Calibrator dipeptide for phosphorylated TAU (p-TAU) 217 immunoassays

Abstract

The presently claimed and described technology provides a calibrator dipeptide for use in immunoassay methods for detecting phosphorylated tau (p-tau)217.

Description

Attorney Docket No. 772157: DABX-002PCTCALIBRATOR DIPEPTIDE FOR PHOSPHORYLATED TAU (P-TAU) 217 IMMUNOASSAYS CROSS REFERENCE TO RELATED APPLICATION

[0001] The present patent application claims the priority benefit of U. S. Provisional Patent Application Serial No. 63 / 751,113, filed January 29, 2025, the contents of which are hereby incorporated by reference in its entirety into this disclosure.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on January 29, 2025, is named 68617_Sequence.xml and is 14,557 bytes in size.BACKGROUND

[0003] Dementia is one of the costliest conditions to society. In 2022, the total national cost of caring for people living with Alzheimer’s Disease (AD) and other dementias was projected to reach $321 billion, and an estimated 6.5 million Americans aged 65 and older were estimated to be living with AD. By 2060, the number of people aged 65 and older with AD is projected to reach 13.8 million, barring the development of medical breakthroughs to prevent, slow or cure AD. 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 analyzing assays and can efficiently perform clinical analysis on a large number of samples, with multiple tests being run concurrently or within short time intervals.

[0005] Plasma phosphorylated tau (p-tau) biomarkers, including p-taul81, p-tau217, and p-tau231, have shown strong diagnostic performance in identifying AD and distinguishing it from other neurodegenerative conditions. These biomarkers, detectable through blood tests,Attorney Docket No. 772157: DABX-002PCTare promising for early and non-invasive AD diagnosis due to their high specificity and sensitivity. It has been observed that certain different p-tau variants correlates with different stages of AD pathology, providing valuable information about disease progression and enhancing the potential for more targeted therapeutic interventions.

[0006] Immunoassays instruments are widely used in clinical and research settings for the detection and quantification of target molecules in various samples (e.g., serum, plasma, whole blood). Before a sample in an immunoassay can be evaluated, calibration is required. The process of calibration generally involves using an immunoassay calibrator (e.g., a recombinant protein or synthetic peptide) as a reference point for quantifying the amount of a target molecule in a sample. Often, a series of dilutions of the calibrator of known concentrations are prepared. Each of the dilutions is measured using the immunoassay, and the resulting signals are plotted against the corresponding known concentrations. A curve generated from the plot is then used to correlate the signal produced by the sample to the concentration of the target molecule in the sample.

[0007] A need exists for optimized calibration strategies to aid in optimal assay performance.BRIEF SUMMARY

[0008] One aspect of the disclosure is a calibrator dipeptide including: a first peptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 9; a second peptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 10; and a linker connecting the first peptide to the second peptide.

[0009] In an aspect, the first peptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively 99% identical toAttorney Docket No. 772157: DABX-002PCTthe amino acid sequence set forth in SEQ ID NO: 9; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0010] In an aspect, the first peptide comprises at least about a ten amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 9.

[0011] In an aspect, the second peptide comprises an amino acid sequence at least at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 10; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0012] In an aspect, the second amino acid chain comprises at least a ten amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 10.

[0013] In an aspect, the first peptide and the second peptide are covalently connected via the linker.

[0014] In an aspect, the linker comprises a polymer or an amino acid linker.

[0015] In an aspect, the linker is a polymer and comprises polyethylene glycol (PEG), poly(vinyl pyrrolidone) (PVP), poly(2-oxazoline)s, polysarcosine (pSar), zwitterionic polymers, polyacrylamides, polyamino acids, or a combination thereof.

[0016] In an aspect, the linker is a polymer and comprises PEG4, PEG2, PEG3, PEG5, PEG6, or a combination thereof.

[0017] In an aspect, linker is a polymer and comprises PEG4.

[0018] In an aspect, the linker is an amino acid linker and comprises a cysteine-cysteine linker or a histidine-histidine linker.

[0019] One aspect of the disclosure is a calibrator dipeptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NOs: 11 or 12.

[0020] In an aspect, the calibrator dipeptide comprises an amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at leastAttorney Docket No. 772157: DABX-002PCT96% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0021] In an aspect, the calibrator dipeptide comprises an amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0022] In an aspect, the calibrator dipeptide is formulated into a composition.

[0023] In an aspect, the composition is a liquid, an aqueous preparation, a solution, a suspension, a power, or a lyophilized preparation.

[0024] In an aspect, the composition comprises an ionic surface charge modifier, a stabilizer, a salt, a buffer, an excipient, water, a solvent, or combinations thereof.

[0025] In an aspect, the calibrator dipeptide is used to calibrate an automated analyzer. In an aspect, the automated analyzer is a clinical chemistry analyzer or an immunoassay analyzer.

[0026] One aspect of the disclosure is a set of standard solutions for preparing a calibration curve, the set of standard solutions comprising: at least a first solution comprising the calibrator dipeptide of any one of embodiments having a first concentration of the calibrator dipeptide; and at least a second solution comprising the calibrator dipeptide having a second concentration of the calibrator dipeptide; and wherein the first concentration is greater than the second concentration.

[0027] In an aspect, the first concentration of calibrator dipeptide is at least IX greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 2X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 3X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibratorAttorney Docket No. 772157: DABX-002PCTdipeptide is at least 4X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 5X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 6X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 7X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 8X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 9X greater than the second concentration of calibrator dipeptide; or alternatively the first concentration of calibrator dipeptide is at least 10X greater than the second concentration of calibrator dipeptide.

[0028] In an aspect, the first concentration of calibrator dipeptide is at least 100 fg / mL; alternatively the first concentration of calibrator dipeptide is at least 500 fg / mL; alternatively the first concentration of calibrator dipeptide is at least 1 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 2 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 5 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 10 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 20 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 50 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 100 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 200 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 500 pg / mL; or alternatively the first concentration of calibrator dipeptide is at least 1 ng / mL.

[0029] One aspect of the disclosure is a method for detecting phosphorylated tau (p-tau) 217 in a sample from a subject using an immunoassay analyzer, the method comprising: calibrating the immunoassay analyzer using at least a first solution comprising the calibrator dipeptide of any one of the embodiments having a first concentration of the calibrator dipeptide and at least a second solution comprising the calibrator dipeptide having a second concentration of the calibrator dipeptide; wherein the first concentration is greater than the second concentration and wherein the calibration comprises generating a calibration curve; performing a quantitative immunoassay on a portion of the sample from the subject, wherein the quantitative immunoassay generates at least one sample value; and determining a concentration of p-tau 217 in the sample by comparing the at least one generated sample value to the calibration curve.

[0030] In an aspect, the first concentration of the calibrator dipeptide is at least 1X greater than the second concentration of the calibrator dipeptide; alternatively the firstAttorney Docket No. 772157: DABX-002PCTconcentration of the calibrator dipeptide is at least 2X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 3X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 4X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 5X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 6X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 7X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 8X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 9X greater than the second concentration of the calibrator dipeptide; or alternatively the first concentration of the calibrator dipeptide is at least 10X greater than the second concentration of the calibrator dipeptide.

[0031] In an aspect, the first concentration of the calibrator dipeptide is at least 100 fg / mL; alternatively the first concentration of the calibrator dipeptide is at least 500 fg / mL; alternatively the first concentration of the calibrator dipeptide is at least 1 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 2 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 5 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 10 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 20 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 50 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 100 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 200 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 500 pg / mL; or alternatively the first concentration of the calibrator dipeptide is at least 1 ng / mL.

[0032] In an aspect, the method further comprises: mixing the first solution with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a first reaction mixture; mixing the first reaction mixture with a reagent comprising a detection molecule, generating a first detection mixture; detecting, using a detector arrangement, a reaction in the first detection mixture to generate a first calibrator dipeptide value; mixing the second solution with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a second reaction mixture; mixing the second reaction mixture with a reagent comprising a detection molecule, generating a second detection mixture; detecting, using a detectorAttorney Docket No. 772157: DABX-002PCTarrangement, a reaction in the second detection mixture to generate a second calibrator dipeptide value; and wherein the first calibrator dipeptide value and the second calibrator dipeptide value are used to generate the calibration curve.

[0033] In an aspect, the method further comprises: aspirating, using a sample pipettor, a portion of the sample from a sample vessel and dispensing the aspirated 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; detecting, using the detector arrangement, a presence of a reaction in a reaction vessel; and generating the at least one sample value based on the reaction detected in the reaction vessel.

[0034] In an aspect, the method further comprises: mixing the sample with a reagent comprising at least one affinity molecule that binds to at least one portion of p-tau217, generating a reaction mixture; mixing the reaction mixture with a reagent comprising a detection molecule, generating a detection mixture; detecting, using a detector arrangement, a reaction in the detection mixture; and generating the at least one sample value based on the reaction detected in the detection mixture.

[0035] These and other advantages, aspects, and novel features of the present disclosure, as well as details of illustrated embodiments thereof, will be more fully understood from the following description and drawings.BRIEF DESCRIPTION OF THE FIGURES

[0036] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0037] FIG. 1 is a calibration curve plot constructed using exemplary calibrator dipeptides of varying concentrations using an exemplary p-tau217 assay and an exemplary immunoassay analyzer as disclosed herein.

