Assay methods for predicting alzheimer's disease using APOE and tau

Abstract

The presently described and claimed technology relates immunoassay methods of assessing a subject's Alzheimer's Disease status and zygosity for the APOE ε4 allele.

Description

Attorney Docket No. 2024-25047-P-WO (68618WO01)ASSAY METHODS FOR PREDICTING ALZHEIMER’S DISEASE USING APOE AND TAU BACKGROUND

[0001] 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.

[0002] 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.

[0003] 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, are 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.

[0004] APOE4 (apolipoprotein E4) zygosity is a well-established genetic predictor of AD risk. Individuals with one copy of the APOE4 allele (heterozygous) have an elevated risk of developingAD, while those with two copies (homozygous) have an even higher risk and often experience earlier onset. APOE4 influences AD risk by affecting amyloid-beta accumulation and neuroinflammation, contributing to the pathology of AD. This genetic marker is used in research and clinical settings to assess AD risk and inform decisions on preventive or therapeutic approaches.

[0005] However, the APOE4 genotype, especially in individuals with one or two copies of the ε4 allele, is linked to a heightened risk of Amyloid-Related Imaging Abnormalities (ARIA) when undergoing certain Alzheimer’s disease (AD) treatments, particularly those targeting amyloidbeta. ARIA are adverse events often reported in clinical trials for disease-modifying therapies (DMTs), especially for AD treatments involving amyloid-beta (A ) targeting therapies. ARIA can manifest as ARIA-E (edema or swelling) or ARIA-H (hemorrhage or bleeding), both detectable on MRI scans. Due to the risks associated with ARIA, monitoring for these abnormalities is crucial in clinical trials and later in clinical practice when administering these DMTs. Effective triaging for DMT eligibility requires identifying patients' risk factors for ARIA, such as age, genetic predispositions (e.g., APOE e4 status), and baseline MRI findings, to ensure the safe and effective delivery of these therapies. Due to increased risk of ARIA, APOE4 carriers may require more intensive monitoring, particularly during the first year of treatment, to detect and manage ARIA events early.

[0006] A need exists for a diagnostic method that is sensitive and specific enough to assess a subject’s Alzheimer’s Disease status and zygosity for the APOE s4 allele.BRIEF SUMMARY

[0007] One aspect of the disclosure includes an immunoassay method of assessing a subject’s Alzheimer’s Disease status and zygosity for the APOE e4 allele, the method comprising: contacting a first portion of at least one biological sample from the subject with first and second apolipoprotein (ApoE) antibodies, wherein the second ApoE antibody is specific to apolipoprotein E4 isoform (ApoE4), generating a first reaction mixture; conducting a detection reaction in the first reaction mixture generating a first signal; detecting the first signal; contacting a second portion of the at least one biological sample from the subject with third and fourth ApoE antibodies capable of binding to multiple ApoE isoforms, wherein the third or fourth ApoE antibody and the first ApoE antibody bind to a same epitope of ApoE, generating a second reaction mixture; conductinga detection reaction in the second reaction mixture generating a second signal; detecting the second signal; calculating a ratio of the first signal to the second signal; assigning the subject an APOE s4 genotype based on comparison of the ratio to a ratio established in a reference population; performing a quantitative immunoassay on a third portion of the at least one sample from the subject to determine a concentration of phosphorylated tau isoform in the sample; and assessing the Alzheimer’s Disease status of the subject based on the comparison of the determined concentration to a concentration value established in a reference population.

