An automated and high-throughput analyzer method for a brain derived TAU (BD-TAU) immunoassay

Abstract

The presently described and claimed technology relates to high-throughput automated methods for detecting brain derived tau (BD-tau) in a biological sample using an immunoassay analyzer.

Description

Attorney Docket No. 773988: DABX-945PCTAN AUTOMATED AND HIGH-THROUGHPUT ANALYZER METHOD FOR A BRAIN DERIVED TAU (BD-TAU) IMMUNOASSAYRELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U. S. Provisional Patent Application Ser. No. 63 / 763,425, filed February 26, 2025, U. S. Provisional Patent Application Ser. No. 63 / 847,788, filed July 21, 2025, U. S. Provisional Patent Application Ser. No.63 / 878,509, filed September 9, 2025, U. S. Provisional Patent Application Ser. No. 63 / 894,909, filed October 7, 2025, the content of each is hereby incorporated by reference in its entirety into this disclosure.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on July 18, 2025, is named DACT-945.xml and is 9,913 bytes in size.BACKGROUND

[0003] Dementia is one of the costliest conditions for 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): S 188-S 196. 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] Assays 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. Automated analyzers are commonly used in clinical chemistry, immunoassay, hematology, and other biological sampling and analyzingAttorney Docket No. 773988: DABX-945PCTassays and can efficiently perform clinical analysis on a large number of samples, with multiple tests being run concurrently or within short time intervals.

[0005] Neuro-biomarkers, such as the tau protein, have 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.BRIEF SUMMARY

[0006] One aspect of the disclosure includes a method for quantitatively determining an amount of brain-derived tau (BD-tau) in at least one biological sample using a high-throughput immunoassay analyzer, the method comprising: aspirating a portion of the at least one biological sample from a sample vessel and dispensing the aspirated biological sample into a first reaction vessel of the immunoassay analyzer, wherein the immunoassay analyzer comprises: a pipettor arrangement comprising at least a first reagent pipettor and a second reagent pipettor; and a detector arrangement; detecting, using the detector arrangement, a presence of a reaction in the first reaction vessel; and quantitatively determining an amount of BD-tau in the biological sample based on the presence of the reaction; and wherein the second reagent pipettor is configured to dispense reagents into a second reaction vessel simultaneously as the first reagent pipettor dispenses fluid into the first reaction vessel.

[0007] In an aspect, the method further includes aspirating, using the first reagent pipettor, a portion of a fluidic substance from a first reagent vessel and dispensing said fluidic substance into the first reaction vessel, wherein the fluidic substance from the first reagent vessel comprises at least one capture antibody capable of binding to at least one portion BD-tau; aspirating, using the first reagent pipettor, a portion of a fluidic substance from a second reagent vessel and dispensing said fluidic substance into the first reaction vessel, wherein the fluidic substance from the second reagent vessel comprises at least one detector affinity molecule specific to BD-tau; and dispensing a substrate into the first reaction vessel.

[0008] In an aspect, the first reagent vessel and second reagent vessel are housed in a reagent pack. In an aspect, the first reagent vessel and second reagent vessel comprise an elastomeric self-sealing membrane. In an aspect, each reagent vessel is configured to store a volume of reagent required for at least about 20 instances of a BD-tau assay.

[0009] In an aspect, the method is configured to analyze at least about 200 biological samples / hr.; alternatively the method is configured to analyze at least about 300 biological samples / hr.; alternatively the method is configured to analyze at least about 400 biological samples / hr.; alternatively the method is configured to analyze at least about 450 biologicalAttorney Docket No. 773988: DABX-945PCTsamples / hr.; or alternatively the method is configured to analyze at least about 500 biological samples / hr.

[0010] 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.

[0011] 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.

[0012] In an aspect, the reaction in the first reaction vessel generates a chemiluminescent signal, wherein the chemiluminescent signal quantitatively corresponds to a concentration of BD-tau in the plasma sample.

[0013] In an aspect, the reaction in the first reaction vessel generates a chemiluminescent signal when a substrate formulation is added to the reaction vessel, wherein the substrate formulation comprises:a chemiluminescent compound of the formula A or a salt thereof:whereinA is Ci -ehaloalky 1, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NCfr substituents;Ri is selected from the group consisting of Cs-uaryl, Ci-6 alkyl, Ci-6 haloalkyl, and C5-14 aralkyl groups;Attorney Docket No. 773988: DABX-945PCTR7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or R11-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;R15 is C1-6 alkyl;each M is independently selected from the group consisting of H, an alkali metal, alkaline earth metal, transition metal, ammonium, phosphonium, organic amine salt, and an amino acid salt; Z is O or S; andn is 0, 1, or 2;a cationic aromatic compound (CAC);a background reducing agent; andan ether-linked nonionic surfactant or a hydrophilic polymer.

[0014] 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.

[0015] In an aspect, the immunoassay analyzer further comprises a third reagent pipettor, and wherein the second reagent pipettor and third reagent pipettor are configured to dispense reagents into the second reaction vessel and a third reaction vessel simultaneously.

[0016] In an aspect, the fluidic substances dispensed into the first reaction vessel, second reaction vessel, and / or third reaction vessel are agitated via an ultrasonic mixing module.

[0017] 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 functionalities and / or assay functionalities of the immunoassay analyzer.

[0018] In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

[0019] 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.

[0020] In an aspect, 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, alternativelyAttorney Docket No. 773988: DABX-945PCTconfigured to perform at least four wash actions, or alternatively configured to perform at least five wash actions.

[0021] One aspect of the disclosure includes a method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising aspirating, using a sample pipettor, a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer; wherein the immunoassay analyzer comprises: a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor; and a detector arrangement; wherein the sample pipettor is configured to aspirate and / or dispense less than about 10 pL, alternatively about 2.0 pL to about 9.9 pL; detecting, using the detector arrangement, a presence of a reaction in a reaction vessel; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the reaction vessel.

[0022] One aspect of the disclosure includes a method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising: aspirating, using a sample pipettor, a portion of the biological sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of the immunoassay analyzer; wherein the immunoassay analyzer comprises: a reagent pack comprising a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about 20 instances of a BD-tau assay; a pipettor arrangement comprising at least one reagent pipettor and at a sample pipettor; and a detector arrangement; aspirating, using the reagent pipettor, a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into a reaction vessel of the immunoassay analyzer, generating a first reaction mixture; aspirating, using the reagent pipettor, a portion of a second reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a second reaction mixture; detecting, using the detector arrangement, a reaction in the reaction vessel; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the reaction vessel.

[0023] One aspect of the disclosure includes a method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising: mixing the biological sample with a reagent comprising at least one affinity molecule that binds to at least one portion BD-tau generating a first reaction mixture; mixing the first reaction mixture with a reagent comprising a detection molecule, generating a detection mixture; wherein the immunoassay analyzer comprises an ultrasonic mixing module, and the first reaction mixture and / or the detection mixture are agitated via the ultrasonic mixing module; detecting, using a detectorAttorney Docket No. 773988: DABX-945PCTarrangement, a reaction in the detection mixture; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the detection mixture.

[0024] In an aspect, the BD-tau is BD-pTau217. In an aspect, the at least one capture antibody capable of binding to at least one portion of BD-tau or the at least one affinity molecule that binds to at least one portion BD-tau is an anti-pTau217 antibody. In an aspect, the anti-pTau217 antibody is coupled to at least one paramagnetic particle. In an aspect, the at least one detector affinity molecule specific to BD-tau or detection molecule is an anti-Tau Exon 4 / 5 junction antibody conjugated to alkaline phosphatase.

[0025] In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

[0026] 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 DRAWINGS

[0027] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0028] FIG. 1 is a graph depicting an assay of the present invention’s performance in discriminating between amyloid (“AM”) negative healthy individuals “Normal” (n=12 on Dxl 9000, n=9 on Access 2) or patients diagnosed with FTD “FTD” (n=6) versus AM positive patients diagnosed with Alzheimer’s Disease “AD” (n=l 7), in K2 EDTA plasma.

[0029] FIGs 2A — 2D are graphs depicting Plasma p-Tau217 levels (FIG. 2A), BD p-Tau217 levels (FIG.2C), and their respective Aβ42 ratios (FIGs.2B and 2D) for individuals classified by AD neuropathological changes (ADNC) using assays of the present invention.

[0030] FIGs. 3A and 3B are graphs depicting receiver operating characteristic (ROC) curves of plasma biomarkers for ADNC classification analyzed using assays of the present invention. Letters in parentheses indicate significant differences (DeLong, Holm-adjusted p<0.5). Significantly different from a, P-tau217; b, P-tau217 / Ap42; c, BD P-tau217; d, BD P-tau217 / Ap42

[0031] FIGs 4A-4C are graphs depicting mean fold-changes in plasma biomarker levels across ADNC groups analyzed using assays of the present invention.Attorney Docket No. 773988: DABX-945PCT

[0032] FIGs. 5A and 5B are schematics illustrating one non-limiting embodiment of a BD-pTau217 assay.

