Detecting interferon gamma ("IFN-γ") using chemiluminescent dioxetane compounds

Abstract

The presently claimed and described technology provides methods for chemiluminescence-based assays for detecting interferon-gamma (IFN-γ) in a biological sample employing a dioxetane compound.

Description

Attorney Docket No. P2023-3979-WO01 (67724WO01) DETECTING INTERFERON GAMMA (“IFN-γ”) USING CHEMILUMINESCENT DIOXETANE COMPOUNDS RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 625,509, filed January 26, 2024, the content of which is hereby incorporated by reference in its entirety into this disclosure. INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on January 20, 2025, is named 67724WO01_seq.xml and is 1,896 bytes in size. BACKGROUND

[0003] Tuberculosis (“TB”) remains on the deadliest diseases in the world. TB is caused by a bacterium called Mycobacterium tuberculosis. In 2021, about a quarter of the global population is estimated to have been infected with TB bacteria, an estimated 10.6 million people fell ill with tuberculosis (TB) worldwide and 1.3 million people died. World Health Organization, Tuberculosis, https: / / www.who.int / news-room / fact-sheets / detail / tuberculosis. Treating TB has good results and will reduce the progression of the disease. Control of TB relies on early detection and treatment of TB cases.

[0004] Interferon-gamma (IFN-γ) is a cytokine released by T cells or peripheral blood mononuclear cells (PBMC) upon stimulation with pathogenic antigens. Current diagnostic methods for the identification of TB include interferon-gamma release assays, which involve detecting TB infection by measuring the release of IFN-γ when stimulated with a TB-specific antigen in vitro. Generally, a higher level of IFN-γ released in response to the antigen suggests a current or past TB infection. Conventional IFN-γ release assays require 16 - 24 hours of incubation time with the antigen for T-cells to produce amounts of IFN-γ detectable by the assays with sufficient sensitivity and reliability to diagnose TB.

[0005] A need exists for an IFN-γ release assay that can detect smaller amounts of IFN-γ after shorter incubation times with sufficient sensitivity to aid physicians and neurologists in diagnosing patients with TB earlier. BRIEF SUMMARY

[0001] One aspect of the invention is a method of detecting Interferon gamma (“IFN-γ”) in a biological sample, the method comprising: exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a dioxetane compound or salt thereof of the formula

[0002] wherein each of R1 and R2 areC10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring; wherein each of R3 and R6 are independently C1-C10 alkyl, C6-C10 aryl, or heteroaryl; wherein the 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 to a calibration curve to quantify the level of the IFN-γ in the biological sample.

[0003] One aspect of the disclosure is a method of detecting IFN-γ in a biological sample, the method comprising: exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the IFN-γ in the biological sample.

[0004] One aspect of the invention is a method for measuring a cell-mediated immune response to an antigen in a biological sample from a subject, the method comprising: incubating thebiological sample with a cytokine release agent, producing an incubation mixture, wherein the incubation generates IFN-γ; and performing the method for detecting IFN-γ released from the biological sample.

[0005] In an aspect, the antigen is a tuberculosis antigen.

[0006] In an aspect, the cytokine release agent comprises at least one peptide, antigen, or mitogen.

[0007] In an aspect, the biological sample comprises T cells and / or peripheral blood mononuclear cells. In an aspect, the biological sample is incubated with the cytokine release agent for about 16 hours or less, alternatively about 15 hours or less, alternatively about 14 hours or less, alternatively about 13 hours or less, alternatively about 12 hours or less, alternatively about 11 hours or less, alternatively about 10 hours or less, alternatively about 9 hours or less, alternatively about 8 hours or less, alternatively about 7 hours or less, alternatively about 6 hours or less, alternatively about 5 hours or less, alternatively about 4 hours or less, alternatively about 3 hours or less, alternatively about 2 hours or less, alternatively about 1 hour or less, or alternatively about 30 minutes or less.

[0008] In an aspect, the biological sample is incubated with the cytokine release agent in a vessel, tube, vial, well, or plate.

[0009] In an aspect, the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 1, alternatively at least about 1.5, alternatively at least about 2, alternatively at least about 2.5, alternatively at least about 3, alternatively at least about 3.5, alternatively at least about 4, alternatively at least about 4.5, alternatively at least about 5, alternatively at least about 10, alternatively at least about 15, 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 400, or alternatively at least about 600.

[0010] In an aspect, the method provides a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, alternatively 4% or less, alternatively 3% or less, alternatively 2% or less, alternatively 1.5% or less over two or more tests.

[0011] In an aspect, the affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

[0012] In an aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of IFN-γ.

[0013] In an aspect, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

[0014] In an aspect, the capture antibody and / or enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

[0015] In an aspect, the enzyme comprises an alkaline phosphatase (AP).

[0016] In an aspect, the dioxetane compound is a compound of a formula:

[0017] or a salt thereof

[0018] R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring;

[0019] R3is C1-C10alkyl, C6-C10 aryl, or heteroaryl;

[0020] R5is H or C1-C10 alkyl; and

[0021] X is a phosphate.

[0022] In an aspect, the dioxetane compound is a compound of a formula:

[0023] or a salt thereof

[0025] wherein each of and R11is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10aryl;

[0026] R3is C1-C10alkyl, C6-C10aryl or heteroaryl;

[0027] R5is H or C1-C10 alkyl; and

[0028] X is a phosphate.

[0029] In an aspect, the dioextane compound has a formula o .

[0030] In an aspect, the dioxetane compound is a compound of:

[0031] Formula I

[0032] independently C3-C10alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring;

[0033] R3is C1-C10 alkyl, C6-C10 aryl, or heteroaryl;

[0034] R4is C2-C10 alkenyl;

[0035] R5is H or C1-C10 alkyl; and

[0036] X is a phosphate.

[0037] In an aspect, the dioxetane compound is a compound of Formula II or a salt thereof:

[0038] Formula IIH, halogen, C1-C10alkyl, C2-C10alkenyl, or C6-C10 aryl;

[0041] R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0042] R4is C2-C10alkenyl;

[0043] R5is H or C1-C10 alkyl; and

[0044] X is a phosphate.

[0045] In an aspect, the dioxetane compound is a compound of Formula III or a salt thereof:

[0046] Formula III

[0047] w tly H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10aryl;

[0048] R3is C1-C10alkyl, C6-C10aryl or heteroaryl;

[0049] R5is H or C1-C10 alkyl; and

[0050] X is a phosphate.

[0051] In an aspect, the dioxetane compound is a compound of Formula IV or a salt thereof:

[0052] Formula IV

[0053]

[0054] R5is H or C1-C10 alkyl; and

[0055] X is a phosphate.

[0056] In an aspect, the wherein the dioxetane compound is

[0057]

[0058]

[0059] In an aspect, the dioxetane compound is 4-methoxy-4-(3-phosphatephenyl)spiro[1,2- dioetane-3,2’-adamantane] or a salt thereof.

[0060]

[0061] In an aspect, the signal produced is a least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method, wherein the substrate formulation comprises a compound of the following formula:

[0062] .

[0063] formulation further comprises at least one phosphonium surfactant.

[0064] In an aspect, the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

[0065] In an aspect, the small molecule phosphonium surfactant is a compound having the formula:

[0066]

[0067] each independently C1-C10 alkyl;

[0068] R15 is arylalkyl; and

[0069] X- is a counterion.

[0070] In an aspect, the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

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

[0074] In an aspect, the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

[0075] In an aspect, the reagent vessels comprise an elastomeric self-sealing membrane.

[0076] In an aspect, the reagent pack further comprises containment walls arranged between the reagent vessels.

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

[0078] In an aspect, the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

[0079] In an aspect, the pipettor arrangement further comprises at least a fourth reagent pipettor.

[0080] In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

[0081] In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

[0082] In an aspect, the method is configured to analyze at least about 200 biological samples / hr. In an aspect, the method is configured to analyze at least about 300 biological samples / hr. In an aspect, the method is configured to analyze at least about 400 biological samples / hr.

[0083] In an aspect, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated byaspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

[0084] In an aspect, 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.

[0085] In an aspect, incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0086] In an aspect, 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, 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.

[0087] In an aspect, 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.

[0088] In an aspect, 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.

[0089] In an aspect, the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time,and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

[0090] In an aspect, the immunoassay analyzer further comprises an ultrasonic mixing module.

