Improving signal-to-noise ratio in immunoassays

Abstract

The present disclosure includes specific steps in immunoassays, such as chemiluminescence-based immunoassays, that increase assay sensitivity based on various metrics, including increased signal-to-noise ratio and reduced detection limit. One of these enhanced strategies involves movement and potential isolation of a solid phase in a trigger reagent prior to addition of a second trigger reagent for chemiluminescence.

Description

[0001] Public domain

[0002] The present disclosure relates to immunoassays and methods and systems thereof.This strategy is particularly advantageous for chemiluminescence-based immunoassays, such as those involving acridinium chemiluminescence.

[0003] background

[0004] Immunoassays for detecting target analytes in biological samples are usually operated by using a solid support with an analyte or a binding partner for the analyte fixed thereon. In a competitive immunoassay format, the target analyte in the biological sample competes for binding to a conjugate with a detectable label and / or binding partner. In a sandwich assay format, the target analyte (and usually an antigen) is bound to two layers of a binding partner (e.g., an antibody) for the analyte, one of which may be fixed and the other conjugated to a label. Extracting the solid phase after competitive binding and measuring the amount of the label attached thereto in a controlled manner can give information about the amount of analyte in the biological sample.

[0005] The amount of the marker measured generally depends on what the marker itself is. For example, a chemiluminescent assay involves the conjugation of a chemiluminescent moiety to an analyte for competitive binding. The chemiluminescence can be measured after the solid phase is collected to determine the amount of the labeled conjugate that has competitively bound to the solid support. By adding one or more triggering reagents to the chemiluminescent sample, chemiluminescence can be induced, resulting in an amount of light output related to the amount of the chemiluminescent moiety in the sample. Acridinium-based conjugates, such as acridinium esters and acridinium sulfonamides, generally rely on triggering with alkaline peroxides. These can be added sequentially in the form of two triggering reagents: a first triggering reagent comprising an acid such as H2O2 and a second alkaline triggering reagent (such as sodium hydroxide).

[0006] These assays have inherent limitations associated with them. Various factors can reduce the signal-to-noise ratio, leading to assay inconsistencies and less accurate results. Assays also have inherent limits of detection (LOD), where analyte concentrations below a certain level cannot be distinguished from noise associated with the assay itself.

[0007] There is a continuing need for methods and systems for immunoassays and sample detection that improve the signal-to-noise ratio, providing more accurate and robust immunoassays.

[0008] Overview

[0009] In accordance with the foregoing and other purposes, the present disclosure provides systems and methods that provide benefits to immunoassays, for example, methods and systems for immunoassays and sample detection that improve signal-to-noise ratios, providing more accurate and robust immunoassays. For example, using the systems and methods described herein improves signal-to-noise ratios or reduces detection limits compared to otherwise identical assay protocols. The present disclosure is based in part on increasing particle migration through a chemiluminescent triggering reagent prior to applying a second chemiluminescent reagent.

[0010] Methods are provided, such as methods for immunoassays or methods for initiating or inducing chemiluminescence, which may include: a) adding a first chemiluminescent reagent (e.g., a triggering reagent, such as an acid, which may include hydrogen peroxide, nitric acid, or a combination thereof) to a reaction vessel containing magnetizable particles (e.g., paramagnetic particles, superparamagnetic particles, ferromagnetic particles, ferrimagnetic particles); and b) moving the magnetizable particles to different locations in the reaction vessel (e.g., as measured relative to the container of the reaction vessel) through the first chemiluminescent reagent. For example, in embodiments, the magnetizable particles may be moved by applying a magnetic field to the reaction vessel, which may result in sequestration and migration of the particles or substantially all of the particles in the first chemiluminescent reagent that induces chemiluminescence. In some embodiments, the magnetizable particles or substantially all of the magnetizable particles are sequestered in the reaction vessel during or after the adding step by applying a magnetic field to the reaction vessel. In various embodiments, during the adding step, the magnetizable particles or substantially all of the magnetizable particles are not sequestered in the reaction vessel (e.g., the particles become suspended in the first chemiluminescent reagent, and no magnetic field is applied to the reaction vessel).

[0011] By adding these methods and systems to a chemiluminescent immunoassay, the measurement capability is generally improved. In particular, the particle migration step can occur sequentially or before promoting and inducing chemiluminescence by adding a second chemiluminescent reagent such as a base (e.g., an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide). In some embodiments, the method can further include: c) separating the liquid medium from the magnetizable particles after moving the magnetizable particles; and / or d) adding the second chemiluminescent reagent to the separated liquid medium (e.g., in another reaction vessel) or to the separated magnetizable particles. In some embodiments, the method can include: c) adding the second chemiluminescent reagent to the magnetizable particles and optionally to the existing liquid medium (e.g., the first chemiluminescent reagent) after moving the magnetizable particles. The movement and isolation capabilities provided by the magnetizable particles can be used at any reaction location (e.g., any chemiluminescent reagent addition location or any particle movement location). For example, in some embodiments, during the addition of the second chemiluminescent reagent, the magnetizable particles can be clustered in the reaction vessel (e.g., the reaction vessel has a magnet close to it to induce clustering or accumulation). In various embodiments, the reaction vessel is symmetrical about the main longitudinal axis during the addition of the second chemiluminescent reagent (e.g., cylindrical, such as a cuvette or test tube), and the second chemiluminescent reagent is added at a position other than the main longitudinal axis. In embodiments, this can reduce the fragmentation of particle clusters when adding the second chemiluminescent reagent.

[0012] Also provided are systems for inducing a chemiluminescent reaction from a chemiluminescent sample comprising magnetizable particles (eg, paramagnetic particles, superparamagnetic particles, ferromagnetic particles, ferrimagnetic particles). For example, the system may include: an array of consecutive reaction positions for chemiluminescent samples, such that the chemiluminescent sample (or a medium derived therefrom) can be sequentially placed at each reaction position (e.g., by moving a reaction container (e.g., a cuvette, a test tube) containing a material such as a magnetizable particle and / or a liquid medium between each reaction position, by aspirating the liquid medium in a first reaction container and depositing it into a second reaction container); the array of consecutive reaction positions includes: a first chemiluminescent reagent addition position, wherein the first chemiluminescent reagent can be added to the chemiluminescent sample; one or more (e.g., one to ten, one to five, two, three, four, five, six, seven, eight, nine, ten) particle movement positions, wherein a magnetic field different from that of a previous position (e.g., a first chemiluminescent reagent addition position, a previous particle movement position) can be applied to the chemiluminescent sample to induce movement of the magnetizable particles in the first chemiluminescent reagent; a second chemiluminescent reagent addition position, wherein a second chemiluminescent reagent is added to the chemiluminescent sample (or a portion thereof or a portion derived therefrom) after one or more particle movement positions, so as to induce a chemiluminescent reaction by the chemiluminescent sample (or a portion thereof or a portion derived therefrom).

[0013] In embodiments, the disclosed system can place a reaction vessel and / or sample at an indicated position along a reaction process or protocol. The migration of particles through a first chemiluminescent reagent can be considered a reaction position. For example, a reaction vessel can travel along a track, wherein different elements such as a magnetic field (including an alternating magnetic field at a continuous reaction position) can be applied to an indicated sample in the reaction vessel.

[0014] In various embodiments, the array can include a liquid transfer station, wherein after one or more particle transfer stations, the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration with a pipette); and a second chemiluminescent reagent station adds a second chemiluminescent reagent to the separated liquid sample (e.g., in a reaction vessel different from the reaction vessel used in the first chemiluminescent station, such as a cuvette or test tube). In some embodiments, the second chemiluminescent reagent is added to the chemiluminescent sample containing the magnetizable particles (e.g., in the same reaction vessel, such as a cuvette or test tube, to which the first chemiluminescent sample is added).

[0015] In embodiments, during the addition of the first chemiluminescent reagent, a sample such as a biological sample may be contained in a reaction vessel (e.g., a cuvette, a test tube), and during the addition of the first chemiluminescent reagent addition position, the reaction vessel has a magnet adjacent thereto (e.g., so as to isolate the magnetizable particles along the inner wall of the reaction vessel). In some embodiments, moving the magnetizable particles occurs by moving the reaction vessel relative to the magnet (e.g., rotating, for example, 170-190° or 180°). In various embodiments, moving the magnetizable particles or substantially all of the magnetizable particles occurs by moving the reaction vessel into different magnetic fields generated by different magnet groups. In some embodiments, moving the magnetizable particles occurs by moving the magnet relative to the sample (e.g., the sample contained in the reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field generated by one or more magnets near the sample (e.g., changing electrical parameters, such as the voltage or current of the electromagnet).

