Detecting phosphorylated TAU (p-TAU) 217 using chemiluminescent dioxetane compounds

Abstract

The presently claimed and described technology provides methods for chemiluminescence-based assays for detecting phosphorylated tau (p-tau)217 in a biological sample employing 1,2 dioxetane compounds.

Description

Attorney Docket No. P2024-0015-WO01 (67794WO01) DETECTING PHOSPHORYLATED TAU (P-TAU) 217 USING CHEMILUMINESCENT DIOXETANE COMPOUNDS RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 625,435, 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 67794WO01_seq.xml and is 2,166 bytes in size. BACKGROUND

[0003] Dementia is one of the costliest conditions to society. In 2022, the total national cost of caring for people living with Alzheimer’s Disease (AD) and other dementias was projected to reach $321 billion, and an estimated 6.5 million Americans aged 65 and older were estimated to be living with AD. Am J Manag Care. 2022;28(suppl 10):S188-S196. By 2060, the number of people 65 and older with AD is projected to reach 13.8 million, barring the development of medical breakthroughs to prevent, slow or cure AD. Alzheimer’s Association. 2022 Alzheimer’s Disease Facts and Figures. Alzheimers Dement 2022;18. Dementia and AD are costly to society because affected individuals often require long-term care and support, and available treatments aim to manage symptoms and delay progression, rather than to reverse the underlying disease process. Accordingly, AD is impacting a growing population that is currently underserved with treatment options. Novel treatments for AD are emerging and driving an increased need for testing and diagnosis.

[0004] Tau phosphorylated at threonine 217 (“p-tau217”) has been proposed as a biomarker to aid in identifying patients having, or at risk of having, AD or neurodegenerative diseases, and to select patients for disease modifying treatments. p-tau217 measurements have been shown to be capable of distinguishing between AD and non-AD patients.

[0005] Immunoassays are an important analytical tool for the identification and detection of specific substances in a sample and are routinely used to detect and quantify clinically important blood proteins. Many medical decisions are based on the diagnostic results from these assays, making sensitivity and specificity extremely important. Chemiluminescence-based immunoassays can offer a rapid response to the presence of analytes and excellent sensitivity because unlike fluorescence and absorption-based assays, no light excitation is required. A need exists for assay methods that allow for more sensitive detection of analytes and a higher signal to background ratio than prior substrates, which is sensitive and specific enough to distinguish between AD and non- AD patients, including in combination with other biomarkers or other clinical information, to aid, physicians and neurologists in diagnosing patients with AD and other dementias. BRIEF SUMMARY

[0006] One aspect of the invention is a method of detecting phosphorylated tau at threonine-217 (“ptau-217”) in a biological sample, the method comprising exposing the biological sample to a capture antibody configured to bind to at least one portion of ptau-217, 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; 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 ptau-217 in the biological sample.

[0007] In an aspect, the quantitation detection limit (LOQ) for measuring ptau-217 is equal to or less than about 12 pg / mL, alternatively equal to or less than about 4 pg / mL, alternatively equal to or less than about 3 pg / mL, wherein the detected p-tau217 has a concentration above the LOQ.

[0008] In an aspect, the detected ptau-217 has a concentration that is at least 1X greater than the LOQ, alternatively at least 2X greater than the LOQ, or alternatively at least 3X greater than the LOQ.

[0009] 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.

[0010] In an aspect, the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of ptau-217.

[0011] In an aspect, the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

[0012] In an aspect, the capture antibody and / or enzyme-conjugated affinity molecule is conjugated to at least one magnetic bead.

[0013] In an aspect, the enzyme comprises an alkaline phosphatase (AP).

[0014] In an aspect, the dioxetane compound is a compound of Formula I or a salt thereof:

[0015] Formula I

[0016] independently C3-C10 alkyl, or R1 and R2 taken together with the carbon to which they are attaches provide a C5-C10 cycloalkyl ring;

[0017] R3 is C1-C10 alkyl, C6-C10 aryl, or heteroaryl;

[0018] R4 is C2-C10 alkenyl;

[0019] R5 is H or C1-C10 alkyl; and

[0020] X is a phosphate.

[0021] In an aspect, the dioxetane compound is a compound of Formula II or a salt thereof:

[0022] Formula IIindependently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0025] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0026] R4 is C2-C10 alkenyl;

[0027] R5 is H or C1-C10 alkyl; and

[0028] X is a phosphate.