[0091] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0092] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

[0093] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

[0094] In an aspect, the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

[0095] In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

[0096] In an aspect, the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

[0097] In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

[0098] In an aspect, the biological sample is from a subject infected with or suspected of being infected with tuberculosis (TB).

[0099] In an aspect, the tuberculosis is caused by Mycobacterium tuberculosis.

[0100] In an aspect, the method can differentiate between latent TB infection and active TB.

[0101] One aspect of the disclosure is a kit for carrying out any one of the disclosed methods comprising: at least one incubation vessel; at least one cytokine release agent; a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay; and the substrate formulation.

[0102] In an aspect, the kit further comprises instructions for use for performing any one of the methods disclosed.

[0103] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings. BRIEF DESCRIPTION OF THE FIGURES

[0104] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0105] FIG. 1 is a calibration curve plot constructed for an INFγ assay using calibrators and an exemplary substrate according to an aspect of this disclosure.

[0106] FIG. 2 is a calibration curve plot constructed for an INFγ assay using calibrators and a commercially available substrate. DETAILED DESCRIPTION I. Introduction

[0107] Disclosed herein are immunoassay methods, reagents, kits, and compounds for detecting IFN-γ in a biological sample using chemiluminescent dioxetanes.

[0108] Interferons (IFNs) are cytokines, a class of small secreted proteins produced by immune cells such as T-cells and NK cells. IFNs are divided into 3 types: IFN of type I (-α, -β, -κ, -ε, -ω, - τ), type II (-γ), and type III (-λ1-4, -£1-3). IFN-γ plays crucial roles in antimicrobial, antiviral, and antitumor responses by activating effector immune cells and enhancing antigen presentation. In the first few hours of TB infection, type I and II INFs are produced.

[0109] The amino acid sequence of IFN-γ is thought to be SEQ ID NO: 1 listed in Table 1. IFN-γ may refer to a full-length IFN-γ protein, a variant of IFN-γ, a fragment of IFN-γ, and post- translationally modified forms of IFN-γ.

[0110] Table 1 SEQ ID NO Amino Acid Sequence 1 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADN GTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKE DMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPA AKTGKRKRSQMLFRGRRASQ

[0111] TB is a bacterial infection caused by the Mycobacterium tuberculosis (M. tuberculosis) bacterium. While M. tuberculosis generally attacks the lungs, the infection can spread to any part of the body. A person infected with M. tuberculosis may develop an active TB infection or a latent TB infection, which is a lack of TB symptoms. Most conventional IFN-γ assays are not sensitive enough to distinguish between active TB and latent TB infections. Furthermore, after biological sample collection, the sample has to be cultured prior to performing the IFN-γ assay. During the cell culturing, cells, such as peripheral blood mononuclear cells (PBMC) or T cells, are purified from the biological samples. These cells are then stimulated with an antigen for at least 16-24 hours to release a detectable amount of IFN-γ. Increasing the sensitivity of IFN-γ assays may provide a clinically significant method for identifying active TB and latent TB infections. Additionally, a substrate which has increased analytical sensitivity as compared to substrates used in conventional IFN-γ assays may allow for detection and quantification of small concentrations of IFN-γ. As less IFN-γ would be required for a detection, the cell incubation time could be reduced and the assay speed and throughput could be increased.

[0112] In some instances, methods disclosed herein are for detecting IFN-γ in a biological sample comprise: exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a dioxetane compound or salt thereof of the formula

[0113] wherein each of R1 and R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring; wherein each of R3 and R6 are independently C1-C10 alkyl, C6-C10 aryl, or heteroaryl; wherein the 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 to a calibration curve to quantify the level of the IFN-γ in the biological sample. In some instances, methods disclosed herein are for measuring a cell-mediated immune response to an antigen in a biological sample and comprise incubating the biological sample with a cytokine release agent, producing an incubation mixture, wherein the incubation generates IFN- γ; and performing the method for detecting IFN-γ released from the biological sample.

[0114] In some instances, methods disclosed herein are performed on an immunoassay analyzer disclosed herein, such as an automated analyzer. Automated analyzers are commonly used in clinical chemistry, immunoassay, hematology, and other biological sampling and analyzing applications. Automated analytical equipment, such as automated analytical chemistry instruments, automated analytical immunoassay instruments, automated analytical hematology instruments, etc., can efficiently perform clinical analysis on a large number of samples, with multiple tests being run concurrently or within short time intervals. Automated analytical instruments are particularly well-suited for high-volume and mid-volume testing environments.

[0115] In some instances, methods disclosed herein can measure lower levels of IFN-γ in blood, obtaining lower limits of detection (LoD) and limits of quantification (LoQ) than conventional methods, including in ranges that were previously undetectable, allowing smaller differences in analyte to become more apparent for physicians interpreting results. The LoD is the lowest concentration that the assay can detect the analyte within a certain degree of confidence. It may also be defined as the lowest concentration that can be distinguished from the background reliably. The LoQ is the lowest concentration of analyte that can be quantified reliably. Reliably, or a reliable way, refers to the presence of a suitable precision, trueness, and / or reproducibility.

[0116] In some instances, the signal produced by assay methods disclosed herein is up to 20 times greater, alternatively up to 25 times greater, alternatively up to 30 times greater, or 35 times greater than immunoassay methods where conventional or known substrate formulations are used. In someinstances, methods disclosed herein result in an enhanced ratio of signal produced by the analyte to background noise when compared to conventional methods. When a detector, such as a luminometer, is used the signal, or luminescence output, is measured relative to the light output of the detector. In these instances, the signal read-out values is given as Relative Light Units (“RLU”). II. Definitions

[0117] 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 belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0118] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0119] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0120] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments or aspects does not imply that other embodiments or aspects are not useful and is not intended to exclude other embodiments or aspects from the scope of the invention.

[0121] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0122] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus,the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0123] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of 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 of 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".

[0124] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0125] Reference throughout this specification to "one aspect," "an aspect," "certain aspects," or "some aspects," "one embodiment," "an embodiment," "certain embodiment," or "some embodiment," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0126] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, theterm "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0127] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0128] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0129] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0130] The invention is defined in the claims. However, below is a non-exhaustive listing of non- limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0131] One aspect of the invention is a method for quantitatively assessing a degree of ovarian aging in a subject using an immunoassay analyzer. The term "immunoassay" may be a laboratorymethod 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 of antibodies with antigens, and because of the degree of selectivity for the analyte (either antigen or antibody), an immunoassay can be used to quantitatively determine very low concentrations of analyte in a test sample. An "immunoassay analyzer" can include an instrument on which immunoassays have been automated. Various immunoassay analyzer are commercially available including the Dxl®system (Beckman Coulter, CA), the ADVIA®CENTAUR®systems (Siemens Healthcare, Germany), the COBAS®system (Roche Diagnostic, Germany), the ARCHITECT®system (Abbott, IL), the VITROS®system (Ortho-clinical Diagnostic, NJ), and the VIDAS®system (Biomerieux, France). In some aspects, the immunoassay analyzer is a high-throughput immunoassay analyzer.

[0132] As used herein, the terms "subject", "individual", and "patient" are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are 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., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child). II. Systems and Reagents for Detecting Analytes Using Chemiluminescent Dioxetanes

[0133] In an embodiment, the method includes detecting the presence of an IFN-γ in a biological sample obtained from a subject using an immunoassay analyzer, including exposing the biological sample to an enzyme-conjugated affinity molecule and a substrate formulation comprising dioxetane compound or salt thereof of the formula

[0134]

[0135] wherein each of R1and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring; wherein each of R3and R6are independently C1-C10alkyl, C6-C10aryl, or heteroaryl. In an embodiment, the substrate formulation further includes at least one phosphonium surfactant. In an aspect of the methods described herein, the reaction between the enzyme-conjugated affinity molecule and the substrateformulation generates a chemiluminescent detection signal which can be recorded and compared to quantify the level of analyte in the biological sample. In an embodiment, the method includes aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of an immunoassay analyzer. a. Reagents

[0136] In some instances, “two-site” or “sandwich” immunoassays employ a first antibody or antibody fragment, which is described as the “capture” antibody, is bound to a solid support, such as magnetic beads or particles disclosed herein, using procedures known in the art. Further, a second antibody or antibody fragment, which is described as the “detection” antibody, is coupled or conjugated with a label, such as the enzymes disclosed herein, using procedures known in the art. The label produces a detectable signal when provided with substrate(s), so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte.

