Compositions and methods for analyte detection using bioluminescence

Abstract

To enable creation of better assays for use in the field and in clinical settings.SOLUTION: Provided herein are systems and methods for the detection of an analyte or analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and / or quantifying a target analyte using a bioluminescent complex comprising substrates, peptides, and / or polypeptides capable of generating a bioluminescent signal that correlates to the presence and absence, or an amount of the target analyte.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 832,052, filed April 10, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0002] Provided herein are systems and methods for detecting one or more analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and / or quantifying target analytes using bioluminescent conjugates comprising substrates, peptides, and / or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte. [Background technology]

[0003] Biological processes depend on covalent and non-covalent interactions between molecules, macromolecules, and molecular complexes. To understand such processes and develop techniques and compounds to manipulate them for research, clinical, and other practical applications, tools are needed that can detect and monitor these interactions and / or the components involved in them. Studying these interactions requires high sensitivity, especially under physiological conditions (e.g., normal expression levels for monitoring protein interactions).

[0004] The creation of better assays for use in field and clinical settings remains an area of ​​urgent need. Speed, sensitivity, selectivity, robustness, simplicity, quantitative versus qualitative functionality, and cost are all important factors that influence the validity of a diagnostic bioassay and, therefore, its practicality and adoption in related industries. Rapid diagnostic tests are not only relevant in clinical settings, but can also be directly applicable to environmental, industrial, and consumer situations. Summary of the Invention

[0005] Provided herein are compositions and formulations comprising a luminescent substrate and a target analyte binding agent comprising a target analyte binding element and one of the polypeptide components of a bioluminescent complex or the peptide components of a bioluminescent complex.

[0006] According to these embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:5, at least 60% sequence identity to SEQ ID NO:9, or at least 60% sequence identity to SEQ ID NO:12.

[0007] In some embodiments, the peptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:10, at least 60% sequence identity to SEQ ID NO:11, at least 60% sequence identity to SEQ ID NO:13, or at least 60% sequence identity to SEQ ID NO:14.

[0008] In some embodiments, the composition comprises a complementary peptide or polypeptide component of a bioluminescent complex, wherein the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of the target analyte.

[0009] In some embodiments, a composition comprising a luminescent substrate and a target analyte binding agent is combined in a dry formulation, and the complementary peptide or polypeptide component of the bioluminescent complex is included in a liquid formulation, which is added to the dry formulation and, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0010] In some embodiments, a composition comprising a luminescent substrate, a target analyte binding agent, and the complementary peptide or polypeptide components of a bioluminescent complex are combined into a dry formulation, which upon rehydration forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0011] In some embodiments, the complementary peptide or polypeptide component comprises a second target analyte binding element that forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0012] In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:6, and the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to SEQ ID NO:10.

[0013] In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:6, and the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to SEQ ID NO:14.

[0014] Embodiments of the present disclosure also include compositions comprising a dry formulation that includes: (a) a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:9; and (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:10.

[0015] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0016] In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0017] Embodiments of the present disclosure also include compositions comprising a dry formulation that includes: (a) a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:12; and (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:14.

[0018] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0019] In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0020] Embodiments of the present disclosure also include compositions comprising a dry formulation that includes: (a) a first target analyte binding agent comprising a first target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO: 13; (b) a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 15; and (c) a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO: 12.

[0021] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0022] In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0023] Embodiments of the present disclosure also include compositions comprising: (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:9; and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:10 or SEQ ID NO:11.

[0024] Embodiments of the present disclosure also include compositions comprising: (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO:10 or SEQ ID NO:11; and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO:9.

[0025] Embodiments of the present disclosure also include compositions comprising: (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:12; and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:14.

[0026] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0027] In some embodiments, the liquid formulation further comprises a luminescent substrate.

[0028] In some embodiments, the liquid formulation further comprises a sample containing a target analyte, and the bioluminescent analyte detection complex is formed by combining the dry formulation and the liquid formulation in the presence of the target analyte.

[0029] In some embodiments, the composition further comprises a second complementary peptide or polypeptide component of a bioluminescent complex, and the target analyte binding agent, the first complementary peptide or polypeptide component of the bioluminescent complex, and the second complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of the target analyte.

[0030] In some embodiments, the composition comprising the target analyte binding agent is contained in a dry formulation, and the first complementary peptide or polypeptide component and the second complementary peptide or polypeptide of the bioluminescent complex are contained in a liquid formulation, which is added to the dry formulation and, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0031] In some embodiments, a composition comprising a target analyte binding agent and either a first or second complementary peptide or polypeptide component is combined into a dry formulation, and the first or second complementary peptide or polypeptide component not present in the dry formulation is included in a liquid formulation, which is added to the dry formulation and, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0032] In some embodiments, the target analyte binding agent, the first complementary peptide or polypeptide component, and the second complementary peptide or polypeptide component are combined in a dry formulation that, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0033] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0034] In some embodiments, the liquid formulation further comprises a luminescent substrate.

[0035] In some embodiments, the liquid formulation further comprises a sample containing a target analyte, wherein a bioluminescent analyte detection complex is formed upon combining the dry formulation and the liquid formulation in the presence of the target analyte.

[0036] In some embodiments, either the first or second complementary peptide or polypeptide component comprises a second target analyte binding element that, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0037] In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:6, and either the first or second complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to either SEQ ID NO:13 or SEQ ID NO:15.

[0038] Embodiments of the present disclosure also include compositions comprising: (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:6; and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15, and a second complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0039] Embodiments of the present disclosure also include (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:6, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15; and (b) a liquid formulation comprising a second complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0040] Embodiments of the present disclosure also include (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:6, and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15; and (b) a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0041] Embodiments of the present disclosure also include (a) a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 15, and (b) a liquid formulation comprising a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO: 6.

[0042] Embodiments of the present disclosure also include (a) a dry formulation comprising a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO:6; and (b) a liquid formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO:13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:15.

[0043] Embodiments of the present disclosure also include compositions comprising a dry formulation including a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO:13, a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:15, and a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO:6.

[0044] In some embodiments, the dry formulation further comprises a luminescent substrate.

[0045] In some embodiments, the liquid formulation further comprises a luminescent substrate.

[0046] In some embodiments, the liquid formulation further comprises a sample containing a target analyte, and the bioluminescent analyte detection complex is formed by combining the dry formulation and the liquid formulation in the presence of the target analyte.

[0047] In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the target analyte binding agent is contacted with one or more of the complementary peptide or polypeptide components of the bioluminescent complex, as compared to the bioluminescent signal generated by the target analyte binding agent and the luminescent substrate alone.

[0048] In some embodiments, the target analyte is a target antibody.

[0049] In some embodiments, the target analyte binding agent comprises an element that non-specifically binds to an antibody.

[0050] In some embodiments, the target analyte binding agent comprises an entity that specifically binds to an antibody.

[0051] In some embodiments, the targeting antibody is an antibody against a pathogen, a toxin, or a therapeutic biologic.

[0052] In some embodiments, the target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, Protein A, the Ig binding domain of Protein A, Protein G, the Ig binding domain of Protein G, Protein A / G, the Ig binding domain of Protein A / G, Protein L, the Ig binding domain of Protein L, Protein M, the Ig binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.

[0053] In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW, 1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.

[0054] In some embodiments, the composition further comprises a polymer.

[0055] In some embodiments, the polymer is a natural biopolymer. In some embodiments, the natural biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof. In some embodiments, the natural biopolymer is pullulan.

[0056] In some embodiments, the polymer is a cyclic glycopolymer or a derivative thereof, hi some embodiments, the polymer is hydroxypropyl β-cyclodextrin.

[0057] In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is poloxamer 188.

[0058] In some embodiments, the composition further comprises a substance that reduces self-luminescence.

[0059] In some embodiments, the substance that reduces self-luminescence is ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, a thiourea, or the like.

[0060] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiments, the surfactant is selected from polysorbate 20, polysorbate 40, and polysorbate 80.

[0061] In some embodiments, the compositions are used in conjunction with an analyte detection platform to detect an analyte in a sample.

[0062] In some embodiments, the sample is selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.

[0063] Embodiments of the present disclosure also include methods of detecting an analyte in a sample comprising combining any of the compositions described above with a sample containing the target analyte.

[0064] In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.

[0065] In some embodiments, the method further comprises quantifying the bioluminescent signal generated from the analyte detection complex.

[0066] In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte.

[0067] In some embodiments, one or more of the components of the composition exhibits enhanced stability within the composition compared to the component alone in solution.

[0068]

[0006] Embodiments of the present disclosure also include systems and methods for detecting one or more analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and / or quantifying target analytes using bioluminescent conjugates that include substrates, peptides, and / or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.

[0069] Embodiments of the present disclosure include lateral flow detection systems. According to these embodiments, the system includes an analytical membrane including a detection region and a control region. In some embodiments, the detection region includes a first target analyte-binding agent immobilized in the detection region, a conjugate pad including a second target analyte-binding agent, and a sample pad. In some embodiments, the first target analyte-binding agent and the second target analyte-binding agent form a bioluminescent analyte detection complex in at least one detection region when the target analyte is detected in the sample.

[0070] In some embodiments, the first target analyte binding agent comprises a target analyte binding element and is non-luminescent. In some embodiments, the second target analyte binding agent comprises a target analyte binding element and a bioluminescent polypeptide. In some embodiments, the bioluminescent polypeptide has at least 60% sequence identity to SEQ ID NO:5.

[0071] In some embodiments, the first target analyte-binding agent comprises a target analyte-binding element and a polypeptide component of a bioluminescent complex, and the second target analyte-binding agent comprises a target analyte-binding element and a peptide component of a bioluminescent complex. In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the first target analyte-binding agent contacts the second target analyte-binding agent, compared to the bioluminescent signal generated by the first target analyte-binding agent and the luminescent substrate alone.

[0072] In some embodiments, the first target analyte-binding agent comprises a target analyte-binding element and a peptide component of a bioluminescent complex, and the second target analyte-binding agent comprises a target analyte-binding element and a polypeptide component of a bioluminescent complex. In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the first target analyte-binding agent contacts the second target analyte-binding agent, compared to the bioluminescent signal generated by the first target analyte-binding agent and the luminescent substrate alone.

[0073] In some embodiments, a polypeptide component of a bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 6. In some embodiments, a polypeptide component of a bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 10. In some embodiments, a polypeptide component of a bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 12. In some embodiments, a polypeptide component of a bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 14.

[0074] In some embodiments, the first target analyte-binding agent comprises a target analyte-binding element and a first peptide component of a ternary bioluminescent complex, and the second target analyte-binding agent comprises a target analyte-binding element and a second peptide component of the ternary bioluminescent complex. In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the first target analyte-binding agent contacts the second target analyte-binding agent and a polypeptide component of the ternary bioluminescent complex, compared to the bioluminescent signal generated by (i) the first target analyte-binding agent, the second target analyte-binding agent, and / or the polypeptide component and (ii) the luminescent substrate alone.

[0075] In some embodiments, a first peptide component of the ternary bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 11. In some embodiments, a second first peptide component of the ternary bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 13. In some embodiments, a polypeptide component of the ternary bioluminescent complex has at least 60% sequence identity to SEQ ID NO: 12.

[0076] In some embodiments, the target analyte is a target antibody. In some embodiments, the first target analyte binding element comprises an agent that non-specifically binds to the antibody. In some embodiments, the second target analyte binding element comprises an agent that specifically binds to the target antibody. In some embodiments, the target antibody is an antibody against a pathogen, a toxin, or a therapeutic biologic.

[0077] In some embodiments, the target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, Protein A, the Ig binding domain of Protein A, Protein G, the Ig binding domain of Protein G, Protein A / G, the Ig binding domain of Protein A / G, Protein L, the Ig binding domain of Protein L, Protein M, the Ig binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.

[0078] In some embodiments, the system further comprises a luminescent substrate. In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminescent substrate is applied to the system as part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is applied to the system as part of a composition comprising the luminescent substrate and a substance that reduces autoluminescence, such as ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, or a thiourea.

[0079] In some embodiments, the composition is applied to at least one of a sample pad, a conjugation pad, a detection area, and a control area.

[0080] In some embodiments, the analytical membrane comprises multiple detection regions, each detection region comprising a different target analyte binding agent having a different target analyte binding element.

[0081] In some embodiments, the system further comprises a device for detecting or quantifying a bioluminescent signal from the analyte detection complex.

[0082] Embodiments of the present disclosure also include a conjugate pad comprising at least one target analyte binding agent. According to these embodiments, the at least one target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide comprising at least 60% sequence identity to SEQ ID NO:5, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:9, a peptide comprising at least 60% sequence identity to SEQ ID NO:10, a peptide comprising at least 60% sequence identity to SEQ ID NO:11, a peptide comprising at least 60% sequence identity to SEQ ID NO:13, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:12, a peptide comprising at least 60% sequence identity to SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0083] In some embodiments, the target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide of SEQ ID NO:5, a polypeptide of SEQ ID NO:9, a peptide of SEQ ID NO:10, a peptide of SEQ ID NO:11, a peptide of SEQ ID NO:13, a polypeptide of SEQ ID NO:12, a peptide of SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0084] In some embodiments, the conjugate pad further comprises a luminescent substrate. In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminescent substrate is contained on or within the conjugate pad as part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is applied to the system as part of a composition comprising the luminescent substrate and a substance that reduces self-luminescence, such as ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, or a thiourea.

[0085] Embodiments of the present disclosure also include analytical membranes comprising a detection region and a control region. According to these embodiments, the detection region comprises at least one target analyte binding agent immobilized therein.

[0086] In some embodiments, at least one target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide comprising at least 60% sequence identity to SEQ ID NO:5, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:9, a peptide comprising at least 60% sequence identity to SEQ ID NO:10, a peptide comprising at least 60% sequence identity to SEQ ID NO:11, a peptide comprising at least 60% sequence identity to SEQ ID NO:13, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:12, a peptide comprising at least 60% sequence identity to SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0087] In some embodiments, the target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide of SEQ ID NO:5, a polypeptide of SEQ ID NO:9, a peptide of SEQ ID NO:10, a peptide of SEQ ID NO:11, a peptide of SEQ ID NO:13, a polypeptide of SEQ ID NO:12, a peptide of SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0088] In some embodiments, the analytical membrane further comprises a plurality of detection regions, each detection region comprising a different target analyte binding agent having a different target analyte binding element. In some embodiments, the analytical membrane further comprises a luminescent substrate. In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.

[0089] In some embodiments, the luminescent substrate is reversibly conjugated to the conjugate pad as part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is part of a composition comprising the luminescent substrate and a substance that reduces self-luminescence, such as ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, or a thiourea.

[0090]

[0010] Embodiments of the present disclosure also include solid-phase detection platforms that include a detection region. According to these embodiments, the detection region includes at least one target analyte-binding agent conjugated to the detection region. In some embodiments, the at least one target analyte-binding agent includes a target analyte-binding element and one of a bioluminescent polypeptide comprising at least 60% sequence identity to SEQ ID NO:5, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:9, a peptide comprising at least 60% sequence identity to SEQ ID NO:10, a peptide comprising at least 60% sequence identity to SEQ ID NO:11, a peptide comprising at least 60% sequence identity to SEQ ID NO:13, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:12, a peptide comprising at least 60% sequence identity to SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0091] In some embodiments, the target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide of SEQ ID NO:5, a polypeptide of SEQ ID NO:9, a peptide of SEQ ID NO:10, a peptide of SEQ ID NO:11, a peptide of SEQ ID NO:13, a polypeptide of SEQ ID NO:12, a peptide of SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0092] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to the detection region, the first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:6, and a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO:10.

[0093] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to a detection region, the first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO: 10, and a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO: 6.

[0094] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to the detection region, the first target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO: 11; a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO: 13; and a polypeptide applied to the detection region, the polypeptide comprising at least 60% sequence identity to SEQ ID NO: 12.

[0095] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to a detection region, the first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:6, and a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:14.

[0096] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to a detection region, the first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO: 14, and a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO: 6.

[0097] In some embodiments, the detection platform comprises a first target analyte binding agent conjugated to a detection region, the first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide of at least 60% sequence identity to SEQ ID NO: 5, and a second target analyte binding agent applied to the detection region, the second target analyte binding agent comprising a target analyte binding element and a fluorophore activatable by energy transfer from the bioluminescent polypeptide.

[0098] In some embodiments, the detection platform comprises a first target analyte binding agent applied to a detection region, the first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide of at least 60% sequence identity to SEQ ID NO: 5, and a second target analyte binding agent conjugated to the detection region, the second target analyte binding agent comprising a target analyte binding element and a fluorophore activatable by energy transfer from the bioluminescent polypeptide.

[0099] In some embodiments, the detection platform further comprises a plurality of detection regions, each detection region comprising a different target analyte binding agent having a different target analyte binding element. In some embodiments, the detection platform further comprises a control region. In some embodiments, the detection platform further comprises a luminescent substrate. In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the luminescent substrate is reversibly conjugated to the conjugate pad as part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is part of a composition that includes the luminescent substrate and a substance that reduces self-luminescence, such as ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, or a thiourea.

[0100]

[0010] Embodiments of the present disclosure also include a liquid-phase detection platform comprising at least one detection reservoir and a lyophilized tablet (lyocake). According to these embodiments, the lyocake comprises a target analyte-binding element and a target analyte-binding agent comprising one of a bioluminescent polypeptide comprising at least 60% sequence identity to SEQ ID NO:5, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:9, a peptide comprising at least 60% sequence identity to SEQ ID NO:10, a peptide comprising at least 60% sequence identity to SEQ ID NO:11, a peptide comprising at least 60% sequence identity to SEQ ID NO:13, a polypeptide comprising at least 60% sequence identity to SEQ ID NO:12, a peptide comprising at least 60% sequence identity to SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0101] In some embodiments, the target analyte binding agent comprises a target analyte binding element and one of a bioluminescent polypeptide of SEQ ID NO:5, a polypeptide of SEQ ID NO:9, a peptide of SEQ ID NO:10, a peptide of SEQ ID NO:11, a peptide of SEQ ID NO:13, a polypeptide of SEQ ID NO:12, a peptide of SEQ ID NO:14, or a fluorophore activatable by energy transfer from Oplophorus luciferase.

[0102] In some embodiments, the lyocake comprises a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:6, and a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO:10.

[0103] In some embodiments, the lyocake comprises a first target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO:11, a second target analyte binding agent comprising a target analyte binding element and a peptide comprising at least 60% sequence identity to SEQ ID NO:13, and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:12.

[0104] In some embodiments, the lyocake comprises a first target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:6, and a second target analyte binding agent comprising a target analyte binding element and a polypeptide comprising at least 60% sequence identity to SEQ ID NO:14.

[0105] In some embodiments, the lyocake comprises a first target analyte binding agent comprising a target analyte binding element and a bioluminescent polypeptide having at least 60% sequence identity to SEQ ID NO:5, and a second target analyte binding agent comprising the target analyte binding element and a fluorophore activatable by energy transfer from the bioluminescent polypeptide.

[0106] In some embodiments, the detection platform comprises a 96-well microtiter plate containing a plurality of detection reservoirs and at least two different target analyte binding agents comprising different target analyte binding elements.

[0107] In some embodiments, the lyocake comprises a luminescent substrate, hi some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives.

[0108] In some embodiments, the lyocake comprises a luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof.

[0109] In some embodiments, the lyocake comprises a luminescent substrate and a substance that reduces autoluminescence, such as ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, or a thiourea.

[0110] In some embodiments, the detection platform further comprises at least one sample, in some embodiments, the sample is selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.

[0111]

[0010] Embodiments of the present disclosure also include methods for detecting an analyte in a sample using the lateral flow assay system described above. According to these embodiments, the method includes applying a sample to a sample pad and facilitating flow of the sample from the sample pad to a conjugate pad and then from the conjugate pad to a detection zone and a control zone on an analytical membrane. In some embodiments, the first target analyte binding agent, the second target analyte binding agent, and the target analyte form an analyte detection complex in at least one detection zone when the target analyte is detected in the sample.

[0112] In some embodiments, the sample is a sample from a subject selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, tissue, and saliva. In some embodiments, the sample is selected from a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.

[0113] In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex, hi some embodiments, the method further comprises diagnosing the subject from whom the sample was obtained as having or not having the disease based on the detection of the analyte.

[0114]

[0010] Embodiments of the present disclosure also include methods for detecting an analyte in a sample using the solid-phase detection platform described above. According to these embodiments, the method includes exposing the sample to a detection region and a control region. In some embodiments, at least one target analyte binding agent and at least one target analyte form an analyte detection complex in the at least one detection region when the target analyte is detected in the sample.

[0115] In some embodiments, the sample is a sample from a subject selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, tissue, and saliva. In some embodiments, the sample is selected from a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.

[0116] In some embodiments, the method further comprises quantifying a bioluminescent signal generated from the analyte detection complex, hi some embodiments, the method further comprises diagnosing the subject from whom the sample was obtained as having or not having the disease based on the detection of the analyte.

[0117] Embodiments of the present disclosure also include methods of preparing a substrate for use in a bioluminescence assay. According to these embodiments, the method includes applying a solution onto the substrate. In some embodiments, the solution contains at least one target analyte binding agent, including a target analyte-binding element and one of a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the method includes drying the substrate containing the solution.

[0118] In some embodiments, the solution further comprises a complementary peptide or polypeptide component of a bioluminescent complex, hi some embodiments, the target analyte binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of the target analyte.

[0119] In some embodiments, the solution comprises a protein buffer and at least one excipient. In some embodiments, the solution comprises a luminescent substrate.

[0120] In some embodiments, the substrate containing the dried solution is W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, Ahlstrom A-226 paper, M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester paper, or a combination thereof. In some embodiments, the substrate is a mesh comprising plastic, nylon, metal, or a combination thereof.

[0121] In some embodiments, drying the substrate containing the solution comprises drying for about 30 minutes to 2 hours at a temperature of about 30° C. to 40° C. In some embodiments, drying the substrate containing the solution comprises lyophilizing and / or freezing the substrate.

[0122] In some embodiments, the method further comprises drying at least one target analyte binding agent and / or complementary peptide or polypeptide component of the bioluminescent complex onto a first substrate, and drying the luminescent substrate onto a second substrate.

[0123] According to these embodiments, a bioluminescent signal is generated by exposing the target analyte to a substrate containing the solution, and in some embodiments, the bioluminescent signal is proportional to the concentration of the target analyte.

[0124] In some embodiments, at least one target analyte binding agent and / or complementary peptide or polypeptide component of a bioluminescent complex exhibits enhanced stability when dried onto a substrate.

[0125] Embodiments of the present disclosure include compositions comprising a luminescent substrate, a target analyte binding agent comprising a target analyte binding element and a polypeptide component of a bioluminescent complex, and a complementary polypeptide component of the bioluminescent complex, wherein the target analyte binding agent and the complementary polypeptide component of the bioluminescent complex are capable of forming a bioluminescent analyte detection complex in the presence of the target analyte.

[0126] In some embodiments, the composition further comprises a second target analyte binding agent comprising a second target analyte binding member and a second polypeptide component of a bioluminescent complex.

[0127] In some embodiments, the first and second target analyte binding agents bind to different portions of the same target analyte.

[0128] In some embodiments, the first and second polypeptide components of the bioluminescent complex bind to complementary polypeptide components of the bioluminescent complex to form a bioluminescent analyte detection complex in the presence of a target analyte.

[0129] In some embodiments, the first and second polypeptide components are linked to a modified dehalogenase capable of forming a covalent bond with a haloalkane substrate.

[0130] In some embodiments, the first and second target analyte binding members comprise haloalkane substrates.

[0131] In some embodiments, the first or second polypeptide components of the first and second target analyte binding agents comprise at least 60% sequence identity to SEQ ID NO:10, at least 60% sequence identity to SEQ ID NO:11, at least 60% sequence identity to SEQ ID NO:13, or at least 60% sequence identity to SEQ ID NO:15.

[0132] In some embodiments, the complementary polypeptide component comprises at least 60% sequence identity to SEQ ID NO:6, at least 60% sequence identity to SEQ ID NO:9, or at least 60% sequence identity to SEQ ID NO:12.

[0133] In some embodiments, the target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, Protein A, the Ig binding domain of Protein A, Protein G, the Ig binding domain of Protein G, Protein A / G, the Ig binding domain of Protein A / G, Protein L, the Ig binding domain of Protein L, Protein M, the Ig binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.

[0134] In some embodiments, the target analyte is an antibody, the target analyte binding element of the first target analyte binding agent comprises an antigen recognized by the antibody, and the target analyte binding element of the second target analyte binding agent comprises an Fc binding region.

[0135] In some embodiments, the first and / or second target analyte binding agent further comprises a fluorophore attached to the first and / or second polypeptide component of the bioluminescent complex.

[0136] In some embodiments, one or more components of the composition are in the form of a lyophilized tablet (lyocake) that is capable of forming a bioluminescent complex when reconstituted in solution for detecting and / or quantifying a target analyte.

[0137] In some embodiments, the composition is included in a liquid phase detection platform capable of detecting and / or quantifying a target analyte.

[0138] In some embodiments, the polypeptide components and luminescent substrate are in the form of a lyophilized tablet (lyocake) that is capable of forming a bioluminescent complex when reconstituted in solution for detecting and / or quantifying a target analyte.

[0139] Embodiments of the present disclosure also include methods of detecting an analyte in a sample comprising combining any of the compositions described above with a sample containing the target analyte.

[0140] In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex.

[0141] In some embodiments, the method further comprises quantifying the bioluminescent signal generated from the analyte detection complex.