[0038] FIG. 2 is a box and whisker plot showing p-Tau217 concentrations associated with AD-positive versus normal samples analyzed using an exemplary p-tau217 assay and an exemplary immunoassay analyzer as disclosed herein.

[0039] FIG. 3 is a plot depicting within-run CV was calculated from harmonized dose response and plotted relative to mean dose-response of samples analyzed using an exemplary p-tau217 assay and an exemplary immunoassay analyzer as disclosed herein.Attorney Docket No. 772157: DABX-002PCTDETAILED DESCRIPTIONI. Introduction

[0040] 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.

[0041] 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.

[0042] Table 1: Exemplary Tau IsoformsSEQ IDAmino Acid Sequence Identifier NO MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTOEPESGKVVOE GFLREPGPPGLSHQLMSGMPGAPLLPEGPREATR OPSGTGPEDTEGGRHAPELLKHOLLGDLHQEGPP LKGAGGKERPGSKEEVDEDRDVDESSPQDSPPSK ASPAQDGRPPQTAAREATSIPGFPAEGAIPLPVDF LSKVSTEIPASEPDGPSVGRAKGODAPLEFTFFIVE ITPNVQKEQAHSEEHLGRAAFPGAPGEGPEARGP SLGEDTKEADLPEPSEKQPAAAPRGKPVSRVPOL1 KARMVSKSKDGTGSDDKKAKTSTRSSAKTLKNR PNS-tau PCLSPKHPTPG S SDPLIQP S SP A VCPEPPS SPKYV S S VTSRTGSSGAKEMKLKGADGKTKIATPRGAAPPG QKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSG YSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTP PKSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLK HQPGGGKVQIINKKLDLSNVQSKCGSKDNIKIIVP GGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGG QVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNK KIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDT SPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLAttorney Docket No. 772157: DABX-002PCTSEQ IDAmino Acid Sequence Identifier NO MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKD GTGSDDKKAKGADGKTKIATPRGAAPPGQKGQA NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGStau-441 2 PGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSS(2N4R) AKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGG GKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSV QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVK SEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHK LTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS NVSSTGSIDMVDSPQLATLADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMII QDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVT QARMVSKSKDGTGSDDKKAKGADGKTKIATPRG AAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVtau-3523 AWRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKI(0N3R) GSTENLKFIQPGGGKVQIVYKPVDLSKVTSKCGSL GNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDN ITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIV YKSPWSGDTSPRHLSNVSSTGSIDMVDSPQLATL ADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVS KSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQ KGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGY SSPGSPGTPGSRSRTPSLPTPPTREPKKVAWRTPP tau-381 4KSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLK (1N3R) HQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKP GGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGG GNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVS GDTSPRFILSNVSSTGSIDMVDSPQLATLADEVSAS LAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKD GTGSDDKKAKGADGKTKIATPRGAAPPGQKGQAtau-410 5 NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGS(2N3R) PGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSS AKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGG GKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQV EVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRAttorney Docket No. 772157: DABX-002PCTSEQ IDAmino Acid Sequence Identifier NO HLSNVSSTGSIDMVDSPQLATLADEVSASLAKQG L MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVT QARMVSKSKDGTGSDDKKAKGADGKTKIATPRG AAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKS GDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKV AWRTPPKSPSSAKSRLQTAPVPMPDLKNVKSKI tau-3836GSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSK (0N4R) DNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNI HHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITFI VPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKS PVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADE VSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVS KSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQ KGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGY SSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPtau-4127 KSPSSAKSRLQTAPVPMPDLKNVKSKIGSTENLK(1N4R) HQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVP GGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGG QVEVKSEKLDFKDRVQSKIG SLDNITHVPGG GNK KIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDT SPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0043] 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 non-phosphorylated tau or non-p-tau. Phosphorylated tau (also referred to as p-tau or ptau) may refer to a tau isoform where at least a residue is phosphorylated. In some embodiments, the residue is an amino acid, multiple amino acids, or a sequence of amino acids. For example, p-tau-217 can refer to a tau isoform where the amino acid residue 217 is phosphorylated, p- tau- 181 can refer to a tau isoform where the amino acid residue 181 is phosphorylated, p-tau 205 can refer to a tau isoform where the amino acid residue 205 is phosphorylated, p-tau 231 can refer to a tau isoform where the amino acid residue 231 is phosphorylated, and the like. Table 2 lists an exemplary phosphorylated tau isoform (e.g., tau-441 where the amino acid residue 217 is phosphorylated). The phosphorylated threonine residue is underlined and starred.Attorney Docket No. 772157: DABX-002PCT

[0044] Table 2: Exemplary Phosphorylated Tau IsoformSEQ IDAmino Acid Sequence Identifier NO MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMH QDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDA KSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKD GTGSDDKKAKGADGKTKIATPRGAAPPGQKGQA tau-441 with NATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGS amino acid 8 PGT*PGSRSRTPSLPTPPTREPKKVAVVRTPPKSPS residue 217 SAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPG phosphorylate GGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGS d VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEV KSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETH KLTFRENAKAKTDFIGAEIVYKSPVVSGDTSPRFILSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

[0045] 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.

[0046] In some embodiments, the p-tau includes amino acid residues with phosphorylation in the proline-rich domain of tau, such as threonine 217, threonine 181, threonine 205, threonine 231, threonine 153, threonine 175, threonine 212, serine 184, serine 185, serine 191, serine 198, serine 199, serine 202, serine 208, serine 210, serine 214, serine 235, serine 237, serine 238, and tyrosine 197.

[0047] In some embodiments, the p-tau includes amino acid residues with phosphorylation within other tau domains, such as tyrosine 18, serine 46, threonine 50, serine 69, threonine 71, serine 113, threonine 123, serine 258, serine 262, serine 289, serine 356, tyrosine 394, serine 396, serine 400, threonine 403, serine 404, serine 409, serine 412, serine 413, threonine 414, serine 416, serine 422, threonine 427, serine 433, and serine 435.

[0048] Phosphorylated tau has been proposed as providing a more accurate identification of AD due to the presence of hyperphosphorylated tau in neurofibrillary tangles. Wattmo, C., et al. Cerebro-spinal fluid biomarker levels: phosphorylated tau (T) and total tau (N) as markers for rate of progression in Alzheimer’s disease. BMC Neurol 20, 10 (2020). InAttorney Docket No. 772157: DABX-002PCTparticular, tau phosphorylated at threonine 217 (p-tau217) has been shown to have diagnostic value in predicting Alzheimer’s Disease. Plasma levels of p-tau217 have been found to be clinically significant in an early, specific and accurate diagnosis of AD, including the manifestation and progression of AD. Telser, J et al Clin Chim Acta. 2022 Jun 1:531:100-111.II. Definitions

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be presentAttorney Docket No. 772157: DABX-002PCTdepending upon whether or not they materially affect the activity or action of the listed elements.

[0055] 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”.

[0056] 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.

[0057] 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.

[0058] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term “about” as used in connection with a numerical value throughoutAttorney Docket No. 772157: DABX-002PCTthe 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] As used herein, the terms "subject", "individual", and "patient" are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).Attorney Docket No. 772157: DABX-002PCT

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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 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, protein biomarkers of reproductive health, cancer, cardiac health, and blood viruses.. AnAttorney Docket No. 772157: DABX-002PCT"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.

[0067] 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.

[0068] 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” 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.

[0069] As used herein, the term “epitope” refers to a binding site recognized by an affinity molecule. 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.

[0070] Affinity molecules include, but are not limited to, antibodies (including monoclonal antibodies, polyclonal antibodies, antibody fragments, 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 chainAttorney Docket No. 772157: DABX-002PCTvariable 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] 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. Calibrator Dipeptides

[0073] Tau is one of the proteins thought to be closely associated with the hallmark abnormalities of AD - neurofibrillary tangles composed of hyperphosphorylated tau aggregates and amyloid plaques. Amyloid PET scans - which allow for the visualization of amyloid buildup in the brain - have been used to confirm AD diagnoses and distinguish it from other types of dementia. Measurements of tau and phosphorylated tau in CSF has also been demonstrated to serve as indicators of AD. Assay targeting phosphorylated tau in plasma, including those targeting p-taul81, p-tau217 and p-tau231, have also demonstrated the ability to detect and to differentiate Alzheimer’s disease from other neurodegenerative diseases, showing correlation to CSF tau and Amyloid PET in diagnosing AD.

[0074] Calibrator dipeptides are molecules generally comprising two amino acids, amino acid chains, or peptides, linked together. These synthetic calibrator dipeptides may be used as reference standards in various laboratory techniques or to calibrate instruments, such as automated analyzers, which helps to ensure reproducible results. One aspect of the disclosure includes a calibrator dipeptide that is used in conjunction with a p-Tau217 diagnostic plasma assay.

[0075] One embodiment includes a calibrator dipeptide with a first peptide and a second peptide, with a linker connecting the first peptide to the second peptide. In some aspects, the linker comprises a polymer or an amino acid linker. In some embodiments, the amino acid linkers include a cysteine-cysteine linker or a histidine-histidine linker. In some embodiments, the polymer linkers include polyethylene glycol (PEG), poly( vinyl pyrrolidone) (PVP), poly(2-oxazoline)s, polysarcosine (pSar), zwitterionic polymers, polyacrylamides, polyamino acids, or a combination thereof. In some aspects, the linker includes Polyethylene Glycol 4 (PEG4),Attorney Docket No. 772157: DABX-002PCTPolyethylene Glycol 2 (PEG2), Polyethylene Glycol 3 (PEG3), Polyethylene Glycol 5 (PEG5), Polyethylene Glycol 6 (PEG6), or combinations thereof.