[0137] In an aspect, the capture antibody and / or the affinity molecule is conjugated to a magnetic bead or a magnetic particle. In some instances, 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 (~20-30 nm), such as magnetite. On the surface, the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding. Antibodies targeting the analyte of interest or capture antibody can be covalently coupled to the surface of the magnetic bead. In some embodiments, there is about 2 µg or antibody per mg of magnetic bead. In some embodiments, there is about 3 µg or antibody per mg of magnetic bead. In some embodiments, there is about 4 µg or antibody per mg of magnetic bead. In some embodiments, there is about 5 µg or antibody per mg of magnetic bead. In some embodiments, there is about 6 µg or antibody per mg of magnetic bead. In some embodiments, there is about 7 µg or antibody per mg of magnetic bead. In some embodiments, there is about 8 µg or antibody per mg of magnetic bead. In some embodiments, there is about 9 µg or antibody per mg of magnetic bead. In some embodiments, there is about 10 µg or antibody per mg of magnetic bead. In some embodiments, there is about 12 µg or antibody per mg of magnetic bead. In some embodiments, there is about 13 µg or antibody per mg of magnetic bead. In some embodiments, there is about 14 µg or antibody per mg of magnetic bead. In some embodiments, there is about 15 µg or antibody per mg of magnetic bead.

[0138] In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. The term “chemiluminescent compound” refers to a compound that produces chemiluminescence in the presence of a phosphatase enzyme and oxygen under appropriate conditions as provided herein. In a non-limiting example, the substrate formulation includes a dioxetane compound or salt thereof of the formula

[0139]

[0140] and R2are independently C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring; wherein each of R3and R6are independently C1-C10alkyl, C6-C10aryl, or heteroaryl. Chemiluminescent compounds useful in the present formulations are capable of generating chemiluminescence when contacted with an alkaline phosphatase. Such compounds can be synthesized as described in U.S. Patent Application No. 2022 / 0390459 which is incorporated by reference herein.

[0141] At operation, the chemiluminescent substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The light production is inversely proportional to the concentration of free analyte in the sample. The amount of analyte in the sample is then determined from a stored, multi-point calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0142] In an exemplary method of producing light from the reaction of the chemiluminescent substrate with a phosphatase enzyme (e.g., detection antibody), the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffer solution at a pH between 7 and 12, 8 and 11, or preferably between 8.5 and 10. The enzyme is preferably an alkaline phosphatase or an alkaline phosphatase conjugate.

[0143] In an embodiment, the dioxetane compound is a compound of a formula:or a salt thereof nd R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;

[0146] R3 is C1-C10 alkyl, C6-C10 aryl, or heteroaryl;

[0147] R5 is H or C1-C10 alkyl; and

[0148] X is a phosphate.

[0149] In an embodiment, the dioxetane compound is a compound of a formula:

[0150] or a salt thereofR11 is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0153] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0154] R5 is H or C1-C10 alkyl; and

[0155] X is a phosphate.

[0156] In an embodiment, the dioextane compound has a formula of .an dioxetane compound is a compound of Formula I or a salt thereof:

[0158] Formula I

[0159] are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;

[0160] R3 is C1-C10 alkyl, C6-C10 aryl, or heteroaryl;

[0161] R4 is C2-C10 alkenyl;

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

[0163] X is a phosphate.

[0164] In an embodiment, the dioxetane compound is a compound of Formula II or a salt thereof: IIC1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0167] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0168] R4 is C2-C10 alkenyl;

[0169] R5 is H or C1-C10 alkyl; and

[0170] X is a phosphate.

[0171] In an embodiment, the dioxetane compound is a compound of Formula III or a salt thereof: Formula IIIH, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0174] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0175] R5 is H or C1-C10 alkyl; and

[0176] X is a phosphate.

[0177] In an embodiment, the dioxetane compound is a compound of Formula IV or a salt thereof:

[0178] Formula IV

[0179] wherein R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0180] R5 is H or C1-C10 alkyl.

[0181] In an embodiment, the 1,2 the dioxetane compound is

[0182]

[0183] or a salt thereof.

[0184] In an embodiment, the dioxetane compound is 4-methoxy-4-(3- phosphatephenyl)spiro[1,2-dioetane-3,2’-adamantane] or a salt thereof.

[0185] The substrate also includes at least one phosphonium surfactant. In some embodiments, the phosphonium surfactant operates as a luminescence enhancer. In certain embodiments, the phosphonium surfactant comprises small-molecule phosphonium surfactants or polymeric phosphonium surfactants.

[0186] In an embodiment, the small molecule phosphonium surfactant is a compound having the formula:

[0187]

[0188] each independently C1-C10 alkyl;

[0189] R15 is arylalkyl; and

[0190] X- is a counterion.

[0191] In an embodiment, the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

[0194] In certain embodiments, the substrate formulation further comprises a magnesium (II) salt.

[0195] Another aspect of the disclosure includes a kit for performing any of the disclosed methods. The kit may include a reagent pack. In some aspects, the reagent pack includes several reagent formulations. In one aspect, the reagent pack includes a first reagent formulation which comprises the capture antibody. In an embodiment, the capture antibody is conjugated to magnetic beads. In one aspect, the reagent pack includes reagent formulation which comprises the enzyme-conjugated affinity molecule. In an embodiment, the enzyme-conjugated affinity molecule is conjugated to magnetic beads. The reagent pack may also include other formulations which comprise buffers and / or salts needed for the methods. In some aspects, the kit may include the substrate formulation, calibrators, and / or wash buffers. In some aspects, the kit includes at least one incubation vessel and at least one cytokine release agent. The kit may also include instructions for performing any one of the disclosed methods. b. Analyzer Systems

[0196] In an aspect, an immunoassay analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analytic unit, an incubator station, a washing station, a read station, and reagent storage. In addition, the immunoassay analyzer may include a pipettor arrangement with at least one sample pipettor and at least one reagent pipettor, and at least one transport device. In an aspect, the transport device includes mechanisms, such as pick-and-place grippers, which are used to transport sample and reaction vessels among the various modules of the immunoassay analyzer.

[0197] The immunoassay analyzer may include a container carriage device which is configured to hold and carry the containers at various locations in the instrument so that the analytic unit, incubator station, wash station, and read station can use the containers in various manners. Examples of container carriage devices include vessel racks (e.g., a sample rack, a reagent rack, and a diluent rack), the sample presentation unit, vessel carriage units (e.g., a sample carriage unit, a reaction vessel carriage unit, and a reagent carriage unit), vessel transfer units (e.g., a sample transfer unit, a reagent transfer unit, an incubator transfer unit, and an reaction vessel transfer unit), and vessel holding plates or wheels (e.g., a sample wheel, an incubator, and a wash wheel), which are described herein.

[0198] The read station may include a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector,a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.

[0199] In an aspect, primary sample containers can be placed into an onload section (e.g., individually or on racks) of the sample presentation unit. In certain 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 certain embodiments, each sample rack can hold at least one sample vessel, alternatively at least two sample vessels, alternatively at least three sample vessels, alternatively at least four sample vessels, alternatively at least five sample vessels, alternatively at least six sample vessels, or alternatively at least seven sample vessels. In a specific embodiment, the sample presentation unit holds about 140 sample vessels. In a specific embodiment, the sample presentation unit has 20 sample racks with each rack holding seven sample vessels.

[0200] In certain embodiments, the sample vessels include a barcode label that uniquely identifies the sample vessel in the immunoassay analyzer. The barcode label also may include alphanumeric characters that correspond to the barcode identification information. The immunoassay analyzer may include at least optical reader, such as a barcode scanner. In one embodiment, the optical readers are area scan cameras that provide a two-dimensional image of the barcode and / or sample vessel. In another embodiment, optical readers are area scan cameras that provide a three- dimensional image of the barcode and / or sample vessel.