[0016] In embodiments, the method and system are most beneficial when chemiluminescence is triggered and / or promoted by adding a variety of chemiluminescent reagents. For example, acridinium chemiluminescence generally involves the addition of a first chemiluminescent reagent (e.g., an acid in a solvent, such as hydrogen peroxide, nitric acid, or a combination thereof), which induces oxidation of the acridinium system. The reaction can be accelerated by adding a second chemiluminescent reagent such as a base, which changes the pH of the system to an alkaline state, forming an alkaline peroxide sufficient to trigger and induce chemiluminescence. In various embodiments, the first chemiluminescent reagent is acidic (e.g., a reagent comprising an acid such as hydrogen peroxide, nitric acid, or a combination thereof), and optionally includes a detergent (e.g., a cationic detergent, such as a quaternary nitrogen or phosphorus-based salt). In some embodiments, the second chemiluminescent reagent is alkaline (e.g., a reagent comprising a base such as an alkali metal hydroxide (e.g., sodium hydroxide)), and optionally includes a detergent (e.g., a cationic detergent, such as a quaternary nitrogen or phosphorus-based salt).

[0017] In embodiments, the methods and systems of the present disclosure can be used in an immunoassay format for detecting an analyte of interest. For example, the immunoassay can involve a competitive heterogeneous assay in which a chemiluminescent conjugate competes with an analyte in a biological sample for binding sites on a solid phase magnetizable particle. For example, the method may further comprise forming a sample by mixing the magnetizable particles having a molecule capable of forming a binding complex with the target analyte or its binding partner immobilized thereon with a biological sample (e.g., serum, urine) and an assay reagent comprising a chemiluminescent conjugate (e.g., an acridinium compound, such as an acridinium ester or an acridinium sulfonamide) capable of forming a binding complex with the molecule immobilized on the magnetizable particles; and optionally incubating the biological sample, the magnetizable particles and the chemiluminescent conjugate; isolating the magnetizable particles in the reaction vessel by applying a magnetic field to the reaction vessel; adding a wash buffer (e.g., a buffer solution that may contain one or more salts, such as sodium chloride and sodium azide, a detergent, such as a cationic detergent, a buffer, such as a phosphate, a blocking agent, such as bovine serum albumin (BSA), or a combination thereof) to the magnetizable particles; and separating the liquid medium (e.g., from the biological sample, from the assay reagent, from the first chemiluminescent reagent, from the second chemiluminescent reagent, from the wash buffer, from a combination thereof) from the magnetizable particles (e.g., the isolated magnetizable particles).

[0018] In various embodiments, the wash buffer is added when the magnetizable particles are dispersed throughout the liquid medium. In some embodiments, the wash buffer is added when the magnetizable particles are isolated (e.g., by applying a magnetic field to the reaction vessel). For example, the method may include at least two wash buffer additions, including a first wash buffer addition, wherein the magnetizable particles are dispersed throughout the liquid medium when the wash buffer is added; and a second wash buffer addition, wherein the magnetizable particles are isolated during the wash buffer addition. In some embodiments, the method further includes measuring the chemiluminescent light output (e.g., using a photomultiplier tube, such as a photometer) after adding the second chemiluminescent reagent.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 1A and 1B ) shows the process involved in adding an assay reagent ( Figure 1A ) and aspirating the liquid medium and adding reagents to those magnetizable particles (e.g. Figure 1B ) The top image in each individual step is a view of the reaction vessel from the side, and the bottom image is a view of the reaction vessel from the top.

[0021] Figure 2 Exemplary particle migration induced by rotation of a reaction vessel is provided.

[0022] Figure 3 An exemplary particle migration involving the use of a second magnet is shown.

[0023] Figure 4 A schematic diagram of particle motion involving multiple magnets is provided.

[0024] Figure 5 Figure 5A , 5B 5C) shows the reaction position associated with adding a second chemiluminescent reagent. The addition of the second chemiluminescent reagent can occur near the PMT (e.g., in a luminometer) for subsequent measurement of chemiluminescence (e.g., the rightmost inset in each figure).

[0025] Figure 6 is a flow chart illustrating the steps of an exemplary immunoassay of the present disclosure.

[0026] Figure 7 Fig. 7A and 7B ) provides an array (or sample holder) and a linear ( Figure 8 A) and ring ( Figure 8 B) Elevation view of the sequence of reaction vessels through which the format passes.

[0027] Figure 8The measurement results of the three assay protocols at two different analyte concentrations were compared.

[0028] Figure 9 Fig.9A and 9B ) showed that particle migration enabled detection below the detection limit of the standard assay performed at 0, 0.003 and 0.006 ng / ML. Fig.9A Concentrations measured using the disclosed immunoassay were compared, showing that measurement resolution and complete separation from noise (0 ng / mL) can be achieved below the detection limit of the standard assay (0.006 ng / mL). Fig. 9B The same measurements using the standard assay protocol are shown below its detection limit, highlighting that no statistical separation can be achieved between measurements with noise when run on the standard assay.

[0029] Figure 10 Fig. 10A and 10B ) shows that particle migration enables detection below the detection limit of the standard assay performed at 0 and 0.004 ng / mL. Fig. 10A Concentrations measured using the disclosed immunoassay were compared, showing that measurement resolution and complete separation from noise (0 ng / mL) can be achieved below the detection limit of the standard assay (0.006 ng / mL). Fig. 10B The same measurements using the standard assay protocol are shown below its detection limit, highlighting that no statistical separation can be achieved between measurements with noise when run on the standard assay.

[0030] Fig.11 An exemplary block diagram of a computer system 1100 suitable for performing the methods of the present disclosure on a chemical analyzer is depicted. DETAILED DESCRIPTION OF THE INVENTION

[0032] Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the present disclosure, and the present disclosure can be implemented in various forms. In addition, each example given in conjunction with the various embodiments of the present disclosure is intended to be illustrative rather than restrictive.

[0033] Unless otherwise provided, all terms used herein are intended to have their ordinary meaning in the art. Unless otherwise defined, all concentrations are in weight percent of the designated component relative to the total weight of the subject composition.

[0034] As used herein, "a" or "an" shall mean one or more. As used herein, when used in conjunction with the word "comprising", the word "a" or "an" shall mean one or more than one. As used herein, "another" shall mean at least a second or more.

[0035] The use of the term "at least one" will be understood to include one and any number greater than one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, an amount of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. Furthermore, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z.

[0036] The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing between two or more items and, unless expressly stated otherwise, is not meant to imply any order or sequence or importance, for example, of one item relative to another or any order of addition.

[0037] The term "or" is used in the claims to mean an inclusive "and / or" unless explicitly stated to refer to only alternatives or unless the alternatives are mutually exclusive. For example, the condition "A or B" satisfies any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0038] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. For example, the appearance of the phrase "in some embodiments" or "an example" in various places in the specification does not necessarily refer to the same embodiment. In addition, all references to one or more embodiments or examples should be interpreted as non-limiting to the claims.

[0039] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the composition / device / apparatus, the method used to determine the value, or the variation that exists between the subjects of study. For example, but not as a limitation, when the term "about" is used, a specified value can vary from the specified value by plus or minus 20%, or 15%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, as such variations are appropriate for performing the disclosed methods and as understood by those of ordinary skill in the art.

[0040] The term "antibody" is used in the broadest sense herein and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the desired analyte binding biological activity (e.g., but not limited to Fab, Fab', F(ab')2, Fv, scFv, Fd, diabodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of an intact antibody), antibody surrogate proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. Antibodies can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0041] Detection agent: As used herein, the term "detection agent" refers to any element, molecule, functional group, compound, fragment, or moiety that can be detected. In some embodiments, the detection agent is provided or utilized alone. In some embodiments, the detection agent is provided and / or utilized in conjunction with (e.g., linked to) another agent. Examples of detection agents include, but are not limited to, various ligands, radionuclides (e.g., 3 H. 14 C. 18 F. 19 F. 32 P. 35 S. 135 I. 125 I. 123 I. 64 Cu, 187 Re, 111 In, 90 Y. 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (such as acridinium esters, stable dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes, colorimetric markers (such as dyes, colloidal gold, etc.), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0042] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherently present therein.

[0043] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will appreciate that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0044] As used herein, the term "substantially" means that the subsequently described event or situation occurs completely or that the subsequently described event or situation occurs to a large extent or in scope. For example, when related to a particular event or situation, the term "substantially" means that the subsequently described event or situation occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are closely adjacent to each other, but not 100% adjacent, or that a portion of one of the two items is not 100% adjacent to the other item, but that the other item is closely adjacent.