[0033] FIG. 6 are plots illustrating the comparison of a BD-pTau217 assay to an p-Tau217 assay.

[0034] FIGs. 7A-7B are box plots (FIG. 7A) and Receiver Operating Characteristic (ROC) analysis (FIG. 7B) illustrating amyloid pathology differentiation.

[0035] FIG. 8 is a plot illustrating the detection capability of the BD-pTau217 assay.

[0036] FIG. 9 is a plot illustrating the linearity of the BD-pTau217 assay.DETAILED DESCRIPTIONI. Introduction

[0037] Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that affects millions of individuals. Without being limited by any particular theory, individuals with AD are thought to have amyloid accumulation in the brain which damages the synaptic function and forms neurofibrillary tangles. Tau protein abnormalities, such as the hyperphosphorylation of tau, are thought to be a hallmark feature of AD.

[0038] Tau is a heterogeneous neuron-specific, axon- enriched, microtubule-associated protein, transcribed by the microtubule-associated protein tau (MAPT) gene. Alternative splicing of the microtubule-associated protein tau (MAPT) gene results in the expression of numerous isoforms of Tau, of which six main isoforms are thought to exist in the human brain. These isoforms - also referred to as brain-derived tau - range in length from 352 to 441 amino acids. Sequences of and common designators for each isoform are listed in Table 1.

[0039] The peripheral nervous system (PNS)-tau isoform (also referred to as “Big tau” or “peripheral tau”) is an isoform of tau preferentially expressed in peripheral tissues. Without being limited by any theory, Big tau is distinguishable from brain-derived isoforms of tau by the presence of a large peptide insert resulting from the transcription of an extra exon (exon 4a) of the MAPT gene. The amino acid sequence of Big Tau is listed in Table 1 where the amino acid sequence encoded by the 4a exon is underlined. The exon 4a insert breaks the junction between the domains encoded by exons 4 and 5, making it unique to Big Tau, as the domains expressed by exons 4 and 5 are continuous in the six brain derived tau isoforms. The glutamine (Q) and alanine (A) bolded refers to the connective portion between the amino acidAttorney Docket No. 773988: DABX-945PCTsequence encoded by exon 4 and the amino acid sequence encoded by exon 5. This may also be referred to as the “exon 4-5 connective portion.”

[0040] The longest brain-derived tau isoform, 2N4R (441 amino acids in length) contains 2 amino-terminal inserts (2N) and 4 microtubule-binding repeats (4R). The two amino-terminal inserts are encoded by two alternatively spliced exons, E2 and E3, and encode 29 amino acids each. The other brain-derived tau isoforms arise from alternative splicing of exon 2, 3 and 10. These isoforms may differ in either 0, 1 or 2 inserts of the 29 amino acid amino-terminal part and three or four microtubule-binding repeats. Brain-derived tau isoforms differ from the PNS-tau isoform in that they lack the 4a exon. For example, as compared to 2N4R, the PNS-tau isoform includes an insertion between amino acids 124 of 2N4R and 125 of 2N4R. Nonlimiting examples of other brain-derived tau isoforms include, the 0N3R isoform (352 amino acids in length), the 0N4R isoform (383 amino acids in length), the 1N3R isoform (383 amino acids in length), the 1N4R (412 amino acids in length), and the 2N3R isoform (410 amino acids in length).

[0041] Table 1: Exemplary Tau IsoformsSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPT AEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAG IGDTPSLEDEAAGHVTQEPESGKVVOEGFLREPGPPG LSHQLMSGMPGAPLLPEGPREATRQPSGTGPEDTEG GRHAPELLKHOLLGDLHOEGPPLKGAGGKERPGSKE EVDEDRDVDESSPODSPPSKASPAQDGRPPOTAARE ATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDGPSVGR AKGODAPLEFTFHVEITPNVOKEQAHSEEHLGRAAF PGAPGEGPEARGPSLGEDTKEADLPEPSEKOPAAAPR1 GKPVSRVPOLKARMVSKSKDGTGSDDKKAKTSTRS PNS-tau SAKTLKNRPCLSPKHPTPGSSDPLIQPSSPAVCPEPPSS PKYVSSVTSRTGSSGAKEMKLKGADGKTKIATPRGA APPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDR SGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAWRTP PKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQ PGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKS EKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTF RENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLAttorney Docket No. 773988: DABX-945PCTSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPT AEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAG IGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKA KGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPA PKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLP2 TPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDL tau-441 (2N4R)KNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQS KCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSL GNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNIT HVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSP VVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVS ASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQ KGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSS PGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPPKSPS3 tau-352 (0N3R)SAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGG KVQIVYKPVDLSKVTSKCGSLGNIFIFIKPGGGQVEVK SEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLT FRENAKAKTDHGAEIVYKSPWSGDTSPRHLSNVSS TGSIDMVDSPQLATLADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPT AEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGT GSDDKKAKGADGKTKIATPRGAAPPGQKGQANATR IPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGS4 RSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTA tau-381 (1N3R)PVPMPDLKNVKSKIGSTENLKHQPGGGKVQIVYKPV DLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDR VQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKT DHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSP QLATLADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPT AEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAG IGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKA KGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPA PKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLP5 tau-410 (2N3R)TPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMPDL KNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSKVTS KCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGS LDNITFIVPGGGNKKIETFIKLTFRENAKAKTDFIGAEI VYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLA DEVS ASLAKQGLAttorney Docket No. 773988: DABX-945PCTSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKAEEAGIGDTPSLEDEAAGIIVTQARM VSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQ KGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSS PGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPPKSPS6 SAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGG tau-383 (0N4R)KVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIV YKPVDLSKVTSKCGSLGNIIIIIKPGGGQVEVKSEKLD FKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENA KAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSID MVDSPQLATLADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD QEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTPT AEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGT GSDDKKAKGADGKTKIATPRGAAPPGQKGQANATR IPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGS RSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTA7 tau-412 (1N4R)PVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKL DLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKV TSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKI GSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGA EIVYKSPVVSGDTSPRIILSNVSSTGSIDMVDSPQLAT LADEVSASLAKQGL

[0042] Tau is thought to hold at least 80 potential serine, threonine, or tyrosine phosphorylation sites. If a tau isoform does not include phosphorylation, it may be referred to as nonphosphorylated tau or non-p-tau. Phosphorylated tau or p-tau may also refer to a full-length tau protein phosphorylated at one or more amino acid residues, a variant of a tau protein phosphorylated at one or more amino acid residues, a fragment of a tau protein phosphorylated at one or more amino acid residues, and post-translationally modified forms of tau protein phosphorylated at one or more amino acid residues. In some embodiments, a p-tau includes 1, 2, 3, 4, 5, or more phosphorylated serine, threonine, and / or tyrosine residues.

[0043] Several diagnostic tools are being developed to detect tau. Cerebrospinal fluid tau (total and phosphorylated) has been extensively studied as a biomarker for identifying patients with Alzheimer’s disease as increased levels of tau isoforms and p-tau in the CSF are associated with the development of Alzheimer’s disease. It has been observed that CSF total tau immunoassays, but not plasma total tau immunoassays, agree with PET and other neuropathological evidence of Alzheimer’s disease. Further it has been hypothesized that aAttorney Docket No. 773988: DABX-945PCTsignificant portion of tau in plasma originates from peripheral sources, and that immunoassays capable of selectively measuring brain derived isoforms of tau in blood could produce improved performance in identifying patients with Alzheimer’s disease.

[0044] A need exists for an objective, standardized immunoassay method that employs an automated analyzer method that can screen a biological sample for BD-tau. The present invention fulfills a need in the art by providing methods for detecting BD-tau in a biological sample using an immunoassay analyzer.II. Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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' isAttorney Docket No. 773988: DABX-945PCTmeant 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.

[0051] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) }. In other words, "x, y and / or z" means "one or more of x, y and z".

[0052] 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.

[0053] 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.

[0054] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood asAttorney Docket No. 773988: DABX-945PCTbeing 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 depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 hereinAttorney Docket No. 773988: DABX-945PCTincludes 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).

[0059] 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. In some instances, methods disclosed herein are performed on an automated analyzer, such as a clinical chemistry analyzer or an immunoassay analyzer.

[0060] A “clinical chemistry analyzer” includes an automated analyzer that utilizes spectrometry, chromatography, photometry, or potentiometry principles to measure or calculate the concentration of certain substances. Some clinical chemistry analyzers apply the photoelectric colorimetric principle and employ at least one chemical reaction to measure or calculate the concentration of certain substances. Clinical chemistry analyzers can be used to evaluate a variety of samples, including, but not limited to, samples of serum, plasma, urine and / or other body fluids. Substances analyzed through these instruments include, for example, certain metabolites, electrolytes, proteins, and / or drugs. Various clinical chemistry analyzers are commercially available including the DxC® systems (Beckman Coulter, CA) and the AU® systems (Beckman Coulter, CA). In some aspects, the clinical chemistry analyzer is a high-throughput clinical chemistry analyzer.

