be capable of binding to a second epitope of the analyte. In certain embodiments, the type of epitope of the first epitope of the analyte and the second epitope of the analyte may be the same type of epitope. In certain embodiments, the type of epitope of the first epitope of the analyte and the second epitope of the analyte may be two different epitope types. In certain embodiments, the type of epitope of the first epitope of the analyte and the second epitope of the analyte may share portions of their sequence identify, for example at least 75%, 80%, 85%, 90%, or at least 95% shared sequence identity; or at least 75%, 80%, 85%, 90%, or at least 95% shared sequences with their sequences but not necessarily at the same positions relative to the terminal portions of the sequences. In certain embodiments, the at least one peptide-tagged agent and the at least one detectable agent may be the same type of antibody. In certain embodiments, the at least one peptide-tagged agent and the at least one detectable agent may be different types of antibodies. In certain embodiments, the first epitope of the analyte and/or the second epitope of the analyte is a phospho-epitope. In certain embodiments, the first epitope of the analyte and the second epitope of the analyte may overlap in a common portion of the analyte. In certain embodiments, the first epitope of the analyte and the second epitope of the analyte may not overlap but may be in close proximity to each other, for example within 50 nm of one another, for example within 25 nm, within 10 nm, within 5 nm, within 1 nm, or within 5 Angstroms of one anthor. In certain embodiments, the first epitope of the analyte and the second epitope of the analyte may be distal from one another, for example at least 1 nm apart from one another, for example at least 2 nm, 5 nm, 10 nm, 25 nm, or at least 50 nm apart from one another. In certain embodiments, the first epitope of the analyte and the second epitope of the analyte may be in the range of 10 Angstroms-10 nm of one another, for example 10 Angstroms-5 nm, 10 Angstroms-3 nm, or in the range of 25 Angstroms-3 nm of one another. In certain embodiments, the first epitope of the analyte and the second epitope of the analyte do not overlap and may be sufficiently distal to each such that no steric interactions or substantially no steric interactions occur between the at least one peptide-tagged agent and the at least one detectable agent. In certain embodiments, the first and second epitopes may be bound by the at least one peptide-tagged agent and the at least one detectable agent, respectively.
[0151] Certain embodiments may provide an assay platform for detecting an analyte. In certain embodiments, the assay platform may comprise, for example, a permeable zone that may be configured, for example, to transport at least a portion of a sample that may comprise the analyte, and optionally a further permeable zone which may be configured, for example, to receive at least a portion of the sample. In certain embodiments, the permeable zone may comprise, for example, a plurality of peptide-tagged agents. In certain embodiments, the plurality of peptide-tagged agents may be capable of forming an analyte complex that may comprise at least one of the plurality of peptide-tagged agents and the analyte. In certain embodiments, the further permeable zone may be configured to receive at least a portion of the portion of the sample from the permeable zone. In certain embodiments, the further permeable zone may comprise, for example, a plurality of anti-peptide agents present, for example, at a ratio of at least 100:1, one a weight:weight and/or mole:mole basis, relative to the plurality of peptide-tagged agents. In certain embodiments, the plurality of anti-peptide agents may be configured, for example, to immobilize the at least one of the plurality of peptide-tagged agents. In certain embodiments, the plurality of anti-peptide agents may be configured, for example, to immobilize the analyte complex (for example, the plurality of anti-peptide agents may be configured, for example, to immobilize the analyte complex with a probability of .01-99.95%, for example a probability of 10-99.95%, 10-25%, 10-50%, 25-75%, 50%-75%, or with a probability of 75-99.95%). In certain further embodiments, the plurality of anti-peptide agents may be configured, for example, to immobilize the at least 5% of the plurality of peptide-tagged agents, for example at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or at least 99.9% of the plurality of peptide-tagged agents, or all of the plurality of peptide-tagged agents. In certain embodiments, the plurality of anti-peptide agents may be configured, for example, to immobilize in the range of 5%-100% of the plurality of peptide-tagged agents, for example in the range of 10%-95%, 10%-75%, 10%-50%, 25%-95%, 25%-75%, 20%-50%, 50%-100%, 50%-75%, 75%-100%, or to immobilize in the range of 75%-99.95% of the plurality of peptide-tagged agents.