[0201] After being fed into the onload section, the sample containers may be moved into a presentation section of the sample presentation unit. In certain embodiments, the sample presentation unit is maintained at between about 4.5°C to 14°C. The transfer station may receive primary sample containers from the sample presentation unit from a transport device. Primary sample containers delivered to the transfer station from the transport device may be initially processed in different ways. For example, the primary sample container may temporarily remain at the transfer station while the sample provided within is aspirated by the sample pipettor at the transfer station. Following such aspiration, the primary sample container may be expelled from the automated analyzer at the offload station of the sample presentation unit or may be passed on to the transport device for further processing. The instrument may also contain a sample retention unit configured to receive sample retention vessels and store, analyze, or otherwise process samples retained within the sample retention vessels. A “primary sample container” loaded into the automated analyzer with a sample may also be considered a “sample retention vessel” when placed in a sample retention unit, such as a storage unit. As used herein, “containers” or “vessels” are analogous, and can be of various types, such as specimen tubes (also referred to herein as sample tubes) and pipettor tips, such as micro or disposable tips. In certain embodiments, the vessels are tubes with diameters between about 12mm to about 16mm and / or heights between about 75mm to about 100mm. In certain embodiments, the vessels are cups with volumes of about 0.5mL, alternatively about 1.0mL, alternatively about 1.5mL, alternatively about 2.0mL, alternatively about 2.5mL, or alternatively about 3.0mL.

[0202] In an aspect, the analytic unit is configured to receive and analyze samples. In an aspect, the analytic unit configured to perform an immunoassay. In certain embodiments, the analytic unit includes the pipettor arrangement. The pipettor arrangement may be configured to aliquot, aspirate, and dispense fluidic substances into various vessels, including, but not limited to sample vessels, diluent vessels, reagent vessels, and reaction vessels. Fluidic substances are substances that have fluidic characteristics. In some embodiments, the fluidic substance is a single fluidic substance. In other embodiments, the fluidic substance is a mixture of a plurality of substances.

[0203] The pipettor arrangement may contain at least one, two, three, or four reagent pipettors used to mix reagents with sample aliquots for an assay. The pipettor arrangement may also contain at least one, two, three, or four sample pipettors used to transfer sample aliquots for an assay. In some instances, the pipettor arrangement contains one sample pipettor. In some instances, thepipettor arrangement contains one sample pipettor and four reagent pipettors. In an aspect, the reagent pipettors may be arranged as dual reagent pipetting stations and are independent to each other, each having its own fluid pumps and valves, wash towers, reaction vessel carriages, and pipettor. A sample aliquot may be transferred from a sample retention vessel into a reaction vessel using the sample pipettor in order to mix the sample aliquot with one or more reagents. In an aspect, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 10 μL. In an embodiment, the at least one reagent pipettor and at least one sample pipettor 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, alternatively between than about 9.9 μL and 2.0 μL.

[0204] In an aspect, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are selectively and / or simultaneously operated. In certain embodiments, the sample pipettor, the first reagent pipettor, the second reagent pipettor, the third reagent pipettor, and / or the fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

[0205] The claimed configuration allows for the simultaneous performance of at least two assays for a plurality of biological samples, alternatively at least three assays for a plurality of biological samples, or at least four assays for a plurality of biological samples. Depending on the analysis desired the biological samples may be from the same subject or from multiple subjects. In some aspects, the method further comprises a plurality of sample vessels. In some embodiments, the method comprises at least one sample vessel, at least two sample vessels, at least three sample vessels, at least four sample vessels, at least five sample vessels, at least ten sample vessels, at least twenty sample vessels, at least fifty sample vessels, at least one hundred sample vessels, at least two hundred sample vessels, at least three hundred sample vessels, at least four hundred sample vessels, or at least five hundred sample vessels. In some aspects, the sample vessels are housed within the immunoassay analyzer.

[0206] In a non-limiting example, if multiple assays for multiple biomarkers are being performed, the first pipettor may be configured to aspirate a reagent from a reagent vessel comprising a firstcapture antibody and dispense into a first reaction vessel, while a second pipettor may be configured to simultaneously aspirate a reagent from a reagent vessel comprising a second capture antibody and dispense into a second reaction vessel. Depending on the analysis desired, the third and fourth pipettor may also be configured to simultaneously aspirate a reagent from a reagent vessel comprising a third and fourth affinity molecule and dispense into a third and fourth reaction vessel.

[0207] The disclosed simultaneous and / or selective operation of the pipettors allows for a high- throughput analysis. In some embodiments, the method is configured to analyze at least about 200 plasma samples / hr., alternatively at least about 300 plasma samples / hr., alternatively at least about 400 plasma samples / hr., alternatively at least about 440 plasma samples / hr., or alternatively at least 500 plasma samples / hr.

[0208] In an aspect, the immunoassay analyzer includes a reagent pack configured to hold a plurality of reagent vessels. A “reagent pack” may include any suitable container that can store a reagent. An example of a reagent pack can include a generally rectangular elongated body formed to include multiple reagent vessels including one or more large reagent vessels, and one or more relatively smaller reagent vessels, as well as features to facilitate handling and automation. US Patent No. 9,519,000, which is incorporated by reference in its entirety herein, discloses non- limiting examples of a reagent pack that may be used in an aspect of the invention.

[0209] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances.

[0210] In some embodiments a reagent pack may supplied with empty or partially filled reagent vessels, to which reagents are subsequently transferred from bulk containers, such as bottles. Individual reagent vessels may differ in dimension to accommodate the requirements of an assay type. Factors that can determine the size of a reagent vessel include the number of uses desired for the reagent pack type, concentration-dependent stability issues with reagent components, and the need to minimize the volume of the final reaction mixture. As noted above, in some embodiments, each reagent pack can include a large reagent vessel and a plurality of small reagent vessels. Each reagent vessel may be large enough to accommodate a microtip or disposable tip (i.e., dispo-tip) of a reagent pipettor used to remove a volume of reagent for use in an assay. In certain embodiments, the reagent pack may be maintained at a temperature of between about 4°C to 10°C.

[0211] A “reagent vessel” may refer to a vessel, unit, fluid container, or the like that is configured to store reagents. In an embodiment, the reagent vessels include an elastomeric self-sealing membrane. An elastomeric self-sealing membrane may be a polymer, such as polypropylene, which is able to regain its original shape when pierced. For some embodiments, the elastomeric membrane can be a thermoplastic elastomer with hardness of 30-40 durometer (Shore) A. In other embodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A. Elastomers deform sufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.

[0212] The elastomeric membrane can be large enough to provide adequate compression without bottoming on the sealing portion of the vessel. The hardness and dimensions can cooperate to allow the elastomeric membrane to the sealing portion with reasonable sealing force. In some embodiments, the elastomeric membrane diameter is small enough so that, when compressed by engagement of the pipettor tip, it conforms to the sealing portion without contacting the wall of the pipettor tip. This advantageously concentrates sealing force to the sealing portion of vessel and distributes sealing force evenly to prevent leaks. In some embodiments, the sealing force is about 44 newtons (about 9.9 lbs.) and produces a pressure on the sealing surface of about 300 (about 43.5 pounds per square inch) to about 1000 kPa (145.0 pounds per square inch).

[0213] In methods where multiple samples are analyzed, the elastomeric self-sealing membrane allows for aspiration of the reagents without concern of evaporation. In some embodiments, the reagent pack further includes containment walls arranged between the reagent vessels. In an aspect, the reagent pack may include at least one reagent vessel, alternatively at least two reagent vessels, alternatively at least three reagent vessels, alternatively at least four reagent vessels, alternatively at least five reagent vessels, or alternatively at least ten reagent vessels. In an embodiment, the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0214] At operation, a fluidic substance is dispensed to a reaction vessel. Examples of the fluidic substance include a sample, diluent, reagent, substrate, or any combination thereof, as described herein. In some embodiments, the reaction vessel already contains other fluidic substances, suchas a sample, and after a fluidic substance is dispensed to a reaction vessel, the fluidic substance is mixed with the other fluidic substances in the reaction vessel. The mixing can be performed with a stirrer in direct contact with the fluidic substances, an ultrasonic probe in direct or indirect contact with the fluidic substances, or any other suitable mixing apparatus. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For instance, a reagent pipettor may be outfitted with a tip that allows it to perform ultrasonic mixing of a reagent in a reagent pack before aspirating it for transport to a reaction vessel, thereby ensuring that the aspirated reagent would not be impacted by any settling that may have taken place in the reagent pack. Sample pipettors may similarly be specialized.

