Methods to amplify immunoassay signals

Abstract

This document discloses methods using modified liposomes or carrier proteins comprising (i) acridinium ester (AE), and (ii) a first reagent encapsulated by the liposomes, and / or (iii) a second reagent on the surface of the liposomes or carrier protein. Specifically, the disclosed methods provide methods for labeling targets of interest, assaying biological samples against target antigens, and detecting target antigens in biological samples. This document further discloses methods for increasing the signal intensity detected by imaging modalities.

Description

Technical Field

[0001] This article discloses a method and kit for amplifying immunoassay labeling and detecting analytes in samples using liposomes encapsulating hydrophilic acridinium esters and products containing acridinium ester proteins.

[0002] background

[0003] Immunoassays remain the preferred method in clinical laboratories for analyzing a wide range of analytes, particularly complex heterogeneous molecules. A lack of immunoassay signal and sensitivity, or low immunoassay signal and sensitivity, can be a major obstacle to accurate diagnosis and disease prediction. There has been a persistent need in the field for improved immunoassay labeling methods that provide rapid and reliable results, which would benefit both patients and healthcare providers.

[0004] Overview

[0005] This document discloses a method for detecting an analyte in a sample. The method includes (a) combining a sample with a conjugation reagent, a linker reagent, an amplification reagent, and optionally a capture binding pair of the analyte in a culture medium; and (b) examining the culture medium for a bound analyte comprising an analyte bound to the conjugation reagent, the conjugation reagent binding to a linker reagent bound to the amplification reagent, wherein the conjugation reagent comprises a detection binding pair of the analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first and second small molecules.

[0006] This document discloses a kit comprising (a) a conjugation reagent; (b) a linker reagent; (c) an amplification reagent; and optionally, a capture binding pair, wherein the conjugation reagent comprises a detection binding pair of a target analyte and a first small molecule, wherein the linker reagent comprises a binding pair of the small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first small molecule and the second small molecule.

[0007] In some implementations, the detection binding coupler of the analyte comprises an antibody that specifically binds to the analyte.

[0008] In some embodiments, the first and second small molecules comprise biotin. In additional embodiments, the binding coupler of the first and second small molecules comprises streptavidin.

[0009] In some embodiments, the first and second small molecules comprise fluorescein. In additional embodiments, the binding coupler of the first and second small molecules comprises an antifluorescein antibody.

[0010] In some embodiments, the analyte capture and binding pair further comprises a support. In further embodiments, the support is a non-magnetic particle, a magnetic particle, a plate, or a tube.

[0011] In some embodiments, the conjugation reagent further comprises a labeling reagent.

[0012] In some embodiments, the connector reagent comprises a labeling reagent.

[0013] In some embodiments, the labeling reagent comprises acridine ester (AE).

[0014] In some embodiments, the liposomes have a diameter of about 20 nm to about 1000 nm.

[0015] In some implementations, the encapsulated AE has a size of at least 1 x 10 -8 mol / L to at least 1 x 10 -6 Concentration in the range of mol / L.

[0016] In some embodiments, the liposomes encapsulate approximately 1,000 to approximately 100,000,000,000 hydrophilic AE molecules.

[0017] In some embodiments, the carrier protein comprises bovine serum albumin (BSA).

[0018] In some embodiments, the carrier protein binds at least 1 to about 100 AE molecules.

[0019] In some embodiments, the disclosed method further includes a washing step prior to the step of examining the culture medium for the bound analyte.

[0020] In other embodiments of the disclosed method, the sample, the conjugation reagent, the connector reagent, the amplification reagent, and optionally, the capture-binding partner are combined simultaneously or sequentially. Brief description of the attached diagram

[0022] The overview and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the disclosed apparatus, systems, and methods are shown in the drawings for the purpose of illustrating the invention; however, the apparatus, systems, and methods are not limited to the specific embodiments disclosed. In the figures:

[0023] Figure 1 This is a schematic diagram illustrating an example of cross-linking based on the linker protein streptavidin. Ag = antigen; Ab = antibody; AE = acridine ester.

[0024] Figure 2 These are a series of schematic diagrams illustrating how cross-linking with the adaptor protein streptavidin can be used in the compositions and methods of this disclosure.

[0025] Figure 3 This is a schematic diagram illustrating an example of cross-linking between adaptor protein-based anti-fluorescein antibodies. Ag = antigen; Ab = antibody; AE = acridine ester.

[0026] Figure 4 These are a series of schematic diagrams illustrating how cross-linking with antifluorescein antibodies can be used in the compositions and methods of this disclosure.

[0027] Figure 5 is a series of diagrams depicting the DLS of biotinylated monolayer liposome vesicles (LUV) ± avidin and luciferinized (FL) liposome vesicles LUV ± anti-luciferin polyclonal antibody (anti-FL). Crosslinking of individual LUVs is observed. The anti-FL antibody can be monoclonal or polyclonal. F1 indicates the first fraction collected from the purification. FTIC and FL are similar compounds.

[0028] Figure 6 These are images and tables illustrating the assays described in this disclosure. In assay A (Assay A in the table), biotinylated vesicles of collected AEs can bind to DYNAL. ® M270 (Thermo Fisher Scientific) beads (magnetic latex beads coated with streptoacidin). As shown in the figure, the detector is a Berthold Autolumat Plus LB 953 (with a magnetic rack on top for manual measurement). The binding, displayed by the output in relative optical units (RLU), is proportional to the amount of biotinylated vesicles added to collect AE. In Experiment B (Experiment B in the table), reducing the number of M270 beads decreased the output signal.