[0045] As used herein, the phrase "associated with" includes direct association of two moieties with each other as well as indirect association of two moieties with each other. Non-limiting examples of association include covalent binding of one moiety to another through a direct bond or through a spacer group, non-covalent binding of one moiety to another directly or through a specific binding pair member bound to the moiety, mixing of one moiety into another (e.g., by dissolving one moiety in the other or by synthesis), and coating one moiety on another.

[0046] The term "biological fluid sample" as used herein should be understood to include any liquid test sample that can be obtained from a patient and used according to the present disclosure. Examples of available biological fluid samples include, but are not limited to, whole blood or any portion thereof (i.e., plasma or serum), serum, EDTA plasma, lithium heparin plasma, combinations thereof, and the like.

[0047] As used herein, the term "volume" as it relates to a liquid test sample used in accordance with the present disclosure generally refers to a liquid test sample volume of about 0.1 μl to about 100 μl, or about 1 μl to about 75 μl, or about 2 μl to about 60 μl, or a value less than or equal to about 50 μl, or the like.

[0048] The term "specific binding partner", as used herein specifically (but not by way of limitation) in the term "target analyte specific binding partner", will be understood to refer to any molecule capable of specifically associating with a target analyte. For example, but not limited to, the binding partner may be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), a combination or derivative thereof, and any other molecule capable of specifically binding to a target analyte.

[0049] The term "immunoassay" as used herein refers to an assay for determining the presence of an analyte in a biological sample by reacting the sample with an antibody (or fragment thereof) that specifically binds to the analyte, wherein the reaction is carried out under conditions such that an immune complex is formed between the antibody (or fragment thereof) and the analyte for a period of time such that an immune complex is formed between the antibody (or fragment thereof) and the analyte. In embodiments, a quantitative assay of such immune complexes may then be performed.

[0050] As used herein, all numerical ranges include all possible values ​​disclosed between the endpoints and the disclosed values. The exact values ​​of all half-integer values ​​are also considered to be specifically disclosed and as limitations of all subsets of the disclosed ranges. For example, a range of 0.1% to 3% specifically discloses percentages of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. In addition, a range of 0.1 to 3% includes subsets of the original range, including 0.5% to 2.5%, 1% to 3%, 0.1% to 2.5%, etc. It should be understood that the sum of all weight % of a single component does not exceed 100%.

[0051] In the context of a composition, "consisting essentially of" means that the ingredients include only the listed components, as well as normal impurities present in commercially available materials and any other additives present at a level that does not affect the operation of the embodiments disclosed herein (e.g., at a level of less than 5 wt %, or less than 1 wt %, or even 0.5 wt %). A composition comprising the indicated components may consist primarily of the indicated components (e.g., the indicated components have the highest weight percentage in the composition, such as greater than 40 wt %, greater than 50 wt %, greater than 60 wt %, greater than 70 wt %, greater than 80 wt %, greater than 90 wt %), consist essentially of the indicated components, and consist of the indicated components.

[0052] The present disclosure is based in part on adding steps to immunoassay protocols that can enhance those assays. Typically, these steps involve the migration (and possible isolation or re-isolation) of magnetizable particles through a first chemiluminescent reagent, such as a triggering reagent (e.g., an acidic reagent comprising H2O2), prior to the addition of a second chemiluminescent reagent (e.g., an alkaline reagent such as an alkali metal hydroxide in a solvent).

[0053] Now refer to Figure 1 ( Figure 1A and 1B ) - magnetizable particles having molecules capable of forming binding complexes with target analytes or their binding partners immobilized thereon, examples of isolated magnetizable particles can be seen, showing a view of the reactor from the side and a view of the reactor from the top in three stages (or reaction positions in the system). Figure 1AA sequential progression from incubation of the sample and freely dispersed reagents (left), migration of the magnetizable particles in a magnetic field (center) and isolation (right) is shown. A reaction vessel 1 comprises a chemiluminescent sample 2 formed by mixing a biological sample (e.g., a biological fluid sample such as blood, serum, saliva or urine) with magnetizable particles having an analyte immobilized thereon or a binding partner for a target analyte in the biological sample, and one or more detection agents such as a chemiluminescent compound including an acridinium compound such as an acridinium ester or an acridinium sulfonamide capable of forming a complex with the immobilized binding partner. The magnetizable particles may be suspended in a freely dispersed state in a liquid medium 2 comprising a liquid component of the biological sample and an assay reagent (e.g., a solvent for the chemiluminescent compound). In the depicted embodiment, the acridinium compound and the analyte in the sample competitively bind to the binding partner on the particles for further analysis in an assay. In some embodiments, the chemiluminescent sample 2 has been further processed with one or more washes using a wash buffer, as described in U.S. Pat. No. 6,143,578, which is incorporated herein by reference in its entirety, and particularly with respect to reaction sites involving sample preparation, washing, and resuspension wash sites. In some embodiments, the methods and systems of the present disclosure may employ a resuspension wash, which may involve aspirating the liquid phase containing the unbound components of the assay while the bound components are held in place by a magnet, followed by reintroduction of the wash solution into the reaction vessel.

[0054] Removing the liquid phase can result in a portion of the liquid phase in any reaction vessel or at any reaction location remaining in the reaction vessel. For example, if the reaction vessel is pumped to remove the liquid phase, some portion of the liquid phase can remain in the reaction vessel, as understood in the case of pumping. In various embodiments, pumping can occur without taking further steps to dry any of the reaction locations shown. In various embodiments, during the process of removing the liquid phase (e.g., pumping), more than 70% or more than 80% or more than 90% or more than 95% or more than 99% of the liquid phase can be removed.

[0055] from Figure 1A As can be seen in FIG, after binding and / or washing, the reaction vessel 1 can be exposed to a magnetic field generated by a magnet 10, resulting in the accumulation of magnetizable particles for subsequent preparation of a chemiluminescent sample for chemiluminescence initiation (and light detection). In the depicted embodiment, the magnetizable particles are attracted to the magnet 10, so that the concentration of magnetizable particles 2 formed near the magnet 10 is increased compared to the concentration formed in the rest of the medium 3. Finally, the particles are isolated 4 after exposure to the magnet 10 for an appropriate period of time, separated from the liquid medium 5.

[0056] Isolation does not require isolation of all magnetizable particles in the sample. For example, isolation may involve the accumulation of more than 60% or more than 70% or more than 80% or more than 90% or more than 95% or more than 99% of the magnetizable particles in the sample. After isolation, the liquid medium 5 can be removed from the reaction vessel 1 by an aspirator 20 (e.g. a tube, a pipette), leaving the magnetizable particles 4 isolated near the magnet 10 in the reaction vessel 1. These magnetizable particles can be regarded as a chemiluminescent sample for subsequent initiation of a chemiluminescent reaction and final measurement of the chemiluminescence. Figure 1B In the rightmost reaction position shown, a nozzle 25 is oriented above a reaction vessel 1 containing isolated magnetizable particles (or chemiluminescent samples). The nozzle 25 is in fluid communication with a reservoir of a first chemiluminescent reagent (e.g., an initiator comprising H2O2, nitric acid, or a combination thereof and a solvent) and adds the chemiluminescent reagent to the reaction vessel via a spray 27, forming a deposit 15 of the first chemiluminescent reagent in the reaction vessel. The nozzle 25 is positioned to centrally add the first chemiluminescent reagent 27 by spreading on the main longitudinal axis 6 of the reaction vessel 1. The deposit 15 can initiate the process of reacting with the chemiluminescent compound immobilized on the magnetizable particles 4 to initiate the chemiluminescent reaction. For example, when the chemiluminescent particles are acridinium compounds, the first chemiluminescent reagent can include an acid (e.g., hydrogen peroxide, nitric acid, a combination thereof) and initiate an oxidation reaction in the chemiluminescent reaction scheme. In various embodiments, the first chemiluminescent reagent can cleave or release some of the chemiluminescent compound immobilized on the particles.

[0057] It has been found that the migration or movement of particles through the deposit 15 of the first chemiluminescent reagent enhances the immunoassay including the systems and methods described herein. For example, it is shown herein that the migration of particles through the first chemiluminescent reagent improves the signal-to-noise ratio of the chemiluminescent output (e.g., compared to the otherwise identical method or system not involving particle migration). Without wishing to be bound by theory, the migration of the first chemiluminescent reagent can shorten the exposure of particles to reagents (e.g., acids) and / or reduce the amount of non-specific binding released from beads. In some embodiments, the signal-to-noise ratio can be improved by more than 1.2 (e.g., more than 1.3, more than 1.4, 1.1-5, 1.1-4.5) compared to the otherwise identical assay without any bead migration in the first chemiluminescent reagent.