[0215] The transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator. In some embodiments, the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator. Further, the transfer unit can transfer one or more reaction vessels from the incubator to the reagent carriage unit. The transfer unit can also remove from the reaction vessels that have been read or completed the incubator.

[0216] The incubator is thermally controlled to maintain a predetermined temperature. In some embodiments, the incubator is maintained about 30 ˚C to 40 ˚C. In other embodiments, the incubator is maintained about 37 ˚C to ensure immunological reaction and enzyme reaction, for example. By way of example, the incubator performs assay incubation.

[0217] In some embodiments, the transfer unit transfers incubated reaction vessels from the incubator to the wash unit, transfers assay reaction vessels from the wash unit to the incubator, transfers reaction vessels containing substrate from the wash unit to the incubator for substrate incubation or enzyme reaction, transfers washed reaction vessels from the incubator to detector arrangement after substrate incubation, and transfers the reaction vessels that have been read or completed from the detector arrangement to the incubator. The used reaction vessels can be delivered to a waste location.

[0218] At operation, a sample and a reagent are dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, the sample and the reagent interact. The resulting “first reaction mixture” is a result of the incubation between the sample and the reagent. The reagent may include a specific-binding reagent, such as a capture antibody specific to the analyte being analyzed by the immunoassay analyzer. In a non-limiting example, theincubation 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.

[0219] At operation, a reagent, which is different from the reagent used in the first reaction mixture, is added to the first reaction mixture and mixed. The mixture is then transferred to the incubator. During incubation, the first reaction mixture and the reagent interact. The resulting “second reaction mixture” is a result of an incubation between the first reaction mixture and the reagent. The reagent may include an affinity molecule, which may be an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen. In certain 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 at least one portion of an analyte or at least one portion of the capture antibody. In a non-limiting example, 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.

[0220] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the immunoassay analyzer. In an embodiment, the wash station is configured to wash away at least some of the unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The wash station may be configured to perform a set number of wash actions depending on the assay. The wash station may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions. In some embodiments, the wash station is a thermally controlled device to separatebound or free analytes from particles after incubation. In some embodiments, the wash unit is maintained about 30 ˚C to 40 ˚C. In other embodiments, the wash unit is maintained about 37 ˚C to ensure enzyme reaction, for example, U.S. Patent Publication No. 2022 / 0357352, which is incorporated by reference in its entirety herein, discloses configurable wash processes according to an aspect of the invention.

[0221] The wash station may comprise a washing arrangement which may be configured to provide a base number of wash series (or wash actions) for each reaction vessel and optionally provide an additional number(s) of wash actions. The additional number(s) of wash actions may include one, a plurality, or all of a potential number of wash actions. An additional number of wash actions beyond the base number of wash actions may be specified for certain assays in an assay protocol file. The washing arrangement may include cleaning dispense nozzle (or probe) which dispenses a rinsing fluid and a cleaning aspiration nozzle (or probe) which aspirates the unreacted components. At operation, a base number of wash actions performed may be one, two, three, four, or five and the additional number of wash action(s) may be one, two, three, four or five. At operation, a base number of wash actions performed may be three and the additional number of wash action(s) may be one or two. In this embodiment, the base number of wash actions may be performed if three probes dispense buffer solution once per vessel and the three probes aspirate the at least some of the unreacted components some of the buffer solution, and / or the at least some of the unreacted reagents once per vessel. According to the principles of the present disclosure, certain probe(s) may be selectively used to dispense clean buffer solution into the vessel and aspirate the at least some of the unreacted components of the sample, some of the buffer solution, and / or the at least some of the unreacted reagents from the vessel to perform the additional wash action(s).

[0222] In an aspect, the immunoassay analyzer further comprises a washing arrangement. In an embodiment, the washing arrangement is configured to wash away at least some of the unreacted components from the sample, first reagent, second reagent, or substrate formulation 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 fluid, etc.) and unreacted sample. The washing arrangement may be configured to perform a set number of wash actions depending on the assay. The washing arrangement may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, thewashing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

[0223] In an embodiment, the first reaction mixture or second reaction mixture is subjected to a magnetic field. The magnetic beads do not exhibit bead-to-bead attraction, only migrating when a magnetic field is applies. Captured analytes or targets are separated from the mixture and magnetization may be used to retain desired components within a reaction vessel.

[0224] At operation, the vessel containing the first reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0225] At operation, the vessel containing the second reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series.As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle.

[0226] In an aspect, the immunoassay analyzer includes a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses non- limiting examples of a detector that may be used in an aspect of the invention.

[0227] At operation, a substrate is dispensed into the second reaction mixture and mixed. After a certain reaction time necessary for the substrate and the second reaction mixture to interact. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle. The resulting detection mixture is transferred to the detector arrangement.

[0228] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions.

[0229] In certain embodiments, the transport device includes three pick-and-place grippers, where a first pick-and-place gripper may be used to transport sample containers among the onload section, the transfer station, and reagent pipetting stations. A second pick-and-place gripper may be used to transport reaction vessels between the reagent pipetting stations and the incubator station or read station. A third pick-and-place gripper may be used to transport reaction vessels between the incubator station and the wash station or read station. A detailed description of theconfigurations and functions of one embodiment of the vessel pick-and-place grippers is provided in U.S. Patent No. 7128874 and is incorporated herein in its entirety by reference. However, it should be understood that other pick-and-place mechanism that are capable of transporting sample and reaction vessels among the various modules of the immunoassay analyzer is also contemplated for the purpose of the present invention.

[0230] In an aspect, the immunoassay analyzer includes a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis. In some embodiments, the machine vision apparatus utilizes one or more image capture units to determine whether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether the sample is appropriately prepared so that the analytic unit produces a reliable result. An exemplary machine vision apparatus is described in U.S. Patent No. 11,263,433, which is incorporated by reference herein.

[0231] In some aspects, the machine vision apparatus operates to detect a volume of a fluidic substance in a container and determine whether the volume held in the container is appropriate as targeted. As described herein, this volume detection is configured to detect a volume at a dispense tip using the dispense tip image capture unit, and a volume at a vessel using a vessel image capture unit.

[0232] In some aspects, the machine vision apparatus operates to detect any interferents, which can interfere with an analytic procedure and may generate incorrect results in the dispense tip. As described herein, this dispense tip evaluation is configured to determine a quality of a fluidic substance at a dispense tip using a dispense tip image capture unit, and an alignment of the dispense tip with respect to the dispense tip image capture unit.

[0233] In some aspects, the machine vision apparatus operates to determine a particle concentration in a fluidic substance contained in a vessel, such as a reaction vessel, a sample vessel,a dilution vessel, a cuvette, or any suitable type of vessel, which is used throughout the process in the immunoassay analyzer. In some embodiments, this reaction vessel particle concentration check uses the vessel image capture unit.

[0234] The dispense tip image capture unit operates to capture images of dispense tips in one or more locations. In some embodiments, the dispense tip image capture unit is fixed at a particular location in the instrument. In other embodiments, the dispense tip image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of dispense tip image capture units. As described herein, the dispense tip image capture unit can include a camera unit.

[0235] The vessel image capture unit operates to capture images of vessels in one or more locations. In some embodiments, the vessel image capture unit is fixed at a particular location in the immunoassay analyzer. In other embodiments, the vessel image capture unit is movably disposed in the immunoassay analyzer, which can move either independently from other components of the immunoassay analyzer or together with one or more components of the immunoassay analyzer. Some embodiments of the immunoassay analyzer include a plurality of vessel image capture units. As described herein, the vessel tip image capture unit includes a camera unit.

[0236] The carriage image capture unit operates to capture images of container carriage devices with or without containers in one or more locations. In some embodiments, the carriage image capture unit is fixed at a particular location in the instrument. In other embodiments, the carriage image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of carriage image capture units.

[0237] All of the units of the immunoassay analyzer are connected to a controller, which can perform block control of all of the analyzer functions by using, for example, a microcomputer. The controller may contain subunits such as a data processing unit, a communication interface, and others. A controller in accordance with an exemplary embodiment of the present technology may comprise a data processor, a non-transitory computer-readable medium, and a data storage coupledto the data processor. The non-transitory computer-readable medium may comprise 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.