[0061] In some aspects, the clinical chemistry analyzer may be used with an assay, such as an immunochemical assay. As used herein, the term “immunochemical assay” may be a laboratory method that utilizes chemical reactions between enzyme-conjugated antibodies or antigens to determine the amount of an analyte in a sample. In some aspects, the immunochemical assay is a colorimetric immunochemical assay, wherein a reaction between a chemical entity and an enzyme-conjugated antibody or antigen results in a detectable color change.

[0062] An “immunoassay analyzer” includes an automated analyzer that primarily relies on immune-based techniques (e.g. chemiluminescent or fluorescent immunoassays) to detect presence or concentration of an analyte in a solution. Immunoassay analyzers can be used toAttorney Docket No. 773988: DABX-945PCTevaluate a variety of samples, including, but not limited to, samples of serum, plasma, urine and / or other body fluids. Substances analyzed through these instruments include, for example, protein biomarkers of reproductive health, cancer, cardiac health, and blood viruses. An "immunoassay analyzer" can include an instrument on which immunoassays have been automated. Various immunoassay analyzer are commercially available including the Dxl® systems (Beckman Coulter, CA). In some aspects, the immunoassay analyzer is a high-throughput immunoassay analyzer.

[0063] As used herein, 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. In some aspects, the immunoassay is a luminescent immunoassay, wherein a reaction between a substrate and an enzyme- conjugated antibody or antigen results in a detectable luminescence. In some aspects, the luminescent immunoassay is a chemiluminescent assay.

[0064] Both the immunochemical assays and immunoassays may be “two-site” or “sandwich” assays which employ a first antibody or antibody fragment, which is described as the “capture” antibody, that 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” or “signal” 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 it interacts with a substrate or substrates, so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte. Other types of immunochemical assays and immunoassays include competitive assays and antibody detection assays.

[0065] 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 signsAttorney Docket No. 773988: DABX-945PCTof 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.

[0066] An "effective amount" includes a "therapeutically effective amount" and a "prophy lactically 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.

[0067] 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.III. Brain Derived (BD) Tau Assay Methods for Predicting the Likelihood of Developing a Neurodegenerative Disease

[0068] The use of tau to assess the pathology of neurodegenerative diseases, such as AD, has continued to evolve. For example, BD-tau has been proposed as a general indicator of neurodegeneration and levels are significantly elevated in patients with AD as compared to age-matched controls. BD-tau measurements can be utilized alongside Amyloid pathology (A) and Tau pathology (T) as key indicators of Alzheimer’s disease (AD)-associated neurodegeneration (N). This combined approach allows for a more comprehensive assessment of the disease process, helping to identify individuals who may be at an increased risk of experiencing accelerated cognitive decline. By integrating BD-tau measurements with established biomarkers of AD pathology, researchers and clinicians can enhance earlyAttorney Docket No. 773988: DABX-945PCTdetection efforts, improve diagnostic accuracy, and potentially guide more targeted interventions aimed at slowing disease progression.

[0069] In some instances, methods disclosed herein employ capture antibodies or affinity molecules specific to BD-tau and detection antibodies specific to the N-terminus of BD-tau.

[0070] Affinity molecules include, but are not limited to, antibodies (including monoclonal antibodies, polyclonal antibodies, synthetic antibody mimics, and the like), aptamers, affimers, DARPins, oligonucleotides, peptides, and antigens.

[0071] As used herein, the term “antibody” or “antibodies” refers to a binding protein, immunoglobulin, or glycoprotein that maintains antigen-binding properties. An antibody often comprises a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen. Within each variable domain are three complementarity determining regions (CDR) which form loops in the heavy chain variable domain (VH) and light chain variable domain (VL) that contact the surface of the antigen. Antibodies herein also include “antigen binding portion” or fragments of the antibody that are capable of binding to the antigen.

[0072] As used herein, the term “epitope” refers to a binding site recognized by an antibody. Epitopes may include any molecule or grouping thereof, including, but not limited to, amino acid side chains, sugars, and lipids, and can have a specific three-dimensional structure or conformation.

[0073] Some aspects of the method include an antibody that specifically recognizes at least one brain-derived tau isoform but does not bind to, for example, the PNS-tau isoform. As used herein, the phrase “specifically recognizes brain-derived tau isoforms” or “specific to brain-derived tau isoforms” refers to an interaction of an antibody with a peptide, epitope, or binding region common to, or present in, brain-derived tau isoforms and not with a peptide, epitope, or binding region which is not common to, or not present in, brain-derived tau isoforms. For example, a capture antibody specific to brain-derived tau isoforms may recognize the exon 4-5 connective portion but will not recognize a PNS-tau isoform or a tau isoform comprising the amino acid sequence encoded by exon 4a.

[0074] In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 124 and 125 of SEQ ID NO: 2. In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 66 and 67 of SEQ ID NO: 3. InAttorney Docket No. 773988: DABX-945PCTsome embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 95 and 96 of SEQ ID NO: 4. In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 124 and 125 of SEQ ID NO: 5. In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 66 and 67 of SEQ ID NO: 6. In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope which comprises at least amino acids 95 and 96 of SEQ ID NO: 7.

[0075] In some embodiments, the capture antibody specific to brain-derived tau isoforms binds to or recognizes an epitope comprising a phosphorylated tau (p-tau) reside. In some instances, plasma p-tau may be used in an assay to detect AD pathology. In some embodiments, the epitope comprising a phosphorylated tau reside comprises phosphorylated threonine 217 (p-Tau217). p-Tau217 is a post-translational modification strongly associated with pathological tau species in AD and related tauopathies.

[0076] Without being bound to any specific theory, a brain-derived phosphorylated threonine 217 (BD p-Tau217) assay allows for enhanced discrimination between pathological and non-pathological forms. In some aspects, the use of a BD p-Tau217 assay allows for the capture of only brain-derived tau with the disease-linked phosphorylation pattern. In some embodiments, a BD p-Tau217 assay may have improved signal-to-noise ratios and / or generate AD-specific signals, potentially increasing diagnostic accuracy. This selectivity also improves the utility of diagnostic assays and therapeutic interventions.

[0077] Some aspects of the method include an antibody that specifically recognizes multiple isoforms of tau. As used herein, the phrase “specifically recognizes multiple isoforms of tau” or “specific to multiple isoforms of tau” refers to an interaction of an antibody with peptide, epitope, or binding region that binds to tau irrespective of phosphorylation state. In some aspects, the antibody is characterized by its ability to bind both phosphorylated and unphosphorylated tau, both non-pathological and pathological forms and conformations of tau, and misfolded / aggregated forms of tau.

[0078] In an aspect, the disclosure provides for a method for quantitatively determining an amount of brain-derived tau (BD-tau) in at least one biological sample using a high-throughput immunoassay analyzer. Non-limiting examples of the biological sample include serum, whole blood, plasma, and / or cerebral spinal fluid. Depending on the analysis desired, the method may include introducing a capture antibody capable of binding to at least one portion BD-tau and a detector affinity molecule specific to BD-tau to the biological sample. In some embodiments,Attorney Docket No. 773988: DABX-945PCTthe capture antibody may be specific to multiple isoforms of tau or to specific brain-derived isoforms of tau. In some embodiments, a reaction may be generated after the introduction of the capture antibody capable and / or detector affinity molecule. In some embodiments, a reaction may be generated after the introduction of a substrate formulation.

[0079] In an aspect, the method further includes quantitatively determining an amount of BD-tau in the biological sample based on the presence of the reaction. In some aspects, the reaction is a chemiluminescent reaction, an electrochemiluminescence reaction, an electrogenerated chemiluminescence reaction, a photoluminescence reaction, or a bioluminescence reaction. In some embodiments, the chemiluminescent reaction is a dioxetane -based reaction, a luminolbased reaction, an acridinium ester-based reaction, a peroxyoxalate reaction, a luciferin-luciferase reaction, a metal-catalyzed reaction, a halogen-based reaction, or a hydrazine -based reaction. In some embodiments, the detector affinity molecule is conjugated to an enzyme, such alkaline phosphatase or horseradish peroxidase. In this embodiment, a substrate for the enzyme is added to the reaction mixture triggering a chemiluminescent reaction. The detector affinity molecule may be an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

[0080] In some aspects, the amount of BD-tau may be used to assign a subject a degree of likelihood that the subject will develop a neurodegenerative disease and / or assess an accuracy of diagnosis of a neurodegenerative disease.