[0152] In certain embodiments, the permeable zone may be distinguishable from another portion of the assay platform, for example the further permeable zone. For example, in certain embodiments, distinguishable features of the permeable zone may be inclusive of, but not limited to, any one or more of the following features: comprising a plurality of peptide-tagged agents; configured to be the portion of the apparatus to which the sample is introduced; configured to be the portion of the apparatus to which the analyte is introduced; configured to bring the analyte into contact with the plurality of peptite-tagged agents and/or detectable agents; configured to communicate at least a portion of the sample (and/or a portion of the plurality of peptide-tagged agents, detectable agents, analyte, and/or analyte complex) by passive transport, capillary action, wetting, wicking, or a combination of two or more thereof; comprising a material that may be, for example, absorbent, fibrous, or porous; and comprising a release zone configured to introduce at least a portion of the peptide-tagged agents and/or detectable agents into the sample. Other distinguishing features of the permeable zone are contemplated herein. In certain further embodiments, the another portion, for example the further permeable zone, may not comprise one or more of the foregoing distinguishable features.
[0153] In certain embodiments, the further permeable zone may be distinguishable from another portion of the assay platform, for example the permeable zone. For example, in certain embodiments, distinguishable features of the further permeable zone may be inclusive of, but not limited to, any one or more of the following features: comprising a plurality of anti-peptide agents; configured to receive at least a portion of the sample (and/or a portion of the plurality of peptide-tagged agents, detectable agents, analyte, and/or analyte complex) by passive transport, capillary action, wetting, wicking, or a combination of two or more thereof; comprising a detection zone that may be configured to be the portion of the apparatus to which the analyte complex may be immobilized; configured to be the portion of the apparatus to which the analyte may be detected; comprising a material that may be, for example, absorbent, fibrous, or porous; and comprising a membrane, inclusive of, for example, a nitrocellulose membrane. Other distinguishing features of the further permeable zone are contemplated herein.
[0154] In certain embodiments, the permeable zone and the further permeable zone may be non-overlapping. In other embodiments, at least a portion of the permeable zone and at least a portion of the further permeable zone may overlap. In certain embodiments, the permeable zone may be contained within the further permeable zone. In certain embodiments, the further permeable zone may be contained within the permeable zone.
[0155] Certain embodiments may provide a lateral flow device. In certain embodiments, the lateral flow device may comprise, for example, a flow path that may be defined by, for example, at least a portion of a permeable sub-assembly of the lateral flow device. In certain embodiments, the lateral flow device may further comprise, for example, a release zone that may comprise, for example, a plurality of peptide-tagged agents. In certain embodiments, the lateral flow device may further comprise, for example, a detection zone that may comprise, for example, a plurality of anti-peptide agents that may be present, for example, in the flow path at a ratio of at least 100:1, on a weight and/or mole:mole basis, relative to the plurality of peptide-tagged agents. In certain embodiments, the release zone may be configured, for example, to release at least a portion of said plurality of peptide-tagged agents into the flow path. In certain embodiments, at least one of said plurality of anti-peptide agents may be a binding partner with at least one of said plurality of peptide-tagged agents.
[0156] In certain embodiments, the permeable sub-assembly may comprise one or more permeable members, for example, 1, 2, 3, 4, or 5 permeable members. Examples of suitable permeable members are inclusive of, for example, for example, a permeable strip, a fibrous pad, and a porous membrane. In certain embodiments, one or more of the permeable members may be capable of being wetted. In certain embodiments, one or more of the permeable members may comprise matrix of solid material, such as fibers or particles, interspersed with open spaces. In certain embodiments, the open spaces may comprise, for example, pores, channels, microchannels, spaces between fibers, cusps formed by adjacent and/or packed particles (for example, particles such as microspheres), and the like. Other types of open spaces are contemplated herein. In certain embodiments, a sample, for example a liquid, suspension, or solution, may be introduced to a portion of the permeable sub-assembly and transported through the open spaces of at least a further portion of the sub-assembly by passive transport, capillary action, wetting, wicking, or a combination of two or more thereof.
[0157] In certain embodiments, the flow path may be characterized by the flux, or migration, of a sample being transported through at least a portion of the permeable sub-assembly. In certain embodiments, the flow path may characterized by the general, average, or net direction of flow resulting from transport of the sample and/or portions thereof through the open spaces in at least a portion of the sub-assembly. In certain embodiments, the flow path may not be uniform in a cross-section of the permeable sub-assembly. For example, in certain embodiments, a sample may flow or migrate more quickly through larger open spaces and/or near a boundary of the permeable sub-assembly, for example near or at a surface, for example a top surface, or an edge of the permeable sub-assembly.
[0158] In certain embodiments, at least a portion of the flow path may be approximately parallel, linear, convergent, or divergent. In certain embodiments, at least a portion of the flow path may be a radial flow path.