[0008] One aspect of the disclosure includes an immunoassay method of assessing a subject’s Alzheimer’s Disease status and zygosity for the APOE s4 allele, the method comprising: exposing a first portion of at least one biological sample from the subject to a first capture antibody capable of binding to Apolipoprotein E (ApoE) and a first detector affinity molecule specific to apolipoprotein E4 isoform (ApoE4), generating a first reaction mixture; conducting a detection reaction in the first reaction mixture wherein the first detector affinity molecule generates a first detection signal; recording the first detection signal; exposing a second portion of the at least one biological sample from the subject to a second capture antibody capable of binding to ApoE and a second detector affinity molecule capable of binding to multiple isoforms of ApoE, generating a second reaction mixture, wherein the first capture antibody and the second capture antibody bind to the same epitope of ApoE; conducting a detection reaction in the second reaction mixture wherein the second detector affinity molecule generates a second detection signal; recording the second detection signal; calculating a ratio of the recorded first detection signal to the recorded second detection signal to generate an ApoE4 comparison value and, predicting, based on the ApoE4 comparison value, whether the subject is heterozygous or homozygous for the APOE s4 allele; exposing a third portion of the at least one biological sample from the subject to a third capture antibody capable of binding to tau, generating a third reaction mixture; conducting a detection reaction in the third reaction mixture; quantitatively determining an amount of a phosphorylated tau isoform in the plasma based on the presence of the reaction in the third reaction mixture; correlating the third detection signal to a concentration of a phosphorylated tau isoform; and assessing the subject’s Alzheimer’s Disease status based on the concentration of the phosphorylated tau isoform.

[0009] In an aspect, the first capture antibody and the second capture antibody are the same.|

[0010] In an aspect, third capture antibody capable of binding to tau binds to a tau epitope comprising an isoform subject to phosphorylation when the isoform is phosphorylated.

[0011] In an aspect, the method further comprises exposing the third portion of the at least one biological sample to a third detector affinity molecule prior to conducting the detection reaction.

[0012] In an aspect, the phosphorylated tau isoform is threonine 217.

[0013] In an aspect, assessing the subject’s Alzheimer’s Disease status comprises comparing the concentration of the phosphorylated tau isoform to concentrations of phosphorylated tau established in a reference population of plasma samples, wherein each of the reference population samples have been classified as positive or negative for Alzheimer’s disease based on a correlation to tau levels measured via positron emission tomography (PET) or cerebrospinal fluid (CSF) analysis.

[0014] In an aspect, the subject has mild cognitive impairment and / or is 55 years or older.

[0015] In an aspect, assessing the subject’s Alzheimer’s Disease status comprises diagnosing the subject with Alzheimer’s disease.

[0016] In an aspect, if the concentration of the phosphorylated tau isoform is equal to or less than a reference range established in the reference population, the subject is determined to not have Alzheimer’s disease.

[0017] In an aspect, if the concentration of the phosphorylated tau isoform is higher than a reference signal established in the reference population, the subject is determined to have Alzheimer’s disease.

[0018] In an aspect, the quantitation detection limit (LOQ) for measuring the phosphorylated tau isoform is equal to or less than about 3 pg / mL, alternatively equal to or less than about 2 pg / mL, equal to or less than about 1 pg / mL, equal to or less than about 0.6 pg / mL, and wherein the detected phosphorylated tau isoform has a concentration above the LOQ.

[0019] In an aspect, the detected phosphorylated tau isoform has a concentration that is at least IX greater than the LOQ; alternatively at least 2X greater than the LOQ; or alternatively at least 3X greater than the LOQ.

[0020] In an aspect, the detected phosphorylated tau isoform exhibits a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

[0021] In an aspect, the method further comprises assessing a degree of likelihood that the subject will be at risk of developing Amyloid-Related Imaging Abnormalities (ARIA) based on the whether the subject is heterozygous or homozygous for the APOE s4 allele.

[0022] In an aspect, the degree of likelihood that the subject will develop ARIA is low risk, medium risk, or high risk.

[0023] In an aspect, the subject is recommend further treatment.

[0024] In an aspect, the further treatment comprises qualifying the subject for a clinical trial, assessing the risks associated with the subject entering the clinical trial, and / or increasing safeguards for the subject in clinical trial.

[0025] In an aspect, the further treatment comprises assessing a subject’s eligibility for a diseasemodifying therapy (DMT), administering a DMT and / or providing monitoring or additional treatments in conjunction with the DMT.

[0026] In an aspect, the DMT is selected from the group consisting of anti-amyloid therapies, anti-tau therapies, immunotherapies, vaccines, BACE inhibitors, anti-inflammatory drugs, neurotrophic drugs, and combinations thereof.

[0027] In an aspect, the detector affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, an antibody fragment, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

[0028] In an aspect, the capture antibody, the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity molecule is conjugated to at least one magnetic bead.