[0142] In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte. [Brief explanation of the drawings]

[0143] [Figure 1]FIG. 1 shows a representative schematic diagram of a lateral flow assay for detecting and / or quantifying target analyte(s) in a sample based on bioluminescent complex formation, according to one embodiment of the present disclosure. [Figure 2] FIG. 1 shows a representative schematic diagram of a solid-phase detection platform for detecting and / or quantifying a target analyte in a sample based on bioluminescent complex formation, according to one embodiment of the present disclosure. [Figure 3] Representative images are shown showing that the components of the bioluminescent complex produce detectable bioluminescence after being applied to a solid support substrate (e.g., a membrane), dried, and stored at room temperature. [Figure 4] Representative images are shown showing that components of the bioluminescent complex produce detectable bioluminescence after application to membrane and paper-based solid support substrates. [Figure 5] A representative assay schematic (left) and a representative graph (right) are shown demonstrating the ability of components of a bioluminescent complex used as reporters on a target analyte-binding agent to detect a target analyte. [Figure 6] FIG. 1 shows a representative diagram of an assay platform that uses components of a bioluminescent complex as reporters on a target analyte-binding agent to detect the target analyte. [Figure 7] 1 shows representative stability studies of an assay platform that uses components of a bioluminescent complex as a reporter on a target analyte binder to detect a target analyte according to one embodiment of the present disclosure (A at 4°C; B at 25°C; C at 37°C; D at 37°C with the addition of NanoLuc; and E at 4°C and 37°C with the addition of HiBiT). [Figure 8] FIG. 1 shows a representative test of storage conditions for an assay platform that uses components of a bioluminescent complex as a reporter on a target analyte binder to detect a target analyte according to one embodiment of the present disclosure (A, 4° C. and 25° C.; B, sucrose-based protein buffer at 4° C. and 25° C.). [Figure 9]Representative images of the solid-phase assay platform (A) are shown, demonstrating the generation of a bioluminescent signal and detection of the target analyte in a complex sampling environment (B: whole blood and C: serum). [Figure 10] Figure 1 shows that the RLU signal obtained from Whatman 903 paper spots after rehydration with assay buffer can be measured either quantitatively (A) or qualitatively (B). [Figure 11] A representative graph is shown demonstrating the ability of the high-affinity dipeptide Pep263 to form a bioluminescent complex (Pep263 is a peptide comprising the β9 and β10 strands of the NanoTrip complex; see, e.g., U.S. Patent Application No. 16 / 439,565 (PCT / US2019 / 036844), the entire contents of which are incorporated herein by reference). [Figure 12] Representative results of a solid-phase assay are shown, showing the qualitative assessment of bioluminescence from paper punches placed in a standard microtiter plate using a standard camera on an iPhone (e.g., iPhone 6S) or an imager (e.g., LAS4000). [Figure 13] Quantitative analysis of the same solid-phase assay as shown in Figure 12 is shown, but luminescence was detected using a luminometer after 3 days of storage at 25°C. [Figure 14] Quantitative time courses of the same solid-phase assays shown in Figures 12-13 are shown, demonstrating the stability of all proteins in the experimental conditions at all temperatures tested over this time frame. [Figure 15] Representative RLU signal kinetic results collected on day 0 of an accelerated stability study conducted under two buffer conditions at 25°C and 60°C are shown. [Figure 16] FIG. 15 shows the time course results of an accelerated stability study of proteins placed using the conjugation buffer conditions defined. [Figure 17] 1 shows a comparison of the effect of buffer conditions on luminescence from NanoLuc dried onto a nitrocellulose membrane. [Figure 18]The effects of membrane blocking and sucrose pretreatment on lateral flow assays performed in a running buffer of 20× SSC, 1% BSA (pH 7.0), and 10 μM N205 (Live Cell Substrate; LCS) are shown. [Figure 19] 1 shows the effect of membrane blocking and sucrose pretreatment in a lateral flow assay performed in a running buffer of 0.01 M PBS, 1% BSA (pH 7.0), and 10 μM Permeable Cell Substrate (PCS). [Figure 20] 1 shows the effect of membrane blocking and sucrose pretreatment on a lateral flow assay performed with 5x LCS dilution buffer plus running buffer of 5x LCS diluted to 1X with PBS. [Figure 21] 1 shows the effect of membrane properties on bioluminescent reagent absorption and capillary action in a lateral flow assay. [Figure 22] Bioluminescence signals from NanoBiT / HiBiT complements on nitrocellulose (left) and Whatman grade 541 (right) paper (A) and edited images from the corresponding movie taken over the entire exposure time (B). [Figure 23] Bioluminescent signal from NanoBiT / HiBiT complement on Whatman 903 paper is shown with additional substrate and liquid spike at 20 minutes. [Figure 24] Bioluminescent signal from NanoBiT / HiBiT complement on Whatman 903 paper is shown. [Figure 25] Figure 25C shows the bioluminescence signals obtained from LgTrip and substrate reconstituted with dipeptide on Whatman 903 paper prepared with (B) or without (A) BSA, and Figure 25C shows the maximum RLU signal for each concentration tested in Figure 25B. [Figure 26]The bioluminescence signal obtained from the dipeptide-based reconstitution of LgTrip and substrate from the lyocake (A) is shown along with the titration of the dipeptide, and B shows the maximum RLU signal obtained for each concentration tested in A. [Figure 27] Bioluminescence signals from three different solid-phase materials (Whatman 903, Ahlstrom 237, and Ahlstrom 6613H) obtained from reconstitution by addition of dipeptides to dried LgTrip and substrate or by addition of NanoLuc to dried LgTrip and substrate are shown. [Figure 28] Figure 1 shows the bioluminescence signal generated from Whatman 903 spots containing Lg / Trip / substrate and stored at ambient conditions for 25 days. The spots were exposed to 1 nM of the dipeptide in PBS. [Figure 29] Bioluminescence signals (RLU) of NanoLuc (A), LgBiT (B), and LgTrip (C) dried onto Whatman 903 paper using various protein buffer formulations and reconstituted with furimazine are shown. [Figure 30] Figure 29 shows the bioluminescence signal (Bmax) of NanoLuc (A), LgBiT (B), and LgTrip (C) dried onto Whatman 903 paper using various protein buffer formulations and reconstituted with furimazine. [Figure 31] FIG. 29 shows the background bioluminescence levels of LgBiT (A) and LgTrip (B) dried onto Whatman 903 paper using various protein buffer formulations and reconstituted with furimazine. [Figure 32A] Figure 1 shows the bioluminescence signal of NanoLuc (RLU signal kinetics after reconstitution with furimazine) after drying Whatman 903 paper with various protein buffer formulations and storing at 60°C for 6 days, followed by reconstitution with furimazine. [Figure 32B]Figure 1 shows the bioluminescence signal of LgBiT (RLU signal kinetics after reconstitution with furimazine) dried on Whatman 903 paper with various protein buffer formulations, stored at 60°C for 6 days, and then reconstituted with furimazine. [Figure 32C] Figure 1 shows the bioluminescence signal of LgTrip (RLU signal kinetics after reconstitution with furimazine) after drying Whatman 903 paper with various protein buffer formulations and storing at 60°C for 6 days, followed by reconstitution with furimazine. [Figure 32D] Bioluminescence signal (Bmax) of NanoLuc dried onto Whatman 903 paper with various protein buffer formulations and stored at 60° C. for 6 days, followed by reconstitution with furimazine, is shown. [Figure 32E] Bioluminescence signal (Bmax) of LgBiT dried onto Whatman 903 paper with various protein buffer formulations and stored at 60°C for 6 days, followed by reconstitution with furimazine, is shown. [Figure 32F] Figure 1 shows the bioluminescence signal (Bmax) of LgTrip dried onto Whatman 903 paper with various protein buffer formulations, stored at 60°C for 6 days, and then reconstituted with furimazine. [Figure 33] A representative embodiment of an all-in-one lyophilized cake ("lyocake") or tablet containing all the reagents necessary to perform an analyte detection test supporting several types of assay formats, including cuvettes, test tubes, large volume bottles, snap test type assays, etc. [Figure 34] Compiled images corresponding to a movie taken over the entire exposure time show the bioluminescent signal due to substrate migration across a lateral flow strip containing NanoLuc. [Figure 35] Compiled images corresponding to a movie taken over the entire exposure time show the bioluminescent signal due to the movement of NanoLuc across the lateral flow strip. [Figure 36]Shown are various tracers generated by coupling fumonisin B1 to a peptide tag (e.g., comprising SEQ ID NO: 10) via a biotin / streptavidin linkage, a HaloTag linkage, or directly (e.g., via a sulfo-SE label as described in U.S. Patent Application No. 16 / 698,143 (PCT / US2019 / 063652), incorporated herein by reference), which can be used in competitive binding assays according to the materials and methods described herein. [Figure 37] An exemplary competitive binding assay is shown in which various concentrations of unlabeled fumonisin B1 disrupt the bioluminescent complex, resulting in reduced light emission and the ability to detect / quantitate the amount of fumonisin B1 in a sample. [Figure 38] Shown is the bioluminescence signal obtained from a lyophilized cake containing LgBiT and substrate when reconstituted with the dipeptide in PBS (A); B shows the maximum RLU signal obtained for each concentration tested in A. [Figure 39] Bioluminescence signals obtained from reconstitution of lyophilized LgBiT or LgTrip 3546 directly into standard 96-well plates with or without substrate are shown, and reconstitution was performed with the dipeptide in PBS with or without substrate. [Figure 40A] Bioluminescence signals obtained from LgBiT-Protein G, SmBiT-TNFα, and substrate complementation on Whatman 903 paper spots after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 40B] Bioluminescence signals obtained from LgBiT-Protein G, SmBiT-TNFα, and substrate complementation on Whatman 903 paper spots after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 40C] Bioluminescence signals obtained from LgBiT-Protein G, SmBiT-TNFα, and substrate complementation in the lyocake format after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 41A]Bioluminescence signals obtained from LgTrip, SmTrip9-Protein G, HiBiT-TNFα, and substrate complementation on Whatman 903 paper spots after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 41B] Bioluminescence signals obtained from LgTrip, SmTrip9-Protein G, HiBiT-TNFα, and substrate complementation in the lyocake format after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 41C] Bioluminescence signals obtained from LgTrip, SmTrip9-Protein G, HiBiT-TNFα, and substrate complementation in the lyocake format after reconstitution with various concentrations of the target analyte Remicade in PBS are shown. [Figure 42A] 1 shows the bioluminescent signal resulting from complementation of the bioluminescent complex dried in a substrate-free form. When the substrate is added separately, a bioluminescent signal is generated in the presence of the analyte. [Figure 42B] The bioluminescent signal obtained from complementation of the bioluminescent complex dried in a substrate-free form (mesh-based lyocake) is shown. Substrate is added separately, resulting in the generation of a bioluminescent signal in the presence of analyte. [Figure 42C] The bioluminescent signal obtained from complementation of the bioluminescent complex dried in a substrate-free form (mesh-based lyocake) is shown. Substrate is added separately, resulting in the generation of a bioluminescent signal in the presence of analyte. [Figure 42D] Shown is the bioluminescent signal resulting from complementation of the bioluminescent complex dried in substrate-free form (mesh-based film). Substrate is added separately, resulting in the generation of a bioluminescent signal in the presence of analyte. [Figure 42E] Shown is the bioluminescent signal resulting from complementation of the bioluminescent complex dried in substrate-free form (mesh-based film). Substrate is added separately, resulting in the generation of a bioluminescent signal in the presence of analyte. [Figure 43]Lyophilized cake formation and colorimetric pH of four different furimazine substrate formulations are shown. [Figure 44] Figure 1 shows the kinetic activity performance of various furimazine (Fz) substrate formulations in the presence of purified NanoLuc (Nluc) enzyme. [Figure 45] Figure 1 shows the activity performance of furimazine substrate formulations stored at 60°C for the indicated days. [Figure 46] Figure 1 shows the thermal stability over time in days of various furimazine substrate formulations maintained at ambient temperature (A) or 60°C (B), analyzed by HPLC for absolute furimazine concentration remaining after reconstitution with PBS (pH 7.0) containing 0.01% BSA. [Figure 47] 1 shows the amount of furimazine remaining 12 days after reconstitution with water for various furimazine substrate formulations analyzed by HPLC demonstrating liquid stability. [Figure 48] 1 shows a schematic diagram of a homogeneous three-component immunoassay for the analyte interleukin-6 (IL-6). [Figure 49] An example of an SDS-PAGE gel of antibodies labeled with the tripartite HaloTag fusion protein is shown. Variants of SmTrip9 or SmTrip10 were fused to HaloTag, expressed, purified, and used to label mouse anti-human IL-6 antibodies. [Figure 50] Signal kinetics of a solution-based homogeneous three-component IL-6 immunoassay with and without IL-6 (A is raw RLU, and B is fold response). [Figure 51] FIG. 1 shows the dose-response curve of recombinant human IL-6 in a solution-based homogeneous IL-6 ternary immunoassay (A is logarithmic graph; B is linear graph). [Figure 52] Figure 1 shows the IL-6 immunoassay performance and storage stability of lyophilized cake products (A; #1 and #2) and various formulated single-reagent lyophilized cakes without (Fz; B) and with (Fz; C) furimazine reconstituted after storage at ambient temperature for the indicated days. [Figure 53]Cake appearance (A) and performance (B) and storage stability of a formulated lyophilized single-reagent IL-6 tripartite immunoassay stored for 90 days in ambient storage are shown. [Figure 54] Figure 1 shows the signal kinetics of a single reagent lyophilized three-component IL-6 immunoassay after reconstitution. [Figure 55] 1 shows the suitability of a lyophilized single-reagent IL-6 immunoassay with a complex human matrix. [Figure 56] A lyophilized single-reagent IL-6 tripartite immunoassay in a pre-filled 96-well microtiter plate (A) and a rhIL-6 dose-response curve using the lyophilized single-reagent IL-6 tripartite immunoassay assay plate after reconstitution (B) are shown. [Figure 57] Assay performance of a solution-based IL-6 ternary immunoassay in a single formulation vehicle (A) and in various formulation solutions (B). [Figure 58] 1 shows a schematic diagram of a homogeneous three-component immunoassay for the model analyte cardiac troponin I. [Figure 59] Dose-response curves for a solution-based homogeneous cardiac troponin I ternary immunoassay using recombinant human cardiac troponin I are shown in raw RLU (A) and signal over background (B). [Figure 60] Assay performance of the single-reagent combination lyophilized troponin cardiac I three-component immunoassay after reconstitution with 10% normal pooled human serum diluted in PBS containing 0.01% BSA or common serum diluent is shown in raw RLU. [Figure 61] Figure 1 shows raw RLU results of background signal for a solution-based homogeneous IL-6 three-component immunoassay in the presence of human serum using an assay buffer of PBS containing 0.01% BSA (A) and a common serum diluent (B). [Figure 62]Figure 1 shows raw Bmax RLU results of a solution-based homogeneous IL-6 three-component immunoassay in the presence of 50 ng / ml rhIL-6 in the presence of human serum using an assay buffer of PBS containing 0.01% BSA (A) and common serum diluent (B). [Figure 63] Figure 1 shows the signal-to-background results of a solution-based homogeneous IL-6 three-component immunoassay performed with increasing amounts of normal pooled human serum (A and C) or normal pooled human plasma (B and D) in the presence or absence of 50 ng / ml rhIL-6, using either PBS or General Serum Diluent containing 0.01% BSA as the assay buffer, and NanoGlo (Promega Catalog No. N113) (C and D) or Live Cell (Promega Catalog No. N205) substrate (A and B). [Figure 64] Figure 1 shows the signal-to-background results of a solution-based homogeneous IL-6 three-component immunoassay in the presence or absence of 50 ng / ml rhIL-6, with increasing amounts of normal pooled human serum and pooled human serum depleted of endogenous IgG, using common serum diluent as the assay buffer. [Figure 65] Shown are the background RLU (A), Bmax RLU (B), and resulting signal-to-background (C) results of a solution-based homogeneous IL-6 three-component immunoassay using increasing amounts of the human blood chemistry panel components provided in the VeriChem Matrix Plus Chemistry Reference Kit in the presence or absence of 50 ng / ml rhIL-6. [Figure 66] Shown are the background RLU (A), Bmax RLU (B), and resulting signal-to-background (C) results of a solution-based homogeneous IL-6 three-component immunoassay using increasing amounts of pooled normal human urine and NanoGlo (Promega Cat. No. N113) or Live Cell (Promega Cat. No. N205) substrate in the presence or absence of 50 ng / ml rhIL-6. [Figure 67]1 shows the raw RLU activity assay responses of reconstituted lyophilized formulated furimazine tested with purified NanoLuc enzyme (Nluc) (A), formulated LgTrip polypeptide (SEQ ID NO: 12) tested with purified dipeptide (SEQ ID NO: 14) (B), and formulated furimazine and LgTrip polypeptide (SEQ ID NO: 12) tested with purified dipeptide (SEQ ID NO: 14) along with analysis of thermal stability of lyophilized vials (C). [Figure 68] Schematic diagram of the homogeneous three-component immunoassay for three anti-TNFα biologics: Remicade, Enbrel, and Humira. [Figure 69] Assay performance is shown in raw RLU for a solution-based homogeneous three-component (LgTrip 3546 + SmTrip9 pep521 + SmTrip10) immunoassay for the quantification of the anti-TNFα biologics Remicade, Humira, and Enbrel. [Figure 70] Kinetic assay performance of reconstituted, formulated, lyophilized single-reagent immunoassays for the detection of Remicade using NanoTrip (ternary NanoLuc; A) and NanoBiT (B) is shown as raw RLU. [Figure 71]

[0049] Figure 1 shows the thermal stability at ambient temperature of single-reagent lyophilized NanoBiT ("Bits") and NanoTrip ("Trips"; ternary NanoLuc) immunoassay systems for detecting Remicade. Lyocakes were reconstituted at the indicated time points in the absence or presence of 100 nM Remicade, and the resulting raw RLUs were analyzed. [Figure 72]Representative results are shown using the NanoBiT system to detect Remicade. The formulation components were separated into two individual cakes prior to assay: (A) Image of two separate lyophilized components, one containing the LgBiT-TNFα fusion protein and furimazine (yellow), and the other containing the SmBiT-Protein G fusion protein (white). (B) Image of the two lyophilized components in Figure 72A after manual recombination. (C) Image of the reconstituted lyophilized component. (D) Kinetic bioluminescence RLU signals obtained in the presence of increasing amounts of Remicade. [Figure 73] Kinetic bioluminescence RLU signals obtained in the presence of increasing amounts of Remicade using the Dual Lyophilized NanoTrip Immunoassay System, whereby TNFα plus furimazine and Protein G fusion proteins were formulated, lyophilized separately, and then combined prior to reconstitution. [Figure 74] FIG. 1 shows a schematic diagram of the homogeneous NanoTrip (three-component NanoLuc) cell-based immunoassay system for detecting anti-EGFR biologics (e.g., panitumumab). [Figure 75] 1 shows the dose-response curve for panitumumab using the homogeneous cell-based NanoTrip immunoassay system for anti-EGFR biologics. [Figure 76] 1 shows the dose-response curves of panitumumab using the homogeneous cell-based NanoTrip immunoassay system for anti-EGFR biologics testing different variants of SmTrip9 (SEQ ID NO: 13) fused to Protein G. [Figure 77A] 1 shows a dose-response curve for Remicade using the homogeneous solution-based NanoTrip immunoassay system for anti-TNFα biologics, testing various variants of SmTrip9 (SEQ ID NO: 13) fused to Protein G. [Figure 77B] 1 shows the dose-response curve for Remicade using the lyophilized NanoTrip immunoassay system for anti-TNFα biologics. [Figure 78] 1 shows a schematic diagram of a three-component IL-6 immunoassay system using an antibody directly labeled with a reactive peptide (e.g., SEQ ID NO: 18). [Figure 79] 1 shows a denaturing SDS-PAGE gel analysis of directly labeled antibody conjugates. [Figure 80] Shown are raw RLU outputs obtained from IL-6 titrations in the presence of anti-IL-6 antibody pairs directly labeled with reactive peptides HW-0984 (SEQ ID NO: 20), HW-1010 (SEQ ID NO: 24), and HW-0977 (SEQ ID NO: 18). [Figure 81] Shown are the raw RLU outputs obtained from IL-6 titrations in the presence of an anti-IL-6 antibody pair directly labeled with the reactive peptides HW-0984 (SEQ ID NO: 20) and HW-1053 (SEQ ID NO: 19). [Figure 82] Shown are raw RLU outputs obtained from IL-6 titrations in the presence of anti-IL-6 antibody pairs labeled with reactive peptides HW-1042 (SEQ ID NO: 20), HW-1050 (SEQ ID NO: 27), HW-1052 (SEQ ID NO: 25), HW-1043 (SEQ ID NO: 24) and HW-1055 (SEQ ID NO: 25). [Figure 83] 1 shows the raw RLU outputs obtained from IL-6 titrations in the presence of individual anti-IL-6 antibodies directly labeled with reactive peptides HW-0977 (SEQ ID NO: 18), HW-0984 (SEQ ID NO: 20), HW-1010 (SEQ ID NO: 24), HW-1042 (SEQ ID NO: 20), HW-1050 (SEQ ID NO: 27), HW-1052 (SEQ ID NO: 25), HW-1053 (SEQ ID NO: 19), HW-1043 (SEQ ID NO: 24), and HW-1055 (SEQ ID NO: 25). [Figure 84] 1 shows raw RLU outputs obtained from IL-6 titration in the presence of LgTrip 5146 (SEQ ID NO: 451) and anti-IL-6 antibody pairs labeled with reactive peptides HW-1050 (SEQ ID NO: 27), HW-1043 (SEQ ID NO: 24), and HW-0977 (SEQ ID NO: 18). [Figure 85]FIG. 1 shows a schematic diagram of a three-component IL-6 immunoassay model using an antibody directly labeled with a reactive peptide containing a fluorophore, allowing BRET between luciferase and the labeled antibody. [Figure 86] IL-6 induced BRET between complementary three-component luciferases and fluorophores on a labeled anti-IL-6 antibody. [Figure 87] Luminescence obtained from the luminescent substrates N113 Fz (A), JRW-1404 (B), and JRW-1482 (C) in complex matrices is shown. DETAILED DESCRIPTION OF THE INVENTION

[0144] Embodiments of the present disclosure provide systems and methods for detecting one or more analytes in a sample. In particular, the present disclosure provides compositions, assays, and methods for detecting and / or quantifying target analytes using bioluminescent conjugates that include substrates, peptides, and / or polypeptides capable of generating a bioluminescent signal that correlates to the presence, absence, or amount of the target analyte.

[0145] Most rapid diagnostic bioassays are based on immunological principles. Some embodiments of the present disclosure combine immunoassay-based concepts with the advantages of bioluminescence, including a wider linear range and extremely low background. However, despite these advantages, bioluminescence-based point-of-care immunoassays have not yet been commercially available. This may be due in part to the low signal of many currently available luciferases, inherently limiting their usefulness in immunoassays. Furthermore, when bioluminescent signal output is conditionally configured (e.g., via complementation or bioluminescence resonance energy transfer (BRET)), the signal can be further reduced. Many currently available luciferases also have poor tolerance or sensitivity to certain assay conditions, such as high temperatures, suboptimal buffer compositions, and complex sample matrices, requiring specialized chemistries for adaptation to point-of-care devices.

[0146]

[0004] Embodiments of the present disclosure also address the need for an "all-in-one" assay format for analyte detection, which has not been developed or described in the prior art until the present application. For example, Tenda, K. et al. (Angew. Chem. Int. Ed. 57, 15369-15373 (2018)) disclose a paper device in which the substrate and bioluminescent components are dried onto separate sections of paper rather than being included together in a single-format system. Furthermore, Yu, Q. et al. (Science 361, 1122-1126 (2018)) disclose that, although the bioluminescent components can be dried together, rather than drying the substrate and bioluminescent components in a single-format system, the substrate is mixed separately with the analyte of interest and then added to the paper. As further described herein, embodiments of the present disclosure provide methods, compositions, and systems that include all necessary components of a bioluminescent detection complex (except the analyte of interest) in a single-format (e.g., "all-in-one") system. This is in contrast to currently available systems that include at least one of the necessary bioluminescent components in a separate format / solution. Accordingly, embodiments of the present disclosure provide surprising and unexpected advantages over currently available bioluminescent analyte detection systems.

[0147] To address the need for a bioluminescence-based point-of-care immunoassay platform that is not necessarily limited to the use of typical immunoassay reagents, embodiments of the present disclosure include the use of the NanoLuc® bioluminescence platform, which includes compositions and methods for constructing bioluminescent complexes from two or more peptide and / or polypeptide components. In some embodiments, the peptide and / or polypeptide components are not fragments of existing proteins (e.g., are not complementary subsequences of known polypeptide sequences), but confer bioluminescent activity through structural complementarity, as further described herein (see, e.g., WO / 2014 / 151736 (International Application No. PCT / US2014 / 026354) and U.S. Patent Application No. 16 / 439,565 (PCT / US2019 / 036844), which are incorporated by reference in their entireties). In some embodiments, the peptide and / or polypeptide components are non-luminescent in the absence of complementarity and / or enhance the bioluminescence of the peptide or polypeptide components in the presence of complementarity. In some embodiments, target analyte binding agents are labeled with various components of the bioluminescent conjugates described herein without impairing the binding ability of the binding agent to the target analyte. The components of the bioluminescent conjugates of the present disclosure are configured to be compatible with currently available point-of-care devices and systems, such as lateral flow devices, paper-based spot tests, dipstick tests, lab-on-a-chip, microfluidic devices, and pre-filled 96-well microtiter plates.

[0148] For example, embodiments of the present disclosure incorporate NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, Nano-Glo (e.g., NANOGLO Live Cell Substrate or NANOGLO LCS (Promega Category Nos. N205 and N113)), NanoBRET, etc.) into target analyte detection assays that can be incorporated into solid-phase assays or devices, including plastics, matrices, and membranes of various compositions, and / or used in other assay formats, such as lyophilized cakes or tablets for liquid-phase assays, all of which function reliably in complex sampling environments (e.g., blood components, food matrices, soil samples, stool, urine, water, and other human and animal biological samples). In some embodiments, the NanoLuc®-based reporter system is incorporated into lateral flow assay (LFA) technologies, paper spot tests, and similar devices. LFA is a popular point-of-care technology used to measure a variety of target analytes, including, but not limited to, antibodies, bacterial and viral antigens, metabolites, proteins, etc. As shown in Figure 1, the LFA, combined with NanoLuc®-based reporter technology, provides a multiplexed viral infection detection assay for point-of-care detection of antiviral antibodies. The only currently available, approved emergency immunoassay for detecting Zika exposure is a conventional plate-based multistep sandwich ELISA that detects the presence of anti-Zika IgM in blood samples. In contrast to this system, the NanoLuc®-based bioluminescent reporter platform, with its multiplexing capabilities, can rapidly detect multiple antibodies in a sample, regardless of whether the antibodies recognize multiple different epitopes on the same virus or multiple different epitopes on two or more viruses. The ability to rapidly and sensitively detect and identify viral infections through bioluminescence can aid in treatment decisions.In addition to antibodies and antigens, the small size of the component peptides of the bioluminescent complexes described herein allows for the detection of many other target analytes using alternative binders and materials, including, but not limited to, DARPins, aptamers, oligonucleotide probes, peptide nucleic acids (PNAs), and locked nucleic acid assays (LNAs).

[0149] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0150] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0151] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," and "contain(s)," and variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Many embodiments herein are described using the open term "comprising." Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments and may alternatively be claimed or described using such language. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0152] The recitation of numerical ranges herein expressly contemplates each intervening number to the same precision, for example, in the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are also expressly contemplated.

[0153] "Bioluminescence" refers to the production and emission of light by a chemical reaction catalyzed or enabled by an enzyme, protein, protein complex, or other biological molecule (e.g., a bioluminescent complex). In typical embodiments, a substrate of a bioluminescent entity (e.g., a bioluminescent protein or bioluminescent complex) is converted by the bioluminescent entity to an unstable form, after which the substrate emits light.

[0154] "Complementarity" refers to the property that two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) can hybridize, dimerize, or otherwise form a complex with one another. For example, a "complementary peptide and polypeptide" can combine to form a complex. Complementary elements may require assistance (e.g., from interacting elements) to form the complex, such as positioning the elements in a conformation appropriate for complementarity, colocalizing the complementary elements, lowering the interaction energy due to complementarity, etc.

[0155] A "complex" refers to an assembly or aggregation of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with one another. In one embodiment, "contact" or more specifically "direct contact" means that two or more molecules are in sufficient proximity such that non-covalent attractive interactions, such as van der Waals forces, hydrogen bonding, ionic and hydrophobic interactions, dominate the interaction of the molecules. In such embodiments, a complex of molecules (e.g., peptides and polypeptides) forms under assay conditions such that the complex is thermodynamically favored (e.g., compared to the unaggregated or uncomplexed states of its constituent molecules). As used herein, the term "complex," unless otherwise specified, refers to an assembly of two or more molecules (e.g., peptides, polypeptides, or combinations thereof).

[0156] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence and exhibits an altered domain structure compared to the original or parent antibody. A derivative can still adopt the typical domain organization and amino acid sequence found in a natural antibody and is capable of specifically binding to a target (antigen). Typical examples of antibody derivatives are antibodies bound to other polypeptides, rearranged antibody domains, or antibody fragments. A derivative may also contain at least one additional compound, such as a protein domain, linked by a covalent or non-covalent bond. Linkage may be based on gene fusion according to methods known in the art. The additional domain present in an antibody-containing fusion protein may be linked by a flexible linker, advantageously a peptide linker, containing multiple hydrophilic peptide-bonded amino acids of sufficient length to connect the C-terminus of the additional protein domain to the N-terminus of the antibody or vice versa. The antibody may be linked to an effector molecule, e.g., a biologically active substance (e.g., a cytokine or growth hormone), chemical agent, peptide, protein, or drug, that has a suitable conformation for biological activity or selective binding to a solid support.

[0157] A "fragment" refers to a peptide or polypeptide that results from cleavage or "fragmentation" of a larger whole entity (e.g., protein, polypeptide, enzyme, etc.), or that is prepared to have the same sequence as such. A fragment is thus a subsequence of the whole entity (e.g., protein, polypeptide, enzyme, etc.) from which it is made and / or engineered. A peptide or polypeptide that is not a subsequence of an existing whole protein is not a fragment (e.g., not a fragment of an existing protein). A peptide or polypeptide that is "not a fragment of an existing bioluminescent protein" is an amino acid chain that (1) physically exists prior to the design and / or synthesis of the peptide or polypeptide, and (2) is not a subsequence of a protein (e.g., natural or synthetic) that exhibits substantial bioluminescent activity.