[0238] The data processor may include any suitable data computation device or combination of such devices. An exemplary data processor may comprise one or more microprocessors working together to accomplish a desired function. The data processor may include a CPU that comprises at least one high-speed data processor adequate to execute program components for executing 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’s and Sony’s Cell processor; Intel’s Celeron, Itanium, Pentium, Xeon, and / or XScale; Apple M1, and / or the like processor(s).

[0239] The computer-readable medium and the data storage may be any suitable device or devices that can store electronic data. Examples of memories may comprise, for example, one or more memory chips, disk drives, etc. Such memories may operate using any suitable electrical, optical, and / or magnetic mode of operation.

[0240] The computer-readable medium may comprise code, executable by the data processor to perform any suitable method. For example, the computer-readable medium may comprise code, executable by the processor, to cause the controller to operate on a pre-determined schedule. In some embodiments of the presently claimed technology, the pre-determined schedule is a constituent test.

[0241] In an aspect the cycle time of an immunoassay analyzer described herein is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of an immunoassay analyzer to complete its tasks necessary for generating a result. In certain 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. At operation, the sample pipettor can complete its tasks in 8 seconds and the reagent pipettor can complete its tasks in 32 seconds. In some aspects, a task for the pipettor is defined as the time to aspirate and dispense a sample or reagent, inclusive of the time required to move from and return to the starting position. In this embodiment, to maintain a higher throughput, four reagent pipettors are present in theimmunoassay analyzer (32 seconds / 4 = 8 seconds). If the incubation of one of the reaction mixtures is 5 minutes, at least 38 incubation positions are needed to support this incubation time (300 seconds / 8 seconds = 37.5 seconds). In certain 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.

[0242] In an aspect, the time to first result (TTFR) of an immunoassay analyzer described herein is about 60 minutes or less. The “TTFR” is a measure of time from when the sample is aspirated to when the presence and / or concentration of an analyte is determined. In certain embodiments the TTFR is about 60 minutes or less, alternatively 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 an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents 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 instances of an assay. In some embodiments, each reagent pack includes reagent for about 50 instances of an assay. In some embodiments, each reagent pack includes reagent for about 250 instances of an assay. In some embodiments, each reagent pack includes reagent for about 500 instances of an assay. In some embodiments, each reagent pack includes reagent for about 650 instances of an assay. In some embodiments, the reagent pack may include a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about one instance of an assay. In some embodiments, the reagent pack is configured to store a volume of reagent required for up to 650 instances of an assay. In a non-limiting example, the reagent pack includes at least three reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured tostore a volume of reagent required for up to 650 instances of an assay. In a non-limiting example, the reagent pack includes at least four reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured to store a volume of reagent required for up to 650 instances. In a non-limiting example, the reagent pack includes at least five reagent vessels, wherein each reagent vessel is independently configured to store a volume of reagent required for at least about one instance of an assay. In an embodiment, each reagent vessel is independently configured to store a volume of reagent required for up to 650 instances. III. Assay Methods Using Chemiluminescent Dioxetanes

[0244] In an aspect of the methods disclosed herein, IFN-γ is detected in a biological sample. In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid. In some aspects, the biological samples comprises T-cells, NK cells, or a combination thereof. In an aspect, the method includes exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture. In some embodiments, the IFN-γ must be released from the biological sample prior exposure to the capture antibody. In some aspects, this includes first incubating the biological sample with a cytokine release agent, producing an incubation mixture, wherein the incubation generates IFN-γ. In certain embodiments, when combined with the methods of this disclosure, this method may be used to measure a cell-mediated immune response to an antigen in a biological sample from a subject. In these aspects, the antigen may be tuberculosis antigen.

[0245] Depending on the analysis and / or assay, the cytokine release agent may include at least one peptide, antigen, or mitogen. In some aspects, the cytokine release agent includes two or more peptides, antigens, or mitogens; alternatively three or more peptides, antigens, or mitogens; alternatively four or more peptides, antigens, or mitogens; alternatively five or more peptides, antigens, or mitogens; alternatively six or more peptides, antigens, or mitogens; or alternatively seven or more peptides, antigens, or mitogens. A “peptide” is at least two amino acids linked by a peptide bond. A peptide may also include a polypeptide. An “antigen” is a molecule, such as a protein, peptide, polysaccharide, lipid, or nucleic acids, which can bind a specific antibody or T-cell receptor. A “mitogen” is a protein or peptide that induces cell division or enhances the rate of division within a cell.

[0246] The biological sample may incubated with the cytokine release agent for a time sufficient to generate a detectable amount of IFN-γ. In an aspect, the biological sample is incubated with the cytokine release agent for about 16 hours or less, alternatively about 15 hours or less, alternatively about 14 hours or less, alternatively about 13 hours or less, alternatively about 12 hours or less, alternatively about 11 hours or less, alternatively about 10 hours or less, alternatively about 9 hours or less, alternatively about 8 hours or less, alternatively about 7 hours or less, alternatively about 6 hours or less, alternatively about 5 hours or less, alternatively about 4 hours or less, alternatively about 3 hours or less, alternatively about 2 hours or less, alternatively about 1 hour or less, or alternatively about 30 minutes or less. In an aspect, the biological sample and cytokine release agent are incubated in a vessel prior to introduction to the automated analyzer. In another aspect, the biological sample and cytokine release agent are incubated are incubated on the automated analyzer. In this aspect, the sample pipettor may aspirate and dispense an aliquot of the biological sample into a vessel and the reagent pipettor may aspirate and dispense an aliquot of the cytokine release agent into the vessel. The resulting incubation mixture may then be transferred to the incubator. In some aspects, the incubation occurs in a tube, vial, well, or plate.

[0247] The “first reaction mixture” is a result of an incubation between the mixture comprising IFN-γ released from the biological sample and the capture antibody. In certain embodiments, the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture antibody from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. During the incubation, the sample and the capture antibody interact. In an embodiment, the capture antibody binds to a portion of the analyte. In a non-limiting example, 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.

[0248] In an embodiment, the method includes exposing the first reaction mixture to an enzyme- conjugated antibody or an enzyme-conjugated antigen, generating a second reaction mixture. In an aspect, the enzyme comprises an alkaline phosphatase (AP). The “second reaction mixture” isa result of an incubation between the first reaction mixture and the enzyme-conjugated antibody or an enzyme-conjugated antigen. In certain embodiments, the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated antibody or an enzyme-conjugated antigen from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. During the incubation, the first reaction mixture and the enzyme- conjugated antibody or an enzyme-conjugated antigen interact. In an embodiment, the enzyme- conjugated antibody is configured to bind at least one portion of an analyte. In another embodiment, the enzyme-conjugated antigen is configured to bind at least one portion of the capture antibody. In an 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.

[0249] In an embodiment, the method exposing the second reaction mixture to a substrate formulation comprising a dioxetane compound. In an embodiment, the substrate formulation further comprises at least one phosphonium surfactant. In an embodiment, a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel. In an aspect, 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, 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.

[0250] In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. In certain embodiments, the reaction between the enzyme-conjugated antibody or the enzyme-conjugated antigen and the substrate formulation generates a chemiluminescent detection signal.

[0251] At operation, the substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The light generated (e.g., the detection signal) is recorded and the amount of analyte in the sample is then determined from a stored, multi- point calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0252] In an exemplary method of producing light from the reaction of the substrate with a phosphatase enzyme, the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffer solution at a pH between 7 and 12, 8 and 11, or preferably between 8.5 and 10.

[0253] In an aspect, capture antibody, enzyme-conjugated antibody and / or enzyme-conjugated antigen is conjugated to a magnetic bead or a magnetic particle. In an embodiment, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection. Magnetization may be used to retain desired components within a reaction vessel. In certain embodiments, the reaction vessel is moved near one or more magnets after the introduction of the first reagent or second reagent. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The washing arrangement is used to wash the reaction vessel for a predetermined number to times. While washing the magnet(s) retain the magnetic bead(s) or magnetic particle(s) while the unreacted components are washed away. In certain embodiments, increasing the numbers of wash actions may result in a better signal to noise ratio and increase the sensitivity of the assay.

[0254] In an aspect, the method is performed using an immunoassay analyzer herein and the cycle time is about 45 seconds or less. In certain 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.