[0029] Figure 7 This table demonstrates the enhancement of signal output by adding the adaptor protein streptavidin. In Experiment A (Experiment A in the table), anti-FL paramagnetic particles (pmps) were used to capture biotinylated and fluoresceinized vesicles collecting AEs. The addition of the adaptor protein streptavidin generated and amplified the signal. In Experiment B (Experiment B in the table), increased concentration of the adaptor protein led to increased signal amplification. Experiments were conducted at room temperature (RT).

[0030] Figure 8 This is a diagram depicting the competitive binding of fluorescein and fluoresceinized AE vesicles to anti-FL pmp.

[0031] Figure 9 This is a description in Biacore ® A diagram illustrating the scaled-up protocol demonstrated on an optical biosensor from General Electric Healthcare. Using a sensor chip immobilized with fluoresceinized BSA, a protein hapten (anti-FL Mab (2H1) conjugated to neutral avidin) was first added to the chip, followed by two injections of biotinylated microvesicles (Stage 1 scale-up). Additional neutral avidin was introduced to allow binding of even more biotinylated microvesicles (Stage 2 scale-up). Finally, Stage 3 scale-up was a simple repetition of Stage 2 scale-up.

[0032] Figure 10 This is a series of schematic diagrams and tables illustrating the setup of the assay disclosed herein on the Siemens Centaur automated system. TSH = Thyroid-stimulating hormone; TSH1, TSH5, and TSH10 (in the table below) = TSH standards at increasing concentrations; lite reagent (LR) = AE-labeled streptavidin. Here, particles immobilized with anti-TSH Pab (polyclonal antibody) can bind to the TSH antigen, which subsequently forms a sandwich structure with biotinylated anti-TSH Mab (monoclonal antibody). The addition of AE-labeled streptavidin generates a signal.

[0033] Figure 11 This is a series of diagrams and tables illustrating the introduction of adaptor proteins and AE-labeled amplifiers on the Siemens Centaur automated system. Adaptor protein = unlabeled streptavidin in the helper well (AW); Amplifier = AE-BSA-Biotin; Top panel = Control; Bottom panel = Biotinylated anti-TSH Mab+ amplifier in the LR well. The adaptor protein streptavidin (without AE labeling in this case) is in the AW.

[0034] Detailed description of the illustrative implementation plan

[0035] The methods of this disclosure can be more readily understood by taking into account the accompanying drawings, which form part of this disclosure, and the following detailed description. It should be understood that the methods of this disclosure are not limited to the specific methods described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed methods.

[0036] It should be understood that, for clarity, certain features of the disclosed methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods described in the context of a single embodiment may also be provided separately or in any sub-combination.

[0037] definition

[0038] 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 to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. The following terminology will be used in describing and claiming protection for this invention.

[0039] It should also be understood that the terminology used herein is for the purpose of describing specific implementations only and is not intended to be limiting.

[0040] As used herein, the singular forms “a” ("a", "an") and “the” ("the") include plural indicators unless the context explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly indicates otherwise.

[0041] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent in the context of its use. As used herein, when referring to measurable values ​​such as amount, concentration, duration, etc., the term “about” is intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, of the specified value, because such variations are suitable for implementing the methods of this disclosure.

[0042] As disclosed herein, the term acridine ester refers to any acridine ester that can be encapsulated in liposomes and can generate a chemiluminescent signal.

[0043] As used herein, the term "analyte" is a broad term and is used in its usual sense, including but not limited to, referring to a detectable component or target of interest in a sample, such as a substance or chemical component in biological fluids (e.g., blood, interstitial fluid, cerebrospinal fluid, lymph, or urine). Analytes can include naturally occurring substances, man-made substances, metabolites, and / or reaction products. Examples of analytes include, but are not limited to, single or multiple epitopes, antigenic or haptenic ligands, or nucleic acids such as DNA or RNA.

[0044] As used herein, the terms “solid support,” “support structure,” and “substrate” are used interchangeably and refer to a material or group of materials having one or more rigid or semi-rigid surfaces. There are no limitations on the shape or size of the support structure. In many embodiments, the solid support will take the form of beads (e.g., silica beads, magnetic beads, paramagnetic beads, etc.), resin, gel, microspheres, or other geometries.

[0045] As used herein, a "functional group" refers to a chemical group within a molecule that is responsible for a characteristic chemical reaction. Exemplary functional groups include, but are not limited to, those containing oxygen, nitrogen, phosphorus, or sulfur atoms, such as primary amines, carboxyl groups, carbonyl groups, aldehydes, mercapto groups, hydroxyl groups, and esters. As used herein, a functional group is reactive to another group if two groups can react to form a covalent bond.

[0046] A "connector" is a molecule that connects two other molecules by covalent bonds or by ionic bonds, van der Waals bonds or hydrogen bonds. For example, a nucleic acid molecule that hybridizes with a complementary sequence at the 5' end and another complementary sequence at the 3' end, thereby connecting two non-complementary sequences.

[0047] A "crosslinking agent" is a connector that covalently links two other molecules.

[0048] As used in this article, the term “linklification” refers to a signal amplification scheme for a given labeler.

[0049] As used herein, the term "liposome" refers to an artificially formed vesicle or capsule consisting of a membrane comprising at least one lipid bilayer. This term is understood to exclude naturally occurring vesicles or other naturally occurring membrane substances isolated from cells or biological samples containing cells. The terms "vesicle" and "liposome" may be used herein as synonyms for an artificially formed capsule comprising at least one lipid bilayer membrane. For example, a large, artificially formed monolayer liposome vesicle or "LUV" is referred to as a vesicle, but for the purposes of this patent application, it is also referred to as a liposome.