[0158] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody that contains at least a portion of the antigen-binding or variable region. Antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134, incorporated herein by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain and pepsin digestion of antibodies), by recombinant DNA techniques, or by chemical polypeptide synthesis. For example, a "Fab" fragment contains one light chain and one C heavy chain. H1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. An "Fab'" fragment contains one light chain and a C H1 Domain and C H2 The Fab' fragment comprises one heavy chain and one light chain containing an additional constant region extending between the heavy and light chains. An interchain disulfide bond can form between the two heavy chains of the Fab' fragment to form an "F(ab')2" molecule. The "Fv" fragment contains the variable regions from both the heavy and light chains but does not contain the constant regions. The single-chain Fv (scFv) fragment contains the heavy and light chain variable regions connected by a flexible linker to form a single polypeptide chain containing an antigen-binding region. Exemplary single-chain antibodies are described in detail in WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, which are incorporated herein by reference in their entireties. In certain instances, a single variable region (e.g., heavy or light chain variable region) may have the ability to recognize and bind to an antigen. Other antibody fragments will be understood by those skilled in the art.

[0159] As used herein, an "isolated polynucleotide" can mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by its origin, is not associated with all or a portion of a polynucleotide with which the "isolated polynucleotide" is found in nature, is operably linked to a polynucleotide with which it is not naturally linked, or does not occur in nature as part of a larger sequence.

[0160] "Non-luminescent" refers to an entity (e.g., a peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting detectable amounts of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it exhibits no detectable luminescence in a given assay. As used herein, the term "non-luminescent" is synonymous with the term "substantially non-luminescent." For example, a non-luminescent polypeptide is substantially non-luminescent, e.g., 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10) luminescent, compared to a complex of the polypeptide and its non-luminescent complementary peptide. 3 times 1×10 4 times 1×10 5 times 1×10 6 times 1×10 7 In some embodiments, an entity is "non-luminescent" if any light emission is small enough so as not to produce an interfering background in a particular assay.

[0161] "Non-luminescent peptides" and "non-luminescent polypeptides" either exhibit substantially no luminescence (e.g., in the presence of a substrate) or, when compared to a significant signal (e.g., a luminescent complex) in a typical instrument (e.g., a luminometer) under standard conditions (e.g., physiological conditions, assay conditions, etc.), the luminescence is below noise or is 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10 3 times 1×10 4 times 1×10 5 times 1×10 6 times 1×107 The term "bioluminescent complex" refers to peptides and polypeptides that exhibit an amount (e.g., 1 / 2 fold) of light emitted by the non-luminescent peptides and polypeptides. In some embodiments, such non-luminescent peptides and polypeptides assemble according to the criteria described herein to form a bioluminescent complex. As used herein, a "non-luminescent element" refers to a non-luminescent peptide or polypeptide. The term "bioluminescent complex" refers to a complex formed by the assembly of two or more non-luminescent peptides and / or polypeptides. A bioluminescent complex catalyzes or enables the conversion of the substrate of the bioluminescent complex into an unstable form, after which the substrate emits light. When not complexed, the two non-luminescent elements that form a bioluminescent complex may also be referred to as a "non-luminescent pair." When a bioluminescent complex is formed by three or more non-luminescent peptides and / or polypeptides, the uncomplexed components of the bioluminescent complex may be referred to as "non-luminescent groups."

[0162] As used herein, unless otherwise specified, "peptide" and "polypeptide" refer to a polymeric compound in which two or more amino acids are linked through a backbone by peptide amide bonds (--C(O)NH--). The term "peptide" typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), while the term "polypeptide" typically refers to long amino acid polymers (e.g., chains having more than 25 amino acids).

[0163] A "pre-existing protein" refers to an amino acid sequence that physically existed prior to a particular event or date. A "peptide that is not a fragment of a pre-existing protein" is a short chain of amino acids that is not a fragment or subsequence of a protein (e.g., synthetic or natural) that physically existed prior to the design and / or synthesis of the peptide.

[0164] As used herein, the terms "sample," "test sample," "specimen," "sample from a subject," and "patient sample" may be used interchangeably and may refer to a sample of blood, e.g., whole blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample may be used directly as obtained from the patient, or may be pretreated by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to alter the characteristics of the sample in some manner discussed herein or otherwise known in the art.

[0165] "Sequence identity" refers to the degree to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have monomer subunits of the same sequence composition. The term "sequence similarity" refers to the degree to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymeric sequences. For example, similar amino acids share the same biophysical characteristics and can be classified into, for example, acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) families. "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid is present in both sequences, a similar amino acid is present in both sequences) to obtain the number of matching positions; (3) dividing the number of matching positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the percent sequence identity or sequence similarity. For example, if peptides A and B are both 20 amino acids long and have identical amino acids at all but one position, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C.For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0166] As used interchangeably herein, the terms "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals and humans. In some embodiments, the subject may be human or non-human. The subject or patient may be undergoing multiple treatments. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, e.g., chimpanzees and other ape and monkey species; livestock such as cows, sheep, pigs, goats, llamas, camels, and horses; domestic mammals such as dogs and cats; and laboratory animals, including rodents such as mice, rats, rabbits, and guinea pigs. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, regardless of gender, are intended to be included within the scope of this term.

[0167] A "subsequence" refers to a peptide or polypeptide that has 100% sequence identity with another larger peptide or polypeptide. A subsequence is an exact sequence match for a portion of a larger chain of amino acids.

[0168] As used herein, "substantially" means that the recited characteristics, parameters, and / or values ​​need not be achieved exactly, but rather that deviations or variations, including, for example, tolerances, measurement errors, measurement precision limits, and other factors known in the art, may occur in amounts that do not interfere with the function of the characteristics they are intended to provide. A substantially absent characteristic or property (e.g., substantially non-luminescent) can be within noise, below background, below the detection capability of the assay used, or a very small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of the characteristic of interest (e.g., the luminescence intensity of a bioluminescent protein or bioluminescent complex).

[0169] The term "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence due to amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. "SNP" refers to a variant that is a single nucleotide polymorphism. Representative examples of "biological activity" include the ability to bind to a specific antibody or the ability to stimulate an immune response. The term "variant" is also used herein to describe a protein containing an amino acid sequence that is substantially identical to a reference protein that retains at least one biological activity. Conservative amino acid substitutions (e.g., replacing one amino acid with a different amino acid with similar properties, such as hydrophilicity, degree and distribution of charged regions, etc.) are recognized in the art as typically involving minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of an amino acid. The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in a peptide allows for the calculation of the peptide's maximum local average hydrophilicity, a useful index that has been reported to correlate with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Substitutions with amino acids whose hydrophilicity values ​​are within ±2 of each other can be performed. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this finding, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0170] As used herein, "target analyte" or "analyte" refers to a substance in a sample that can be detected, quantified, measured, tested, and / or monitored, often as part of a method for evaluating a process or condition (e.g., a diagnostic or prognostic assay). Target analytes can include, but are not limited to, proteins, peptides, polypeptides, enzymes, cofactors, nucleotides, polynucleotides, DNA, RNA, small molecule compounds, antibodies, and any variations, combinations, and derivatives thereof.

[0171] As used herein, a "target analyte-binding agent" refers to an agent capable of binding to a target analyte. In some embodiments, a target analyte-binding agent includes an agent capable of binding to multiple substances, such as a target analyte and a solid support. In some embodiments, a target analyte-binding agent includes an agent that binds to both the target analyte and a different peptide / polypeptide (e.g., via a target analyte-binding element) to form a target analyte detection complex (e.g., generate a bioluminescent signal). In some embodiments, a target analyte-binding agent can include a target analyte-binding element capable of binding to a group or class of analytes (e.g., protein L binding to an antibody), while in other embodiments, a target analyte-binding agent can include a target analyte-binding element capable of binding to a specific analyte (e.g., an antigen binding to a monoclonal antibody). The target analyte binding agent can be an antibody, an antibody fragment, a receptor domain that binds to a target ligand, a protein or protein domain that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), a binding domain of a protein that binds to an immunoglobulin (e.g., Protein A, Protein G, Protein A / G, Protein L, Protein M), an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a purified protein or protein domain (either the analyte itself or a protein that binds to the analyte), and an analyte-binding domain(s) of a protein, etc. Table A provides a list of exemplary binding moieties that can be used singly or in various combinations in the methods, systems, and assays (e.g., immunoassays) herein.

[0172] Table 1: Exemplary target analyte binding agents. TIFF2025133108000001.tif187159 TIFF2025133108000002.tif67163

[0173] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The meaning and scope of terms should be clear; however, in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0174] 2. Bioluminescence The present disclosure includes bioluminescent polypeptides, bioluminescent complexes and components thereof, as well as materials and methods related to bioluminescence resonance energy transfer (BRET).

[0175] In some embodiments, provided herein are solid-phase and / or lateral flow assays, devices, and systems incorporating bioluminescent polypeptide components and / or bioluminescent conjugates (of non-luminescent peptide(s) and / or non-luminescent polypeptide components) based (e.g., structurally, functionally, etc.) on NanoLuc® luciferase, a luciferase from Oplophorus gracilirostris (Promega Corporation; U.S. Patent Nos. 8,557,970; 8,669,103; incorporated herein by reference in their entireties), NanoBiT (U.S. Patent No. 9,797,889; incorporated herein by reference in their entireties), or NanoTrip (U.S. Patent Application No. 16 / 439,565; and U.S. Provisional Application No. 62 / 941,255; both of which are incorporated herein by reference in their entireties). As described below, in some embodiments, the compositions, assays, devices, methods, and systems herein incorporate commercially available NanoLuc®-based technologies (e.g., NanoLuc® luciferase, NanoBRET, NanoBiT, NanoTrip, NanoGlo, etc.), while in other embodiments, various combinations, variations, or versions of commercially available NanoLuc®-based technologies are employed.

[0176] a.NanoLuc PCT Application No. PCT / US2010 / 033449, U.S. Patent No. 8,557,970, PCT Application No. PCT / 2011 / 059018, and U.S. Patent No. 8,669,103 (each of which is incorporated by reference in its entirety for all purposes) describe compositions and methods comprising bioluminescent polypeptides. Such polypeptides find use in embodiments herein and can be used with the compositions, assays, devices, systems, and methods described herein.

[0177] In some embodiments, the compositions, assays, devices, systems, and methods provided herein comprise SEQ ID NO:5 or a bioluminescent polypeptide having at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range therebetween) sequence identity to SEQ ID NO:5.

[0178] In some embodiments, any of the above-described bioluminescent polypeptides are linked (e.g., fused, chemically linked, etc.) to a binding member or other component of the assays and systems described herein.

[0179] In some embodiments, any of the foregoing bioluminescent polypeptides, or a fusion or conjugate thereof (e.g., with a binding element, etc.), is immobilized to a portion of a device described herein (e.g., a detection or control region of a lateral flow assay, a solid-phase detection element, etc.).

[0180] b.NanoBiT PCT Application No. PCT / US14 / 26354 and U.S. Pat. No. 9,797,889 (each of which is incorporated herein by reference in its entirety for all purposes) describe compositions and methods for constructing bioluminescent conjugates, and such conjugates, as well as their peptide and polypeptide components, find use in embodiments herein and can be used with the assays and methods described herein.

[0181] In some embodiments, provided herein are non-luminescent (NL) polypeptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:9, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:6.

[0182] In some embodiments, provided herein are non-luminescent (NL) peptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:10, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:8.

[0183] In some embodiments, provided herein are NL peptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:8.

[0184] In some embodiments, any of the aforementioned NL peptides or NL polypeptides are linked (e.g., by fusion, chemical linkage, etc.) to a binding element or other component of the compositions, assays, devices, methods, and systems described herein.

[0185] In some embodiments, any of the aforementioned NL peptides or NL polypeptides, or fusions or conjugates thereof (e.g., with binding elements, etc.), are immobilized to a portion of a device described herein (e.g., a detection or control area of ​​a lateral flow assay, a solid-phase detection element, etc.).

[0186] In some embodiments, provided herein is a lateral flow detection system comprising: an analytical membrane including a detection region and a control region, wherein the detection region comprises a first target analyte-binding agent immobilized in the detection region; a conjugate pad including a second target analyte-binding agent; and a sample pad, wherein the first target analyte-binding agent comprises a first target analyte-binding element and a first NanoBiT-based NL peptide or NL polypeptide component (described above); and the second target analyte-binding agent comprises a second target analyte-binding element and a complementary NanoBiT-based NL peptide or NL polypeptide component (described above). In some embodiments, the first target analyte-binding agent and the second target analyte-binding agent form an analyte detection complex in at least one detection region when the target analyte is detected in the sample. In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the first target analyte-binding agent is contacted with the second target analyte-binding agent, as compared to the bioluminescent signal generated by the second target analyte-binding agent or the first target analyte-binding agent and the luminescent substrate alone.

[0187] In some embodiments, provided herein is a solid-phase detection system comprising a solid-phase substrate comprising a first target analyte-binding agent and a second target analyte-binding agent, wherein the first target analyte-binding agent comprises a first target analyte-binding element and a first NanoBiT-based NL peptide or NL polypeptide component (described above), and the second target analyte-binding agent comprises a second target analyte-binding element and a complementary NanoBiT-based NL peptide or NL polypeptide component (described above). In some embodiments, the first target analyte-binding agent and the second target analyte-binding agent form an analyte detection complex on the solid-phase substrate when the target analyte is detected in a sample. In some embodiments, the bioluminescent signal generated in the presence of a luminescent substrate is substantially increased when the first target analyte-binding agent contacts the second target analyte-binding agent, compared to the bioluminescent signal generated by the second target analyte-binding agent or the first target analyte-binding agent and the luminescent substrate alone.

[0188] c.NanoTrip U.S. Patent Application No. 16 / 439,565 (PCT / US2019 / 036844) and U.S. Provisional Application No. 62 / 941,255 (both of which are incorporated by reference in their entirety for all purposes) describe compositions, systems, and methods for constructing bioluminescent conjugates. Such conjugates, and their peptide and polypeptide components, find use in embodiments herein and can be used with the assays and methods described herein.

[0189] In some embodiments, provided herein are non-luminescent (NL) polypeptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:12, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:9.

[0190] In some embodiments, provided herein are non-luminescent (NL) peptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:11, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:8.

[0191] In some embodiments, provided herein are NL peptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:13, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:7.

[0192] In some embodiments, provided herein are NL peptides that have at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or any range therebetween) sequence identity to SEQ ID NO:14, but less than 100% (e.g., <99%, <98%, <97%, <96%, <95%, <94%, <93%, <92%, <91%, <90%) sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:8.

[0193] In some embodiments, any of the aforementioned NanoTrip-based NL peptides or NL polypeptides are linked (e.g., by fusion, chemical linkage, etc.) to a binding element or other component of the compositions, methods, devices, assays, and systems described herein.

[0194] In some embodiments, any of the aforementioned NanoTrip-based NL peptides or NL polypeptides, or fusions or conjugates thereof (e.g., with binding elements, etc.), are immobilized to a portion of a device described herein (e.g., a detection or control area of ​​a lateral flow assay, a solid-phase detection element, etc.).

[0195] In some embodiments, provided herein is a lateral flow detection system comprising: an analytical membrane including a detection region and a control region, wherein the detection region comprises a first target analyte-binding agent immobilized in the detection region; a conjugate pad including a second target analyte-binding agent; and a sample pad, wherein the first target analyte-binding agent comprises a first target analyte-binding element and a first NanoTrip-based NL peptide (described above); and the second target analyte-binding agent comprises a second target analyte-binding element and a complementary NanoTrip-based NL peptide (described above). In some embodiments, the first target analyte-binding agent and the second target analyte-binding agent form an analyte detection complex in at least one detection region in the presence of the NanoTrip-based NL polypeptide component (described above) when the target analyte is detected in the sample. In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the first target analyte binding agent is contacted with the second target analyte binding agent in the presence of the NanoTrip-based NL polypeptide component, compared to the bioluminescent signal generated by the second target analyte binding agent or the first target analyte binding agent and the luminescent substrate alone.

[0196] In some embodiments, provided herein is a solid-phase detection system comprising a solid phase (e.g., a paper substrate) comprising a first target analyte-binding agent and a second target analyte-binding agent, wherein the first target analyte-binding agent comprises a first target analyte-binding element and a first NanoTrip-based NL peptide (described above), and the second target analyte-binding agent comprises a second target analyte-binding element and a complementary second NL NanoTrip-based peptide (described above). In some embodiments, the first target analyte-binding agent and the second target analyte-binding agent form an analyte detection complex in the presence of a NanoTrip-based NL polypeptide (described above) when the target analyte is detected in a sample. In some embodiments, the bioluminescent signal generated in the presence of a luminescent substrate is substantially increased when the first target analyte-binding agent contacts the second target analyte-binding agent and the NanoTrip-based NL polypeptide, compared to the bioluminescent signal generated by the second target analyte-binding agent or the first target analyte-binding agent and the luminescent substrate alone.

[0197] d. NanoBRET Bioluminescence resonance energy transfer (BRET) compositions, systems, and methods (e.g., incorporating NanoLuc®-based technology) have been described, as disclosed in PCT Application No. PCT / US13 / 74765 and U.S. Patent Application No. 15 / 263,416 (herein incorporated by reference in their entireties for all purposes), and such compositions, systems, and methods, as well as bioluminescent polypeptides and their fluorophore-binding components, find use in embodiments herein and can be used with the compositions, systems, devices, assays, and methods described herein.

[0198] In some embodiments, any of the NanoLuc®-, NanoBiT-, and / or NanoTrip-based peptides, polypeptides, complexes, fusions, and conjugates (described in sections a-c above) may find use in BRET-based applications using the compositions, assays, methods, devices, and systems described herein. For example, in certain embodiments, a first target analyte binding agent comprises a first target analyte binding element and a NanoLuc®-, NanoBiT-, and / or NanoTrip-based polypeptide, peptide, or complex, and a second target analyte binding agent comprises a second target analyte binding element and a fluorophore (e.g., a fluorescent protein, a small molecule fluorophore, etc.), wherein the emission spectrum of the NanoLuc®-, NanoBiT-, and / or NanoTrip-based polypeptide, peptide, or complex overlaps with the excitation spectrum of the fluorophore. In some embodiments, NanoLuc®-based, NanoBiT-based, and / or NanoTrip-based polypeptides, peptides, or complexes can be prepared in lyophilized form, which may or may not contain a luminescent substrate (e.g., furimazine).

[0199] In some embodiments, the target analyte binding agent comprises a target analyte binding element and a fluorophore activatable by energy transfer from a bioluminescent polypeptide.

[0200] As used herein, the term "energy acceptor" refers to any small molecule (e.g., a chromophore), macromolecule (e.g., an autofluorescent protein, a phycobiliprotein, a nanoparticle, an interface, etc.), or molecular complex that generates a readily detectable signal in response to energy absorption (e.g., resonance energy transfer). In certain embodiments, the energy acceptor is a fluorophore or other detectable chromophore.Suitable fluorophores include xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, Texas Red, etc.), cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, etc.), naphthalene derivatives (e.g., dansyl and prodan derivatives), oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, etc.), pyrene derivatives (e.g., cascade blue), oxazine derivatives (e.g., Nile red, Nile blue, cresyl violet, oxazine 170, etc.), acridine derivatives (e.g., proflavine, acridine orange, acridine yellow, etc.), arylmethine derivatives (e.g., auramine, crystal violet, malachite green, etc.), tetrapyrrole derivatives (e.g., porphine, phthalocyanine, bilirubin, etc.), CF dyes (Biotium), BODIPY (Invitrogen), ALEXA These include, but are not limited to, FLuoR (Invitrogen), DYLIGHT FLUOR (Thermo Scientific, Pierce), ATTO and TRACY (Sigma Aldrich), FluoProbes (Interchim), DY and MEGASTOKES (Dyomics), SULFO CY dyes (CYANDYE, LLC), SETAU AND SQUARE DYES (SETABioMedicals), QUASAR and CAL FLUOR dyes (Biosearch Technologies), SURELIGHT DYES (APC, RPE, PerCP, phycobilisome) (Columbia Biosciences), APC, APCXL, RPE, BPE (Phyco-Biotech), autofluorescent proteins (e.g., YFP, RFP, mCherry, mKate), quantum dot nanocrystals, and the like. In some embodiments, the fluorophore is a rhodamine analog (e.g., a carboxyrhodamine analog), such as those described in U.S. Patent Application No. 13 / 682,589, the entire contents of which are incorporated herein by reference.

[0201] e.HALOTAG Some embodiments herein include a capture protein capable of forming a covalent bond with a capture ligand. The capture protein can be linked to a first component (e.g., a peptide component of a bioluminescent complex), and the capture ligand can be linked to a second component (e.g., a target analyte-binding component (e.g., an antibody or antigen-binding protein)), where the first and second components are linked to each other through the formation of a covalent bond. In some embodiments, linking the first and second components generates a target analyte-binding agent. In some embodiments, two or more target analyte-binding agents so formed can bind to complementary polypeptide components (e.g., LgTrip) to form a bioluminescent complex in the presence of an analyte (e.g., a target antigen recognized by the target analyte-binding component) (see, e.g., Figures 48 and 58). In some embodiments, the capture ligand is a haloalkane (also known as an "alkyl halide"). In some embodiments, the capture ligand is a chloroalkane. In some embodiments, the capture ligand is -AX. In some embodiments, X is Cl. In some embodiments, -AX is -(CH)Cl. When the capture ligand is a haloalkane, the capture protein is typically a dehalogenase enzyme modified to form a covalent bond with its substrate (see, e.g., U.S. Patent Nos. 7,425,436; 7,429,472; 7,867,726; 7,888,086; 7,935,803; RE42,931; 8,168,405; 8,202,700; 8,257,939; which are incorporated herein by reference in their entireties).

[0202] One such modified dehalogenase is the commercially available HALOTAG protein (SEQ ID NO: 720). In some embodiments, the capture protein comprises a polypeptide with at least 70% sequence identity (e.g., 75% identity, 80% identity, 85% identity, 90% identity, 95% identity, 98% identity, 99% identity) to SEQ ID NO: 720. Some embodiments comprise a fusion protein of a capture protein (e.g., HALOTAG) and another peptide / polypeptide element (e.g., a binding moiety, a peptide / polypeptide component of a bioluminescent complex, etc.). In some embodiments, the capture ligand comprises an alkyl chain (e.g., (CH2) 4-24 In some embodiments, the other end of the alkyl chain is attached to a linker or another element (e.g., a peptide, an analyte, etc.). The linker can include an alkyl chain or a substituted alkyl chain (e.g., C=O, NH, S, O, carbamate, ethylene, etc.), such as those disclosed in U.S. Patent Application No. 14 / 207,959, which is incorporated herein by reference.

[0203] 3. Compositions and Formulations Embodiments of the present disclosure include compositions and formulations comprising one or more of the peptide and / or polypeptide components of the bioluminescent complexes provided herein. According to these embodiments, the compositions and formulations of the present disclosure may include a luminescent substrate and / or various other components. The compositions and methods provided herein can be used to formulate shelf-stable liquid formulations (e.g., for use in liquid-phase assay formats) and shelf-stable dry formulations (e.g., for use in solid-phase assay formats) that are capable of generating a luminescent signal in the presence of an analyte of interest, even after extended storage. As described in more detail below, the compositions and formulations of the present disclosure may include one or more components of NanoLuc, NanoBiT, NanoTrip, and NanoBRET, as well as various luminescent substrates (e.g., furimazine) described herein.

[0204] In contrast to many currently available fluorescent and colorimetric assays, the compositions and formulations of the present disclosure provide a means for performing bioassays in which one or more of the peptide and / or polypeptide components of the bioluminescent complex are present in a stable dry formulation that can be reconstituted, for example, in a solution containing a complementary peptide / polypeptide and / or luminescent substrate, such that the bioluminescent complex forms in the presence of an analyte of interest. In some embodiments, the compositions and formulations of the present disclosure provide a means for performing robust solid-phase bioassays in which the bioluminescent signal generated is quantitative and proportional to the concentration of the analyte of interest.

[0205] In some embodiments, compositions and formulations of the present disclosure comprise a luminescent substrate and a target analyte binding agent comprising a target analyte binding element and a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:6, at least 60% sequence identity to SEQ ID NO:9, or at least 60% sequence identity to SEQ ID NO:12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 70% sequence identity to SEQ ID NO:6, at least 70% sequence identity to SEQ ID NO:9, or at least 70% sequence identity to SEQ ID NO:12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 80% sequence identity to SEQ ID NO:6, at least 80% sequence identity to SEQ ID NO:9, or at least 80% sequence identity to SEQ ID NO:12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 85% sequence identity to SEQ ID NO:6, at least 85% sequence identity to SEQ ID NO:9, or at least 85% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 90% sequence identity to SEQ ID NO:6, at least 90% sequence identity to SEQ ID NO:9, or at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 95% sequence identity to SEQ ID NO:6, at least 95% sequence identity to SEQ ID NO:9, or at least 95% sequence identity to SEQ ID NO:12.

[0206] In other embodiments, the peptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO: 10, at least 60% sequence identity to SEQ ID NO: 11, at least 60% sequence identity to SEQ ID NO: 13, or at least 60% sequence identity to SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 70% sequence identity to SEQ ID NO: 10, at least 70% sequence identity to SEQ ID NO: 11, at least 70% sequence identity to SEQ ID NO: 13, or at least 70% sequence identity to SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 80% sequence identity to SEQ ID NO: 10, at least 80% sequence identity to SEQ ID NO: 11, at least 80% sequence identity to SEQ ID NO: 13, or at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 85% sequence identity to SEQ ID NO: 10, at least 85% sequence identity to SEQ ID NO: 11, at least 85% sequence identity to SEQ ID NO: 13, or at least 85% sequence identity to SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 90% sequence identity to SEQ ID NO: 10, at least 90% sequence identity to SEQ ID NO: 11, at least 90% sequence identity to SEQ ID NO: 13, or at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, the peptide component of the target analyte binding agent comprises at least 95% sequence identity to SEQ ID NO: 10, at least 95% sequence identity to SEQ ID NO: 11, at least 95% sequence identity to SEQ ID NO: 13, or at least 95% sequence identity to SEQ ID NO: 14.

[0207] In some embodiments, the composition or formulation includes a complementary peptide or polypeptide component of a bioluminescent complex. According to these embodiments, the target analyte-binding agent and the complementary peptide or polypeptide component of the bioluminescent complex can form a bioluminescent analyte detection complex in the presence of a target analyte. In some embodiments, a composition including a luminescent substrate and a target analyte-binding agent can be combined into a dry formulation, and the complementary peptide or polypeptide component of the bioluminescent complex can be formulated as a liquid formulation. In some embodiments, the liquid formulation is added to the dry formulation and, upon rehydration, forms a bioluminescent analyte detection complex in the presence of a target analyte. In other embodiments, a composition or formulation including a luminescent substrate, a target analyte-binding agent, and the complementary peptide or polypeptide component of the bioluminescent complex is combined into a dry formulation, and the dry formulation, upon rehydration, forms a bioluminescent analyte detection complex in the presence of a target analyte.

[0208] In some embodiments, the complementary peptide or polypeptide component comprises a second target analyte-binding element that forms a bioluminescent analyte detection complex in the presence of the target analyte. In some embodiments, the polypeptide component of the target analyte-binding agent comprises at least 60% sequence identity to SEQ ID NO:6, and the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to SEQ ID NO:10. In some embodiments, the polypeptide component of the target analyte-binding agent comprises at least 60% sequence identity to SEQ ID NO:6, and the complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to SEQ ID NO:14.

[0209] Embodiments of the present disclosure also include compositions or formulations, including a dry formulation comprising a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO: 9, and a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 10. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0210] Embodiments of the present disclosure also include compositions comprising a dry formulation comprising a first target analyte binding agent comprising a first target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO: 12, and a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 14. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0211] Embodiments of the present disclosure also include compositions comprising a dry formulation including a first target analyte binding agent comprising a first target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO: 13, a second target analyte binding agent comprising a second target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 15, and a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO: 12. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the composition further comprises a liquid formulation comprising the target analyte.