[0255] In an aspect, the method is performed using an immunoassay analyzer disclosed herein and the time to first result (TTFR) is about 60 minutes or less. In certain 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, alternativelyabout 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.

[0256] In an aspect, the disclosed methods include the detection of IFN-γ using an immunoassay. Depending on the analysis desired, different enzyme-conjugated affinity molecules may be used in the IFN-γ assay. For example, the affinity molecule may be an antibody, wherein the antibody is configured to bind at least one portion of IFN-γ. In another non-limiting example, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody. In some aspects, IFN-γ commercial assays were employed per manufacturers’ protocol.

[0257] In some aspects of the method, the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 1, alternatively at least about 1.5, alternatively at least about 2, alternatively at least about 2.5, alternatively at least about 3, alternatively at least about 3.5, alternatively at least about 4, alternatively at least about 4.5, alternatively at least about 5, alternatively at least about 10, alternatively at least about 15, 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 400, or alternatively at least about 600.

[0258] In some aspects, the method provides a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, alternatively 4% or less, alternatively 3% or less, alternatively 2% or less, alternatively 1.5% or less over two or more tests.

[0259] In some aspects, the IFN-γ immunoassay is used to identify patients infected with M. tuberculosis. In some aspects, the IFN-γ immunoassay is used to differentiate between active TB infection and latent TB infection. In some aspects, the method may further include administering an effective amount of a pharmaceutical composition to the subject and / or identifying a course of treatment (or treatment protocol) based on the IFN-γ alone or in conjunction with risk assessment, radiography, or other medical and diagnostic evaluations.

[0260] The terms "treat", "treating", or "treatment" refer to administering to a subject a compound or pharmaceutical composition to partially or completely alleviate, inhibit, ameliorate, or relieve the condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a condition are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular condition refers to any lessening, whether permanentor temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a condition in the subject; arrest, inhibit, or slow the progression of the condition in the subject; and / or decrease the number, frequency, or severity of clinical symptoms of the condition in the subject. A “treatment protocol” is a protocol or regime developed regarding specific therapies (including pharmaceuticals or therapeutic interventions) for treatment. A “therapeutic intervention” refers to a clinical intervention intended to manage a disease, condition, disorder or injury and avoid further clinical interventions.

[0261] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / or substantially lessening the chances of a condition in a subject. The effective amount of the pharmaceutical composition may be administered orally or via intravenous injection. The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular composition, its mode of administration, its mode of activity, and the like.

[0262] Additional examples are provided below.

[0263] EXAMPLES

[0264] Example 1: INFγ Enhanced Signal Generation

[0265] A series of INFγ calibrators were used to compare the difference in the chemiluminescence produced by a substrate formulation comprising a 1,2 dioxetane compound of Formula I herein and a phosphonium surfactant according to an aspect of the disclosure (Substrate 1) and an alkaline phosphatase chemiluminescent substrate comprising the(Substrate 2). The calibrator levels were 0.0050 international , 0.10 IU / mL, 0.20 IU / mL, 0.40 IU / mL, 0.080 IU / mL, 0.181 IU / mL, 0.260 IU / mL, 0.680 IU / mL, 2.9 IU / mL, 6.10 IU / mL, and 12.2 IU / mL. Two control samples (0 IU / mL) were also analyzed. Eachcalibrator and control underwent four runs. A calibrator with a concentration of 0.0025 IU / mL was evaluated only using Substrate 1.

[0266] The INFγ enhanced signal generation was performed using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor; (ii) reagent packs configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; (iv) the capability to perform over 5 wash cycles per reaction vessel; and (v) a luminometer.

[0267] Paramagnetic particle conjugated with INFγ antibodies capable of binding to an epitope INFγ were pipetted into a reaction vessel using one of four reagent pipettors along with a buffer. A sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a first reaction mixture. A magnetic field was applied to the reaction vessel and the first reaction mixture was washed using a wash buffer to remove any unreacted components. A secondary antibody capable of binding a different epitope of INFγ conjugated to alkaline phosphatase (“ALP”) was added to the reaction vessel containing the first reaction mixture. The reaction vessel was mixed ultrasonically and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. Substrate 1 was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer. This process was repeated for Substrate 2.

[0268] Test results were determined automatically by the system software. Detection of analyte in the sample was determined from the measured light production by means of the stored calibration data. Signal generated by the assay was measured in relative light units (RLUs). Table 2 details the mean signal generated by performing the assay with Substrate 1 versus Substrate 2 for each calibrator level, as well as the corresponding standard deviation (SD) and coefficient of variation (CV). The table also details the signal-to-noise ratio (Sx / S0) for Substrate 1 and Substrate 2 and the ratio of signals produced by Substrate 1 and Substrate 2. Table 3 details the mean signal generated by Substrate 1 and Substrate 2, subtracting background signal, and the ratio of signals produced by Substrate 1 and Substrate 2.

[0269] Table 2: Signal Produced by INFγ Immunoassay (Calibrator Levels) Substrate 1Substrate 2Calibrator Concentration Mean Mean Sub1:Sub2 (IU / mL) (RLU) SD CV Sx / S0 (RLU) SD CV Sx / S0 ratio 0 176,548 14,523 8.2% 1.00 6,987 160 2.3% 1.0025.30 168,363 2,496 1.5% 6,885 93 1.4%24.50.0025 176,620 6,213 3.5% 1.00 0.0050 214,967 12,617 5.9% 1.22 8,330 19 0.2% 1.1925.80.10 242,702 3,774 1.6% 1.37 9,930 12 0.1% 1.4224.40.20 327,747 7,609 2.3% 1.86 12,582 306 2.4% 1.8026.00.40 489,256 23,590 4.8% 2.77 17,234 235 1.4% 2.4728.40.080 820,994 13,727 1.7% 4.65 27,36 252 0.9% 3.8730.40.181 1,695,314 71,334 4.2% 10 55,16 555 1.0% 830.80.260 2,134,629 75,325 3.5% 12 67,119 1,201 1.8% 1031.80.680 5,928,755 262,700 4.4% 34 188,348 1,461 0.8% 2731.52.9 17,536,624 550,717 3.1% 99 539,329 8,800 1.6% 7732.56.10 50,194,206 884,621 1.8% 284 1,576,720 12,599 0.8% 22631.812.20 98,629,468 3,797,778 3.9% 559 3,105,723 39,227 1.3% 44531.8

[0270] Table 3: Signal Produced by INFγ Immunoassay Subtracting Background (Calibrator Levels) Substrate 1 Substrate 2 Calibrator Concentration Mean Mean (IU / mL) (RLU) (RLU) Sub1:Sub2 ratio 0 0 0 0.0025 72 0.0050 38,419 1,343 28.6 0.10 66,154 2,943 22.5 0.20 151,199 5,595 27.0 0.40 312,708 10,247 30.5 0.080 644,446 20,49 32.1 0.181 1,518,766 48,29 31.6 0.260 1,958,081 60,132 32.6 0.680 5,752,207 181,361 31.7 2.9 17,360,076 532,342 32.6 6.10 50,17,658 1,569,733 31.9 12.20 98,452,920 3,098,736 31.8

[0271] A calibration curve was generated and corresponding concentrations were calculated for each calibrator based on calibration curve fit. The results are also plotted in FIG. 1 (Substrate 1) and FIG. 2 (Substrate 2).

[0272] From the results shown in Tables 2 and 3 and FIGs. 1 and 2, Substrate 1 was found to generate higher signal at all calibrator concentrations as compared to Substrate 2.

[0273] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0274] It will be understood that while the invention has been described in conjunction with the detailed description thereof, 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

CLAIMS What is claimed is:

1. A method of detecting Interferon gamma (“IFN-γ”) in a biological sample, the method comprising: exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture; exposing the second reaction mixture to a substrate formulation comprising a dioxetane compound or salt thereof of the formula wherein each of R1and R2areC10 alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring; wherein each of R3and R6are independently C1-C10alkyl, C6-C10aryl, or heteroaryl; wherein the 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 to a calibration curve to quantify the level of the IFN- γ in the biological sample.

2. A method of detecting IFN-γ in a biological sample, the method comprising: exposing a mixture comprising IFN-γ released from the biological sample to a capture antibody configured to bind to at least one portion of IFN-γ, generating a first reaction mixture; exposing the first reaction mixture to an enzyme-conjugated affinity molecule, forming a second reaction mixture;exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and at least one phosphonium surfactant; recording the detection signal generated by the reaction; and comparing the recorded signal to a calibration curve to quantify the level of the IFN- γ in the biological sample.