[0081] The disclosed methods provide distinct advantages to several methodologies used to assessing a subject’s likelihood of developing a neurodegenerative disease, including diagnosing AD, such as PCR, cerebrospinal fluid (CSF) analysis, and positron emission tomography (PET) scans. Each of these approaches presents distinct disadvantages that can impact their effectiveness and applicability in clinical settings.

[0082] PCR, while effective for genotyping, does not provide information on the actual expression levels of the Tau protein, potentially missing functional insights important for understanding disease risk. Additionally, there is a lack of standardization across different assays, which can affect the reliability and comparability of results between laboratories.

[0083] The procedure to obtain CSF is invasive, requiring a lumbar puncture that can cause discomfort, pain, and carries risks such as headaches or infections. This invasiveness may limit the willingness of patients to undergo frequent testing, which is often necessary for monitoring disease progression or treatment efficacy. Moreover, CSF collection requires specialized medical settings and trained personnel, making it less accessible and more costly compared toAttorney Docket No. 773988: DABX-945PCTblood-based assays. Variability in CSF biomarker levels due to factors like hydration status and diurnal fluctuations can also impact the consistency and reliability of results.

[0084] PET scans are expensive and require access to specialized imaging facilities, limiting their availability to larger medical centers and increasing the overall cost of diagnosis. Additionally, PET scans involve exposure to radioactive tracers, raising concerns about radiation safety, especially with repeated scans needed for longitudinal studies or treatment monitoring. The procedure is time-consuming and may be uncomfortable for patients, particularly those with cognitive impairments or movement difficulties. Furthermore, interpreting PET scan results requires highly trained specialists, which can introduce variability and subjectivity in diagnosis.

[0085] In some instances, methods disclosed herein are performed on an automated analyzer disclosed herein, such as a clinical chemistry analyzer or an immunoassay 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.

[0086] In certain embodiments, the immunoassay analyzer used in the disclosed method includes an automated pipetting system configured to aspirate and dispense liquids into designated reaction vessels. The analyzer comprises at least a first reagent pipettor and a second reagent pipettor, each capable of aspirating fluid from independent reagent vessels or reservoirs. The system also includes a detector arrangement, which may be based on chemiluminescent, fluorescent, absorbance, or electrochemiluminescent detection, among others.

[0087] The analyzer may contain multiple reaction vessels, which can be cuvettes, wells, tubes, or integrated reaction compartments of a disposable assay cartridge. The analyzer is designed so that different pipettors may operate simultaneously. For example, while the first reagent pipettor is dispensing fluid into a first reaction vessel, the second reagent pipettor may dispense another reagent into a second reaction vessel. This parallel functionality supports high-throughput processing.Attorney Docket No. 773988: DABX-945PCT

[0088] The method may begin by aspirating a portion of a biological sample, such as plasma, serum, cerebrospinal fluid, whole blood extract, or another clinically relevant sample, from a sample vessel. This may occur using a dedicated sample probe or pipettor integrated into the analyzer. The aspirated biological sample is dispensed into a first reaction vessel, where BD-tau detection will occur.

[0089] The analyzer may automatically track sample identification, aspirated volumes, reaction timing, incubation conditions, and assay progression, ensuring that each reaction vessel is processed under the appropriate protocol.

[0090] In certain embodiments, after the biological sample is introduced into the first reaction vessel, the first reagent pipettor aspirates a fluidic substance from a first reagent vessel and dispenses it into the reaction vessel. This first fluidic substance includes at least one capture antibody capable of binding to a portion of BD-tau. The capture antibody may be specific for BD-tau generally, or, in some embodiments, may specifically recognize BD-pTau217, a phosphorylated tau epitope that is highly enriched in brain-derived tau fragments. In these embodiments, the capture antibody may be an anti-pTau217 antibody.

[0091] The capture antibody may be free in solution or coupled to a solid support, such as a paramagnetic particle. In certain embodiments, paramagnetic microparticles facilitate automated separation, washing, and signal development within the analyzer.

[0092] After addition of the capture reagent, the first reagent pipettor may also aspirate a second fluidic substance from a second reagent vessel and dispense it into the reaction vessel. This second fluidic substance includes a detector affinity molecule specific to BD-tau. In certain embodiments, the detector affinity molecule is an antibody that recognizes the Tau Exon 4 / 5 junction, which is known to be enriched in brain-derived tau isoforms relative to peripheral tau. In some embodiments, this antibody is conjugated to alkaline phosphatase (ALP) or another enzyme that participates in a detectable reaction when introduced to a suitable substrate.

[0093] After the capture and detector reagents have incubated with the sample, allowing the formation of a capture antibody-BD-tau-detector antibody sandwich complex, a substrate is added to the reaction vessel. The substrate may be enzymatic, chemiluminescent, fluorescent, or electrochemiluminescent depending on the labeling chemistry. The addition of the substrate initiates a detectable reaction that correlates with the amount of BD-tau present.Attorney Docket No. 773988: DABX-945PCT

[0094] Following reagent additions and appropriate incubations and wash steps (if required by the assay format), the analyzer uses its detector arrangement to measure the presence and magnitude of the reaction in the first reaction vessel. The detector arrangement may monitor luminescence, fluorescence, absorbance, or other measurable emissions resulting from the interaction between the detector affinity molecule and the substrate.

[0095] The intensity of the measured signal is compared against calibration curves, reference standards, or internal controls to quantitatively determine the amount of BD-tau in the biological sample. In embodiments where the method measures BD-pTau217 specifically, the readout correlates with the concentration of the BD-pTau217 isoform.

[0096] The analyzer’s software may automatically compute sample concentrations, correct for background signals, flag out-of-range values, and store assay data in laboratory information systems.

[0097] The disclosed methods are compatible with a variety of high-throughput analyzers used in centralized clinical laboratories. It can be adapted to different tau assay formats, including two-step, three-step, or one-step sandwich immunoassays, depending on instrumentation capabilities.

[0098] The method may be suitable for quantifying BD-tau or BD-pTau217 in patient samples for research, diagnosis, prognostic monitoring, or therapeutic response assessment.

[0099] Another aspect of the disclosure includes a kit for performing any of the disclosed methods. The kit may include a reagent pack. 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.

[0100] A “reagent vessel” may refer to a vessel, unit, fluid container, well, or the like that is configured to store reagents. In some aspects, the reagent pack comprises enough reagent vessels to perform an assay for BD-tau. 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 leastAttorney Docket No. 773988: DABX-945PCTfive reagent vessels, or alternatively at least ten 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.

[0185] 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.

[0101] 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 deformAttorney Docket No. 773988: DABX-945PCTsufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.

[0102] 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).

[0103] In an aspect, the capture antibody is conjugated to a magnetic bead or a magnetic particle. In some instances, magnetic beads (also known as magnetic particles, paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (-20-30 nm), such as magnetite. On the surface, the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding. Antibodies targeting the analyte of interest or capture antibody can be covalently coupled to the surface of the magnetic bead. In some embodiments, there is about 2 pg or antibody per mg of magnetic bead. In some embodiments, there is about 3 pg or antibody per mg of magnetic bead. In some embodiments, there is about 4 pg or antibody per mg of magnetic bead. In some embodiments, there is about 5 pg or antibody per mg of magnetic bead. In some embodiments, there is about 6 pg or antibody per mg of magnetic bead. In some embodiments, there is about 7 pg or antibody per mg of magnetic bead. In some embodiments, there is about 8 pg or antibody per mg of magnetic bead. In some embodiments, there is about 9 pg or antibody per mg of magnetic bead. In some embodiments, there is about 10 pg or antibody per mg of magnetic bead. In some embodiments, there is about 12 pg or antibody per mg of magnetic bead. In some embodiments, there is about 13 pg or antibody per mg of magnetic bead. In some embodiments, there is about 14 pg or antibody per mg of magnetic bead. In some embodiments, there is about 15 pg or antibody per mg of magnetic bead.

[0104] In an aspect, the substrate formulation is configured to produce a colorimetric response. These substrates produce a visible color based on a chemical reaction between an analyte andAttorney Docket No. 773988: DABX-945PCTa reagent. At operation, this substrate is added to a vessel with a reaction mixture and the light generated is measured with a photometer. Depending on the analysis desired, a first substrate formulation may be added to the first reaction mixture, and a second substrate formulation may be added to a third reaction mixture. In some aspects, the first substrate formulation and the second substrate formulation are the same.

[0105] 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. Chemiluminescent compounds useful in the present formulations are capable of generating chemiluminescence when contacted with an alkaline phosphatase.