[0212] Embodiments of the present disclosure also include compositions comprising a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:9, and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:10 or SEQ ID NO:11.

[0213] Embodiments of the present disclosure also include compositions comprising a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO:10 or SEQ ID NO:11, and a liquid formulation containing a second target analyte binding agent comprising a target analyte binding element and a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO:9.

[0214] Embodiments of the present disclosure also include compositions comprising: a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO: 12; and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 14. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a sample comprising the target analyte. According to these embodiments, a bioluminescent analyte detection complex is formed when the dry formulation and the liquid formulation are combined in the presence of the target analyte.

[0215] In some embodiments, the composition further comprises a second complementary peptide or polypeptide component of the bioluminescent complex. According to these embodiments, the target analyte-binding agent, the first complementary peptide or polypeptide component of the bioluminescent complex, and the second complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of the target analyte.

[0216] In some embodiments, the composition comprising the target analyte binding agent is prepared as a dry formulation. In some embodiments, the first complementary peptide or polypeptide component and the second complementary peptide or polypeptide of the bioluminescent complex are prepared as a liquid formulation. According to these embodiments, the liquid formulation can be added to the dry formulation, and upon rehydration, promotes the formation of a bioluminescent analyte detection complex in the presence of the target analyte.

[0217] In some embodiments, a composition comprising a target analyte-binding agent and either a first or second complementary peptide or polypeptide component is combined into a dry formulation, and the first or second complementary peptide or polypeptide component not present in the dry formulation is made into a liquid formulation that can be added to the dry formulation and, upon rehydration, promotes the formation of a bioluminescent analyte detection complex in the presence of the target analyte.

[0218] In some embodiments, the target analyte binding agent, the first complementary peptide or polypeptide component, and the second complementary peptide or polypeptide component are combined in a dry formulation that, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a sample containing the target analyte, and the bioluminescent analyte detection complex is formed upon combining the dry formulation and the liquid formulation in the presence of the target analyte.

[0219] In some embodiments, either the first or second complementary peptide or polypeptide component comprises a second target analyte binding element that, upon rehydration, forms a bioluminescent analyte detection complex in the presence of the target analyte.

[0220] In some embodiments, the polypeptide component of the target analyte binding agent comprises at least 60% sequence identity to SEQ ID NO:12, and either the first or second complementary peptide or polypeptide component of the bioluminescent complex comprises at least 60% sequence identity to either SEQ ID NO:13 or SEQ ID NO:15.

[0221] Embodiments of the present disclosure also include compositions comprising: a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:12; and a liquid formulation comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15, and further comprising a second complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0222] Embodiments of the present disclosure also include dry formulations comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:12, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15, and further include liquid formulations comprising a second complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0223] Embodiments of the present disclosure also include dry formulations comprising a first target analyte binding agent comprising a target analyte binding element and a polypeptide component having at least 60% sequence identity to SEQ ID NO:12, and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15, and liquid formulations comprising a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:13 or SEQ ID NO:15.

[0224] Embodiments of the present disclosure also include dry formulations comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO: 13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 15, and further include liquid formulations comprising a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO: 12.

[0225] Embodiments of the present disclosure also include a dry formulation comprising a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO:12, and a liquid formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO:13, and a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO:15.

[0226] Embodiments of the present disclosure also include compositions comprising a dry formulation comprising a first target analyte binding agent comprising a target analyte binding element and a peptide component having at least 60% sequence identity to SEQ ID NO: 13; a second target analyte binding agent comprising a target analyte binding element and a complementary peptide component having at least 60% sequence identity to SEQ ID NO: 15; and a complementary polypeptide component having at least 60% sequence identity to SEQ ID NO: 12. In some embodiments, the dry formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a luminescent substrate. In some embodiments, the liquid formulation further comprises a sample comprising the target analyte, and a bioluminescent analyte detection complex is formed by combining the dry formulation and the liquid formulation in the presence of the target analyte.

[0227] In some embodiments, the bioluminescent signal generated in the presence of the luminescent substrate is substantially increased when the target analyte binding agent is contacted with one or more of the complementary peptide or polypeptide components of the bioluminescent complex, as compared to the bioluminescent signal generated by the target analyte binding agent and the luminescent substrate alone.

[0228] In some embodiments, the target analyte is a target antibody. In some embodiments, the target analyte binding agent comprises an element that nonspecifically binds to the antibody. In some embodiments, the target analyte binding agent comprises an element that specifically binds to the antibody. In some embodiments, the target antibody is an antibody against a pathogen, a toxin, or a therapeutic biologic.

[0229] In some embodiments, the target analyte binding element is selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, Protein A, the Ig binding domain of Protein A, Protein G, the Ig binding domain of Protein G, Protein A / G, the Ig binding domain of Protein A / G, Protein L, the Ig binding domain of Protein L, Protein M, the Ig binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.

[0230] In some embodiments, the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, and other coelenterazine analogs or derivatives. In some embodiments, the coelenterazine analog or derivative is a precursor luminescent substrate, such as those disclosed in U.S. Pat. No. 9,487,520, which is incorporated herein by reference. In some embodiments, the coelenterazine analog or derivative is Enduazine (Promega Corporation) or Vivazine (Promega Corporation).

[0231] In some embodiments, the composition further comprises a polymer. In some embodiments, the polymer is a natural biopolymer. In some embodiments, the natural biopolymer is selected from pullulan, trehalose, maltose, cellulose, dextran, and any combination thereof. In some embodiments, the natural biopolymer is pullulan. In some embodiments, the polymer is a cyclic sugar polymer or a derivative thereof. In some embodiments, the polymer is hydroxypropyl β-cyclodextrin.

[0232] In some embodiments, the polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the synthetic polymer is a block copolymer comprising at least one poly(propylene oxide) block and at least one poly(ethylene oxide) block. In some embodiments, the synthetic polymer is poloxamer 188.

[0233] In some embodiments, the composition further comprises a buffer, a surfactant, a reducing agent, a salt, a radical scavenger, a chelating agent, a protein, or any combination thereof. In some embodiments, the surfactant is selected from polysorbate 20, polysorbate 40, and polysorbate 80.

[0234] In some embodiments, the composition further comprises a substance that reduces autoluminescence. In some embodiments, the substance is ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, a thiourea, or the like. In some embodiments, the substance is a thionucleoside as disclosed in U.S. Pat. No. 9,676,997, which is incorporated herein by reference. In some embodiments, the substance is a thiourea, the use of which to reduce autoluminescence is disclosed in U.S. Pat. Nos. 7,118,878, 7,078,181, and 7,108,996, which are incorporated herein by reference.

[0235] In some embodiments, the composition is used in conjunction with an analyte detection platform to detect an analyte in a sample, in some embodiments, the sample is selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, saliva, a tissue sample, a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.

[0236] Embodiments of the present disclosure also include methods for detecting an analyte in a sample, comprising combining any of the compositions described above with a sample containing the target analyte. In some embodiments, detecting the target analyte in the sample comprises detecting a bioluminescent signal generated from the analyte detection complex. In some embodiments, the method further comprises quantifying the bioluminescent signal generated from the analyte detection complex. In some embodiments, the bioluminescent signal generated from the analyte detection complex is proportional to the concentration of the analyte. In some embodiments, one or more of the components of the composition exhibit improved stability within the composition compared to the component alone in solution.

[0237] Various embodiments of the compositions and formulations described above exhibit improved stability, as shown in the Examples and Figures. For example, when prepared as a dry formulation such as a lyocake, dried onto a substrate or matrix (e.g., Whatman 903, Ahlstrom 237, and Ahlstrom 6613H; wells of a 96-well plate, nylon mesh), or dried in various protein buffer formulations, with or without a luminescent substrate, the disclosed compositions and formulations exhibit improved stability when stored for extended periods of time. As provided herein, the disclosed compositions and formulations are capable of generating a luminescent signal in the presence of a target analyte even after extended storage. In some embodiments, the disclosed compositions and formulations exhibit improved stability compared to compositions and formulations containing the same or similar components of a bioluminescent complex (e.g., complementary peptide / polypeptide, luminescent substrate) but formulated without one or more of the other components of the formulation and / or not formulated according to the methods described herein.

[0238] In some embodiments, the compositions and formulations of the present disclosure exhibit enhanced stability for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 12 months, and up to 1 year. In some embodiments, the compositions and formulations of the present disclosure exhibit improved stability at temperatures ranging from about 0°C to 65°C, about 4°C to 65°C, about 10°C to 65°C, about 15°C to 65°C, about 15°C to 65°C, about 20°C to 65°C, about 25°C to 65°C, about 30°C to 65°C, about 35°C to 65°C, about 37°C to 65°C, about 40°C to 65°C, about 45°C to 65°C, about 50°C to 65°C, about 55°C to 65°C, about 60°C to 65°C, about 4°C to 55°C, about 10°C to 50°C, about 15°C to 45°C, and about 20°C to 40°C.

[0239] 4. Detection Assays and Systems Embodiments of the present disclosure include compositions, systems, assays, and methods for detecting one or more analytes in a sample. In accordance with these embodiments, exemplary assays and devices for use with various embodiments herein are described below. The following devices and assays should not be considered limiting of the full scope of the systems, assays, and methods described herein.

[0240] a. Lateral flow assay In certain embodiments, the present disclosure provides compositions and materials for performing lateral flow assays (e.g., lateral flow immunoassays). Lateral flow assays are based on the principles of immunochromatography and can be used to detect, quantify, test, measure, and monitor a variety of analytes, including, but not limited to, analytes related to ovulation monitoring, detection / diagnosis of infectious diseases / infectious organisms, analysis of drugs of abuse, detection / quantification of analytes important to human physiology, security screening, veterinary testing, agricultural applications, environmental testing, product quality assessment, etc.

[0241] As shown in FIG. 1 , embodiments of the present disclosure include a lateral flow detection system (100) for detecting and / or quantifying a target analyte based on bioluminescent complex formation. In some embodiments, the lateral flow assay system of the present disclosure includes an analytical membrane (105) divided into one or more detection regions (110) and one or more control regions (115). One or more detection regions may contain a target analyte binding agent immobilized in a portion of the detection region to prevent displacement when lateral flow through the analytical membrane is promoted. The lateral flow assay system of the present disclosure may also include a conjugate pad (120) containing the target analyte binding agent. In some embodiments, the target analyte binding agent is contained within the conjugate pad, but flows from the conjugate pad across the analytical membrane toward the detection and control regions upon lateral flow. The lateral flow assay system of the present disclosure may also include a sample pad (125) located at the distal end of the lateral flow assay system (e.g., opposite the absorbent pad). A sample containing (or capable of containing) the target analyte is applied to the sample pad. In some embodiments, the lateral flow assay system also includes a wick pad (130) at the end of the device distal to the sample pad, which generates capillary flow of the sample from the sample pad through the conjugate pad, analytical membrane, detection region, and control region.

[0242] According to these embodiments, when a sample is added to the sample pad, lateral flow is promoted, causing a target analyte in the sample to contact a first target analyte-binding agent in the conjugate pad, and then lateral flow causes the target analyte and first target analyte-binding agent to contact a second target analyte-binding agent immobilized in a detection zone of the analytical membrane. The presence and / or amount of the target analyte is then determined based on detection of the analyte in the detection zone (e.g., in the presence of a luminescent substrate of a bioluminescent complex) and / or comparison to a control.

[0243] In some embodiments, the lateral flow systems utilize one or more NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, NanoBRET, etc.) for detection of bound target analytes.

[0244] In an exemplary embodiment, as shown in FIG. 1 , the target analyte is an antibody produced in a subject in response to infection / exposure to an infectious organism. The first target analyte-binding agent includes both a target analyte-binding element that binds to the antibody (e.g., a nonspecific antibody binder (e.g., Protein L)) and a first peptide or polypeptide that can interact with a different peptide or polypeptide to generate a bioluminescent signal (e.g., a NanoBiT nonluminescent peptide or polypeptide or a variant thereof (e.g., one of SEQ ID NOS: 9-11 or 12 / 14)). The second target analyte-binding agent can include a target analyte-binding element that binds to the antibody, e.g., an epitope of an antigen recognized by the antibody, and a second peptide or polypeptide that can interact with the first peptide or polypeptide to generate a bioluminescent signal (e.g., a NanoBiT nonluminescent peptide or polypeptide or a variant thereof (e.g., one of SEQ ID NOS: 9-11 or 12 / 14)). If the bioluminescent complex remains in the detection zone, the bioluminescent signal can be detected and / or quantified (e.g., in the presence of a luminescent substrate for the bioluminescent complex), thus indicating the presence / amount of antibody in the sample.

[0245] As shown in Figure 1, lateral flow assays of the present disclosure can be configured to test a single sample from a subject for multiple different analytes, such as antibodies raised against different diseases / microorganisms (e.g., multiplexed). According to these embodiments, the analytical membrane can include multiple detection regions, each detection region containing a different target analyte binding agent with a different target analyte binding element (e.g., a different disease antigen).

[0246] In another lateral flow embodiment, different from that shown in FIG. 1 , the target analyte is an antibody produced in a subject in response to infection / exposure to an infectious organism. The first target analyte-binding agent comprises both a target analyte-binding element that binds to the antibody (e.g., an epitope of an antigen recognized by the antibody) and a bioluminescent polypeptide (e.g., NanoLuc or a variant thereof (e.g., SEQ ID NO: 5, SEQ ID NO: 6)). The second target analyte-binding agent can comprise a target analyte-binding element that binds to the antibody, such as a nonspecific antibody binder (e.g., Protein L). Detection of bioluminescence in the detection zone (e.g., in the presence of a luminescent substrate for the bioluminescent complex) then indicates that both target analyte-binding agents have bound to the target analyte, and thus indicates the presence of the target analyte in the sample.

[0247] In another exemplary alternative embodiment, the target analyte is an antibody produced in a subject in response to infection / exposure to an infectious organism. The first target analyte binding agent includes both a target analyte binding element (e.g., a non-specific antibody binding agent (e.g., Protein L), a target-specific (e.g., antibody) binding agent) that binds to the antibody and a first non-luminescent (NL) peptide tag (e.g., SEQ ID NO: 13 or 11, or a variant thereof) that can interact with a second non-luminescent (NL) peptide (e.g., SEQ ID NO: 11 or 13, or a variant thereof) and a non-luminescent (NL) polypeptide (e.g., SEQ ID NO: 12, or a variant thereof) to generate a bioluminescent signal. The second target analyte binding agent includes a target analyte binding element that binds to the antibody (e.g., a target-specific (e.g., antibody) binding agent, a non-specific antibody binding agent (e.g., Protein L)) and a second NL peptide tag (e.g., SEQ ID NO: 11 or 13, or a variant thereof). Formation of a bioluminescent complex in the presence of the NL polypeptide component (e.g., SEQ ID NO: 12, or a variant thereof) and a luminescent substrate in the detection zone indicates the presence of the target analyte in the sample. The antibody in the sample is detected / quantified by detecting and / or quantifying the bioluminescent signal.

[0248] Additional alternatives to the exemplary embodiments described above are also contemplated, such as alternative binding agents, target analytes, detectable elements, and ordering of various components (e.g., non-specific binder / target-specific binder, target-specific binder / non-specific binder, target-specific binder / target-specific binder, etc.) described herein, and embodiments incorporating various combinations of components are within the scope of the present invention.

[0249] In some embodiments, the target analyte is not an antibody, but rather a small molecule, peptide, protein, carbohydrate, lipid, etc. In some embodiments, the lateral flow assays and systems described above are configured for the detection of any such target analyte (e.g., using one or more NanoLuc®-based technologies (e.g., NanoBiT, NanoTrip, NanoBRET, etc.)).

[0250] b. Solid-phase assay Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample. According to these embodiments, the present disclosure provides compositions and materials for performing solid-phase assays (e.g., solid-phase platforms for performing immunoassays). Solid-phase detection platforms are generally the simplest form of immunoassays and can be used to detect, quantify, test, measure, and monitor a variety of analytes, including, but not limited to, ovulation monitoring, detection / diagnosis of infectious diseases / infectious organisms, analysis of drugs of abuse, detection / quantification of analytes important to human physiology, veterinary testing, security screening, agricultural applications, environmental testing, product quality assessment, and the like. In contrast to lateral flow assays, solid-phase detection platforms do not involve facilitating the flow of assay reagents through a membrane, but instead typically include a solid support to which the assay components are bound or contained (e.g., dipstick or spot tests).

[0251] As shown in FIG. 2 , embodiments of the present disclosure include a solid-phase detection platform (200) for detecting and / or quantifying a target analyte based on bioluminescent complex formation. In some embodiments, the solid-phase detection platform of the present disclosure includes one or more detection regions (205) and one or more control regions (210) to which a sample is applied. In some embodiments, one or more detection regions include a target analyte-binding agent conjugated within and / or to a portion of the detection region. The solid-phase detection platform of the present disclosure may also include a solid support (215) to which the detection region and the control region are attached, separating the regions from each other, and to which a sample can be applied to the detection region and the control region (e.g., a dipstick test).

[0252] According to these embodiments, the sample or a portion of the sample is applied to the detection and control regions of the solid phase assay platform, whereby the target analyte is contacted with the target analyte binding agent (220) conjugated to and / or within the detection region under conditions whereby a binding event and / or immobilization of the target analyte on the solid phase (e.g., immobilization of a bioluminescent entity and formation of a bioluminescent complex) is detectable, thereby indicating the presence of the analyte in the sample.

[0253] In some embodiments, the solid-phase assay platform includes a first target analyte-binding agent (e.g., a target-specific binding agent (e.g., a target-specific antibody, an antigen of the target antibody, etc.)) immobilized to the solid phase. A second target analyte-binding agent (e.g., a target-specific binding agent (e.g., a target-specific antibody, an antigen of the target antibody, etc.), a non-specific binder (e.g., Protein L)) linked to a bioluminescent polypeptide (e.g., SEQ ID NO: 5 or a variant thereof) is added to the solid phase along with the sample (e.g., simultaneously, sequentially, etc.). Both target analyte-binding agents bind to the target analyte, and the bioluminescent polypeptide is immobilized on the solid phase. Detection / quantification of bioluminescence on the solid phase (e.g., after a washing step) indicates the presence / amount of the target analyte in the sample. In some cases, the first target analyte-binding agent is conjugated to a detection zone, and the second target analyte-binding agent (linked to the bioluminescent polypeptide) is applied to the detection zone with or without a sample. In some cases, a second target analyte-binding agent is conjugated to the detection zone, and a first target analyte-binding agent (linked to a bioluminescent polypeptide) is applied to the detection zone, with or without a sample. According to these embodiments, immobilization of bioluminescence in the detection zone can be detected and / or quantified in the presence of a luminescent substrate (described in more detail below), thus indicating the presence (or absence) of the antibody in the sample.

[0254] In an alternative embodiment, a solid-phase assay platform detects target analytes using a two-component complementation approach in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide / polypeptide components (e.g., the NanoBiT system). Multiple configurations of solid-phase assays and systems utilizing the two-component complementation approach are within the scope of this specification. For example, an exemplary system may include (i) a first target analyte-binding agent linked to a first NL peptide or NL polypeptide (e.g., SEQ ID NO: 9 or 10 or a variant thereof) capable of interacting with high affinity with a second, different NL polypeptide or NL peptide (e.g., SEQ ID NO: 10 or 9 or a variant thereof) to generate a bioluminescent signal, and (ii) a second target analyte-binding agent linked to a complementary NL polypeptide or NL peptide, where the second target analyte-binding agent is immobilized on a solid phase. Upon binding of the target analyte-binding agent to the target analyte, a bioluminescent complex is formed on the solid phase, and a bioluminescent signal is detectable / quantifiable in the presence of a luminescent substrate (described in more detail below).

[0255] In other embodiments, the solid-phase assay platform detects target analytes using a ternary complementation approach in which a bioluminescent complex is formed upon binding of two non-light-emitting (NL) peptide and polypeptide components (e.g., the NanoTrip system). In some embodiments, the solid-phase assay platform includes: (i) a first target analyte-binding agent that includes both a target analyte-binding element (e.g., a general or specific) and an NL peptide (e.g., SEQ ID NO: 11 or 13) capable of forming a ternary bioluminescent complex (e.g., a NanoTrip complex); (ii) a second target analyte-binding agent that includes both a target analyte-binding element (e.g., a specific) and an NL peptide (e.g., SEQ ID NO: 11 or 13) capable of forming a ternary bioluminescent complex (e.g., a NanoTrip complex); (iii) the NL polypeptide component of the ternary bioluminescent complex (e.g., a NanoTrip complex); and (iv) a luminescent substrate. In some cases, a first target analyte-binding agent is conjugated to the detection zone and a second target analyte-binding agent is applied to the detection zone with or without a sample. In some cases, a second target analyte-binding agent is conjugated to the detection zone and the first target analyte-binding agent is applied to the detection zone with or without a sample. When a bioluminescent complex is formed in the detection zone, a bioluminescent signal is detected and / or quantified, thereby indicating the presence (or absence) of the antibody in the sample.

[0256] In other embodiments, the solid-phase assay platform includes (i) a first target analyte-binding agent comprising a target analyte-binding element and a NanoLuc®-based peptide or polypeptide, (ii) a target analyte-binding agent comprising a target analyte-binding element and a fluorophore, and (iii) optionally, an additional peptide / polypeptide component that forms a bioluminescent complex (e.g., in embodiments where the NanoLuc®-based peptide or polypeptide is not a bioluminescent polypeptide, e.g., non-luminescent), such that upon binding of the first and second target analyte-binding agents to the target analyte in the sample, light emission from the NanoLuc®-based component (e.g., the NanoLuc® protein or bioluminescent complex) excites the fluorophore (e.g., via BRET) in the presence of any additional components required for bioluminescence (e.g., a luminescent substrate, a complementary component, etc.). In some cases, the first target analyte-binding agent is conjugated to a detection region, and the second target analyte-binding agent is applied to the detection region with or without a sample. In some cases, a second target analyte binding agent is conjugated to the detection region and the first target analyte binding agent is applied to the detection region with or without sample.

[0257] As shown in Figure 2, the solid-phase platforms of the present disclosure can be configured to test a single sample from a subject for multiple different analytes, such as antibodies raised against different diseases / microorganisms (e.g., multiplexing). According to these embodiments, the solid-phase platform can include multiple detection regions, each detection region containing a different target analyte binding agent with a different target analyte binding element (e.g., a different disease antigen).

[0258] In some embodiments, the solid-phase platform of the present disclosure can include multiple detection regions, such as, for example, one or more wells of a microtiter plate. In such embodiments, one or more different target analyte-binding agents can be conjugated (e.g., coated) to the wells of the microtiter plate along with one or more other detection reagents (e.g., a second target analyte-binding agent, a luminescent substrate, an assay buffer, etc.) required to perform a particular bioluminescence assay. In some embodiments, one or more of the other detection reagents (reagents not conjugated to the microtiter plate) required to perform the assay can be added to the wells of the microtiter plate in the form of a lyophilized cake (lyocake) or tablet and reconstituted as part of the bioluminescence assay.

[0259] c. Liquid-phase assay Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample. According to these embodiments, the present disclosure provides compositions and materials for performing liquid-phase assays (e.g., liquid-based formats for performing immunoassays in solution). Liquid-phase detection platforms can be used for the detection, quantification, testing, measurement, and monitoring of various analytes, including, but not limited to, analytes related to ovulation monitoring, detection / diagnosis of infectious diseases / infectious organisms, analysis of drugs of abuse, detection / quantification of analytes important to human physiology, veterinary testing, security screening, agricultural applications, environmental testing, product quality assessment, and the like. In contrast to lateral flow assays and solid-phase detection platforms, liquid-phase detection platforms typically include a container for a solution / liquid in which a reaction involving detection reagents occurs, rather than one or more of the detection reagents being conjugated to a solid support or membrane to facilitate detection.

[0260] For example, as shown in Figure 33, embodiments of the liquid phase platform of the present disclosure can include one or more components of a bioluminescent complex in a tablet or lyophilized cake that can be reconstituted in a solution (e.g., a buffer) to facilitate detection of an analyte. In some embodiments, the tablet or lyophilized cake can contain all the reagents necessary to carry out a reaction to detect an analyte. Such lyophilized cakes or tablets are compatible with many different assay formats, including, but not limited to, cuvettes, wells of a microtiter plate (e.g., a 96-well microtiter plate), test tubes, large-volume bottles, SNAP assays, etc.

[0261] In some embodiments, the liquid phase assay platform comprises a lyocake or tablet comprising one or more of a first target analyte binding agent (e.g., a target-specific binding agent (e.g., a target-specific antibody, an antigen of the target antibody, etc.)), a second target analyte binding agent (e.g., a target-specific binding agent (e.g., a target-specific antibody, an antigen of the target antibody, etc.)) conjugated to a bioluminescent polypeptide (e.g., SEQ ID NO: 5 and variants thereof), and a non-specific binder (e.g., Protein L). Detection / quantification of bioluminescence in solution indicates the presence / amount of the target analyte in the sample.

[0262] In some embodiments, solution-phase assay platforms detect target analytes using a two-component complementation approach in which a bioluminescent complex is formed upon binding of two non-luminescent (NL) peptide / polypeptide components (e.g., the NanoBiT system). Multiple configurations of solution-phase assays and systems utilizing the two-component complementation approach are within the scope of this specification. For example, an exemplary system may include (i) a first target analyte-binding agent linked to a first NL peptide or NL polypeptide (e.g., SEQ ID NO: 9 or 10 or a variant thereof) capable of interacting with high affinity with a second, different NL polypeptide or NL peptide (e.g., SEQ ID NO: 10 or 9 or a variant thereof) to generate a bioluminescent signal, and (ii) a second target analyte-binding agent linked to a complementary NL polypeptide or NL peptide. Upon binding of the target analyte-binding agent to the target analyte, a bioluminescent complex is formed in solution, and in the presence of a luminescent substrate (described in more detail below), the bioluminescent signal is detectable / quantifiable.

[0263] In other embodiments, the solution-phase assay platform detects target analytes using a ternary complementation approach in which a bioluminescent complex is formed upon binding of two non-light-emitting (NL) peptide and polypeptide components (e.g., the NanoTrip system). In some embodiments, the solution-phase assay platform includes: (i) a first target analyte-binding agent that includes both a target analyte-binding element (e.g., a general or specific) and an NL peptide (e.g., SEQ ID NO: 11 or 13) capable of forming a ternary bioluminescent complex (e.g., a NanoTrip complex); (ii) a second target analyte-binding agent that includes both a target analyte-binding element (e.g., a specific) and an NL peptide (e.g., SEQ ID NO: 11 or 13) capable of forming a ternary bioluminescent complex (e.g., a NanoTrip complex); (iii) the NL polypeptide component of the ternary bioluminescent complex (e.g., a NanoTrip complex); and (iv) a luminescent substrate. Once the bioluminescent complex is formed in solution, the bioluminescent signal is detected and / or quantified, thereby indicating the presence (or absence) of the antibody in the sample.

[0264] In other embodiments, the solution-phase assay platform includes (i) a first target analyte-binding agent comprising a target analyte-binding element and a NanoLuc®-based peptide or polypeptide; (ii) a target analyte-binding agent comprising a target analyte-binding element and a fluorophore; and (iii) optionally, an additional peptide / polypeptide component that forms a bioluminescent complex (e.g., in embodiments where the NanoLuc®-based peptide or polypeptide is not a bioluminescent polypeptide, e.g., is non-luminescent), such that upon binding of the first and second target analyte-binding agents to a target analyte in a sample, light emission from the NanoLuc®-based component (e.g., the NanoLuc® protein or the bioluminescent complex) excites the fluorophore (e.g., via BRET) in the presence of any additional components required for bioluminescence (e.g., a luminescent substrate, a complementary component, etc.).