[0050] Poly(ethylene glycol), commonly known as PEG, refers to oligomers that form straight chains of ethylene oxide. PEG molecules can be straight-chain or branched, wherein each molecule has at least two, and usually three or more, PEG branches or arms derived from a central core group.

[0051] The term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabody"), Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv), camel antibodies, and humanized antibodies. As envisioned herein, antibodies conjugated to quantum dots and support structures can specifically or non-specifically recognize and / or bind to analytes, thereby enabling qualitative and quantitative analysis of the analytes.

[0052] As used herein, the terms "comprising," "including," "containing," and "characterized in" are interchangeable, inclusive, open-ended, and do not exclude additional, unlisted elements or method steps. Any reference to the term "comprising" herein, particularly in the description of the components of a composition or the elements of an apparatus, should be understood to cover compositions and methods that are substantially composed of the listed components or elements, as well as compositions and methods composed of the listed components or elements.

[0053] As used herein, the term "composed of" excludes any element, step, or component not specified in the claims.

[0054] "Disease" refers to any symptom or lesion that impairs or interferes with the normal function of cells, tissues, or organs.

[0055] "Detection" refers to identifying the presence, absence, or quantity of a target (e.g., an analyte to be detected).

[0056] When these terms are used interchangeably herein, “individual,” “patient,” or “subject” includes members of any animal species, including but not limited to birds, humans, and other primates, as well as other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Preferably, a subject is a human.

[0057] As used herein, the terms "treatment" and "treating" refer to methods used to obtain a beneficial or desired outcome, including but not limited to therapeutic and / or preventative benefits. For example, the term treatment includes administering a medication before or after the onset of a disease or lesion to prevent or eliminate all symptoms of the disease or lesion. As another example, administering a medication after the clinical manifestation of a disease to combat its symptoms constitutes "treatment" of the disease.

[0058] Therapeutic benefit refers to the eradication or improvement of the underlying lesion being treated, or the improvement of one or more physiological symptoms associated with the underlying lesion, thereby observed improvement in a patient, even though the patient may still have the underlying lesion. For preventative benefit, the composition may be administered to patients at risk of developing a specific disease, or to patients who have reported one or more physiological symptoms of a disease, even if a diagnosis of the disease may not yet have been made.

[0059] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, a range description should be considered as specifically disclosing all possible subranges and the individual numerical values ​​within those ranges, and, where appropriate, the partial integers of the numerical values ​​within the ranges. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing, for example, subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0060] Various terms are used throughout the specification and claims to relate to the aspects described. Unless otherwise stated, these terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0061] Detailed description

[0062] This article provides a method for amplifying immunoassay labels and detecting analytes in samples using liposomes encapsulating hydrophilic acridine esters (AEs) or articles containing proteins carrying AEs for signal amplification purposes.

[0063] The disclosed method for detecting an analyte in a sample comprises: (a) combining the sample with a conjugation reagent, a linker reagent, an amplification reagent, and optionally a capture binding pair of the analyte in a culture medium; and (b) examining the culture medium for a bound analyte comprising an analyte bound to the conjugation reagent, the conjugation reagent being bound to a linker reagent bound to the amplification reagent, wherein the conjugation reagent comprises a detection binding pair of the analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first and second small molecules.

[0064] In some embodiments, the detection binding coupler of the analyte comprises an antibody that specifically binds to the analyte. The antibody may be a monoclonal antibody, an antibody fragment, a bispecific or multispecific antibody, a dimer, tetramer, or multimer antibody, or a single-chain antibody capable of specifically binding to the analyte.

[0065] In some embodiments, the analyte may be an antigen derived from a biological sample. In some embodiments, the biological sample may be, but is not limited to, whole blood, serum, plasma, urine, saliva, semen, or cerebrospinal fluid.

[0066] In some embodiments, the methods of this disclosure can be used for a variety of assays. These assays include biochemical assays, such as immunoassays, clinical chemistry assays, or other medical or diagnostic tests. In some embodiments, the assays may include sandwich assays or in situ hybridization assays.

[0067] In some embodiments, the methods of this disclosure include a reaction mixture for a biochemical assay. The mixture may contain one or more reagents or buffer solutions for the assay, and a biological sample.

[0068] In some embodiments, the amplification reagent comprising liposomes or carrier proteins is added in suspension form to the biological sample, reagent, or reaction mixture used for the biochemical assay. In some embodiments, the amplification reagent may be restored from a "dry form" in the biological sample, reagent, or reaction mixture, or in one or more components of the reaction mixture used for the biochemical assay.

[0069] In some embodiments, the first and second small molecules comprise biotin, avidin, or avidin derivatives (e.g., neutral avidin). In other embodiments, the binding coupler of the first and second small molecules further comprises streptavidin.

[0070] In some embodiments, the first and second small molecules comprise fluorescein. In other embodiments, the binding coupler of the first and second small molecules further comprises an antifluorescein antibody.

[0071] In some embodiments, the first and second small molecules are conjugated to a polypeptide, an antibody or its antigen-binding fragment, an aptamer, an affinity compound, an affinity polymer, a carbohydrate, polyethylene glycol (PEG), or a PEGylated polypeptide. In some embodiments, the PEGylated polypeptide includes a PEGylated antibody or a PEGylated biotin.

[0072] In some embodiments, the carrier proteins of this disclosure include small or large proteins (MW > 100 kD) or polymers that can conjugate with analytes. Suitable carrier proteins include, but are not limited to, chitin, chitosan, gelatin, albumin, bovine serum albumin (BSA), ferritin, α1-macroglobulin, and thyroglobulin. The carrier protein can be a synthetic polymer, such as polyvinyl alcohol, polyacrylate, polysulfonate, polyamide, polyester, and polyether.