[0159] In certain embodiments, the release zone may comprise a portion of the permeable sub-assembly. In certain further embodiments, the plurality of peptide-tagged agents may be dried on, bound, releasably bound, or unbound to at least a portion of the permeable sub-assembly. In embodiments in which at least a portion of the plurality of anti-peptide agents may be unbound to the sub-assembly, the plurality of peptide-tagged agents may be introduced to a portion of the sub-assembly with the sample, or introduced separately from the sample and become dissolved or suspended in the sample in the open spaces of the permeable sub-assembly. In certain embodiments in which the plurality of peptide-tagged agents is bound or releasably bound in the release zone, the release zone may be configured to further release at least a portion of said plurality of peptide-tagged agents into the flow path. In certain embodiments, at least a portion of the plurality of peptide-tagged agents may migrate, dissolve, disconnect, and/or diffuse from at least a portion of the permeable sub-assembly, for example the release zone. In certain embodiments, at least a portion of the plurality of peptide-tagged agents may migrate, dissolve, disconnect, and/or diffuse from at least a portion of the permeable sub-assembly after contact with the sample and/or a solvent.
[0160] In certain embodiments, said plurality of anti-peptide agents may be configured, for example, to immobilize at least a fraction of the at least a portion of said plurality of peptide-tagged agents configured to be released, for example released in the detection zone of the sub-assembly. In certain further embodiments, at least one of the at least a portion of said plurality of peptide-tagged agents configured to be released may be capable of binding with an analyte prior to being immobilized by said plurality of anti-peptide agents.
[0161] In certain embodiments, the detection zone may comprise a portion of the permeable sub-assembly. In certain further embodiments, the plurality of anti-peptide agents may be bound to at least a portion of the permeable sub-assembly. In certain embodiments, the plurality of anti-peptide agents may be passively bound. In certain embodiments, the plurality of anti-peptide agents may be bound by non-covalent interactions. Examples of suitable interactions include, for example, electrostatic interactions, hydrophilic interactions, hydrophobic interactions, ionic electrostatic interactions, van de Waals interactions, or combinations of two or more such interactions. In some embodiments, the anti-peptide agent may be actively bound, for example covalently bound, to the solid substrate.
[0162] In certain embodiments, at least a portion of the permeable sub-assembly may comprise a plurality of linkers which may facilitate covalent bonding of the plurality of anti-peptide agents. Suitable linkers may include, for example, glutathione, maleic anhydride, a metal chelate, or meleimide. In certain further embodiments, the plurality of anti-peptide agents may be exposed to at least a portion of the open spaces in at least a portion of the permeable sub-assembly, for example the open spaces present in the detection zone.
[0163] In certain further embodiments, at least a portion of the plurality of detectable agents may be dried on, bound, releasably bound, or unbound to at least a portion the permeable sub-assembly. In certain embodiments in which at least a portion of the plurality of detectable agents may be unbound to the sub-assembly, the detectable agent may be introduced to a portion of the sub-assembly with the sample, or introduced separately from the sample and become dissolved or suspended in the sample on the permeable sub-assembly. In certain embodiments in which the plurality of detectable agents is bound or releasably bound in the release zone, the release zone may be configured to further release at least a portion of said plurality of detectable agents into the flow path. In certain further embodiments, at least a portion of the plurality of detectable agents may be capable of migrating, dissolving, disconnecting, or diffusing from at least a portion of the permeable sub-assembly, for example the release zone. In certain embodiments, at least a portion of the plurality of detectable agents may migrate, dissolve, disconnect, and/or diffuse from at least a portion of the permeable sub-assembly after contact with the sample and/or a solvent.
[0164] Certain embodiments may provide an assembly. In certain embodiments, the assembly may comprise, for example, a permeable release member that may have a plurality of peptide-tagged agents deposited on at least a portion thereof and, optionally, a plurality of detectable agents deposited on at least a portion thereof. In certain embodiments, the assembly may further comprise, for example, a permeable detection member that may be in fluid communication with the permeable release member. In certain embodiments, the permeable detection member may have a plurality of anti-peptide agents bound to at least a portion thereof.
[0165] In certain embodiments, the permeable release member may absorb at least a portion of the plurality of peptide-tagged agents and/or at least a portion of the plurality of detectable agents. In certain embodiments, the permeable release member may allow release of at least a portion of the plurality of peptide-tagged agents and/or at least a portion of the plurality of detectable agents. In certain embodiments, the permeable release member may provide structural support for at least a portion of the assembly. In certain embodiments, at least a portion of the plurality of peptide-tagged agents and/or at least a portion of the plurality of detectable agents may be striped onto the permeable release member. In certain embodiments, at least a portion of the plurality of peptide-tagged agents and/or at least a portion of the plurality of detectable agents may be dried onto the permeable release member. As explained herein, the striped and/or dried peptide-tagged agents and/or detectable agents may migrate, dissolve, disconnect, and/or diffuse from at least a portion of the permeable release member into a sample or a solvent. In certain embodiments, at least a portion of the plurality of peptide-tagged agents and/or plurality of detectable agents may be able to passively mix with a liquid sample present on the permeable release member. In certain embodiments, any one or more of the types of analyte complexes contemplated herein may be able to be formed on the permeable release pad.
