Assay method using chemiluminescent dioxetane compounds

Abstract

The technology claimed and described herein provides a chemiluminescence-based assay method using a 1,2-dioxane compound. In some cases, the analyte is cardiac troponin I (TNI), thyroid-stimulating hormone (TSH), or procalcitonin (PCT). The method disclosed herein comprises: exposing a biological sample to a capture antibody configured to bind to at least a portion of the analyte disclosed herein, thereby generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, thereby forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising the 1,2-dioxane compound disclosed herein; wherein a reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of the analyte in the biological sample.

Description

[0001] Related applications

[0002] This patent application claims priority to U.S. Provisional Patent Application Serial No. 63 / 625,439, filed January 26, 2024, the contents of which are hereby incorporated herein by reference in their entirety.

[0003] By incorporating it into the electronic sequence list

[0004] This application contains a sequence list that has been submitted in a computer-readable format and is hereby incorporated in its entirety by means of its inclusion. The computer-readable file, created on January 20, 2025, is named 67670WO01_seq.xml and has a size of 3,860 bytes. Background Technology

[0005] Immunoassays are essential analytical tools for identifying and detecting specific substances in samples, and are commonly used to detect and quantify clinically important blood proteins. Many medical decisions are based on the diagnostic results of these assays, making sensitivity and specificity extremely important. Chemiluminescence-based immunoassays can provide rapid responses to the presence of analytes and offer excellent sensitivity because, unlike fluorescence and absorption-based assays, they do not require photoexcitation. There is a need for assays that allow for more sensitive detection of analytes than existing substrates and with a higher signal-to-background ratio. Summary of the Invention

[0006] One aspect of the present invention is a method for detecting an analyte in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least a portion of the analyte to generate a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme-conjugated antigen to generate a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant; wherein a reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of the analyte in the biological sample.

[0007] One aspect of the present invention is a method for detecting an analyte in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least a portion of the analyte to generate a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated antibody or an enzyme-conjugated antigen to generate a second reaction mixture; and exposing the second reaction mixture to a substrate formulation comprising a compound of formula I or a salt thereof.

[0008] Formula I

[0009] Each of R1 and R2 is independently a C3-C10 alkyl group, or R1 and R2 together with the carbon to which they are attached provide a C5-C10 cycloalkyl ring;

[0010] R3 is a C1-C10 alkyl, C6-C10 aryl, or heteroaryl group;

[0011] R4 is a C2-C10 alkenyl group;

[0012] R5 is H or a C1-C10 alkyl group; and

[0013] X is a phosphate;

[0014] The reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate preparation generates a chemiluminescent detection signal; the detection signal generated by the reaction is recorded; and the recorded signal is compared with a calibration curve to quantify the level of the analyte in the biological sample.

[0015] In one aspect, the substrate formulation further comprises at least one phosphocation surfactant.

[0016] In one aspect, the enzyme-conjugated antibody is configured to bind to at least a portion of the analyte. In another aspect, the enzyme-conjugated antigen is configured to bind to at least a portion of the capture antibody.

[0017] One aspect of the present invention is a method for detecting cardiac troponin I (TNI) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least a portion of TNI to generate a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule to form a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant; wherein a reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of TNI in the biological sample.

[0018] On one hand, the concentration of the quantified TNI level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

[0019] On one hand, the ratio of the signal generated by cutting the enzyme to the background noise is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800, alternatively at least about 900, alternatively at least about 1,000, alternatively at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, alternatively at least about 5,000, alternatively at least about 6,000, alternatively at least about 7,000, alternatively at least about 8,000, alternatively at least about 9,000, alternatively at least about 10,000, alternatively at least about 15,000, alternatively at least about 20,000, or alternatively at least about 25,000.

[0020] In one aspect, the affinity molecule is an antibody, monoclonal antibody, polyclonal antibody, synthetic antibody mimic, aptamer, affixer, DARPin, oligonucleotide, peptide, or antigen. In another aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of a TNI.

[0021] One aspect of the present invention is a method for detecting thyroid-stimulating hormone (TSH) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least a portion of TSH to generate a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule to form a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant; wherein a reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of TSH in the biological sample.

[0022] On one hand, the concentration of the quantified TSH level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

[0023] On one hand, the ratio of the signal generated by cutting the enzyme to the background noise is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 4. 00, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, or at least about 8,000.

[0024] In one aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of the TSH.

[0025] One aspect of this disclosure is a method for detecting procalcitonin (PCT) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least a portion of PCT to generate a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule to form a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant; wherein a reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of PCT in the biological sample.

[0026] In one respect, the limit of quantitation (LoQ) of the determination is about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

[0027] In one respect, the coefficient of variation (CV) of the method is 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

[0028] In one respect, the concentration of the detected PCT level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

[0029] On one hand, the ratio of the signal generated by cutting the enzyme to the background noise is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000.

[0030] In one aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of PCT.

[0031] In one aspect, the affinity molecule is an antigen, wherein the antigen is configured to bind to at least a portion of the capture antibody.

[0032] In one aspect, the capture antibody and / or the enzyme-conjugated affinity molecule are conjugated to at least one magnetic bead.

[0033] In one respect, the enzyme comprises alkaline phosphatase (AP).

[0034] In one aspect, the generated signal is at least about 10 times, alternatively at least about 20 times, or alternatively at least about 30 times greater than a signal generated by a method in which the substrate formulation comprises a compound of the following formula:

[0035] .

[0036] In one respect, the 1,2-dioxane compound is a compound of formula I or a salt thereof:

[0037] Formula I

[0038] Where R 1 and R 2 Each of them is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon it is attached to, it provides C5-C. 10 cycloalkyl ring;

[0039] R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl;

[0040] R 4 It is C2-C 10 alkenyl;

[0041] R 5 Is it H or C1-C? 10 Alkyl; and

[0042] X is phosphate.

[0043] In one respect, the 1,2-dioxane compound is a compound of formula II or a salt thereof:

[0044] Formula II

[0045] Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl;

[0046] R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl;

[0047] R 4 It is C2-C 10 alkenyl;

[0048] R 5 Is it H or C1-C? 10 Alkyl; and

[0049] X is phosphate.

[0050] In one respect, the 1,2-dioxane compound is a compound of formula III or a salt thereof:

[0051] Formula III

[0052] Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl;

[0053] R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl;

[0054] R 5 Is it H or C1-C? 10 Alkyl; and

[0055] X is phosphate.

[0056] In one respect, the 1,2-dioxane compound is a compound of formula IV or a salt thereof:

[0057] Formula IV

[0058] Where R 3It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl;

[0059] R 5 Is it H or C1-C? 10 Alkyl group; and X is a phosphate.

[0060] In one respect, the 1,2-dioxane compound is

[0061] ,

[0062] Or its salt.

[0063] In one respect, the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

[0064] In one respect, the phosphocation surfactant is selected from the group consisting of small molecule phosphocation surfactants and polymeric phosphocation surfactants.

[0065] On one hand, the small molecule phosphocation surfactant is a compound having the following formula:

[0066]

[0067] R12-R14 are each independently a C1-C10 alkyl group;

[0068] R15 is an aralkyl group; and

[0069] X - It is a counter ion.

[0070] In one aspect, the polymeric phosphocationic surfactant comprises repeating units (A), repeating units (B), or both:

[0071]

[0072] Bu3 is tributyl, and Oct3 is trioctyl.

[0073] In one aspect, the substrate formulation further comprises a magnesium(II) salt.

[0074] In one aspect, the method is determined and performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent kit configured to contain a plurality of reagent containers, wherein each reagent container is configured to store a certain volume of reagent required for at least one of the assays, wherein at least one reagent contains the capture antibody and at least one reagent contains the enzyme-conjugated affinity molecule; a pipette device configuration comprising at least one reagent pipette and at least one sample pipette; and a detector device configuration.

[0075] In one aspect, the reagent container includes an elastomeric self-sealing membrane.

[0076] In one aspect, the reagent package further includes a containment wall disposed between the reagent containers.

[0077] In one aspect, the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0078] In one aspect, the pipette device is configured to include at least a first reagent pipette, a second reagent pipette, a third reagent pipette, and at least one sample pipette.

[0079] In one aspect, the pipette device configuration further includes at least a fourth reagent pipette.

[0080] In one respect, the first reagent pipette, the second reagent pipette, the third reagent pipette, and / or the fourth reagent pipette operate selectively and / or simultaneously.

[0081] In one aspect, the first reagent pipette, the second reagent pipette, the third reagent pipette, and / or the fourth reagent pipette are configured to engage the dispensing tip before aspiration.

[0082] In one respect, the method is configured to analyze at least approximately 200 biological samples per hour.

[0083] In one respect, the method is configured to analyze at least approximately 300 biological samples per hour.

[0084] In one respect, the method is configured to analyze at least approximately 400 biological samples per hour.

[0085] In one aspect, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample container and dispensing the aspirated biological sample into the reaction vessel of the immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent containing the enzyme-conjugated affinity molecule from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; and the detection mixture is generated by aspirating the substrate preparation and dispensing the aspirated substrate preparation into the reaction vessel.

[0086] In one respect, the incubation time of the first reaction mixture is at least about 30 minutes, or alternatively at least about 40 minutes, or alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

[0087] In one respect, the incubation time of the second reaction mixture is at least about 2 minutes, or alternatively at least about 5 minutes, or alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0088] On one hand, the test mixture is incubated for at least 20 seconds, or alternatively at least 30 seconds, or alternatively at least 40 seconds, or alternatively at least 50 seconds, or alternatively at least 60 seconds, or alternatively at least 70 seconds, or alternatively at least 80 seconds, or alternatively at least 90 seconds, or alternatively at least 100 seconds, or alternatively at least 110 seconds, or alternatively at least 120 seconds, or alternatively at least 130 seconds, or alternatively at least 140 seconds, or alternatively at least 150 seconds, or alternatively at least 160 seconds, or alternatively at least 170 seconds, or alternatively at least 180 seconds, or alternatively at least 190 seconds, or alternatively at least 200 seconds, or alternatively at least 210 seconds, or alternatively at least 220 seconds, or alternatively at least 230 seconds, or alternatively at least 240 seconds, or alternatively at least 250 seconds, or alternatively at least 260 seconds, or alternatively at least 270 seconds, or alternatively at least 280 seconds.

[0089] On the one hand, the cycle time is approximately 45 seconds or less, alternatively approximately 40 seconds or less, alternatively approximately 35 seconds or less, alternatively approximately 30 seconds or less, alternatively approximately 25 seconds or less, alternatively approximately 20 seconds or less, or alternatively approximately 15 seconds.

[0090] On the one hand, the time to first result (TTFR) is approximately 60 minutes or less, alternatively approximately 55 minutes or less, alternatively approximately 50 minutes or less, 45 minutes or less, alternatively approximately 40 minutes or less, alternatively approximately 35 minutes or less, alternatively approximately 30 minutes or less, alternatively approximately 25 minutes or less, alternatively approximately 20 minutes or less, alternatively approximately 15 minutes or less, or alternatively approximately 10 minutes or less.

[0091] In one aspect, the detector device comprises a photodetector configured to sense photons emitted from a measurement reaction within a time period; an analog circuit configured to provide an analog signal based on the photons emitted from the measurement reaction within the time period; and a counter circuit configured to provide a photon count based on the photons emitted from the measurement reaction within the time period.

[0092] In one aspect, the immunoassay analyzer further includes an ultrasonic mixing module. In another aspect, the first reaction mixture, the second reaction mixture, and / or the detection mixture are stirred via the ultrasonic mixing module.

[0093] In one aspect, the first reaction mixture, the second reaction mixture, and / or the detection mixture contain unreacted components, and the immunoassay analyzer further includes a washing device configuration, wherein the washing device configuration is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

[0094] In one aspect, the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field before the at least one washing action is performed.

[0095] In one aspect, the immunoassay analyzer further includes: a machine vision device comprising an image capture device and an image interpretation device, the image capture device and the image interpretation device being configured to monitor the instrument functions and / or assay functions of the immunoassay analyzer. In one aspect, the instrument functions are selected from the group consisting of: optical sensors, pressure sensors, and thermistors. In one aspect, the assay functions are selected from the group consisting of: sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

[0096] In one respect, the biological sample is serum, whole blood, plasma, and / or cerebrospinal fluid.

[0097] These and other advantages, aspects and novel features of this disclosure, as well as the details of the embodiments shown therein, will be more fully understood from the following description and accompanying drawings. Attached Figure Description

[0098] Embodiments of this disclosure will now be described by way of example only with reference to the accompanying drawings, in which:

[0099] Figure 1 It is a signal comparison between a commercially available substrate according to one aspect of this disclosure and an exemplary substrate.

[0100] Figure 2 The calibration curve is constructed according to one aspect of this disclosure using calibrators and commercially available substrates and exemplary substrates for the determination of troponin I (TNI).

[0101] Figure 3 The calibration curve is constructed according to one aspect of this disclosure using calibrators and commercially available substrates and exemplary substrates for the determination of thyroid-stimulating hormone (TSH).

[0102] Figure 4 The calibration curve is constructed according to one aspect of this disclosure using calibrators and commercially available substrates and exemplary substrates for the determination of procalcitonin (PCT). Detailed Implementation

[0103] I. Introduction

[0104] This article discloses immunoassay methods, reagents, kits, and compounds for detecting analytes in biological samples using chemiluminescent dioxane.

[0105] In some cases, the analyte is cardiac troponin I (TNI). TNI is considered an indicator of myocardial injury. Elevated TNI levels have been associated with pathologies including, but not limited to, congestive heart failure, acute and chronic trauma, cardioversion, hypertension, hypotension, arrhythmias, pulmonary embolism, severe asthma, sepsis, critical illness, myocarditis, stroke, non-cardiac surgery, extreme sports, drug toxicity (doxorubicin, 5-fluorouracil, herceptin, snake venom), end-stage renal disease, and rhabdomyolysis with cardiac injury. Those skilled in the art understand that troponins (I, C, and T) are members of a protein complex that regulates calcium-mediated interactions between actin and myosin within muscle cells, and understand that at least three isoforms of troponin I exist: one associated with fast-twitch skeletal muscle, one with slow-twitch skeletal muscle, and one with cardiac muscle. Cardiac-specific TNI isotypes are believed to have a molecular weight of approximately 24,000 Da and a 31-amino acid post-translational tail at the N-terminus of the molecule. In some cases, the amino acid sequence of cardiac TNI is SEQ ID NO: 1. TNI can refer to full-length cardiac troponin I, variants of cardiac troponin I, fragments of cardiac troponin I, and post-translational modified forms of cardiac troponin I.

