Improving signal-to-noise in immunoassays

Abstract

The present disclosure includes specific steps in immunoassays, such as chemiluminescence-based immunoassays, that increase assay sensitivity based on various measures, including increasing signal-to-noise and decreasing detection limits. One strategy for these enhancements involves the movement, and potentially sequestration, of a solid phase in one trigger reagent prior to the addition of a second trigger reagent for chemiluminescence.

Description

[Technical Field]

[0001] The present disclosure relates to immunoassays, and methods and systems therefor. These approaches are particularly advantageous for chemiluminescence-based immunoassays, such as those involving acridinium chemiluminescence. [Background technology]

[0002] Immunoassays for detecting an analyte of interest in a biological sample often work by using a solid support with the analyte or its binding partner immobilized thereon. In competitive immunoassay formats, the analyte of interest in the biological sample competes for binding with a conjugate having a detectable label and / or binding partner. In sandwich assay formats, the analyte of interest (and typically an antigen) binds to two layers of binding partners for the analyte (e.g., antibodies), one of which can be immobilized and the other conjugated to a label. Extraction of this solid phase after competitive binding and controlled measurement of the amount of label attached thereto can provide information about the amount of analyte in the biological sample.

[0003] Measuring the amount of label typically depends on the identity of the label itself. For example, chemiluminescent assays involve conjugation of a chemiluminescent moiety to an analyte in competitive binding. After collection of the solid phase, chemiluminescence can be measured to determine the amount of labeled conjugate competitively bound to the solid support. Chemiluminescence can be induced by adding one or more trigger reagents to the chemiluminescent sample, and the resulting amount of light output correlates to the amount of chemiluminescent moiety in the sample. Acridinium-based conjugates, such as acridinium esters and acridinium sulfonamides, typically rely on alkaline hydrogen peroxide triggering. These can be added sequentially in the form of two trigger reagents: a first trigger reagent containing an acid, such as HO, and a second basic trigger reagent (such as sodium hydroxide).

[0004] These assays have inherent limitations. Various factors can reduce the signal-to-noise ratio, leading to assay inconsistency and imprecise results. Assays also have an inherent limit of detection (LOD), below which analyte concentrations cannot be distinguished from the noise associated with the assay itself. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a continuing need for methods and systems for immunoassays and sample detection that increase signal-to-noise to provide more accurate and robust immunoassays. [Means for solving the problem]

[0006] In accordance with these and other objectives, the present disclosure provides systems and methods that provide benefits to immunoassays, such as methods and systems for immunoassays and sample detection that increase signal-to-noise to provide more accurate and robust immunoassays. For example, using the systems and methods described herein, signal-to-noise is increased or detection limits are reduced compared to otherwise identical assay protocols. The present disclosure is based, in part, on the addition of particle movement through a chemiluminescent trigger reagent prior to the addition of a second chemiluminescent reagent.

[0007] Methods, such as those for use in immunoassays or for initiating or inducing chemiluminescence, are provided, which can include: a) adding a first chemiluminescent reagent (e.g., a trigger reagent such as an acid, which can 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 through the first chemiluminescent reagent to different locations within the reaction vessel (e.g., measured relative to the volume of the reaction vessel). For example, in embodiments, the magnetizable particles can be moved by application of a magnetic field to the reaction vessel, which can result in segregation and movement of the particles or substantially all of the particles within the first chemiluminescent reagent, which initiates chemiluminescence. In some embodiments, the magnetizable particles or substantially all of the magnetizable particles are sequestered within the reaction vessel during or after the adding step by applying a magnetic field to the reaction vessel. In various implementations, the magnetizable particles or substantially all of the magnetizable particles are not sequestered within the reaction vessel during the addition step (e.g., the particles are suspended within the first chemiluminescent reagent and no magnetic field is applied to the reaction vessel).

[0008] The addition of these methods and systems to chemiluminescent immunoassays typically increases measurement capabilities. In particular, the particle movement step can be performed sequentially or can be performed before the promotion and induction of chemiluminescence by the addition of a second chemiluminescent reagent, such as a base (e.g., an alkaline 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 a second chemiluminescent reagent to the separated liquid medium (e.g., in a separate reaction vessel) or the separated magnetizable particles. In some embodiments, the method can include: c) adding a second chemiluminescent reagent to the magnetizable particles and, optionally, to the liquid medium (e.g., the first chemiluminescent reagent) after moving the magnetizable particles. It is possible to utilize the movement and segregation capabilities provided by magnetizable particles at any reaction location, such as any chemiluminescent reagent addition location or any particle movement location. For example, in some embodiments, the magnetizable particles can be clustered within the reaction vessel during addition of the second chemiluminescent reagent (e.g., the reaction vessel has a magnet in its vicinity to induce clustering or accumulation). In various embodiments, the reaction vessel during addition of the second chemiluminescent reagent is symmetrical about its major longitudinal axis (e.g., cylindrical, such as a cuvette or tube), and the second chemiluminescent reagent is added at a location other than the major longitudinal axis. In embodiments, this can reduce breakup of particle clusters when the second chemiluminescent reagent is added.

[0009] Also provided is a system for inducing a chemiluminescent reaction from a chemiluminescent sample containing magnetizable particles (e.g., paramagnetic particles, superparamagnetic particles, ferromagnetic particles, ferrimagnetic particles). For example, the system can include: an array of successive reaction locations for the chemiluminescent sample, 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., cuvette, tube) containing materials such as magnetizable particles and / or liquid medium between each reaction location, aspirating the liquid medium in a first reaction vessel, and depositing it in a second reaction vessel); the array of successive reaction locations can include: a first chemiluminescent reagent addition location at which a first chemiluminescent reagent can be added to the chemiluminescent sample; and a previous location (e.g., a first The particle movement position includes one or more (e.g., 1 to 10, 1 to 5, 2, 3, 4, 5, 6, 7, 8, 9, 10) particle movement positions at which a magnetic field different from that of the 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; and a second chemiluminescent reagent addition position at which, after one or more particle movement positions, a second chemiluminescent reagent is added to the chemiluminescent sample (or a portion or a portion derived therefrom) to induce a chemiluminescent reaction from the chemiluminescent sample (or a portion or a portion derived therefrom).

[0010] In embodiments, the disclosed system can position reaction vessels and / or samples at indicated locations along a reaction progression or plan. The movement of particles through a first chemiluminescent reagent can be considered a reaction location. For example, a reaction vessel can progress along a track, and different elements, such as a magnetic field (including alternating magnetic fields at successive reaction locations), can be applied to indicated samples within the reaction vessel.

[0011] In various implementations, the array can include a liquid transfer position, following one or more particle movement positions, where the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration with a pipette); a second chemiluminescent reagent vessel position adds a second chemiluminescent reagent to the separated liquid sample (e.g., in a reaction vessel such as a cuvette or tube different from the reaction vessel used in the first chemiluminescent position). In some embodiments, the second chemiluminescent reagent is added to the chemiluminescent sample containing magnetizable particles (e.g., in the same reaction vessel such as a cuvette or tube to which the first chemiluminescent sample was added).

[0012] In embodiments, a sample, such as a biological sample, can be contained in a reaction vessel (e.g., a cuvette, a tube) during addition of a first chemiluminescent reagent, the reaction vessel having a magnet adjacent thereto (e.g., to sequester magnetizable particles along an interior wall of the reaction vessel) during addition of the first chemiluminescent reagent. In some embodiments, moving the magnetizable particles occurs by moving the reaction vessel relative to the magnet (e.g., rotating, e.g., 170-190° or 180°). In various implementations, moving the magnetizable particles, or substantially all of the magnetizable particles, occurs by moving the reaction vessel into a different magnetic field produced by a different set of magnets. In some embodiments, moving the magnetizable particles occurs by moving a magnet relative to the sample (e.g., contained within a reaction vessel) and / or adjusting (e.g., increasing, decreasing) a magnetic field provided by one or more magnets proximate the sample (e.g., changing an electrical parameter such as voltage or current to an electromagnet).