[0166] In certain embodiments, the permeable release member may comprise a bibulous, hydrophilic material, such as, for example, an absorbent material. Suitable permeable release member materials may comprise, for example, cotton linter; cellulosic materials, or materials made of cellulose together with a polymeric fibrous material, such as polyamide or rayon fibers; and glass fiber material. For example, suitable materials may include cotton linter paper, such as S&S 903 and S&S GB002 (available from Schleicher and Schuell, Inc., Keene, N.H.), and BFC 180 (available from Whatman, Fairfield, N.J.); cellulosic materials, such as Grade 939 made of cellulose with polyamide, Grade 989 made of cellulose blend fiber, and Grade 1278 and Grade 1281 made of cellulose and rayon with polyamide (available from Ahlstrom Corporation, Mt. Holly Springs, Pa.); and glass fiber, such as Lydall borosilicate (available from Lydall, Inc., Rochester, N.H.).
[0167] In certain embodiments, the permeable release member may be blocked to prevent non-specific binding. In certain embodiments, the permeable release member may be coated with an aqueous solution containing bovine serum albumin (BSA) and a nonionic surfactant, such as polyethylene glycol, for example the polyethylene glycol sold under the trade name Triton X-100 (available from Rohm & Haas Co., Philadelphia, Pa.), for example, a combination of about 3% BSA and about 0.1% Triton X-100.
[0168] In certain embodiments, the permeable detection member may be formed of a substance which may permit binding of the plurality of anti-peptide agents thereto. In certain embodiments, the permeable detection member may comprise a polymeric material, for example a microporous film or membrane which may permit at least a portion of the anti-peptide agents to be passively bound or absorbed thereon. In such embodiments, it may not be necessary to bind the plurality of anti-peptide agents to the permeable detection member by chemical or physical fixation. In other embodiments, the plurality of anti-peptide agents may be bound to the permeable detection member by chemical or physical fixation.
[0169] In certain embodiments, suitable permeable detection member materials may comprise, for example, a microporous polymeric film of nitrocellulose, nylon, charge-modified nylon, polyvinylidine fluoride, polyethersulfone, or similar materials, or combinations of such materials. In certain embodiments, suitable permeable detection member materials may have a pore size in a range of between 0.1 μm and 20 μm, for example in a range of between 1 μm and 3 μm, 3 μm and 10 μm, 10 μm and 20 μm, or in a range of between 5 μm and 20 μm.
[0170] In certain embodiments, the permeable detection member may comprise a nitrocellulose membrane, inclusive of, for example, a nitrocellulose membrane having added detergents and/or surfactants. In certain embodiments, the permeable detection member may comprise a nitrocellulose membrane, inclusive of, for example, a membrane comprising nitrocellulose alone or a mixed ester of nitrocellulose, such as in combination with an ester of nitric acid and/or other acids. In certain further embodiments, a nitrocellulose membrane permeable detection member may be coated or laminated onto a translucent or transparent polymeric film to provide physical support for the membrane. In certain embodiments, the permeable detection member may comprise a nitrocellulose polymer which has been cast onto a polyester film, such as MYLAR®. In certain embodiments, the permeable detection member may comprise a nitrocellulose membrane laminated onto a polyester film, or laminated onto another backing materials besides polyester. Suitable backing materials may include, for example, pre-laminated or pre-cast planar sheets.
[0171] In certain embodiments, the permeable release member and the permeable detection member may be joined by overlapping a downstream edge of the permeable release member over an upstream edge of the permeable detection member, then adhering the resulting biphasic material to a clear polymer film or sheet, thereby holding the two members in place. In other embodiments, the permeable release member and the permeable detection member may be joined at a non-overlapping butt-joint, and adhered to a clear polymer film or sheet, thereby holding the two members in place. In certain embodiments, the joined members may provide a continuous flow path for transport of the sample, peptide-tagged agent, detectable agent, analyte, analyte complex, or any combination of the foregoing. Transport may comprise, for example, passive transport, capillary action, wetting, wicking, or a combination of two or more thereof. In certain embodiments, the joined members are in fluid communication.