[0106] Table 1

[0107]

[0108] In some cases, the analyte is thyroid-stimulating hormone (TSH). Those skilled in the art understand that TSH has clinical utility for assessing thyroid status, including in combination with other thyroid hormones. TSH should be understood as a glycoprotein hormone composed of two non-covalently bound subunits: an α subunit, which is substantially identical to the α subunits of human luteinizing hormone (hLH), human follicle-stimulating hormone (hFSH), and human chorionic gonadotropin (hCG); and a β subunit, which is responsible for immunological and biological specificity. In some cases, TSH (including in combination with thyroid hormones or antibodies) is used to: 1) detect or rule out hypothyroidism or hyperthyroidism; 2) monitor T4 replacement therapy in hypothyroidism or antithyroid therapy in hyperthyroidism; 3) monitor TSH suppression in patients with thyroid cancer receiving thyroxine therapy; and 4) assess response to TRH stimulation tests. In some cases, the amino acid sequence of TSH is the amino acid sequence of SEQ ID NO: 2 listed in Table 2. TSH can refer to full-length thyroid-stimulating hormone, thyroid-stimulating hormone variants, thyroid-stimulating hormone fragments, and post-translational modifications of thyroid-stimulating hormone.

[0109] Table 2

[0110]

[0111] In some cases, the analyte is procalcitonin (PCT). PCT levels are considered to be correlated with the severity of bacterial infections and are thought to be useful in assessing patients who may have sepsis or septic shock. It is believed that in healthy individuals, PCT (the pro-hormone of calcitonin) is produced in thyroid C cells and subsequently converted to calcitonin in the thyroid gland, with very little PCT entering circulation. However, in individuals with systemic inflammation or bacterial infections, PCT levels rise in circulation in response to bacterial endotoxins and inflammatory cytokines. PCT is also considered specific for bacterial infections and can aid in the differential diagnosis between non-bacterial diseases, bacterial diseases, and sepsis.

[0112] PCT is believed to consist of 116 amino acids with a molecular weight of approximately 13 kDa. In some cases, the amino acid sequence of PCT is the amino acid sequence of SEQ ID NO: 3 listed in Table 3. PCT can refer to full-length procalcitonin, variants of procalcitonin, fragments of procalcitonin, and post-translational modifications of procalcitonin.

[0113] Table 3

[0114]

[0115] In some cases, the methods disclosed herein include: exposing a biological sample to a capture antibody configured to bind to at least a portion of an analyte disclosed herein, thereby generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, thereby forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound disclosed herein; wherein a reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; recording the detection signal generated by the reaction; and comparing the recorded signal with a calibration curve to quantify the level of the analyte in the biological sample.

[0116] In some cases, the methods disclosed herein are performed on the immunoassay analyzers disclosed herein, such as automated analyzers. Automated analyzers are commonly used in clinical chemistry, immunoassays, hematology, and other biological sampling and analysis applications. Automated analytical devices (such as automated analytical chemistry instruments, automated analytical immunoassay instruments, automated analytical hematology instruments, etc.) can efficiently perform clinical analysis on large volumes of samples, running multiple tests simultaneously or within short time intervals. Automated analyzers are particularly suitable for high- and medium-capacity testing environments.

[0117] In some cases, the methods disclosed herein can measure lower levels of analytes in blood (e.g., TNI, PCT, TSH) with lower limits of detection (LoD) and quantitation (LoQ) than conventional methods, including those previously undetectable, thus allowing smaller differences in the analyte to be more apparent to physicians for interpretation. LoD is the lowest concentration of an analyte that can be detected within a certain confidence level. It can also be defined as the lowest concentration that can be reliably distinguished from background. LoQ is the lowest concentration of an analyte that can be reliably quantified. "Reliably" or "in a reliable manner" means that appropriate precision, accuracy, and / or reproducibility are present.

[0118] In some cases, the signal generated by the assay methods disclosed herein is up to 35 times greater than that of immunoassay methods using conventional or known substrate preparations. In some cases, the methods disclosed herein improve the signal-to-background-noise ratio generated by the analyte compared to conventional methods. When a detector (such as a luminometer) is used, the signal or luminescence output is measured relative to the detector's light output. In these cases, the signal readout value is given in relative light units (“RLU”).

[0119] II. Definition

[0120] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein pertain. Unless otherwise specified, any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method described at the time of filing of this disclosure.

[0121] For any method disclosed herein that includes discrete steps, the steps can be performed in any feasible order. Furthermore, where appropriate, any combination of two or more steps can be performed simultaneously.

[0122] All headings are for the reader's convenience and should not limit the meaning of the text following them unless otherwise specified.

[0123] The terms "preferred" and "ideally" refer to embodiments of the invention that may provide certain benefits in certain circumstances. However, other embodiments may also be preferred in the same or other circumstances. Furthermore, the description of one or more preferred embodiments or aspects does not imply that other embodiments or aspects are useless, and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0124] The term "comprises" and its variations are not intended to be limiting in their use in the specification and claims. Such terms are to be understood as implying the inclusion of the stated steps or elements or groups of steps or elements, but not excluding any other steps or elements or groups of steps or elements.

[0125] The phrase "consisting of" means including and limited to anything following the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the listed element is necessary or mandatory, and no other elements may be present. The phrase "consisting essentially of" means including any element listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activities or actions specified in this disclosure of the listed element. Therefore, the phrase "consisting essentially of" indicates that the listed element is necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they substantially affect the activities or actions of the listed element.

[0126] Unless the context explicitly states otherwise, the singular forms “a / an” and “the” include plural indicators. These articles refer to one or more (i.e., at least one). As used herein, unless the content explicitly states otherwise, the term “or” is generally used in its usual meaning, including “and / or”. The term “and / or” means any one or more items in a list connected by “and / or”. As an example, “x and / or y” means any element in the three-element set {(x), (y), (x,y)}. In other words, “x and / or y” means “one or both of x and y”. As another example, “x, y and / or z” means any element in the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, “x, y and / or z” means “one or more of x, y and z”.

[0127] When a range is given, the endpoints include all numbers contained within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise specified or clearly apparent from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges in different embodiments of this disclosure may take any particular value or subrange within the stated range, accurate to one-tenth of the lower limit unit of the range, unless the context explicitly specifies otherwise. In this document, a “maximum” number (e.g., at most 50) includes the number (e.g., 50). The terms “in the range” or “within the range” (and similar statements) include the endpoints of the stated range.

[0128] Throughout this specification, references to "an aspect," "one aspect," "certain aspects," "some aspects," "an embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., mean that a particular feature, configuration, composition, or characteristic described in connection with that aspect is included in at least one aspect of this disclosure. Therefore, the appearance of such phrases throughout this specification does not necessarily refer to the same embodiment of this disclosure. Furthermore, specific features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0129] Unless otherwise specified, all figures representing amounts of components, molecular weights, etc., used in the specification and claims should be understood to be modified by the term "about" in all cases. As used herein, with respect to a measured quantity, the term "about" refers to the variation in the measured quantity expected by a person skilled in the art when performing the measurement and taking appropriate precautions commensurate with the purpose of the measurement and the accuracy of the measuring equipment used. The term "about," used throughout the specification and claims in conjunction with numerical values, indicates an accuracy range familiar and acceptable to a person skilled in the art. Typically, such an accuracy range is + / - 10%. Therefore, unless otherwise specified, the numerical parameters shown in the specification and claims are approximations that can vary depending on the desired properties sought to be obtained according to the invention. At least, without attempting to limit the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0130] While the wide range of numerical ranges and parameters described in this invention are approximations, the values ​​illustrated in the specific examples are reported as precisely as possible. However, all values ​​inherently contain a range that is necessarily generated by the standard deviation present in their respective test measurements.

[0131] The term “exemplary” means as a non-limiting instance, example, or illustration. As used herein, the terms “e.g.” and “for example” list one or more non-limiting aspects, instances, examples, or illustrations.

[0132] As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or extent of the characteristic or property of interest. Biological and chemical phenomena rarely (if at all) proceed to completion and / or progress to fullness or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the inherent lack of completeness in many biological and chemical phenomena. For example, "substantially" may mean at least about 20%, alternatively at least about 10%, or alternatively at least about 5% of the characteristic or property of interest.

[0133] The invention is defined in the claims. However, the following is a non-exhaustive list of non-limiting exemplary aspects. Any one or more features of these aspects may be combined with any one or more features of another instance, embodiment, or aspect described herein.

[0134] One aspect of the present invention is a method for quantitatively assessing the degree of ovarian aging in a subject using an immunoassay analyzer. The term "immunoassay" can refer to a laboratory method that uses one or more antibodies or antigens to determine the amount of an analyte in a sample. It can be based on the interaction between the antibody and the antigen, and due to the degree of selectivity for the analyte (antigen or antibody), immunoassays can be used to quantitatively determine very low concentrations of the analyte in a test sample. "Immunoassay analyzer" can include instruments in which immunoassays have been automated. Various immunoassay analyzers are commercially available, including Dxl. ® Systems (Beckman Coulter, CA), ADVIA ® CENTAUR ® Systems (Siemens Healthcare, Germany), COBAS ® System (Roche Diagnostics, Germany) , ARCHITECT ® Systems (Abbott, IL), VITROS ® Systems (Ortho-clinical Diagnostic, NJ) and VIDAS ® The system (biomerieux, France). In some respects, the immunoassay analyzer is a high-throughput immunoassay analyzer.

[0135] As used herein, the terms “subject,” “individual,” and “patient” are interchangeable and refer to vertebrates, preferably mammals. For example, in the context of this disclosure, mammals include humans, non-human primates, domesticated animals (such as dogs, cats, sheep, cattle, goats, pigs, horses, etc.), laboratory animals (such as mice, rats, rabbits, guinea pigs, etc.), and captive animals (such as animals in zoos). As used herein, the term “animal” includes humans. The term “subject” may also include patients suffering from a disease, i.e., animals. In exemplary aspects, a subject, individual, or patient refers to a person (e.g., a man, woman, or child).

[0136] II. Systems and reagents for detecting analytes using chemiluminescent dioxane.

[0137] In one embodiment, the method includes detecting the presence of an analyte in a biological sample obtained from a subject using an immunoassay analyzer. The method includes exposing the biological sample to an enzyme-conjugated antibody or enzyme-conjugated antigen and a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant. In one aspect of the method described herein, the reaction between the enzyme-conjugated antibody or enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal, which can be recorded and compared to quantify the analyte level in the biological sample. In one embodiment, the method includes aspirating a portion of the biological sample from a sample container and dispensing the aspirated sample into the reaction vessel of the immunoassay analyzer.

[0138] a. reagent

[0139] In some cases, "two-site" or "sandwich" immunoassays employ a first antibody or antibody fragment (described as a "capture" antibody) bound to a solid carrier (such as magnetic beads or particles disclosed herein) using procedures known in the art. Additionally, a second antibody or antibody fragment (described as a "detection" antibody) is coupled or conjugated to a tag (such as an enzyme disclosed herein) using procedures known in the art. When provided with a substrate, the tag generates a detectable signal such that the amount of signal measured corresponds to the amount of detective antibody bound to the analyte.

[0140] In one aspect, capture antibodies and / or affinity molecules are conjugated to magnetic beads or magnetic particles. In some cases, the magnetic beads (also known as magnetic particles, paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (about 20-30 nm) (such as magnetite). On the surface, the magnetic beads are encapsulated with, for example, polymers, protein A, protein G, protein L, secondary antibodies, or epoxy resins. Surface modification of the coating minimizes any nonspecific protein binding. Antibodies targeting the analyte of interest or capture antibodies may be covalently coupled to the surface of the magnetic beads. In some embodiments, about 2 µg of antibody is present per mg of magnetic beads. In some embodiments, about 3 µg of antibody is present per mg of magnetic beads. In some embodiments, about 4 µg of antibody is present per mg of magnetic beads. In some embodiments, about 5 µg of antibody is present per mg of magnetic beads. In some embodiments, about 6 µg of antibody is present per mg of magnetic beads. In some embodiments, about 7 µg of antibody is present per mg of magnetic beads. In some embodiments, about 8 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 9 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 10 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 12 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 13 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 14 µg of antibody is present per mg of magnetic beads. In some embodiments, approximately 15 µg of antibody is present per mg of magnetic beads.

[0141] In one aspect, substrate formulations are configured to produce chemiluminescence. These substrates can generate light and thereby provide detection corresponding to the amount of analyte captured. The term "chemiluminescent compound" refers to a compound that produces chemiluminescence in the presence of phosphatase and oxygen under suitable conditions as provided herein. In a non-limiting example, the substrate formulation comprises a 1,2-dioxane compound and at least one phosphocation surfactant. Chemiluminescent compounds that can be used in formulations of the present invention are capable of producing chemiluminescence upon contact with alkaline phosphatase. Such compounds can be synthesized as described in U.S. Patent Application No. 2022 / 0390459, which is incorporated herein by reference.

[0142] In operation, a chemiluminescent substrate is added to a container containing a second reaction mixture, and the light generated by the reaction is measured using a luminometer. The amount of light generated is inversely proportional to the concentration of the free analyte in the sample. The amount of analyte in the sample is then determined based on a stored multi-point calibration curve. In one embodiment, the detector can generate an output signal that can be processed to produce a relative light unit (“RLU”) value (i.e., an output response) indicating the measurement result. For example, a larger RLU value indicates more light compared to a smaller RLU value, which indicates a greater amount of analyte in the biological sample.