[0076] In an aspect, the first peptide is at least 75% identical to an amino acid sequence of a first epitope of Tau and second peptide at least 75% identical to an amino acid sequence of a second epitope of Tau.

[0077] In some aspects, the first peptide is at least 80% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 81% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 82% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 83% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 84% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 85% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 86% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 87% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 88% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 89% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 90% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 91% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 92% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 93% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 94% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 95% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 96% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 97% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 98% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 99% identical to the amino acid sequence of a first epitope of Tau. In some aspects, the first peptide is at least 100% identical to the amino acid sequence of a first epitope of Tau.

[0078] In some aspects, wherein the first epitope of Tau is within 40 amino acids of the N-terminus of Tau. In a non-limiting example, the first epitope of Tau comprises amino acids 6-15, 11-20, 16-25, 21-30, 26-35, or 31-40 of SEQ ID NO: 2 or amino acids 1-20, 6-25, 11-30, 16-35, or 21-40 of SEQ ID NO: 2.Attorney Docket No. 772157: DABX-002PCT

[0079] In some aspects, the second peptide is at least 80% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 81% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 82% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 83% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 84% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 85% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 86% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 87% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 88% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 89% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 90% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 91 % identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 92% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 93% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 94% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 95% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 96% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 97% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 98% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 99% identical to the amino acid sequence of a second epitope of Tau. In some aspects, the second peptide is at least 100% identical to the amino acid sequence of a second epitope of Tau.

[0080] In some aspects, the second epitope of Tau comprises phosphorylated threonine 217 and in certain embodiments the second epitope of Tau is within 20 amino acids of phosphorylated threonine 217.

[0081] In a non-limiting examples second epitope of Tau includes amino acids 209-218, 210-219, 211 -220, or 212-221 of SEQ ID NO: 8 or 199-218, 200-219, 201 -220, or 202-221 of SEQ ID NO: 8.

[0082] Table 3 lists non-limiting examples of calibrator dipeptide sequences.Attorney Docket No. 772157: DABX-002PCT

[0083] Table 3: Calibrator Dipeptide SequencesSEQ ID NO Amino Acid Sequence9 GTYGLGDRKDQGGYTMHQDQ10 SPGTPGSRSRTPSLP(phosphoT)PPTR11 GTYGLGDRKDQGGYTMHQDQ-PEG 4- SPGTPGSRSRTPSLP(phosphoT)PPTR12 Ac-GTYGLGDRKDQGGYTMHQDQ-PEG 4-SPGTPGSRSRTPSLP(phosphoT)PPTR-Amide

[0084] In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 81% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 82% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 83% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 84% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 86% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 87% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is atAttorney Docket No. 772157: DABX-002PCTleast 92% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of the calibrator dipeptide has an amino acid sequence that is at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

[0085] In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 10 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 11 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 12 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 13 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 14 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 15 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 16 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibrator dipeptide comprises at least about a 17 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the first peptide of a calibratorAttorney Docket No. 772157: DABX-002PCTdipeptide comprises at least about a 18 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 9.

[0086] In some embodiments, the first peptide comprises amino acids 1-10, 5-15, or 10-20 ofSEQ ID NO: 2.

[0087] In some embodiments, the first peptide has N-terminal acetylation. In some embodiments, the N-terminal region is within 40 amino acids of the N-terminus. In some embodiments, the first peptide of the calibrator dipeptide includes amino acids 6-15, 11-20 16-25, 21-30, 26-35, or 31-40 of SEQ ID NO: 2 or amino acids 1-20, 6-25, 11-30, 16-35, or 21-40 ofSEQ ID NO: 2.

[0088] In some embodiments, the calibrator dipeptide includes a second peptide of residues from the (p-tau)217 region of Tau. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 81 % identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 82% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 83% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 84% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 86% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 87% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of theAttorney Docket No. 772157: DABX-002PCTcalibrator dipeptide has an amino acid sequence that is at least 91 % identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of the calibrator dipeptide has an amino acid sequence that is at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

[0089] In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 10 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 11 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 12 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 13 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 14 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 15 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 16 amino acid sequenceAttorney Docket No. 772157: DABX-002PCTidentical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 17 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the second peptide of a calibrator dipeptide comprises at least about a 18 amino acid sequence identical to a section of the amino acid sequence set forth in SEQ ID NO: 10.

[0090] In some embodiments, the second peptide includes amino acids 7-17, 8-18, 9-19, or 10-20 of SEQ ID NO: 10.

[0091] In some embodiments, the second chain has C-terminal amidation. In one embodiment, the second chain includes amino acids within 40 amino acids of tau when the threonine 217 is phosphorylated. In another embodiment, the second chain contains a sequence of amino acids from within 20 amino acids of tau when the threonine 217 is phosphorylated. In another embodiment, the second chain of the calibrator dipeptide includes amino acids 209-218, 210-219, 211-220, or 212-221 of SEQ ID NO: 8 or amino acids 199-218, 200-219, 201-220, or 202-221 of SEQ ID NO: 8.

[0092] One aspect of the disclosure includes a calibrator dipeptide that has an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NOs: 11 or 12.

[0093] In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 81 % identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 82% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 83% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 84% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 86% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 87% identicalAttorney Docket No. 772157: DABX-002PCTto the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

[0094] In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 80 % identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 81 % identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 82% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 83% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 84% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQAttorney Docket No. 772157: DABX-002PCTID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 86% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 87% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 88% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the calibrator dipeptide has an amino acid sequence that is at least 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0095] In various aspects, the calibrator dipeptide may be formulated into a composition. In some embodiments, is a liquid, an aqueous preparation, a solution, a suspension, a power, or a lyophilized preparation. One example of a preparation where the calibrator dipeptide may be present in both solution and suspension is a liquid preparation comprising ammonium sulfate in a concentration such that the dipeptide calibrator is in solution and in suspension. In other embodiments, the calibrator dipeptide composition can be made by aggregating, precipitating, or crystallizing the calibrator from a liquid preparation. The calibrator dipeptide so prepared can be suspended or solvated in a suitable buffer for use in an immunoassay.Attorney Docket No. 772157: DABX-002PCT

[0096] The calibrator dipeptide may be present in any suitable concentration. Suitable concentrations include a concentration from about 1 fg / mL to 1 mg / mL. For example, the levels of p-tau217 in blood may be in the AttoMole, Milli-intemational Unit (mIU), pg, or fg range. As such, the suitable concentrations for the calibrator dipeptide composition should include concentrations in those ranges.

[0097] In some embodiments, the calibrator dipeptide composition is used to create a set of standard solutions for preparing a calibration curve. In an aspect, a first aliquot of a calibrator dipeptide composition or a first solution comprising the calibrator dipeptide having a first concentration is retrieved. This concentration is the highest concentration and may also be referred to as the “stock” solution. A second aliquot of a calibrator dipeptide composition or a second solution comprising the calibrator dipeptide having a second concentration is also retrieved. The second solution may be formed by diluting the stock solution or it may be provided in a kit which contains multiple concentrations of the same calibrator dipeptide composition. In some aspects, the calibration curve is prepared using at least 3 solutions of the calibrator dipeptide, each having a different concentration. In some aspects, the calibration curve is prepared using at least 4 solutions of the calibrator dipeptide, each having a different concentration. In some aspects, the calibration curve is prepared using at least 5 solutions of the calibrator dipeptide, each having a different concentration. In some aspects, the calibration curve is prepared using at least 6 solutions of the calibrator dipeptide, each having a different concentration.

[0098] A kit according to an aspect of this disclosure may include calibrators in liquid preparations that are either concentrated or dilute. The kit can also comprise two or more containers, wherein the concentration of calibrator dipeptide in the containers varies. The kit can include two or more calibrators dipeptide in a single container, with each calibrators dipeptide independently at the same or different concentrations.

[0099] The kit can further comprise instructions for its use in any suitable format. The instructions can comprise any of the teachings disclosed herein, as well as information known in the art for carrying out, for example, tests using the components of the kits described herein.

[0100] Depending on the calibration curve desired, the first concentration of calibrator dipeptide may be at least IX greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 2X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixthAttorney Docket No. 772157: DABX-002PCTconcentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 3X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 4X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 5X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 6X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 7X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 8X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 9X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide. In an aspect, the first concentration of calibrator dipeptide is at least 10X greater than the second concentration (or third concentration, fourth concentration, fifth concentration, sixth concentration, etc.) of calibrator dipeptide.

[0101] In an aspect, the first concentration of calibrator dipeptide is at least 100 fg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 500 fg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 1 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 2 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 5 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 10 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 20 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 50 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 100 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 200 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 500 pg / mL. In an aspect, the first concentration of calibrator dipeptide is at least 1 ng / mL.