[0265] The liquid-phase platforms of the present disclosure can be configured to test a single sample from a subject for multiple different analytes, such as antibodies raised against different diseases / microorganisms (e.g., multiplexing). In some embodiments, one or more of the detection reagents needed to perform a bioluminescent reaction to detect / quantify the analyte are present in one or more containers (e.g., individual wells of a 96-well plate) of the particular assay platform being used, e.g., as lyocakes or tablets reconstituted in a buffer solution. In other embodiments, one or more sample solutions are already present in the container, and one or more lyocakes or tablets are added to the container and hydrated to facilitate the bioluminescent reaction. According to these embodiments, the liquid-phase platform can include multiple containers containing different target analyte binding agents with different target analyte binding elements (e.g., different disease antigens).

[0266] d. Other assays Embodiments of the present disclosure include compositions, assays, systems, devices, and methods for detecting one or more analytes in a sample using other assay platforms known in the art. For example, target analytes can be detected and / or measured using the bioluminescent polypeptides and / or complexes described herein in the context of microfluidic and / or chip-based assays. Because microfluidic systems integrate a variety of operations for manipulating fluids, such as chemical or biological samples, these systems are applicable to many different fields, such as biological and medical diagnostics. One type of microfluidic device is a microfluidic chip. Microfluidic chips can contain microscale features (or microfeatures), such as channels, valves, pumps, and / or reservoirs, that store fluids, deliver fluids to various locations on the chip, and / or react with fluidic reagents.

[0267] Microfluidic chips or lab-on-a-chip (LOC) devices constructed with bioluminescent polypeptides and / or conjugates, including peptides and polypeptides capable of generating a bioluminescent signal in the presence of a target analyte, offer increased flexibility for automation, integration, miniaturization, and multiplexing. For example, microfluidic chip-based pathogen detection typically uses microscale or nanoscale reaction chambers, allowing for miniaturization and portability of the device, which is particularly advantageous for point-of-care testing. LOC technology allows for the integration of sample preparation, amplification, and signal detection, reducing the time required to generate results. High throughput and low sample and reagent consumption make this technology flexible and relatively cost-effective. Nucleic acid-based microfluidic pathogen detection for detecting bacteria, viruses, and fungi does not require PCR or real-time PCR for amplification, a distinct advantage of the bioluminescent conjugates of the present disclosure.

[0268] 5. Assay Compositions, Components, and Methods of Preparation Embodiments of the present disclosure also include methods for producing assay platforms for use with bioluminescent peptides and polypeptides to detect target analytes. While assay platforms can vary depending on various factors, such as the analyte to be detected, the complexity of the sampling environment, and diagnostic parameters, the compositions, materials, and methods of the present disclosure are applicable to most currently available assay platforms, such as solid-phase assays, lateral flow assays, and microfluidic-based assays.

[0269] a. Luminescent substrate In some embodiments, methods for manufacturing assay platforms of the present disclosure include the use of luminescent substrates. Luminescent substrates, such as coelenterazine and its analogs and derivatives, can degrade during storage, resulting in loss of the substrate before addition or use in a biological assay. Such degradation can result in temperature-dependent instability of the luminescent substrate in solution over time. This degradation wastes the luminescent substrate and reduces the sensitivity and reproducibility of luminescence measurements obtained from biological assays utilizing the degraded luminescent substrate.

[0270] Provided herein are compositions comprising a luminescent substrate, such as coelenterazine or an analog or derivative thereof. Exemplary coelenterazine analogs include coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, and JRW-1744.

[0271] In some embodiments, the substrate is coelenterazine and has the following structure: JPEG2025133108000003.jpg5869 Exemplary coelenterazine analogs include coelenterazine-h (2-deoxycoelenterazine or 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), coelenterazine-hh (dideoxycoelenterazine or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), on), furimazine (8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one), JRW-0238 (8-benzyl-2-(furan-2-ylmethyl)-6-(3-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1404 (8-benzyl-6-(2-fluoro-3-hydroxyphenyl)-2-(furan-2-yl methyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1482 (6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one), JRW-1667 (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-a]pyrazin-3(7H)-one). and JRW-1743 (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazin-3(7H)-one), having the following structure: JPEG2025133108000004.jpg145164

[0272] Additional exemplary coelenterazine analogs include coelenterazine-n, coelenterazine-f, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, coelenterazine-i, coelenterazine-icp, coelenterazine-v, 2-methylcoelenterazine, and the like. In some embodiments, the compound is a compound selected from the group consisting of WO2003 / 040100; U.S. Patent Publication No. 2008 / 0248511 (e.g., paragraph

[0086] ); U.S. Patent No. 8,669,103; WO2012 / 061529; U.S. Patent Publication No. 2017 / 0233789; U.S. Patent No. 9,924,073; U.S. Patent Publication No. 2018 / 0030059; U.S. Patent No. 10,000,500; U.S. Patent Publication No. 2018 / 0155350; U.S. Patent Application No. 16 / 399,410 (PCT / US2019 / 029975); U.S. Patent Application No. 16 / 548,214 (PCT / US2019 / 047688); U.S. Patent Publication No. 2014 / 0227759; U.S. Patent No. 9,840,730; U.S. Patent No. 7,268,229; U.S. Patent No. 7,537,912; U.S. Patent No. 8,809,529; U.S. Patent No. 9,139,836; U.S. Patent No. 10,077,244; U.S. Patent No. 9,487,520; U.S. Patent No. 9,924,073; U.S. Patent No. 9,938,564; U.S. Patent No. 9,951,373; U.S. Patent No. 10,280,447; U.S. Patent No. 10,308,975; U.S. Patent No. 10,428,075, the disclosures of which are incorporated herein by reference in their entireties. In some embodiments, the coelenterazine analog comprises a precursor substrate such as those described in U.S. Patent Publication Nos. 2008 / 0248511; 2012 / 0707849; 2014 / 0099654; U.S. Patent Nos. 9,487,520; 9,927,430; and 10,316,070, which are incorporated herein by reference in their entireties. In some embodiments, the compound is furimazine. In some embodiments, the compound is JRW-0238. In some embodiments, the compound is JRW-1743. In some embodiments, the compound is JRW-1744.

[0273] Provided herein is a composition for fabricating a bioluminescent target analyte detection platform, comprising a luminescent substrate, such as coelenterazine or an analog or derivative thereof, and a polymer or paper / fiber substrate. Compositions that stabilize and / or improve the reconstitution efficiency of luminescent substrates, such as coelenterazine or an analog or derivative thereof, are described in U.S. Patent Application No. 16 / 592,310 (PCT / US2019 / 054501), the entire contents of which are incorporated herein by reference. In some embodiments, the composition stabilizes the compound against degradation. In some embodiments, the composition stabilizes the compound against degradation compared to a composition that does not contain the polymer or paper / fiber substrate. In some embodiments, the polymer or paper / fiber substrate reduces or inhibits the formation of one or more degradation products from the compound. In some embodiments, the composition improves the reconstitution efficiency or reconstitution rate of the substrate.

[0274] Furthermore, embodiments of the present disclosure include means for stabilizing (e.g., increasing storage stability) the compositions further described herein. In some embodiments, increasing the storage stability of the compositions provided herein includes methods and compositions for stabilizing a luminescent substrate. The luminescent substrate may be, but is not limited to, coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, a derivative thereof, an analog thereof, or any combination thereof. The composition may include a luminescent substrate, a thionucleoside, and an organic solvent. The composition may or may not include a luminescent enzyme. As provided in U.S. Pat. No. 9,676,997, incorporated herein by reference, the thionucleoside may be a compound of formula (I) or a tautomer thereof:

[0275] JPEG2025133108000005.jpg3079

[0276] During the ceremony,

[0277] R 1is hydrogen, alkyl, substituted alkyl, alkyl-aryl, alkyl-heteroaryl, cycloalkyl, aryl, heteroaryl, carboxylic acid, ester, NR a R b , imine, hydroxyl, or oxo;

[0278] R 2 is hydrogen, NR a R b , imine, alkyl, or aryl;

[0279] R a and R b are each independently hydrogen, alkyl, or aryl.

[0280] In some embodiments, the compound of Formula (I) is selected from the group consisting of ATT (6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one); 3-(4-amino-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid; tetrahydro-2-methyl-3-thioxo-1,2,4-triazine-5,6-dione; 4-((2-furylmethylene)amino)-3-mercapto-6-methyl-1,2,4-triazine- 5(4H)-one;6-Benzyl-3-sulfanyl-1,2,4-triazin-5-ol;4-Amino-3-mercapto-6-methyl-1,2,4-triazin-5(4H)-one;3-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)propanoic acid;(E)-6-Methyl-4-((thiophen-2-ylmethylene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one;(E)-6-Methyl -4-((3-nitrobenzylidene)amino)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; (E)-4-((4-(diethylamino)benzylidene)amino)-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; ATCA ethyl ester; TAK-0021, TAK-0020, TAK-0018, TAK-000 as described in U.S. Pat. No. 9,676,997 (incorporated herein by reference). 9, TAK-0014, TAK-0007, TAK-0008, TAK-0003, and TAK-0004; 3-thioxo-6-(trifluoromethyl)-3,4-dihydro-1,2,4-triazin-5(2H)-one; 6-cyclopropyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; 6-(hydroxymethyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one; or any combination thereof.

[0281] In some embodiments, the thionucleoside may stabilize the luminescent substrate against decomposition over time, in the presence or absence of light, and / or at different temperatures. The thionucleoside may stabilize the luminescent substrate against decomposition into one or more degradation products over time, in the presence or absence of light, and / or at different temperatures. Thus, the inclusion of a thionucleoside in the compositions described further herein may stabilize the luminescent substrate against decomposition by inhibiting or reducing the formation of one or more degradation products compared to compositions that do not contain the thionucleoside. This allows the luminescent substrate to be stored or incubated for a period of time at a particular temperature, in the presence and / or absence of light, without significant degradation of the luminescent substrate prior to use in an assay. According to these embodiments, the inclusion of a thionucleoside in the compositions described herein can enhance the storage stability of the composition. These embodiments also relate to methods for stabilizing a luminescent substrate. Such methods may stabilize the luminescent substrate against decomposition and / or inhibit or reduce the formation of one or more degradation products. The method can include contacting a luminescent substrate with an effective amount of a thionucleoside (e.g., 225 mM) in the presence of an organic solvent. The contacting step can include forming the composition described above.

[0282] In some embodiments, one or more of the non-luminescent (NL) peptide / polypeptide components that form the bioluminescent complexes described above, with or without a luminescent substrate, can be included as part of a composition such as a lyophilized powder. These compositions, with or without other components, can be applied directly to a portion of the detection platform or can be reconstituted as part of a separate solution that is applied to the detection platform.

[0283] Coelenterazine and its analogs and derivatives can suffer from challenges associated with reconstitution into buffer systems used in many assays, such as the bioluminescence assays described herein. For example, coelenterazine, or its analogs or derivatives, such as furimazine, can dissolve slowly and / or unevenly in buffers (e.g., due to the heterogeneous microcrystalline nature of the solid material). While dissolution in an organic solvent before dilution with a buffer can produce faster and more uniform results, coelenterazine compounds can suffer from instability in organic solutions during storage, including both thermal and photoinstability. In some embodiments, the composition further comprises a polymer. As further described herein, the presence of a polymer can stabilize the compound against degradation and can improve the solubility of the compound in water or aqueous solutions.

[0284] The polymer can be a natural biopolymer or a synthetic polymer. In some embodiments, the polymer is a natural biopolymer. Suitable natural biopolymers are carbohydrates, including disaccharides (e.g., trehalose and maltose) and polysaccharides (e.g., pullulan, dextran, and cellulose). Mixtures of natural biopolymers can also be used. In some embodiments, the polymer is pullulan, which is a polysaccharide containing maltotriose repeating units. Maltotriose is a trisaccharide containing three glucose units linked via α-1,4 glycosidic bonds. The maltotriose units within a pullulan polymer are linked to each other via α-1,6 glycosidic bonds.

[0285] In some embodiments, the polymer is a synthetic polymer. The synthetic polymer can be a homopolymer, copolymer, or block copolymer (e.g., a diblock copolymer, a triblock copolymer, etc.). Non-limiting examples of suitable polymers include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and poly(D,L-lactide-co-PPO-co-D,L-lactide).L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), poly(ethylene glycol), poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes (e.g., polyethylene and polypropylene), polyalkylene glycols (e.g., poly(ethylene glycol) (PEG)), polyalkylene terephthalates (e.g., poly(ethylene terephthalate)), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters (e.g., poly(vinyl acetate)), polyvinyl halides (e.g., poly(vinyl chloride) (PVC)), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses (e.g., alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, etc.), esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, etc.), polymers of acrylic acid (“polyacrylic acid”) (e.g., poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polydioxanone and its copolymers (e.g., polyhydroxyalkanoates, polypropylene fumarate), polyoxymethylene, poloxamer, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), trimethylene carbonate, and mixtures and copolymers thereof.

[0286] In some embodiments, the composition further comprises a paper substrate. As further described herein, the presence of the paper substrate can stabilize the compound against degradation and improve the solubility of the compound in aqueous solution. Exemplary paper substrates include, but are not limited to, Whatman brand paper (e.g., W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, etc.), Ahlstrom paper (e.g., A-226 paper, etc.), M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester paper, and combinations thereof.

[0287] In addition to the compound and the polymer and / or paper substrate, the composition may include additional components such as a buffer, a surfactant, a salt, a protein, or any combination thereof. For example, the composition may include a buffer such as a phosphate buffer, a borate buffer, an acetate buffer, or a citrate buffer, or other common buffers such as bicine, tricine, tris(hydroxymethyl)aminomethane (Tris), N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino)ethanesulfonic acid (MES), etc.

[0288] In some embodiments, the composition may contain a surfactant. Exemplary surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and zwitterionic surfactants. For example, the surfactant may be a nonionic surfactant such as sorbitan 20.

[0289] In some embodiments, the composition may include salts such as sodium chloride, potassium chloride, magnesium chloride, and the like.

[0290] In some embodiments, the composition may include a protein. For example, the composition may include a carrier protein to prevent surface adsorption of a luminescent enzyme that may be added in a downstream assay. In some embodiments, the protein may be bovine serum albumin (BSA).

[0291] In some embodiments, the composition may include a substance that reduces autoluminescence. In some embodiments, the substance is ATT (6-aza-2-thiothymine), a derivative or analog of ATT, a thionucleoside, a thiourea, or the like. In some embodiments, the substance is a thionucleoside as disclosed in U.S. Pat. No. 9,676,997, which is incorporated herein by reference. In some embodiments, the substance is a thiourea, the use of which to reduce autoluminescence is disclosed in U.S. Pat. Nos. 7,118,878, 7,078,181, and 7,108,996, which are incorporated herein by reference.

[0292] The composition may be in the form of a lyophilized powder. Such a composition can be prepared by drying a mixture of the components of the composition. For example, the composition can be prepared by dissolving a compound in a solvent (e.g., an organic solvent) to form a first solution, adding a polymer to the first solution to form a second solution, and then drying the second solution to obtain the composition. In some embodiments, the drying step can include lyophilization, which can provide the composition in the form of a powder. In some embodiments, the drying step can include air-drying, which can provide the composition in the form of a malleable disk.

[0293] In some embodiments (e.g., compositions including a paper substrate as well as a polymer), the composition is in the form of a solution. When the composition is a solution, it can have a pH of about 5.5 to about 8.0, e.g., about 6.5 to about 7.5. In some embodiments, the composition has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0294] b. Lateral flow component In some embodiments, the present disclosure provides a method for manufacturing a lateral flow assay platform that includes a conjugate pad, an analytical membrane, a sample pad, and other components (e.g., an absorbent pad) necessary to facilitate lateral flow across the membrane. For example, the conjugate pad can include at least one target analyte binding agent reversibly conjugated to the conjugate pad, such that when lateral flow is applied, the target analyte binding agent can migrate from the conjugate pad to the analytical membrane, allowing the target analyte binding agent to bind to the target analyte and form a bioluminescent complex. In some embodiments, the target analyte binding agent comprises a target analyte binding element that facilitates binding to the target analyte, and a bioluminescent polypeptide or a component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and variants thereof), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), a NL peptide of SEQ ID NO: 10 (SmBiT), a NL peptide of SEQ ID NO: 11 (HiBiT), a NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), a NL peptide of SEQ ID NO: 13 (SmTrip), a NL peptide of SEQ ID NO: 14 (β9 / β10 dipeptide), or a variant thereof. In some embodiments, the target analyte binding agent comprises a fluorophore that is activatable by energy transfer (e.g., from a bioluminescent polypeptide or a component of a bioluminescent complex).

[0295] In some embodiments, the conjugate pad comprises a first target analyte-binding agent. In some embodiments, the first target analyte-binding agent comprises a first target analyte-binding element and a first bioluminescent polypeptide or a first component of a bioluminescent complex (e.g., an NL peptide or NL polypeptide). In some embodiments, the target analyte-binding agent is stored on or within the conjugate pad such that it remains on the conjugate pad until it migrates laterally through the device.

[0296] In some embodiments, the conjugate pad comprises a luminescent substrate, such as coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, precursor substrates, and / or other substrates described herein (e.g., coelenterazine analogs or derivatives). In some embodiments, the luminescent substrate is reversibly conjugated to the conjugate pad. In some embodiments, the luminescent substrate is dried onto or within the conjugate pad. In some embodiments, the luminescent substrate is part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof (e.g., as described in detail above and / or in U.S. Provisional Application No. 62 / 740,622). In some embodiments, the luminescent substrate is applied as part of a composition or solution, such as a protein buffer. In some embodiments, the protein buffer comprises 20 mM NaPO; 5% w / v BSA; 0.25% v / v Tween 20; 10% w / v sucrose. In some embodiments, the luminescent substrate is added to the protein buffer and allowed to dry on a substrate or matrix (e.g., filter paper or membrane) at 37°C for 1 hour. In other embodiments, the luminescent substrate is applied as a separate reagent that is part of an assay or system.

[0297] In some embodiments, the assay platform includes an analytical membrane including a detection region and a control region to facilitate detection of a bioluminescent complex indicative of detection of a target analyte. The detection region may include at least one target analyte binding agent immobilized therein such that it is not displaced by application of lateral flow through the membrane. In some embodiments, the analytical membrane includes at least one target analyte binding agent. In some embodiments, the target analyte binding agent includes a target analyte binding element and a bioluminescent polypeptide or a first component of a bioluminescent complex (e.g., an NL peptide or NL polypeptide).

[0298] In some embodiments, the analytical membrane comprises multiple detection regions, each detection region comprising a different target analyte binding agent that comprises a different target analyte binding element (eg, multiplex capability).

[0299] In some embodiments, the analytical membrane comprises a luminescent substrate, such as coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, a coelenterazine analog or derivative, a precursor substrate, or another substrate described herein (e.g., a coelenterazine analog or derivative). In some embodiments, the luminescent substrate is reversibly conjugated to and / or contained on / within the analytical membrane as part of a composition, e.g., comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is applied as part of a composition or solution, such as a protein buffer. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 5% w / v BSA; 0.25% v / v Tween 20; 10% w / v sucrose. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 5% w / v BSA; 0.25% v / v Tween 20; 5% w / v pullulan. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% w / v BSA; 0.25% v / v Tween 20. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% w / v Prionex; 0.25% v / v Tween 20. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% w / v BSA, 5 mM ATT. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% v / v Prionex, 5 mM ATT. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid. In some embodiments, the protein buffer comprises 20 mM Na3PO4; 1-5% w / v Prionex, 5 mM ATT, 5 mM ascorbic acid.In some embodiments, the protein buffer comprises 20 mM NaPO; 1-5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid. In some embodiments, the protein buffer comprises 1-5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid. In some embodiments, the luminescent substrate is added to the protein buffer and allowed to dry on a substrate or matrix (e.g., filter paper or membrane) at 37°C for 1 hour. In other embodiments, the luminescent substrate is applied as a separate reagent that is part of the assay or system.

[0300] c. solid phase component In some embodiments, the present disclosure provides methods for manufacturing a solid-phase detection platform (e.g., a dipstick assay or spot test) that includes a detection region and a control region. In some embodiments, the detection region includes at least one target analyte binding agent conjugated to the detection region. In some embodiments, the detection region includes at least one target analyte binding agent that is not conjugated to the detection region. Such an unconjugated binding agent may be added to the detection region (e.g., with the sample or as part of a detection reagent) or may be present on or in the detection region unconjugated. In some embodiments, the unconjugated binding agent comprises a target analyte-binding element and a bioluminescent polypeptide or a component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and its variants), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), a NL peptide of SEQ ID NO: 10 (SmBiT), a NL peptide of SEQ ID NO: 11 (HiBiT), a NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), a NL peptide of SEQ ID NO: 13 (SmTrip), a NL peptide of SEQ ID NO: 14 (β9 / β10 dipeptide), or a variant thereof.

[0301] In some embodiments, the solid-phase detection platform includes multiple detection regions, each detection region containing a different target analyte-binding agent that includes a different target analyte-binding element (e.g., multiplexing capability). In some embodiments, one or more different target analyte-binding agents can be conjugated (e.g., coated) to a well of a microtiter plate along with one or more other detection reagents required to perform a particular assay (e.g., a second target analyte-binding agent, a luminescent substrate, an assay buffer, etc.). In other embodiments, the detection reagent can be applied as a separate reagent that is part of the assay method or system (e.g., as part of a lyocake or tablet and reconstituted as part of the assay).

[0302] The detection platform may also include a luminescent substrate such as coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, precursor substrates, or other substrates described herein (e.g., coelenterazine analogs or derivatives). In some embodiments, the luminescent substrate is reversibly conjugated to the detection zone. In some embodiments, the luminescent substrate is stably stored on or within the detection zone. In some embodiments, the luminescent substrate is part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is applied as part of a composition or solution such as a protein buffer, a detection reagent, or with the sample. In some embodiments, the protein buffer comprises 20 mM NaPO; 5% w / v BSA; 0.25% v / v Tween 20; 10% w / v sucrose. In some embodiments, the luminescent substrate is added to the protein buffer and allowed to dry onto a substrate or matrix (e.g., filter paper, membrane, individual wells of a microtiter plate) for 1 hour at 37°C. In other embodiments, the luminescent substrate is applied as a separate reagent that is part of the assay method or system (e.g., as part of a lyocake or tablet and reconstituted as part of the assay).

[0303] Embodiments of the present disclosure also include methods for making a substrate or matrix for use in a bioluminescent assay. According to these embodiments, the method includes generating a solution or liquid formulation containing at least one target analyte-binding agent, the target analyte-binding agent comprising a target analyte-binding element and one of a polypeptide component of a bioluminescent complex or a peptide component of a bioluminescent complex. In some embodiments, the solution includes a protein buffer and at least one excipient, including but not limited to, a surfactant, a reducing agent, a salt, a radical scavenger, a chelator, a protein, or any combination thereof. In some embodiments, the solution includes a complementary peptide or polypeptide component of a bioluminescent complex, whereby the target analyte-binding agent and the complementary peptide or polypeptide component of the bioluminescent complex form a bioluminescent analyte-detection complex in the presence of the target analyte. In some embodiments, the solution includes a luminescent substrate.

[0304] The method includes forming a solution or liquid formulation and then applying the solution to a surface of a substrate or matrix. In some embodiments, the substrate or matrix is ​​W-903 paper, FTA paper, FTA Elute paper, FTA DMPK paper, Ahlstrom A-226 paper, M-TFN paper, FTA paper, FP705 paper, Bode DNA collection paper, nitrocellulose paper, nylon paper, cellulose paper, Dacron paper, cotton paper, and polyester paper, or a combination thereof. In other embodiments, the substrate or matrix is ​​a mesh comprising plastic, nylon, metal, or a combination thereof.

[0305] Method embodiments also include drying the substrate or matrix after applying the solution to the substrate or matrix. In some embodiments, drying the substrate or matrix containing the solution includes drying the substrate or matrix at a temperature of about 30°C to 65°C, about 30°C to 60°C, about 30°C to 55°C, about 30°C to 50°C, about 30°C to 45°C, or about 30°C to 40°C. In some embodiments, the matrix or substrate is dried for about 15 minutes to 8 hours, about 30 minutes to 7 hours, about 45 minutes to 6 hours, about 1 hour to 5 hours, about 2 hours to 4 hours, about 30 minutes to 2 hours, or about 30 minutes to 1 hour. In some embodiments, drying the substrate containing the solution includes lyophilizing and / or freezing the substrate.

[0306] In some embodiments, the method comprises drying at least one target analyte binding agent and / or complementary peptide or polypeptide component of a bioluminescent complex on a first substrate and drying a luminescent substrate on a second substrate. In some embodiments, at least one target analyte binding agent and / or complementary peptide or polypeptide component of a bioluminescent complex is dried on a paper-based substrate and the luminescent substrate is dried on a mesh (see, e.g., Figures 42A-42E).

[0307] According to these embodiments, the substrate or matrix can be used in a bioluminescent assay to detect a target analyte. For example, a bioluminescent signal can be generated when the substrate or matrix containing the solution is exposed to the target analyte. In some embodiments, the bioluminescent signal is proportional to the concentration of the target analyte. In some embodiments, as further described herein, at least one target analyte-binding agent and / or complementary peptide or polypeptide component of the bioluminescent complex exhibits improved stability when dried on the substrate.

[0308] d. Liquid phase components In some embodiments, the present disclosure provides methods for manufacturing a liquid-phase detection platform (as described herein) that includes one or more detection and control regions (e.g., wells of a 96-well microtiter plate). For example, as shown in FIG. 33, embodiments of the liquid-phase platform of the present disclosure can include one or more components of the bioluminescent complexes described herein in a tablet or lyophilized cake that can be reconstituted in a solution (e.g., a buffer) to facilitate detection of the analyte. In some embodiments, the tablet or lyophilized cake can contain all the reagents necessary to perform a reaction to detect the analyte and is included as part of the liquid-phase detection platform (e.g., present in one or more wells of a 96-well microtiter plate). Such lyophilized cakes or tablets are compatible with many different assay formats, including, but not limited to, cuvettes, wells of a microtiter plate (e.g., a 96-well microtiter plate), test tubes, large-volume bottles, SNAP assays, etc.

[0309] In some embodiments, one or more components of a bioluminescent complex described herein can be added to the detection region and / or can be pre-existing in the detection region in the presence or absence of a sample. The detection reagent can then be reconstituted (e.g., rehydrated) as part of performing detection of an analyte in a sample. In some embodiments, the detection reagent comprises a target analyte-binding element and a bioluminescent polypeptide or component of a bioluminescent complex, such as a bioluminescent polypeptide of SEQ ID NO: 5 (NanoLuc and its variants), a non-luminescent (NL) polypeptide of SEQ ID NO: 9 (LgBiT), a NL peptide of SEQ ID NO: 10 (SmBiT), a NL peptide of SEQ ID NO: 11 (HiBiT), a NL polypeptide of SEQ ID NO: 12 (LgTrip-3546), a NL peptide of SEQ ID NO: 13 (SmTrip), a NL peptide of SEQ ID NO: 14 (β9 / β10 dipeptide), or a variant thereof.

[0310] Liquid-phase detection platforms can also include a luminescent substrate, such as coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, JRW-0238, JRW-1404, JRW-1482, JRW-1667, JRW-1743, JRW-1744, other coelenterazine analogs or derivatives, precursor substrates, or other substrates described herein (e.g., coelenterazine analogs or derivatives). In some embodiments, the luminescent substrate is part of a composition comprising the luminescent substrate and a polymer selected from pullulan, trehalose, maltose, cellulose, dextran, polystyrene, poly(meth)acrylate, and any combination thereof. In some embodiments, the luminescent substrate is applied as part of a composition or solution, such as a protein buffer, a detection reagent, or with the sample. In some embodiments, the luminescent substrate is applied as a separate reagent that is part of an assay or system, and in other embodiments, it is part of a lyocake or tablet that contains one or more detection reagents.

[0311] 6.Target analyte Embodiments of the present disclosure find use in detecting / quantitating target analytes and include target analyte binding agents capable of binding to or interacting with target analytes via a target analyte binding element. In some embodiments, the target analyte binding agent comprises a target analyte binding element capable of binding to a group or class of analytes (e.g., protein L, which generally binds antibodies); such binding elements may be referred to herein as "non-specific" or the like. In other embodiments, the target analyte binding agent comprises a target analyte binding element capable of binding to a specific analyte (e.g., an antigen binding to a monoclonal antibody); such binding elements may be referred to herein as "target-specific" or the like.