[0013] In embodiments, the methods and systems are most beneficial when chemiluminescence is triggered and / or enhanced by the addition of multiple chemiluminescent reagents. For example, acridinium chemiluminescence typically involves adding a first chemiluminescent reagent (e.g., an acid such as hydrogen peroxide, nitric acid, or a combination thereof in a solvent), thereby inducing oxidation of the acridinium system. This reaction can be accelerated by adding a second chemiluminescent reagent, such as a base, thereby changing the pH of the system to an alkaline state and forming sufficient alkaline hydrogen peroxide to trigger and induce chemiluminescence. In various implementations, the first chemiluminescent reagent is acidic (e.g., a reagent containing 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 basic (e.g., a reagent containing a base such as an alkaline hydroxide (e.g., sodium hydroxide)) and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen- or phosphorus-based salt).

[0014] In embodiments, the disclosed methods and systems can be used in immunoassay formats for the detection of 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 solid-phase magnetizable particles. For example, this method can include forming a sample by combining magnetizable particles bearing a molecule capable of forming a binding complex with the analyte of interest or its binding partner immobilized thereon with a biological sample (e.g., serum, urine) and an assay reagent including 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; optionally incubating the biological sample, magnetizable particles, and chemiluminescent conjugate; and reacting the chemiluminescent conjugate by applying a magnetic field to the reaction vessel. The method can further include isolating the magnetizable particles in a reaction 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, or from a combination thereof) from the magnetizable particles (e.g., the isolated magnetizable particles).

[0015] In various implementations, 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 sequestered (e.g., by application of a magnetic field to the reaction vessel). For example, the method can include at least two wash buffer additions, including a first wash buffer addition in which the magnetizable particles are dispersed throughout the liquid medium when the wash buffer is added; and a second wash buffer addition in which the magnetizable particles are sequestered during the wash buffer addition. In some embodiments, the method further includes measuring chemiluminescent light output (e.g., with a photomultiplier tube, such as in a luminometer) following the addition of the second chemiluminescent reagent. [Brief explanation of the drawings]

[0016] [Figure 1A] 1A-1B illustrate the progression of a reaction involving the sequestration of magnetizable particles after addition of assay reagents to a biological sample (FIG. 1A) and the aspiration of liquid medium and the addition of reagents to those magnetizable particles (e.g., FIG. 1B). At each individual step, the top image is a view of the reaction vessel from the side, and the bottom image is a view of the reaction vessel from above. [Figure 1B] 1A-1B illustrate the progression of a reaction involving the sequestration of magnetizable particles after addition of assay reagents to a biological sample (FIG. 1A) and the aspiration of liquid medium and the addition of reagents to those magnetizable particles (e.g., FIG. 1B). At each individual step, the top image is a view of the reaction vessel from the side, and the bottom image is a view of the reaction vessel from above. [Figure 2] FIG. 1 provides an exemplary particle movement induced by rotation of a reaction vessel. [Figure 3] FIG. 10 illustrates an exemplary particle movement involving the use of a second magnet. [Figure 4] FIG. 1 is a schematic diagram of particle movement involving multiple magnets. [Figure 5A]10A-10C show reaction locations associated with the addition of a second chemiluminescent reagent, which can be performed proximal to a PMT (e.g., in a luminometer) for subsequent measurement of chemiluminescence (e.g., right-most panel in each image). [Figure 5B] 10A-10C show reaction locations associated with the addition of a second chemiluminescent reagent, which can be performed proximal to a PMT (e.g., in a luminometer) for subsequent measurement of chemiluminescence (e.g., right-most panel in each image). [Figure 5C] 10A-10C show reaction locations associated with the addition of a second chemiluminescent reagent, which can be performed proximal to a PMT (e.g., in a luminometer) for subsequent measurement of chemiluminescence (e.g., right-most panel in each image). [Figure 6] 1 is a flow chart showing the steps of an exemplary immunoassay of the present disclosure. [Figure 7] (FIGS. 7A and 7B) Elevation views showing an array (or sample holder) and a series of reaction vessels passing through the array in a linear (FIG. 8A) and ring (FIG. 8B) format. [Figure 8] FIG. 1 compares measurements of three assay protocols at two different analyte concentrations. [Figure 9A] Figure 9A shows that particle migration allows detection below the detection limit of the standard assay performed at 0, 0.003, and 0.006 ng / mL. Figure 9A compares concentrations measured using the immunoassay of the present disclosure and shows that below the detection limit of the standard assay (0.006 ng / mL), measurement resolution can be achieved and complete separation from noise can be achieved (0 ng / mL). Figure 9B shows the same measurement using the standard assay protocol below its detection limit, highlighting the inability to achieve statistical separation between measurements due to noise when performed with the standard assay. [Figure 9B]Figure 9A shows that particle migration allows detection below the detection limit of the standard assay performed at 0, 0.003, and 0.006 ng / mL. Figure 9A compares concentrations measured using the immunoassay of the present disclosure and shows that below the detection limit of the standard assay (0.006 ng / mL), measurement resolution can be achieved and complete separation from noise can be achieved (0 ng / mL). Figure 9B shows the same measurement using the standard assay protocol below its detection limit, highlighting the inability to achieve statistical separation between measurements due to noise when performed with the standard assay. [Figure 10A] Figure 10A shows that particle migration allows detection below the detection limit of the standard assay performed at 0 and 0.004 ng / mL. Figure 10A compares concentrations measured using the immunoassay of the present disclosure and shows that below the detection limit of the standard assay (0.006 ng / mL), measurement resolution can be achieved and complete separation from noise can be achieved (0 ng / mL). Figure 10B shows the same measurement using the standard assay protocol below its detection limit, highlighting the inability to achieve statistical separation between measurements due to noise when performed with the standard assay. [Figure 10B] Figure 10A shows that particle migration allows detection below the detection limit of the standard assay performed at 0 and 0.004 ng / mL. Figure 10A compares concentrations measured using the immunoassay of the present disclosure and shows that below the detection limit of the standard assay (0.006 ng / mL), measurement resolution can be achieved and complete separation from noise can be achieved (0 ng / mL). Figure 10B shows the same measurement using the standard assay protocol below its detection limit, highlighting the inability to achieve statistical separation between measurements due to noise when performed with the standard assay. [Figure 11] FIG. 11 is an exemplary block diagram of a computer system 1100 suitable for executing the methods of the present disclosure on a chemical analyzer. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed embodiments of the present disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely exemplary of the present disclosure, which may be embodied in various forms. Additionally, each of the examples given in connection with various embodiments of the present disclosure are intended to be illustrative, not limiting.

[0018] All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of weight percent of the specified component relative to the total weight of the subject composition unless otherwise defined.

[0019] As used herein, "a" or "an" means one or more. As used herein, when used in connection with the word "comprising," the words "a" or "an" mean one or more. As used herein, "another" means at least a second or more.

[0020] Use of the term "at least one" will be understood to include 1 as well as any quantity greater than 1, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend up to 100 or 1000 or more, depending on the term that accompanies it; in addition, a quantity of 100 / 1000 should not be considered limiting, and higher limits may provide satisfactory results. Additionally, 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.

[0021] The use of ordinal numbers (i.e., "first," "second," "third," "fourth," etc.) is intended only to distinguish between two or more items and is not meant to imply, for example, any sequence or order or importance of one item relative to another, or any additional order, unless expressly stated otherwise.

[0022] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless explicitly stated to refer to alternatives only, or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by 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).

[0023] Any reference herein to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, configuration, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, appearances of the phrases "in some embodiments" or "one example" in various places herein do not necessarily all refer to the same embodiment. Moreover, all references to one or more embodiments or examples should not be construed as limiting the scope of the claims.