[0058] Figure 2An exemplary step of inducing particle migration is provided. After preparing the isolated particles 4 in the first chemiluminescent reagent 15 (e.g., a trigger reagent, such as an acid reagent including hydrogen peroxide), the reaction vessel can move in a magnetic field so that the magnetizable particles are subjected to different forces (e.g., magnetic force, normal force). For example, the reaction vessel can be rotated 16, for example, 180 °. This motion causes the offset of the magnetic field and the normal force that the magnetizable particles are subjected to. By rotating the reaction vessel and exposing the particles to different forces, the particles move through the first chemiluminescent reagent as shown in arrow 17, causing re-accumulation in the reaction vessel near the magnet.

[0059] Any reaction location or step that involves exposing the magnetizable particles to an altered magnetic field (compared to the immediately preceding step or reaction location) to promote migration through the first chemiluminescent reagent can be used. For example, in some embodiments, the system and method can involve exposing the reaction vessel to different magnets, the different magnets having, for example, different magnetic fields generated by different magnets (e.g., electromagnets) or different positioning of one or more magnets relative to the reaction vessel and / or the magnetizable particles (e.g., isolated magnetizable particles isolated by a preceding step, freely dispersed magnetizable particles from a preceding step). In some embodiments, the system and method can induce migration by exposing the reaction vessel to two or more magnets (e.g., electromagnets). For example, the magnetic field can be changed by independently moving two or more (e.g., two to four) magnets relative to the reaction vessel and / or applying different electrical properties (e.g., current, voltage) to any electromagnets used to change the magnetic field. Reference is now made to Figure 3 --exist Figure 1B or Figure 2 The possible particle migration position after the final reaction position of the reaction vessel, which involves isolation from the magnet 10. In the depicted particle migration reaction position, the magnet 10 moves away from the reaction vessel, reducing the magnetic field contribution from the magnet. The second magnet 12 is close to the opposite edge of the reaction vessel. The magnetic field change induced by this change causes the previously isolated particles to move toward the magnet 12 located on the opposite side of the reaction indicated by the arrow 18 (for example, the opposite side measured in a plane perpendicular to the longitudinal axis). In some embodiments, the process is repeated one or more times, wherein at each subsequent particle migration position, a magnet is brought closer (and / or the field strength on the reaction vessel is increased), and the second magnet is moved farther (and / or the field strength on the reaction vessel is reduced). In some embodiments, the magnets are on opposite sides. In various embodiments, the field strength of two or more magnets is changed independently, wherein the magnets are distributed around the periphery of the reaction vessel.

[0060] like Figure 4As shown, the accumulated particles 4 can be pre-accumulated at this position due to the interaction with the magnetic field (e.g., magnet 10, magnet 12). The reaction vessel can then enter a reaction position, where the magnetizable particles are exposed to a second magnet 13, which is positioned in a manner to induce movement as shown by arrow 19 through the first chemiluminescent reagent. Magnets 10 and 13 can be moved close to the reaction vessel or away from the reaction vessel in order to control the movement of particles through the first chemiluminescent reagent. In some embodiments, magnets 10 and 13 are, for example, in fixed positions, and the magnetic field from each magnet is independently controlled by changing its electrical properties (e.g., current, voltage) to induce particle migration through the liquid medium. The migration step and the reaction position (e.g., prepared by applying different magnetic fields relative to the reaction vessel) can be performed one or more times (e.g., two or more times, three or more times, four or more times, five or more times, 2 to 10 times) before adding the second chemiluminescent reagent. For example, as Figure 4 As shown, after interacting with the magnet 13 in the final step indicated on the right, the reaction vessel and the magnetizable particles can migrate a second time through the liquid medium 15 by being exposed to a magnetic field from the magnet 10 in a subsequent step (e.g. by bringing the magnet 10 closer to the reaction vessel and / or moving the magnet 13 further away from the reaction vessel).

[0061] Exemplary chemiluminescence induction by adding a second chemiluminescent reagent can be Figure 5A . On the left is shown the magnetizable particles 4 having migrated through the first chemiluminescent reagent deposit 15, ending up isolated adjacent the magnet 12. In the depicted embodiment, the first chemiluminescent reagent deposit 15 is aspirated from the reaction vessel (middle), the reaction vessel is moved to the photometer, and a second chemiluminescent reagent 32 (e.g., a base) is deposited 33 to promote chemiluminescence from the magnetizable particles 4. In some embodiments, the liquid medium 33 contains both the first chemiluminescent reagent and the second chemiluminescent reagent (e.g., the first chemiluminescent reagent is not aspirated before adding the second chemiluminescent reagent, the first chemiluminescent reagent is not completely aspirated before adding the second chemiluminescent reagent). Figure 5B , a process is shown in which aspiration of the first chemiluminescent reagent deposit 15 is not performed and a second chemiluminescent reagent 32 is added directly from an off-center nozzle 30 into the reaction vessel 1 to form a liquid deposit 34. The liquid deposit 34 can be a combination of a first chemiluminescent reagent (e.g., an acid), a second chemiluminescent reagent (e.g., a base), and any chemiluminescent compounds (e.g., acridinium) that are removed from the particles during the addition of these reagents (e.g., by cleaving ester or sulfonamide groups during chemiluminescence).

[0062] from Figure 5A and 5BIt can be seen that the second chemiluminescent reagent can be added at a position outside the main longitudinal axis of the reaction vessel (or off-center addition) through the nozzle 30. In particular, in embodiments where the final chemiluminescent sample measured includes isolated magnetizable particles, such off-center addition of the second chemiluminescent (e.g., base) reagent can reduce disaggregation of the isolated particles during reagent addition compared to central addition. Such reduction in disaggregation can improve assay consistency and accuracy.

[0063] Chemiluminescence can also be induced by a liquid sediment aspirated from the reaction vessel 110 that does not contain magnetizable particles. Figure 5C , the liquid deposit 15 formed by adding the first chemiluminescent reagent to the magnetizable particles and their subsequent particle movement is used to measure chemiluminescence and evaluate the concentration of the analyte in the original biological sample. The liquid deposit 15 (or a portion thereof) can be aspirated from the reaction vessel 1 by an aspirator 22 and separated from the magnetizable particles. Subsequently, the liquid deposit 15 can be added to a new reaction vessel 24 by, for example, a nozzle 26, which is operably connected to the aspirator 22 and sprayed 28 into the reaction vessel 24, so as to form a liquid deposit 16 in the new reaction vessel. The liquid deposit 16 can be all or a portion of the material from the liquid deposit 15 (e.g., a specific volume of the liquid deposit 15, such as 1 μL to 100 mL). Before adding the liquid deposit 16, the new reaction vessel 24 can be positioned in a photometer. Subsequently, a nozzle 30 can be positioned above the reaction vessel 24 so that a second chemiluminescent reagent (e.g., a base) is sprayed 32 onto the liquid deposit 16 to induce chemiluminescence. Typically, a second chemiluminescent reagent is added to a reaction vessel in a luminometer for chemiluminescence measurements.

[0064] exist Figure 6 ( Figure 6 An exemplary flow chart of the process employed by embodiments of the present disclosure is shown in Figures 6A and 6B. Each step can be viewed as an independent reaction location where the system of the present disclosure can be operated to induce the indicated reaction conditions (e.g., adding reagents, isolating particles, moving particles throughout the indicated reagents). Steps 100-120 involve steps for processing and reacting a biological sample to form a chemiluminescent sample for subsequent spectral analysis (e.g., as shown in Figures 6A and 6B). Figure 1A and 1B ). As can be seen, the particles can be washed one or more times with the wash buffer.

[0065] After the magnetizable particles are prepared, a first chemiluminescent reagent may be added at step 130. Step 140 may involve Figure 2-4The one or more particles shown in the figure move. These steps may include moving one or more magnets relative to the reaction vessel, for example one to five times. After the particle migration, a second chemiluminescent reagent may be added to induce (or promote) chemiluminescence. The second chemiluminescent reagent may be added to a sample, for example, in a photometer. Particle migration may occur one or more times as shown by arrow 142. In some embodiments, a second chemiluminescent reagent may be added to a reaction vessel containing magnetizable particles and chemiluminescent reagents that have moved through the first chemiluminescent reagent (e.g., step 150). In some embodiments, a portion or all of the liquid medium in the reaction vessel may be aspirated, and a second chemiluminescent material may be added to the first reaction vessel (e.g., step 145 followed by step 150). In some embodiments, after aspirating the first chemiluminescent reagent, a second chemiluminescent reagent may be added to a reaction vessel containing magnetizable particles (e.g., step 145 followed by step 155). For example, the first chemiluminescent reagent through which the particles have migrated at step 150 can be aspirated into a new reaction vessel at step 145, the new reaction vessel can be placed in a luminometer, the second chemiluminescent reagent can be added to the first chemiluminescent reagent at step 155, and the chemiluminescence can be measured at step 160.