[0312] In some embodiments, target analyte-binding agents and corresponding target analyte-binding elements are generated to detect one or more analytes associated with a disease state or environmental condition. The target analyte-binding elements can be independently selected from the group consisting of antibodies (e.g., polyclonal, monoclonal, and / or recombinant), antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, scFv, Fd, variable light chain, variable heavy chain, diabody, scFv, etc.), Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, oligonucleotide probes, peptide nucleic acids, DARPins, aptamers, affimers, purified proteins (e.g., either the analyte itself or a protein that binds to the analyte), and analyte-binding domain(s) of a protein.

[0313] In some embodiments, the target analyte binding element comprises an antigen or epitope recognized by an antibody (target analyte), e.g., an antibody produced by a subject in response to an immunogenic response to a pathogen and may indicate that the subject is infected with that pathogen. In some embodiments, the target analyte is an antibody to Zika virus, dengue virus, West Nile virus, yellow fever virus, and / or chikungunya virus, and the target analyte binding element is an immunogenic epitope specifically recognized by the antibody. In some embodiments, the target analyte is an antibody to hepatitis A, B, C, D, or E. In some embodiments, the target analyte is an antibody to mumps, measles, rubella, RSV, EBV, herpes, influenza, varicella-zoster, prenatal Zika, or parainfluenza types 1, 2, or 3. In some embodiments, the target analyte is an antibody to arbovirus, HIV, prenatal hepatitis, CMV, hantavirus, poliovirus, or parvovirus. In some embodiments, the target analyte is an antibody against a tick-borne disease (e.g., Lyme disease). In some embodiments, the target analyte is an antibody against Bordetella pertussis, Streptococcus pneumoniae, Chlamydia, Streptococcus, M. pneumoniae, S. pneumoniae, Shigella-producing bacteria, E. coli, Enterobacter, syphilis, or gonorrhea. In some embodiments, the target analyte is an autoantibody against ANA, cardiolipin, celiac disease, insulin, GAD65, IA-2, reticulin, thyroglobulin, RNP, neutrophil cytoplasm, thyrotropin receptor, thyroid peroxidase, platelet antibodies, PLAR2, myocardium, GBM, tissue transglutaminase, or thyroid-stimulating antibodies. In some embodiments, the target analyte is a toxin or an antibody against a toxin (e.g., diphtheria, tetanus). In some embodiments, the target analyte is derived from a parasite or is an antibody against a parasite (eg, Trichinella spiralis, Trichinellosis, Trypanosoma cruzi, Toxoplasma gondii).In some embodiments, the target analyte is a therapeutic biologic or an antibody to a therapeutic biologic (e.g., vedolizumab, adalimumab, infliximab, certilizumab, entanercept, Opdivo, Keytruda, ipilimumab, ustekinumab, secukinumab, guselkumab, tocilizumab, rituximab, panitumumab, trastuzumab, cetuximab, ofatumumab, eptratuzumab, abatacept, tofacitinib).

[0314] Other target analytes include known biomarkers associated with pathogenic organisms such as viruses, bacteria, protozoa, prions, fungi, parasitic nematodes, or other microorganisms. Disease biomarkers can include markers of the pathogenic organism itself and / or markers of a subject's response to infection by the pathogenic organism. Diseases that can be detected using the assays and methods of the present disclosure include any of the following: Acinetobacter infection (Acinetobacter baumannii), actinomycosis (Actinomyces sraelii, Actinomyces gerencseriae, and Propionibacterium propionicus), African sleeping sickness or African trypanosomiasis (Trypanosoma brucei), AIDS (HIV), amebiasis (Entamoeba histolytica), anaplasmosis (Anaplasma species), Angiostrongylus, Anisakiasis, Anthrax (Bacillus anthracis), Arcanobacterium haemolyticum infection (Arcanobacterium haemolyticum), Argentine Teaguan fever (Junin virus), Ascariasis (Ascaris lumbricoides, aspergillosis (Aspergillus species), astrovirus infections (family Astroviridae), babesiosis (Babesia species), cereus infection (Bacillus cereus), bacterial pneumonia (multiple bacteria), bacteroides infections (Bacteroides species), balantidiosis (Balantidium coli), bartonellosis (Bartonella), Baylissascaris infection (Baylisascaris species), BK virus infection (BK virus), black sand cricket (Piedraia hortae), blastocystosis (Blastocystis species), blastomycosis (Blastomyces dermatitidis), Bolivian hemorrhagic fever (Machupo virus), Brazilian hemorrhagic fever (Sabia virus), brucellosis (Brucella species), bubonic plague (Yersinia pestis), Burkholderia infections (usually Burkholderiacepacia and other Burkholderia species), Buruli ulcer (Mycobacterium ulcerans), calicivirus infections (family Caliciviridae), campylobacteriosis (Campylobacter species), candidiasis (usually Candida albicans and other Candida species), Carrion's disease (Bartonella bacilliformis), cat scratch disease (Bartonella henselae), cellulitis (usually group A Streptococcus and Staphylococcus), Chagas' disease (Trypanosoma cruzi), chancroid (Haemophilus ducreyi), chickenpox (varicella-zoster virus or VZV), chikungunya (alphavirus), chlamydiosis (Chlamydia trachomatis), cholera (Vibrio cholerae), chromoblastomycosis (usually Fonsecaea pedrosoi), chytridiomycosis (Batrachochytrium dendrabatidis), clonorchiasis (Clonorchis sinensis), Clostridium difficile enteritis (Clostridium difficile), coccidioidomycosis (Coccidioides immitis and Coccidioides posadasii), Colorado tick fever (Colorado tick fever virus or CTFV), common cold (usually rhinoviruses and coronaviruses), Creutzfeldt-Jakob disease (PRNP), Crimean-Congo hemorrhagic fever (Crimean-Congo hemorrhagic fever virus), cryptococcosis (Cryptococcus neoformans), cryptosporidiosis (Cryptosporidium species), cutaneous larva migrans (usually Ancylostoma braziliense; several other parasites), cyclosporiasis (Cyclospora cayetanensis), cytocysticercosis (Taenia solium), cytomegalovirus infection (cytomegalovirus), dengue fever (dengue virus: DEN-1, DEN-2, DEN-3 and DEN-4), desmodesmus infection (Green algae Desmodesmus armatus), diamoebiasis (Dientamoebafragilis, diphtheria (Corynebacterium diphtheriae), diphyllobothriasis (Diphyllobothrium), dracunculus worm disease (Dracunculus medinensis), Ebola hemorrhagic fever (Ebola virus or EBOV), echinococcosis (Echinococcus species), ehrlichiosis (Ehrlichia species), pinworm infection (Enterobius vermicularis), enterococcal infection (Enterococcus species), enterovirus infection (Enterovirus species), epidemic typhus (Rickettsia prowazekii), erythema infectiosum (parvovirus B19), exanthema subitum (human herpesvirus 6 or HHV-6; human herpesvirus 7 or HHV-7), fascioliasis (Fasciola hepatica and Fasciola gigantica), fascioliasis (Fasciolopsis buski), fatal familial insomnia (PRNP), filariasis (superfamily Filarioidea), Fusobacterial infection (Fusobacterium species), gas gangrene (usually Clostridium perfringens; other Clostridium species), geotrichum (Geotrichum candidum), Gerstmann-Sträussler-Scheinker syndrome (PRNP), giardiasis (Giardia lamblia), glanders (Burkholderia mallei), gnathostomiasis (Gnathostoma spinigerum and Gnathostoma hispidum), gonorrhea (Neisseria gonorrhoeae), granuloma venereum (Klebsiella granulomatis), group A streptococcal infection (Streptococcus pyogenes), group B streptococcal infection (Streptococcus agalactiae), Haemophilus influenzae infection (Haemophilus influenza), hand, foot and mouth disease (enteroviruses, mainly Coxsackie A virus and enterovirus 71 or EV71), hantavirus pulmonary syndrome (Sin Nombre virus), heartland virus disease (Heartland virus), Helicobacter pylori infection (Helicobacter pylori), hemolytic uremic syndrome (Escherichia coli)O157:H7, O111, and O104:H4), hemorrhagic fever with renal syndrome (family Bunyaviridae), hepatitis A (hepatitis A virus), hepatitis B (hepatitis B virus), hepatitis C (hepatitis C virus), hepatitis D (hepatitis D virus), hepatitis E (hepatitis E virus), herpes simplex (herpes simplex viruses 1 and 2 (HSV-1 and HSV-2)), histoplasmosis (Histoplasma capsulatum), hookworm infection (Ancylostoma duodenale and Necator americanus), human bocavirus infection (human bocavirus or HBoV), human ehrlichiosis (Ehrlichia ewingii), human granulocytic anaplasmosis (Anaplasma phagocytophilum), human metapneumovirus infection (human metapneumovirus or hMPV), human monocytic ehrlichiosis (Ehrlichia chaffeensis), human papillomavirus (HPV) infection (human papillomavirus or HPV), human parainfluenza virus infection (human parainfluenza virus or HPIV), membrane-like tapeworm disease (Hymenolepis nana and Hymenolepis diminuta), infectious mononucleosis due to Epstein-Barr virus infection (Epstein-Barr virus or EBV), influenza (family Orthomyxoviridae), isosporosis (Isospora belli), Kingella kingae infection (Kingella kingae), kuru (PRNP), Lassa fever (Lassa virus), Legionnaires' disease (Legionella pneumophila), Legionellosis (Legionella pneumophila), leishmaniasis (Leishmania species), leprosy (Mycobacterium leprae and Mycobacterium lepromatosis), leptospirosis (Leptospira species), listeriosis (Listeria monocytogenes), Lyme disease (Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii), lymphatic filariasis (Wuchereria bancrofti and Brugiamalayi), lymphocytic choriomeningitis (lymphocytic choriomeningitis virus or LCMV), malaria (Plasmodium species), Marburg hemorrhagic fever (Marburg virus), measles (measles virus), Middle East respiratory syndrome (Middle East respiratory syndrome coronavirus), melioidosis (Burkholderia pseudomallei), meningococcal disease (Neisseria meningitidis), fluke disease (usually Metagonimus yokagawai), microsporidiosis (Microsporidia phylum), molluscum contagiosum (molluscum contagiosum virus or MCV), monkeypox (monkeypox virus), mumps (mumps virus), typhus (Rickettsia typhi), mycoplasma pneumonia (Mycoplasma pneumoniae), mycetoma (many bacterial (Actinomycetoma) and fungal (Eumycetoma) species), myiasis (parasitic dipteran fly larvae), neonatal conjunctivitis (most commonly Chlamydia trachomatis and Neisseria gonorrhoeae), norovirus (Norovirus), nocardiosis (usually Nocardia asteroides and other Nocardia species), onchocerciasis (Onchocerca volvulus), opisthorchiasis (Opisthorchis viverrini and Opisthorchis felineus), paracoccidioidomycosis (Paracoccidioides brasiliensis), paragonimiasis (usually Paragonimus westermani and other Paragonimus species), pasteurellosis (Pasteurella species), head lice (Pediculus humanus) capitis), body lice (Pediculus humanus corporis), pubic lice (Phthirus pubis), whooping cough (Bordetella pertussis), plague (Yersinia pestis), pneumococcal infection (Streptococcus pneumoniae), pneumocystis pneumonia (Pneumocystisjirovecii), pneumonia (multiple causes), poliomyelitis (poliovirus), Prevotella infection (Prevotella species), primary amebic meningoencephalitis (usually Naegleria fowleri), Progressive multifocal leukoencephalopathy (JC virus), psittacosis (Chlamydophila psittaci), Q fever (Coxiella burnetiid), rabies (Rabies virus), relapsing fever (Borrelia hermsii, Borrelia recurrentis, and other Borrelia species), respiratory syncytial virus infection (respiratory syncytial virus (RSV)), rhinosporidiosis (Rhinosporidium seeberi), rhinovirus infection (rhinovirus), rickettsial infection (Rickettsia species), rickettsialpox (Rickettsia akari), Rift Valley fever (Rift Valley fever virus), Rocky Mountain spotted fever (Rickettsia rickettsia), rotavirus infection (rotavirus), rubella (Rubella virus), salmonellosis (Salmonella species), severe acute respiratory syndrome (SARS coronavirus), scabies (Sarcoptes scabiei), scarlet fever (group A Streptococcus species), schistosomiasis (Schistosoma species), septicemia (multiple causes), shigellosis (Shigella species), shingles (varicella-zoster virus or VZV), smallpox (variola major or variola minor), sporothrix (Sporothrix schenckii), staphylococcal food poisoning (Staphylococcus species), staphylococcal infections (Staphylococcus species), strongyloidiasis (Strongyloides stercoralis), subacute sclerosing panencephalitis (measles virus), syphilis (Treponema pallidum), tapeworm disease (Taenia species), tetanus (Clostridium tetani), tinea barbae (usually Trichophyton species), tinea capitis (usually Trichophyton tonsurans), tinea corporis (usually Trichophyton species), tinea cruris (usually Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes), tinea manus (Trichophyton rubrum), tinea nigricans (usually Hortaeawerneckii), tinea pedis (usually Trichophyton species), tinea unguium (usually Trichophyton species), tinea versicolor (Malassezia species), toxocariasis (Toxocara canis or Toxocara cati), toxoplasmosis (Toxoplasma gondii), trachoma (Chlamydia trachomatis), trichinosis (Trichinella spiralis), trichomoniasis (Trichomonas vaginalis), whipworm (Trichuris trichiura), tuberculosis (usually Mycobacterium tuberculosis), tularemia (Francisella tularensis), typhoid fever (Salmonella enterica subsp. enterica, serovar typhi), typhus (Rickettsia), Ureaplasma urealyticum infection (Ureaplasma urealyticum), valley fever (Coccidioides immitis or Coccidioides posadasii), Venezuelan equine encephalitis (Venezuelan equine encephalitis virus), Venezuelan hemorrhagic fever (Guanarito virus), Vibrio vulnificus infection (Vibrio vulnificus), Vibrio parahaemolyticus enteritis (Vibrio parahaemolyticus), viral pneumonia (multiple viruses), West Nile fever (West Nile virus), Trichosporon beigelii, Yersinia pseudotuberculosis, Yersinia enterocolitica, yellow fever (Yellow fever virus), Zygomycosis (Mucorales order (Mucorrosis) and Entomophthorales order (Entomophthorrosis)), and Zika fever (Zika virus).

[0315] 7. Methods of Detection, Quantitation, and Diagnosis Embodiments of the present disclosure include methods for detecting and / or quantifying a target analyte in a sample using an assay platform (e.g., a solid-phase detection platform or a lateral flow assay) that uses a bioluminescent polypeptide or bioluminescent conjugate (and its components; e.g., a non-luminescent peptide or polypeptide) to detect the target analyte. Embodiments also include methods for diagnosing a disease state or assessing an environmental condition based on detecting and / or quantifying a target analyte in a sample.

[0316] In some embodiments, a method for detecting an analyte in a sample includes using a lateral flow assay system or solid-phase detection platform described herein. According to these embodiments, the method includes applying a sample to a sample pad and facilitating sample flow from the sample pad to a conjugate pad and then from the conjugate pad to a detection zone and a control zone on an analytical membrane. The method may include a first target analyte binding agent, a second target analyte binding agent, and a target analyte, which form an analyte detection complex in at least one detection zone when the target analyte is detected in the sample. In some embodiments, the method includes one or more steps of sample addition, reagent (e.g., detection reagent) addition, washing, waiting, etc.

[0317] In some embodiments, the sample is a biological sample from a subject, such as blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, and saliva. In other embodiments, the sample is a sample from a natural or industrial environment, such as a water sample, soil sample, plant sample, food sample, beverage sample, oil, and industrial fluid sample. The method includes detecting a target analyte in the sample by detecting a bioluminescent signal generated from the analyte detection complex. In some embodiments, the target analyte in the sample is quantified based on the bioluminescent signal generated from the analyte detection complex. In some embodiments, the method includes diagnosing the subject from whom the sample was obtained as having or not having a disease based on the detection of the analyte.

[0318] 8. Competition Some embodiments herein utilize competition between a labeled analyte and a target analyte in a sample to detect / quantitate the target analyte in a sample. Exemplary embodiments include the use of (i) an analyte (e.g., identical to or similar to the target analyte) labeled with a detectable element described herein (e.g., NanoLuc®-based technology (e.g., NanoLuc, NanoBiT, NanoTrip, NanoBRET, or a component (e.g., peptide, polypeptide, etc.) or variant thereof)) and (ii) a binding moiety for the target analyte (e.g., fused or linked to a second detectable element described herein (e.g., NanoLuc®-based technology (e.g., NanoLuc, NanoBiT, NanoTrip, NanoBRET, or a component (e.g., peptide, polypeptide, etc.) or variant thereof)). In the absence of target analyte in the sample, the detectable element generates a detectable signal (e.g., via complementarity between the detectable elements, BRET, etc.) and is generated by the system. When the system is exposed to a sample (e.g., a biological sample, an environmental sample, etc.), the bioluminescent signal is reduced (the labeled analyte dissociates from the complex) in the presence of the target analyte in the sample.

[0319] Various embodiments herein utilize such competitive immunoassays to detect small molecules. In some embodiments, the target small molecule is a toxin (e.g., mycotoxin, etc.), a metabolite (e.g., amino acid, glucose molecule, fatty acid, nucleotide, cholesterol, steroid, etc.), a vitamin (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin H, or vitamin K, etc.), a coenzyme or cofactor (e.g., coenzyme A, coenzyme B, coenzyme M, coenzyme Q, cytidine triphosphate, acetyl coenzyme A, reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine (NAD+), nucleotide adenosine monophosphate, nucleotide adenosine triphosphate, glutathione, heme, lipoamide, molybdopterin, 3'-phosphoadenosine-5'-phosphosulfate, pyrroloquinoline quinone, tetrahydrobiopterin, etc.), a biomarker, or antigens (e.g., erythropoietin (EPO), ferritin, folate, hemoglobin, alkaline phosphatase, transferrin, apolipoprotein E, CK, CKMB, parathyroid hormone, insulin, cholesteryl ester transfer protein (CETP), cytokines, cytochrome c, apolipoprotein AI, apolipoprotein AII, apolipoprotein BI, apolipoprotein B-100, apolipoprotein B48, apolipoprotein CII, apolipoprotein CIII, apolipoprotein E, triglycerides, HD cholesterol, LDL cholesterol, lecithin cholesterol acyltransferase, paraoxonase, alanine aminotransferase (ALT), aspartate transferase (AST), CEA, HER-2, bladder tumor antigen, thyroglobulin, alpha-fetoprotein, PSA, CA125, CA19.9, CA15).3. leptin, prolactin, osteopontin, CD98, fascin, troponin I, CD20, HER2, CD33, EGFR, VEGFA, etc.), drugs (cannabinoids (e.g., tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN)), opioids (e.g., heroin, opium, fentanyl, etc.), stimulants (e.g., cocaine, amphetamine, methamphetamine, etc.), club drugs (e.g., MDMA, flunitrazepam, gamma-hydroxybutyric acid, etc.), dissociative drugs (e.g., ketamine, phencyclidine, These include drugs such as salvia and dextromethorphan, hallucinogens (e.g., LSD, mescaline, and psilocybin), explosives (e.g., 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and pentaerythritol tetranitrate (PETN)), and toxic chemicals (e.g., tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), 2-(dimethylamino)ethyl N,N-dimethylphosphoramidofluoridate (GV), VE, VG, VM, VP, VR, VS, or VX nerve gases).

[0320] In some embodiments, small molecule detection immunoassays, such as those exemplified in Example 5, are performed in solid phase, lateral flow, and other assays and devices described herein. [Example]

[0321] 9. Working Example It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily adaptable and obvious and can be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having thus described the present disclosure in detail, the present disclosure will be more clearly understood by reference to the following examples, which are intended merely to illustrate certain aspects and embodiments of the present disclosure and should not be construed as limiting the scope of the present disclosure. The disclosures of all journal articles, U.S. patents, and publications mentioned herein are hereby incorporated by reference in their entirety.

[0322] The present disclosure has multiple aspects, exemplified by the following non-limiting examples.

[0323] Example 1 solid phase material As shown in Figure 3, components of the bioluminescent complexes of the present disclosure generate detectable bioluminescence after application to a solid support substrate (e.g., a membrane). Antibodies labeled with NanoLuc® components (e.g., target analyte binders) were applied to blocked (Buffer 1; top two membranes in the left and right panels) or unblocked (Buffer 2; bottom two membranes in the left and right panels) membranes, which were then dried at room temperature with nitrogen or at 37°C without nitrogen. Using an Imagequant LAS4000 imaging platform (1-second exposure), detectable bioluminescence was generated under these conditions. These results demonstrate that components of the bioluminescent complexes of the present disclosure can be successfully used in solid-phase and lateral flow assay platforms, which may involve drying and application of reagents to solid-phase materials and exposure to various temperature and processing conditions.

[0324] As shown in Figure 4, the components of the bioluminescent complex generate detectable bioluminescence after application to membrane- and paper-based solid support matrices. A composition containing a buffer, a substrate (e.g., furimazine), and two complementary components of the bioluminescent complex (e.g., HiBiT and LgBiT) was applied to a nitrocellulose membrane (the three left panels) or filter paper (Whatman 541, shown in the middle three panels; Whatman 903, shown in the right three panels). These components were then dried and shipped at 4°C. After 24 hours, they were examined using the LAS4000 imaging platform (30-second and 5-minute exposures). Under these conditions, detectable bioluminescence was generated, with the filter paper matrix producing a brighter bioluminescent signal than the nitrocellulose membrane. Detectable bioluminescent signals were also obtained with matrices made of glass fiber and synthetic fibers (e.g., Ahlstrom grade 8950) (data not shown), demonstrating the compatibility of the disclosed bioluminescent complex components with a variety of matrix materials.

[0325] Example 2 Detection of target analytes by bioluminescent conjugates As shown in Figure 5, components of the bioluminescent complexes of the present disclosure (e.g., non-luminescent peptides and polypeptides) can be used as target analyte binding agents to recognize target analytes. For example, as shown in Figure 5 (left panel), a polyclonal goat anti-mouse IgG3 antibody (e.g., a target analyte-binding element) was conjugated to components of the bioluminescent complex (e.g., LgBiT and SmBiT). In the presence of a target analyte (e.g., mouse IgG3), a bioluminescent complex was formed, and a bioluminescent signal was generated from the complementary interaction of the components of the bioluminescent complex (Figure 5 right panel), with the signal increasing as the concentration of the target analyte increased. These results demonstrate the feasibility of detecting target analytes using components of the bioluminescent complexes of the present disclosure.

[0326] As shown in Figure 6, an embodiment of the present disclosure includes a solid-phase assay platform that uses components of a bioluminescent complex as target analyte-binding agents to recognize target analytes. Four test spots were prepared on Whatman 903 filter paper as shown, followed by the addition of target analytes (Figure 6, top panel). In one embodiment, 20 ng of goat anti-mouse conjugated to a component of a bioluminescent complex (e.g., SmBiT) and 20 ng of goat anti-mouse conjugated to a complementary component of the bioluminescent complex (e.g., LgBiT) were each prepared in 5 μl of protein buffer (20 mM Na3PO4; 5% w / v BSA; 0.25% v / v Tween 20; 10% w / v sucrose) and dried at 37°C for 1 hour at the indicated locations on the paper. Additionally, 5 μl of a 5 mM furimazine solution in ethanol was applied to the designated spots under high vacuum for 15 minutes (Figure 6, top panel). The prepared spots were then stored at 4°C for one week. As shown, in the presence of a target analyte (e.g., mouse IgG3; spot #2), a bioluminescent complex was formed, and a bioluminescent signal was generated from the complementary interaction of the components of the bioluminescent complex (Figure 6, bottom panel). While a background bioluminescent signal was generated in the absence of the target analyte (spot #4), the signal generated in the presence of the target analyte and a luminescent substrate (e.g., furimazine) was substantially increased compared to the signal generated by the luminescent substrate alone (compare spots #2 and #4).

[0327] Additional tests were performed on the stability of the substrate and protein, as shown in Figures 7A-7E. These tests were performed as described above, with the additional step of adding a fully functional bioluminescent conjugate (e.g., NanoLuc) after the addition of the target analyte to test the stability of the luminescent substrate. As shown in Figures 7A-7C, the components of the bioluminescent conjugate lose activity when stored at higher temperatures (e.g., 37°C) for 2 weeks. The loss of bioluminescent signal does not appear to be due to instability or destruction of the luminescent substrate, as a signal is still generated upon addition of a fully functional bioluminescent conjugate (e.g., NanoLuc) (Figure 7D). Furthermore, to test whether destruction of one or more components of the bioluminescent conjugate is responsible for the decrease in bioluminescent signal, a non-antibody conjugate component (e.g., HiBiT), which is not affected by storage conditions, was added. As shown in Figure 7E, addition of the non-antibody conjugate component generates a bioluminescent signal at 4°C but not at 37°C, indicating that degradation of the complementary component of the bioluminescent complex (e.g., LgBiT) likely leads to loss of signal.

[0328] Additional storage condition tests were performed and are shown in Figures 8A-8B. These tests were performed as described above, except that the test spots were stored for a total of 3 months. As shown in Figure 8A, although there was some loss of activity, a detectable bioluminescent signal was still produced in the presence of target analyte at both 4°C and 25°C after 3 months of storage. Addition of a fully functional bioluminescent conjugate (e.g., NanoLuc) produced a signal (Figure 8B), although the signal appeared to be dependent on the use of a protein buffer (compare spots #1 and #2), suggesting that the protein buffer stabilizes the luminescent substrate.

[0329] Example 3 Detection of target analytes in complex sampling environments Figures 9A-9C are representative images of a solid-phase assay platform (e.g., spot test) testing the feasibility of bioluminescent complex formation and analyte detection in complex sampling environments. As shown in Figure 9A, a luminescent substrate and two complementary components of a bioluminescent complex (HiBiT and LgBiT) were applied to Whatman 903 filter paper. Each component also contained a target analyte binding element (polyclonal anti-mouse IgM) as described above and was stored at 4°C for 6 weeks. A whole blood sample collected in EDTA (Figure 9B) and a 100% serum sample (Figure 9C) were each spiked with 10 pg of mouse IgG3 (target analyte) and applied to the spot shown in Figure 9A. Corresponding control samples were not spiked with mouse IgG3. These results demonstrate the feasibility of detecting target analytes using the components of the bioluminescent complexes of the present disclosure in complex sampling environments.

[0330] Example 4 Qualitative and quantitative evaluation Figures 10A-10B contain representative results of the solid-phase assay, demonstrating that bioluminescent signals can be assessed both quantitatively (Figure 10A) and qualitatively (Figure 10B). As shown in Figure 10A, 10 μM of luminescent substrate (e.g., furimazine) was applied to filter paper and placed in a microtiter plate. PBS assay buffer containing NanoLuc® enzyme was then added, and bioluminescent signals were assessed quantitatively (Figure 10A, right panel) and qualitatively (Figure 10B). Furthermore, bioluminescent signals were effectively assessed using a luminometer (Figure 10B, left panel) and a smartphone (Figure 10B, right panel).

[0331] These results demonstrate that the assays and methods of the present disclosure can include comparing the level of bioluminescence corresponding to detection of a target analyte with various control samples, facilitating rapid quantitative and qualitative assessment. For example, the assay format can include multiple control samples containing various concentrations of the target analyte, which can serve as standards against which test samples can be evaluated.

[0332] These methods allow for both quantitative and qualitative assessment of bioluminescent signals using high-affinity dipeptides capable of forming bioluminescent complexes with LgBiT or LgTrip. The results shown in Figures 11A-11B are representative graphs (Figure 11A: RLU; Figure 11B: S / B) demonstrating the ability of the high-affinity dipeptide pep263 to form bioluminescent complexes with LgBiT and LgTrip. The high-affinity dipeptide pep263 comprises the β9 and β10 strands of the NanoTrip complex. (See, e.g., U.S. Patent Application No. 16 / 439,565 (PCT / US2019 / 036844) and U.S. Provisional Application No. 62 / 941,255, both of which are incorporated herein by reference in their entireties.)