[0024] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the variation of the method used to determine the value, or the variation that exists among study subjects. For example, without limitation, when the term "about" is used, the specified value may vary by ±20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent from the specified value, with such variations being appropriate to practice the disclosed methods, as would be understood by one of ordinary skill in the art.

[0025] The term "antibody" is used herein in the broadest sense and refers to, for example, intact monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof (including, 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 replacement proteins or peptides (i.e., artificial binding proteins / peptides), and combinations or derivatives thereof, that exhibit the desired biological activity of analyte binding. 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).

[0026] Detection Agent: As used herein, the term "detection agent" refers to any element, molecule, functional group, compound, fragment, or moiety that is capable of being detected. In some embodiments, a detection agent is provided or utilized alone. In some embodiments, a detection agent is provided and / or utilized in conjunction with (e.g., conjugated to) another chemical entity. Examples of detection agents include, but are not limited to: various ligands, radioisotopes (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 (e.g., acridinum esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticle (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (e.g., dyes, gold colloids, etc.), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0027] As used in this specification and claims, the word "comprising" (and all forms of comprising, such as "comprise" and "comprises"), "having" (and all forms of having, such as "have" and "has"), "including" (and all forms of including, such as "includes" and "include"), or "containing" (and all forms of containing, such as "contains" and "contain") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements and may include other elements that are not expressly recited or that are essentially absent.

[0028] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" includes at least one of: A, B, C, AB, AC, BC, or ABC, and is intended to also include BA, CA, CB, CBA, BCA, ACB, BAC, or CAB where order is important in the particular context. Continuing with this example, combinations involving repeats of one or more items or terms are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that typically, no limitation exists regarding the number of items or terms in any combination, unless otherwise apparent from the context.

[0029] As used herein, the term "substantially" means that the subsequently described event or circumstance occurs entirely, or that the subsequently described event or circumstance occurs to a significant extent or degree. For example, when relating to a particular event or circumstance, the term "substantially" means that the subsequently described event or circumstance occurs with at least an 80% probability, or at least an 85% probability, or at least a 90% probability, or at least a 95% probability. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent to each other, or that a portion of one of two items is close to the other item but not 100% adjacent to the other item.

[0030] As used herein, the phrase "associated with" includes both 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 moiety by a direct bond or through a spacer group, non-covalent binding of one moiety to another moiety either directly or by a specific binding pair member attached to the two moieties, incorporation of one moiety into another moiety, such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety onto another moiety.

[0031] As used herein, the term "biological fluid sample" will be understood to include any liquid test sample obtained from a patient and that can be utilized in accordance with the present disclosure. Examples of biological fluid samples that can be utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), serum, EDTA plasma, lithium heparin plasma, combinations thereof, and the like.

[0032] As used herein, the term "volume," when referring to a liquid test sample utilized by the present disclosure, typically refers to the volume of the liquid test sample, such as in the range of about 0.1 μl to about 100 μl, or in the range of about 1 μl to about 75 μl, or in the range of about 2 μl to about 60 μl, or a value of about 50 μl or less.

[0033] The term "specific binding partner," as used herein, particularly but not exclusively 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, a binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic substrate), combinations or derivatives thereof, and any other molecule capable of specifically binding to a target analyte.

[0034] As used herein, the term "immunoassay" 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, the reaction being carried out for a time and under conditions that allow the formation of an immune complex between the antibody (or fragment thereof) and the analyte. In embodiments, a quantitative determination of such immune complexes can then be performed.

[0035] As used herein, all ranges of numerical values ​​include the endpoints and all possible values ​​between the disclosed values. The exact values ​​of all half-integer numerical values ​​are also contemplated as limits for all subsets of the disclosed ranges, as if they were specifically disclosed. For example, a range of 0.1% to 3% specifically discloses percentages of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. Additionally, 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 is understood that the sum of all weight percentages of the individual components does not exceed 100%.

[0036] In the context of compositions, "consist essentially of" means that the components include only the recited components, along with normal impurities present in commercially available materials and other additives present at levels that do not affect the operation of the embodiments disclosed herein, e.g., less than 5% by weight, or less than 1% by weight, or even less than 0.5% by weight. A composition that includes the recited components can consist primarily of the recited components, can consist essentially of the recited components, or can consist of the recited components (e.g., the recited components have the highest weight percentage in the composition, such as greater than 40% by weight, greater than 50% by weight, greater than 60% by weight, greater than 70% by weight, greater than 80% by weight, greater than 90% by weight, etc.).

[0037] The present disclosure is based, in part, on the addition of steps to immunoassay protocols so that those assays can be enhanced. Typically, these steps involve the movement (and possible sequestration or re-sequestration) of magnetizable particles through a first chemiluminescent reagent, such as a trigger reagent (e.g., an acidic reagent including HO), prior to the addition of a second chemiluminescent reagent (e.g., a basic reagent such as an alkaline hydroxide in a solvent).

[0038] Referring now to Figure 1 (Figures 1A and 1B), which shows magnetizable particles bearing molecules capable of forming binding complexes with an analyte of interest or its binding partner immobilized thereon, an example of magnetizable particle sequestration can be seen, showing a side view of the vessel and a top view of the vessel in three stages (or reaction locations within the system). Figure 1A shows the sequential progression from incubation of freely dispersed sample and reagents (left), to movement of magnetizable particles in a magnetic field (center), and sequestration (right). Reaction vessel 1 contains chemiluminescent sample 2 formed by mixing a biological sample, such as a biological fluid sample such as blood, serum, saliva, or urine, with magnetizable particles bearing an analyte in the biological sample or a binding partner for the analyte of interest immobilized thereon, and one or more detection agents, such as a chemiluminescent compound comprising an acridinium compound, such as an acridinium ester or acridinium sulfonamide, capable of forming a complex with the immobilized binding partner. The magnetizable particles can be suspended in a freely dispersed state in a liquid medium in 2, which includes the biological sample and the liquid components of the assay reagents (e.g., a solvent for the chemiluminescent compound). In the illustrated embodiment, the acridinium compound and analyte in the sample competitively bind to binding partners on the particles for further analysis in the assay. In some embodiments, the chemiluminescent sample 2 is further processed by one or more washes with a wash buffer, as described in U.S. Pat. No. 6,143,578, incorporated herein by reference in its entirety, particularly in connection with the reaction positions with the sample preparation position, wash position, and resuspension wash position. In some embodiments, the disclosed methods and systems can employ a resuspension wash, which can involve aspirating the liquid phase containing unbound components of the assay while holding the bound components in place with a magnet, followed by reintroducing the wash solution into the reaction vessel.

[0039] As a result of removing the liquid phase, a portion of the liquid phase in any reaction vessel or at any reaction location may remain in the reaction vessel. For example, if a reaction vessel is aspirated to remove the liquid phase, some portion of the liquid phase may remain in the reaction vessel, as understood by the aspirate. In various implementations, the aspirate may be performed without requiring an additional step to dry any indicated reaction location. In various implementations, more than 70%, or more than 80%, or more than 90%, or more than 95%, or more than 99% of the liquid phase may be removed during removal (e.g., aspirate) of the liquid phase.

[0040] 1A, following binding and / or washing, reaction vessel 1 can be exposed to a magnetic field generated by magnet 10 to accumulate magnetizable particles for subsequent preparation of a chemiluminescent sample for use in chemiluminescence initiation (and light detection). In the embodiment shown, the magnetizable particles are attracted to magnet 10, and thus a higher concentration of magnetizable particles 2 forms near magnet 10 compared to that formed in the remainder of medium 3. Finally, after a suitable period of exposure to magnet 10, particles 4 become sequestered and separate from liquid medium 5.