[0143] In an exemplary method for generating light by the reaction of a chemiluminescent substrate with a phosphatase (e.g., a detection antibody), the reaction is carried out at a temperature of 5°C to 50°C, preferably 20°C to 40°C, in an aqueous buffer solution with a pH of 7 to 12, 8 to 11, or preferably 8.5 to 10. The enzyme is preferably an alkaline phosphatase or an alkaline phosphatase conjugate.

[0144] In one embodiment, the 1,2-dioxane compound is a compound of formula I or a salt thereof:

[0145] Formula I

[0146] Each of R1 and R2 is independently a C3-C10 alkyl group, or R1 and R2 together with the carbon to which they are attached provide a C5-C10 cycloalkyl ring;

[0147] R3 is a C1-C10 alkyl, C6-C10 aryl, or heteroaryl group;

[0148] R4 is a C2-C10 alkenyl group;

[0149] R5 is H or a C1-C10 alkyl group; and

[0150] X is phosphate.

[0151] In one embodiment, the 1,2-dioxane compound is a compound of formula II or a salt thereof:

[0152] Formula II

[0153] Each of R10 and R11 is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0154] R3 is a C1-C10 alkyl, C6-C10 aryl, or heteroaryl group;

[0155] R4 is a C2-C10 alkenyl group;

[0156] R5 is H or a C1-C10 alkyl group; and

[0157] X is phosphate.

[0158] In one embodiment, the 1,2-dioxane compound is a compound of formula III or a salt thereof:

[0159] Formula III

[0160] Each of R10 and R11 is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0161] R3 is a C1-C10 alkyl, C6-C10 aryl, or heteroaryl group; and

[0162] R5 is H or a C1-C10 alkyl group; and

[0163] X is phosphate.

[0164] In one embodiment, the 1,2-dioxane compound is a compound of formula IV or a salt thereof:

[0165] Formula IV

[0166] Wherein R3 is a C1-C10 alkyl, C6-C10 aryl, or heteroaryl; and

[0167] R5 is H or a C1-C10 alkyl group.

[0168] In one embodiment, the 1,2-dioxane compound is

[0169] ,

[0170] Or its salt.

[0171] In one embodiment, the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

[0172] The substrate also includes at least one phosphonic surfactant. In some embodiments, the phosphonic surfactant acts as a luminescence enhancer. In some embodiments, the phosphonic surfactant comprises a small molecule phosphonic surfactant or a polymeric phosphonic surfactant.

[0173] In one embodiment, the small molecule phosphocation surfactant is a compound having the following formula:

[0174]

[0175] R12-R14 are each independently a C1-C10 alkyl group;

[0176] R15 is an aralkyl group; and

[0177] X - It is a counter ion.

[0178] In one embodiment, the polymeric phosphocationic surfactant comprises repeating unit (A), repeating unit (B), or both:

[0179]

[0180] Bu3 is tributyl, and Oct3 is trioctyl.

[0181] In some embodiments, the substrate formulation further comprises a magnesium(II) salt.

[0182] Another aspect of this disclosure includes a kit for performing any of the methods disclosed. The kit may include a reagent package. In some aspects, the reagent package includes a plurality of reagent formulations. In one aspect, the reagent package includes a first reagent formulation containing a capture antibody. In one embodiment, the capture antibody is conjugated to magnetic beads. In one aspect, the reagent package includes a reagent formulation containing an enzyme-conjugated affinity molecule. In one embodiment, the enzyme-conjugated affinity molecule is conjugated to magnetic beads. The reagent package may also include other formulations containing buffers and / or salts required for the methods. The kit may also include instructions for performing any of the methods disclosed. In some aspects, the kit may include substrate formulations, calibrators, and / or wash buffers.

[0183] b. Analyzer system

[0184] In one aspect, the immunoassay analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analysis unit, an incubator station, a washing station, a reading station, and a reagent storage unit. Furthermore, the immunoassay analyzer may include a pipette device configuration having at least one sample pipette and at least one reagent pipette; and at least one transport device. In one aspect, the transport device includes mechanisms, such as pick-and-place grippers, for transporting samples and reaction containers between the various modules of the immunoassay analyzer.

[0185] Immunoassay analyzers may include container rack assemblies configured to hold and carry containers at various locations within the instrument, allowing the containers to be used in various ways by analysis units, incubator stations, washing stations, and reading stations. Examples of container rack assemblies include container racks (e.g., sample racks, reagent racks, and diluent racks) described herein, sample presentation units, container rack units (e.g., sample rack units, reaction container rack units, and reagent rack units), container transfer units (e.g., sample transfer units, reagent transfer units, incubator transfer units, and reaction container transfer units), and container holding plates or wheels (e.g., sample wheels, incubators, and washing wheels).

[0186] The readout station may include a detector device configuration. In one aspect, the detector device configuration may include a detector configured to detect light or emission (e.g., chemiluminescence). The detector may be a emission detector, a chemiluminescence detector, a luminometer, or a photomultiplier tube-based detection instrument. In one embodiment, the detector includes a light detector configured to sense photons emitted from a measurement reaction over a time period; analog circuitry configured to provide an analog signal based on the photons emitted from the measurement reaction over the time period; and a counter circuitry configured to provide a photon count based on the photons emitted from the measurement reaction over the time period. U.S. Patent No. 11,604,146 discloses non-limiting examples of detectors that may be used in one aspect of the present invention, which is incorporated herein by reference in its entirety.

[0187] On one hand, the main sample container may be placed in the loading portion of the sample presentation unit (e.g., placed separately or placed on a shelf). In some embodiments, the sample presentation unit has at least one sample rack, alternatively at least two sample racks, alternatively at least three sample racks, alternatively at least four sample racks, alternatively at least five sample racks, alternatively at least six sample racks, alternatively at least seven sample racks, alternatively at least eight sample racks, alternatively at least nine sample racks, alternatively at least ten sample racks, alternatively at least 11 sample racks, alternatively at least 12 sample racks, alternatively at least 13 sample racks, alternatively at least 14 sample racks, alternatively at least 15 sample racks, alternatively at least 16 sample racks, alternatively at least 17 sample racks, alternatively at least 18 sample racks, alternatively at least 19 sample racks, or alternatively at least 20 sample racks. In some embodiments, each sample rack may hold at least one sample container, alternatively at least two sample containers, alternatively at least three sample containers, alternatively at least four sample containers, alternatively at least five sample containers, alternatively at least six sample containers, or alternatively at least seven sample containers. In a specific embodiment, the sample presentation unit holds approximately 140 sample containers. In another specific embodiment, the sample presentation unit has 20 sample racks, each rack accommodating seven sample containers.

[0188] In some embodiments, the sample container includes a barcode label that uniquely identifies the sample container within the immunoassay analyzer. The barcode label may also include alphanumeric characters corresponding to the barcode identification information. The immunoassay analyzer may include at least an optical reader, such as a barcode scanner. In one embodiment, the optical reader is a region scanning camera that provides a two-dimensional image of the barcode and / or the sample container. In another embodiment, the optical reader is a region scanning camera that provides a three-dimensional image of the barcode and / or the sample container.

[0189] After being fed into the loading section, the sample container can be moved into the delivery section of the sample delivery unit. In some embodiments, the sample delivery unit is maintained between approximately 4.5°C and 14°C. A transfer station can receive the main sample container from the sample delivery unit from the transport device. The main sample container delivered from the transport device to the transfer station can initially be handled in different ways. For example, the main sample container can be temporarily held at the transfer station while the sample provided inside is aspirated by a sample pipette at the transfer station. After such aspiration, the main sample container can be discharged from the automated analyzer at the unloading station of the sample delivery unit, or it can be transferred to the transport device for further processing. The instrument may also include a sample retention unit configured to receive a sample retention container and store, analyze, or otherwise process the sample retained in the sample retention container. When placed in a sample retention unit (such as a storage unit), the “main sample container” with the sample loaded into the automated analyzer can also be considered a “sample retention container”. As used herein, "receptacle" or "container" is similar and can be of various types, such as sample tubes (also referred to herein as sample tubes) and pipette tips, such as micropipettes or disposable tips. In some embodiments, the container is a tube with a diameter between about 12 mm and about 16 mm and / or a height between about 75 mm and about 100 mm. In some embodiments, the container is a cup with a volume of about 0.5 mL, alternatively about 1.0 mL, alternatively about 1.5 mL, alternatively about 2.0 mL, alternatively about 2.5 mL, or alternatively about 3.0 mL.

[0190] In one aspect, the analytical unit is configured to receive and analyze samples. In another aspect, the analytical unit is configured to perform an immunoassay. In some embodiments, the analytical unit includes a pipette device configuration. The pipette device configuration can be configured to aliquot, aspirate, and dispense fluid substances into various containers, including but not limited to sample containers, diluent containers, reagent containers, and reaction containers. The fluid substance is a substance having fluid properties. In some embodiments, the fluid substance is a single fluid substance. In other embodiments, the fluid substance is a mixture of multiple substances.

[0191] The pipette device configuration may include at least one, two, three, or four reagent pipettes for mixing reagents with sample aliquots for determination. The pipette device configuration may also include at least one, two, three, or four sample pipettes for transferring sample aliquots for determination. In some cases, the pipette device configuration includes one sample pipette. In some cases, the pipette device configuration includes one sample pipette and four reagent pipettes. In one aspect, the reagent pipettes may be arranged as dual reagent pipetting stations and are independent of each other, each pipetting station having its own fluid pump and valve, washing column, reaction vessel holder, and pipette. Sample aliquots can be transferred from the sample retention container to the reaction vessel using the sample pipettes to mix the sample aliquots with one or more reagents. In one aspect, at least one reagent pipette and at least one sample pipette are configured to aspirate and / or dispense less than about 10 µL. In one embodiment, at least one reagent pipette and at least one sample pipette are configured to aspirate and / or dispense less than about 9.9 µL, alternatively less than about 9.5 µL, alternatively less than about 8.0 µL, alternatively less than about 7.0 µL, alternatively less than about 6.0 µL, alternatively less than about 5.0 µL, alternatively less than about 4.0 µL, alternatively less than about 3.0 µL, alternatively less than about 2.0 µL, or alternatively between about 9.9 µL and 2.0 µL.

[0192] In one aspect, the first reagent pipette, the second reagent pipette, the third reagent pipette, and / or the fourth reagent pipette operate selectively and / or simultaneously. In some embodiments, the sample pipette, the first reagent pipette, the second reagent pipette, the third reagent pipette, and / or the fourth reagent pipette are configured to engage the dispensing tip prior to aspiration.

[0193] The claimed configuration allows for the simultaneous execution of at least two assays on multiple biological samples, or alternatively, at least three or four assays on multiple biological samples. Depending on the desired analysis, the biological samples may be from the same subject or from multiple subjects. In some aspects, the method further comprises multiple sample containers. In some embodiments, the method comprises at least one sample container, at least two sample containers, at least three sample containers, at least four sample containers, at least five sample containers, at least ten sample containers, at least twenty sample containers, at least fifty sample containers, at least one hundred sample containers, at least two hundred sample containers, at least three hundred sample containers, at least four hundred sample containers, or at least five hundred sample containers. In some aspects, the sample containers are housed within an immunoassay analyzer.

[0194] In a non-limiting example, if multiple assays targeting multiple biomarkers are being performed, a first pipette may be configured to aspirate reagent from a reagent container containing a first capture antibody and dispense it into a first reaction container, while a second pipette may be configured to simultaneously aspirate reagent from a reagent container containing a second capture antibody and dispense it into a second reaction container. Depending on the desired analysis, a third and fourth pipette may also be configured to simultaneously aspirate reagent from reagent containers containing a third affinity molecule and a fourth affinity molecule and dispense it into a third reaction container and a fourth reaction container, respectively.

[0195] The disclosed pipette's simultaneous and / or selective operation allows for high-throughput analysis. In some embodiments, the method is configured to analyze at least about 200 plasma samples / hour, alternatively at least about 300 plasma samples / hour, alternatively at least about 400 plasma samples / hour, alternatively at least about 440 plasma samples / hour, or alternatively at least about 500 plasma samples / hour.

[0196] In one aspect, an immunoassay analyzer includes a reagent kit configured to contain a plurality of reagent containers. A "reagent kit" can include any suitable container capable of storing reagents. Examples of reagent kits can include a generally rectangular, elongated body formed to include a plurality of reagent containers (including one or more large reagent containers and one or more relatively smaller reagent containers) and features for ease of handling and automation. U.S. Patent No. 9,519,000 discloses a non-limiting example of a reagent kit that can be used in one aspect of the invention, which is incorporated herein by reference in its entirety.

[0197] In one embodiment, the reagent pack may be configured to contain a sufficient volume of reagent for multiple assays. In some embodiments, each reagent pack includes reagent for about 20 to about 100 assays, and in some cases about 50 assays.

[0198] In some embodiments, the reagent kit may be equipped with an empty or partially filled reagent container, from which the reagent is subsequently transferred from a mass container (e.g., a vial) to the reagent container. The size of individual reagent containers may vary to suit the requirements of the assay type. Factors that may determine the size of the reagent container include the expected number of uses for the reagent kit type, concentration-dependent stability concerns regarding the reagent components, and the need to minimize the volume of the final reaction mixture. As described above, in some embodiments, each reagent kit may include a large reagent container and multiple smaller reagent containers. Each reagent container may be large enough to accommodate a micropipette tip or disposable pipette tip (i.e., a dispo-tip) for removing a volume of reagent for the assay. In some embodiments, the reagent kit may be maintained at a temperature between approximately 4°C and 10°C.

[0199] "Reagent container" can refer to a container, unit, fluid containment, etc., configured to store reagents. In one embodiment, the reagent container includes an elastomeric self-sealing membrane. The elastomeric self-sealing membrane can be a polymer, such as polypropylene, capable of returning to its original shape when the polymer is punctured. In some embodiments, the elastomeric membrane can be a thermoplastic elastomer with a hardness of 30-40 Shore A. In other embodiments, the hardness can be 20-50 Shore A, or about 30 Shore A. The elastomer deforms sufficiently to form a tight seal with the base of the container. Thermoplastic elastomers are advantageous because they are compatible with plastic injection molding processes.