[0102] The calibrator dipeptide composition may include an ionic surface charge modifier that comprises a charged polymer. The ionic surface charge modifier may compriseAttorney Docket No. 772157: DABX-002PCTa cationic detergent, a zwitterion, a fatty acid, a charged lipid, a phospholipid, a sulfolipid, an anionic detergent, or the salt of a fatty acyl sulfate anion. The ionic surface charge modifier may be derived from natural sources, or synthetic. In a specific embodiment, the ionic surface charge modifier is sodium dodecyl sulfate (SDS). In various embodiments, two or more ionic surface charge modifiers (such as those selected from the aforementioned group) may be used together in equal or varying ratio with respect to one another.

[0103] In various embodiments, the calibrator dipeptide composition comprises at least one stabilizer. Suitable stabilizers include common stabilizers such as, for example, glycerol: albumins such as bovine serum albumin and human albumin; amino acids such as arginine; commercially available stabilizers such as GUARD CHOICE, a proprietary formulation. The stabilizer can be added, if desired, after the dipeptide has been exposed to the ionic surface charge modifier. In various embodiments where the calibrator dipeptide is heated in the presence of the ionic surface charge modifier, the stabilizer may be present while the calibrator dipeptide is exposed in a denatured state to the ionic surface charge modifier. In other embodiments, the stabilizer is added after the calibrator dipeptide has been exposed in a denatured state to the ionic surface charge modifier.

[0104] In some aspects, the calibrator dipeptide is used to calibrate an automated analyzer. In certain embodiments, the automated analyzer may be a clinical chemistry or an immunoassay analyzer.

[0105] When being used to calibrate an automated analyzer, the calibrator dipeptide or a composition or solution containing the calibrator dipeptide may be used in conjunction with an immunoassay, such as a p-tau217 immunoassay.

[0106] One aspect of the disclosure includes calibrating the immunoassay analyzer using at least a first solution comprising the calibrator dipeptide having a first concentration of the calibrator dipeptide and at least a second solution comprising the calibrator dipeptide having a second concentration of the calibrator dipeptide; wherein the first concentration is greater than the second concentration. In this aspect, the calibration comprises generating a calibration curve.

[0107] A quantitative immunoassay may then be performed on a portion of the sample from the subject, wherein the quantitative immunoassay generates at least one sample value. The concentration of p-tau 217 in the sample may then be determined by comparing the at least one generated sample value to the calibration curve.Attorney Docket No. 772157: DABX-002PCT

[0108] In an embodiment, at least one concentration of a calibrator dipeptide or a composition or solution containing the calibrator dipeptide is exposed to a capture affinity molecule capable of binding tau or capable of recognizing an epitope of tau forming a reaction mixture. In some aspects, the capture affinity molecule only binds to the calibrator dipeptide when it comprises a residue subject to phosphorylation when the residue is phosphorylated. In some aspects, the residue is an amino acid residue. In some aspects, the amino acid residue is located in the proline -rich domain of tau. In some embodiments, the amino acid residue is threonine 217, threonine 181, threonine 205, threonine 231, threonine 153, threonine 175, threonine 212, serine 184, serine 185, serine 191, serine 198, serine 199, serine 202, serine 208, serine 210, serine 214, serine 235, serine 237, serine 238, tyrosine 197, tyrosine 18, serine 46, threonine 50, serine 69, threonine 71, serine 113, threonine 123, serine 258, serine 262, serine 289, serine 356, tyrosine 394, serine 396, serine 400, threonine 403, serine 404, serine 409, serine 412, serine 413, threonine 414, serine 416, serine 422, threonine 427, serine 433, or serine 435.

[0109] In some embodiments, the capture affinity molecule only binds to the calibrator dipeptide when the threonine 217 is phosphorylated (i.e., p-tau217).

[0110] In an embodiment, at least a portion of the reaction mixture is exposed to a detector affinity molecule, wherein the detector affinity molecule is capable of binding or recognizing an epitope of tau comprising the N-terminus of tau. A detection reaction is then conducted, which generates a detection signal which is then recorded.

[0111] In an embodiment, a first solution containing a first concentration of calibrator dipeptide is mixed with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a first reaction mixture. The first reaction mixture is mixed with a reagent comprising a detection molecule, generating a first detection mixture. Using a detector arrangement, a reaction in the first detection mixture is detected and generates a first calibrator dipeptide value.

[0112] A second solution containing a second concentration of calibrator dipeptide is mixed with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a second reaction mixture. The second reaction mixture is mixed with a reagent comprising a detection molecule, generating a second detection mixture. Using a detector arrangement, a reaction in the second detection mixture is detected and to generates a second calibrator dipeptide value. The first calibrator dipeptide value and the second calibrator dipeptide value are then used to generate the calibration curve. DependingAttorney Docket No. 772157: DABX-002PCTon the calibration curve desired, this method may then be repeated for two or more, or three or more, or several different concentrations of the calibrator dipeptide to generate a suitable calibration curve. For example, values may be generated for several concentrations of calibrator dipeptide, including, but not limited a third concentration of calibrator dipeptide, a fourth concentration of calibrator dipeptide, a fifth concentration of calibrator dipeptide, or a second concentration of calibrator dipeptide.

[0113] In some aspects, the reaction generated when the reaction mixture is mixed with the reagent comprising a detection molecule 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 luminol-based reaction, a 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 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 second reaction mixture triggering a chemiluminescent reaction.IV. Automated Analyzer and Systems for Detecting p-tau217

[0114] In an embodiment, the method includes detecting a presence of p-tau217 in one or more samples obtained from the subject using an immunoassay analyzer and performing a quantitative immunoassay on a portion of the sample from the subject to determine a concentration of p-tau 217 in the sample. In an aspect of the methods disclosed herein, the immunoassay analyzer may be calibrated using the calibrator dipeptide prior to performing the quantitative immunoassay.

[0115] In an aspect, an immunoassay analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analytic unit, an incubator station, a washing station, a read station, and reagent storage. In addition, the immunoassay analyzer may include a pipettor arrangement with at least one sample pipettor and at least one reagent pipettor, and at least one transport device. In an aspect, the transport device includes mechanisms, such as pick-and-place grippers, which are used to transport sample and reaction vessels among the various modules of the immunoassay analyzer.

[0116] The immunoassay analyzer may include a container carriage device which is configured to hold and carry the containers at various locations in the instrument so that the analytic unit, incubator station, wash station, and read station can use the containers in variousAttorney Docket No. 772157: DABX-002PCTmanners. 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.

[0117] The read station may include a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.

[0118] 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.Attorney Docket No. 772157: DABX-002PCT

[0119] In certain embodiments, the sample vessels include a barcode label that uniquely identifies the sample vessel in the immunoassay analyzer. The barcode label also may include alphanumeric characters that correspond to the barcode identification information. The immunoassay analyzer may include at least optical reader, such as a barcode scanner. In one embodiment, the optical readers are area scan cameras that provide a two-dimensional image of the barcode and / or sample vessel. In another embodiment, optical readers are area scan cameras that provide a three-dimensional image of the barcode and / or sample vessel.

[0120] After being fed into the onload section, the sample containers may be moved into a presentation section of the sample presentation unit. In certain embodiments, the sample presentation unit is maintained at between about 4.5°C to 14°C. The transfer station may receive primary sample containers from the sample presentation unit from a transport device. Primary sample containers delivered to the transfer station from the transport device may be initially processed in different ways. For example, the primary sample container may temporarily remain at the transfer station while the sample provided within is aspirated by the sample pipettor at the transfer station. Following such aspiration, the primary sample container may be expelled from the automated analyzer at the offload station of the sample presentation unit, or may be passed on to the transport device for further processing. The instrument may also contain a sample retention unit configured to receive sample retention vessels and store, analyze, or otherwise process samples retained within the sample retention vessels. A “primary sample container” loaded into the automated analyzer with a sample may also be considered a “sample retention vessel” when placed in a sample retention unit, such as a storage unit. As used herein, “containers” or “vessels” are analogous, and can be of various types, such as specimen tubes (also referred to herein as sample tubes) and pipettor tips, such as micro or disposable tips. In certain embodiments, the vessels are tubes with diameters between about 12mm to about 16mm and / or heights between about 75mm to about 100mm. In certain embodiments, the vessels are cups with volumes of about 0.5mL, alternatively about 1.0mL, alternatively about 1.5mL, alternatively about 2.0mL, alternatively about 2.5mL, or alternatively about 3.0mL.

[0121] In an aspect, the analytic unit is configured to receive and analyze samples. In an aspect, the analytic unit configured to perform an immunoassay. In certain embodiments, the analytic unit includes the pipettor arrangement. The pipettor arrangement may be configured to aliquot, aspirate, and dispense fluidic substances into various vessels, including, but not limited to sample vessels, diluent vessels, reagent vessels, and reaction vessels. Fluidic substances are substances that have fluidic characteristics. In some embodiments, the fluidicAttorney Docket No. 772157: DABX-002PCTsubstance is a single fluidic substance. In other embodiments, the fluidic substance is a mixture of a plurality of substances.