[0041] Sequestration does not require the isolation of all magnetizable particles in the sample. For example, sequestration can involve the accumulation of more than 60%, 70%, 80%, 90%, 95%, or 99% of the magnetizable particles in the sample. Following sequestration, 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 sequestrated near the magnet 10 within the reaction vessel 1. These magnetizable particles can be considered the chemiluminescent sample used for subsequent initiation of the chemiluminescent reaction and for final chemiluminescence measurement. In the rightmost reaction position shown in Figure 1B, the nozzle 25 is aimed above the reaction vessel 1 to contain the sequestrated magnetizable particles (or chemiluminescent sample). Nozzle 25 is in fluid communication with a reservoir of a first chemiluminescent reagent (e.g., HO, nitric acid, or a combination thereof, and a trigger agent including a solvent) and adds the chemiluminescent reagent to the reaction vessel by spraying 27 to form a first chemiluminescent reagent deposit 15 within the reaction vessel. Nozzle 25 is positioned to centrally add first chemiluminescent reagent 27 by dispersing it along the major longitudinal axis 6 of reaction vessel 1. Deposit 15 can initiate a reaction with chemiluminescent compounds immobilized on magnetizable particles 4 to initiate the chemiluminescent reaction process. For example, when the chemiluminescent particles are acridinium compounds, the first chemiluminescent reagent can include an acid (e.g., hydrogen peroxide, nitric acid, or a combination thereof) to initiate an oxidation reaction in a chemiluminescent reaction scheme. In various implementations, the first chemiluminescent reagent can cleave or liberate some chemiluminescent compounds immobilized on the particles.

[0042] Particle migration or movement through the first chemiluminescent reagent deposit 15 has been found to provide enhancements to immunoassays including the systems and methods described herein. For example, particle migration through the first chemiluminescent reagent has been shown herein to increase the signal-to-noise of the chemiluminescent output (e.g., compared to an otherwise identical method or system without particle migration). Without wishing to be bound by theory, migration through the first chemiluminescent reagent can reduce particle exposure to reagents (e.g., acid) and / or reduce the amount of nonspecific binding released from the beads. In some embodiments, the signal-to-noise can be improved by more than 1.2 (e.g., more than 1.3, more than 1.4, 1.1 to 5, 1.1 to 4.5) compared to an otherwise identical assay without bead migration within the first chemiluminescent reagent.

[0043] Figure 2 provides an exemplary process for inducing particle migration. Following preparation of sequestered particles 4 in a first chemiluminescent reagent 15 (e.g., a trigger reagent such as an acidic reagent containing hydrogen peroxide), the reaction vessel can be moved within 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, e.g., 180° (16). This movement induces changes in the magnetic field and normal force experienced by the magnetizable particles. By rotating the reaction vessel and exposing the particles to different forces, the particles travel through the first chemiluminescent reagent, as indicated by arrow 17, and consequently re-accumulate within the reaction vessel proximal to the magnet.

[0044] To facilitate migration through the first chemiluminescent reagent, a reaction station or step can be used that involves exposing the magnetizable particles to an altered magnetic field (compared to the immediately preceding step or reaction station). For example, in some embodiments, systems and methods can involve exposing the reaction vessel to a different magnetic field, e.g., resulting from a different magnet (e.g., electromagnet), or to a different magnet having a different arrangement of one or more magnets relative to the reaction vessel and / or the magnetizable particles (e.g., isolated magnetizable particles isolated from a previous step, freely dispersed magnetizable particles from a previous step). In some embodiments, systems and methods can induce migration by exposing the reaction vessel to two or more magnets (e.g., electromagnets). The magnetic field can be altered, e.g., by independently moving two or more (e.g., two to four) magnets relative to the reaction vessel and / or by applying different electrical properties (e.g., current, voltage) to any electromagnets used to modify the magnetic field. Referring now to FIG. 3, possible particle movement positions after the final reaction station of FIG. 1B or FIG. 2 involving isolation from magnet 10 are shown. At the particle transfer reaction position shown, magnet 10 is moved away from the reaction vessel, reducing the magnetic field contribution from this magnet. A second magnet 12 is moved proximal to the opposite end of the reaction vessel. The magnetic field change induced by this change causes the previously isolated particles to move toward magnet 12 located on the opposite side of the reaction (e.g., opposite as measured in a plane perpendicular to the longitudinal axis), as indicated by arrow 18. In some embodiments, this process is repeated one or more times, with one magnet being moved closer (and / or the magnetic field strength above the reaction vessel being increased) and a second magnet being moved away (and / or the magnetic field strength above the reaction vessel being decreased) at each subsequent particle transfer position. In some embodiments, the magnets are positioned on opposite sides. In various implementations, the magnetic field strength of two or more magnets is changed independently, and the magnets are distributed around the reaction vessel.

[0045] As shown in FIG. 4, accumulated particles 4 can have previously accumulated at this location through interaction with a magnetic field (e.g., magnet 10, magnet 12). The reaction vessel can then enter a reaction location where the magnetizable particles are exposed to a second magnet 13 positioned to induce movement, indicated by arrow 19, through the first chemiluminescent reagent. Magnets 10 and 13 can be moved closer to or farther from the reaction vessel to control particle movement through the first chemiluminescent reagent. In some embodiments, magnets 10 and 13 are in fixed positions, for example, and the magnetic field from each magnet is independently controlled by altering an electrical property (e.g., current, voltage) relative to the magnetic field to induce particle movement through the liquid medium. The movement process and reaction location (e.g., prepared by application of different magnetic fields to the reaction vessel) can occur one or more times (e.g., two or more, three or more, four or more, five or more, 2-10 times) before the addition of the second chemiluminescent reagent. For example, as shown in FIG. 4, following interaction with magnet 13 in the final step shown on the right, the reaction vessel and magnetizable particles can be moved a second time within liquid medium 15 by exposure to a magnetic field from magnet 10 in a later step (e.g., by moving magnet 10 closer to the reaction vessel and / or moving magnet 13 away from the reaction vessel).

[0046] An exemplary induction of chemiluminescence by the addition of a second chemiluminescent reagent can be seen in FIG. 5A. As shown on the left, magnetizable particles 4 move through first chemiluminescent reagent deposit 15 and end up isolated proximal to magnet 12. In the embodiment shown, first chemiluminescent reagent deposit 15 is aspirated from the reaction vessel (center), the reaction vessel is moved into a luminometer, and second chemiluminescent reagent 32 (e.g., a base) is deposited (33) to promote chemiluminescence from magnetizable particles 4. In some embodiments, liquid medium 33 includes both the first and second chemiluminescent reagents (e.g., the first chemiluminescent reagent is not aspirated before the addition of the second chemiluminescent reagent, and the first chemiluminescent reagent is not completely aspirated before the addition of the second chemiluminescent reagent). 5B shows a process in which the first chemiluminescent reagent deposit 15 is not aspirated and a second chemiluminescent reagent 32 is added directly into the reaction vessel 1 through an off-center nozzle 30 to form a liquid deposit 34. The liquid deposit 34 can be a combination of the first chemiluminescent reagent (e.g., an acid), the second chemiluminescent reagent (e.g., a base), and a chemiluminescent compound (e.g., an acridinium) that was removed from the particles during the addition of these reagents (e.g., due to cleavage of an ester or sulfonamide group during the chemiluminescence process).

[0047] 5A and 5B, the second chemiluminescent reagent can be added through nozzle 30 at a location other than the major longitudinal axis of the reaction vessel (i.e., off-center addition). Particularly in embodiments in which the final chemiluminescent sample to be measured contains sequestered magnetizable particles, this off-center addition of the second chemiluminescent reagent (e.g., base) can reduce disaggregation of the sequestered particles during reagent addition compared to a central addition. This reduced disaggregation can increase the consistency and accuracy of the assay.