[0172] In certain embodiments, at least one of the permeable release member and the permeable detection member has at least one flow channel spanning at least a portion of the respective member with a smallest cross-sectional width in a range of 3-20 times a diameter of a sphere having a molecular volume of the analyte complex. In certain embodiments, the at least one flow channel has a smallest cross-section width of less than 10, from 10 to 20, or greater than 20 times a diameter of a sphere having a molecular volume of the analyte complex.
[0173] A schematic view of a representative lateral flow detection apparatus 100 is illustrated in
[0174] A schematic view of solvent-driven lateral flow detection apparatus 200 is illustrated in
[0175] A schematic view of multi-purpose lateral flow test kit 300 configurable to detect any one of three types of analytes is illustrated in
[0176] A schematic view of representative radial flow detection apparatus 400 for the detection of eight (8) different analytes is illustrated in
[0177] A schematic view of a representative multi-analyte lateral flow detection apparatus 500 for the detection of six (6) different analytes is illustrated in
EXAMPLES
Lateral Flow Test Strip Preparation
[0178] Release Pads: Millipore GFDX glass fiber pads were blocked with blocking buffer (10 mM borate, 3% BSA, 1% PVP-40, 0.25% Tx-100), dried, and stored desiccated. The dried glass fiber pads were then sprayed with detectable agent and, optionally, a peptide-tagged agent, in accordance with Table 1 and then dried at approximately 40° C. and stored desiccated.
[0179] Detection Membranes: Sartorius Unisart CN140 nitrocellulose membranes were striped with an immobilization agent in accordance with Table 1 using a Biodot frontline dispenser at a dispense rate of about 1 pL/cm, dried, and stored dessicated. The nitrocellulose membranes were then blocked with blocking buffer (10mM phosphate, 0.1% sucrose, 0.1% BSA, 0.2% PVP-40, pH 7.2), dried at approximately 40° C., and stored desiccated.
TABLE-US-00001 TABLE 1 Conjugate Pad and Detection Membrane Preparations Detection Conjugate Pad Membrane Prepa- Peptide-Tagged Detectable Immobilization ration Agent Agent Agent A 6.5 ng anti-hCG-β 250 ng anti- 2000 ng mouse tagged with a plurality hCG-α monoclonal IgG1 of peptide tags conjugated to immobilization agent (sequence: colloidal gold (capable of CDYKDDDDK immobilizing the (SEQ ID NO: 8)) tagged anti-hCG- β) B (no Capture Agent 500 ng anti- 500 ng anti-hCG-β was placed hCG-α (capable of directly Conjugate Pad) conjugated to immobilizing hCG) colloidal gold
[0180] Lamination and Assembly: Each lateral flow test strip comprised: (1) a conjugate pad having a 2 mm overlap with the top of one end of a detection membrane, (2) a wick pad having a 2 mm overlap with the top of the opposite end of the detection membrane, and (3) an adhesive backing card onto which the conjugate pad—detection membrane—wick pad configuration was affixed. 160 test strips were used (80 each of based on Preparation A and Preparation B, respectively) in the following Example.
[0181] Example 1: A series of hCG detection experiments were conducted at the concentrations shown in Table 2. In each experiment, 40 pL of hCG solution was deposited on the conjugate pad and allowed to run for 8 minutes. Afterwards, a wash solution was deposited on the conjugate pad and allowed to run for 7 minutes. Results were obtained by detection of detectable agent present in the test zone with an ESE TS012 Gold Reader and reported in Table 2.
TABLE-US-00002 TABLE 2 Detection Results for Example 1 Lateral Flow Test Strip Based Lateral Flow Test Strip Based On On Preparation A Preparation B Experiment Concentration Average Standard Average Standard No. hCG, mIU/mL Signal* Deviation Signal/Noise Signal* Deviation Signal/Noise 1 0 8.3 4.96 1.00 16.4 9.99 1.00 2 0.25 13.9 5.82 1.67 14.2 7.51 0.87 3 0.5 16.5 6.41 1.99 20.0 5.16 1.22 4 1 16.2 6.71 1.95 13.0 6.28 0.80 5 2 33.3 12.60 4.01 19.3 6.75 1.18 6 5 71.9 17.45 8.66 49.4 7.59 3.02 7 10 112.4 15.01 13.54 90.0 7.22 5.51 8 15 164.3 20.42 19.80 121.5 14.89 7.43 9 25 197.7 32.45 23.82 177.3 24.34 10.84 10 50 349.8 35.40 42.14 279.5 11.87 17.10 11 100 451.5 65.08 54.39 389.1 20.75 23.80 *Each experiment was repeated 4-8 times resulting in a total of 80 experiments each for Lateral Flow Test Strip Preparation A and Preparation B, respectively.
[0182] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0183] While certain embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.