[0122] The pipettor arrangement may contain at least one, two, three, or four reagent pipettors used to mix reagents with sample aliquots for an assay. The pipettor arrangement may also contain at least one, two, three, or four sample pipettors used to transfer sample aliquots for an assay. In some instances, the pipettor arrangement contains one sample pipettor. In some instances, the pipettor arrangement contains one sample pipettor and four reagent pipettors. In an aspect, the reagent pipettors may be arranged as dual reagent pipetting stations and are independent to each other, each having its own fluid pumps and valves, wash towers, reaction vessel carriages, and pipettor. A sample aliquot may be transferred from a sample retention vessel into a reaction vessel using the sample pipettor in order to mix the sample aliquot with one or more reagents. In an aspect, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 10 μL. In an embodiment, the at least one reagent pipettor and at least one sample pipettor are configured to aspirate and / or dispense less than about 9.9 μL, alternatively less than about 9.5 μL, alternatively less than about 8.0 μL, alternatively less than about 7.0 μL, alternatively less than about 6.0 μL, alternatively less than about 5.0 μL, alternatively less than about 4.0 μL, alternatively less than about 3.0 μL, alternatively less than about 2.0 μL, alternatively between than about 9.9 μL and 2.0 μL.

[0123] 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.

[0124] In an aspect, the immunoassay analyzer includes a reagent pack configured to hold a plurality of reagent vessels. A “reagent pack” may include any suitable container that can store a reagent. An example of a reagent pack can include a generally rectangular elongated body formed to include multiple reagent vessels including one or more large reagent vessels, and one or more relatively smaller reagent vessels, as well as features to facilitate handling and automation. US Patent No. 9,519,000, which is incorporated by reference in its entirety herein, discloses non- limiting examples of a reagent pack that may be used in an aspect of the invention.Attorney Docket No. 772157: DABX-002PCT

[0125] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances.

[0126] In some embodiments a reagent pack may supplied with empty or partially filled reagent vessels, to which reagents are subsequently transferred from bulk containers, such as bottles. Individual reagent vessels may differ in dimension to accommodate the requirements of an assay type. Factors that can determine the size of a reagent vessel include the number of uses desired for the reagent pack type, concentration-dependent stability issues with reagent components, and the need to minimize the volume of the final reaction mixture. As noted above, in some embodiments, each reagent pack can include a large reagent vessel and a plurality of small reagent vessels. Each reagent vessel may be large enough to accommodate a microtip or disposable tip (i.e., dispo-tip) of a reagent pipettor used to remove a volume of reagent for use in an assay. In certain embodiments, the reagent pack may be maintained at a temperature of between about 4°C to 10°C.

[0127] A “reagent vessel” may refer to a vessel, unit, fluid container, or the like that is configured to store reagents. In an embodiment, the reagent vessels include an elastomeric selfsealing membrane. An elastomeric self-sealing membrane may be a polymer, such as polypropylene, which is able to regain its original shape when pierced. For some embodiments, the elastomeric membrane can be a thermoplastic elastomer with hardness of 30-40 durometer (Shore) A. In other embodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A. Elastomers deform sufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.

[0128] 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.) andAttorney Docket No. 772157: DABX-002PCTproduces 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).

[0129] In methods where multiple samples are analyzed, the elastomeric self-sealing membrane allows for aspiration of the reagents without concern of evaporation. In some embodiments, the reagent pack further includes containment walls arranged between the reagent vessels. In an aspect, the reagent pack may include at least one reagent vessel, alternatively at least two reagent vessels, alternatively at least three reagent vessels, alternatively at least four reagent vessels, alternatively at least five reagent vessels, or alternatively at least ten reagent vessels. In an embodiment, the immunoassay analyzer further includes a reagent storage unit, wherein the reagent pack is housed in the reagent storage unit.

[0130] At operation, a fluidic substance is dispensed to a reaction vessel. Examples of the fluidic substance include a sample, diluent, reagent, substrate, or any combination thereof, as described herein. In some embodiments, the reaction vessel already contains other fluidic substances, such as a sample, and after a fluidic substance is dispensed to a reaction vessel, the fluidic substance is mixed with the other fluidic substances in the reaction vessel. The mixing can be performed with a stirrer in direct contact with the fluidic substances, an ultrasonic probe in direct or indirect contact with the fluidic substances, or any other suitable mixing apparatus. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For instance, a reagent pipettor may be outfitted with a tip that allows it to perform ultrasonic mixing of a reagent in a reagent pack before aspirating it for transport to a reaction vessel, thereby ensuring that the aspirated reagent would not be impacted by any settling that may have taken place in the reagent pack. Sample pipettors may similarly be specialized.

[0131] The transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator. In some embodiments, the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator. Further, the transfer unit can transfer one or more reaction vessels from the incubator to the reagent carriage unit. The transfer unit can also remove from the reaction vessels that have been read or completed the incubator.

[0132] 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. 772157: DABX-002PCT

[0133] 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.

[0134] At operation, a sample and a reagent are dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, the sample and the reagent interact. The resulting “first reaction mixture” is a result of the incubation between the sample and the reagent. The reagent may include a specific-binding reagent, such as an affinity molecule specific to the analyte being analyzed by the immunoassay analyzer. In a non- limiting example, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0135] At operation, a reagent, which is different from the reagent used in the first reaction mixture, is added to the first reaction mixture and mixed. The mixture is then transferred to the incubator. During incubation, the first reaction mixture and the reagent interact. The resulting “second reaction mixture” is a result of an incubation between the first reaction mixture and the reagent. The reagent may include a detection molecule, such as an alkaline phosphatase (AP)-conjugated secondary antibody, or a labeled antibody. In a nonlimiting example, the incubation time of the second reaction mixture is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0136] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the immunoassay analyzer. In an embodiment, the wash station is configured to wash away at least some of the unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The wash station may be configured to perform a set number of wash actions depending on the assay. The wash station may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at leastAttorney Docket No. 772157: DABX-002PCTthree wash actions, alternatively configured to perform at least four wash actions, or alternatively configured to perform at least five 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.

[0137] 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).

[0138] In an aspect, the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle. In some instances, magnetic beads (also known as magnetic particles, paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (~20-30 nm), such as magnetite. On the surface, the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding. Antibodies targeting the analyte of interest can be covalently coupled to the surface of the magnetic bead. In some embodiments,Attorney Docket No. 772157: DABX-002PCTthere is about 2 μg or antibody per mg of magnetic bead. In some embodiments, there is about 3 μg or antibody per mg of magnetic bead. In some embodiments, there is about 4 μg or antibody per mg of magnetic bead. In some embodiments, there is about 5 μg or antibody per mg of magnetic bead. In some embodiments, there is about 6 μg or antibody per mg of magnetic bead. In some embodiments, there is about 7 μg or antibody per mg of magnetic bead. In some embodiments, there is about 8 μg or antibody per mg of magnetic bead. In some embodiments, there is about 9 μg or antibody per mg of magnetic bead. In some embodiments, there is about 10 μg or antibody per mg of magnetic bead. In some embodiments, there is about 12 μg or antibody per mg of magnetic bead. In some embodiments, there is about 13 μg or antibody per mg of magnetic bead. In some embodiments, there is about 14 μg or antibody per mg of magnetic bead. In some embodiments, there is about 15 μg or antibody per mg of magnetic bead.

[0139] 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.

[0140] At operation, the vessel containing the first reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound- free cleaning aspiration nozzle.

[0141] At operation, the vessel containing the second reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid andAttorney Docket No. 772157: DABX-002PCTaspirating 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.

[0142] At operation, a substrate is dispensed into the second reaction mixture and mixed. After a certain reaction time necessary for the substrate and the second reaction mixture to interact. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspirating. The reaction vessel may undergo a series of wash process which may include at least two series of dispensing the rinsing fluid and aspirating the uncollected fluid components, alternatively at least three series, alternatively at least four series, alternatively at least five series. As a result, an unreacted substance or substances in the vessel is removed (e.g., rinsed away) by the bound-free cleaning aspiration nozzle. The resulting detection mixture is transferred to the detector arrangement.

[0143] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions. In some instances, “two-site” or “sandwich” immunoassays employ a first antibody or antibody fragment, which is described as the “capture” antibody, is bound to a solid support, such as magnetic beads or particles disclosed herein, using procedures known in the art. Further, a second antibody or antibody fragment, which is described as the “detection” antibody, is coupled or conjugated with a label, such as the enzymes disclosed herein, using procedures known in the art. The label produces a detectable signal when provided with substrate(s), so that the amount of signal measured corresponds to the amount of detection antibody that is bound to the analyte.

[0144] 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. 7,128,874 and is incorporated herein in its entirety by reference. However, it should be understood that other pick-and-placeAttorney Docket No. 772157: DABX-002PCTmechanism that are capable of transporting sample and reaction vessels among the various modules of the immunoassay analyzer is also contemplated for the purpose of the present invention.

[0145] In an aspect, the immunoassay analyzer includes a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis. In some embodiments, the machine vision apparatus utilizes one or more image capture units to determine whether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether the sample is appropriately prepared so that the analytic unit produces a reliable result. An exemplary machine vision apparatus is described in U.S. Patent No. 11,263,433, which is incorporated by reference herein.

[0146] 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.

[0147] In some aspects, the machine vision apparatus operates to detect any interferents, which can interfere with an analytic procedure and may generate incorrect results in the dispense tip. As described herein, this dispense tip evaluation is configured to determine a quality of a fluidic substance at a dispense tip using a dispense tip image capture unit, and an alignment of the dispense tip with respect to the dispense tip image capture unit.

[0148] 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.