[0048] Chemiluminescence can also be induced from a liquid sediment aspirated from a reaction vessel that does not contain magnetizable particles. Referring now to FIG. 5C, the liquid sediment 15 formed from the addition of a first chemiluminescent reagent to the magnetizable particles and subsequent particle movement is used to measure chemiluminescence and assess the concentration of an analyte in the original biological sample. The liquid sediment 15 (or a portion thereof) can be aspirated from the reaction vessel 1 through an aspirator 22 and separated from the magnetizable particles. The liquid sediment 15 can then be added to a new reaction vessel 24, for example, through a nozzle 26 operatively connected to the reaction vessel 24 via the aspirator 22 and a spray 28, to form a liquid sediment 16 within the new reaction vessel. The liquid sediment 16 can be all of the material from the liquid sediment 15, or a portion of it (e.g., a specific volume of liquid sediment 15, such as 1 μL to 100 mL). The new reaction vessel 24 can be placed in a luminometer prior to the addition of the liquid sediment 16. A nozzle 30 can then be positioned above the reaction vessel 24 to spray 32 a second chemiluminescent reagent (e.g., a base) onto the liquid deposit 16 to induce chemiluminescence. Typically, the second chemiluminescent reagent is added to the reaction vessel in a luminometer for chemiluminescence measurement.

[0049] An exemplary flow diagram for this process used by embodiments of the present disclosure is shown in Figure 6 (Figures 6A and 6B). Each step can be considered an independent reaction location, and the system of the present disclosure can operate to initiate the indicated reaction conditions (e.g., add reagents, isolate particles, move particles across the indicated reagents). Steps 100-120 involve processing the biological sample and reacting with the biological sample to form a chemiluminescent sample for subsequent spectroscopic analysis (e.g., as shown in Figures 1A and 1B). As can be seen, the particles can be washed one or more times with a wash buffer.

[0050] Following preparation of the magnetizable particles, a first chemiluminescent reagent can be added in step 130. Step 140 can involve one or more particle movements, as shown in FIGS. 2-4. These steps can involve moving one or more magnets relative to the reaction vessel, for example, one to five times. Following particle movement, a second chemiluminescent reagent can be added to induce (or enhance) chemiluminescence. The second chemiluminescent reagent can be added to the sample, for example, in a luminometer. Particle movement can occur one or more times, as indicated by arrow 142. In some embodiments, a second chemiluminescent reagent can be added to the reaction vessel containing the magnetizable particles and chemiluminescent reagent that have passed through the first chemiluminescent reagent (e.g., step 150). In some embodiments, some or all of the liquid medium in the reaction vessel can be aspirated, and a second chemiluminescent reagent can be added to the first reaction vessel (e.g., step 145 followed by step 150). In some embodiments, following the aspiration of the first chemiluminescent reagent, a second chemiluminescent reagent can be added to the reaction vessel containing the magnetizable particles (e.g., step 145 followed by step 155). For example, the first chemiluminescent reagent through which the particles were displaced in step 150 can be aspirated into a new reaction vessel in step 145, the new reaction vessel can be placed in a luminometer, the second chemiluminescent reagent can be added to the first chemiluminescent reagent in step 155, and the chemiluminescence can be measured in step 160.

[0051] A magnet is generally a device capable of producing a magnetic field that can affect magnetizable particles. For example, the magnets can be a pair of adjacent magnets with opposite polarities. The magnets can be, for example, electromagnets.

[0052] Referring now to FIG. 7 (FIGS. 7A and 7B), an exemplary array can be seen. Positions A-P can be considered reaction positions used to perform the indicated steps described herein. A reaction vessel can be inserted into a slot (e.g., a slot occurring at each position) and translated along the track. The assay can involve lateral translation of the sample (e.g., by movement along the track to each position), thereby enabling the indicated reaction position or condition. In some embodiments, the next reaction position may not involve lateral translation of the sample. For example, in FIG. 7A, reaction positions A-P represent a series of positions along a linear path at which a sample can be manipulated. At reaction position A, a biological sample and assay reagents including a chemiluminescent conjugate having an analyte or a binding partner for the analyte immobilized thereon and magnetizable particles can be added to the reaction vessel. The reaction vessel can be moved along the path to position B, where the magnetizable particles begin to be sequestered by magnet 40, as shown, for example, in FIG. 1A. Each translational movement through positions B-E can involve segregation of magnetizable particles. At some positions, for example, position F, the liquid medium can be removed, and at position G, a wash buffer can be added. Position H can include magnet 42 separated further from the reaction vessel, allowing the magnetizable particles to be dispersed and resuspended or redispersed in the wash buffer. Positions I-N can involve additional wash steps (e.g., position M). Position N can seek wash buffer from the reaction vessel. At position O, a first chemiluminescent reagent (e.g., a trigger reagent such as an acid) can be added to the reaction vessel. At position P, magnets 44 and / or 46 can affect segregation of the magnetizable particles (e.g., depending on the relative location of the magnets to the reaction vessel, the magnetic field strength of the magnets, or a combination thereof). At position P, the presence of two magnets can allow the sample to undergo one or more particle movement positions. For example, movement from position O to P can induce a first particle movement.Manipulation of the relative locations of magnets 44 and 46 can similarly induce one or more particle movement reaction positions without any translational movement of the reaction vessel. In certain embodiments, the particle movement reaction positions can involve the re-accumulation of magnetizable particles within the reaction vessel. Following the particle movement step, the reaction vessel at position P can be moved (e.g., by a translation arm) into a luminometer where a second chemiluminescent reagent is added and chemiluminescence is measured. In various implementations, 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. FIG. 7B provides a similar embodiment of a track for potential reaction positions in a circular carousel or ring format, where the sample is exposed to magnets 50, 52, 54, and 56 as it is moved around the ring. Following the reaction vessel's passage through positions A-P, chemiluminescence can ultimately be induced by the addition of a second chemiluminescent reagent to the appropriate medium. The addition can be done proximate to a photomultiplier tube for light collection, such as in a luminometer. The processes described herein can be produced by multiple paths, such as adjacent carousels, each providing the reaction location shown. In various implementations, the system includes a diverter for moving reaction vessels between adjacent carousels.

[0053] 11, there is shown 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 can include an analytical instrument 1160 for determining the level of one or more analytes of the present disclosure, such as one or more biomarkers, in a sample.

[0054] 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 on storage device 1130, including receiving or sending information via input / output device 1140.

[0055] 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.

[0056] 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 a non-transitory computer-readable medium according to the present disclosure.

[0057] In an embodiment, input / output device(s) 1140 provide input / output operations to system 1100. In one embodiment, input / output device(s) 1140 include a keyboard and / or a pointing device. In one embodiment, input / output device(s) 1140 include a display unit for displaying a graphical user interface.

[0058] 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 located 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.

[0059] Additionally, non-transitory computer-readable media are provided that include executable instructions that, when executed, cause a processor to perform operations, including the methods provided herein. For example, non-transitory computer-readable media are provided that include executable instructions that, when executed, cause a processor to perform operations, including the methods described herein. In embodiments, non-transitory computer-readable media include hard drives, external hard drives, disks, CDs, DVDs, etc. that store data. In embodiments, software located within physical media is suitable for use herein.

[0060] In some embodiments, a non-transitory computer-readable medium is provided that includes executable instructions that, when executed, cause a processor to perform operations comprising a method, the method including 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 through the first chemiluminescent reagent to different locations within the reaction vessel (e.g., measured relative to a container of the reaction vessel).

[0061] In embodiments, the present disclosure includes one or more articles of manufacture, such as a system or components thereof, including a non-transitory computer-readable medium encoded with instructions, the instructions configured to cause one or more processors to perform the methods of embodiments 1-19 described below.

[0062] In some embodiments, a non-transitory computer-readable medium is provided that contains executable instructions that, when executed, cause a processor to perform operations including one or more of embodiments 1-19 described below.

[0063] Embodiment 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 through the first chemiluminescent reagent to different locations within the reaction vessel (e.g., measured relative to a reservoir of the reaction vessel).