[0333] Additionally, Figure 12 shows representative results of a solid-phase assay in which bioluminescence from paper punches placed on a standard microtiter plate was qualitatively assessed using a standard camera on an iPhone or an imager (e.g., LAS4000). This spot test assay assessed the functional stability of different LgBiT components dried onto Whatman 903 paper. Whatman 903 Protein Saver Spot Cards (1 / 8-inch punch holes) were used with the following protein buffer: 20 mM NaPO, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose. A 1000x NanoLuc® stock solution was diluted 1:1000 in protein buffer. Approximately 5 μL of this reaction solution was applied to spot 1. For HT-LgBiT complexes, approximately 5 μL of 106.8 nM protein was used per spot. Approximately 20 μM stock protein was diluted 1:100 in protein buffer. Approximately 534 μL of the stock was diluted to 466 μL with protein buffer. Approximately 5 μL of this conjugation solution was added to spot 2. For the LgTrip(2098) conjugate, approximately 5 μL of 106.8 nM protein was used per spot. Approximately 9.6 μM protein stock was obtained by diluting approximately 11.6 μL of stock with 988 μL of protein buffer to 1 mL of 106.8 nM solution. Approximately 5 μL of this conjugation solution was added to spot 3. For the LgTrip(3546) conjugate, approximately 5 μL of 106.8 nM protein was obtained per spot. Approximately 94 μM protein stock was obtained by diluting approximately 1.13 μL of LgTrip stock into 998.87 μL of protein buffer. Approximately 5 μL of this conjugation solution was added to spot 4. After all the proteins were added, the samples were dried at 30°C for 1 hour, 4°C, 25°C, and 37°C.

[0334] The method for assessing RLU activity in these experiments included imaging at 25°C and 37°C on day 6 (after 1 or 2 days at 4°C) for all, at 4°C on day 8 for LgTrip 3546, and at day 9 for NanoLuc, LgBiT, and LgTrip 2098. Furimazine was tested at 50 μM, and approximately 1.2 μM of the dipeptide was used for NanoBiT and NanoTrip experiments. All spots were placed on a plate containing substrate reagent, images were taken with an iPhone and a LAS4000 imaging system, and then inserted into a plate reader. NanoGlo Live Cell Substrate catalog number N205B (lot 189096) was used with assay buffer (1x PBS, pH 7.0).

[0335] Figures 13A-13B show quantitative analyses of the same solid-phase assay shown in Figure 12, but luminescence was detected using a luminometer on day 3 at 25°C (Figure 13A: RLU; Figure 13B: S / B). These quantitative data support the qualitative data in Figure 12. The materials and methods used for Figure 12 were the same as those used for Figures 13A-13B (e.g., adding 1 μM dipeptide + 50 μM live cell substrate in PBS, pH 7.0, and reading on a luminometer). In some cases, high background of LgBiT can reduce the S / B ratio.

[0336] Figures 14A-14C show the quantitative time course of the same solid-phase assay shown in Figures 12-13, demonstrating the stability of all proteins in this time frame under all experimental conditions at all temperatures tested. max RLU values ​​are shown over time (0-60 days). These quantitative data are consistent with Figures 12 and 13, demonstrating stability for all conjugates tested and at all temperatures tested over this time frame. The materials and methods used for Figure 12 were the same as those used for Figures 14A-14C.

[0337] Example 5 Buffer composition Experiments were also conducted to test the short-term or accelerated stability of the complexes over a 0-90 min period in different buffer compositions. The method involved using a concentration of approximately 1.068 nM of each protein, absorbed onto a Whatman 903 paper spot (1 / 8 inch), and dried. Protein samples were prepared and dried onto the paper spot in either protein buffer or PBS buffer (see individual figures for the specific buffer compositions used). Stock concentrations were 1000x (0.4 mg / mL) for NanoLuc, 20 μM for LgBiT-1672-11s-His, and 94 μM for LgTrip(3546). The protein buffer consisted of 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, and 10% w / v sucrose. Luminescence activity was tested using the dipeptide spiked with furimazine (final [dipeptide] = 1 nM; final [furimazine] = 50 μM) in 100 μl of assay buffer PBS (pH 7.0). Samples were read at time 0 (freshly removed from 4°C) and then placed at 60°C and 25°C for continued testing. A 1000x stock solution of NanoLuc was diluted 1:1000 in protein buffer (1 mL), or 10 μL of stock was diluted in 990 μL of protein buffer to yield a 1.068 nM stock (see individual figures for specific buffer compositions used). Approximately 5 μL of each concentration was added to paper spots for testing. For each protein tested (LgBiT and LgTrip), appropriate dilutions were made in each buffer to ensure approximately 5 μL of 1.068 nM protein was used per spot. After all proteins were added, the samples were dried at 35°C for 1 hour, and 40 spots were prepared for each condition and temperature.

[0338] Figures 15A-15D show representative results collected on day 0 of accelerated stability studies conducted under two buffer conditions at 25°C and 60°C (Figures 15A and 15C use protein buffer, while Figures 15B and 15D use PBS). These data indicate that the tested conjugates are less tolerant to PBS than protein buffer as a buffer condition for incorporation into Whatman 903 paper. Buffer conditions appear to affect stability even at early time points. In some cases, LgTrip 3546 exhibited superior activity, suggesting somewhat greater chemical stability than NanoLuc and LgBiT under these conditions.

[0339] Figures 16A-16B show the results of the accelerated stability study shown in Figure 15, but over a time course of 0 to 50 days. Figure 16A shows the results for samples tested in protein buffer at 25°C, and Figure 16B shows the results for samples tested in protein buffer at 60°C. The same materials and methods were used as in Figure 15. These results indicate that the conjugate is stable at these conditions (25°C and 60°C) for up to at least 50 days.

[0340] Figure 17 compares the effect of buffer conditions on luminescence from NanoLuc® dried onto a nitrocellulose membrane to assess its stability in a lateral flow assay. Four different conditions were tested: Condition 1: PBS (pH 7.4) containing mouse anti-Hum+IgG-Nluc; Condition 2: PBS (pH 7.4) containing IgG-Nluc; Condition 3: Loading buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) containing mouse anti-Hum+IgG-Nluc; and Condition 4: Loading buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose) containing IgG-Nluc. Each condition was applied to the membrane and allowed to dry at either room temperature or 37°C.

[0341] For these experiments, the following solutions were prepared: (1) 5 μl of mouse / anti-human in 995 μl loading buffer (0.1 M PBS, pH 7.4); (2) 5 μl of anti-mouse-NanoLuc in 995 μl loading buffer (0.1 M PBS, pH 7.4); (3) 5 μl of mouse / anti-human in protein buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose); and (4) 5 μl of anti-mouse-NanoLuc in 995 μl protein buffer (20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose). Approximately 0.5 ml of solution (1) was loaded onto an airbrush and applied to the left side of the nitrocellulose strips (strips 1 and 2). The strips were allowed to dry at room temperature or 37°C for 1 hour. Approximately 0.5 ml of solution (2) was applied to the surface of either Strip 1 or Strip 2 and allowed to dry at room temperature or 37°C, forming conditions 1 and 2, respectively. Approximately 0.5 ml of solution (3) was loaded into an airbrush and applied to the left side of the nitrocellulose strips (Strips 3 and 4). The strips were allowed to dry at room temperature or 37°C for 1 hour. Approximately 0.5 ml of solution (4) was applied to the surface of either Strip 3 or Strip 4 and allowed to dry at room temperature or 37°C, forming conditions 3 and 4, respectively. For imaging, a 1x solution of substrate was prepared (4 ml PBS + 1 ml Nano-Glo LCS Dilution Buffer + 50 μl Nano-Glo Live Cell Substrate), and each strip was overlaid with 1 ml of substrate solution. Imaging was then initiated immediately thereafter.

[0342] These data demonstrate that the buffer formulation is critical for activity on lateral flow membranes. In conditions 1–4, where the protein was simply applied to the membrane in PBS, very little light was observed when the membrane was exposed to freshly prepared Nano-Glo Live Cell Substrate. In contrast, proteins prepared in loading buffers containing additional components such as Na3PO4, BSA, Tween 20, and sucrose exhibited significant luminescence output. This demonstrates that the specific loading buffer used to add the protein to the membrane surface is important for stability and functionality (Figure 17).

[0343] Example 6 Lateral Flow Assay Components Experiments were conducted to test different membrane blocking agents and assay running buffers to promote proper migration of proteins and targets during lateral flow assays. Four strips were used, and each design (with or without sucrose and blocking agent) is shown as a schematic diagram below the left-most image in Figure 18. Briefly, strip 1 contained a blocked membrane with sucrose pretreatment on a conjugation pad, strip 2 contained a blocked membrane without sucrose pretreatment on a conjugation pad, strip 3 contained an unblocked membrane with sucrose pretreatment on a conjugation pad, and strip 4 contained an unblocked membrane without sucrose pretreatment on a conjugation pad.

[0344] The blocking buffer consisted of 20 mM Tris (pH 7.4) containing 1% w / v polyvinyl alcohol. The conjugation pretreatment contained 30% w / v sucrose in DI water. The conjugation pad was made of Ahlstrom Grade 8950 (chopped glass with binder, 50 g / m). 2) and the membrane was nitrocellulose. To block, the membrane was immersed in blocking buffer at room temperature for 30 minutes, then removed from the buffer, washed with DI water, and dried at 35°C for 30 minutes. As a secondary pretreatment, a sucrose solution was applied to the membrane pad near the location where the conjugation reagent (substrate) would be applied. The membrane was dried at 35°C for 1 hour. To prepare the protein, approximately 5 μL of anti-mouse-NanoLuc was added to 995 μL of protein buffer. Approximately 1 ml of the protein solution was placed in an airbrush and thinly coated onto the conjugation pad. This was allowed to dry at 35°C for 1 hour. The strip was then attached to a mounting board. Furthermore, for Figures 18-20, the following buffer compositions were tested: Buffer 1 consisted of 20X SSC, 1% BSA (pH 7.0) + 10 μM LCS (Figure 18). Buffer 2 consisted of 0.01M PBS, 1% BSA (pH 7.0) + 10 μM PCS (Figure 19). Buffer 3 consisted of 5x LCS dilution buffer + 5x LCS diluted to 1x with PBS (Figure 20).

[0345] Figure 18 shows the effect of membrane blocking and sucrose pretreatment on a lateral flow assay performed with a running buffer of 20X SSC, 1% BSA (pH 7.0) + 10 μM LCS. Figure 19 shows the effect of membrane blocking and sucrose pretreatment on a lateral flow assay performed with a running buffer of 0.01 M PBS, 1% BSA (pH 7.0) + 10 μM Permeable Cell Substrate (PCS). Figure 20 shows the effect of membrane blocking and sucrose pretreatment on a lateral flow assay performed with a running buffer of 5X LCS dilution buffer + 5X LCS diluted to 1X with PBS. These data demonstrate that membrane treatment and protein buffer affect the flow of assay fluid through the conjugation pad and across the lateral flow membrane.

[0346] Experiments were also conducted to evaluate different membranes and membrane properties in the context of a lateral flow assay, such as the effect of membrane properties on absorption and capillarity. Figure 21 shows the effect of membrane properties on bioluminescent reagent absorption and capillarity in a lateral flow assay. Membranes with different pore sizes were tested for flow efficiency. Each membrane was pretreated with 30% w / v sucrose in approximately the bottom third of the strip, without blocking. Other materials included conjugation pads (Ahlstrom grade 8950, chopped glass with binder, 50 g / m). 2 ); sample pad (cellulose glass fiber CFSP203000 (Millipore)); and absorbent pad (cotton linter, grade 238 (Ahlstrom)). The following membrane conditions were tested: 1. Nitrocellulose FF170HP (Ahlstrom) 2. Nitrocellulose Hi-Flow Plus HFC18002 (Millipore) - 180 seconds / 4 cm 3. Nitrocellulose Hi-Flow Plus HFC13502 (Millipore) - 135 seconds / 4 cm 4. Nitrocellulose Hi-Flow Plus HFC09002 (Millipore) - 90 seconds / 4 cm 5. Nitrocellulose Hi-Flow Plus HFC12002 (Millipore) - 120 seconds / 4 cm 6. Nitrocellulose Hi-Flow Plus HFC07502 (Millipore) - 75 seconds / 4 cm 7. Nitrocellulose FF170HP (Ahlstrom) - negative control.

[0347] The running buffer consisted of 5x LCS dilution buffer plus 5x LCS diluted to 1x with PBS. The membrane was pretreated by applying a 30% sucrose solution to the membrane, covering approximately 1.5 cm of the bottom of the strip, and drying at 35°C for 1 hour. The protein was prepared by adding approximately 5 μL of anti-mouse NanoLuc to 995 μL of protein buffer. Approximately 1 mL of the protein solution was added to an airbrush and used to lightly coat the conjugation pad. This was allowed to dry at 35°C for 1 hour. The negative control for these experiments contained protein buffer without protein and was applied with an airbrush in the same manner as the test conditions. The strip was attached to a cardboard mount. The conjugation pad, sample pad, and wick pad were cut to 2 cm x 1 cm. The sample pad and conjugation pad overlapped by approximately 1.8 cm. The overall dimensions of the strip were approximately 6 cm x 1 cm.

[0348] The imaging program was configured to take 5-second exposure images every 30 seconds for a total of approximately 10 minutes. If NanoLuc still appeared to be flowing through the membrane, imaging was repeated. Images were overlaid into a movie using ImageJ. The final image included in Figure 21 is the cumulative signal of all images taken over time.

[0349] These results show that, based on the conditions used in these experiments, strips 4 and 6 (boxed in Figure 21) resulted in the most complete transfer of NanoLuc from the conjugation pad to the sample reservoir.

[0350] Example 7 Formation of bioluminescent complexes Experiments were performed to evaluate bioluminescent complex formation on membranes and filter paper in the presence of various reagents. The experiments were designed and performed according to the following schematic diagram, which shows four different test conditions. JPEG2025133108000006.jpg87153

[0351] For these experiments, 2.5 μL of HaloTag-HiBiT was added to 498 μL of protein buffer. Approximately 5 μL of this solution was spotted onto quadrants 1, 3, and 4 (see schematic diagram above) of both the membrane and the filter paper and allowed to dry at 37°C for 1 hour. Approximately 2.5 μL of ATG-1672-11S-6His was diluted with 498 μL of protein buffer, and approximately 5 μL was spotted directly onto quadrants 2, 3, and 4 (see schematic diagram above) of the nitrocellulose membrane and the filter paper. The membrane was allowed to dry at room temperature for 1 hour. Furimazine was prepared as a 5 mM stock solution in EtOH. Approximately 5 μL of this solution was spotted onto quadrants 1, 2, and 3 of both the membrane and the filter paper and immediately placed under high vacuum for 15 minutes. Approximately 2.5 μL of stock protein (20 μM) was diluted into 498 μL of NanoGLO buffer without substrate. Approximately 5 μL was added to the four quadrants described above and then read on a luminometer.

[0352] Figures 22A-22B show the bioluminescent signal from NanoBiT / HiBiT complements on nitrocellulose (left) and Whatman grade 541 (right) paper (Figure 22A) and corresponding edited images from a movie taken over the entire exposure time (movie available upon request). Images were taken over increasing exposure times (1 s, 3 s, 10 s, 30 s, 1 min, 2 min, 3 min, 4 min, 5 min, 10 min) starting at 1 s and ending with a 10 min exposure after addition of the reagents indicated in 26, for a total time (26 min).

[0353] These results suggest that filter paper may provide an increased signal compared to membranes. Also, quadrant 4 conditions did not produce detectable luminescence, which may indicate that complex formation was prevented by one or more of the other reagents present.

[0354] Experiments were conducted to assess the effect of increasing substrate concentration on complex formation. Figure 23 shows the bioluminescence signal from NanoBiT / HiBiT complementation on Whatman Grade 903 paper, spiked with additional substrate and liquid at 20 minutes. Figure 23 shows a representative edited image from the corresponding movie taken over the entire exposure time (movie available upon request). Approximately 2.5 μl of purified LgBiT or HiBiT was diluted in 498 μl 1X LCS buffer and added directly to the filter paper (conditions consistent with quadrant 1) in a volume of 10 μL (2:1 LgBiT to HiBiT ratio). The original substrate was NanoBRET NanoGlo (5 μl added at 5 mM), and the additional immersion substrate was NanoBRET NanoGlo (5 mM stock) diluted 1:5 with 1X NanoGlo buffer diluted 1X with PBS. Approximately 500 μl was added to cover the filter paper. Images were taken at all times with repeated 30 second exposures.

[0355] Spiking with an excess volume of additional substrate (furimazine) showed that the signal returned, suggesting that as the components begin to move in the additional fluid, more complexes can form due to increased mobility. This experiment also shows that the enzyme remains active, and that the concentration of substrate is a limiting factor, but can be improved by adding excess substrate.

[0356] Figure 24 shows the bioluminescence signal from NanoBiT / HiBiT complement on Whatman 903 paper, but not on Whatman 541 paper, under experimental conditions consistent with the schematic above (quadrant 1-4 in Figure 22). Near the end of the experiment, buffer was added to rehydrate the membrane. Figure 24 shows a representative edited image from the corresponding movie taken over the entire exposure time (movie available upon request). Quadrant 2 appears to yield the strongest luminescence signal.

[0357] Example 8 Spot test using LgTrip and substrate Experiments were first conducted to assess the feasibility of "all-in-one" spots by testing a paper matrix containing LgTrip 3546 and furimazine, to which an analyte of interest (e.g., a dipeptide) could be added. Figures 25A-25C show the bioluminescence signal on Whatman 903 paper obtained from reconstituting LgTrip 3546 and the substrate with a dipeptide in the presence (Figure 25B) and absence (Figure 25A) of BSA, along with a serial dilution of the dipeptide with BSA (Figure 25C). Two sets of spots were generated. Each spot consisted of the following components: 1) 5 mM ATT, 5 mM ascorbic acid, 5 μM LgTrip 3546, and 1 mM furimazine; 2) 5% BSA, 5 mM ATT, 5 mM ascorbic acid, 5 μM LgTrip 3546, and 1 mM furimazine.

[0358] To prepare the spots, vials containing 200 μL of 5 μM LgTrip 3546, 5 mM ATT, and 5 mM ascorbic acid were prepared. Approximately 5 μL of this solution was added to each spot, and the spots were then dried at 35°C for 1 hour. After drying, a 1 mM stock of furimazine was prepared in ethanol. Approximately 5 μL of this solution was added to each spot, and the spots were then dried at 35°C for an additional 30 minutes. To measure luminescence, a 1.2 mM dipeptide stock solution was diluted to 1 e with PBS (pH 7.0) at the time of testing. -10 Approximately 100 μL of each dipeptide stock was added to the 96-well plate containing the spots, and kinetic measurements were started immediately.

[0359] These data demonstrate that a stable, concentration-dependent response was observed upon addition of the dipeptide (Figure 25). This experiment demonstrates that a paper format containing LgTrip 3546 and a substrate can be prepared and then reconstituted in a buffered aqueous medium containing a potential analyte of interest (e.g., a dipeptide). Different materials were then tested with the substrate and LgTrip 3546. Either a new dipeptide was added at 1 nM to test the activity of NanoTrip and the substrate, or a new Nluc was added to isolate the substrate. Figure 27 shows the bioluminescence signal obtained from reconstitution of LgTrip 3546 and the substrate with the dipeptide or addition of NanoLuc to dried LgTrip 3546 and the substrate on three different solid-phase materials (Whatman 903, Ahlstrom 237, and Ahlstrom 6613H). Ahlstrom 6613H appears to have a negative effect on signal output over time, as the luminescence signal appears to decrease under both conditions. Overall, the stability of the assay components can be affected by the composition of the solid matrix material in which they are embedded.

[0360] Figure 28 shows the bioluminescence signal from Whatman 903 paper containing both LgTrip 3546 and substrate and stored at ambient conditions for 25 days. At the time of testing, the spots were exposed to 1 nM dipeptide in PBS. Overall, this experiment demonstrates that the signal from the material does not significantly fade even after extended storage at ambient temperature.

[0361] Example 9 Lyophilized cake containing LgTrip and substrate Figures 26A-26B show the bioluminescence signals obtained from the dipeptide-based reconstitution of LgTrip 3546 and substrate from lyocakes (Figure 26A), along with the dipeptide titration summary data (Figure 26B). To prepare the lyocakes, 5% w / v pullulan was added to water containing 26.3 mM ATT and 11.3 mM ascorbic acid (Solution 1). Solution 1 was then dispensed into snap-cap vials in a volume of 35 μL. Approximately 10 μL of 95 μM LgTrip 3546 protein was then added to each vial and mixed by pipetting (Solution 2). A 10 mM furimazine stock solution was prepared in ethanol, and 5 μL of this solution was added to each vial and mixed (Solution 3). The vials containing Solution 3 were placed on dry ice to freeze for 1 hour and then lyophilized overnight. To measure luminescence, a 1.2 mM dipeptide stock in water was diluted to 1 e with PBS (pH 7.0) at the time of testing. -10 Approximately 100 μL of each dipeptide stock was added to a lyophilized vial containing IgTrip 3546 and substrate, briefly pipetted to mix, then placed in a 96-well plate and kinetic measurements were immediately initiated.

[0362] These data demonstrate that a stable, concentration-dependent bioluminescent response was observed upon addition of the dipeptide (Figure 26). This experiment demonstrates that solid-format lyophilized cakes or tablets containing LgTrip 3546 and substrate can be prepared and then reconstituted in aqueous media containing potential analytes of interest (e.g., dipeptides).

[0363] Example 10 Protein Buffer Formulation For Figures 29-33, experiments were performed to test the compatibility of protein components with different protein buffer formulations according to the experimental design shown in the schematic diagram below. JPEG2025133108000007.jpg101164

[0364] For these experiments, Whatman 903 Protein Saver Spot Cards were used with the following protein buffer formulations: Protein Buffer 1: 20 mM NaPO, 5% w / v BSA, 0.25% v / v Tween 20, 10% w / v sucrose Protein buffer 2: 20 mM NaPO, 0.25% v / v Tween 20, 10% w / v sucrose Protein Buffer 3: 20 mM NaPO, 5% w / v BSA, 0.25% v / v Tween 20 Protein Buffer 4: 20 mM Na3PO4, 5% w / v BSA, 0.25% v / v Tween 20, 2.5% pullulan Protein buffer 5: 20 mM Na3PO4, 0.25% v / v tween 20, 2.5% pullulan.

[0365] For NanoLuc, a 1000x stock solution was diluted 1:1000 in protein buffer (1 mL). For the 1.068 nM stock solution, 3 μl was diluted in 297 μl of protein buffer. Approximately 5 μL of each concentration was spotted onto filter paper. For LgBiT-1672-11s-His, 5 μL of 1.068 nM protein was used per spot. Approximately 10 μL was diluted in 990 μL of protein buffer to obtain a 2e -7Approximately 100 μL of a 100 nM protein solution was then prepared, and approximately 10 μL of the stock was diluted into 990 μL of protein buffer to create a 1 nM stock. Approximately 5 μL of each concentration was spotted onto filter paper. For LgTrip 3546, approximately 5 μL of 1.068 nM protein was used per spot. Approximately 1.1 μL of LgBiT-1672 stock was diluted into 998.94 μL of protein buffer. Approximately 3 μL of the stock was diluted into 297 μL of protein buffer. Approximately 5 μL of each concentration was spotted onto filter paper. After all the protein was added, the samples were dried at 30°C for approximately 1 hour. Approximately 40 spots were made for each condition (see schematic above). Spots were tested for baseline on day 0, then placed at 60°C and tested after 6 days. RLU activity was tested by adding 1 nM of the high affinity dipeptide plus 50 μM live cell substrate in PBS (pH 7.0).

[0366] Figures 29A-29C show the bioluminescence signal, measured in RLU, for NanoLuc (Figure 29A), LgBiT-1672 (Figure 29B), and LgTrip 3546 (Figure 29C) in various protein buffer formulations as described above. Figures 30A-30C show the bioluminescence signal, measured in RLU, for NanoLuc (Figure 30A), LgBiT-1672 (Figure 30B), and LgTrip 3546 (Figure 30C) in various protein buffer formulations. max Figure 1 shows the bioluminescence signal measured at 1000 kJ / min. Collectively, these data suggest that BSA is a significant component in the protein buffer formulations tested, with NanoLuc and LgTrip 3546 showing the greatest reduction in RLU (Buffers 2 and 5).

[0367] Experiments were also conducted to evaluate the background bioluminescence levels at the various protein buffer compositions described above. Figures 31A-31B show the background bioluminescence levels at various protein buffer compositions for LgBiT-1672 (Figure 31A) and LgTrip 3546 (Figure 31B). These data suggest that BSA or pullulan are important components of the protein buffer formulation for LgBiT-1672 to minimize background bioluminescence, but appear to have little effect on the background levels of LgTrip 3546 under these conditions.

[0368] In Figures 32A-32F, the kinetics of the above conditions were evaluated after addition of the dipeptide and substrate in PBS. More specifically, Figures 32A-32F show the bioluminescence signals (Figures 32A-32C are RLU; Figures 32D-32F are BLU) of NanoLuc® (Figures 32A and 32D), LgBiT-1672 (Figures 32B and 32E), and LgTrip 3546 (Figures 32C and 32F) in various protein buffer formulations after 6 days at 60°C. max ) These data show that under these conditions, the protein is stable and remains active after 6 days at 60°C, suggesting that BSA is a critical component in all protein buffer formulations. Additionally, Figure 33 includes a representative embodiment of an all-in-one lyophilized cake ("lyocake") or tablet containing all the reagents necessary to perform an analyte detection test supporting several types of assay formats, including, but not limited to, cuvettes, test tubes, large volume bottles, snap test type assays, etc.

[0369] Example 11 Lateral flow assay For Figures 34 and 35, lateral flow assays were performed using the information obtained in the experiments described above and according to the experimental design shown in the schematic diagram below. JPEG2025133108000008.jpg108138

[0370] Materials used in these experiments included conjugation pads (Ahlstrom grade 8950, chopped grass with binder, 50 g / m 2 The strip contained a sample pad (cellulose glass fiber CFSP203000 (Millipore)), an absorbent pad (cotton linter, grade 238 (Ahlstrom)), a membrane (nitrocellulose Hi-Flow Plus HFC07502 (Millipore), Strip Test 2 #6), and running buffer (5x LCS dilution buffer + 5x LCS diluted to 1x with PBS). The membrane was prepared by applying a 30% sucrose solution to the membrane, covering approximately 1.5 cm of the bottom of the strip. The membrane was dried at 35°C for 1 hour. The strips were first cut to 4.5 cm x 1 cm.

[0371] Protein preparation was carried out according to the following conditions: Condition 1: 5 μL of mouse anti-NanoLuc antibody was diluted with 995 μL of protein buffer and evenly applied to the entire conjugation pad with an airbrush and dried in an oven at 37 °C. 2.5 μL of mouse antibody was diluted with 0.5 mL of protein buffer and applied directly to the membrane. Condition 2: 2.5 μL of NanoLuc was diluted with 0.5 mL of protein buffer and applied directly to the membrane. The membrane was dried at 37°C for 1 hour. Condition 3: The entire membrane was directly treated with 5 μL of NanoLuc diluted to 1 mL with protein buffer. The mixture was evenly applied with an airbrush and allowed to dry at 37°C for 1 hour. Condition 4: 2.5 μL of mouse anti-NanoLuc antibody in 997 μL of protein buffer. Applied evenly with an airbrush across the entire conjugation pad. Allowed to dry at 37°C for 1 hour. Condition 5: 1 μL of mouse anti-NanoLuc antibody in 999 μL of protein buffer. Applied evenly across the conjugation pad with an airbrush. Allowed to dry at 37°C for 1 hour.

[0372] The strips were assembled on a backing card with the conjugation pad, sample pad, and wick pad cut to 1 cm x 1 cm. Once assembled, the strips were cut in half lengthwise to a final dimension of 4.5 cm x 0.5 cm. For imaging analysis, approximately 250 μl of 1X LCS buffer + LCS was diluted in PBS. Images were taken with a 5-second exposure and a 5-second wait time between images. Representative images are edited images from the corresponding movie taken over the entire exposure time (movie available upon request). The total read time was 2:40 min.