[0106] In a non-limiting example, the substrate formulation includes a chemiluminescent compound of formula I or a salt thereof:

[0108] wherein

[0109] A is Ci -ehaloalky 1, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NO2 substituents;

[0110] Ri is selected from the group consisting of Cs-uaryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;

[0111] R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or R11-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;

[0112] R15 is C1-6 alkyl;Attorney Docket No. 773988: DABX-945PCT

[0113] each M is independently selected from the group consisting of H, an alkali metal, alkaline earth metal, transition metal, ammonium, phosphonium, organic amine salt, and an amino acid salt;

[0114] Z is O or S; and

[0115] n is 0, 1, or 2;

[0116] a cationic aromatic compound (CAC);

[0117] a background reducing agent; and

[0118] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0119] 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. Pat. Nos. 6,545,727, 6,690,571, 6,139,782, 6,218,137, 6,270,695, 6,296,787, and 10,703,971 each of which is incorporated by reference herein.

[0120] At operation, this substrate is added to a vessel with a reaction mixture and light generated by the reaction is measured with a luminometer. In some aspects, the first substrate formulation and the second substrate formulation are the same.

[0121] 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.

[0122] 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.

[0123] In one aspect, the substrate formulation comprises 0.01 mM-50 mM compound I, 0.01-200 pM cationic aromatic compound, 1 pM -10 mM background reducing agent, 0.5-20 g / L ether-linked non-ionic surfactant or hydrophilic polymer, 0.01-10 g / L anionic surfactant, and an amine buffer at from 0.025M to 0.65M and at pH 7-12.

[0124] In one aspect, the substrate formulation comprises 0.5 mM-10 mM compound I, 0.5-50 pM cationic aromatic compound, 10 uM-1000 pM background reducing agent, 0.1 to 10 g / LAttorney Docket No. 773988: DABX-945PCTether-linked non-ionic surfactant or hydrophilic polymer, 0.1 to 5 g / L anionic surfactant, and an amine buffer at from 0.05M to 0.5M and at pH 8-11.

[0125] In one aspect, the substrate formulation comprises 0.1 mM-5 mM compound I, 0.1-25 pM cationic aromatic compound, 50 to 500 pM background reducing agent, 0.2 to 5 g / L ether-linked non-ionic surfactant or hydrophilic polymer, 0.1 to 5 g / L anionic surfactant, and an amine buffer at from 0.1M-0.4M and at pH 8-11.

[0126] In one aspect, the compound I has the formulaIV. Automated Analyzer

[0127] In an aspect, an automated 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 automated 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 automated analyzer.

[0128] The automated 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.

[0129] The read station may include a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect colorimetric response, light orAttorney Docket No. 773988: DABX-945PCTluminescence. The detector may be a photometer, luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument.

[0130] In an embodiment, the detector includes a photometer (or photometry unit) configured to emit light of a predetermined wavelength toward a reaction vessel and receive the light that transmits through the reaction mixture. 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 Nos.5,134,216 and 11,604,146, which are incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.

[0131] In an aspect, primary sample containers can be placed into an onload section (e.g., individually or on racks) of the sample presentation unit. In certain embodiments, the sample presentation unit has at least one sample rack, alternatively at least two sample racks, alternatively at least three sample racks, alternatively at least four sample racks, alternatively at least five sample racks, alternatively at least six sample racks, alternatively at least seven sample racks, alternatively at least eight sample racks, alternatively at least nine sample racks, alternatively at least ten sample racks, alternatively at least 11 sample racks, alternatively at least 12 sample racks, alternatively at least 13 sample racks, alternatively at least 14 sample racks, alternatively at least 15 sample racks, alternatively at least 16 sample racks, alternatively at least 17 sample racks, alternatively at least 18 sample racks, alternatively at least 19 sample racks, or alternatively at least 20 sample racks. In certain embodiments, each sample rack can hold at least one sample vessel, alternatively at least two sample vessels, alternatively at least three sample vessels, alternatively at least four sample vessels, alternatively at least five sample vessels, alternatively at least six sample vessels, or alternatively at least seven sample vessels. In a specific embodiment, the sample presentation unit holds about 140 sample vessels. In a specific embodiment, the sample presentation unit has 20 sample racks with each rack holding seven sample vessels.

[0132] 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 an optical reader, such as a barcode scanner. In one embodiment, the optical readers are area scan cameras that provide a two-dimensional imageAttorney Docket No. 773988: DABX-945PCTof 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.

[0133] 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 l. OmL, alternatively about 1.5mL, alternatively about 2.0mL, alternatively about 2.5mL, or alternatively about 3.0mL.

[0134] In an aspect, the analytic unit is configured to receive and analyze samples. In an aspect, the analytic unit configured to perform an assay, such as an immunochemical assay or 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.

[0135] 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 alsoAttorney Docket No. 773988: DABX-945PCTcontain 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 of 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 pL. 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 pL, alternatively less than about 9.5 pL, alternatively less than about 8.0 pL, alternatively less than about 7.0 pL, alternatively less than about 6.0 pL, alternatively less than about 5.0 pL, alternatively less than about 4.0 pL, alternatively less than about 3.0 pL, alternatively less than about 2.0 pL, alternatively between than about 9.9 pL and 2.0 pL.

[0136] 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.

[0137] The claimed configuration allows for the simultaneous performance of at least two assays for a plurality of biological samples, alternatively at least three assays for a plurality of biological samples, or at least four assays for a plurality of biological samples. Depending on the analysis desired, the biological samples may be from the same subject or from multiple subjects. In some aspects, the method further comprises a plurality of sample vessels. In some embodiments, the method comprises at least one sample vessel, at least two sample vessels, at least three sample vessels, at least four sample vessels, at least five sample vessels, at least ten sample vessels, at least twenty sample vessels, at least fifty sample vessels, at least one hundred sample vessels, at least two hundred sample vessels, at least three hundred sample vessels, at least four hundred sample vessels, or at least five hundred sample vessels. In some aspects, the sample vessels are housed within the immunoassay analyzer.

[0138] 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 vesselAttorney Docket No. 773988: DABX-945PCTcomprising 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.

[0139] 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 biological samples / hr., alternatively at least about 300 biological samples / hr., alternatively at least about 400 biological samples / hr., alternatively at least about 440 biological samples / hr., or alternatively at least 500 biological samples / hr.

[0140] At operation, a biological sample and a reagent comprising capture antibody configured to bind to BD-tau are dispensed into a reaction vessel and mixed. 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.

[0141] The mixture is then transferred to an incubator. 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 by the incubator. 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.Attorney Docket No. 773988: DABX-945PCT

[0142] During the incubation, the biological sample and the capture antibody interact. The resulting “first reaction mixture” is a result of the incubation between the sample and the reagent.

[0143] 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.

[0144] At operation, a first portion of the first reaction mixture and a reagent comprising an enzyme-conjugated detection affinity molecule configured bind BD-tau at a different binding site than the capture antibody is dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, the sample and the enzyme-conjugated detection affinity molecule interact. The resulting “second reaction mixture” is a result of the incubation between the enzyme-conjugated detection affinity molecule and the reagent.

[0145] In a non-limiting example, the incubation time of the first and / or second reaction mixtures 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.

[0146] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the automated analyzer. In an embodiment, the wash station is configured to wash away at least some of the unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The wash station may be configured to perform a set number of wash actions depending on the assay. The wash station may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions,Attorney Docket No. 773988: DABX-945PCTalternatively 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.

[0147] The wash station may comprise a washing arrangement which may be configured to provide a base number of wash series (or wash actions) for each reaction vessel and optionally provide an additional number(s) of wash actions. The additional number(s) of wash actions may include one, a plurality, or all of a potential number of wash actions. An additional number of wash actions beyond the base number of wash actions may be specified for certain assays in an assay protocol file. The washing arrangement may include cleaning dispense nozzle (or probe) which dispenses a rinsing fluid and a cleaning aspiration nozzle (or probe) which aspirates the unreacted components. At operation, a base number of wash actions performed may be one, two, three, four, or five and the additional number of wash action(s) may be one, two, three, four or five. At operation, a base number of wash actions performed may be three and the additional number of wash action(s) may be one or two. In this embodiment, the base number of wash actions may be performed if three probes dispense buffer solution once per vessel and the three probes aspirate the at least some of the unreacted components some of the buffer solution, and / or the at least some of the unreacted reagents once per vessel. According to the principles of the present disclosure, certain probe(s) may be selectively used to dispense clean buffer solution into the vessel and aspirate the at least some of the unreacted components of the sample, some of the buffer solution, and / or the at least some of the unreacted reagents from the vessel to perform the additional wash action(s).

[0148] 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 beAttorney Docket No. 773988: DABX-945PCTconfigured 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.

[0149] 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.

[0150] 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) 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 aspiration. The reaction vessel may undergo a series of wash processes 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.

[0151] At operation, a substrate is dispensed into the second reaction mixture. After mixing the substrate and the second reaction mixture are allowed 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 aspiration. The reaction vessel may undergo a series of wash processes 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 unreactedAttorney Docket No. 773988: DABX-945PCTsubstance 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.

[0152] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions. 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 mechanisms that are capable of transporting sample and reaction vessels among the various modules of the automated analyzer are also contemplated for the purpose of the present invention.