[0066] Magnets are generally those devices capable of generating a magnetic field that may affect magnetizable particles. For example, the magnets may be a pair of adjacent magnets of opposite polarity. The magnets may be, for example, electromagnets.

[0067] Now refer to FIG. 7 ( Fig. 7A and 7B ), an exemplary array can be seen. Position AP can be considered a reaction position for performing the indicated steps described herein. A reaction vessel can be inserted into a slot (e.g., a slot present at each position) and translated along a track. The assay can involve lateral translation of the sample (e.g., by moving along the track to a corresponding position), which allows for indicated reaction positions or conditions. In some embodiments, subsequent reaction positions may not involve lateral translation of the sample. For example, at Fig. 7A , reaction positions AP illustrate a series of positions along a linear tract where samples can be manipulated. At reaction position A, a biological sample and an assay reagent including a chemiluminescent conjugate and a magnetizable particle having an analyte or a binding partner for the analyte immobilized thereon can be added to a reaction vessel. The reaction vessel can be moved along the tract to position B, where the magnetizable particles begin to be isolated by a magnet 40, e.g., Figure 1AAs shown. The translation through positions BE can each involve the isolation of magnetizable particles. At some positions, such as at position F, liquid culture medium can be removed, and at position G, washing buffer can be added. Position H, including a magnet 42 further separated from the reaction vessel, can disperse the magnetizable particles and resuspend or redisperse in washing buffer. Position IN can involve additional washing steps (for example, at position M). Position N can draw washing buffer from the reaction vessel. At position O, a first chemiluminescent reagent (for example, a trigger reagent, such as acid) can be added to the reaction vessel. At position P, the isolation of magnetizable particles can be affected by magnets 44 and / or 46 (for example, depending on their relative position to the reaction vessel, depending on their field strength, and a combination thereof). At position P, the presence of two magnets enables the sample to undergo one or more particle movement positions. For example, the movement from position O to P can cause the first particle to move. The manipulation of the relative positions of magnets 44 and 46 can also cause one or more particle motion reaction positions without any translational movement of the reaction vessel. In certain embodiments, any particle movement reaction position may involve re-accumulation of magnetizable particles in the reaction vessel. After the particle migration step, the reaction vessel at position P may be moved (e.g., with a translation arm) to a luminometer, where a second chemiluminescent reagent is added and chemiluminescence is measured. In various embodiments, liquid medium is aspirated from the reaction vessel at position P and added to a new reaction vessel already placed or to be placed in the luminometer for chemiluminescence measurement. Figure 7B Similar embodiments are provided for tracks of potential reaction positions in a circular turntable or ring format, wherein as the sample moves around the ring, it is exposed to magnets 50, 52, 54, and 56. After the reaction vessel moves through position AP, chemiluminescence can be ultimately induced by adding a second chemiluminescent reagent to an appropriate medium. The addition can occur near a photomultiplier tube for light collection, such as in a photometer. The steps described herein can be generated by multiple channels, such as adjacent turntables, each turntable providing any indicated reaction position. In various embodiments, the system includes a diverter for moving reaction vessels between adjacent turntables.

[0068] Now refer to Fig.11 , shows a block diagram of a computer system 1100 that can be used in the operation of embodiments of the present disclosure. The system 1100 includes a processor 1110, a memory 1120, a storage device 1130, and an input / output device 1140. Each of the components 1110, 1120, 1130, and 1140 are interconnected using a system bus 1150. The system may include an analytical device 1160 for determining the level of one or more analytes of the present disclosure (such as one or more biomarkers in a sample).

[0069] In an embodiment, processor 1110 is capable of processing instructions for execution within system 1100. In one embodiment, processor 1110 is a single-threaded processor. In another embodiment, processor 1110 is a multi-threaded processor. Processor 1110 is capable of processing instructions stored in memory 1120 or stored on storage device 1130, including instructions for receiving or sending information through input / output device 1140.

[0070] In an embodiment, memory 1120 stores information within system 1100. In one embodiment, memory 1120 is a computer readable medium. In one embodiment, memory 1120 is a volatile memory unit. In another embodiment, memory 1120 is a non-volatile memory unit.

[0071] In an embodiment, storage device 1130 is capable of providing mass storage for system 1100. In one embodiment, storage device 1130 is a computer-readable medium or includes non-transitory computer-readable media in accordance with the present disclosure.

[0072] In an embodiment, input / output device 1140 provides input / output operations for system 1100. In one embodiment, input / output device 1140 includes a keyboard and / or a pointing device. In one embodiment, input / output device 1140 includes a display unit for displaying a graphical user interface.

[0073] The system 1100 can be used to build a database. In an embodiment, the method of the present disclosure is performed in the system 1100 disposed in a chemical analyzer. For example, a computer program product can include instructions that cause the processor 1110 to perform the steps of the method of the present disclosure.

[0074] In addition, a non-transitory computer-readable medium containing executable instructions is provided, which, when executed, causes a processor to perform operations including the methods provided herein. For example, a non-transitory computer-readable medium containing executable instructions, which, when executed, causes a processor to perform operations including the methods described herein. In an embodiment, the non-transitory computer-readable medium includes a hard drive, an external hard drive, an optical disk, a CD, a DVD, etc. storing data. In an embodiment, the software disposed in the physical medium is applicable to this invention.

[0075] In some embodiments, a non-transitory computer-readable medium containing executable instructions is provided, which when executed causes a processor to perform operations including a method, the method comprising a process sequence, such as: a) adding a first chemiluminescent reagent (e.g., a trigger reagent, such as an acid) to a reaction vessel containing magnetizable particles (e.g., paramagnetic particles); and b) moving the magnetizable particles to different positions in the reaction vessel (e.g., as measured relative to the container of the reaction vessel) through the first chemiluminescent reagent.

[0076] In an embodiment, the present disclosure includes one or more articles of manufacture, such as a system or a component thereof, which includes a non-transitory computer-readable medium having instructions encoded thereon, the instructions being configured to cause one or more processors to perform the methods of Embodiments 1-19 as described below.

[0077] In some embodiments, a non-transitory computer-readable medium containing executable instructions that, when executed, cause a processor to perform operations including one or more of embodiments 1-19 described below.

[0078] Implementation

[0079] Embodiment 1: A method embodiment comprising: a) adding a first chemiluminescent reagent (e.g., a trigger reagent such as an acid) to a reaction vessel containing magnetizable particles (e.g., paramagnetic particles); and b) moving the magnetizable particles to different locations in the reaction vessel (e.g., as measured relative to the vessel of the reaction vessel) through the first chemiluminescent reagent.

[0080] Embodiment 2. A method according to embodiment 1, wherein the magnetizable particles are moved by applying a magnetic field to the reaction vessel.

[0081] Embodiment 3. The method according to embodiment 1 or embodiment 2, wherein the magnetizable particles are isolated in the reaction vessel during or after the adding step by applying a magnetic field to the reaction vessel.

[0082] Embodiment 4. The method according to any one of embodiments 1-3, wherein during the adding step, the magnetizable particles are not isolated in the reaction vessel (e.g., the particles become suspended in the first chemiluminescent reagent and no magnetic field is applied to the reaction vessel).

[0083] Embodiment 5. The method according to any one of embodiments 1 to 4, further comprising: c) separating the liquid medium from the magnetizable particles after moving the magnetizable particles; and / or d) adding a second chemiluminescent reagent to the separated liquid medium (e.g. in another reaction vessel) or to the separated magnetizable particles.

[0084] Embodiment 6. The method according to any one of embodiments 1 to 4, further comprising: c) adding a second chemiluminescent reagent to the magnetizable particles and optionally to the liquid medium present (eg, the first chemiluminescent reagent) after moving the magnetizable particles.

[0085] Embodiment 7. A method according to Embodiment 6, wherein during the addition of the second chemiluminescent reagent, the magnetizable particles are clustered in the reaction vessel (eg, the reaction vessel has a magnet proximate thereto to induce clustering or accumulation).

[0086] Embodiment 8. A method according to Embodiment 7, wherein the reaction vessel is symmetrical about the main longitudinal axis during the addition of the second chemiluminescent reagent (e.g., cylindrical, such as a cuvette or test tube), and the second chemiluminescent reagent is added at a position other than the main longitudinal axis.