[0029] In an aspect, the dioxetane compound is a compound of Formula III or a salt thereof:

[0030] Formula III

[0031] H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0032] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0033] R5 is H or C1-C10 alkyl; and

[0034] X is a phosphate.

[0035] In an aspect, the dioxetane compound is a compound of Formula IV or a salt thereof:

[0036] Formula IV

[0037] aryl or heteroaryl;

[0038] R5 is H or C1-C10 alkyl;

[0039] and X is a phosphate.

[0040] In an aspect, the wherein the dioxetane compound is

[0041]

[0042]

[0043] In an aspect, the dioxetane compound is 4-methoxy-4-(3-phosphatephenyl)spiro[1,2- dioetane-3,2’-adamantane] or a salt thereof.

[0044] 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:

[0045] .

[0046] surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

[0047] In an aspect, the small molecule phosphonium surfactant is a compound having the formula:

[0048]

[0049] are each independently C1-C10 alkyl;

[0050] R15 is arylalkyl; and

[0051] X- is a counterion.

[0052] In an aspect, the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

[0053]

[0054]

[0055] In an aspect, the substrate formulation further comprises a magnesium (II) salt.

[0056] 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.

[0057] In an aspect, the reagent vessels comprise an elastomeric self-sealing membrane.

[0058] In an aspect, the reagent pack further comprises containment walls arranged between the reagent vessels.

[0059] In an aspect, the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0060] 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.

[0061] In an aspect, the pipettor arrangement further comprises at least a fourth reagent pipettor.

[0062] In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

[0063] 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.

[0064] 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.

[0065] 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 by aspirating the substrate formulation and dispensing the aspirated substrate formation into the reaction vessel.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] In an aspect, the immunoassay analyzer further comprises an ultrasonic mixing module.

[0073] In an aspect, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0074] 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,

[0075] In an aspect, 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.

[0076] 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.

[0077] 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.

[0078] In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

[0079]

[0080] 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.

[0081] In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

[0082] In an aspect, the method further comprises detecting at least one additional biomarker is selected from the group consisting of Aβ peptides, Neurofilament light chain (NfL), total tau (t- tau), p-tau231, p-tau181, and combinations thereof.

[0083] 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

[0084] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0085] FIG. 1 is a calibration curve plot constructed for a p-tau217 assay using calibrators and a commercially available substrate and an exemplary substrate according to an aspect of this disclosure.

[0086] FIG. 2 is a plot of the signal generated by performing the assay with the a commercially available substrate on the x-axis as compared to the signal generated by the an exemplary substrate according to an aspect of this disclosuron the y-axis for calibrators (minus background signal) and patient samples. DETAILED DESCRIPTION I. Introduction

[0087] Disclosed herein are immunoassay methods, reagents, kits, and compounds for detecting ptau-217 in a biological sample using chemiluminescent dioxetanes.

[0088] Tau is a heterogeneous neuron-specific, axon-enriched, microtubule-associated protein, found to be dysregulated in AD and other tauopathies. Six isoforms of tau protein are generated from the TAU gene with alternative splicing. The isoforms have different amino-terminal inserts and different numbers of tandem repeats near the carboxy terminus, ranging in length from 352 to 441 amino acids. Hyperphosphorylation of tau is thought to be a hallmark feature of AD, and phosphorylation of tau at threonine 217 (pTau 217) is of particular interest to those skilled in the art as an early marker of Alzheimer’s development.

[0089] The amino acid sequence of the 2N4R isoform of tau, tau-441, is listed in Table 1. pTau- 217 can refer to a form of tau-441 where residue 217 is phosphorylated, but also any other tau isoforms where residue 217 is phosphorylated. pTau217 may refer to a full-length tau proteinphosphorylated at residue 217, a variant of a tau protein phosphorylated at residue 217, a fragment of a tau protein phosphorylated at residue 217, and post-translationally modified forms of tau protein phosphorylated at residue 217.