[0064] Embodiment 2. The method of embodiment 1, wherein the magnetizable particles are set in motion by application of a magnetic field to the reaction vessel.

[0065] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the magnetizable particles are sequestered within the reaction vessel during or after the adding step by a magnetic field to the reaction vessel.

[0066] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the magnetizable particles are not sequestered within the reaction vessel during the adding step (e.g., the particles are suspended within the first chemiluminescent reagent and no magnetic field is applied to the reaction vessel).

[0067] Embodiment 5. The method of any one of embodiments 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 separated liquid medium (e.g., in a separate reaction vessel) or to the separated magnetizable particles.

[0068] Embodiment 6.c) The method of any one of embodiments 1 to 4, further comprising adding a second chemiluminescent reagent to the liquid medium (e.g., the first chemiluminescent reagent) optionally present on the magnetizable particles after moving the magnetizable particles.

[0069] Embodiment 7. The method of embodiment 6, wherein the magnetizable particles are clustered within the reaction vessel during addition of the second chemiluminescent reagent (e.g., the reaction vessel has a magnet in its proximity to induce clustering or accumulation).

[0070] Embodiment 8. The method of embodiment 7, wherein the reaction vessel during addition of the second chemiluminescent reagent is symmetrical about a major longitudinal axis (e.g., cylindrical, such as a cuvette or tube), and the second chemiluminescent reagent is added at a position other than along the major longitudinal axis.

[0071] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the sample is contained within a reaction vessel (e.g., a cuvette, a tube) during addition of the first chemiluminescent reagent, and the reaction vessel has a magnet adjacent to it (e.g., to sequester magnetizable particles along the interior wall of the reaction vessel) during addition of the first chemiluminescent reagent addition location.

[0072] Embodiment 10. The method of embodiment 9, wherein moving the magnetizable particles occurs by movement of the reaction vessel relative to the magnet (e.g., rotating, for example, 170-190° or 180°).

[0073] Embodiment 11. The method of embodiment 9 or 10, wherein moving the magnetizable particles occurs by moving the reaction vessel into different magnetic fields produced by different sets of magnets.

[0074] Embodiment 12. The method of any one of embodiments 1 to 11, wherein moving the magnetizable particles occurs by moving a magnet relative to the sample (e.g., contained within a reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field provided by one or more magnets in proximity to the sample (e.g., changing an electrical parameter such as voltage or current to an electromagnet).

[0075] Embodiment 13. The method of any one of embodiments 1 to 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 comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0076] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the second chemiluminescent reagent is basic (e.g., a reagent containing a base such as an alkaline hydroxide (e.g., sodium hydroxide)) and optionally contains a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0077] Embodiment 15. Forming a sample by mixing magnetizable particles having a molecule capable of forming a binding complex with an analyte of interest or its binding partner immobilized thereon with a biological sample (e.g., serum, urine) and an assay reagent including 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; optionally incubating the biological sample, magnetizable particles, and chemiluminescent conjugate; and applying a magnetic field to the reaction vessel to form a magnetically coupled chemiluminescent conjugate within the reaction vessel. 15. The method of any one of embodiments 1 to 14, further comprising: isolating the magnetizable particles; adding a wash buffer (e.g., a buffer solution that may include 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, or from a combination thereof) from the magnetizable particles (e.g., the isolated magnetizable particles).

[0078] Embodiment 16. The method of embodiment 15, wherein the wash buffer is added when the magnetizable particles are dispersed throughout the liquid medium.

[0079] Embodiment 17. The method of embodiment 15, wherein the wash buffer is added when the magnetizable particles are in isolation (e.g., by application of a magnetic field to the reaction vessel).

[0080] Embodiment 18. The method of any one of embodiments 15-17, wherein the method comprises at least two wash buffer additions, including a first wash buffer addition in which the magnetizable particles are dispersed throughout the liquid medium as the wash buffer is added; and a second wash buffer addition in which the magnetizable particles are sequestered during the wash buffer addition.

[0081] Embodiment 19. The method of any one of embodiments 1-18, wherein the method further comprises measuring chemiluminescent light output (e.g., by a photomultiplier tube, such as in a luminometer) following the addition of the second chemiluminescent reagent.

[0082] Embodiment 20. A system for inducing a chemiluminescent reaction from a chemiluminescent sample containing magnetizable particles (e.g., paramagnetic particles), comprising: an array of successive reaction locations for the chemiluminescent sample, such that the chemiluminescent sample (or medium derived therefrom) can be sequentially placed at each reaction location (e.g., by movement of a reaction vessel (e.g., cuvette, tube) containing materials such as magnetizable particles and / or liquid medium between each reaction location, aspiration of liquid medium in a first reaction vessel, and deposition into a second reaction vessel); said array of successive reaction locations comprising: a first chemiluminescent reagent at which a first chemiluminescent reagent can be added to the chemiluminescent sample; A system comprising: a drug addition position; one or more (e.g., 1 to 10, 1 to 5, 2, 3, 4, 5, 6, 7, 8, 9, 10) particle movement positions that can apply a magnetic field different from that of a previous position (e.g., a first chemiluminescent reagent addition position, a previous particle movement position) to the chemiluminescent sample to induce movement of magnetizable particles in the first chemiluminescent reagent; and a second chemiluminescent reagent addition position that adds a second chemiluminescent reagent to the chemiluminescent sample (or a portion or a part derived therefrom) after one or more particle movement positions to induce a chemiluminescent reaction from the chemiluminescent sample (or a portion or a part derived therefrom).

[0083] Embodiment 21. The system of embodiment 20, wherein the array includes a liquid transfer position where, following one or more particle movement positions, the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration using a pipette); and a second chemiluminescent reagent vessel position where a second chemiluminescent reagent is added to the separated liquid sample (e.g., in a reaction vessel such as a cuvette or tube different from the reaction vessel used in the first chemiluminescent position).

[0084] Embodiment 22. The system of embodiment 20, wherein the second chemiluminescent reagent is added to the chemiluminescent sample containing magnetizable particles (e.g., in the same reaction vessel, such as a cuvette or tube, to which the first chemiluminescent sample was added).

[0085] Embodiment 23. The system of embodiment 22, wherein the magnetizable particles are clustered within the reaction vessel at a second chemiluminescent reagent addition location (e.g., the reaction vessel at the second chemiluminescent location has a magnet in its vicinity to induce clustering).

[0086] Embodiment 24. The system of embodiment 23, wherein the reaction vessel at the second chemiluminescence location is symmetrical about its major longitudinal axis (e.g., cylindrical, such as a cuvette or tube), and the second chemiluminescent reagent is added at a location other than the major longitudinal axis.

[0087] Embodiment 25. A system described in any one of embodiments 20 to 24, wherein the chemiluminescent sample is contained in a reaction vessel (e.g., a cuvette, a tube) at a first chemiluminescent reagent addition position, and the first chemiluminescent reagent is added to the reaction vessel.

[0088] Embodiment 26. The system of embodiment 25, wherein the reaction vessel has a magnet adjacent to it in the first chemiluminescent reagent addition position (e.g., to sequester magnetizable particles along the interior wall of the reaction vessel).

[0089] Embodiment 27. The system of embodiment 25 or 26, wherein at least one of the different magnetic fields at at least one particle motion location is generated on the chemiluminescent sample by rotating the reaction vessel (e.g., by rotating 170-190° or 180°).

[0090] Embodiment 28. A system described in any one of embodiments 25 to 27, wherein at least one of the different magnetic fields at at least one particle movement location is generated by moving the reaction vessel into a different magnetic field generated by a different set of magnets.