[0200] The elastomeric membrane can be large enough to provide sufficient compression without bottoming out the container's sealing portion. The stiffness and size can be matched to allow the elastomeric membrane to reach the sealing portion with a reasonable sealing force. In some embodiments, the diameter of the elastomeric membrane is small enough that when compressed by engagement with the pipette tip, it adheres to the sealing portion without contacting the wall of the pipette tip. This advantageously concentrates the sealing force on the container's sealing portion and distributes the sealing force evenly, thereby preventing leakage. In some embodiments, the sealing force is about 44 Newtons (about 9.9 pounds) and generates a pressure of about 300 kPa (about 43.5 pounds per square inch) to about 1000 kPa (145.0 pounds per square inch) on the sealing surface.

[0201] In methods for analyzing multiple samples, elastomeric self-sealing membranes allow reagent aspiration without concern for evaporation. In some embodiments, the reagent pack further includes a baffle wall disposed between the reagent containers. In one aspect, the reagent pack may include at least one reagent container, alternatively at least two reagent containers, alternatively at least three reagent containers, alternatively at least four reagent containers, alternatively at least five reagent containers, or alternatively at least ten reagent containers. In one embodiment, the immunoassay analyzer further includes a reagent storage unit, in which the reagent pack is housed.

[0202] During operation, a fluid substance is dispensed into the reaction vessel. Examples of fluid substances include samples, diluents, reagents, substrates, or any combination thereof as described herein. In some embodiments, the reaction vessel already contains other fluid substances, such as samples, and after the fluid substance is dispensed into the reaction vessel, it is mixed with other fluid substances in the reaction vessel. Mixing can be performed using a stirrer in direct contact with the fluid substance, an ultrasonic probe in direct or indirect contact with the fluid substance, or any other suitable mixing device. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For example, a reagent pipette may be equipped with a tip to allow the reagent pipette to ultrasonically mix the reagent in the reagent package before aspirating it for transport to the reaction vessel, thereby ensuring that the aspirated reagent is not affected by any sedimentation that may occur in the reagent package. Similarly, a sample pipette may be dedicated to this purpose.

[0203] The transfer unit transfers reaction vessels to an incubator station including an incubator and removes reaction vessels from the incubator station including an incubator. In some embodiments, the transfer unit transfers one or more pipette-loaded reaction vessels from a reagent rack unit to an incubator. Additionally, the transfer unit can transfer one or more reaction vessels from an incubator to a reagent rack unit. The transfer unit can also remove an incubator from a read or completed reaction vessel.

[0204] The incubator is thermally controlled to maintain a predetermined temperature. In some embodiments, the incubator is maintained at about 30°C to 40°C. In other embodiments, the incubator is maintained at about 37°C to ensure, for example, immune and enzymatic responses. As an example, the incubator is used for assay incubation.

[0205] In some embodiments, the transfer unit transfers incubated reaction containers from the incubator to the washing unit, the assay reaction container from the washing unit to the incubator, the reaction container containing substrate from the washing unit to the incubator for substrate incubation or enzyme reaction, the washed reaction container after substrate incubation is transferred from the incubator to the detector device configuration, and the read or completed reaction container is transferred from the detector device configuration to the incubator. Used reaction containers may be delivered to a waste location.

[0206] In operation, the sample and reagents are dispensed into a reaction vessel and mixed. The mixture is then transferred to an incubator. During incubation, the sample and reagents interact. The resulting “first reaction mixture” is the result of the incubation between the sample and reagents. The reagents may include specific binding reagents, such as capture antibodies that are specific to analytes analyzed by an immunoassay analyzer. In non-limiting examples, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0207] In operation, a reagent different from that used in the first reaction mixture is added to and mixed with the first reaction mixture. The mixture is then transferred to an incubator. During incubation, the first reaction mixture and the reagent interact. The resulting "second reaction mixture" is the result of incubation between the first reaction mixture and the reagent. The reagent may include an affinity molecule, which is an antibody, monoclonal antibody, polyclonal antibody, synthetic antibody mimic, aptamer, affimer, DARPin, oligonucleotide, peptide, or antigen. In some embodiments, the affinity molecule is an enzyme-conjugated antibody, an enzyme-conjugated antigen, an alkaline phosphatase (AP)-conjugated secondary antibody, or a labeled antibody. The affinity molecule may be configured to bind to at least a portion of the analyte or at least a portion of the capture antibody. In non-limiting examples, the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0208] The washing station receives and supports the reaction vessel thereon, enabling various aspects of the diagnostic process using an immunoassay analyzer. In one embodiment, the washing station is configured to wash away at least some unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles and / or fluids, etc.) and unreacted samples. The washing station may be configured to perform a predetermined number of washing actions according to the assay. The washing station may also be configured to perform a predetermined number of washes in a predetermined sequence. In some embodiments, the washing station is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions. In some embodiments, the washing station is a thermally controlled device to separate bound or free analytes from particles after incubation. In some embodiments, the washing unit is maintained at about 30°C to 40°C. In other embodiments, the washing unit is maintained at about 37°C to ensure enzyme reaction; for example, U.S. Patent Publication No. 2022 / 0357352 discloses a configurable washing process according to an aspect of the invention, which is incorporated herein by reference in its entirety.

[0209] The washing station may include a washing apparatus configuration that can be configured to provide a basic number of washing sequences (or washing actions) per reaction vessel, and optionally provide an additional number of washing actions. The additional number of washing actions may include one, multiple, or all of the possible number of washing actions. An additional number of washing actions beyond the basic number may be specified for certain assays in the assay protocol document. The washing apparatus configuration may include a cleaning dispensing nozzle (or probe) for dispensing rinsing fluid and a cleaning aspiration nozzle (or probe) for aspirating unreacted components. In operation, the basic number of washing actions performed may be one, two, three, four, or five, and the additional number of washing actions may be one, two, three, four, or five. In operation, the basic number of washing actions performed may be three, and the additional number of washing actions may be one or two. In this embodiment, the basic number of washing actions can be performed if three probes dispense buffer solution to each vessel once, and three probes aspirate at least some unreacted components, some buffer solution, and / or at least some unreacted reagents from each vessel once. Based on the principles of this disclosure, certain probes can be selectively used to dispense cleaning buffer solution into a container and aspirate at least some unreacted components, some buffer solution, and / or at least some unreacted reagents from the container to perform additional washing actions.

[0210] In one aspect, the immunoassay analyzer further includes a washing device configuration. In one embodiment, the washing device configuration is configured to wash away at least some unreacted components from the sample, first reagent, second reagent, or substrate preparation in the first reaction mixture, second reaction mixture, and / or detection mixture. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluids, etc.) and unreacted samples. The washing device configuration may be configured to perform a predetermined number of washing actions according to the assay. The washing device configuration may also be configured to perform a predetermined number of washes in a predetermined sequence. In some embodiments, the washing apparatus is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

[0211] In one embodiment, a first or second reaction mixture is subjected to a magnetic field. The magnetic beads do not exhibit bead-to-bead attraction but migrate only when the magnetic field is applied. The captured analyte or target is separated from the mixture, and magnetization can be used to retain the desired component within the reaction vessel.

[0212] During operation, a container containing a first reaction mixture is moved in the vicinity of one or more magnets. The magnets attract magnetic beads to one or more sides of the reaction container. The reaction container is then subjected to a washing process, wherein a cleaning dispensing nozzle dispenses rinsing fluid, and a cleaning aspiration nozzle aspirates unreacted components. The aspiration nozzles may be washed with a probe washer before and / or after aspiration. The reaction container may undergo a series of washing processes, which may include at least two series of dispensing rinsing fluid and aspiration of uncollected fluid components, alternatively at least three series, alternatively at least four series, or alternatively at least five series. Thus, one or more unreacted substances in the container are removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzles.

[0213] During operation, a container containing a second reaction mixture is moved in the vicinity of one or more magnets. The magnets attract magnetic beads or particles to one or more sides of the reaction container. The reaction container is then subjected to a washing process, wherein a cleaning dispensing nozzle dispenses rinsing fluid and a cleaning aspiration nozzle aspirates unreacted components. The aspiration nozzles may be washed with a probe washer before and / or after aspiration. The reaction container may undergo a series of washing processes, which may include at least two series of dispensing rinsing fluid and aspiration of uncollected fluid components, alternatively at least three series, alternatively at least four series, or alternatively at least five series. Thus, one or more unreacted substances in the container are removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzles.

[0214] In one aspect, the immunoassay analyzer includes a detector device configuration. In another aspect, the detector device configuration may include a detector configured to detect light or luminescence (e.g., chemiluminescence). The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier tube-based detection instrument. In one embodiment, the detector includes a light detector configured to sense photons emitted from the assay reaction over a time period; analog circuitry configured to provide an analog signal based on the photons emitted from the assay reaction over the time period; and a counter circuitry configured to provide a photon count based on the photons emitted from the assay reaction over the time period. U.S. Patent No. 11,604,146 discloses non-limiting examples of detectors that may be used in one aspect of the present invention, which is incorporated herein by reference in its entirety.

[0215] In operation, the substrate is dispensed into the second reaction mixture and mixed. After a certain reaction time required for the substrate and the second reaction mixture to interact, the reaction vessel is then subjected to a washing process, wherein a cleaning dispensing nozzle dispenses flushing fluid and a cleaning aspiration nozzle aspirates unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after aspiration. The reaction vessel may undergo a series of washing processes, which may include at least two series of dispensing flushing fluid and aspiration of uncollected fluid components, alternatively at least three series, alternatively at least four series, or alternatively at least five series. Thus, one or more unreacted substances in the vessel are removed (e.g., rinsed away) by the binding-free cleaning aspiration nozzle. The resulting detection mixture is transferred to a detector device configuration.

[0216] The determination of features and / or characteristics described herein may benefit from one or more additional washing actions.

[0217] In some embodiments, the transport device includes three pick-and-place holders, wherein a first pick-and-place holder can be used to transport sample containers between the loading section, transfer station, and reagent pipetting station. A second pick-and-place holder can be used to transport reaction containers between the reagent pipetting station and incubator station or reading station. A third pick-and-place holder can be used to transport reaction containers between the incubator station and washing station or reading station. A detailed description of the configuration and function of one embodiment of a container pick-and-place holder is provided in U.S. Patent No. 7,128,874, and is incorporated herein by reference in its entirety. However, it should be understood that other pick-and-place mechanisms capable of transporting samples and reaction containers between various modules of an immunoassay analyzer are also contemplated for the purposes of this invention.

[0218] In one aspect, the immunoassay analyzer includes a machine vision device comprising an image capture unit and an image interpretation unit configured to monitor the instrumentation and / or assay functions of the immunoassay analyzer. In some embodiments, the instrumentation functions may include optical sensors, pressure sensors, and thermistors. In some embodiments, the assay functions may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision device operates to evaluate the preparation of a sample for subsequent analysis. In some embodiments, the machine vision device utilizes one or more image capture units to determine whether a sample has been properly prepared for analysis. As described herein, the machine vision device provides a direct and simple measurement of the volume or integrity of a sample to determine whether the sample has been properly prepared, thereby enabling the analysis unit to produce reliable results. Exemplary machine vision devices are described in U.S. Patent No. 11,263,433, which is incorporated herein by reference.

[0219] In some respects, machine vision devices operate to detect the volume of fluid material in a container and determine whether the volume held in the container is suitable as a target. As described herein, such volume detection is configured to use a dispensing tip image capture unit to detect the volume at the dispensing tip and a container image capture unit to detect the volume at the container.

[0220] In some respects, machine vision equipment operates to detect any interfering elements that may disrupt the analysis process and potentially produce incorrect results at the dispensing tip. As described herein, this dispensing tip evaluation is configured to use a dispensing tip image capture unit and the alignment of the dispensing tip relative to the dispensing tip image capture unit to determine the quality of the fluid material at the dispensing tip.

[0221] In some aspects, machine vision equipment operates to determine the particle concentration in a fluid substance contained in a container (such as a reaction vessel, sample container, dilution vessel, cuvette, or any suitable type of container) used throughout the immunoassay analyzer process. In some embodiments, such reaction vessel particle concentration checks utilize a container image capture unit.

[0222] The dispensing tip image capture unit operates to capture images of the dispensing tip at one or more locations. In some embodiments, the dispensing tip image capture unit is fixed at a specific location within the instrument. In other embodiments, the dispensing tip image capture unit is movably disposed within the instrument, and the dispensing tip image capture unit can move independently of other components of the instrument or move with one or more components of the instrument. Some embodiments of the instrument include multiple dispensing tip image capture units. As described herein, the dispensing tip image capture unit may include a camera unit.

[0223] A container image capture unit operates to capture images of containers at one or more locations. In some embodiments, the container image capture unit is fixed at a specific location within the immunoassay analyzer. In other embodiments, the container image capture unit is movably disposed within the immunoassay analyzer, and the container image capture unit can move independently of other components of the immunoassay analyzer or move together with one or more components of the immunoassay analyzer. Some embodiments of the immunoassay analyzer include multiple container image capture units. As described herein, the container tip image capture unit includes a camera unit.

[0224] The carriage image capture unit operates to capture images of a receptacle carriage assembly, with or without receptacles, at one or more locations. In some embodiments, the carriage image capture unit is fixed at a specific location within the instrument. In other embodiments, the carriage image capture unit is movably disposed within the instrument, and the dispensing tip image capture unit can move independently of other components of the instrument or move with one or more components of the instrument. Some embodiments of the instrument include multiple carriage image capture units.

[0225] All units of the immunoassay analyzer are connected to a controller, which can perform block control of all analyzer functions using, for example, a microcomputer. The controller may contain sub-units, such as a data processing unit, a communication interface, etc. According to an exemplary embodiment of the invention, the controller may include a data processor, a non-transitory computer-readable medium, and a data storage device coupled to the data processor. The non-transitory computer-readable medium may contain code executable by the data processor to perform the functions described herein. The data processor may store, for example, data for processing samples, sample data, or data for analyzing sample data.