[0090] Table 1 SEQ ID NO Amino Acid Sequence 1 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQ EGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPTA EDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGD TPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKT KIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEP PKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVA VVRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTE NLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPG GGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEK LDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTD HGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADE VSASLAKQGL

[0091] AD is a progressive neurodegenerative disorder that affects millions of individuals. Individuals with AD have amyloid accumulation in the brain which damages the synaptic function and forms neurofibrillary tangles. While total tau is useful as a biomarker for neurodegeneration, phosphorylated tau has been proposed as providing a more accurate identification of AD due to the presence of hyperphosphorylated tau in neurofibrillary tangles. There are several variants of phosphorylated tau, many of which can be detected in cerebrospinal fluid (CSF). However, the removal of CSF requires a lumbar puncture, which may be painful and presents several risks for complications. Phosphorylated tau at Thr217 (p-tau217) is a blood-based biomarker. The bolded Thr in Table 1 is the phosphorylation site in p-tau217. Plasma levels of p-tau217 have been found to be clinically significant in an early, specific and accurate diagnosis of AD, including the manifestation and progression of AD.

[0092] In some instances, methods disclosed herein comprise: exposing the biological sample to a capture antibody configured to bind to at least one portion of ptau-217, 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 formulationcomprising a 1,2 dioxetane compound and at least one phosphonium surfactant; 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 ptau-217 in the biological sample.

[0093] 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.

[0094] In some instances, methods disclosed herein can measure lower levels of p-tau217 in plasma, 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.

[0095] In some instances, the signal produced by assay methods disclosed herein is up to 35 times greater than immunoassay methods where conventional or known substrate formulations are used. In some instances, 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

[0096] 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 methodsdescribed 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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 joinedby "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".

[0103] 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.

[0104] 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.

[0105] 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, the term "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 dependingupon 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] The term "immunoassay" may be a laboratory method that uses one or more antibodies or antigens to determine the amount of an analyte in a sample. It can be based on the interaction 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.

[0111] 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

[0112] In an embodiment, the method includes detecting the presence of an p-tau217 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 a 1,2 dioxetane compound and at least one phosphonium surfactant. In an aspect of the methods described herein, the reaction between the enzyme-conjugated affinity molecule and the substrate formulation 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

[0111] 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.

[0112] 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 surroundedby 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.

[0113] 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 1,2 dioxetane compound and at least one phosphonium surfactant. 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.

[0114] 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, alarger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0115] 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.

[0116] In an embodiment, the 1,2 dioxetane compound is a compound of Formula I or a salt thereof:

[0117] Formula I

[0118] 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;

[0119] R3 is C1-C10 alkyl, C6-C10 aryl, or heteroaryl;

[0120] R4 is C2-C10 alkenyl;

[0121] R5 is H or C1-C10 alkyl; and

[0122] X is a phosphate.

[0123] In an embodiment, the 1,2 dioxetane compound is a compound of Formula II or a salt thereof: II

[0125] wherein each of R10 and R11 is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0126] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl;

[0127] R4 is C2-C10 alkenyl;

[0128] R5 is H or C1-C10 alkyl; and

[0129] X is a phosphate.

[0130] In an embodiment, the 1,2 dioxetane compound is a compound of Formula III or a salt thereof:H, halogen, C1-C10 alkyl, C2-C10 alkenyl, or C6-C10 aryl;

[0133] R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0134] R5 is H or C1-C10 alkyl; and

[0135] X is a phosphate.

[0136] In an embodiment, the 1,2 dioxetane compound is a compound of Formula IV or a salt thereof:

[0137] Formula IV

[0138] wherein R3 is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and

[0139] R5 is H or C1-C10 alkyl.

[0140] In an embodiment, the 1,2 the dioxetane compound is

[0141]

[0142]

[0143] In an embodiment, the 1,2 dioxetane compound is 4-methoxy-4-(3- phosphatephenyl)spiro[1,2-dioetane-3,2’-adamantane] or a salt thereof.

[0144] 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.

[0145] In an embodiment, the small molecule phosphonium surfactant is a compound having the formula:

[0146]

[0147] wherein R12-R14 are each independently C1-C10 alkyl;

[0148] R15 is arylalkyl; and

[0149] X- is a counterion.

[0150] In an embodiment, the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:

[0153] In certain embodiments, the substrate formulation further comprises a magnesium (II) salt.