[0073] In some embodiments, the carrier protein includes bovine serum albumin (BSA).

[0074] In some implementations, the labeling reagent comprises acridine esters (AEs). AEs are stable compounds that offer superior immunoassay performance in the form of increased sensitivity compared to radioisotopes. The use of AEs can be beneficial for a variety of applications, such as labeling ligands or analytes (e.g., antigens); labeling specific binding partners of ligands or analytes (e.g., corresponding antibodies); or labeling nucleic acids and molecules containing nucleic acids.

[0075] In some embodiments, the carrier protein binds at least 1 to at least about 10 AE molecules, at least 1 to at least about 20 AE molecules, at least 1 to at least about 30 AE molecules, at least 1 to at least about 40 AE molecules, at least 1 to at least about 50 AE molecules, at least 1 to at least about 60 AE molecules, at least 1 to at least about 70 AE molecules, at least 1 to at least about 80 AE molecules, at least 1 to at least about 90 AE molecules, at least 1 to at least about 100 AE molecules, at least 1 to at least about 200 AE molecules, at least 1 to at least about 300 AE molecules, at least 1 to at least about 400 AE molecules, at least 1 to at least about 500 AE molecules, at least 1 to at least about 600 AE molecules, at least 1 to at least about 700 AE molecules, at least 1 to at least about 800 AE molecules, at least 1 to at least about 900 AE molecules, and at least 1 to at least about 1000 AE molecules.

[0076] In some implementations, the carrier protein binds at least 1 to about 100 AE molecules.

[0077] In some embodiments, the labeling agent encapsulated by liposomes is a hydrophilic acridine ester (AE). The hydrophilic nature of AE makes it suitable for encapsulation in liposomes without leakage through the liposome wall. A detailed description of hydrophilic AEs can be found in the art, for example in U.S. Patent No. 5,656,426 A, the disclosure of which is incorporated herein by reference in its entirety.

[0078] In some embodiments, the concentration of the hydrophilic AE encapsulated by liposomes is at least 1.10. -10mol / L to at least 1.10 -9 mol / L, at least 1.10 -9 mol / L to at least 1.10 -8 mol / L, at least 1.10 -8 mol / L to at least 1.10 -7 mol / L, at least 1.10 -7 mol / L to at least 1.10 -6 mol / L, at least 1.10 -6 mol / L to at least 1.10 -5 mol / L, at least 1.10 -5 mol / L to at least 1.10 -4 mol / L, at least 1.10 -4 mol / L to at least 1.10 -3 mol / L, at least 1.10 -3 mol / L to at least 1.10 - 2 mol / L, and at least 1.10 -2 mol / L to at least 1.10 -1 mol / L. In other embodiments, the hydrophilic AE has a concentration of at least 1.10 mol / L. -8 mol / L to at least 1.10 -6 Concentration in the range of mol / L.

[0079] In some embodiments, the liposomes may encapsulate at least 10 to at least 100 hydrophilic AE molecules, at least 100 to at least 1,000 hydrophilic AE molecules, at least 1,000 to at least 10,000 hydrophilic AE molecules, at least 10,000 to at least 100,000 hydrophilic AE molecules, at least 100,000 to at least 1,000,000 hydrophilic AE molecules, at least 1,000,000 to at least 10,000,000 hydrophilic AE molecules, at least 10,000,000 to at least 100,000,000 hydrophilic AE molecules, at least 100,000,000 to at least 1,000,000,000 hydrophilic AE molecules, at least 1,000,000,000 to at least 10,000,000,000 hydrophilic AE molecules, at least 10,000,000,000 to at least 100,000,000,000 hydrophilic AE molecules, and at least 100,000,000,000 to at least 1,000,000,000,000 hydrophilic AE molecules. In other embodiments, the modified liposomes comprise at least about 1,000 to at least about 100,000,000,000 hydrophilic AE molecules.

[0080] In further embodiments, the liposomes can have various sizes. In some embodiments, the diameter of the liposomes is from about 20 nm to about 1000 nm. In some embodiments, the diameter of the liposomes is about 20 nm to about 30 nm; about 30 nm to about 40 nm; about 40 nm to about 50 nm; about 50 nm to about 60 nm; about 60 nm to about 70 nm; about 70 nm to about 80 nm; about 80 nm to about 90 nm; about 90 nm to about 100 nm; about 100 nm to about 110 nm; about 110 nm to about 120 nm; about 120 nm to about 130 nm; about 130 nm to about 140 nm; about 140 nm to about 150 nm; about 150 nm to about 160 nm; about 160 nm to about 170 nm; about 170 nm to about 180 nm; about 180 nm to about 190 nm; about 190 nm to about 200 nm; about 200 nm to about 250 nm; about 250 nm to about 300 nm; about 350 nm to about 400 nm; about 400 nm to about 450 nm; about 450 nm to about 500 nm. nm; about 500 nm to about 550 nm; about 550 nm to about 600 nm; about 600 nm to about 650 nm; about 650 nm to about 700 nm; about 700 nm to about 750 nm; about 750 nm to about 800 nm; about 800 nm to about 850 nm; about 850 nm to about 900 nm; about 900 nm to about 950 nm; and about 950 nm to about 1000 nm. In other embodiments, the diameter of the liposomes is about 10 nm to about 500 nm. In yet another embodiment, the diameter of the liposomes is about 30 nm to about 100 nm.