[0029] In an aspect, the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity molecule is conjugated to an enzyme.

[0030] In an aspect, the enzyme comprises horseradish peroxidase or alkaline phosphatase.

[0031] In an aspect, the method is performed using an 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.

[0032] In an aspect, the detector arrangement comprises a luminometer, an electrochemiluminescence (ECL) detector, a photomultiplier tube (PMT) detector, a photometer, a fluorometer, or a bioluminescence detector.

[0033] In an aspect, wherein the first detection signal, the second detection signal, and / or the third detection signal are luminescent signals, eletrochemiluminescent signals, or chemiluminescent signals.

[0034] In an aspect, the first detection signal, the second detection signal, and / or the third detection signal are generated via a detection reaction between the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity and a substrate formulation, wherein the substrate formulation comprises:

[0035] a chemiluminescent compound of the formula A or a salt thereof:

[0036]

[0037] wherein

[0038] A is Ci-ehaloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NO2 substituents;

[0039] Ri is selected from the group consisting of Cs-uaryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;

[0040] R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or R11-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;

[0041] R15 is C1-6 alkyl;

[0042] 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;

[0043] Z is O or S; and

[0044] n is 0, 1, or 2;

[0045] a cationic aromatic compound (CAC);

[0046] a background reducing agent; and

[0047] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0048] In an aspect, the immunoassay analyzer further comprises at least one reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assays.

[0049] In an aspect, the immunoassay analyzer further comprises at least two reagent packs configured to hold a plurality of reagent vessels, wherein the first reagent pack comprises a reagent vessel comprising the first capture antibody, a reagent vessel comprising the first detector affinity molecule, and a reagent vessel comprising the second detector affinity molecule, and the second reagent pack comprises a reagent vessel comprising the third capture antibody and a reagent comprising the third detector affinity molecule.

[0050] In an aspect, the immunoassay analyzer further comprises an ultrasonic mixing module.

[0051] In an aspect, the immunoassay analyzer further comprises: a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

[0052] In an aspect, the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

[0053] In an aspect, the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

[0054] In an aspect, the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, and a third reagent pipettor. In an aspect, the pipettor arrangement further comprises at least a fourth reagent pipettor. In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor is selectively and / or simultaneously operated. In an aspect, the first reagent pipettor, second reagent pipettor, third reagent pipettor, and / or fourth reagent pipettor are configured to engage a dispense tip prior to aspiration.

[0055] In an aspect, the first reaction mixture, the second reaction mixture, and / or the third reaction mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement, wherein the washing arrangement is configured to perform atleast one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

[0056] In an aspect, the first reaction mixture, the second reaction mixture, and / or the third reaction mixture is subjected to a magnetic field prior to performing the at least one wash action.

[0057] In an aspect, the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid. In an aspect, the biological sample volume is less than about 10 pL, alternatively between about 2 µL to about 9.9 µL. In an aspect, the method is configured to analyze at least about 200 biological samples / hr; alternatively at least about 300 biological samples / hr; or alternatively at least about 400 biological samples / hr.

[0058] 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

[0059] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0060] FIG.1 is a graph showing the role of APOE genotypes in Alzheimer’s Disease monitoring.

[0061] FIG.2 is a graph of patient samples analyzed using a method according to an aspect of this disclosure and stratified according to ApoE4 genotype.

[0062] FIG.3 is a graph depicting discrimination of amyloid positive patient samples and negative patient samples analyzed using an exemplary p-Tau217 assay.DETAILED DESCRIPTIONI. Introduction

[0063] AD is a progressive neurodegenerative disorder that affects millions of individuals. It is thought that individuals with AD have amyloid accumulation in the brain which damages thesynaptic function and forms neurofibrillary tangles. Apolipoprotein E (ApoE), encoded by the APOE gene, has been shown to have an essential role in lipid metabolism. In the central nervous system, APOE is mainly produced by astrocytes and microglia, and transports cholesterol to neurons via APOE receptors.