[0153] In an aspect the cycle time of an automated 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,Attorney Docket No. 773988: DABX-945PCTalternatively at least 125 incubation positions, alternatively at least 150 incubation positions, alternatively at least 175 incubation positions, or alternatively at least 200 incubation positions.

[0154] In an aspect, the time to first result (TTFR) of an automated analyzer described herein is about 60 minutes or less. The “TTFR” is a measure of time from when the sample is aspirated to when the presence and / or concentration of an analyte is determined. In certain embodiments the TTFR is about 60 minutes or less, alternatively about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0155] In an aspect, the automated 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 automated analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis. In some embodiments, the machine vision apparatus utilizes one or more image capture units to determine whether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether the sample is appropriately prepared so that the analytic unit produces a reliable result. An exemplary machine vision apparatus is described in U. S. Patent No. 11,263,433, which is incorporated by reference herein.

[0156] 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.

[0157] 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 fluidicAttorney Docket No. 773988: DABX-945PCTsubstance 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.

[0158] 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.

[0159] 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.

[0160] The vessel image capture unit operates to capture images of vessels in one or more locations. In some embodiments, the vessel image capture unit is fixed at a particular location in the immunoassay analyzer. In other embodiments, the vessel image capture unit is movably disposed in the immunoassay analyzer, which can move either independently from other components of the immunoassay analyzer or together with one or more components of the immunoassay analyzer. Some embodiments of the immunoassay analyzer include a plurality of vessel image capture units. As described herein, the vessel tip image capture unit includes a camera unit.

[0161] 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.

[0162] All of the units of the automated 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,Attorney Docket No. 773988: DABX-945PCTand 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.

[0163] 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 Ml, and / or the like processor(s).

[0164] 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.

[0165] 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.

[0166] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0167] EXAMPLES

[0168] Example 1: Exemplary BD-tau assayAttorney Docket No. 773988: DABX-945PCT

[0169] The assay 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.

[0170] The reagent pack includes 5 reagent vessels. The first reagent vessel includes capture antibodies capable of binding to multiple isoforms of BD-tau conjugated to paramagnetic particles (“BD-tau capture antibodies”). The second reagent vessel includes BD-tau antibodies conjugated to alkaline phosphatase (“BD-tau detector affinity molecule”).

[0171] The BD-tau capture antibodies from the first reagent vessel were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. A magnetic field was applied to the reaction vessel and the incubated mixture was washed using a wash buffer to remove any unreacted components.

[0172] The BD-tau detector affinity molecule was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a reaction mixture. A magnetic field was applied to the reaction vessel and the reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0173] The test results were determined automatically by the system software. The signal generated by the assay was measured in relative light units (RLUs).

[0174] Example 2: Sensitivity Analysis

[0175] Studies were performed to estimate the limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) for an exemplary BD-Tau assay on an exemplary immunoassay analyzer.

[0176] The LoB study was performed using an exemplary immunoassay analyzer with a single reagent lot and a single calibrator lot. Two sets of SO calibrator matrix and wash buffer were analyzed over 3 days, with 5 replicates per sample and a single run per day.

[0177] LoD and LoQ analysis were performed by monitoring the dose variance of a panel of 9 native K2 EDTA samples over 5 days on the exemplary immunoassay analyzer. The studyAttorney Docket No. 773988: DABX-945PCTwas performed using a single reagent lot and a single calibrator lot. All samples were analyzed over 5 days, with 9 replicates per sample and a single run per day. The results of the sensitivity study are listed in Table 2.

[0178] Table 2: SensitivityParameter pg / mLLimit of Blank (LoB) 0.034Limit of Detection (LoD) 0.057Limit of Quantitation (LoQ) 0.131

[0179] Example 3: Discrimination between AD, frontotemporal dementia (FTD), and healthy individuals using BD-tau assay.

[0180] A study was performed to assess the discrimination between AD, FTD, and healthy individuals using the exemplary BD-Tau assay. The study was run on two exemplary immunoassay analyzers, with a single reagent and calibrator lot, according to the method of Example 1.

[0181] K2 EDTA plasma and serum samples were collected from patients diagnosed with AD (MMSE scores ranging from 1-29, 59-86 years old), age-matched healthy individuals (55-68 years old), and FTD (MMSE scores ranging from 16-25, 50-67 years old). Each sample was tested in replicates of two. AD patients were amyloid (AM) positive, FTD and healthy individuals were AM negative. AM positivity was determined based on results from the Lumipulse G pTau 217 / p-Amyloid 1-42 plasma ratio assay as shown in FIG. 1 and Table 3. As shown in FIG. 1, the p -values observed ( O.20; p=0.095) indicate that the BD-tau assay can generate statistically significant results that allow for the discrimination between AD and FTD or healthy AM negative individuals. Additionally, the example demonstrates discrimination comparable to literature, and in both K2 EDTA plasma and serum.

[0182] Table 3Median AD (pg / mL) Median FTD (pg / mL) Median Normal (pg / mL)5.9 2.6 2.5Attorney Docket No. 773988: DABX-945PCT

[0183] Example 4: Exemplary BD p-tau217 assay

[0184] The assay 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.

[0185] The reagent pack includes 5 reagent vessels. The first reagent vessel includes capture antibodies conjugated to paramagnetic particles specific to isoforms of tau phosphorylated at threonine 217 (“p-Tau217 capture antibodies”). The second reagent vessel includes antibodies conjugated to alkaline phosphatase specific to BD-tau (“BD-tau detector affinity molecule”).

[0186] The p-Tau217 capture antibodies from the first reagent vessel were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. A magnetic field was applied to the reaction vessel and the incubated mixture was washed using a wash buffer to remove any unreacted components.

[0187] The BD-tau detector affinity molecule was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a reaction mixture. A magnetic field was applied to the reaction vessel and the reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0188] The test results were determined automatically by the system software. The signal generated by the assay was measured in relative light units (RLUs).

[0189] Example 5: Comparison of p-Tau217 and BD p-Tau217 immunoassays

[0190] End-of-life plasma samples from 269 participants (mean age 85.5 ± 8.5; MMSE 22.5 ± 7.5; 44.9% female) were analyzed for p-Tau217, BD p-Tau217, BD-Tau, and Aβ42 RUO using an exemplary immunoassay analyzer. The BD p-Tau217 was analyzed using the method of example 4 and the BD-Tau was analyzed using the method of example 1. The remaining biomarkers were analyzed using commercially available assays and methods. Ratios of p-Tau217 / Aβ42 and BD p-Tau217 / Aβ42 were also evaluated. Biomarker levels were examined by Alzheimer’s disease neuropathological change (ADNC; not AD, low, intermediate, high), and discriminative accuracy was assessed with ROC area under the curve (AUC). An ADNCAttorney Docket No. 773988: DABX-945PCTscore for AD neuropathologic change incorporates histopathologic assessments of amyloid β deposits, staging of neurofibrillary tangles, and scoring of neuritic plaques.

[0191] Plasma p-Tau217, BD p-Tau217, and their respective Aβ42 ratios differed significantly across ADNC groups (FIGs. 2A-2D), with strong discriminative accuracy for distinguishing none / low vs. intermediate / high ADNC (AUCs 0.83-0.88) and none vs. high ADNC (AUCs 0.96-0.99) (FIG. 3A and 3B), with BD p-Tau217 / Aβ42 yielding the highest point estimates. Fold-change analyses (FIGs. 4A - 4C) showed the largest increases for BD p-Tau217 (6.91) and BD p-Tau217 / Aβ42 (7.97), compared with p-Tau217 (3.26) and p-Tau217 / Aβ42 (3.76), in high ADNC vs. not AD.

[0192] This is further highlighted by the Table 4-6

[0193] Table 4: Not AD + Low ADNC vs. Int + High ADNCScenario Lower Upper PLR PPV NLR NPV Sensitivity Specificity Indeterminate Cutoff Cutoff (%) (%) (PPA, %) (NPA, %) (%) pTau217Maximum PLR < 6*StandaloneBD- pTau217Standalone 0.19 0.645 11.6 94.3 0.14 82.9 96.3 94.7 49.1 pTau217 / Aβ42 0.0131 0.0352 9.2 93.4 0.1 87.5 96.9 94.7 52.7 BD- pTau217 / Aβ42 0.0071 0.0163 10.4 93.8 0.13 84 95.1 93 35.7

[0194] As shown in Table 4, BD-pTau217 improves PLR performance compared to pTau217, pTau217 / Aβ42 improves PLR performance to achieve clinical targets, and BD-pTau217 / Aβ42 reduces the indeterminate zone compared to BD-pTau217.