3. A method for measuring a cell-mediated immune response to an antigen in a biological sample from a subject, the method comprising: incubating the biological sample with a cytokine release agent, producing an incubation mixture, wherein the incubation generates IFN-γ; and performing the method of claim 1 or claim 2 to detect IFN-γ released from the biological sample.

4. The method of claim 3, wherein the antigen is a tuberculosis antigen.

5. The method of any one of claims 1 to 4, wherein the cytokine release agent comprises at least one peptide, antigen, or mitogen.

6. The method of any one of claims 1 to 5, wherein the biological sample comprises T cells and / or peripheral blood mononuclear cells.

7. The method of any one of claims 3 to 6, wherein the biological sample is incubated with the cytokine release agent for about 16 hours or less, alternatively about 15 hours or less, alternatively about 14 hours or less, alternatively about 13 hours or less, alternatively about 12 hours or less, alternatively about 11 hours or less, alternatively about 10 hours or less, alternatively about 9 hours or less, alternatively about 8 hours or less, alternatively about 7 hoursor less, alternatively about 6 hours or less, alternatively about 5 hours or less, alternatively about 4 hours or less, alternatively about 3 hours or less, alternatively about 2 hours or less, alternatively about 1 hour or less, or alternatively about 30 minutes or less.

8. The method of any one of claims 3 to 6, wherein the biological sample is incubated with the cytokine release agent in a vessel, tube, vial, well, or plate.

9. The method of any one of claims 1 to 8, wherein the ratio of the signal produced by cleavage of the enzyme to the background noise is at least about 1, alternatively at least about 1.5, alternatively at least about 2, alternatively at least about 2.5, alternatively at least about 3, alternatively at least about 3.5, alternatively at least about 4, alternatively at least about 4.5, alternatively at least about 5, alternatively at least about 10, alternatively at least about 15, 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 400, or alternatively at least about 600.

10. The method of any one of claims 1 to 9, wherein the method provides a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, alternatively 4% or less, alternatively 3% or less, alternatively 2% or less, alternatively 1.5% or less over two or more tests.

11. The method of any one of claims 1 to 10, wherein the affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

12. The method of claim 11, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of IFN-γ.

13. The method of claim 11, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

14. The method of any one of claims 1 to 13, wherein the capture antibody and / or enzyme- conjugated affinity molecule is conjugated to at least one magnetic bead.

15. The method of any one of claims 1 to 14, wherein the enzyme comprises an alkaline phosphatase (AP).

16. The method of any one of claims 1 to 15, wherein the dioxetane compound is a compound of a formula: or a salt thereof wherein each of R1C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring; R3is C1-C10alkyl, C6-C10 aryl, or heteroaryl; R5is H or C1-C10 alkyl; and X is a phosphate.

17. The method of any one of claims 1 to 16, wherein the dioxetane compound is a compound of a formula:or a salt thereof wherein each , halogen, C1-C10alkyl, C2-C10alkenyl, or C6-C10 aryl; R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl; R5is H or C1-C10alkyl; and X is a phosphate.

18. The method of any one of claims 1 to 17, wherein the dioextane compound has a formula of .

19. The method of any one of claims 1 to 18, wherein the dioxetane compound is a compound of Formula I or a salt thereof: Formula Iwherein each of R1 and R2 are independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring; R3is C1-C10alkyl, C6-C10 aryl, or heteroaryl; R4is C2-C10 alkenyl;R5is H or C1-C10 alkyl; and X is a phosphate.

20. The method of any one of claims 1 to 19, wherein the dioxetane compound is a compound of Formula II or a salt thereof: Formula II wherein eachC1-C10alkyl, C2-C10alkenyl, or C6-C10aryl; R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl; R4is C2-C10 alkenyl; R5is H or C1-C10alkyl; and X is a phosphate.

21. The method of any one of claims 1 to 20, wherein the dioxetane compound is a compound of Formula III or a salt thereof: Formula III wherein eachC1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl; R3is C1-C10alkyl, C6-C10aryl or heteroaryl;R5is H or C1-C10alkyl; and X is a phosphate.

22. The method of any one of claims 1 to 21, wherein the dioxetane compound is a compound of Formula IV or a salt thereof: IV wherein R3R5is H or C1-C10alkyl; and X is a phosphate.

23. The method of any one of claims 1 to 22, wherein the wherein the dioxetane compound is24. The method of any one of claims 1 to 23, wherein the dioxetane compound is 4-methoxy- 4-(3-phosphatephenyl)spiro[1,2-dioetane-3,2’-adamantane] or a salt thereof.

25. The method of any one of claims 19 to 24, wherein the signal produced is a least about 10X greater, alternatively at least about 20X greater, or alternatively at least about 30X greater than a signal produced by the method, wherein the substrate formulation comprises a compound of the following formula:.

26. The method of any one of claims 19 to 25, wherein the substrate formulation further comprises at least one phosphonium surfactant.

27. The method of claim 26, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

28. The method of claim 27, wherein the small molecule phosphonium surfactant is a compound having the formula: Wherein R12-R14 are eachalkyl; R15 is arylalkyl; and X- is a counterion.

29. The method of claim 27, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bu3 30. The method of any one of claims 1 to 29, wherein the substrate formulation further comprises a magnesium (II) salt.

31. The method of any one of claims 1 to 30, wherein the method is an assay and is performed using an immunoassay analyzer, wherein the immunoassay analyzer comprises: a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay, wherein at least one reagent comprises the capture antibody and at least one reagent comprises the enzyme-conjugated affinity molecule; a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement.

32. The method of claim 31, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

33. The method of claim 31 or claim 32, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

34. The method of any one of claims 31 to 33, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

35. The method of any one of claims 31 to 34, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, a third reagent pipettor and at least one sample pipettor.

36. The method of claim 35, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

37. The method of claim 35 or claim 36, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

38. The method of any one of claims 35 to 37, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

39. The method of any one of claims 31 to 38, wherein the method is configured to analyze at least about 200 biological samples / hr.

40. The method of any one of claims 31 to 38, wherein the method is configured to analyze at least about 300 biological samples / hr.

41. The method of any one of claims 31 to 38, wherein the method is configured to analyze at least about 400 biological samples / hr.

42. The method of any one of claims 31 to 41, wherein: the first reaction mixture is generated by aspirating a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer and aspirating a portion of a first reagent comprising the capture reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; the second reaction mixture is generated by aspirating a portion of a second reagent comprising the enzyme-conjugated affinity molecule from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel; and a detection mixture is generated by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

43. The method of claim 42, wherein 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.

44. The method of claim 42 or claim 43, wherein incubation time of the second reaction mixture is at least about 2 minutes, alternatively is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

45. The method of any one of claims 42 to 44, 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, alternatively at least 140 seconds, alternatively at least 150 seconds, alternatively at least 160 seconds, alternatively at least 170 seconds, alternatively at least 180seconds, 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.

46. The method of any one of claim 31 to 45, wherein 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.

47. The method of any one of claim 31 to 46, wherein 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.

48. The method of any one of claims 31 to 47, wherein the detector arrangement comprises a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.

49. The method of any one of claim 31 to 48, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

50. The method of claim 49, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

51. The method of any one of claims 31 to 50, wherein the first reaction mixture, second reaction mixture, and / or detection mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

52. The method of claim 51, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to performing the at least one wash action.

53. The method of any one of claims 31 to 52, wherein the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

54. The method of claim 53, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

55. The method of claim 53, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

56. The method of any one of claims 1 to 55, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

57. The method any one of claims 1 to 56, wherein the biological sample is from a subject infected with or suspected of being infected with tuberculosis (TB).

58. The method of claim 57, wherein the tuberculosis is caused by Mycobacterium tuberculosis.

59. The method of claim 57 or claim 58, wherein the method can differentiate between latent TB infection and active TB.

60. A kit for carrying out the method of any one of claims 3 to 57 comprising: at least one incubation vessel; at least one cytokine release agent; a reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assay; and the substrate formulation.

61. The kit of claim 60, further comprising instructions for use for performing the method of any one of claims 2 to 56.

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