[0154] 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. The kit may also include instructions for performing any one of the disclosed methods. In some aspects, the kit may include the substrate formulation, calibrators, and / or wash buffers. b. Analyzer Systems

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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 unitholds about 140 sample vessels. In a specific embodiment, the sample presentation unit has 20 sample racks with each rack holding seven sample vessels.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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, the pipettor 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.

[0163] 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.

[0164] 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 biologicalsamples, 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.

[0165] 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 first capture 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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).

[0172] 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.

[0173] 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, such as 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0178] 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.

[0179] 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 washstation 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 separate bound 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.

[0180] 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 someof 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).

[0181] 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, 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.

[0182] 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.

[0183] 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. Thereaction 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.

[0184] 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.

[0185] 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.

[0186] 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 vesselmay 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.

[0187] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions.

[0188] 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 the configurations 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.

[0189] 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 reliableresult. An exemplary machine vision apparatus is described in U.S. Patent No. 11,263,433, which is incorporated by reference herein.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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 ofvessel image capture units. As described herein, the vessel tip image capture unit includes a camera unit.

[0195] 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.

[0196] 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 coupled to 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.

[0197] 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).

[0198] 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.

[0199] 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.

[0200] 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 the immunoassay 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.

[0201] 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 about25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0202] 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 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 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

[0203] In an aspect of the methods disclosed herein, p-tau217 is detected in a biological sample. In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid. In an aspect, the method includes exposing the biological sample to a capture antibody configured to bind to at least one portion of p-tau217, generating a first reaction mixture.

[0204] The “first reaction mixture” is a result of an incubation between 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.

[0205] 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” is a 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.

[0155] In an embodiment, the method exposing the second reaction mixture to a substrate formulation comprising a 1,2 dioxetane compound and 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 atleast 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0206] 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. Whilewashing 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.

[0207] 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.

[0208] 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, 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.

[0209] In an aspect, the disclosed methods include the detection of p-tau217 using an immunoassay. Depending on the analysis desired, different enzyme-conjugated affinity molecules may be used in the p-tau217 assay. For example, the affinity molecule may be an antibody, wherein the antibody is configured to bind at least one portion of p-tau217. 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, p-tau217 commercial assays were employed per manufacturers’ protocol. A non-limiting assay protocol according to an aspect of the invention is listed in Table 2.

[0210] In some aspects of the method, the quantitation detection limit (LOQ) for measuring ptau- 217 is equal to or less than about 12 pg / mL, alternatively equal to or less than about 4 pg / mL, alternatively equal to or less than about 3 pg / mL, wherein the detected p-tau217 has a concentration above the LOQ.

[0211] In some aspects, the detected ptau-217 has a concentration that is at least 1X greater than the LOQ, alternatively at least 2X greater than the LOQ, or alternatively at least 3X greater than the LOQ.

[0212] In some cases, for patients having AD, or at risk of progressing to AD, the levels of p- tau217 in blood may be in the AttoMole, Milli-international Unit (mIU), pg, or fg range. In some aspects, the p-tau217 immunoassay is used to identify patients having AD, or at risk of progressingto AD; detect abnormal amyloid and tau pathology; monitor the treatment responses to clinical and / or pharmaceutical interventions; and / or monitor the progression of AD. 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 p- tau217.

[0213] 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 permanent or 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.

[0214] 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.

[0215] Additional examples are provided below.

[0216] EXAMPLES

[0217] Example 1: ptau-217 Enhanced Signal Generation

[0218] A series of ptau-217 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 a alkaline phosphatase chemiluminescent substrate comprising the structure (Substrate 2). The calibrator levels were 4 pg / mL (S1), 12 pg / mL108 pg / mL (S4). A control sample (0 pg / mL, S0) was also analyzed. Each calibrator and control were run in duplicates.

[0219] The ptau-217 sensitivity comparison 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.

[0220] Paramagnetic particle conjugated with ptau-217 antibodies capable of binding to an epitope ptau-217 were pipetted into a reaction vessel using one of four reagent pipettors along with a HEPES 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 ptau-217 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 one more time for Substrate 1 and twice for Substrate 2. An exemplary assay protocol is listed in Table 2.