[0195] Table 5: Not AD + Low ADNC vs. Int + High ADNCScenario Lower Upper PLR PPV NLR NPV Sensitivity Specificity Indeterminate Cutoff Cutoff (%) (%) (PPA,%) (NPA,%) (%) pTau217Standalone 0.492 0.999 4.954 87.6 0.147 82.6 92.6 89.5 40.1 BD- pTau217Standalone 0.22 0.46 5.89 88.3 0.143 83 94.5 86 31.4 pTau217 / Aβ42 0.0132 0.0258 5.202 88.1 0.12 85.4 96.3 86 36.5 BD- pTau217 / Aβ42 0.00748 0.0129 5.266 88.3 0.14 83.3 94.5 85.1 28.2

[0196] Table 6: Amyloid: Thai Score 0 & 1 vs 3 & 4Attorney Docket No. 773988: DABX-945PCTScenario Lower Upper PLR PPV NLR NPV Sensitivity Specificity Indeterminate Cutoff Cutoff (%) (%) (PPA,%) (NPA,%) (%) pTau217Standalone 0.444 1.46 8.192 90.2 0.093 90.6 96.6 96.2 62.3 BD- pTau217Standalone 0.238 0.645 10.796 92.4 0.097 90.2 95.9 93.8 39.9 pTau217 / Aβ42 0.0132 0.0313 10.462 92.2 0.099 89 97.3 93.8 48.6 BD- pTau217 / Aβ42 0.00818 0.0148 10.863 92.4 0.104 89.6 95.2 92.3 27.9

[0197] BD p-Tau217 variants demonstrated nearly twice the fold-change of standard p-Tau217 measures in high ADNC, suggesting a stronger dynamic response to AD pathology. These findings highlight the use of the exemplary BD p-Tau217 immunoassay in a clinical setting where diagnostic confidence and / or population-level screening is desired. Additionally, this assay may be used for therapeutic trial applications, including monitoring disease progression or therapeutic response, or future clinical use.

[0198] Example 6: BD-pTau217 assay assessment of comparison assay, imprecision, analytical sensitivity, linearity, analytical specificity, and dilution recovery

[0199] Results were collected for a BD-pTau217 immunoassay developed using an exemplary immunoassay analyzer. The prototype assay was compared to a p-Tau217 Research Use Only (RUO) assay, which measures total circulating p-Tau217 regardless of tissue origin, including evaluation of subjects with and without amyloid pathology. Further, CLSI-based analytical studies are presented for the BD-pTau217 prototype assay to assess imprecision, analytical sensitivity, linearity, and analytical specificity.

[0200] The BD-pTau217 assay is a two-step, two-site immunometric assay and is shown in FIGs. 5A and 5B. The assay uses an antibody with specificity for the Tau Exon 4 and Exon 5 alternative splicing junction in combination with an antibody that recognizes phosphorylated threonine at position 217 on the Tau protein. The antibody specific for Tau phosphorylated at threonine 217 is coupled to paramagnetic particles, while the second antibody specific for a Tau epitope predominantly expressed in the central nervous system is conjugated to alkaline phosphatase. In a first step the anti-pTau217 antibody coupled to paramagnetic particles was incubated with a reaction buffer and a sample or control sample for 21 minutes at 37 °C and then washed. In a second step an anti-Tau Exon 4 / 5 junction antibody conjugated to alkaline phosphatase was added and incubated for 8 minutes at 37 °C. This was then washed and the chemiluminescent substrate was added, followed by detection of the relative light units (RLUs).Attorney Docket No. 773988: DABX-945PCT

[0201] A cohort of N=60 K2 EDTA plasma samples were tested in duplicate using the BD-pTau217 assay and the p-Tau217 RUO assay. Included within the cohort were 30 samples from subjects with Alzheimer’s Disease (AD), 10 samples from subjects with mild cognitive impairment (MCI), and 20 normal samples. Each sample had matched cerebral spinal fluid (CSF) and was tested to assess the presence of amyloid pathology. Method comparison results are shown in FIG. 6 with Passing-Bablok and Bland-Altman analysis (Table 7). Amyloid pathology differentiation is depicted in FIGs. 7A-7B with box plots (FIG. 7A), ratio estimates (Table 8), and Receiver Operating Characteristic (ROC) analysis (FIG. 7B).

[0202] Table 7Passing-Bablok Bland-Altman N R Slope Intercept Mean Bias Slope 95% CI Intercept 95% CI Bias (%) 95% CI60 0.9 0.53 [0.45, 0.64] -0.3 [-0.7, -0.01] -57 [-79, -35]

[0203] Table 8Mean MedianAssay Amyloid Status N Ratio Ratio (pg / mL) (pg / mL)Negative 45 0.348 0.29p-Tau217 2.3 2.5Positive 15 0.798 0.711Negative 45 0.14 0.115BD-pTau217 3 3Positive 15 0.413 0.344

[0204] Imprecision was estimated for the BD-pTau217 prototype assay using an abbreviated study based on CLSI EP05 Evaluation of Precision of Quantitative Measurement Procedures. Ten EDTA plasma samples containing concentrations of BD-pTau217 spanning the analytical measuring range were tested in replicates of five with two runs / day over five total days of testing. Within-run, between-run, between-day, and within-laboratory variance components were calculated with coefficient of variation (%CV). Imprecision results are shown in Table 9.

[0205] Table 9Mean Within-Run Between-Run Between-Day Within-Lab Sample N(pg / mL) %CV %CV %CV %CV 1 50 0.18 20.7 12.3 0 24.12 50 0.032 11.4 3.7 0 12Attorney Docket No. 773988: DABX-945PCT3 50 0.059 6.5 2.9 0 7.1 4 50 0.074 6 2.3 3.4 7.3 5 50 0.124 4.8 1.7 0 5.1 6 49 0.325 3.7 0 1.5 4 7 50 0.653 3.1 0 0.7 3.2 8 50 1.091 2.9 1.2 1.3 3.4 9 50 3.466 3.2 0 0.9 3.410 50 6.675 4.4 0 0.1 4.4

[0206] Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) were estimated for the BD-pTau217 prototype assay using protocols based on CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures. For LoB estimation, 4 blank samples were tested in replicates of five over 3 days. For LoD and LoQ estimation, 10 EDTA plasma samples were tested in replicates of five with two runs / day over five total days of testing. Detection capability results are depicted in FIG. 8 with associated precision profile.

[0207] Linearity was evaluated using a protocol based on CLSI EP06 Evaluation of Linearity of Quantitative Measurement Procedures. Paired low and high EDTA plasma samples covering the full analytical measuring range of the assay were used for linearity determination. In addition to the low and high concentration samples, seven mixtures were tested in this study. These samples were prepared independently by using incrementally larger proportions of the high sample diluted with the low sample, to achieve concentrations that covered the range of the assay. The low sample was run in replicates of eight, and all other samples were run in replicates of four. Linearity results are presented in FIG. 9 and Table 10; linear equation, correlation coefficient and non-linearity estimates are provided.

[0208] Table 10Concentration Non-linearity(pg / mL) (%)0.02 -4.90%1.262 8.30%2.505 4.90%3.747 2.60%4.989 3.20%6.232 -3.10%7.474 -2.50%8.716 -3.90%Attorney Docket No. 773988: DABX-945PCT| 9.959 | -5.80% |

[0209] Assay specificity was evaluated for potentially interfering or cross-reacting substances using a protocol based on CLSI EP07 Interference Testing in Clinical Chemistry. Each substance was dissolved in an appropriate solvent and spiked into an EDTA plasma sample containing BD-pTau217 analyte. Each substance-spiked sample and a matched solvent-spiked control were tested in replicates of four. The magnitude of interference or cross-reactivity was estimated by comparing the test and corresponding control results. Analytical specificity results are shown in Table 11.

[0210] Table 11Concentration Substance Control TestSubstance Concentration Concentration Change Concentration (pg / mL) (pg / mL) (%) 250 mg / L 0.292 -1.20% Galantamine 0.289250 mg / L 0.292 0.285 -2.60% MemantineDonepezil 30 mg / L 0.292 0.282 -3.60%45 mg / L 0.292 -0.30% Rivastigmine 0.29145 mg / L 0.283 0.284 0.40% AripiprazoleIbuprofen 0.268 0.275 2.80% 0.219 mg / mLAc etaminophen 0.156 mg / mL 0.28 -2.30% 0.287Heparin 3.3 U / mL -4.20% 0.291 0.279Hemoglobin 10 mg / mL 0.274 2.70% 0.267Bilirubin (Conj) 0.4 mg / mL 0.292 0.282 -3.50% Bilirubin (Unconj) 0.4 mg / mL 0.278 0.278 -0.10% Triolein 15 mg / mL 0.284 0.281 -1.10% HAMA 1000 ng / mL 0.362 0.341 -5.80% RF 50 IU / mL 0.384 0.383 -0.20% HSA 0.15 g / mL 0.222 -2.30% 0.217100 pg / mL 0.282 0.292 3.60% Non-Phospho Tau

[0211] Two EDTA plasma samples containing high concentrations of analyte were diluted 1:2, 1:5, and 1:10 with a wash buffer. Each neat and diluted sample was tested in replicates of four, and percent recovery was calculated for each sample and dilution factor. Dilution recovery results are depicted in Table 12.Attorney Docket No. 773988: DABX-945PCT

[0212] Table 12Observed ExpectedRecoverySample Dilution Concentration Concentration(pg / mL) (pg / mL) (%)Neat 1.1491:02 0.59 0.574 103%11:5 0.237 0.23 103%1:10 0.119 0.115 104%Neat 3.7141:02 1.887 1.857 102%21:5 0.757 0.743 102%1:10 0.373 0.371 100%

[0213] The BD-pTau217 assay on the exemplary immunoassay analyzer showed significant correlation to the p-Tau217 RUO assay with comparable ability to differentiate the presence of amyloid pathology, as determined by CSF p-Amyloid 42 / 40 ratio, for a cohort. Greater negative bias at low concentrations and increased fold difference of mean / median values reflected selective detection of CNS-enriched Tau species and opportunity to improve differentiation of amyloid positive and negative samples. The BD-pTau217 assay exhibited excellent analytical performance as assessed through studies of imprecision, analytical sensitivity, linearity, analytical specificity, and dilution recovery. All samples from the cohort tested yielded concentrations above the derived LoQ.