[0226] A data processor may include any suitable data computing device or a combination of such devices. An exemplary data processor may include one or more microprocessors that work together to perform a desired function. A data processor may include a CPU containing at least one high-speed data processor sufficient to execute program components for performing user- and / or system-generated requests. The CPU may be a microprocessor such as AMD's Athlon, Duron, and / or Opteron; IBM and / or Motorola's PowerPC; IBM and Sony's Cell processors; Intel's Celeron, Itanium, Pentium, Xeon, and / or XScale; Apple M1 and / or similar processors.

[0227] Computer-readable media and data storage devices can be any suitable one or more devices capable of storing electronic data. Instances of memory can include, for example, one or more memory chips, disk drives, etc. Such memory can be operated using any suitable electrical, optical, and / or magnetic mode of operation.

[0228] A computer-readable medium may contain code that can be executed by a data processor to perform any suitable method. For example, a computer-readable medium may contain code that can be executed by a processor to cause a controller to operate according to a predetermined schedule. In some embodiments of the technology claimed in this invention, the predetermined schedule is a component test.

[0229] In one aspect, the cycle time of the immunoassay analyzer described herein is approximately 45 seconds or less. "Cycle time" is the time required for all modules and / or functions of the immunoassay analyzer to complete the tasks necessary for generating results. In some embodiments, the cycle time is approximately 40 seconds or less, alternatively approximately 35 seconds or less, alternatively approximately 30 seconds or less, alternatively approximately 25 seconds or less, alternatively approximately 20 seconds or less, or alternatively approximately 15 seconds. In operation, the sample pipette can complete its task within 8 seconds, and the reagent pipette can complete its task within 32 seconds. In some aspects, the task of the pipette is defined as the time required to aspirate and dispense a sample or reagent, including the time required to move from the starting position and return to the starting position. In this embodiment, to maintain high throughput, four reagent pipettes are present in the immunoassay analyzer (32 seconds / 4 = 8 seconds). If one of the reaction mixtures is incubated for 5 minutes, then at least 38 incubation positions are required to support that incubation time (300 seconds / 8 seconds = 37.5 seconds). In some embodiments, the immunoassay analyzer has at least 30 incubation positions, alternatively at least 40 incubation positions, alternatively at least 50 incubation positions, alternatively at least 60 incubation positions, alternatively at least 70 incubation positions, alternatively at least 80 incubation positions, alternatively at least 90 incubation positions, alternatively at least 100 incubation positions, alternatively at least 125 incubation positions, alternatively at least 150 incubation positions, alternatively at least 175 incubation positions, or alternatively at least 200 incubation positions.

[0230] In one respect, the time to first result (TTFR) of the immunoassay analyzer described herein is approximately 60 minutes or less. “TTFR” is a measure of the time from sample aspiration to the determination of the presence and / or concentration of the analyte. In some embodiments, TTFR is approximately 60 minutes or less, alternatively approximately 55 minutes or less, alternatively approximately 50 minutes or less, alternatively approximately 45 minutes or less, alternatively approximately 40 minutes or less, alternatively approximately 35 minutes or less, alternatively approximately 30 minutes or less, alternatively approximately 25 minutes or less, alternatively approximately 20 minutes or less, alternatively approximately 15 minutes or less, or alternatively approximately 10 minutes or less.

[0231] In one embodiment, the reagent pack may be configured to contain a sufficient volume of reagent for multiple assays. In some embodiments, each reagent pack includes reagent for about 1, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 assays. In some embodiments, each reagent pack includes reagent for about 50 assays. In some embodiments, each reagent pack includes reagent for about 250 assays. In some embodiments, each reagent pack includes reagent for about 500 assays. In some embodiments, each reagent pack includes reagent for about 650 assays. In some embodiments, the reagent pack may include a plurality of reagent containers, wherein the reagent pack is configured to store a volume of reagent required for at least about one assay. In some embodiments, the reagent pack is configured to store a volume of reagent required for up to 650 assays. In a non-limiting example, the reagent pack includes at least three reagent containers, wherein each reagent container is independently configured to store a volume of reagent required for at least about one assay. In one embodiment, each reagent container is independently configured to store a volume of reagent required for up to 650 measurements. In a non-limiting example, the reagent kit includes at least four reagent containers, each independently configured to store a volume of reagent required for at least about one measurement. In one embodiment, each reagent container is independently configured to store a volume of reagent required for up to 650 measurements. In a non-limiting example, the reagent kit includes at least five reagent containers, each independently configured to store a volume of reagent required for at least about one measurement. In one embodiment, each reagent container is independently configured to store a volume of reagent required for up to 650 measurements.

[0232] III. Determination of dioxane using chemiluminescence

[0233] In one aspect of the methods disclosed herein, an analyte (e.g., TSH, TNI, PCT) is detected in a biological sample. In one aspect, the biological sample is serum, whole blood, plasma, and / or cerebrospinal fluid. In one aspect, the method includes exposing the biological sample to a capture antibody configured to bind to at least a portion of the analyte, thereby generating a first reaction mixture.

[0234] The resulting "first reaction mixture" is the result of incubation between a biological sample and a capture antibody. In some embodiments, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample container and dispensing the aspirated biological sample into a reaction vessel of an immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into a reaction vessel. During incubation, the sample and the capture antibody interact. In one embodiment, the capture antibody binds to a portion of the analyte. In non-limiting examples, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, or alternatively at least about 50 minutes.

[0235] In one embodiment, the method includes exposing a first reaction mixture to an enzyme-conjugated antibody or an enzyme-conjugated antigen to generate a second reaction mixture. In one aspect, the enzyme comprises alkaline phosphatase (AP). The “second reaction mixture” is the result of incubation between the first reaction mixture and the enzyme-conjugated antibody or enzyme-conjugated antigen. In some embodiments, the second reaction mixture is generated by aspirating a portion of a second reagent containing the enzyme-conjugated antibody or enzyme-conjugated antigen from at least one reagent container and dispensing the aspirated reagent into a reaction container. During incubation, the first reaction mixture and the enzyme-conjugated antibody or enzyme-conjugated antigen interact. In one embodiment, the enzyme-conjugated antibody is configured to bind to at least a portion of the analyte. In another embodiment, the enzyme-conjugated antigen is configured to bind to at least a portion of a capture antibody. In one aspect, the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0236] In one embodiment, the method involves exposing a second reaction mixture to a substrate formulation comprising a 1,2-dioxane compound and at least one phosphocation surfactant. In one embodiment, the mixture is detected by aspirating the substrate formulation and dispensing the aspirated substrate formulation into a reaction vessel. On one hand, the test mixture is incubated for at least 20 seconds, or alternatively at least 30 seconds, or alternatively at least 40 seconds, or alternatively at least 50 seconds, or alternatively at least 60 seconds, or alternatively at least 70 seconds, or alternatively at least 80 seconds, or alternatively at least 90 seconds, or alternatively at least 100 seconds, or alternatively at least 110 seconds, or alternatively at least 120 seconds, or alternatively at least 130 seconds, or alternatively at least 140 seconds, or alternatively at least 150 seconds, or alternatively at least 160 seconds, or alternatively at least 170 seconds, or alternatively at least 180 seconds, or alternatively at least 190 seconds, or alternatively at least 200 seconds, or alternatively at least 210 seconds, or alternatively at least 220 seconds, or alternatively at least 230 seconds, or alternatively at least 240 seconds, or alternatively at least 250 seconds, or alternatively at least 260 seconds, or alternatively at least 270 seconds, or alternatively at least 280 seconds.

[0237] In one aspect, the substrate formulation is configured to generate chemiluminescence. These substrates can generate light and thereby provide detection corresponding to the amount of analyte captured. In some embodiments, the reaction between an enzyme-conjugated antibody or an enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal.

[0238] In operation, a substrate is added to a container containing a second reaction mixture, and the light produced by the reaction is measured using a luminometer. The produced light is recorded (e.g., a detection signal), and the amount of analyte in the sample is then determined based on a stored multi-point calibration curve. In one embodiment, the detector may generate an output signal that can be processed to produce a relative light unit (“RLU”) value (i.e., an output response) indicating the measurement result. For example, a larger RLU value indicates more light compared to a smaller RLU value, which indicates a greater amount of analyte in the biological sample.

[0239] In an exemplary method for generating light by the reaction of a substrate with a phosphatase, the reaction is carried out at a temperature of 5°C to 50°C, preferably 20°C to 40°C, in an aqueous buffer solution with a pH of 7 to 12, 8 to 11, or preferably 8.5 to 10.

[0240] In one aspect, the capture antibody, enzyme-conjugated antibody, and / or enzyme-conjugated antigen are conjugated with magnetic beads or magnetic particles. In one embodiment, the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field prior to detection. Magnetization can be used to retain the desired component within the reaction vessel. In some embodiments, after the introduction of the first or second reagent, the reaction vessel is moved near one or more magnets. The one or more magnets attract the magnetic beads or magnetic particles to one or more sides of the reaction vessel. A washing device is configured to wash the reaction vessel a predetermined number of times. During washing, the magnets retain the magnetic beads or magnetic particles while washing away unreacted components. In some embodiments, increasing the number of washing actions can produce a better signal-to-noise ratio and increase the sensitivity of the assay.

[0241] In one aspect, the method is performed using the immunoassay analyzer described herein, and the cycle time is about 45 seconds or less. In some embodiments, the cycle time is about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

[0242] In one aspect, the method is performed using the immunoassay analyzer disclosed herein, and the time to first result (TTFR) is about 60 minutes or less. In some embodiments, the TTFR is about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0243] In one aspect, the disclosed method includes detecting TNI using an immunoassay. Depending on the desired analysis, different enzyme-conjugated affinity molecules can be used in the TNI assay. For example, the affinity molecule can be an antibody configured to bind to at least a portion of the TNI. In another non-limiting example, the affinity molecule is an antigen configured to bind to at least a portion of a capture antibody. In some aspects, commercial TNI assays are employed according to the manufacturer's protocol. A non-limiting assay according to one aspect of the invention is shown in Example 2.

[0244] In some aspects of the method, for TNI assay, the ratio of the signal generated by the cleavage enzyme to the background noise is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, alternatively at least about 800. Alternatives are at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000, at least about 15,000, at least about 20,000, or at least about 25,000. The signal-to-noise ratio (S / S0) gives a measure of the confidence that the difference between the signal and noise and the background is true.

[0245] In some respects, the concentration of the quantified TNI level is at least 1, or alternatively at least 2, or alternatively at least 3 times the LoQ of the method.

[0246] In some aspects, TNI immunoassay is used to diagnose myocardial infarction and / or monitor symptoms of myocardial ischemia; new ischemic changes in an electrocardiogram (ECG); the development of pathological Q waves in an ECG; imaging evidence of new loss of viable myocardium or new regional wall motion abnormalities consistent with an ischemic etiological pattern; and / or to identify coronary thrombosis by angiography or autopsy. In some aspects, TNI immunoassay is used to monitor increases and / or decreases in cardiac Tn values. In some aspects, the method may further include administering an effective amount of the pharmaceutical composition to the subject and / or identifying the treatment process (or treatment regimen) based on cardiac Tn values.

[0247] The terms “treat,” “treating,” or “treatment” refer to the administration of a compound or pharmaceutical composition to a subject to partially or completely alleviate, suppress, improve, or reduce symptoms suffered by the subject. This means any manner in which one or more symptoms of the condition are improved or otherwise beneficially altered. As used herein, improvement of symptoms of a particular condition means any reduction, whether permanent or temporary, persistent or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of this disclosure. For example, treating a subject may mean eliminating or reducing clinical signs of the subject's condition; stopping, suppressing, or slowing the progression of the subject's condition; and / or reducing the number, frequency, or severity of clinical symptoms of the subject's condition. A “treatment regimen” is a protocol or system of specific therapies (including drugs or therapeutic interventions) developed for treatment. A “therapeutic intervention” refers to a clinical intervention designed to manage a disease, condition, symptom, or injury and to prevent further clinical intervention.

[0248] "Effective dose" includes "therapeutic effective dose" and "preventive effective dose." The term "therapeutic effective dose" refers to the amount that effectively treats and / or improves the condition of a subject. The term "preventive effective dose" refers to the amount that is effective in preventing and / or significantly reducing the subject's chance of developing the condition. An effective dose of the pharmaceutical composition can be administered orally or via intravenous injection. The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject based on species, age and general condition, severity of condition, specific composition, mode of administration, mode of activity, etc.

[0249] In one aspect, the disclosed method includes detecting TSH using an immunoassay. Depending on the desired analysis, different enzyme-conjugated affinity molecules can be used in the TSH assay. For example, the affinity molecule can be an antibody configured to bind to at least a portion of TSH. In another non-limiting example, the affinity molecule is an antigen configured to bind to at least a portion of a capture antibody. In some aspects, commercial TSH assays are employed according to manufacturer-specified protocols. A non-limiting assay protocol according to one aspect of the invention is shown in Example 3.

[0250] In some aspects of the method, for TSH assay, the ratio of the signal generated by the cleavage enzyme to the background noise is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, or alternatively at least about 100, 200, or 300. The alternative is at least about 400, the alternative is at least about 500, the alternative is at least about 600, the alternative is at least about 700, the alternative is at least about 800, the alternative is at least about 900, the alternative is at least about 1,000, the alternative is at least about 2,000, the alternative is at least about 3,000, the alternative is at least about 4,000, the alternative is at least about 5,000, the alternative is at least about 6,000, the alternative is at least about 7,000, or the alternative is at least about 8,000.

[0251] In some respects, the concentration of the quantified TSH level is at least 1, or alternatively at least 2, or alternatively at least 3 times the LoQ of the method.

[0252] In some aspects, TSH immunoassay is used to detect TSH levels that are abnormally associated with various thyroid conditions. In some embodiments, TSH is measured in conjunction with thyroid hormones or antibodies to: 1) detect or rule out hypothyroidism or hyperthyroidism; 2) monitor T4 replacement therapy in hypothyroidism or antithyroid therapy in hyperthyroidism; 3) monitor TSH suppression in patients with thyroid cancer receiving thyroxine therapy; and / or 4) assess response to TRH stimulation tests. In some embodiments, TSH assays can differentiate between different levels of TSH suppression associated with Graves' disease and subclinical hyperthyroidism and can aid in the diagnosis of thyroid disorders during pregnancy and postpartum. In some aspects, the method may further include administering an effective amount of a pharmaceutical composition to a subject, alone or in combination with other clinical factors, and / or identifying the treatment process (or regimen) based on TSH values.