[0149] 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 atAttorney Docket No. 772157: DABX-002PCTa 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.

[0150] 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.

[0151] 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.

[0152] All of the units of the immunoassay analyzer are connected to a controller, which can perform block control of all of the analyzer functions by using, for example, a microcomputer. The controller may contain subunits such as a data processing unit, a communication interface, and others. A controller in accordance with an exemplary embodiment of the present technology may comprise a data processor, a non-transitory computer-readable medium, and a data storage coupled to the data processor. The non-transitory computer-readable medium may comprise code, executable by the data processor, to perform the functions described herein. The data processor may store, for example, data for processing samples, sample data, or data for analyzing sample data.

[0153] 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 executeAttorney Docket No. 772157: DABX-002PCTprogram 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).

[0154] 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.

[0155] 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 predetermined schedule. In some embodiments of the presently claimed technology, the predetermined schedule is a constituent test.

[0156] In an aspect the cycle time of an immunoassay analyzer described herein is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of an immunoassay analyzer to complete its tasks necessary for generating a result. In certain embodiments, the cycle time is about 40 seconds or less, alternatively about 35 seconds or less, alternatively about 30 seconds or less, alternatively about 25 seconds or less, alternatively about 20 seconds or less, or alternatively about 15 seconds. At operation, the sample pipettor can complete its tasks in 8 seconds and the reagent pipettor can complete its tasks in 32 seconds. In this embodiment, to maintain a higher throughput, four reagent pipettors are present in the immunoassay analyzer (32 seconds / 4 = 8 seconds). If the incubation of one of the reaction mixtures is 5 minutes, at least 38 incubation positions are needed to support this incubation time (300 seconds / 8 seconds = 37.5 seconds). In certain embodiments, the immunoassay analyzer has at least 30 incubation positions, alternatively at least 40 incubation positions, alternatively at least 50 incubation positions, alternatively at least 60 incubation positions, alternatively at least 70 incubation positions, alternatively at least 80 incubation positions, alternatively at least 90 incubation positions, alternatively at least 100 incubation positions, alternatively at least 125 incubation positions, alternatively at least 150 incubation positions, alternatively at least 175 incubation positions, or alternatively at least 200 incubation positions.

[0157] In an aspect, the time to first result (TTFR) of an immunoassay analyzer described herein is about 60 minutes or less. The “TTFR” is a measure of time from when theAttorney Docket No. 772157: DABX-002PCTsample 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.

[0158] In an embodiment, the reagent pack may be configured to accommodate sufficient volumes of reagents for multiple instances of an assay. In some embodiments, each reagent pack includes reagents for about 20 to about 100 instances of an assay and in some cases about 50 instances. 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 20 instances of an p-tau217 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 20 instances of an p-tau217 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 20 instances of an p-tau217 assay. 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 20 instances of an p-tau217 assay.

[0159] In an aspect, the first reagent includes at least one affinity molecule configured to bind to at least one portion p-tau217. Depending on the assay, the affinity molecule may be an antibody, a monoclonal antibody, a polyclonal antibody, a synthetic antibody mimic, an aptamer, an affimer, DARPins, or oligonucleotides or peptides that bind to at least one epitope of p-tau217. In a non- limiting example the affinity molecule is an antibody and the method further comprises exposing the plasma sample to a first antibody which binds to a first p-tau217 epitope and a second antibody which binds to a second p-tau217 epitope.

[0160] The “first reaction mixture” is a result of an incubation between the sample and the first reagent. During the incubation, the sample and the first reagent interact. In an embodiment, the affinity molecule of the first reagent binds with the p-tau217. In a nonlimiting example, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, or alternatively at least about 50 minutes.Attorney Docket No. 772157: DABX-002PCT

[0161] In an embodiment, the method includes aspirating 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. In an aspect, the second reagent includes a detection molecule. The detection molecule, may be, for example, an alkaline phosphatase (AP)-conjugated secondary antibody.

[0162] The “second reaction mixture” is a result of an incubation between the first reaction mixture and the second reagent. During the incubation, the first reaction mixture and the second reagent interact. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule of the first reagent (which is bound with the p-tau217 in the plasma sample.) In an aspect, the incubation time of the second reaction mixture is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0163] In an embodiment, the method includes aspirating a portion of a substrate formulation from at least one reagent vessel and dispensing the aspirated substrate formulation into the reaction vessel, generating a detection mixture. In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. The term “chemiluminescent compound” refers to a compound that produces chemiluminescence in the presence of a phosphatase enzyme and oxygen under appropriate conditions as provided herein. In a non-limiting example, the substrate formulation includes a chemiluminescent compound of formula I or a salt thereof:

[0165] whereinAttorney Docket No. 772157: DABX-002PCT

[0166] A is C1-6haloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, C1-6alkyl, C1-6alkoxy, C1-6haloalkyl, C(O)R15, CN or NO2 substituents;

[0167] Ri is selected from the group consisting of C5-14aryl, C1-6alkyl, C1-6haloalkyl, and C5-14aralkyl groups;

[0168] R7-R14are independently H, C1-6alkoxy, halo, C1-4alkyl, or R7or R8-R9or R9-R10or R11-R12or R12-R13or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;

[0169] R15is C1-6alkyl;

[0170] 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;

[0171] Z is O or S; and

[0172] n is 0, 1, or 2;

[0173] a cationic aromatic compound (CAC);

[0174] a background reducing agent; and

[0175] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0176] 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. 6,457,727, 6,900,571, 6,139,782, 6,218,137, 6,270,695, 6,296,787, and 10,703,971 each of which is incorporated by reference herein.

[0177] At operation, the chemiluminescent substrate is added to the vessel with the second reaction mixture and light generated by the reaction is measured with a luminometer. The amount of analyte in the sample is then determined from a stored, multi-point calibration curve. In an embodiment, the detector can generate an output signal that can be processed to generate a relative light unit (“RLU”) value (i.e., an output response) indicating a result of the assay. For example, a larger RLU value indicates more light, which indicates a larger amount of the analyte in the biological sample than a smaller RLU value indicates.

[0178] 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 inAttorney Docket No. 772157: DABX-002PCTan 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.

[0179] In one aspect, the substrate formulation comprises 0.01 mM-50 mM compound I, 0.01-200 μM cationic aromatic compound, 1 μM -10 mM background reducing agent, 0.05-20 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.

[0180] In one aspect, the substrate formulation comprises 0.05 mM-10 mM compound I, 0.05-50 μM cationic aromatic compound, 10 μM-1000 μM background reducing agent, 0.1 to 10 g / L ether- linked non-ionic surfactant or hydrophilic polymer, 0.1 to 5 g / L anionic surfactant, and an amine buffer at from 0.05M to 0.5M and at pH 8-11.

[0181] In one aspect, the substrate formulation comprises 0.1 mM-5 mM compound I, 0.1-25 μM cationic aromatic compound, 50 to 500 μM 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.

[0182] In one aspect, the compound I has the formula

[0183]

[0184] In an aspect, the immunoassay analyzer includes a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect light or luminescence, for example chemiluminescence. The detector may be a luminescence detector, a chemiluminescence detector, a luminometer, or a photomultiplier-based detection instrument. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U.S. Patent No. 11,604,146, which is incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.Attorney Docket No. 772157: DABX-002PCT

[0185] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration of p-tau217 in the plasma sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of p-tau217 in the plasma sample. 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 plasma sample than a smaller RLU value indicates.

[0186] The claimed configuration allows for the simultaneous performance of at least two assays for a plurality of plasma samples, alternatively at least three assays for a plurality of plasma samples, or at least four assays for a plurality of plasma samples. Depending on the analysis desired the plasma 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.

[0187] In certain embodiments, at least one of the assays is p-tau217 assay, alternatively at least two of the assays are p-tau217 assays, alternatively at least three of the assays are p-tau217 assays, or alternatively at least four of the assays are p-tau217 assays.

[0188] In certain embodiments, the method may include detecting at least one additional biomarker associated with ovarian aging. In some embodiments, at least one of the assays is p-tau217 assay and at least one of the assays is a second analyte assay, wherein the second analyte is inhibin B or follicle-stimulating hormone (FSH).

[0189] In a non-limiting example, if multiple assays for multiple biomarkers are being performed, the first pipettor may be configured to aspirate a reagent from a reagent vessel comprising a first affinity molecule (e.g., a p-tau217 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 affinity molecule (e.g., second analyte antibody) and dispense into a second reaction vessel. Depending on the analysis desired, the third and fourthAttorney Docket No. 772157: DABX-002PCTpipettor 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.

[0190] The disclosed simultaneous and / or selective operation of the pipettors allows for a high-throughput analysis. In some embodiments, the method is configured to analyze at least about 200 plasma samples / hr., alternatively at least about 300 plasma samples / hr., alternatively at least about 400 plasma samples / hr, alternatively at least about 440 plasma samples / hr. or alternatively at least 500 plasma samples / hr.

[0191] In an aspect of the methods disclosed herein, a biological sample is analyzed for p-tau217 using a high-throughput immunoassay analyzer disclosed herein. In an embodiment, the method includes 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 an immunoassay analyzer. In an aspect, the biological sample is serum, whole blood, and / or plasma.