[0091] Embodiment 29. A system described in any one of embodiments 20 to 28, wherein at least one of the different magnetic fields at at least one particle movement location is generated by moving a magnet relative to the sample (e.g., contained in a reaction vessel) and / or adjusting (e.g., increasing, decreasing) the magnetic field provided from one or more magnets in proximity to the sample (e.g., by changing an electrical parameter such as voltage or current to an electromagnet).

[0092] Embodiment 30. The system of any one of embodiments 20 to 29, wherein the first chemiluminescent reagent is acidic (e.g., a reagent containing 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).

[0093] Embodiment 31. The system of any one of embodiments 20 to 30, wherein the second chemiluminescent reagent is basic (e.g., a reagent containing a base such as an alkaline hydroxide (e.g., sodium hydroxide)) and optionally contains a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

[0094] Embodiment 32. The system further includes reaction locations for forming a chemiluminescent sample from a biological sample, the reaction locations for forming the chemiluminescent sample being: a biological sample addition location where the biological sample is added to a reaction vessel; an assay reagent location where an assay reagent (e.g., an analyte of interest or its binding partner immobilized thereon, a chemiluminescent conjugate such as an acridinium compound including an acridinium ester and an acridinium sulfonamide capable of forming a binding complex with a molecule immobilized on a magnetizable particle, or magnetizable particles having a molecule capable of forming a binding complex with both) is added to a reaction vessel (e.g., a reaction vessel containing a biological sample); and an assay reagent location where an assay reagent (e.g., an analyte of interest or its binding partner immobilized thereon, a chemiluminescent conjugate such as an acridinium ester and an acridinium sulfonamide capable of forming a binding complex with a molecule immobilized on a magnetizable particle, or magnetizable particles having a molecule capable of forming a binding complex with both) is added to a reaction vessel (e.g., a reaction vessel containing a biological sample); 32. The system of any one of embodiments 20 to 31, comprising at least one reaction location selected from: an incubation location for magnetically isolating magnetizable particles from a liquid medium of a biological sample and assay reagents; an isolation location where the magnetizable particles are magnetically isolated from a liquid medium of a biological sample and assay reagents; a separation location where the liquid medium (e.g., from the biological sample, from the assay reagents) is separated from the magnetizable particles (e.g., the isolated magnetizable particles); a washing location where a wash buffer (e.g., a buffer solution that may include 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 wash buffer aspiration location where the wash buffer is separated from the magnetizable particles to form a chemiluminescent sample.

[0095] Embodiment 33. The system of embodiment 32, wherein the system includes a wash position in which the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added.

[0096] Embodiment 34. A system according to embodiment 32, wherein the system includes a wash position in which the magnetizable particles are sequestered during addition of a wash buffer.

[0097] Embodiment 35. A system described in any one of embodiments 32 to 34, wherein the system includes at least two wash positions, including a first wash position in which the magnetizable particles are dispersed throughout the liquid medium as the wash buffer is added; and a second wash position in which the magnetizable particles are sequestered during the addition of the wash buffer.

[0098] Embodiment 36. A system described in any one of embodiments 20 to 35, wherein the system further comprises a photomultiplier tube (e.g., a luminometer) for measuring chemiluminescent light output following addition of the second chemiluminescent reagent.

[0099] Embodiment 37. A system described in any one of embodiments 20 to 35, wherein the system further includes a non-transitory computer-readable medium for performing the methods of the present disclosure, including embodiments 1 to 19.

[0100] Example The following examples illustrate specific aspects of the present description. These examples should not be construed as limiting, but merely provide specific understanding and implementation of the embodiments and various aspects thereof. [Example]

[0101] Chemiluminescent beads made from standards of different concentrations of troponin I (TnI) were prepared. A similar preparation is disclosed in R. Payne, European Heart Journal 38 (2017):ehx502.P2754, incorporated herein by reference, specifically relating to the preparation of beads and the Siemens ADVIA Centaur high-sensitivity troponin I sandwich assay. This standard assay has a detection limit of 0.006 ng / mL, and biological samples must contain at least 0.006 ng / mL to measure a detectable concentration difference. The beads were washed with wash buffer, and a magnetic field was applied for 60 seconds during the wash. The wash buffer was removed, and acid was added. The beads were completely suspended in acid and magnetically isolated. The acidic supernatant was transferred to a new cuvette, and base was added to generate a signal. In repeated experiments, the signal-to-noise ratio was measured at four different TnI concentrations. The results are shown in Table 1, which shows a standard deviation of (±) 1 for the measured S / N.

[0102] [Table 1]

[0103] As can be seen, when the beads are removed before measuring the relative light units, an improvement in signal to noise can be seen.

[0104] The assay was compared to a standard TnI assay (Standard TnI-Ultra assay - base dispersion at center) to measure TnI at concentrations of 0 and 0.024 ng / mL. Table 2 provides a comparison of these results and allows for a comparison of the signal to noise for these measurements, including modifications to this assay involving magnetic transfer and off-center addition of a second chemiluminescent reagent (base).

[0105] [Table 2]

[0106] As can be seen, modifications to the standard assay protocol, including particle movement within the first chemiluminescent reagent (acid), resulted in a decrease in RLU measurements for both signal and noise (↓). However, the decrease in noise was more pronounced, resulting in an increase in signal-to-noise (↑). Furthermore, reading in a new reaction vessel following magnetic switching resulted in an increase in signal-to-noise compared to reading in the same reaction vessel. Figure 8 provides a comparison of the RLU measured for both noise (left for each protocol) and signal (right for each protocol). [Example]

[0107] The increased signal-to-noise of the systems and methods disclosed herein has been shown to reduce the limit of detection (LOD) and enable measurements below the standard assay. TnI standards of 0, 0.003, and 0.006 ng / mL were prepared at or below the limit of detection (LOD) of the standard assay, and the assay was performed using the standard protocol in a sandwich assay format and a similar protocol involving magnetic switching and particle movement by depositing liquid medium in a new cuvette. Table 3 provides the results of this comparison, as well as various statistical parameters of the repeated measurements, including the average (Avg), measured standard deviation (SD), coefficient of variation (percentage of the mean), and signal-to-noise (SNR) at each concentration. Figure 9A shows the average values ​​from the 0, 0.003, and 0.006 ng / mL measurements for the magnetic switch, and Figure 9B shows the average values ​​from these measurements for the standard protocol with statistical values.

[0108] [Table 3]

[0109] Measurements were again performed on the TnI standard at 0 and 0.003 ng / mL (below the limit of detection (LOD) of the standard assay), and the assay was run using the standard protocol in the sandwich assay format discussed above, as well as a similar protocol involving magnetic switching and particle movement by deposition of liquid medium in a new cuvette. Table 4 provides the results of those measurements.

[0110] [Table 4]

[0111] FIG. 10A shows the mean values ​​from 0 and 0.004 ng / mL measurements for the magnetic switch, and FIG. 10B shows the mean values ​​from these measurements for the standard protocol with statistical values. Error bars represent standard deviation. As can be seen, the standard assay cannot distinguish a concentration of 0.004 from a concentration of 0. In contrast, using the method provided herein, the magnetic switch allows measurements below the LOD of the standard assay. RLU values ​​are clearly distinguishable for 0 (mean + 2 SD) and 0.004 (mean - 1 SD) when performed using the magnetic switch in an immunoassay.

[0112] Various changes may be made to the subject matter described above without departing from the scope and spirit of the present disclosure, and it is intended that all subject matter contained in the above description or defined in the appended claims be construed as illustrative and exemplary of the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variations that fall within the scope of the appended claims.

[0113] All documents cited or referenced herein, and all documents cited or referenced within the documents cited herein, along with manufacturer's directions, descriptions, product specifications, and product sheets for any products described herein or in any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of this disclosure.

Claims

1. 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); b) moving the magnetizable particles through a first chemiluminescent reagent to different locations within the reaction vessel (e.g., measured relative to the reservoir of the reaction vessel).