[0253] In one aspect, the disclosed method includes detecting PCT using an immunoassay. Depending on the desired analysis, different enzyme-conjugated affinity molecules can be used in the PCT assay. For example, the affinity molecule can be an antibody configured to bind to at least a portion of PCT. In another non-limiting example, the affinity molecule is an antigen configured to bind to at least a portion of a capture antibody. In some aspects, commercial PCT assays are employed according to the manufacturer's protocol. A non-limiting assay according to one aspect of the invention is shown in Example 4.

[0254] In some respects, for PCT assays, the ratio of signal generated by the cleavage enzyme to background noise is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000. In some respects, the LoQ of PCT assays is about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

[0255] In some respects, PCT measurement, when used in conjunction with other laboratory findings and clinical assessments, helps assess the risk of progression to severe sepsis and septic shock in critically ill patients on the first day of admission to the intensive care unit (ICU).

[0256] In some aspects, the method may further include administering an effective amount of the pharmaceutical composition to the subject and / or identifying the treatment process (or treatment regimen) based on a combination of PCT values ​​and other clinical factors.

[0257] Additional examples are provided below.

[0258] Example

[0259] Example 1: Substrate signal comparison

[0260] A comparison was made between substrate formulations (substrate 1) of 1,2-dioxane compounds comprising formula I herein and phosphocation surfactants according to one aspect of this disclosure, and formulations comprising the structure... The difference in chemiluminescence produced by alkaline phosphatase chemiluminescent substrate (substrate 2) was observed. Signal comparison was performed using a luminometer.

[0261] The substrate 1 test solution was prepared by adding 0.125 mg / mL of substrate 1 to a buffer solution containing 0.25 M 2-amino-2-methyl-1-propanol (AMP) buffer and 0.18 mg / mL magnesium chloride.

[0262] Aliquots of the enzyme were placed in six reaction vessels. Aliquots of substrate 1 test solution were added to five reaction vessels, and aliquots of substrate 2 were added to the sixth reaction vessel. Immediately after injection, the generated chemiluminescence signal was continuously read for 280 seconds.

[0263] like Figure 1 As shown, the signal generated by substrate 1 is 4 to 60 times stronger than that generated by substrate 2. The longer the incubation time after substrate injection, the more signal substrate 1 continues to generate.

[0264] Example 2: TNI Enhancement Signal Generation

[0265] A series of TNI calibrators were used to compare the signals generated by substrate 1 and substrate 2. Calibrator levels were 30 pg / mL (S1), 139 pg / mL (S2), 550 pg / mL (S3), 2,222 pg / mL (S4), 8,967 pg / mL (S5), and 26,224 pg / mL (S6). A control sample (0 pg / mL, S0) was also analyzed.

[0266] TNI sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettes and one sample pipette; (ii) a reagent pack configured to store a certain volume of reagents for at least 50 assays; (iii) an ultrasonic mixer; (iv) the ability of each reaction vessel to perform more than 5 wash cycles; and (v) a luminometer.

[0267] A reagent comprising a conjugate containing an antibody capable of binding to a TNI epitope linked to alkaline phosphatase (“ALP”) is added to a reaction vessel. Sample aliquots are pipetted into the reaction vessel using a sample pipette, and the first reaction mixture is incubated. Paramagnetic particles conjugated with the TNI antibody capable of binding to the TNI epitope are pipetted into the reaction vessel using one of four reagent pipettes. The reaction vessel is then mixed using an ultrasonic mixer and incubated to produce a second reaction mixture. A magnetic field is applied to the reaction vessel, and the first reaction mixture is washed with washing buffer to remove any unreacted components. Substrate 1 is added to the reaction vessel, incubated, and the signal generated by the resulting reaction is read using a luminometer. This process is repeated twice for substrate 1 and once for substrate 2.

[0268] The test results were automatically determined by the system software. The detection of the analyte in the sample was determined based on the measured light production. Table 4 presents the signal collected in relative light units (“RLU”) for each measured sample for each substrate. The signal-to-noise ratio (S / S0) was calculated at each concentration value. Substrate 2 determination and Substrate 1 (Run 1) determination used the same volume of reagent and sample. Substrate 1 (Run 2) determination used half the volume of reagent and sample used in Substrate 1 (Run 1) determination. Substrate 1 (Run 3) determination used one-quarter the volume of reagent and sample used in Substrate 1 (Run 1) determination. Figure 2 The results of each measurement were also plotted.

[0269] Table 4

[0270]

[0271] According to Table 4 and Figure 2The results shown indicate that substrate 1 produces a larger signal at all calibrator concentrations compared to substrate 2. For example, as shown for S4, substrate 1 produces a signal up to 35 times stronger than substrate 2.

[0272] Example 3: TSH Enhancement Signal Generation

[0273] A series of TSH calibrators were used to compare the signals generated by substrate 1 and substrate 2. Calibrator levels were 0.5 µIU / mL (S1), 0.3 µIU / mL (S2), 3 µIU / mL (S3), 15 µIU / mL (S4), and 50.5 µIU / mL (S5). A control sample (0 µIU / mL, S0) was also analyzed.

[0274] TSH sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettes and one sample pipette; (ii) a reagent pack configured to store a certain volume of reagents for at least 50 assays; (iii) an ultrasonic mixer; (iv) the ability of each reaction vessel to perform more than 5 wash cycles; and (v) a luminometer.

[0275] Using one of four reagent pipettes, pipette paramagnetic particles conjugated with TSH antibodies capable of binding to TSH epitopes into the reaction vessel. Use a sample pipette to pipette aliquots of the sample into the reaction vessel. Then, use an ultrasonic mixer to mix the reaction vessel and incubate to produce a first reaction mixture. Apply a magnetic field to the reaction vessel and wash the first reaction mixture with wash buffer to remove any unreacted components. Add secondary antibodies capable of binding to different TSH epitopes conjugated with alkaline phosphatase (“ALP”) to the reaction vessel containing the first reaction mixture. Sonicate the reaction vessel and incubate to produce a second reaction mixture. Apply a magnetic field to the reaction vessel and wash the second reaction mixture five times with wash buffer to remove any unreacted components. Add substrate 1 to the reaction vessel, allow incubation, and read the signal generated by the resulting reaction using a luminometer. Repeat the process for substrate 2.

[0276] The test results were automatically determined by the system software. The detection of the analyte in the sample was determined based on the measured light generation using stored calibration data. Table 5 presents the RLU data collected for each sample tested for each substrate. The signal-to-noise ratio (S / SO) was calculated at each concentration value. Figure 3 The results are also plotted. Substrate 1 determination used one-third the volume of reagents and sample used in substrate 2 determination.

[0277] Table 5

[0278]

[0279] According to Table 5 and Figure 3 The results shown indicate that substrate 1 produces a high signal and a high signal-to-noise ratio (S / SO) at all concentrations.

[0280] Example 4: PCT Enhancement Signal Generation

[0281] A series of dilutions were generated using the PCT calibrator (S1, 8.60 ng / mL) to compare the signals produced by substrate 1 and substrate 2. The calibrator dilutions were 8.60 fg / mL (S1 / 100,000), 0.086 pg / mL (S1 / 10,000), 0.86 pg / mL (S1 / 1,000), 8.60 pg / mL (S1 / 100), and 0.086 ng / mL (S1 / 10). A control sample (0 fg / mL, S0) was also analyzed.

[0282] PCT sensitivity comparison was performed using an exemplary immunoassay analyzer having (i) four reagent pipettes and one sample pipette; (ii) a reagent pack configured to store a certain volume of reagents for at least 50 assays; (iii) an ultrasonic mixer; (iv) the ability of each reaction vessel to perform more than 5 wash cycles; and (v) a luminometer.

[0283] A reagent comprising a conjugate containing an antibody capable of binding to a PCT epitope linked to alkaline phosphatase (“ALP”) is added to a reaction vessel. Sample aliquots are pipetted into the reaction vessel using a sample pipette, and the first reaction mixture is incubated. Paramagnetic particles conjugated with the PCT antibody capable of binding to the PCT epitope are pipetted into the reaction vessel using one of four reagent pipettes. The reaction vessel is then mixed using an ultrasonic mixer and incubated to produce a second reaction mixture. A magnetic field is applied to the reaction vessel, and the second reaction mixture is washed five times with washing buffer to remove any unreacted components. Substrate 1 is added to the reaction vessel, incubated, and the signal generated by the resulting reaction is read using a luminometer. The process is repeated for substrate 2.

[0284] The test results were automatically determined by the system software. The detection of the analyte in the sample was determined based on the measured light production using stored calibration data. Table 6 presents the RLU data collected for each measured sample for each substrate. The measurements included a total of 15 replicates for each calibrator dose. The CV% was calculated at each concentration value. Figure 4 The results were also plotted in the image.

[0285] Table 6

[0286]

[0287] According to Table 6 and Figure 4 The results shown indicate that substrate 1 produces a high signal and a high signal-to-noise ratio (S / SO) at all concentrations.

[0288] The results presented in Table 7 were obtained using the same procedure described above, but with a response time (TTFR) of 55 minutes.

[0289] Table 7

[0290]

[0291] The signal ratio (S1 / S0) between low-concentration calibrator and zero calibrator is used as a substitute for sensitivity assessment.

[0292] A LOQ study was conducted, which involved a total of five replicates over three days using the above procedure on one instrument. The resulting data were used to calculate the LOQ for substrate 1 and substrate 2. The LOQ for each immunoassay analyzer was calculated using five standard deviations of S1 / (RLU S1-RLUS0) of the S0 replicate concentration, and the results are summarized in Table 7. As can be seen from Table 7, the LOQ for substrate 1 is at least approximately 34 fg / mL at a CV of 20%.

[0293] As shown in the example above, the unpredictable sensitivity of substrate 1 is superior to that of commercially available substrates, thus allowing for the sensitivity and precision required to distinguish and quantify different low levels of several different analytes.

[0294] All features disclosed in the specification (including the claims, abstract, and drawings) and all steps in any disclosed method or process may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. Unless otherwise expressly stated, each feature disclosed in this specification (including the claims, abstract, and drawings) may be substituted with alternative features for the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example of a series of equivalent or similar features.

[0295] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for detecting an analyte in a biological sample, the method comprising: The biological sample is exposed to a capture antibody configured to bind to at least a portion of the analyte, thereby generating a first reaction mixture; The first reaction mixture is exposed to an enzyme-conjugated antibody or an enzyme-conjugated antigen to generate a second reaction mixture; The second reaction mixture is exposed to a substrate formulation comprising a 1,2-dioxane compound and at least one phospho-cationic surfactant; The reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate preparation generates a chemiluminescent detection signal. Record the detection signal generated by the reaction; The recorded signal is compared with a calibration curve to quantify the level of the analyte in the biological sample.

2. The method according to claim 1, wherein the 1,2-dioxane compound is a compound of formula I or a salt thereof: Equation I Where R 1 and R 2 Each of them is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon it is attached to, it provides C5-C. 10 cycloalkyl rings; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

3. A method for detecting an analyte in a biological sample, the method comprising: The biological sample is exposed to a capture antibody configured to bind to at least a portion of the analyte, thereby generating a first reaction mixture; The first reaction mixture is exposed to an enzyme-conjugated antibody or an enzyme-conjugated antigen to generate a second reaction mixture; Expose the second reaction mixture to a substrate formulation containing a compound of formula I or a salt thereof: Equation I Where R 1 and R 2 Each of them is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon it is attached to, it provides C5-C. 10 cycloalkyl ring; R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is a phosphate; The reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate preparation generates a chemiluminescent detection signal. Record the detection signal generated by the reaction; The recorded signal is compared with a calibration curve to quantify the level of the analyte in the biological sample.

4. The method of claim 3, wherein the substrate formulation further comprises at least one phosphocation surfactant.

5. The method according to any one of claims 1 to 4, wherein the enzyme-conjugated antibody is configured to bind to at least a portion of the analyte.

6. The method according to any one of claims 1 to 4, wherein the enzyme-conjugated antigen is configured to bind to at least a portion of the capture antibody.

7. The method according to any one of claims 1 to 6, wherein the 1,2-dioxane compound is a compound of formula II or a salt thereof: Formula II wherein R 10 each of R 11 is independently H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, or C6-C 10 aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

8. The method according to any one of claims 1 to 7, wherein the 1,2-dioxane compound is a compound of formula III or a salt thereof: Formula III Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

9. The method according to any one of claims 1 to 8, wherein the 1,2-dioxane compound is a compound of formula IV or a salt thereof: Formula IV wherein R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is a phosphate salt.

10. The method according to any one of claims 1 to 9, wherein the 1,2-dioxane compound is 、 Or its salt.

11. The method according to any one of claims 1 to 10, wherein the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

12. The method according to any one of claims 1 to 11, wherein the phosphocation surfactant is selected from the group consisting of small molecule phosphocation surfactants and polymeric phosphocation surfactants.

13. The method according to claim 12, wherein the small molecule phosphoric acid surfactant is a compound having the following formula: wherein R 12 -R 14 each independently is C1-C 10 alkyl; R 15 is aralkyl; and X - is a counterion.

14. The method of claim 12, wherein the polymeric phosphocationic surfactant comprises repeating unit (A), repeating unit (B), or both: Bu3 is tributyl, and Oct3 is trioctyl.

15. The method according to any one of claims 1 to 14, wherein the substrate formulation further comprises a magnesium(II) salt.

16. The method according to any one of claims 1 to 15, wherein the capture antibody, the enzyme-conjugated antibody, and / or the enzyme-conjugated antigen are conjugated to at least one magnetic bead.

17. The method according to any one of claims 1 to 16, wherein the enzyme comprises alkaline phosphatase (AP).

18. The method according to any one of claims 1 to 17, wherein the method is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: A reagent kit, the reagent kit being configured to contain a plurality of reagent containers, wherein each reagent container is configured to store a certain volume of reagent required for at least one of the assays, wherein at least one reagent contains the capture antibody, and at least one reagent contains the enzyme-conjugated antibody or enzyme-conjugated antigen; A pipette arrangement comprising at least one reagent pipette and at least one sample pipette; And the structure of the detector device.