[0087] Embodiment 9. A method according to any one of embodiments 1-8, wherein during the addition of the first chemiluminescent reagent, a sample is contained in a reaction container (e.g., a cuvette, a test tube), and during the addition of the first chemiluminescent reagent addition position, the reaction container has a magnet adjacent thereto (e.g., so as to isolate magnetizable particles along the inner wall of the reaction container).

[0088] Embodiment 10. The method according to embodiment 9, wherein moving the magnetizable particles occurs by moving (eg rotating, for example, 170-190° or 180°) the reaction vessel relative to the magnet.

[0089] Embodiment 11. The method according to embodiment 9 or 10, wherein moving the magnetizable particles occurs by moving the reaction vessel into different magnetic fields generated by different sets of magnets.

[0090] Embodiment 12. A method according to any one of embodiments 1 to 11, wherein moving the magnetizable particles occurs by moving the magnet relative to the sample (e.g., the sample contained in the reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field generated by one or more magnets near the sample (e.g., changing electrical parameters, such as the voltage or current of the electromagnet).

[0091] Embodiment 13. A method according to any one of embodiments 1-12, wherein the first chemiluminescent reagent is acidic (e.g., a reagent comprising an acid such as hydrogen peroxide, nitric acid, or a combination thereof), and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0092] Embodiment 14. A method according to any one of embodiments 1-13, wherein the second chemiluminescent reagent is alkaline (e.g., a reagent comprising a base such as an alkali metal hydroxide (e.g., sodium hydroxide)) and optionally comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0093] Embodiment 15. The method according to any one of embodiments 1 to 14, further comprising forming a sample by mixing the magnetizable particles having a molecule capable of forming a binding complex with the target analyte or its binding partner immobilized thereon with a biological sample (e.g., serum, urine) and an assay reagent comprising a chemiluminescent conjugate (e.g., an acridinium compound, such as an acridinium ester or an acridinium sulfonamide) capable of forming a binding complex with the molecule immobilized on the magnetizable particles; and optionally incubating the biological sample, the magnetizable particles and the chemiluminescent conjugate; and reacting the sample with the chemiluminescent conjugate by reacting the chemiluminescent conjugate with the target analyte or its binding partner immobilized thereon. The invention relates to a method for isolating magnetizable particles in a reaction vessel by applying a magnetic field to the vessel; adding a wash buffer (e.g., a buffer solution that can contain one or more salts such as sodium chloride and sodium azide, a detergent such as a cationic detergent, a buffer such as a phosphate, a blocking agent such as bovine serum albumin (BSA) or a combination thereof) to the magnetizable particles; and separating the liquid medium (e.g., from the biological sample, from the assay reagent, from the first chemiluminescent reagent, from the second chemiluminescent reagent, from the wash buffer, from a combination thereof) from the magnetizable particles (e.g., the isolated magnetizable particles).

[0094] Embodiment 16. The method according to embodiment 15, wherein a wash buffer is added when the magnetizable particles are dispersed throughout the liquid medium.

[0095] Embodiment 17. A method according to embodiment 15, wherein a wash buffer is added while the magnetizable particles are isolated (e.g. by applying a magnetic field to the reaction vessel).

[0096] Embodiment 18. The method according to any one of embodiments 15 to 17, wherein the method comprises at least two wash buffer additions, comprising a first wash buffer addition, wherein the magnetizable particles are dispersed throughout the liquid medium when the wash buffer is added; and a second wash buffer addition, wherein the magnetizable particles are sequestered during the wash buffer addition.

[0097] Embodiment 19. A method according to any one of embodiments 1-18, wherein the method further comprises measuring the chemiluminescent light output (e.g., using a photomultiplier tube, such as a photometer) after adding a second chemiluminescent reagent.

[0098] Embodiment 20. A system for inducing a chemiluminescent reaction by a chemiluminescent sample comprising magnetizable particles (e.g., paramagnetic particles, superparamagnetic particles, ferromagnetic particles, ferrimagnetic particles), wherein the system comprises: an array of consecutive reaction positions for the chemiluminescent sample, such that the chemiluminescent sample (or a medium derived therefrom) can be sequentially placed in each reaction position (e.g., by moving a reaction vessel (e.g., a cuvette, a test tube) containing a material such as a magnetizable particle and / or a liquid medium between each reaction position, by aspirating the liquid medium in a first reaction vessel and depositing it in a second reaction vessel); the array of consecutive reaction positions comprises: a first chemiluminescent reagent addition position, wherein ... a first chemiluminescent reagent is added to the chemiluminescent sample; one or more (e.g., one to ten, one to five, two, three, four, five, six, seven, eight, nine, ten) particle movement positions, wherein a magnetic field different from that of a previous position (e.g., first chemiluminescent reagent addition position, previous particle movement position) can be applied to the chemiluminescent sample to induce movement of magnetizable particles in the first chemiluminescent reagent; a second chemiluminescent reagent addition position, wherein a second chemiluminescent reagent is added to the chemiluminescent sample (or a portion thereof or a portion derived therefrom) after the one or more particle movement positions, so as to induce a chemiluminescent reaction by the chemiluminescent sample (or a portion thereof or a portion derived therefrom).

[0099] Embodiment 21. A system according to embodiment 20, wherein the array comprises a liquid transfer position, wherein after one or more particle movement positions, the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration with a pipette); and a second chemiluminescent reagent tank position adds a second chemiluminescent reagent to the separated liquid sample (e.g., in a reaction vessel different from the reaction vessel used in the first chemiluminescent position, such as a cuvette or test tube).

[0100] Embodiment 22. A system according to embodiment 20, wherein a second chemiluminescent reagent is added to the chemiluminescent sample comprising the magnetizable particles (e.g., in the same reaction vessel, such as a cuvette or test tube, to which the first chemiluminescent sample is added).

[0101] Embodiment 23. A system according to embodiment 22, wherein the magnetizable particles are clustered in the reaction vessel at the second chemiluminescent reagent addition location (eg, the reaction vessel at the second chemiluminescent location has a magnet proximate thereto to induce clustering).

[0102] Embodiment 24. A system according to embodiment 23, wherein the reaction vessel is symmetrical about the main longitudinal axis at the second chemiluminescent position (e.g., cylindrical, such as a cuvette or test tube), and the second chemiluminescent reagent is added at a position other than the main longitudinal axis.

[0103] Embodiment 25. A system according to any one of embodiments 20-24, wherein at the first chemiluminescent reagent addition position, the chemiluminescent sample is contained in a reaction container (e.g., a cuvette, a test tube), and the first chemiluminescent reagent is added to the reaction container.

[0104] Embodiment 26. A system according to Embodiment 25, wherein during the addition of the first chemiluminescent reagent addition location, the reaction vessel has a magnet adjacent thereto (e.g., to isolate magnetizable particles along the inner wall of the reaction vessel).

[0105] Embodiment 27. A system according to embodiment 25 or 26, wherein at least one different magnetic field is generated on the chemiluminescent sample at at least one particle movement position by rotating the reaction vessel (e.g., moving the rotation by, for example, 170-190° or 180°).

[0106] Embodiment 28. A system according to any one of Embodiments 25-27, wherein at least one different magnetic field at at least one particle movement location is generated by moving the reaction vessel into different magnetic fields generated by different magnet groups.

[0107] Embodiment 29. A system according to any one of embodiments 20-28, wherein at least one different magnetic field at at least one particle movement position is generated by moving the magnet relative to the sample (e.g., the sample contained in a reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field generated by one or more magnets near the sample (e.g., changing electrical parameters, such as the voltage or current of the electromagnet).

[0108] Embodiment 30. A system according to any one of embodiments 20-29, wherein the first chemiluminescent reagent is acidic (e.g., a reagent comprising an acid such as hydrogen peroxide, nitric acid, or a combination thereof) and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0109] Embodiment 31. A system according to any one of embodiments 20-30, wherein the second chemiluminescent reagent is alkaline (e.g., a reagent comprising a base such as an alkali metal hydroxide (e.g., sodium hydroxide)) and optionally comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0110] Embodiment 32. A system according to any one of embodiments 20-31, wherein the system further comprises a reaction position for forming a chemiluminescent sample from a biological sample, wherein the reaction position for forming a chemiluminescent sample comprises at least one reaction position selected from the following: a biological sample addition position, wherein the biological sample is added to a reaction vessel; an assay reagent position, wherein an assay reagent (e.g., a magnetizable particle having a molecule capable of forming a binding complex with a target analyte or its binding partner immobilized thereon, a chemiluminescent conjugate including acridinium compounds, including acridinium esters or acridinium sulfonamides, or both) is added to a reaction vessel (e.g., a reaction vessel containing the biological sample); an incubation position for allowing the analyte from the biological sample and the chemiluminescent compound to competitively bind to the magnetizable particles; an isolation portion, in which the magnetizable particles are magnetically isolated from the liquid medium of the biological sample and the assay reagent; a separation position, in which the liquid medium (e.g., from the biological sample, from the assay reagent) is separated from the magnetizable particles (e.g., the isolated magnetizable particles); a washing position, in which a washing buffer (e.g., a buffer solution that can contain one or more salts such as sodium chloride and sodium azide, a detergent such as a cationic detergent, a buffer such as a phosphate, a blocking agent such as bovine serum albumin (BSA) or a combination thereof) is added to the magnetizable particles; and a washing buffer aspiration position, in which the washing buffer is separated from the magnetizable particles to form a chemiluminescent sample.