[0192] In an embodiment, the method includes aspirating, using a reagent pipettor, a portion of a first reagent from at least one reagent vessel and dispensing the aspirated reagent into the reaction vessel. 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 20 instances of an p-tau217 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 20 instances of an p-tau217 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 20 instances of an p-tau217 assay. 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 20 instances of an p-tau217 assay.

[0193] In an aspect, the reagent pack is configured to store a volume of reagent required for at least about 20 instances of an additional biomarker 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 20 instances of an additional biomarker 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 20 instances of an additional biomarker assay. In a nonlimiting example, the reagent pack includes at least five reagent vessels, wherein each reagentAttorney Docket No. 772157: DABX-002PCTvessel is independently configured to store a volume of reagent required for at least about 20 instances of an additional biomarker assay.

[0194] In an aspect, a first reagent includes at least one affinity molecule configured to bind to at least one portion of p-tau217. In an aspect, a first reagent includes at least one affinity molecule configured to bind to at least one portion of an additional biomarker.

[0195] In a non-limiting example, the incubation time of the first reaction mixture is at least about 30 minutes, alternatively at least about 40 minutes, or alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0196] In a non-limiting example, the affinity molecule is an antibody and the method further comprises exposing the biological sample to a first antibody which binds to a first p-tau217 epitope and a second antibody which binds to a second p-tau217 epitope.

[0197] In a non-limiting example, the affinity molecule is an antibody and the method further comprises exposing the biological sample to a first antibody which binds to a first additional biomarker epitope and a second antibody which binds to a second additional biomarker epitope.

[0198] In certain embodiments, the first antibody is a capture antibody and the second antibody is a detection antibody. In other embodiments, both antibodies are capture antibodies.

[0199] In an embodiment, an p-tau217 antibody is bound to magnetic bead. In an embodiment, an additional biomarker antibody is bound to magnetic bead.

[0200] Magnetic beads comprising said antibody or antibodies are added to the sample in a reaction vessel along. At least one buffer or other reagents may be added as required by the assay. The mixture is incubated for a period of time necessary for the antibody to bind with the targeted epitope forming a first reaction mixture. A magnetic field is applied to the reaction vessel and the isolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0201] In an embodiment, the method includes, 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. In an aspect, a second reagent includes a detection molecule. The detection molecule, may be, for example, a detection antibody, such as alkaline phosphatase (AP)-conjugated secondary antibody. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule of the first reagent (which is bound with the p-tau217 in the biological sample) or binds to a different p-tau217 epitope. In an embodiment, the detection molecule of the second reagent binds with the affinity molecule ofAttorney Docket No. 772157: DABX-002PCTthe first reagent (which is bound with the additional biomarker in the biological sample) or binds to a different additional biomarker epitope. In an aspect, the incubation time of the second reaction mixture is at least about 5 minutes, alternatively at least about 8 minutes, or alternatively at least about 10 minutes.

[0202] In an embodiment, an p-tau217 antibody conjugated to alkaline phosphatase is added to the first reaction mixture in a reaction vessel. The mixture in incubated for a period of time necessary for the antibody to bind with a second p-tau217 epitope, forming a second reaction mixture. In an embodiment, an additional biomarker antibody conjugated to alkaline phosphatase is added to the first reaction mixture in a reaction vessel. The mixture in incubated for a period of time necessary for the antibody to bind with a second additional biomarker epitope, forming a second reaction mixture. A magnetic field is applied to the reaction vessel and the isolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0203] In an embodiment, the method includes dispensing a substrate formulation into the reaction vessel, generating a detection mixture. In certain embodiments, the substrate formulation comprises compound I, a cationic aromatic compound, a background reducing agent, an ether-linked non-ionic surfactant or hydrophilic polymer, an anionic surfactant, and an amine buffer. A magnetic field is applied to the reaction vessel and the isolated, bead-bound proteins are washed using the washing arrangement. A base number (three) of wash actions is performed with optional one or two additional wash actions.

[0204] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration of p-tau217 in the biological sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of p-tau217 in the biological sample.

[0205] In an embodiment, the method further includes detecting, using a detector arrangement, a presence of a reaction in the detection mixture and determining a presence and / or concentration of an additional biomarker in the biological sample based on the presence of the reaction in the detection mixture. In an aspect, the reaction in the detection mixture generates a chemiluminescent signal, wherein the chemiluminescent signal corresponds to the presence and / or concentration of an additional biomarker in the biological sample.

[0206] Both the immunochemical assays and immunoassays may be “two-site” or “sandwich” assays which employ a first antibody or antibody fragment, which is described asAttorney Docket No. 772157: DABX-002PCTthe “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” 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.

[0207] In an embodiment, the high-throughput immunoassay analyzer further includes an ultrasonic mixing module. In certain embodiments, the first reaction mixture, second reaction mixture, and / or detection mixture is agitated via the ultrasonic mixing module.

[0208] In an aspect, the immunoassay analyzer further comprises a washing arrangement. In an embodiment, the washing arrangement is configured to wash away at least some of the unreacted components from the sample, first reagent, second reagent, or substrate formulation in the first reaction mixture, second reaction mixture, and / or detection mixture. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The washing arrangement may be configured to perform a set number of wash actions depending on the assay. The washing arrangement may also be configured to perform a set number of washes within a predetermined sequence. In certain embodiments, the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, or alternatively configured to perform at least five wash actions.

[0209] In an aspect, the affinity molecule and / or the detection molecule is conjugated to a magnetic bead or a magnetic particle. In an embodiment, the first reaction mixture, second reaction mixture, and / or detection mixture is subjected to a magnetic field prior to detection. Magnetization may be used to retain desired components within a reaction vessel. In certain embodiments, the reaction vessel is moved near one or more magnets after the introduction of the first reagent or second reagent. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The washing arrangement is used to wash the reaction vessel for a predetermined number of times. While washing the magnet(s) retain the magnetic bead(s) or magnetic particle(s) while the unreacted componentsAttorney Docket No. 772157: DABX-002PCTare washed away. In certain embodiments, increasing the numbers of wash actions may result in a better signal to noise ratio and increase the sensitivity of the assay.

[0210] In an aspect the cycle time is about 45 seconds or less. The “cycle time” is the time required for all modules and / or functions of the high-throughput 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.

[0211] In an aspect, the time to first result (TTFR) 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 p-tau217 is determined. In certain embodiments the TTFR is about 55 minutes or less, alternatively about 50 minutes or less, alternatively about 45 minutes or less, alternatively about 40 minutes or less, alternatively about 35 minutes or less, alternatively about 30 minutes or less, alternatively about 25 minutes or less, alternatively about 20 minutes or less, alternatively about 15 minutes or less, or alternatively about 10 minutes or less.

[0212] Additional examples are provided below.

[0213] EXAMPLES

[0214] p-Tau217 Calibrator Dipeptide Performance

[0215] Approximately 100 calibrator dipetides were synthesized using a plurality of different peptides from the N-terminal and p-Tau217 regions of Tau with linkers between the peptide sequences. The lengths of the peptides were varied in the calibrator dipetides along with the precise segment of Tau that was replicated. Affinity testing was performed on the calibrator dipeptide. A calibrator dipeptide was then selected for further use.

[0216] The selected calibrator dipeptide was then used to calibrate an immunoassay analyzer prior to assessing an assay according to the present disclosure for its ability to distinguish between Alzheimer’s Disease (AD) confirmed and normal patient samples.

[0217] The samples tested included 26 normal patient samples (8 young <55 years patients; 18 aged healthy patients >55 years) and 40 Alzheimer’s positive patient samples (confirmed by physician; MMSE scores ranging from 2 to 25).

[0218] A p-tau217 assay as described herein was performed on each sample using a Beckman Coulter Dxl® 9000 immunoassay analyzer. Assay methodology is summarized below:Attorney Docket No. 772157: DABX-002PCT

[0219] Using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor configured to aspirate and dispense less than about 10 µL; (ii) reagent packs configured to store volumes of reagents for at least 20 instances of assays; (iii) an ultrasonic mixer; and (iv) the capability to perform over 5 wash cycles per reaction vessel, the following steps were carried out:

[0220] Paramagnetic particle conjugated p-tau217 antibodies capable of binding to an epitope of p-tau217 were pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a first reaction mixture. A magnetic field was applied to the reaction vessel and the first reaction mixture was washed using a wash buffer to remove any unreacted components.

[0221] A p-tau217 antibody capable of binding to the N-terminus of p-tau217 conjugated to alkaline phosphatase (“ALP”) was added to the reaction vessel containing the first reaction mixture. The reaction vessel was mixed ultrasonically and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed using a wash buffer to remove any unreacted components.

[0222] A substrate according to the present disclosure was added to the reaction vessel, and the signal generated from the resulting reaction was read using a luminometer.

[0223] The above steps were performed using calibrator dipeptides of varying concentrations (2 replicates each), and a calibration curve was constructed and reduced with adequate fit. The mean signal response for each of the calibrator levels is shown below:

[0224] Table 4: Calibrator Levels; Mean RLU responsea. Mean RLUb. Calsc. SO d. 6,628e. SI f. 11,770g- S2 h. 20,806i. S3 j. 45,712k. S4 1. 124,109Attorney Docket No. 772157: DABX-002PCT

[0225] The assay was performed on the Normal (4 replicates each) and AD-confirmed (2 replicates each) patient samples, and the calibration curves were used to correlate the resulting signal produced to concentrations. The resulting calibration curve plot is shown in FIG. 1.