[0214] 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.

[0215] 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 appendix and appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 773988: DABX-945PCTCLAIMS1. A method for quantitatively determining an amount of brain-derived tau (BD-tau) in at least one biological sample using a high-throughput immunoassay analyzer, the method comprising:aspirating a portion of the at least one biological sample from a sample vessel and dispensing the aspirated biological sample into a first reaction vessel of the immunoassay analyzer, wherein the immunoassay analyzer comprises:a pipettor arrangement comprising at least a first reagent pipettor and a second reagent pipettor; anda detector arrangement;detecting, using the detector arrangement, a presence of a reaction in the first reaction vessel; andquantitatively determining an amount of BD-tau in the biological sample based on the presence of the reaction; andwherein the second reagent pipettor is configured to dispense reagents into a second reaction vessel simultaneously as the first reagent pipettor dispenses fluid into the first reaction vessel.

2. The method of claim 1, further comprisingaspirating, using the first reagent pipettor, a portion of a fluidic substance from a first reagent vessel and dispensing said fluidic substance into the first reaction vessel, wherein the fluidic substance from the first reagent vessel comprises at least one capture antibody capable of binding to at least one portion BD-tau;aspirating, using the first reagent pipettor, a portion of a fluidic substance from a second reagent vessel and dispensing said fluidic substance into the first reaction vessel, wherein the fluidic substance from the second reagent vessel comprises at least one detector affinity molecule specific to BD-tau; anddispensing a substrate into the first reaction vessel.Attorney Docket No. 773988: DABX-945PCT3. The method of claim 2, wherein BD-tau is BD-pTau217.

4. The method of claim 3, wherein the at least one capture antibody capable of binding to at least one portion of BD-tau is an anti-pTau217 antibody.

5. The method of claim 4, wherein the anti-pTau217 antibody is coupled to at least one paramagnetic particle.

6. The method of any one of claims 2 to 5, wherein the at least one detector affinity molecule specific to BD-tau is an anti-Tau Exon 4 / 5 junction antibody conjugated to alkaline phosphatase.

7. The method of any one of the preceding claims, wherein the first reagent vessel and second reagent vessel are housed in a reagent pack.

8. The method of any one of the preceding claims, wherein the first reagent vessel and second reagent vessel comprise an elastomeric self-sealing membrane.

9. The method of any one of the preceding claims, wherein each reagent vessel is configured to store a volume of reagent required for at least about 20 instances of a BD-tau assay.

10. The method of any one of the preceding claims, wherein the method is configured to analyze at least about 200 biological samples / hr.; alternatively the method is configured to analyze at least about 300 biological samples / hr.; alternatively the method is configured to analyze at least about 400 biological samples / hr.; alternatively the method is configured to analyze at least about 450 biological samples / hr.; or alternatively the method is configured to analyze at least about 500 biological samples / hr.Attorney Docket No. 773988: DABX-945PCT11. The method of any one of the preceding claims, 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.

12. The method of any one of the preceding claims, 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.

13. The method of any one of the preceding claims, wherein the reaction in the first reaction vessel generates a chemiluminescent signal, wherein the chemiluminescent signal quantitatively corresponds to a concentration of BD-tau in the plasma sample.

14. The method of claim 13, wherein the reaction in the first reaction vessel generates a chemiluminescent signal when a substrate formulation is added to the reaction vessel, wherein the substrate formulation comprises:a chemiluminescent compound of the formula A or a salt thereof:Attorney Docket No. 773988: DABX-945PCTwhereinA is Ci -ehaloalky 1, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NCfr substituents;Ri is selected from the group consisting of Cs-uaryl, Ci-6 alkyl, Ci-6 haloalkyl, and C5-14 aralkyl groups;R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or R11-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;R15 is C1-6 alkyl;each M is independently selected from the group consisting of H, an alkali metal, alkaline earth metal, transition metal, ammonium, phosphonium, organic amine salt, and an amino acid salt; Z is O or S; andn is 0, 1, or 2;a cationic aromatic compound (CAC);a background reducing agent; andan ether-linked nonionic surfactant or a hydrophilic polymer.

15. The method of any one of the preceding claims, wherein the detector arrangement comprisesa 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, anda counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time.Attorney Docket No. 773988: DABX-945PCT16. The method of any one of the preceding claims, wherein the immunoassay analyzer further comprises a third reagent pipettor, and wherein the second reagent pipettor and third reagent pipettor are configured to dispense reagents into the second reaction vessel and a third reaction vessel simultaneously.

17. The method of claim 16, wherein the fluidic substances dispensed into the first reaction vessel, second reaction vessel, and / or third reaction vessel are agitated via an ultrasonic mixing module.

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

19. The method of claim 18, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

20. The method of claim 18, 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.

21. The method of any one of the preceding claims, wherein 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, or alternatively configured to perform at least five wash actions.Attorney Docket No. 773988: DABX-945PCT22. A method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising:aspirating, using a sample pipettor, a portion of the biological sample from a sample vessel and dispensing the aspirated biological sample into a reaction vessel of the immunoassay analyzer;wherein the immunoassay analyzer comprises:a pipettor arrangement comprising at least one reagent pipettor and at least one sample pipettor;and a detector arrangement;wherein the sample pipettor is configured to aspirate and / or dispense less than about 10 pL, alternatively about 2.0 pL to about 9.9 pL;detecting, using the detector arrangement, a presence of a reaction in a reaction vessel; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the reaction vessel.

23. The method of claim 22, wherein BD-tau is BD-pTau217.

24. A method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising:aspirating, using a sample pipettor, a portion of the biological sample from a sample vessel and dispensing the aspirated sample into a reaction vessel of the immunoassay analyzer; wherein the immunoassay analyzer comprises:a reagent pack comprising a plurality of reagent vessels, wherein the reagent pack is configured to store a volume of reagent required for at least about 20 instances of a BD-tau assay;a pipettor arrangement comprising at least one reagent pipettor and at a sample pipettor;Attorney Docket No. 773988: DABX-945PCTand a detector arrangement;aspirating, using the reagent pipettor, a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into a reaction vessel of the immunoassay analyzer, generating a first reaction mixture;aspirating, using the reagent pipettor, a portion of a second reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel, generating a second reaction mixture;detecting, using the detector arrangement, a reaction in the reaction vessel; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the reaction vessel.

25. The method of claim 24, wherein BD-tau is BD-pTau217.

26. A method for detecting BD-tau in a biological sample using an immunoassay analyzer, the method comprising:mixing the biological sample with a reagent comprising at least one affinity molecule that binds to at least one portion BD-tau generating a first reaction mixture;mixing the first reaction mixture with a reagent comprising a detection molecule, generating a detection mixture;wherein the immunoassay analyzer comprises an ultrasonic mixing module, and the first reaction mixture and / or the detection mixture are agitated via the ultrasonic mixing module;detecting, using a detector arrangement, a reaction in the detection mixture; and determining a presence and / or concentration of BD-tau in the biological sample based on the reaction detected in the detection mixture.

27. The method of claim 26, wherein BD-tau is BD-pTau217.Attorney Docket No. 773988: DABX-945PCT28. The method of claim 27, wherein the at least affinity molecule capable that binds to at least one portion BD-tau is an anti-pTau217 antibody.

29. The method of claim 28, wherein the anti-pTau217 antibody is coupled to at least one paramagnetic particle.

30. The method of any one of claims 26 to 29, wherein the detection molecule specific to BD-tau is an anti-Tau Exon 4 / 5 junction antibody conjugated to alkaline phosphatase.

31. The method of any one the preceding claims, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

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