[0111] Embodiment 33. A system according to embodiment 32, wherein the system comprises a wash position, wherein the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added.

[0112] Embodiment 34. A system according to embodiment 32, wherein the system comprises a wash position, wherein the magnetizable particles are isolated during the addition of wash buffer.

[0113] Embodiment 35. A system according to any one of embodiments 32 to 34, wherein the system comprises at least two wash positions, including a first wash position, wherein the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added; and a second wash position, wherein the magnetizable particles are isolated during the addition of the wash buffer.

[0114] Embodiment 36. A system according to any one of embodiments 20-35, wherein the system further comprises a photomultiplier tube (e.g., a luminometer) to measure the chemiluminescent light output after adding a second chemiluminescent reagent.

[0115] Embodiment 37. A system according to any one of embodiments 20-35, wherein the system further comprises a non-transitory computer-readable medium for executing the method of the present disclosure including embodiments 1-19. Example

[0116] The following examples illustrate specific aspects of the present specification. The examples should not be interpreted as limiting, as the examples only provide a specific understanding and practice of the embodiments and various aspects thereof.

[0117] Example 1

[0118] Chemiluminescent beads made of troponin I (TnI) standards of different concentrations were prepared. Similar preparations are disclosed in R.Payne European Heart Journal 38 (2017): ehx502.P2754, which is incorporated herein by reference, particularly with respect to bead preparation and Siemens ADVIA Centaur high-sensitivity troponin I sandwich assay. The standard assay has a detection limit of 0.006 ng / mL, wherein biological samples need to have at least 0.006 ng / mL to measure detectable concentration differences. The beads were washed with wash buffer, and a magnetic field was applied for 60 seconds during the washing process. The wash buffer was removed and acid was added. The beads were completely suspended in acid and magnetically isolated. The acid supernatant was transferred to a new cuvette and alkali was added to generate a signal. The signal-to-noise ratio was measured at four different TnI concentrations in repeated experiments. These results are shown in Table 1, which shows the (±) 1 standard deviation of the measured S / N.

[0119] Table 1

[0120]

[0121] It can be seen that an improvement in the signal to noise ratio is seen when the beads are removed prior to relative light unit measurements.

[0122] The assay was compared to a standard TnI assay (std TnI-Ultra assay - base dispersion in the center) measuring TnI at concentrations of 0 and 0.024 ng / mL. Table 2 provides a comparison of these results, enabling comparison of the signal-to-noise ratios of these measurements that included changes to the assay involving magnetic migration and off-center addition of a second chemiluminescent reagent (base).

[0123] Table 2

[0124]

[0125]

[0126] It can be seen that the modification to the standard assay protocol, including the movement of particles in the first chemiluminescent reagent (acid), results in a reduction in both signal and noise in the RLU measurement (↓). However, the reduction in noise is more significant, resulting in an improved signal-to-noise ratio (↑). In addition, the readings in the new reaction vessel after magnetic switching produce an improved signal-to-noise ratio compared to the readings in the same reaction vessel. Figure 8 Comparison of RLU measured for noise (left side of each scheme) and signal (right side of each scheme) is provided.

[0127] Example 2

[0128] It is shown that the improved signal-to-noise ratio of the systems and methods disclosed in the present invention reduces the limit of detection (LOD) and enables measurements to be lower than the standard assay. Standards of TnI were prepared at 0 and 0.003 and 0.006 ng / mL, which are equal to or lower than the limit of detection (LOD) of the standard assay, and were assayed using a standard protocol in a sandwich assay format and a similar protocol involving magnetic switching and particle migration and deposition of liquid media in a new cuvette. Table 3 provides the results of this comparison and various statistical parameters of the replicate measurements, including the mean value (Avg), the measured standard deviation (SD), the coefficient of variation (as a percentage of the mean), and the signal-to-noise ratio (SNR) at each concentration. Fig.9A The average values ​​of the magnetically switched 0, 0.003, and 0.006 ng / mL measurements are plotted. Fig. 9B The mean values ​​of these measurements using a standard protocol for statistical values ​​are plotted.

[0129] Table 3

[0130]

[0131] TnI standards at 0 and 0.003 ng / mL (below the limit of detection (LOD) of the standard assay) were measured again and assayed using the standard protocol in sandwich assay format as discussed above and a similar protocol involving magnetic switching with particle migration and deposition of liquid media in a new cuvette. Table 4 provides the results of these measurements.

[0132] Table 4

[0133]

[0134] Fig. 10A The average values ​​from the 0 and 0.004 ng / mL measurements of magnetic switching are plotted, and Fig. 10BThe mean values ​​of these measurements of the standard scheme using statistical values ​​are plotted. The error bar represents a deviation standard. It can be seen that in the standard assay, a concentration of 0.004 cannot be distinguished from a concentration of 0. On the contrary, using the method provided herein, magnetic switching enables measurement below the LOD of the standard assay. When magnetic switching is used in immunoassay, RLU values ​​are clearly separable for 0 (mean + 2SD) and 0.004 (mean - 1SD).

[0135] Since various changes can be made to the above-mentioned subject matter without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description or defined in the appended claims be interpreted as describing and illustrating the present disclosure. According to the above teachings, many modifications and variations of the present disclosure are possible. Therefore, this specification is intended to cover all such substitutions, modifications and variations that fall within the scope of the appended claims.

[0136] All documents cited or referenced herein, as well as all documents cited or referenced in documents cited herein, and any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated herein by reference are hereby incorporated by reference and may be used in the practice of the present disclosure.

Claims

1. A method comprising: a) adding a first chemiluminescent reagent (e.g. a trigger reagent such as an acid) to a reaction vessel containing magnetizable particles (e.g. paramagnetic particles); and b) moving the magnetizable particles to a different position in the reaction vessel (as measured relative to the volume of the reaction vessel) through the first chemiluminescent reagent.

2. The method according to claim 1, wherein the magnetizable particles are moved by applying a magnetic field to the reaction vessel.

3. The method according to claim 1 or 2, wherein the magnetizable particles are isolated in the reaction vessel during or after the adding step by applying a magnetic field to the reaction vessel.

4. The method according to any one of claims 1 to 3, wherein during the adding step, the magnetizable particles are not isolated in the reaction vessel (e.g., the particles become suspended in the first chemiluminescent reagent and no magnetic field is applied to the reaction vessel).

5. The method according to any one of claims 1 to 4, further comprising: c) separating the liquid medium from the magnetizable particles after moving the magnetizable particles; and d) Adding a second chemiluminescent reagent to the separate liquid medium (eg in another reaction vessel) or to the separate magnetizable particles.

6. The method according to any one of claims 1 to 4, further comprising: c) After moving the magnetizable particles, adding a second chemiluminescent reagent to the magnetizable particles and optionally to the liquid medium present (eg the first chemiluminescent reagent).

7. The method of claim 6, wherein during the addition of the second chemiluminescent reagent, the magnetizable particles are clustered in the reaction vessel (eg, the reaction vessel has a magnet proximate thereto to induce clustering or accumulation).

8. A method according to claim 7, wherein the reaction container is symmetrical around the main longitudinal axis during the addition of the second chemiluminescent reagent (e.g., cylindrical, such as a cuvette or test tube), and the second chemiluminescent reagent is added at a position other than the main longitudinal axis.

9. The method according to any one of claims 1 to 8, wherein during the addition of the first chemiluminescent reagent, the sample is contained in a reaction container (e.g., a cuvette, a test tube), and during the addition of the first chemiluminescent reagent addition position, the reaction container has a magnet adjacent thereto (e.g., so as to isolate magnetizable particles along the inner wall of the reaction container).

10. The method according to claim 9, wherein moving the magnetizable particles occurs by moving (eg rotating, for example, 170-190° or 180°) the reaction vessel relative to the magnet.

11. The method according to claim 9 or 10, wherein moving the magnetizable particles occurs by moving the reaction vessel into different magnetic fields generated by different sets of magnets.