[0226] All patient samples were run on the exemplary assay and were shown to be able to distinguish between Alzheimer’s positive versus normal samples. A box and whisker plot showing p-Tau217 concentrations associated with AD-positive versus normal samples is illustrated in FIG. 2. Alzheimer’s positive population were determined to have a median p- Tau217 concentration of 261 fg / mL, whereas Alzheimer’s normal healthy population were found to have a p-Tau217 concentration of 88 fg / mL, with a median based ratio of AD-positive to normal concentrations of 3.0.

[0227] An analysis of the analytical sensitivity of the exemplary p-Tau217 was also conducted. Within-run CV was calculated from harmonized dose response and plotted relative to mean dose-response, as shown in FIG. 3. A log-log quadratic regression was fit to model overall precision. The LOQs at 20% and 10% CV were then calculated and compared to published performance of other conventional analyzers, as shown below in Table 5.

[0228] Table 5: Within-Run LoQ EstimatesWithin-Run LoQ Estimates (fg / mL)Prototype 20% LoQ 10% LoQDxl® 9000 Immunoassay Analyzer SensitivityPerformancep-Tau217 Assay 19 45Alternative Systems PerformanceConventional 37 60Analyzer 1Conventional 40 135Analyzer 2

[0229] 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 areAttorney Docket No. 772157: DABX-002PCTmutually 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.

Claims

1. Attorney Docket No. 772157: DABX-002PCTCLAIMS1. A calibrator dipeptide comprising:a first peptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 9;a second peptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO: 10; anda linker connecting the first peptide to the second peptide.

2. The calibrator dipeptide of claim 1, wherein the first peptide comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 9; alternatively 99% identical to the amino acid sequence set forth in SEQ ID NO: 9; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 9.

3. The calibrator dipeptide of claim 1 or claim 2, wherein the first peptide comprises at least about a ten amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 9.

4. The calibrator dipeptide of any one of claims 1 to 3, wherein the second peptide comprises an amino acid sequence at least at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 95% identical to the amino acid sequenceAttorney Docket No. 772157: DABX-002PCTset forth in SEQ ID NO: 10; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 10; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 10; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 10.

5. The calibrator dipeptide of any one of claims 1 to 4, wherein the second amino acid chain comprises at least a ten amino acid sequence from the amino acid sequence set forth in SEQ ID NO: 10.

6. The calibrator dipeptide of any one of claims 1 to 5, wherein the first peptide and the second peptide are covalently connected via the linker.

7. The calibrator dipeptide of any one of claims 1 to 6, wherein the linker comprises a polymer or an amino acid linker.

8. The calibrator dipeptide of claim 7, wherein the linker is a polymer and comprises polyethylene glycol (PEG), poly(vinyl pyrrolidone) (PVP), poly(2-oxazoline)s, polysarcosine (pSar), zwitterionic polymers, polyacrylamides, polyamino acids, or a combination thereof.

9. The calibrator dipeptide of claim 8, wherein the linker is a polymer and comprises PEG4, PEG2, PEG3, PEG5, PEG6, or a combination thereof.

10. The calibrator dipeptide of claim 9, wherein linker is a polymer and comprises PEG4.

11. The calibrator dipeptide of claim 7, wherein the linker is an amino acid linker and comprises a cysteine-cysteine linker or a histidine-histidine linker.Attorney Docket No. 772157: DABX-002PCT12. A calibrator dipeptide comprising an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NOs: 11 or 12.

13. The calibrator dipeptide of claim 12, wherein the calibrator dipeptide comprises an amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 11.

14. The calibrator dipeptide of claim 12, wherein the calibrator dipeptide comprises an amino acid sequence is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 12; alternatively at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 12; or alternatively 100% identical to the amino acid sequence set forth in SEQ ID NO: 12.

15. The calibrator dipeptide of any one of claims 1 to 13, wherein the calibrator dipeptide is formulated into a composition.Attorney Docket No. 772157: DABX-002PCT16. The calibrator dipeptide of claim 15, wherein the composition is a liquid, an aqueous preparation, a solution, a suspension, a power, or a lyophilized preparation.

17. The calibrator dipeptide of claim 15 or claim 16, wherein the composition comprises an ionic surface charge modifier, a stabilizer, a salt, a buffer, an excipient, water, a solvent, or combinations thereof.

18. The calibrator dipeptide of any one of claims 1 to 17, wherein the calibrator dipeptide is used to calibrate an automated analyzer.

19. The calibrator dipeptide of claim 18, wherein the automated analyzer is a clinical chemistry analyzer or an immunoassay analyzer.

20. A set of standard solutions for preparing a calibration curve, the set of standard solutions comprising:at least a first solution comprising the calibrator dipeptide of any one of claims 1 to 19 having a first concentration of the calibrator dipeptide; andat least a second solution comprising the calibrator dipeptide having a second concentration of the calibrator dipeptide; andwherein the first concentration is greater than the second concentration.

21. The set of standard solutions of claim 20, wherein the first concentration of calibrator dipeptide is at least IX greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 2X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 3X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 4X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 5X greater than the second concentration of calibrator dipeptide;Attorney Docket No. 772157: DABX-002PCTalternatively the first concentration of calibrator dipeptide is at least 6X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 7X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 8X greater than the second concentration of calibrator dipeptide; alternatively the first concentration of calibrator dipeptide is at least 9X greater than the second concentration of calibrator dipeptide; or alternatively the first concentration of calibrator dipeptide is at least 10X greater than the second concentration of calibrator dipeptide.

22. The set of standard solutions of claim 20 or claim 21, wherein the first concentration of calibrator dipeptide is at least 100 fg / mL; alternatively the first concentration of calibrator dipeptide is at least 500 fg / mL; alternatively the first concentration of calibrator dipeptide is at least 1 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 2 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 5 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 10 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 20 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 50 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 100 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 200 pg / mL; alternatively the first concentration of calibrator dipeptide is at least 500 pg / mL; or alternatively the first concentration of calibrator dipeptide is at least 1 ng / mL.

23. A method for detecting phosphorylated tau (p-tau) 217 in a sample from a subject using an immunoassay analyzer, the method comprising:calibrating the immunoassay analyzer using at least a first solution comprising the calibrator dipeptide of any one of claims 1 to 19 having a first concentration of the calibrator dipeptide and at least a second solution comprising the calibrator dipeptide having a second concentration of the calibrator dipeptide; wherein the first concentration is greater than the second concentration and wherein the calibration comprises generating a calibration curve; performing a quantitative immunoassay on a portion of the sample from the subject, wherein the quantitative immunoassay generates at least one sample value; andAttorney Docket No. 772157: DABX-002PCTdetermining a concentration of p-tau 217 in the sample by comparing the at least one generated sample value to the calibration curve.

24. The method of claim 23, wherein the first concentration of the calibrator dipeptide is at least 1X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 2X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 3X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 4X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 5X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 6X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 7X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 8X greater than the second concentration of the calibrator dipeptide; alternatively the first concentration of the calibrator dipeptide is at least 9X greater than the second concentration of the calibrator dipeptide; or alternatively the first concentration of the calibrator dipeptide is at least 10X greater than the second concentration of the calibrator dipeptide.

25. The method of claim 23 or claim 24, wherein the first concentration of the calibrator dipeptide is at least 100 fg / mL; alternatively the first concentration of the calibrator dipeptide is at least 500 fg / mL; alternatively the first concentration of the calibrator dipeptide is at least 1 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 2 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 5 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 10 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 20 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 50 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 100 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 200 pg / mL; alternatively the first concentration of the calibrator dipeptide is at least 500 pg / mL; or alternatively the first concentration of the calibrator dipeptide is at least 1 ng / mL.Attorney Docket No. 772157: DABX-002PCT26. The method of any one of claims 23 to 25, wherein the method further comprises: mixing the first solution with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a first reaction mixture; mixing the first reaction mixture with a reagent comprising a detection molecule, generating a first detection mixture;detecting, using a detector arrangement, a reaction in the first detection mixture to generate a first calibrator dipeptide value;mixing the second solution with a reagent comprising at least one affinity molecule that binds to at least one portion of the calibrator dipeptide, generating a second reaction mixture; mixing the second reaction mixture with a reagent comprising a detection molecule, generating a second detection mixture;detecting, using a detector arrangement, a reaction in the second detection mixture to generate a second calibrator dipeptide value; andwherein the first calibrator dipeptide value and the second calibrator dipeptide value are used to generate the calibration curve.

27. The method of any one of claims 23 to 26, wherein the method further comprises: aspirating, using a sample pipettor, a portion of the sample from a sample vessel and dispensing the aspirated 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;detecting, using the detector arrangement, a presence of a reaction in a reaction vessel; and generating the at least one sample value based on the reaction detected in the reaction vessel.

28. The method of any one of claims 23 to 26, wherein the method further comprises: mixing the sample with a reagent comprising at least one affinity molecule that binds to at least one portion of p-tau217, generating a reaction mixture;Attorney Docket No. 772157: DABX-002PCTmixing the reaction mixture with a reagent comprising a detection molecule, generating a detection mixture;detecting, using a detector arrangement, a reaction in the detection mixture; and generating the at least one sample value based on the reaction detected in the detection mixture.

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