[0081] In some embodiments, the liposomes that can be used in the disclosed methods include multilayered liposome vesicles (MLVs), small monolayered liposome vesicles (SUVs), large monolayered liposome vesicles (LUVs), and giant monolayered liposome vesicles (GUVs). In some embodiments, the lipid bilayer may comprise sphingolipids, glycerophospholipids, sterols, and sterol derivatives. The sphingolipids to be used may include sphingomyelins and ceramides containing saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. Phospholipids having various head group structures can be used, including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, and phosphatidylserine (PS), containing saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. The sterols and sterol derivatives to be used may include cholesterol, brassosterol, allocholesterol, cholesterol methyl ether, campestanol, campesterol, cholesterol acetate, coprostanol, chain sterol, dehydrochain sterol, dihydrocholesterol, dihydrolanosterol, epicholesterol, lathosterol, lanosterol, sitostanol, stigmasterol, zymostenol, and zymosterol.

[0082] Liposomes that can be used in the disclosed methods may contain modified phospholipids. For example, sphingolipids and glycerophospholipids may be modified with small molecules, polyethylene glycol (PEG), fluorescent molecules, fluorescent PEG, and / or bromine. Sphingolipids and glycerophospholipids, sterols, sterol derivatives, and modified forms of lipids are readily available commercially from various sources, such as Sigma-Aldrich (St. Louis, MO); Invitrogen (Carlsbad, CA); Avanti Polar Lipids (Alabaster, AL); Fisher Scientific (Pittsburgh, PA); and Steraloids (Newport, RI).

[0083] In some implementations, the liposomes are ruptured, and the amount of signal generated by the encapsulated hydrophilic AE is measured.

[0084] In some embodiments, the modified liposomes of this disclosure are used to detect peptides and / or nucleic acids. For example, DNA or RNA probes are labeled using ligands, such as haptens or biotinylated modified nucleotides. The DNA or RNA probe is allowed to hybridize with complementary DNA or RNA and immobilized on a solid support. The immobilized probe then reacts with a modified liposome containing a receptor (e.g., an antibody; or, if the probe is biotinylated, an avidin). The liposome is ruptured, and the amount of signal generated by the encapsulated acridinium ester is measured.

[0085] In some embodiments, the capture-binding coupler used in the method for detecting an analyte in a sample according to this disclosure further includes a support. In further embodiments, the support is a non-magnetic particle, a magnetic particle, a plate, or a tube.

[0086] In some embodiments, the analyte is captured by means known in the art. These means include immunoassay devices and methods that may utilize labeled molecules in various sandwich, competitive, or other assay formats. Such assays will produce a signal indicating the presence or absence of a peptide or polypeptide. Furthermore, the signal intensity may preferably be directly or indirectly correlated (e.g., inversely proportional) with the amount of polypeptide present in the sample. Further suitable methods include measuring peptide- or polypeptide-specific physical or chemical properties, such as their precise molecular weight or NMR spectrum. These methods include, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers, NMR analyzers, or chromatographic devices. Further, methods include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g., Siemens platforms such as ADVIA Centaur). ® XPT, ADVIA Centaur ® XP, ADVIA Centaur ® CP, IMMULITE ® 1000, IMMULITE ® 2000 XPi and Atellaca ® ), enzyme-catalyzed cobalt binding assay (CBA) and latex agglutination assay.

[0087] Specific hybridization can be performed under highly stringent or moderately stringent conditions, depending on the circumstances. In a preferred embodiment, the hybridization conditions for specific hybridization are highly stringent. Specific hybridization is then detected using standard methods, if present. If specific hybridization occurs between the nucleic acid probe and a gene in the test sample, the sequence present in the nucleic acid probe is also present in the subject's mRNA. More than one nucleic acid probe may also be used.

[0088] In some embodiments, the method of this disclosure further includes a washing step prior to the step of examining the culture medium for the bound analyte.

[0089] In other embodiments of the method disclosed herein, the sample, the conjugation reagent, the connector reagent, the amplification reagent, and optionally, the capture binding partner are combined simultaneously or sequentially.

[0090] Reagent test kit

[0091] In certain aspects of the methods of this disclosure, a kit is provided. The kit of this disclosure includes: (a) a conjugation reagent; (b) a linker reagent; (c) an amplification reagent; and optionally, a capture binding pair, wherein the conjugation reagent comprises a detection binding pair of a target analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first and second small molecules.

[0092] The kit disclosed herein can be used to detect the presence of an analyte in a sample. In some embodiments, the analyte will include an antigen, antibody, peptide, or polypeptide of interest.

[0093] In some embodiments, the kit includes a set of probes. The probe set includes a large number or a small number of probes for detecting an analyte of interest (e.g., a peptide). The probe set may also include a large number or a small number of probes that detect peptides that do not provide information about the analyte of interest. Such probes can be used for controls and standardization (e.g., incorporated labels).

[0094] The probe assembly can be a dry mixture or a mixture in solution. In some embodiments, the probe assembly can be immobilized to a solid substrate to form a probe array. The probes can be antibodies, or nucleic acids (e.g., DNA, RNA, chemically modified forms of DNA and RNA), LNAs (locked nucleic acids), or PNAs (peptide nucleic acids), or any other polymeric compound capable of specifically interacting with the analyte of interest.

[0095] It is intended that kits can be designed for the isolation and / or detection of analytes in virtually any sample (e.g., urine, blood, etc.), and various reagents and methods are known in the art with reference to this specification.

[0096] The following examples further illustrate various aspects of the invention. However, they are not intended to limit the teachings or disclosure set forth herein.

[0097] Explanatory implementation plan

[0098] This document provides illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of this disclosure or the appended claims.