[0373] Figure 34 shows the bioluminescent signal due to substrate migration across the lateral flow strip, obtained from edited images corresponding to a movie taken over the entire exposure time. When substrate was added to the sample window of the lateral flow assay cassette, real-time imaging showed substrate migration across the strip, confirming NanoLuc® activity across the test window (Strip #3 in the schematic above). By 70 seconds, the substrate had flowed across the entire sample window.

[0374] Figure 35 shows the bioluminescence signal due to the movement of NanoLuc® across the lateral flow strips (strips #4 and #5 in the schematic above) obtained from edited images corresponding to a movie taken over the entire exposure time. Under these conditions, strip #5 appeared superior to strip #4, as indicated by the fluid flow of NanoLuc® out of the conjugation pad, across the membrane, and toward the strip containing the mouse anti-NanoLuc antibody.

[0375] Example 12 Fumonisin detection During the development of embodiments herein, experiments were performed to demonstrate the use of NanoLuc®-based technology in a competitive immunoassay for the detection of an exemplary small molecule toxin, fumonisin B1. Such assays can be performed with other small molecule targets and target analytes on the devices and systems described herein.

[0376] In an exemplary assay, a tracer was generated by tethering fumonisin B1 to an NL peptide tag (e.g., a peptide tag comprising SEQ ID NO: 10) via a biotin / streptavidin linkage, a HaloTag linkage, or directly (Figure 36). In some embodiments, the tracer can be combined with an anti-fumonisin B1 antibody linked to the polypeptide complement of the NL peptide tag (e.g., a complement comprising SEQ ID NO: 9). A bioluminescent complex can be formed between the peptide tag and the polypeptide component upon binding of the antibody to fumonisin B1. Exposure to various concentrations of unlabeled fumonisin B1 disrupts the bioluminescent complex, reducing light emission and the ability to detect / quantitate the amount of fumonisin B1 in a sample (Figure 37).

[0377] Example 13 Lyophilized cake containing LgBiT and substrate Figures 38A-38B show the bioluminescence signal obtained from the dipeptide-based reconstitution of LgBiT and substrate from lyocakes (Figure 38A), along with the dipeptide titration (Figure 38B). To prepare lyocakes containing LgBiT, 5% w / v pullulan in water containing 5 mM ATT and 5 mM ascorbic acid was prepared (Solution 1). Solution 1 was then dispensed into snap-cap vials in a volume of 45 μL. Approximately 5 μL of 20 μM LgBiT protein was then added to each vial and mixed by pipetting (Solution 2). A 10 mM furimazine stock solution was prepared in ethanol, and 5 μL of this solution was added to each vial and mixed (Solution 3). The vials containing Solution 3 were placed on dry ice to freeze for 1 hour and then lyophilized overnight.

[0378] To measure luminescence, a 1.2 mM dipeptide stock solution was diluted with PBS (pH 7.0) for 1 e at the time of testing. -10 Serial dilutions were made up to M. 100 μl of each dipeptide stock was added to a lyophilized vial containing LgBiT and substrate, mixed by pipetting briefly, then placed in a 96-well plate and kinetic measurements were immediately initiated.

[0379] These data demonstrate that a stable, concentration-dependent bioluminescent reaction was observed upon addition of the dipeptide. This experiment demonstrates that a solid-state format containing LgBiT and a substrate can be prepared and then reconstituted in an aqueous medium containing a potential analyte of interest (e.g., a dipeptide).

[0380] Example 14 Substrate and LgTrip 3546 or LgBiT lyophilization Figure 39 shows the bioluminescence signals obtained from LgBiT or LgTrip 3546 and the dipeptide-reconstituted substrate from lyocakes prepared directly in a standard 96-well tissue culture-treated plate (Costar 3917). To prepare the lyocakes in the plate, 2.5% w / v pullulan in water containing 5 mM ATT and 5 mM ascorbic acid was prepared (Solution 1, pH 6.5). Solution 1 was then dispensed into each well of the plate in a volume of 45 μl. 2.6 μl of 95 μM LgTrip 3546 protein was then added to each vial and mixed by pipetting to form Condition 1 (LgTrip 3546 alone). 5 μl of 20 μM LgBiT protein was then added to each vial and mixed by pipetting to form Condition 2 (LgBiT alone). Then, 5 μl of ethanol was added to each well of conditions 1 and 2 as a vehicle control.

[0381] Conditions 3 (LgTrip 3546 / substrate) and 4 (LgBiT / substrate) were prepared as described above: 2.5% w / v pullulan in water containing 5 mM ATT and 5 mM ascorbic acid was prepared (solution 1, pH 6.5). Solution 1 was then dispensed into each well of the plate in a volume of 45 μl. Approximately 2.6 μl of 95 μM LgTrip 3546 protein or 5 μl of 20 μM LgBiT protein was added to each vial and mixed by pipetting. Approximately 5 μl of 10 mM furimazine in ethanol was then added to each well to form conditions 3 and 4, respectively. The plate was then placed in a cooler with dry ice and frozen for 1 hour, followed by lyophilization overnight.

[0382] To measure luminescence, a 1.2 mM dipeptide stock solution was diluted with PBS (pH 7.0) for 1 e at the time of testing. -9 Fresh NanoGlo® substrate was then added to this stock to give a final concentration of 10 μM substrate. 100 μl of this solution was added to wells containing conditions 1 (LgTrip3546) and 2 (LgBiT). Conditions 3 (LgTrip3546 / substrate) and 4 (LgBiT / substrate) were diluted with 100 μl of 1e in PBS. -9 Only the M dipeptide was added. After testing, the plates were wrapped in tin foil and placed on the bench at ambient temperature.

[0383] This data demonstrates that lyocakes containing either LgBiT or LgTrip 3546 and substrate can be prepared directly in 96-well plates and reconstituted in the presence of the analyte (dipeptide) of interest, yielding stable and robust signals.

[0384] Example 15 Paper-based all-in-one analyte detection system An experiment was conducted to test the effectiveness of a paper-based detection platform containing the NanoBiT (Figures 40A-40B) and NanoTrip (Figure 41A) complementation system. Paper spots were created by punching 1 / 8-inch diameter circles from Whatman 903 spotting paper. These spots were treated with 5 μl of a master mix solution (pH 6.5) containing 5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid, 40 nM LgBiT-Protein G fusion, and 20 nM SmBiT-TNFα in water. The spots were dried at 35°C for 1 hour. A 200 μM furimazine solution was prepared in ethanol, and 5 μl of this solution was added to each spot. The spots were then dried at 35°C for an additional 30-60 minutes. At the time of testing, spots were placed into individual wells of a 96-well NBS plate (Costar 3917) and reconstituted with Opti-MEM assay buffer containing either 0 nM (blank), 1 nM, or 100 nM Remicade.

[0385] Figures 40A-40B include assay results using NanoBiT components. Exposing spots to assay buffer containing 1 nM Remicade increased overall luminescence output compared to the blank condition / control without Remicade. As the concentration of Remicade increased to 100 nM, an increase in signal was observed. As shown in Figure 40B, Remicade was prepared in opti-MEM assay buffer at concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM. At the time of testing, 100 μl of each solution containing Remicade was added to wells of a 96-well plate containing spots, and RLU output was measured.

[0386] A similar experiment was performed using NanoTrip components, as shown in Figure 41A. Spots were created by punching 1 / 8-inch diameter circles from Whatman 903 spotting paper. Each spot was treated with 5 μl of a master mix solution (pH 6.5) containing 5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid, 20 μM LgTrip 3546, 100 nM TNFα-15gs-VSHiBiT, and SmTrip9 Pep521-15gs-Protein G in water. The spots were dried at 35°C for 1 hour. A 200 μM furimazine solution was prepared in ethanol, and 5 μl of this solution was added to each spot. The spots were dried for an additional 30 minutes at 35°C. At the time of testing, spots were placed into individual wells of a 96-well NBS plate (Costar 3917) and reconstituted with opti-MEM assay buffer containing either 0 nM (blank), 1 nM, or 100 nM Remicade. The results are shown in Figure 41A.

[0387] These experiments demonstrate that it is possible to construct an all-in-one paper-based bioluminescence assay platform for detecting an analyte of interest using the complementary systems of both NanoBiT and NanoTrip. Additionally, these experiments demonstrate that it is possible to quantify the amount of analyte present in a sample matrix based on the change in overall luminescence output. Increasing the concentration of the analyte of interest (i.e., Remicade) resulted in a proportional increase in the bioluminescence signal (the bioluminescence signal generated from the analyte-detection complex is proportional to the analyte concentration).

[0388] Example 16 Lyocake-based all-in-one analyte detection system Experiments were also performed to test the efficacy of a lyocake-based detection platform containing the NanoBiT (Figure 40C) and NanoTrip (Figures 41B-41C) complementation systems.

[0389] As shown in Figure 40C, stability conditions were tested when the components of the bioluminescent complex were dried. Approximately 45 μl of the master mix solution was added to a 1.5 mL plastic snap-cap vial. The master mix contained 5% w / v pullulan, 5 mM ATT, 5 mM ascorbic acid, 40 nM LgBiT-Protein G fusion, and 20 nM SmBiT-TNFα (pH 6.5). Approximately 5–10 μl of furimazine substrate in ethanol was added to each vial, mixed, and placed on dry ice for approximately 1 hour. The frozen samples were then lyophilized overnight to form lyo-cakes. At the time of testing, 100 nM and 10 nM Remicade solutions were prepared in Opti-MEM assay buffer. Approximately 100 μl of these solutions were added to the vials containing the NanoBiT cakes, mixed by pipetting, and then transferred to a Costar 3600 96-well plate. A blank control was prepared without the analyte Remicade. The results in Figure 40C show that when all components of a bioluminescent complex, including the substrate, are frozen and stored in the form of a lyocake and then exposed to the analyte of interest, increasing the concentration of the analyte results in a proportional increase in signal.

[0390] In Figures 41B-41C, stability conditions were tested when the components of the bioluminescent complex were dried. Approximately 45 μl of master mix solution was added to a 1.5 mL plastic snap-cap vial. The master mix contained 5% w / v pullulan, 5 mM ATT, 5 mM ascorbic acid, 9 μM LgTrip 3546, 225 nM SmTrip9-Protein G, and 45 nM SmBiT-TNFα (pH 6.5). Approximately 5-10 μl of furimazine substrate in ethanol was added to each vial, mixed, and placed on dry ice for approximately 1 hour. The frozen samples were then lyophilized overnight to form lyocakes. At the time of testing, 100 nM, 10 nM, and 1 nM Remicade solutions were prepared in Opti-MEM assay buffer. Approximately 100 μl of these solutions were added to the vials containing the NanoTrip cakes, mixed by pipetting, and then transferred to a Costar 3600 96-well plate. A blank control was prepared without the analyte Remicade. The results in Figures 41B-41C show that when all components of a bioluminescent complex, including the substrate, are frozen and stored in the form of a lyocake and then exposed to the analyte of interest, increasing the concentration of the analyte results in a proportional increase in signal.

[0391] When the spots were exposed to assay buffer containing 1 nM Remicade, the overall luminescence output increased compared to the blank condition without Remicade. When the Remicade concentration was increased to 100 nM, an increase in signal was observed. These experiments demonstrate that it is possible to construct an all-in-one lyocake-based bioluminescence-based assay platform for detecting analytes of interest using both the NanoBiT and NanoTrip complementation systems. Additionally, these experiments demonstrate that it is possible to quantify the amount of analyte present in a sample matrix based on the change in overall luminescence output. Increasing the concentration of the analyte of interest (i.e., Remicade) resulted in a proportional increase in bioluminescence signal (the bioluminescence signal generated from the analyte-detection complex is proportional to the analyte concentration).

[0392] Example 17 Mesh-based system separating substrate and bioluminescent conjugates for analyte detection Experiments were performed to determine the conditions necessary for bioluminescent signal generation when the peptide and polypeptide components of the bioluminescent complexes provided herein were prepared in a substrate-free format. For example, in one embodiment, a volume of solution (e.g., containing the analyte of interest) is added to a mesh or matrix to which a luminescent substrate has been attached ("caked"). Addition of the solution results in the reconstitution of the substrate on the mesh, which then interacts with the surface of paper containing the dried peptides and polypeptides of the bioluminescent complexes of the present disclosure, generating a bioluminescent signal (Figure 42A). The mesh format does not interfere with the ability to detect a bioluminescent signal; any bioluminescence detected is from the surface of the paper and not from any liquid phase formed during the experiment.

[0393] As shown in Figure 42A, this format can be used to detect bioluminescence. Whatman 903 paper spots were fabricated to have a diameter of approximately 0.25 inches, similar to the nylon mesh. The master mix used to create the paper spots containing the bioluminescent peptide / polypeptide components contained 5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid, and 10 μM NanoLuc (pH 6.5). Approximately 10-20 μl of the master mix was added to the spots and then dried at approximately 35°C for approximately 1 hour. To generate the substrate-containing mesh, an approximately 0.75% aqueous pullulan solution was prepared. Approximately 450 μl of this solution was added to a plastic snap-cap vial. Approximately 50 μl of 10 mM furimazine in EtOH was added to the vial and mixed by pipetting. Approximately 25 μl of this solution was added on top of the mesh spots. The mesh spots were then frozen on dry ice and lyophilized overnight. At the time of testing, a mesh containing the Lyocake substrate was placed on top of the spot containing the NanoLuc® protein. The complete system was then added to the wells of a 96-well Costar 3600 plate. Approximately 10 μl of PBS was then added on top of the mesh to reconstitute the material, and the RLU luminescence output of the plate was read.

[0394] Experiments were also conducted using the LgTrip 3546 bioluminescent component in a mesh-based format. The master mix used to create paper spots containing the bioluminescent peptide / polypeptide component contained 5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid, and 100 nM LgTrip 3546 (pH 6.5). Approximately 10–20 μl of the master mix was added to the spot and then dried at approximately 35°C for approximately 1 hour. To generate meshes containing the substrate, a solution of approximately 0.75% pullulan in water was prepared. Approximately 450 μl of this solution was added to a plastic snap-cap vial. Approximately 50 μl of 10 mM furimazine in EtOH was added to the vial and mixed by pipetting. Approximately 25 μl of this solution was added on top of the mesh spot. The mesh spot was then frozen on dry ice and lyophilized overnight. For testing, dipeptides ranging from 100 nM to 0.1 nM were prepared in PBS. The spots were placed in the wells, and a screen containing the substrate was placed on top of the spots. Approximately 10 μl of a solution containing each concentration of peptide was added to the surface of the screen, and the RLU was recorded (Figures 42B-42C). A blank control did not contain any dipeptide.

[0395] Experiments were also conducted using the LgTrip 3546 bioluminescent component in a mesh-based format to form a pullulan film. The master mix used to create paper spots containing the bioluminescent peptide / polypeptide component contained 5% w / v BSA, 5 mM ATT, 5 mM ascorbic acid, and 100 nM LgTrip 3546 (pH 6.5). Approximately 10–20 μl of the master mix was added to the spot and then dried at approximately 35°C for approximately 1 hour. To generate meshes containing the substrate, an approximately 2.0% aqueous pullulan solution was prepared. Approximately 450 μl of this solution was added to a plastic snap-cap vial. Approximately 50 μl of 10 mM furimazine in EtOH was added to the vial and mixed by pipetting. Approximately 25 μl of this solution was added on top of the mesh spot. The spot was then allowed to dry overnight in the dark under ambient conditions. This method formed a pullulan film that filled the holes in the mesh. At the time of testing, dipeptides ranging from 100 nM to 0.1 nM were prepared in PBS. The spots were placed in the wells, and a screen containing the substrate was placed on top of the spots. Approximately 10 μl of a solution containing each concentration of peptide was added to the surface of the screen, and the RLU was recorded (Figures 42D-42E). A blank control did not contain any dipeptide.

[0396] These experiments demonstrate the feasibility of detecting bioluminescent signals in a mesh-based format that separates the peptide / polypeptide components and the substrate. Additionally, in the context of this format, these experiments demonstrate that increasing the concentration of the analyte of interest (i.e., dipeptide) leads to a proportional increase in bioluminescent signal (the bioluminescent signal generated from the analyte-detection complex is proportional to the concentration of the analyte).

[0397] Example 18 Different formulations of lyophilized substrates are tested for cake appearance, reconstituted kinetic activity performance, and accelerated heat stability. To assess the applicability of lyophilization to the storage of furimazine substrate, formulations containing furimazine were prepared. The 20X stock formulation was as follows:

[0398] Condition 1: 100 μM furimazine, 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w / v, ddH2O (Millipore) in ethanol;

[0399] Condition 3: 100 μM furimazine, 5 mM azothiothymine, 5 mM ascorbic acid, 2.5% pullulan w / v, 20 mM HEPES buffer (pH 8.0), 90 mM glycine, 20 mM histidine, 25 mg / ml sucrose, 0.01% polysorbate 80 in ethanol;

[0400] Condition 5: 40 μM furimazine in 85% ethanol + 15% glycerol, 200 mM MES buffer (pH 6.0), 200 mM hydroxypropyl beta-cyclodextrin (mw 1396 Da), 600 mM sodium ascorbate, 2.5% pullulan w / v; and

[0401] Condition 7: 20 μM furimazine in ethanol, 200 mM MES buffer (pH 6.0), 200 mM hydroxypropyl beta-cyclodextrin (mw 1396 Da), 600 mM sodium ascorbate, 2.5% pullulan w / v.

[0402] A 1 mL aliquot of the 20X stock solution was dispensed into a 10 mL amber glass vial, and a runner stopper was partially inserted into the vial. The vial was loaded into a freeze dryer (Virtis Genesis 12EL freeze dryer) with shelves pre-cooled to 4.7 °C. The product then underwent a 2-hour freezing step at a storage temperature of -50 °C, after which the condensation step began. During this procedure, the condenser temperature ranged from -5 °C to -87 °C. Vacuum was then applied at pressure settings of 75 mTorr and 200 mTorr. Sublimation lasted approximately 7.5 hours, and desorption lasted approximately 16.1 hours. At the end of the freeze-drying process, the vial was backfilled with nitrogen and sealed with a stopper fully inserted at a pressure of approximately 600 Torr.

[0403] Vials were stored at 25°C or 60°C and tested at various time points after lyophilization. For activity-based assays, furimazine cakes were reconstituted in 10 mL of PBS containing 0.01% BSA. Vials were shaken by hand and allowed to equilibrate at room temperature for 5 minutes. 50 μl of reconstituted substrate was added to 50 μl of 1 ng / mL purified NANOLUC enzyme (Promega) reconstituted in the same BSA buffer (final [NanoLuc] = 0.5 ng / mL). Controls used were NANOGLO Live Cell Substrate (Promega catalog no. N205) or NANOGLO substrate (Promega catalog no. N113) according to the manufacturer's protocol, but diluted in PBS containing 0.01% BSA rather than the dilution buffer (Promega) provided with the kit. Assays were performed on solid white nonbinding surface (NBS) plates (Costar) and analyzed on a GLOMAX Discover Multimode Microplate Reader (Promega), collecting total luminescence using either kinetic or endpoint readings, depending on the experiment. For analysis of absolute furimazine, reconstituted samples were analyzed by HPLC, absorbance spectra were obtained at a wavelength of 245 nm, and the absolute amount remaining from day 0 was plotted.

[0404] The appearance of the lyophilized cakes obtained from these formulations is shown in Figure 43, which indicates that all four conditions tested produced intact cakes, although conditions 5 and 7 showed some cracking. pH indicators provided with the vials indicated that the resulting cakes had pH values ​​of approximately 2-3 in condition 1, approximately 7.5 in condition 3, and approximately 6 in conditions 6 and 7. The signal kinetics of reconstituted furimazine, when tested with purified NanoLuc, compared to furimazine in standard organic storage buffers (N113 and N205) kept at -20°C showed no observable degradation in performance due to the formulation buffer and the lyophilization process itself, and conditions 5 and 7 showed improved half-lives (Figure 44).

[0405] Accelerated thermal stability studies demonstrated that furimazine formulated and lyophilized under condition 1 retained activity for 3 months. This contrasts sharply with furimazine stored in organic solvent, which lost all activity in approximately 10 days when stored at this elevated temperature (Figure 45). HPLC analysis of the absolute [furimazine] remaining after storage at 25°C and 60°C confirmed the activity findings, with the formulated and lyophilized substrate containing significantly higher purity furimazine compared to furimazine in standard organic storage buffers (Figures 46A and 46B). To determine the liquid stability of the formulated, lyophilized furimazine, vials were reconstituted with water, kept in solution for 12 days, and then analyzed by HPLC for the total amount of furimazine remaining compared to day 0. Conditions 5 and 7 were found to have superior liquid stability (Figure 47).

[0406] Example 19 Development of a solution-based homogeneous human interleukin-6 tripartite immunoassay using HaloTag-peptide fusions to chemically conjugate monoclonal antibody pairs The basic principle of the homogeneous NanoLuc three-component (NanoTrip) immunoassay is shown in Figure 48. First, using HaloTag® technology, a pair of antibodies targeting non-overlapping epitopes on IL-6 are chemically conjugated to SmTrip9 (SEQ ID NO: 13) or HiBiT (SEQ ID NO: 11). Binding of the labeled antibodies to the IL-6 analyte brings the complementary subunits into close proximity, thereby reconstituting bright luciferase in the presence of LgTrip 3546 protein (SEQ ID NO: 12) and furimazine substrate. This assay is quantitative because the amount of light generated by a standard plate-reading luminometer is directly proportional to the amount of target analyte present.

[0407] Gene fusions containing SmTrip9 variant (SmTrip9 Pep521; SEQ ID NO: 16) or SmTrip10 variant (SmTrip10 Pep289 or VSHiBiT; SEQ ID NO: 17) separated by either a 2X or 3X Gly-Ser-Ser-Gly linker to the amino terminus of HaloTag were transfected into pFN29A HIS6HaloTag T7 Flexi This was achieved using Vector (Promega). A glycerol stock of E. coli expressing the HisTag-HaloTag fusion protein was used to inoculate a 50 mL starter culture, which was grown overnight at 37°C in LB medium containing 25 μg / mL kanamycin. The starter culture was diluted 1:100 into 500 mL of fresh LB medium containing 25 μg / mL kanamycin, 0.12% glucose, and 0.2% rhamnose. The culture was grown at 25°C for 22-24 hours. Cells were pelleted by centrifugation (10,000 rpm) for 30 minutes at 4°C and resuspended in 50 mL of PBS. 1 mL of protease inhibitor cocktail (Promega), 0.5 mL of RQ1 DNase (Promega) and 0.5 mL of 10 mg / mL lysozyme (Sigma) were added, and the cell suspension was incubated on ice for 1 hour with gentle agitation. Cells were lysed by sonication at 15% power for 1.5 minutes with 5-second intervals (3 minutes total), followed by centrifugation at 10,000 rpm for 30 minutes at 4°C. The supernatant was collected, and the protein was purified using a HisTag column (GE) according to the manufacturer's recommended protocol. The protein was eluted with 500 mM imidazole, dialyzed against PBS, and characterized using an SDS-PAGE gel, revealing a purity of >95%. The protein was stored in 50% glycerol at -20°C.

[0408] To chemically conjugate the antibody to the HaloTag-peptide fusion protein, the antibody was buffer-exchanged twice into 10 mM sodium bicarbonate buffer (pH 8.5) using a Zeba spin desalting column (ThermoFisher). The antibody was then primed with 200 μM amine-reactive HaloTag succinimidyl ester (04) ligand (Promega) for 2 hours at 22°C with shaking at 1000 rpm. Unreacted ligand was removed by passing the antibody twice through a Zeba spin column in PBS buffer. The antibody was then covalently labeled overnight with 30 μM HaloTag fusion protein at 4°C with shaking. Excess unreacted HaloTag fusion protein was removed using HaloLink Resin (Promega). The conjugated antibody was characterized using a non-denaturing SDS-PAGE gel. The mouse anti-human IL-6 monoclonal antibodies used in the human IL-6 immunoassay were clone 5IL6 (Thermo catalog no. M620) and clone 505E 9A12 A3 (Thermo catalog no. AHC0662). SDS-PAGE gels run on the labeled antibodies revealed that each antibody was labeled with a variable number of peptide-HaloTag fusion proteins, with the...

Claims

1. A method for detecting a target analyte in a sample, comprising: (a) A sample is (i) a luminescent substrate; (ii) a first target analyte binding agent comprising a first target analyte binding member and a first component of a bioluminescent complex; (iii) a second target analyte binding agent comprising a second target analyte binding member and a second component of a bioluminescent complex, and optionally (iv) a third component of the bioluminescent complex; (b) binding the first and second target analyte binding agents to the target analyte to form a bioluminescent complex; and (c) detecting a bioluminescent signal from the bioluminescent complex; The method, wherein the bioluminescent signal indicates the presence of the target analyte in the sample. (a) the first and second components of the bioluminescent complex have at least 90% sequence identity to SEQ ID NOs: 9 and 10; (b) the first and second components of the bioluminescent complex have at least 90% sequence identity to SEQ ID NOs: 12 and 14; (c) the first, second, and third components of the bioluminescent complex are having at least 90% sequence identity with SEQ ID NOs: 10, 13 and 12; have at least 90% sequence identity with SEQ ID NOs: 12, 13 and 10, or have at least 90% sequence identity with SEQ ID NOs: 10, 12 and 13, or (d) the first, second, and third components of the bioluminescent complex are having at least 90% sequence identity with SEQ ID NOs: 11, 13 and 12; have at least 90% sequence identity with SEQ ID NOs: 12, 13 and 11, or The method of claim 1, having at least 90% sequence identity with SEQ ID NOs: 11, 12 and 13.

3. The method of claim 1, wherein the first target analyte binding agent, the second target analyte binding agent and any third component of the bioluminescent complex form a bioluminescent analyte detection complex in the presence of the target analyte.

4. The method of claim 1, wherein the target analyte is a target antibody.

5. The method of claim 1, wherein the first and second target analyte binding elements are selected from the group consisting of an antibody, a polyclonal antibody, a monoclonal antibody, a recombinant antibody, an antibody fragment, Protein A, the Ig-binding domain of Protein A, Protein G, the Ig-binding domain of Protein G, Protein A / G, the Ig-binding domain of Protein A / G, Protein L, the Ig-binding domain of Protein L, Protein M, the Ig-binding domain of Protein M, an oligonucleotide probe, a peptide nucleic acid, a DARPin, an aptamer, an affimer, a protein domain, and a purified protein.

6. The method of claim 1, wherein the luminescent substrate is selected from coelenterazine, coelenterazine-h, coelenterazine-hh, furimazine, and other coelenterazine analogs or derivatives.

7. The method of claim 1, wherein the sample is selected from blood, serum, plasma, urine, stool, cerebrospinal fluid, interstitial fluid, tissue, and saliva.

8. The method of claim 1, wherein the sample is selected from a water sample, a soil sample, a plant sample, a food sample, a beverage sample, an oil, and an industrial fluid sample.

9. The method of claim 1, further comprising a step of quantifying the bioluminescent signal.

10. The method of claim 1, further comprising a step of diagnosing the subject from whom the sample was obtained as having or not having a disease based on detection of the target analyte.

11. The method of claim 1, wherein the first and / or second target analyte binding agents further comprise a fluorophore bound to the first and / or second components of the bioluminescent complex.

12. The method of claim 11, wherein the fluorophore is activatable by energy transfer from a bioluminescent complex.

13. The method of claim 1, wherein one or more of (i) to (iv) are in the form of a freeze-dried tablet.

14. The method described in claim 1, wherein one or more of (i) to (iv) are provided in a lateral flow assay system or a solid-phase detection system.

15. The method of claim 14, wherein the step of contacting the sample with (i) to (iv) includes applying the sample to a sample pad that promotes the flow of the sample through a lateral flow assay system.

16. The method of claim 14, wherein the step of contacting the sample with (i) to (iv) comprises exposing the sample to a detection region and a control region of a solid-phase detection system.