[0221] Table 2Step Action A) pH 7 HEPES buffer B) Paramagnetic PMPs w / ptau-217 mAB 1 C) Sample (200µL) 2 Incubate 240 minute 3 Wash reaction Anti‐N‐terminal tau-ALP 4 conjugate 5 Incubate 8 minutes 6 Wash, Add Substrate and read

[0222] Test results were determined automatically by the system software. Detection of analyte in the sample was determined from the measured light production. Signal generated by the assay was measured in relative light units (RLUs). The results for each calibrator level is plotted in FIG. 1

[0223] Table 3 and FIG. 1 detail the signal generated by performing the assay with the Substrate 2 versus Substrate 1 for each calibrator level.

[0224] Table 3: Signal Produced by pTau217 Immunoassay (Calibrator Levels) Calibrator Substrate 2 Substrate 1 Dose RLU RLU (pg / mL) S0 0 13,059 307,478 S0 0 13,158 294,866 S1 4 19,336 486,835 S1 4 20,401 462,733 S2 12 35,008 970,762 S2 12 33,369 962,370 S3 36 91,181 2,513,430 S3 36 88,910 2,422,584 S4 108 190,779 5,696,974 S4 108 204,152 5,519,456

[0225] Table 3 details the signal generated by performing the assay with the Substrate 2 versus Substrate 1 on each calibrator level, with the mean background level for each substrate subtracted.

[0226] Table 4: Signal Produced by pTau217 Immunoassay (Calibrator Levels, minus background signal) Calibrator Substrate 2 Substrate 1 Dose (pg / mL) RLU RLU S00-50 6,306 S0050 -6,306 S146,228 185,663 S147,293 161,561 S21221,900 669,590 S21220,261 661,198 S33678,073 2,212,258 S33675,802 2,121,412 S4108177,671 5,395,802 S4108191,044 5,218,284

[0227] From the results shown in Tables 3 and 4 and FIG. 1, Substrate 1 was found to generate higher signal at all calibrator concentrations as compared to Substrate 2.

[0228] Example 2: ptau-217 Enhanced Signal Generation for Patient Samples

[0229] The ptau-217 sensitivity comparison on patient samples 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.

[0230] Paramagnetic particle conjugated with ptau-217 antibodies capable of binding to an epitope ptau-217 were pipetted into a reaction vessel using one of four reagent pipettors along with a HEPES 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 ptau-217 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 bufferto 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 one more time for Substrate 1 and twice for Substrate 2. The results were compared to the control samples as shown in Table 5.

[0231] Table 5: Signal Produced by pTau217 Immunoassay (Patient Samples) Sample ID Substrate 1 Substrate 2 (RLU) (RLU) 82 1,581,072 53,473 82 1,566,428 54,232 104 341,277 14,859 104 360,864 14,425 607 1,809,770 57,685 607 1,983,956 61,786 NormPat1 401,117 15,925 NormPat1 357,812 14,793 QC3 1,334,161 48,657 QC3 1,407,907 49,673

[0232] The results were plotted in FIG. 2 which is a plot of the signal generated by performing the assay with Substrate 2 on the x-axis as compared to the signal generated by Substrate 1 on the y-axis for both the calibrators and the patient samples.

[0233] From the results shown in Table 5 and FIG. 2, Substrate 1 was found to generate higher signal with all samples as compared to Substrate 2.

[0234] 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.

[0235] 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 phosphorylated tau at threonine-217 (“ptau-217”) in a biological sample, the method comprising: exposing the biological sample to a capture antibody configured to bind to at least one portion of ptau-217, 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; 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 ptau- 217 in the biological sample.

2. The method of claim 1, wherein the quantitation detection limit (LOQ) for measuring ptau-217 is equal to or less than about 12 pg / mL, alternatively equal to or less than about 4 pg / mL, alternatively equal to or less than about 3 pg / mL, wherein the detected p-tau217 has a concentration above the LOQ.

3. The method of claim 2, wherein the detected ptau-217 has a concentration that is at least 1X greater than the LOQ, alternatively at least 2X greater than the LOQ, or alternatively at least 3X greater than the LOQ.

4. The method of any one of claims 1 to 3, 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.

5. The method of claim 4, wherein the affinity molecule is an antibody, wherein the antibody is configured to bind at least one portion of ptau-217.

6. The method of claim 4, wherein the affinity molecule is an antigen, wherein the antigen is configured to bind at least one portion of the capture antibody.

7. The method of any one of claims 1 to 6, wherein the capture antibody and / or enzyme- conjugated affinity molecule is conjugated to at least one magnetic bead.