[0099] Implementation Scheme 1. A method for detecting an analyte in a sample, the method comprising: (a) combining the sample with a conjugation reagent, a linker reagent, an amplification reagent, and optionally, a capture binding pair of the analyte, in a culture medium; and (b) examining the culture medium for a bound analyte comprising an analyte bound to the conjugation reagent, the conjugation reagent binding to a linker reagent bound to the amplification reagent, wherein the conjugation reagent comprises a detection binding pair of the analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first small molecule and the second small molecule.

[0100] Implementation Scheme 2. A kit comprising: (a) a conjugation reagent; (b) a linker reagent; (c) an amplification reagent; and optionally, a capture binding pair, wherein the conjugation reagent comprises a detection binding pair of a target analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent encapsulated by liposomes or bound to a carrier protein, wherein the liposomes or carrier protein contain a second small molecule on their surface, and wherein the linker reagent comprises a binding pair of the first small molecule and the second small molecule.

[0101] Implementation Scheme 3. The method of Implementation Scheme 1 or the kit of Implementation Scheme 2, wherein the detection binding coupler of the analyte comprises an antibody that specifically binds to the analyte.

[0102] Implementation Scheme 4. The method or kit according to any one of the preceding implementation schemes, wherein the first small molecule and the second small molecule comprise biotin.

[0103] Implementation Scheme 5. The method or kit according to Implementation Scheme 4, wherein the binding partner of the small molecule comprises streptavidin.

[0104] Implementation Scheme 6. The method or kit of any one of Implementation Schemes 1-3, wherein the first small molecule and the second small molecule comprise fluorescein.

[0105] Implementation Scheme 7. The method or kit of Implementation Scheme 6, wherein the binding coupler of the small molecule comprises an antifluorescein antibody.

[0106] Implementation Scheme 8. The method or kit of any of the preceding implementation schemes, wherein the capture binding couple of the analyte further comprises a support.

[0107] Implementation Scheme 9. The method or kit described in Implementation Scheme 8, wherein the support is a non-magnetic particle, a magnetic particle, a plate, or a tube.

[0108] Implementation Scheme 10. The method of any one of Implementation Schemes 1, 3, 4, 5, 6, 7, 8 and 9, further comprising a washing step prior to the step of examining the culture medium for the bound analytes.

[0109] Implementation Scheme 11. The method or kit of any of the preceding implementation schemes, wherein the conjugation reagent further comprises a labeling reagent.

[0110] Implementation Scheme 12. The method or kit of any of the preceding implementation schemes, wherein the connector reagent comprises a labeling reagent.

[0111] Implementation Scheme 13. The method or kit described in any of the preceding implementation schemes, wherein the carrier protein comprises bovine serum albumin (BSA).

[0112] Implementation Scheme 14. The method or kit of any of the preceding implementation schemes, wherein the labeling reagent comprises acridinium ester (AE).

[0113] Implementation Scheme 15. The method of any one of Implementation Schemes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, wherein the sample, the conjugation reagent, the connector reagent, the amplification reagent, and optionally, the capture binding partner are combined simultaneously or sequentially.

[0114] Implementation Scheme 16. The method or kit of any of the preceding implementation schemes, wherein the diameter of the liposomes is from about 20 nm to about 1000 nm.

[0115] Implementation Scheme 17. The method or kit of Implementation Scheme 14, wherein the encapsulated AE has a content of at least 1 x 10 -8 mol / L to at least 1 x 10 -6 Concentration in the range of mol / L.

[0116] Implementation Scheme 18. The method or kit of Implementation Scheme 14, wherein the liposomes encapsulate about 1,000 to about 100,000,000,000 hydrophilic AE molecules.

[0117] Implementation Scheme 19. The method or kit of Implementation Scheme 14, wherein the carrier protein binds at least 1 to about 100 AE molecules.

[0118] Example

[0119] The following examples are provided to further describe some of the embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0120] Materials and methods

[0121] reagents

[0122] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC); 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC); 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS); porcine brain sphingomyelin (SM); cholesterol (CHOL); 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine-N-[Biotinyl (PEG)-2000](PEG 2000) Biotin-DSPE); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(Biotinyl) (Biotin-DPPE); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(Rhodamine-DPPE); N-(Fluorescence-5-thiocarbamoyl)-1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (Fluorescence-DHPE); N-sulfopropyl-dimethylacridinium ester-N-hydroxysuccinimide (NSP-DMAE) and trimethylsilylpropionate DMAE (TSP-DMAE).

[0123] Lipids are stored at -20°C. Various polycarbonate membrane filters with pore sizes of 30, 50, 100, 200, and 400 nanometers are used.

[0124] Preparation and purification of liposomes encapsulating acridinium esters

[0125] To prepare 4 mM large monolayer liposome vesicles (LUVs) encapsulating acridinium esters, lipids (SM or DPPC or POPC or DOPC or SM / POPC 1 / 1 or DPPC / POPC 1 / 2 or POPC / POPS 3 / 1) were mixed and dried under nitrogen, followed by drying under high vacuum for at least 2 hours. The amount of cholesterol used in the liposomes varied between 0 and 50 mol% depending on the specific experiment. All lipid mixtures contained 0.025 mol% rhodamine PE to track the final concentration of the liposomes. The dried lipid membranes were dispersed at 70 °C in phosphate-buffered saline (PBS, 137 mM NaCl, pH 7.4) containing NSP-DMAE or TSP-DMAE, and then cooled to room temperature before use. The concentrations of NSP-DMAE and TSP-DMAE varied between 0 and 15 mg / mL. The lipid mixture was subjected to 10 freeze-thaw cycles and then extruded through polycarbonate filters with specific pore sizes (e.g., 30 nm, 50 nm, 100 nm, 200 nm, and 400 nm) to obtain uniform liposome sizes. NAP-5 (Sephadex G-25) columns were used to remove any uncollected NSP-DMAE or TSP-DMAE. Dynamic light scattering (DLS) measurements were performed before and after NAP-5 column purification. The particle size distribution of the resulting liposomes showed that the average diameter of the liposomes remained intact after the purification steps.