12. The method according to any one of claims 1 to 11, wherein moving the magnetizable particles occurs by moving a magnet relative to a sample (e.g. a sample contained in a reaction vessel) and / or adjusting (e.g. increasing, decreasing) a magnetic field generated by one or more magnets near the sample (e.g. changing an electrical parameter, such as a voltage or current of an electromagnet).

13. The method of any one of claims 1-12, wherein the first chemiluminescent reagent is acidic (e.g., a reagent comprising an acid such as hydrogen peroxide, nitric acid, or a combination thereof) and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

14. The method of any one of claims 1 to 13, wherein the second chemiluminescent reagent is basic (e.g., a reagent comprising a base such as an alkali metal hydroxide (e.g., sodium hydroxide)) and optionally comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

15. The method according to any one of claims 1 to 14, further comprising forming a sample by mixing magnetizable particles having molecules capable of forming a binding complex with the target analyte or its binding partner immobilized thereon with a biological sample (e.g. serum, urine) and an assay reagent comprising a chemiluminescent conjugate (e.g. an acridinium compound such as an acridinium ester or an acridinium sulfonamide) capable of forming a binding complex with the molecules immobilized on the magnetizable particles; and optionally, incubating the biological sample, the magnetizable particles, and the chemiluminescent conjugate; isolating magnetizable particles in a reaction vessel by applying a magnetic field to the reaction vessel; adding a wash buffer (e.g., a buffer solution that may contain one or more salts such as sodium chloride and sodium azide, detergents such as cationic detergents, buffering agents such as phosphates, blocking agents such as bovine serum albumin (BSA), or combinations thereof) to the magnetizable particles; and The liquid medium (eg, from the biological sample, from the assay reagents, from the first chemiluminescent reagent, from the second chemiluminescent reagent, from the wash buffer, from a combination thereof) is separated from the magnetizable particles (eg, isolated magnetizable particles).

16. The method according to claim 15, wherein a wash buffer is added when the magnetizable particles are dispersed throughout the liquid medium.

17. The method of claim 15, wherein a wash buffer is added while the magnetizable particles are isolated (eg by applying a magnetic field to the reaction vessel).

18. The method according to any one of claims 15 to 17, wherein the method comprises at least two wash buffer additions, comprising a first wash buffer addition, wherein the magnetizable particles are dispersed throughout the liquid medium when the wash buffer is added; and a second wash buffer addition, wherein the magnetizable particles are sequestered during the wash buffer addition.

19. The method according to any one of claims 1 to 18, wherein the method further comprises measuring the chemiluminescent light output (eg, using a photomultiplier tube, such as a luminometer) after adding a second chemiluminescent reagent.

20. A system for inducing a chemiluminescent reaction from a chemiluminescent sample comprising magnetizable particles (e.g. paramagnetic particles), wherein the system comprises: An array of consecutive reaction locations for chemiluminescent samples, such that the chemiluminescent sample (or a medium derived therefrom) can be sequentially placed at each reaction location (e.g. by moving a reaction vessel (e.g., a cuvette, a test tube) containing a material such as magnetizable particles and / or a liquid medium between each reaction location, by aspirating the liquid medium in a first reaction vessel and depositing it in a second reaction vessel); the array of consecutive reaction locations comprising: a first chemiluminescent reagent addition position, wherein the first chemiluminescent reagent can be added to the chemiluminescent sample; one or more (e.g., one to ten, one to five, two, three, four, five, six, seven, eight, nine, ten) particle movement positions, wherein a different magnetic field than a previous position (e.g., a first chemiluminescent reagent addition position, a previous particle movement position) can be applied to the chemiluminescent sample to induce movement of magnetizable particles in the first chemiluminescent reagent; A second chemiluminescent reagent addition position, wherein the second chemiluminescent reagent is added to the chemiluminescent sample (or a portion thereof or a portion derived therefrom) after one or more particles move to a position so as to induce a chemiluminescent reaction by the chemiluminescent sample (or a portion thereof or a portion derived therefrom).

21. A system according to claim 20, wherein the array comprises a liquid transfer station, wherein after one or more particle movement stations, the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration with a pipette); and a second chemiluminescent reagent tank station adds a second chemiluminescent reagent to the separated liquid sample (e.g., in a reaction vessel different from the reaction vessel used in the first chemiluminescent station, such as a cuvette or test tube).

22. The system of claim 20, wherein the second chemiluminescent reagent is added to the chemiluminescent sample comprising the magnetizable particles (e.g., in the same reaction vessel, such as a cuvette or test tube, to which the first chemiluminescent sample is added).

23. The system of claim 22, wherein the magnetizable particles are clustered in a reaction vessel at a second chemiluminescent reagent addition location (eg, a reaction vessel at the second chemiluminescent location has a magnet proximate thereto to induce clustering).

24. A system according to claim 23, wherein the reaction vessel is symmetrical about the main longitudinal axis at the second chemiluminescent position (e.g., cylindrical, such as a cuvette or test tube), and the second chemiluminescent reagent is added at a position other than the main longitudinal axis.

25. The system according to any one of claims 20 to 24, wherein at the first chemiluminescent reagent addition position, the chemiluminescent sample is contained in a reaction container (eg, a cuvette, a test tube), and the first chemiluminescent reagent is added to the reaction container.

26. The system of claim 25, wherein during addition of the first chemiluminescent reagent addition location, the reaction vessel has a magnet adjacent thereto (e.g., to isolate magnetizable particles along an inner wall of the reaction vessel).

27. A system according to claim 25 or 26, wherein at least one different magnetic field is generated on the chemiluminescent sample at at least one particle movement position by rotating the reaction vessel (e.g. moving the rotation by e.g. 170-190° or 180°).

28. A system according to any one of claims 25-27, wherein at least one different magnetic field at at least one particle movement location is generated by moving the reaction container into different magnetic fields generated by different magnet sets.

29. A system according to any one of claims 20-28, wherein at least one different magnetic field at at least one particle movement position is generated by moving the magnet relative to the sample (e.g., the sample contained in a reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field generated by one or more magnets near the sample (e.g., changing electrical parameters, such as the voltage or current of the electromagnet).

30. The system of any one of claims 20-29, wherein the first chemiluminescent reagent is acidic (e.g., a reagent comprising an acid such as hydrogen peroxide, nitric acid, or a combination thereof) and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

31. A system according to any one of claims 20-30, wherein the second chemiluminescent reagent is basic (e.g., a reagent comprising a base such as an alkali metal hydroxide (e.g., sodium hydroxide)) and optionally comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

32. The system of any one of claims 20-31, wherein the system further comprises a reaction site for forming a chemiluminescent sample from a biological sample, wherein the reaction site for forming the chemiluminescent sample comprises at least one reaction site selected from the group consisting of: a biological sample addition location, wherein the biological sample is added to the reaction vessel; an assay reagent position, wherein an assay reagent (e.g., a magnetizable particle having a molecule capable of forming a binding complex with the target analyte or its binding partner immobilized thereon, a chemiluminescent conjugate including acridinium compounds, including acridinium esters or acridinium sulfonamides, or both, capable of forming a binding complex with a molecule immobilized on the magnetizable particle) is added to a reaction vessel (e.g., a reaction vessel containing the biological sample); an incubation location for competitive binding of an analyte from a biological sample and a chemiluminescent compound to the magnetizable particles; an isolation portion in which the magnetizable particles are magnetically isolated from the liquid medium of the biological sample and the assay reagents; a separation location, wherein liquid medium (e.g. from a biological sample, from an assay reagent) is separated from magnetizable particles (e.g. isolated magnetizable particles); a washing position, wherein a wash buffer (e.g., a buffered solution that may contain one or more salts such as sodium chloride and sodium azide, detergents such as cationic detergents, buffering agents such as phosphates, blocking agents such as bovine serum albumin (BSA), or combinations thereof) is added to the magnetizable particles; and A wash buffer aspiration station, wherein the wash buffer is separated from the magnetizable particles to form a chemiluminescent sample.

33. The system of claim 32, wherein the system comprises a wash position, wherein the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added.

34. The system of claim 32, wherein the system comprises a wash position, wherein the magnetizable particles are isolated during addition of wash buffer.

35. The system according to any one of claims 32 to 34, wherein the system comprises at least two washing positions, including a first washing position, wherein the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added; and a second washing position, wherein the magnetizable particles are isolated during the addition of the wash buffer.

36. The system of any one of claims 20-35, wherein the system further comprises a photomultiplier tube (eg, a luminometer) to measure chemiluminescent light output after addition of a second chemiluminescent reagent.

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