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

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

4. 4. The method of claim 1, wherein the magnetizable particles are not sequestered in the reaction vessel during the adding step (e.g., the particles are suspended in the first chemiluminescent reagent and no magnetic field is applied to the reaction vessel).

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

6. c) adding a second chemiluminescent reagent to the magnetizable particles and optionally to the liquid medium (e.g., the first chemiluminescent reagent) present after the magnetizable particles have been moved; The method of any one of claims 1 to 4, further comprising:

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

8. 8. The method of claim 7, wherein the reaction vessel during addition of the second chemiluminescent reagent is symmetrical about a major longitudinal axis (e.g., cylindrical, such as a cuvette or tube), and the second chemiluminescent reagent is added at a position other than along the major longitudinal axis.

9. 9. The method of any one of claims 1 to 8, wherein the sample is contained within a reaction vessel (e.g., a cuvette, a tube) during addition of the first chemiluminescent reagent, and the reaction vessel has a magnet adjacent to it (e.g., to sequester magnetizable particles along an inner wall of the reaction vessel) during addition of the first chemiluminescent reagent addition location.

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

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

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

13. 13. The method of any one of claims 1 to 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 comprises a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

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

15. forming a sample by mixing magnetizable particles having a molecule capable of forming a binding complex with the analyte of interest 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 acridinium sulfonamide) capable of forming a binding complex with the molecule immobilized on the magnetizable particles; and optionally incubating a biological sample, magnetizable particles, and a chemiluminescent conjugate; isolating magnetizable particles within a reaction vessel by applying a magnetic field to the reaction vessel; adding a wash buffer (e.g., a buffer solution that may include one or more salts such as sodium chloride and sodium azide, a detergent such as a cationic detergent, a buffering agent such as a phosphate, a blocking agent such as bovine serum albumin (BSA), or a combination thereof) to the magnetizable particles; 15. The method of any one of claims 1-14, further comprising 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, or from a combination thereof) from the magnetizable particles (e.g., the sequestered magnetizable particles).

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

17. 16. The method of claim 15, wherein the wash buffer is added when the magnetizable particles are in isolation (e.g., by application of a magnetic field to the reaction vessel).

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

19. 19. The method of any one of claims 1 to 18, wherein the method further comprises measuring chemiluminescent light output (e.g., by a photomultiplier tube, such as in a luminometer) following the addition of the second chemiluminescent reagent.

20. 1. A system for inducing a chemiluminescent reaction from a chemiluminescent sample containing magnetizable particles (e.g., paramagnetic particles), comprising: It comprises an array of successive reaction locations for a chemiluminescent sample, so that the chemiluminescent sample (or medium derived therefrom) can be sequentially placed at each reaction location (e.g., by moving a reaction vessel (e.g., cuvette, tube) containing materials such as magnetizable particles and / or liquid medium between each reaction location, aspirating the liquid medium in a first reaction vessel, and depositing it into a second reaction vessel); said array of successive reaction locations comprises: a first chemiluminescent reagent addition location where a first chemiluminescent reagent can be added to the chemiluminescent sample; one or more (e.g., 1 to 10, 1 to 5, 2, 3, 4, 5, 6, 7, 8, 9, 10) particle movement positions that can apply a magnetic field to the chemiluminescent sample that is different from a previous position (e.g., a first chemiluminescent reagent addition position, a previous particle movement position) to induce movement of magnetizable particles in the first chemiluminescent reagent; The system includes a second chemiluminescent reagent addition position that adds a second chemiluminescent reagent to the chemiluminescent sample (or a portion thereof or a portion derived therefrom) after one or more particle movement positions to induce a chemiluminescent reaction from the chemiluminescent sample (or a portion thereof or a portion derived therefrom).

21. The system of claim 20, wherein the array includes a liquid transfer position where the liquid chemiluminescent sample is separated from the magnetizable particles (e.g., by aspiration using a pipette) following one or more particle movement positions; and a second chemiluminescent reagent vessel position where a second chemiluminescent reagent is added to the separated liquid sample (e.g., in a reaction vessel such as a cuvette or tube different from the reaction vessel used in the first chemiluminescent position).

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

23. The system of claim 22, wherein the magnetizable particles are clustered within a reaction vessel at a second chemiluminescent reagent addition position (e.g., the reaction vessel at the second chemiluminescent position has a magnet in its vicinity to induce clustering).

24. 24. The system of claim 23, wherein the reaction vessel at the second chemiluminescence location is symmetrical about a major longitudinal axis (e.g., cylindrical, such as a cuvette or tube), and the second chemiluminescent reagent is added at a location other than the major longitudinal axis.

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

26. 26. The system of claim 25, wherein the reaction vessel has a magnet adjacent thereto in the first chemiluminescent reagent addition position (e.g., to sequester magnetizable particles along an interior wall of the reaction vessel).

27. 27. The system of claim 25 or 26, wherein at least one of the different magnetic fields at at least one particle movement location is generated on the chemiluminescent sample by rotating the reaction vessel (e.g., by rotating, e.g., 170-190° or 180°).

28. 28. The system of any one of claims 25 to 27, wherein at least one of the different magnetic fields at the at least one location of particle movement is produced by moving the reaction vessel into different magnetic fields produced by different sets of magnets.

29. 29. The system of any one of claims 20 to 28, wherein at least one of the different magnetic fields at at least one location of particle movement is produced by moving a magnet relative to the sample (e.g., contained in a reaction vessel) and / or adjusting (e.g., increasing, decreasing) a magnetic field provided by one or more magnets in proximity to the sample (e.g., by changing an electrical parameter such as voltage or current to an electromagnet).

30. 30. The system of any one of claims 20 to 29, wherein the first chemiluminescent reagent is acidic (e.g., a reagent containing 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. 31. The system of any one of claims 20 to 30, wherein the second chemiluminescent reagent is basic (e.g., a reagent containing a base such as an alkaline hydroxide (e.g., sodium hydroxide)) and optionally includes a detergent (e.g., a cationic detergent such as a quaternary nitrogen or phosphorus-based salt).

32. The system further includes a reaction location for forming a chemiluminescent sample from the biological sample, the reaction location for forming the chemiluminescent sample comprising: a biological sample addition location where the biological sample is added to the reaction vessel; an assay reagent location where an assay reagent (e.g., magnetizable particles having molecules capable of forming binding complexes with the analyte of interest or its binding partner immobilized thereon, chemiluminescent conjugates such as acridinium compounds including acridinium esters and acridinium sulfonamides capable of forming binding complexes with molecules immobilized on magnetizable particles, or both) is added to a reaction vessel (e.g., a reaction vessel containing a 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 location where the magnetizable particles are magnetically isolated from the liquid medium of the biological sample and assay reagents; a separation location where the liquid medium (e.g., from a biological sample, from an assay reagent) is separated from the magnetizable particles (e.g., sequestered magnetizable particles); a wash station where a wash buffer (e.g., a buffer solution that can include one or more salts such as sodium chloride and sodium azide, a detergent such as a cationic detergent, a buffering agent 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 system according to any one of claims 20 to 31, comprising at least one reaction location selected from a wash buffer aspiration location for separating wash buffer from magnetisable particles to form a chemiluminescent sample.

33. 33. The system of claim 32, wherein the system includes a wash station in which the magnetizable particles are dispersed throughout the liquid medium when a wash buffer is added.

34. 33. The system of claim 32, wherein the system includes a wash station in which the magnetizable particles are sequestered during addition of a wash buffer.

35. 35. The system of any one of claims 32 to 34, comprising at least two wash positions, including a first wash position where the magnetizable particles are dispersed throughout the liquid medium as a wash buffer is added; and a second wash position where the magnetizable particles are sequestered during the addition of the wash buffer.

36. The system of any one of claims 20 to 35, wherein the system further comprises a photomultiplier tube (e.g., a luminometer) for measuring chemiluminescent light output following addition of the second chemiluminescent reagent.

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