[0126] Liposomes encapsulating AEs were prepared with various functional groups present on the liposome surface. Biotin-DPPE, PEG 2000 biotin-DPPE, or fluorescein-DPPE were added to lipid mixtures, i.e., SM, DPPC, POPC, DOPC, SM / POPC 1 / 1, DPPC / POPC 1 / 2, or POPC / POPS 3 / 1, as described above, with or without cholesterol, and the lipids were then dried under nitrogen. The amount of cholesterol varied between 0 and 50 mol%. The amount of biotin-DPPE, PEG 2000 biotin-DPPE, or fluorescein-DPPE used in the liposomes varied between 0 and 20 mol%. The permeability and hydrophilicity of the liposome surface were particularly enhanced by the addition of polyethylene glycol (PEG).

[0127] Example 1: Features of a Connected System

[0128] This article provides methods and kits for detecting analytes in samples using a connection system.

[0129] like Figure 1As shown, streptavidin can be used as a linker protein. Strepavidin is a tetrameric binding protein capable of binding four biotin molecules and can also be labeled with signal-generating molecules (AEs). The specific assay antibody is biotinylated and can also be labeled with signal-generating molecules ("labeled second Ab"). In this case, the amplifier can be a biotinylated liposome encapsulating AEs ("AE-carrying amplifier") or a hapten, such as AE(n)-BSA-Biotin(n). Typical biotinylated liposomes (100 nm inner diameter) can easily carry more than 1000 AE molecules, while smaller haptens AE(n)-BSA-Biotin(n) can have up to 20-30 AE molecules / BSA. In both cases, the number of biotin molecules can be much smaller, but preferably at least two, to enable cross-linking with the linker protein. Strepavidin links the specific assay antibody to the amplifier and further links the amplifier to more amplifiers in a chain reaction, thereby amplifying the signal in the system. Figure 1 The schematic diagram shows the use of two binding sites on streptavidin, as well as two free sites that can bind two additional amplifying agents.

[0130] like Figure 2 As shown, in some embodiments, the assay system includes a solid-phase compartment containing a capture-binding pair of the analyte and a solid support (solid-phase reagent or "SPR"). The luminescent reagent (LR) compartment of the assay system, which generates a signal, contains an LR antibody or an antibody that is also biotinylated. One or more LR antibodies may or may not be labeled with an AE, but must be biotinylated. Some LR antibodies may benefit from not being labeled with an AE, which is generally more hydrophobic than molecules such as biotin or fluorescein. The LR compartment may also contain a biotinylated amplifier carrying significantly more AE molecules (…). Figure 2 The assay system further includes a third reagent compartment, such as an auxiliary well ("AW"), to contain separately adaptor proteins and / or AE-labeled adaptor proteins, which will be introduced into the assay to mix with the luminescent reagent. The optimal molar ratio of all components involved should be determined experimentally. An example could be 1:1:1 (LR Ab: adaptor protein: amplifier).

[0131] like Figure 3As shown, anti-fluorescein antibodies can be used as adaptor proteins. Anti-fluorescein antibodies (monoclonal or polyclonal) are binary binding proteins capable of binding two fluorescein molecules and can also be used to generate molecular AE labels with signal generation. Specific assay antibodies are fluoresceinized and can also be used to generate molecular AE labels with signal generation (“labeled second Ab”). In this case, the amplifier can be a fluoresceinized liposome encapsulating the AE (“AE-carrying amplifier”) or a hapten, such as AE(n)-BSA-fluorescein(n). The anti-fluorescein antibody links the specific assay antibody to the amplifier and further links the amplifier to more amplifiers in a chain reaction, thereby amplifying the signal in the system.

[0132] like Figure 4 As shown, the luminescent reagent (LR) compartment of the assay system contains an LR antibody or an antibody that is also fluoresceinized. One or more LR antibodies may or may not be labeled with an AE, but must be fluoresceinized. Some specific LR antibodies may benefit from not being labeled with an AE, which is generally more hydrophobic than molecules such as biotin or fluorescein. As shown, the LR compartment now also contains a fluoresceinized amplifier carrying significantly more AE molecules. A new reagent compartment must be added to the assay to contain only an adaptor protein and / or an AE-labeled adaptor protein, which will be introduced into the assay to mix with the luminescent reagent. The molar ratio of all components involved should be determined experimentally. An example could be 1:1:1 (LR Ab: adaptor protein: amplifier).

[0133] Example 2: A linker protein and amplifying agent-based linkage system allows for amplification of immunoassay signals.

[0134] Liposomes of various sizes (20-1000 nm) can be used in the methods and kits disclosed herein. Liposomes encapsulated with AE may also contain additional modifications. These modifications include, but are not limited to, the addition of various functional groups, such as biotin, fluorescein, and / or proteins, to the liposome surface (Figure 5).

[0135] Biotinylated liposomes encapsulating AE (“Biotin-AE vesicles”) can bind to streptavidin-coated magnetic latex particles (Dynal’s M270 particles). Figure 6 The binding shown by the output RLU is proportional to the amount of biotinylated liposomes encapsulating the added AE. Figure 6 Experiment A in the experiment). Reducing the number of M270 particles lowers the output signal ( Figure 6 Experiment B in the experiment).