8. The method of any one of claims 1 to 7, wherein the enzyme comprises an alkaline phosphatase (AP).

9. The method of any one of claims 1 to 8, wherein the dioxetane compound is a compound of Formula I or a salt thereof: Formula Iwherein each of R1and R2are independently C3-C10alkyl, or R1and R2taken together with the carbon to which they are attaches provide a C5-C10cycloalkyl ring; R3is C1-C10 alkyl, C6-C10 aryl, or heteroaryl; R4is C2-C10alkenyl; R5is H or C1-C10alkyl; andX is a phosphate.

10. The method of any one of claims 1 to 9, wherein the dioxetane compound is a compound of Formula II or a salt thereof: Formula II wherein eachC1-C10 alkyl, C2-C10 alkenyl, or C6-C10aryl; R3is C1-C10alkyl, C6-C10aryl or heteroaryl; R4is C2-C10 alkenyl; R5is H or C1-C10 alkyl; and X is a phosphate.

11. The method of any one of claims 1 to 10, wherein the dioxetane compound is a compound of Formula III or a salt thereof: Formula III wherein eachC1-C10alkyl, C2-C10alkenyl, or C6-C10 aryl; R3is C1-C10 alkyl, C6-C10 aryl or heteroaryl; and R5is H or C1-C10alkyl; and X is a phosphate.

12. The method of any one of claims 1 to 11, wherein the dioxetane compound is a compound of Formula IV or a salt thereof: Formula IV wherein R3R5is H or C1-C10 alkyl; and X is a phosphate.

13. The method of any one of claims 1 to 12, wherein the wherein the dioxetane compound is14. The method of any one of claims 1 to 13, wherein the dioxetane compound is 4-methoxy- 4-(3-phosphatephenyl)spiro[1,2-dioetane-3,2’-adamantane] or a salt thereof.

15. The method of any one of claims 9 to 14, 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:.

16. The method of any one of claims 1 to 15, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

17. The method of claim 16, wherein the small molecule phosphonium surfactant is a compound having the formula: Wherein R12-R14are eachR15is arylalkyl; and X- is a counterion.

18. The method of claim 16, wherein the polymeric phosphonoium surfactant comprises repeating unit (A), repeating unit (B), or both:wherein Bu3 19. The method of any one of claims 1 to 18, wherein the substrate formulation further comprises a magnesium (II) salt.

20. The method of any one of claims 1 to 19, 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.

21. The method of claim 20, wherein the reagent vessels comprise an elastomeric self-sealing membrane.

22. The method of claim 20 or claim 21, wherein the reagent pack further comprises containment walls arranged between the reagent vessels.

23. The method of any one of claims 20 to 22, wherein the immunoassay analyzer further comprises a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

24. The method of any one of claims 20 to 23, 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.

25. The method of claim 24, wherein the pipettor arrangement further comprises at least a fourth reagent pipettor.

26. The method of claim 24 or claim 25, wherein the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated.

27. The method of any one of claims 24 to 26, 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.

28. The method of any one of claims 20 to 27, wherein the method is configured to analyze at least about 200 biological samples / hr.

29. The method of any one of claims 20 to 27, wherein the method is configured to analyze at least about 300 biological samples / hr.

30. The method of any one of claims 20 to 27, wherein the method is configured to analyze at least about 400 biological samples / hr.

31. The method of any one of claims 20 to 30, 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.

32. The method of claim 31, 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.

33. The method of claim 31 or claim 32, 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.

34. The method of any one of claims 31 to 33, 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.

35. The method of any one of claim 20 to 34, 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.

36. The method of any one of claim 20 to 35, 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.

37. The method of any one of claims 20 to 36, 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.

38. The method of any one of claim 20 to 37, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

39. The method of claim 38, wherein the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

40. The method of any one of claims 20 to 39, 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.

41. The method of claim 40, 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.

42. The method of any one of claims 20 to 41, 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.

43. The method of claim 42, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

44. The method of claim 42, 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.

45. The method of any one of claim 1 to 44, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

46. The method of any one of claims 1 to 45, wherein the method further comprises detecting at least one additional biomarker is selected from the group consisting of Aβ peptides, Neurofilament light chain (NfL), total tau (t-tau), p-tau231, p-tau181, and combinations thereof.

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