19. The method of claim 18, wherein the reagent container comprises an elastomeric self-sealing membrane.

20. The method of claim 18 or claim 19, wherein the reagent package further comprises a containment wall disposed between the reagent containers.

21. The method according to any one of claims 18 to 20, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is contained in the reagent storage unit.

22. The method according to any one of claims 18 to 21, wherein the pipette device configuration comprises at least a first reagent pipette, a second reagent pipette, a third reagent pipette, and at least one sample pipette.

23. The method of claim 22, wherein the pipette device configuration further comprises at least a fourth reagent pipette.

24. The method according to claim 22 or claim 23, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette operate selectively and / or simultaneously.

25. The method according to any one of claims 22 to 24, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette are configured to engage the dispensing tip before aspiration.

26. The method according to any one of claims 18 to 25, wherein the method is configured to analyze at least about 200 biological samples per hour.

27. The method according to any one of claims 18 to 25, wherein the method is configured to analyze at least about 300 biological samples per hour.

28. The method according to any one of claims 18 to 25, wherein the method is configured to analyze at least about 400 biological samples per hour.

29. The method according to any one of claims 18 to 28, wherein: The first reaction mixture is generated by aspirating a portion of the biological sample from the sample container and dispensing the aspirated biological sample into the reaction vessel of the immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; The second reaction mixture is generated by aspirating a portion of a second reagent containing the enzyme-conjugated antibody or enzyme-conjugated antigen from at least one reagent container and dispensing the aspirated reagent into the reaction container; and The detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formulation into the reaction vessel.

30. The method of claim 29, wherein the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

31. The method of claim 29 or claim 30, wherein the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

32. The method according to any one of claims 29 to 31, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, or alternatively... At least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

33. The method according to any one of claims 18 to 32, wherein the cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

34. The method according to any one of claims 18 to 33, wherein the time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

35. The method according to any one of claims 18 to 34, wherein the detector device configuration comprises A photodetector, configured to sense photons emitted from a measurement reaction within a specified time period, Analog circuitry, configured to provide an analog signal based on the photons emitted from the measured response during the time period, and A counter circuit configured to provide a photon count based on the photons emitted from the measured reaction during the time period.

36. The method according to any one of claims 18 to 35, wherein the immunoassay analyzer further comprises an ultrasound mixing module.

37. The method of claim 36, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are stirred via the ultrasonic mixing module.

38. The method according to any one of claims 29 to 37, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture comprises unreacted components, and the immunoassay analyzer further comprises a washing device configuration. The washing apparatus is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

39. The method of claim 38, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field prior to performing the at least one washing action.

40. The method according to any one of claims 18 to 39, wherein the immunoassay analyzer further comprises: A machine vision device, comprising an image capture device and an image interpretation device, the image capture device and the image interpretation device being configured to monitor the instrument functions and / or assay functions of the immunoassay analyzer.

41. The method of claim 40, wherein the instrument function is selected from the group consisting of: optical sensors, pressure sensors, and thermistors.

42. The method according to claim 41, wherein the measurement function is selected from the group consisting of: sample volume monitoring, total reagent volume monitoring, residual volume monitoring and particle retention monitoring.

43. The method according to any one of claims 1 to 42, wherein the biological sample is serum, whole blood, plasma and / or cerebrospinal fluid.

44. A method for detecting cardiac troponin I (TNI) in a biological sample, the method comprising: The biological sample is exposed to a capture antibody configured to bind to at least a portion of the TNI, thereby generating a first reaction mixture; The first reaction mixture is exposed to an enzyme-conjugated affinity molecule to form a second reaction mixture; The second reaction mixture is exposed to a substrate formulation comprising a 1,2-dioxane compound and at least one phospho-cationic surfactant; The reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; Record the detection signal generated by the reaction; The recorded signal is compared with a calibration curve to quantify the level of TNI in the biological sample.

45. The method of claim 44, wherein the generated chemiluminescent detection signal is at least about 10 times, alternatively at least about 20 times, or alternatively at least about 30 times, a signal generated by a method in which the substrate formulation comprises a compound of the following formula: 。 46. ​​The method of claim 44 or claim 45, wherein the concentration of the quantified TNI level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

47. The method according to any one of claims 44 to 46, wherein the ratio of the signal generated by cleaving the enzyme to background noise is at least about 1, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 100, alternatively at least about 200, alternatively at least about 300, alternatively at least about 400, alternatively at least about 500, alternatively at least about 600, alternatively at least about 700, or alternatively at least about 8. 00, at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000, at least about 15,000, at least about 20,000, or at least about 25,000.

48. The method according to any one of claims 44 to 47, wherein the affinity molecule is an antibody, monoclonal antibody, polyclonal antibody, synthetic antibody mimic, aptamer, affimer, DARPin, oligonucleotide, peptide, or antigen.

49. The method of claim 48, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of TNI.

50. The method of claim 48, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind to at least a portion of the capture antibody.

51. The method according to any one of claims 44 to 50, wherein the capture antibody and / or the enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

52. The method according to any one of claims 44 to 51, wherein the enzyme comprises alkaline phosphatase (AP).

53. The method according to any one of claims 44 to 52, wherein the 1,2-dioxane compound is a compound of formula I or a salt thereof: Equation I Where R 1 and R 2 Each of them is independently C3-C 10 Alkyl, or R 1 and R 2 Together with the carbon it is attached to, it provides C5-C. 10 cycloalkyl ring; R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

54. The method according to any one of claims 44 to 53, wherein the 1,2-dioxane compound is a compound of formula II or a salt thereof: Formula II wherein R 10 each of R 11 is independently H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, or C6-C 10 aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 Is it H or C1-C? 10 Alkyl; and X is phosphate.

55. The method according to any one of claims 44 to 54, wherein the 1,2-dioxane compound is a compound of formula III or a salt thereof: Formula III Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

56. The method according to any one of claims 44 to 55, wherein the 1,2-dioxane compound is a compound of formula IV or a salt thereof: Formula IV wherein R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl, X is phosphate.

57. The method according to any one of claims 44 to 56, wherein the 1,2-dioxane compound is 、 Or its salt.

58. The method according to any one of claims 44 to 57, wherein the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

59. The method according to any one of claims 44 to 58, wherein the phosphocation surfactant is selected from the group consisting of small molecule phosphocation surfactants and polymeric phosphocation surfactants.

60. The method of claim 59, wherein the small molecule phosphoric acid surfactant is a compound having the following formula: R12-R14 are each independently a C1-C10 alkyl group; R15 is an aralkyl group; and X - is a counterion.

61. The method of claim 59, wherein the polymeric phosphocationic surfactant comprises repeating unit (A), repeating unit (B), or both: Bu3 is tributyl, and Oct3 is trioctyl.

62. The method according to any one of claims 44 to 61, wherein the substrate formulation further comprises a magnesium(II) salt.

63. The method according to any one of claims 44 to 62, wherein the method is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: A reagent kit configured to contain a plurality of reagent containers, wherein each reagent container is configured to store a certain volume of reagent required for at least one of the assays, wherein at least one reagent contains the capture antibody and at least one reagent contains the enzyme-conjugated affinity molecule; A pipette device configuration comprising at least one reagent pipette and at least one sample pipette; And the structure of the detector device.

64. The method of claim 63, wherein the reagent container comprises an elastomeric self-sealing membrane.

65. The method of claim 63 or claim 64, wherein the reagent package further comprises a retaining wall disposed between the reagent containers.

66. The method according to any one of claims 63 to 65, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent package is contained in the reagent storage unit.

67. The method according to any one of claims 63 to 66, wherein the pipette device configuration comprises at least a first reagent pipette, a second reagent pipette, a third reagent pipette, and at least one sample pipette.

68. The method of claim 67, wherein the pipette device configuration further comprises at least a fourth reagent pipette.

69. The method of claim 67 or claim 68, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette operate selectively and / or simultaneously.

70. The method according to any one of claims 67 to 69, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette are configured to engage the dispensing tip before aspiration.

71. The method according to any one of claims 67 to 70, wherein the method is configured to analyze at least about 200 biological samples per hour.

72. The method according to any one of claims 67 to 70, wherein the method is configured to analyze at least about 300 biological samples per hour.

73. The method according to any one of claims 67 to 70, wherein the method is configured to analyze at least about 400 biological samples per hour.

74. The method according to any one of claims 63 to 73, wherein: The first reaction mixture is generated by aspirating a portion of the biological sample from the sample container and dispensing the aspirated biological sample into the reaction vessel of the immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; The second reaction mixture is produced by drawing a portion of a second reagent containing the enzyme-conjugated affinity molecule from at least one reagent container and dispensing the drawn reagent into the reaction container; and The detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formulation into the reaction vessel.

75. The method of claim 74, wherein the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

76. The method of claim 74 or claim 75, wherein the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

77. The method according to any one of claims 74 to 76, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, or alternatively... At least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180 seconds, alternatively at least 190 seconds, alternatively at least 200 seconds, alternatively at least 210 seconds, alternatively at least 220 seconds, alternatively at least 230 seconds, alternatively at least 240 seconds, alternatively at least 250 seconds, alternatively at least 260 seconds, alternatively at least 270 seconds, or alternatively at least 280 seconds.

78. The method according to any one of claims 63 to 77, wherein the cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

79. The method according to any one of claims 63 to 78, wherein the time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

80. The method according to any one of claims 63 to 79, wherein the detector device configuration comprises A photodetector, configured to sense photons emitted from a measurement reaction within a specified time period, Analog circuitry, configured to provide an analog signal based on the photons emitted from the measured response during the time period, and A counter circuit configured to provide a photon count based on the photons emitted from the measured reaction during the time period.

81. The method according to any one of claims 63 to 80, wherein the immunoassay analyzer further comprises an ultrasound mixing module.

82. The method of claim 81, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are stirred via the ultrasonic mixing module.

83. The method according to any one of claims 63 to 82, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture comprises unreacted components, and the immunoassay analyzer further comprises a washing device configuration. The washing apparatus is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

84. The method of claim 83, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field prior to performing the at least one washing action.

85. The method according to any one of claims 63 to 84, wherein the immunoassay analyzer further comprises: A machine vision device, comprising an image capture device and an image interpretation device, the image capture device and the image interpretation device being configured to monitor the instrument functions and / or assay functions of the immunoassay analyzer.

86. The method of claim 85, wherein the instrument function is selected from the group consisting of: optical sensors, pressure sensors, and thermistors.

87. The method of claim 85, wherein the measurement function is selected from the group consisting of: sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

88. The method according to any one of claims 44 to 87, wherein the biological sample is serum, whole blood, plasma and / or cerebrospinal fluid.

89. A method for detecting thyroid-stimulating hormone (TSH) in a biological sample, the method comprising: The biological sample is exposed to a capture antibody configured to bind to at least a portion of the TSH, thereby generating a first reaction mixture; The first reaction mixture is exposed to an enzyme-conjugated affinity molecule to form a second reaction mixture; The second reaction mixture is exposed to a substrate formulation comprising a 1,2-dioxane compound and at least one phospho-cationic surfactant; The reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; Record the detection signal generated by the reaction; The recorded signal is compared with a calibration curve to quantify the level of TSH in the biological sample.

90. The method of claim 89, wherein the generated signal is at least about 10 times, alternatively at least about 20 times, or alternatively at least about 30 times greater than a signal generated by a method in which the substrate formulation comprises a compound of the following formula: 。 91. The method of claim 89 or claim 90, wherein the concentration of the quantified TSH level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

92. The method according to any one of claims 89 to 91, wherein the ratio of the signal generated by cleaving the enzyme to background noise is at least about 5, alternatively at least about 10, alternatively at least about 20, alternatively at least about 30, alternatively at least about 40, alternatively at least about 50, alternatively at least about 60, alternatively at least about 70, alternatively at least about 80, alternatively at least about 90, alternatively at least about 100, alternatively at least about 200, or alternatively at least about 3. 00, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, or at least about 8,000.

93. The method according to any one of claims 89 to 92, wherein the affinity molecule is an antibody, monoclonal antibody, polyclonal antibody, synthetic antibody mimic, aptamer, affimer, DARPin, oligonucleotide, peptide, or antigen.

94. The method of claim 93, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of TSH.

95. The method of claim 93, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind to at least a portion of the capture antibody.

96. The method according to any one of claims 89 to 95, wherein the capture antibody and / or the enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

97. The method according to any one of claims 89 to 96, wherein the enzyme comprises alkaline phosphatase (AP).

98. The method according to any one of claims 89 to 97, wherein the 1,2-dioxane compound is a compound of formula I or a salt thereof: Equation I wherein R 1 each of R 2 is independently C3-C 10 alkyl, or R 1 and R 2 together with the carbon to which they are attached provide a C5-C 10 cycloalkyl ring; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 Is it H or C1-C? 10 Alkyl; and X is phosphate.

99. The method according to any one of claims 89 to 98, wherein the 1,2-dioxane compound is a compound of formula II or a salt thereof: Formula II Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 It is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

100. The method according to any one of claims 89 to 99, wherein the 1,2-dioxane compound is a compound of formula III or a salt thereof: Formula III wherein R 10 each of R 11 is independently H, halogen, C1-C 10 alkyl, C2-C 10 alkenyl, or C6-C 10 aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

101. The method according to any one of claims 89 to 100, wherein the 1,2-dioxane compound is a compound of formula IV or a salt thereof: Formula IV wherein R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is a phosphate salt.

102. The method according to any one of claims 89 to 101, wherein the 1,2-dioxane compound is 、 Or its salt.

103. The method according to any one of claims 89 to 102, wherein the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

104. The method according to any one of claims 89 to 103, wherein the phosphocation surfactant is selected from the group consisting of small molecule phosphocation surfactants and polymeric phosphocation surfactants.

105. The method according to claim 104, wherein the small molecule phosphoric acid surfactant is a compound having the following formula: Where R 12 -R 14 Each is C independently 1 -C 10 alkyl; R 15 It is an aralkyl group; and X - is a counterion.