[0136] like Figure 7As shown, the addition of the adaptor protein streptavidin enhances signal output. In Experiment A, anti-FL paramagnetic particles (pmps) were used to capture biotinylated and fluoresceinized liposomes encapsulating AE. The addition of the adaptor protein streptavidin generated and amplified the signal. In Experiment B (Experiment B in the table), increased adaptor protein concentration led to increased signal amplification.

[0137] Furthermore, competitive binding between fluorescein and fluoresceinized AE vesicles with anti-FL paramagnetic particles (pmps) was observed, confirming that fluoresceinized AE-encapsulated liposomes are functional. Figure 8 ).

[0138] The connection system (i.e., amplification system) disclosed herein is practical and appears to have no specific limitations on signal amplification (see [link]). Figure 9 Using a sensor chip immobilized with fluoresceinized BSA, a protein hapten (anti-FL Mab (2H1) conjugated to neutral avidin) was first added to the chip, followed by two injections of biotinylated microvesicles (Stage 1 amplification). Additional neutral avidin was introduced to allow binding of even more biotinylated microvesicles (Stage 2 amplification). Finally, Stage 3 amplification was performed, which was a simple repetition of Stage 2 amplification. Figure 9 ).

[0139] like Figure 10 As shown, the connectivity system of this disclosure can be implemented in automated clinical systems (e.g., systems for tracking thyroid-stimulating hormone (TSH)). Particles immobilized with anti-TSH Pab (polyclonal antibody) bind to the TSH antigen, which subsequently forms a sandwich structure with biotinylated anti-TSH Mab (monoclonal antibody), and the addition of AE-labeled streptavidin generates a signal.

[0140] like Figure 11 As shown, the unlabeled linker protein streptavidin, placed in a separate reagent compartment ("AW" refers to the auxiliary well), allows for the attachment of biotinylated anti-TSH Mab to the amplifying agent (AE-BSA-Biotin) during the assay. This condition was compared to a control in which AE is directly attached to anti-TSH Mab.

[0141] The methods and kits disclosed herein can be implemented using various immunoassay platforms known in the art, such as, but not limited to, Siemens platforms (e.g., ADVIA Centaur). ® XPT, ADVIA Centaur ® XP, ADVIA Centaur ® CP, IMMULITE ®1000, IMMULITE ® 2000 XPi and Atellaca ® ), or General Electric Healthcare platforms (such as Biacore) ® The methods and kits disclosed herein allow for a significant increase in immunoassay signal or relative optical units (RLU) and optimize assay sensitivity.

[0142] The publications of every patent, patent application, and publication cited in this article are incorporated herein in their entirety by reference.

[0143] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the invention, and such changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the invention.

Claims

1. A method for detecting an analyte in a sample, the method comprising: (a) In a culture medium, the sample is simultaneously combined with a conjugation reagent, a linker reagent, an amplification reagent, and a capture-binding pair of the analyte; and (b) Examining the culture medium for a bound analyte comprising an analyte bound to the conjugating reagent, the conjugating reagent binding to a linker reagent bound to the amplifying reagent, wherein the conjugating reagent comprises a detection binding pair of the analyte and a first small molecule, wherein the amplifying reagent comprises a labeling reagent bound to a carrier protein, wherein the carrier protein comprises a second small molecule on its surface, and wherein the linker reagent comprises a binding pair of the first and second small molecules, wherein the labeling reagent comprises an acridinium ester; The first and second small molecules include fluorescein, and the binding coupler of the small molecules includes an antifluorescein antibody.

2. The method of claim 1, wherein the detection binding coupler of the analyte comprises an antibody that specifically binds to the analyte.

3. The method of claim 1, wherein the capture binding couple of the analyte further comprises a support.

4. The method of claim 3, wherein the support is a non-magnetic particle, a magnetic particle, a plate, or a tube.

5. The method of claim 1, further comprising a washing step prior to the step of examining the culture medium for the bound analyte.

6. The method of claim 1, wherein the conjugation reagent further comprises a labeling reagent.

7. The method of claim 1, wherein the connector reagent comprises a marking reagent.

8. The method of claim 1, wherein the carrier protein comprises bovine serum albumin.

9. The method of claim 1, wherein the sample, the conjugation reagent, the connector reagent, the amplification reagent, and the capture binding partner are combined sequentially.

10. The method of claim 1, wherein the carrier protein binds at least 1 to 100 acridine ester molecules.

11. A reagent kit comprising: (a) Conjugation reagent; (b) Connector reagent; (c) Amplification reagents; and (d) Capture binding coupler, wherein the conjugation reagent comprises a detection binding coupler of the target analyte and a first small molecule, wherein the amplification reagent comprises a labeling reagent that binds to a carrier protein, wherein the carrier protein comprises a second small molecule on its surface, and wherein the linker reagent comprises a binding coupler of the first small molecule and the second small molecule, wherein the labeling reagent comprises an acridinium ester. The first and second small molecules include fluorescein, and the binding coupler of the small molecules includes an antifluorescein antibody.

12. The kit of claim 11, wherein the detection binding coupler of the analyte comprises an antibody that specifically binds to the analyte.

13. The kit of claim 11, wherein the capture binding couple of the analyte further comprises a support.

14. The kit of claim 13, wherein the support is a non-magnetic particle, a magnetic particle, a plate, or a tube.

15. The kit of claim 11, wherein the conjugation reagent further comprises a labeling reagent.

16. The kit of claim 11, wherein the adapter reagent comprises a labeling reagent.

17. The kit of claim 11, wherein the carrier protein comprises bovine serum albumin.

18. The kit of claim 11, wherein the carrier protein binds at least 1 to 100 acridine ester molecules.

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