106. The method of claim 104, wherein the polymeric phosphocationic surfactant comprises repeating unit (A), repeating unit (B), or both: Bu3 is tributyl, and Oct3 is trioctyl.

107. The method according to any one of claims 89 to 106, wherein the substrate formulation further comprises a magnesium(II) salt.

108. The method according to any one of claims 89 to 107, wherein the method is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: A reagent kit configured to contain a plurality of reagent containers, wherein each reagent container is configured to store a certain volume of reagent required for at least one of the assays, wherein at least one reagent contains the capture antibody and at least one reagent contains the enzyme-conjugated affinity molecule; A pipette device configuration comprising at least one reagent pipette and at least one sample pipette; And the structure of the detector device.

109. The method of claim 108, wherein the reagent container comprises an elastomeric self-sealing membrane.

110. The method of claim 108 or claim 109, wherein the reagent package further comprises a retaining wall disposed between the reagent containers.

111. The method according to any one of claims 108 to 110, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is contained in the reagent storage unit.

112. The method according to any one of claims 108 to 111, wherein the pipette device configuration comprises at least a first reagent pipette, a second reagent pipette, a third reagent pipette, and at least one sample pipette.

113. The method of claim 112, wherein the pipette device configuration further comprises at least a fourth reagent pipette.

114. The method according to claim 112 or claim 113, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette operate selectively and / or simultaneously.

115. The method according to any one of claims 112 to 114, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette, the fourth reagent pipette and / or are configured to engage the dispensing tip before aspiration.

116. The method according to any one of claims 108 to 115, wherein the method is configured to analyze at least about 200 biological samples per hour.

117. The method according to any one of claims 108 to 115, wherein the method is configured to analyze at least about 300 biological samples per hour.

118. The method according to any one of claims 108 to 115, wherein the method is configured to analyze at least about 400 biological samples per hour.

119. The method according to any one of claims 108 to 118, wherein: The first reaction mixture is generated by aspirating a portion of the biological sample from the sample container and dispensing the aspirated biological sample into the reaction vessel of the immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; The second reaction mixture is produced by drawing a portion of a second reagent containing the enzyme-conjugated affinity molecule from at least one reagent container and dispensing the drawn reagent into the reaction container; and The detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formulation into the reaction vessel.

120. The method of claim 119, wherein the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

121. The method of claim 119 or claim 120, wherein the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

122. The method according to any one of claims 119 to 121, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, or alternatively... The alternative is at least 140 seconds, the alternative is at least 150 seconds, the alternative is at least 160 seconds, the alternative is at least 170 seconds, the alternative is at least 180 seconds, the alternative is at least 190 seconds, the alternative is at least 200 seconds, the alternative is at least 210 seconds, the alternative is at least 220 seconds, the alternative is at least 230 seconds, the alternative is at least 240 seconds, the alternative is at least 250 seconds, the alternative is at least 260 seconds, the alternative is at least 270 seconds, or the alternative is at least 280 seconds.

123. The method according to any one of claims 108 to 122, wherein the cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

124. The method according to any one of claims 108 to 123, wherein the time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

125. The method according to any one of claims 108 to 124, wherein the detector device configuration comprises A photodetector, configured to sense photons emitted from a measurement reaction within a specified time period, Analog circuitry, configured to provide an analog signal based on the photons emitted from the measured response during the time period, and A counter circuit configured to provide a photon count based on the photons emitted from the measured reaction during the time period.

126. The method according to any one of claims 108 to 125, wherein the immunoassay analyzer further comprises an ultrasound mixing module.

127. The method of claim 126, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are stirred via the ultrasonic mixing module.

128. The method according to any one of claims 108 to 127, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture comprises unreacted components, and the immunoassay analyzer further comprises a washing device configuration. The washing apparatus is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

129. The method of claim 128, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field prior to performing the at least one washing action.

130. The method according to any one of claims 108 to 129, wherein the immunoassay analyzer further comprises: A machine vision device, comprising an image capture device and an image interpretation device, the image capture device and the image interpretation device being configured to monitor the instrument functions and / or assay functions of the immunoassay analyzer.

131. The method of claim 130, wherein the instrument function is selected from the group consisting of: optical sensors, pressure sensors, and thermistors.

132. The method of claim 131, wherein the measurement function is selected from the group consisting of: sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

133. The method according to any one of claims 89 to 132, wherein the biological sample is serum, whole blood, plasma and / or cerebrospinal fluid.

134. A method for detecting procalcitonin (PCT) in a biological sample, the method comprising: The biological sample is exposed to a capture antibody configured to bind to at least a portion of PCT, thereby generating a first reaction mixture; The first reaction mixture is exposed to an enzyme-conjugated affinity molecule to form a second reaction mixture; The second reaction mixture is exposed to a substrate formulation comprising a 1,2-dioxane compound and at least one phospho-cationic surfactant; The reaction between the enzyme-conjugated affinity molecule and the substrate formulation generates a chemiluminescent detection signal; Record the detection signal generated by the reaction; The recorded signal is compared with a calibration curve to quantify the level of PCT in the biological sample.

135. The method of claim 134, wherein the generated signal is at least about 10 times, alternatively at least about 20 times, or alternatively at least about 30 times greater than a signal generated by a method in which the substrate formulation comprises a compound of the following formula: 。 136. The method of claim 134 or claim 135, wherein the limit of quantitation (LoQ) of the determination is about 100 fg / mL or less, 50 fg / mL or less, or 34 fg / mL or less.

137. The method according to any one of claims 134 to 136, wherein the coefficient of variation (CV) of the method is 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

138. The method according to any one of claims 135 to 138, wherein the concentration of the detected PCT level is at least 1 times, or alternatively at least 2 times, or alternatively at least 3 times, the limit of quantitation (LoQ) of the method.

139. The method according to any one of claims 134 to 136, wherein the ratio of the signal generated by cutting the enzyme to the background noise is at least about 2,000, alternatively at least about 3,000, alternatively at least about 4,000, or alternatively at least about 5,000.

140. The method according to any one of claims 134 to 139, wherein the affinity molecule is an antibody, monoclonal antibody, polyclonal antibody, synthetic antibody mimic, aptamer, affimer, DARPin, oligonucleotide, peptide, or antigen.

141. The method of claim 140, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind to at least a portion of PCT.

142. The method of claim 140, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind to at least a portion of the capture antibody.

143. The method according to any one of claims 134 to 142, wherein the capture antibody and / or the enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

144. The method according to any one of claims 134 to 143, wherein the enzyme comprises alkaline phosphatase (AP).

145. The method according to any one of claims 134 to 144, wherein the 1,2-dioxane compound is a compound of formula I or a salt thereof: Equation I wherein R 1 each of R 2 is independently C3-C 10 alkyl, or R 1 and R 2 together with the carbon to which they are attached provide a C5-C 10 cycloalkyl ring; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

146. The method according to any one of claims 134 to 145, wherein the 1,2-dioxane compound is a compound of formula II or a salt thereof: Formula II Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl; R 3 It is C1-C 10 Alkyl, C6-C 10 aryl or heteroaryl; R 4 is C2-C 10 alkenyl; R 5 Is it H or C1-C? 10 Alkyl; and X is phosphate.

147. The method according to any one of claims 134 to 146, wherein the 1,2-dioxane compound is a compound of formula III or a salt thereof: Formula III Where R 10 and R 11 Each of these elements is independently H, halogen, C1-C. 10 Alkyl, C2-C 10 alkenyl or C6-C 10 Aryl; R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is phosphate.

148. The method according to any one of claims 134 to 147, wherein the 1,2-dioxane compound is a compound of formula IV or a salt thereof: Formula IV wherein R 3 is C1-C 10 alkyl, C6-C 10 aryl or heteroaryl; R 5 is H or C1-C 10 alkyl; and X is a phosphate salt.

149. The method according to any one of claims 134 to 148, wherein the 1,2-dioxane compound is 、 Or its salt.

150. The method according to any one of claims 134 to 149, wherein the 1,2-dioxane compound is 4-methoxy-4-(3-phosphophenyl)spiro[1,2-dioxane-3,2'-adamantane] or a salt thereof.

151. The method according to any one of claims 134 to 150, wherein the phosphocation surfactant is selected from the group consisting of small molecule phosphocation surfactants and polymeric phosphocation surfactants.

152. The method according to claim 151, wherein the small molecule phosphoric acid surfactant is a compound having the following formula: R12-R14 are each independently a C1-C10 alkyl group; R15 is an aralkyl group; and X - It is a counter ion.

153. The method of claim 151, wherein the polymeric phosphocationic surfactant comprises repeating unit (A), repeating unit (B), or both: Bu3 is tributyl, and Oct3 is trioctyl.

154. The method according to any one of claims 134 to 153, wherein the substrate formulation further comprises a magnesium(II) salt.

155. The method according to any one of claims 134 to 154, wherein the method is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: A reagent kit configured to contain a plurality of reagent containers, wherein each reagent container is configured to store a certain volume of reagent required for at least one of the assays, wherein at least one reagent contains the capture antibody and at least one reagent contains the enzyme-conjugated affinity molecule; A pipette device configuration comprising at least one reagent pipette and at least one sample pipette; And the structure of the detector device.

156. The method of claim 155, wherein the reagent container comprises an elastomeric self-sealing membrane.

157. The method of claim 155 or claim 156, wherein the reagent package further comprises a retaining wall disposed between the reagent containers.

158. The method according to any one of claims 155 to 157, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is contained in the reagent storage unit.

159. The method according to any one of claims 155 to 158, wherein the pipette device configuration comprises at least a first reagent pipette, a second reagent pipette, a third reagent pipette, and at least one sample pipette.

160. The method of claim 159, wherein the pipette device configuration further comprises at least a fourth reagent pipette.

161. The method of claim 159 or claim 160, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette operate selectively and / or simultaneously.

162. The method according to any one of claims 159 to 161, wherein the first reagent pipette, the second reagent pipette, the third reagent pipette and / or the fourth reagent pipette are configured to engage the dispensing tip before aspiration.

163. The method according to any one of claims 155 to 162, wherein the method is configured to analyze at least about 200 biological samples per hour.

164. The method according to any one of claims 155 to 162, wherein the method is configured to analyze at least about 300 biological samples per hour.

165. The method according to any one of claims 155 to 162, wherein the method is configured to analyze at least about 400 biological samples per hour.

166. The method according to any one of claims 155 to 165, wherein: The first reaction mixture is generated by aspirating a portion of the biological sample from the sample container and dispensing the aspirated biological sample into the reaction vessel of the immunoassay analyzer, and by aspirating a portion of a first reagent containing the capture antibody from at least one reagent container and dispensing the aspirated reagent into the reaction vessel; The second reaction mixture is produced by drawing a portion of a second reagent containing the enzyme-conjugated affinity molecule from at least one reagent container and dispensing the drawn reagent into the reaction container; and The detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formulation into the reaction vessel.

167. The method of claim 166, wherein the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, or alternatively at least about 60 minutes.

168. The method of claim 166 or claim 167, wherein the incubation time of the second reaction mixture is at least about 2 minutes, alternatively at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

169. The method according to any one of claims 166 to 168, wherein the detection mixture is incubated for at least 20 seconds, alternatively at least 30 seconds, alternatively at least 40 seconds, alternatively at least 50 seconds, alternatively at least 60 seconds, alternatively at least 70 seconds, alternatively at least 80 seconds, alternatively at least 90 seconds, alternatively at least 100 seconds, alternatively at least 110 seconds, alternatively at least 120 seconds, alternatively at least 130 seconds, or alternatively... The alternative is at least 140 seconds, the alternative is at least 150 seconds, the alternative is at least 160 seconds, the alternative is at least 170 seconds, the alternative is at least 180 seconds, the alternative is at least 190 seconds, the alternative is at least 200 seconds, the alternative is at least 210 seconds, the alternative is at least 220 seconds, the alternative is at least 230 seconds, the alternative is at least 240 seconds, the alternative is at least 250 seconds, the alternative is at least 260 seconds, the alternative is at least 270 seconds, or the alternative is at least 280 seconds.

170. The method according to any one of claims 155 to 169, wherein the cycle time is about 45 seconds or less, alternatively about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds.

171. The method according to any one of claims 155 to 170, wherein the time to first result (TTFR) is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

172. The method according to any one of claims 155 to 171, wherein the detector device configuration comprises A photodetector, configured to sense photons emitted from a measurement reaction within a specified time period, Analog circuitry, configured to provide an analog signal based on the photons emitted from the measured response during the time period, and A counter circuit configured to provide a photon count based on the photons emitted from the measured reaction during the time period.

173. The method according to any one of claims 155 to 172, wherein the immunoassay analyzer further comprises an ultrasound mixing module.

174. The method of claim 173, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are stirred via the ultrasonic mixing module.

175. The method according to any one of claims 155 to 174, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture comprises unreacted components, and the immunoassay analyzer further comprises a washing device configuration. The washing apparatus is configured to perform at least one washing action to wash away at least a portion of the unreacted components, alternatively configured to perform at least two washing actions, alternatively configured to perform at least three washing actions, alternatively configured to perform at least four washing actions, alternatively configured to perform at least five washing actions, alternatively configured to perform at least six washing actions, alternatively configured to perform at least seven washing actions, alternatively configured to perform at least eight washing actions, alternatively configured to perform at least nine washing actions, or alternatively configured to perform at least ten washing actions.

176. The method of claim 175, wherein the first reaction mixture, the second reaction mixture, and / or the detection mixture are subjected to a magnetic field prior to performing the at least one washing action.

177. The method according to any one of claims 155 to 176, wherein the immunoassay analyzer further comprises: A machine vision device, comprising an image capture device and an image interpretation device, the image capture device and the image interpretation device being configured to monitor the instrument functions and / or assay functions of the immunoassay analyzer.

178. The method of claim 177, wherein the instrument function is selected from the group consisting of: optical sensors, pressure sensors, and thermistors.

179. The method of claim 177, wherein the measurement function is selected from the group consisting of: sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

180. The method according to any one of claims 134 to 179, wherein the biological sample is serum, whole blood, plasma and / or cerebrospinal fluid.

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