[0064] The APOE gene has three major allelic variants, s2, s3, and e4, which encode ApoE2, ApoE3, and ApoE4 protein isoforms. The isoforms vary from one to another by one or two amino acids at residues 112 and 158 in the APOE protein. ApoE2 contains 112 / 158 cys / cys; ApoE3 contains 112 / 158 cys / arg; and ApoE4 contains 112 / 158 arg / arg. There are also six different genotypes, APOE2 / 2, APOE2 / 3, APOE3 / 3, APOE2 / 4, APOE3 / 4, and APOE4 / 4. ApoE2 can refer to any full length form of ApoE2, a variant of ApoE2, a fragment of ApoE2, and post-translationally modified forms of ApoE2. ApoE3 can refer to any full length form of ApoE3, a variant of ApoE3, a fragment of ApoE3, and post-translationally modified forms of ApoE3. ApoE4 can refer to any full length form of ApoE4, a variant of ApoE4, a fragment of ApoE4, and post-translationally modified forms of ApoE4. “Total APOE” in the context of a biological sample refers to the total amount of all isoforms, or all major isoforms, of the APOE protein in the sample, including APOE2, APOE3, and APOE4. In some aspects, an antibody that binds to all isoforms, or all major isoforms, of ApoE is referred to as an anti-total-ApoE antibody, a total- ApoE antibody, an anti-PAN-ApoE antibody, or a PAN-ApoE antibody.

[0065] In the brain, ApoE regulates neuronal and synaptic functions. Genome-wide association studies have confirmed that the e4 allele of APOE is the strongest genetic risk factor for both early -onset AD and late onset or sporadic AD. Risk of AD is thought to be increased in individuals with one copy of the s4 allele (e2 / s4; s3 / s4) or two copies (c4 / s4). ApoE4 has been associated with numerous cellular pathways that may influence AD disease progression, such as amyloid clearance, mitochondrial function, autophagy, and inflammation. It is thought that APOE s4 is associated with increased risk of progression from MCI to AD type dementia and impaired memory performance and increased risk of memory decline in middle aged (40-59 years) and elderly (60-85 years) people with MCI.

[0066] The amino acid sequences of exemplary ApoE isoforms are listed in Table 1.

[0067] Table 1iSEQ ID NO Amino Acid Sequence IdentifierKVEQAVETEPEPELRQQTEWQSGQRWELALGRFW ApoE2D YLRWVQTLSEQVQEELLS S QVTQELRALMDETMK ELKAYKSELEEQLTPVAEETRARLSKELQAAQARLG ADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLAS HLRKLRKRLLRDADDLQKCLAVYQAGAREGAERG1 LSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQA WGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKL EEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWA GLVEKVQAAVGTSAAPVPSDNHKVEQAVETEPEPELRQQTEWQSGQRWELALGRFW ApoE3D YLRWVQTLSEQVQEELLS S QVTQELRALMDETMK ELKAYKSELEEQLTPVAEETRARLSKELQAAQARLG ADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLAS2 HLRKLRKRLLRDADDLQKRLAVYQAGAREGAERG LSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQA WGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKL EEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWA GLVEKVQAAVGTSAAPVPSDNH KVEQAVETEPEPELRQQTEWQSGQRWELALGRFW ApoE4 DYLRWVQTLSEQVQEELLSSQVTQELRALMDETMK ELKAYKSELEEQLTPVAEETRARLSKELQAAQARLG ADMEDVRGRLVQYRGEVQAMLGQSTEELRVRLAS3 HLRKLRKRLLRDADDLQKRLAVYQAGAREGAERG LSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQA WGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKL EEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWA GLVEKVQAAVGTSAAPVPSDNH

[0068] There are conventional diagnostic tools capable of detecting ApoE, in particular the ApoE4 isoform, to ascribe a person’s risk or susceptibility for developing Alzheimer’s disease (AD) or other neurological disorders. Without being limited by any theory, as shown in FIG.1, individuals with APOE2 / 2, APOE2 / 3, and APOE3 / 3 genotypes have a low risk for developing AD. While individuals with APOE2 / 4 and APOE3 / 4 have a moderate risk (5 times higher) with and those with APOE4 / 4 having the highest risk (15 times higher).

[0069] 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 neurofibrillaryiOtangles. Tau protein abnormalities, such as the hyperphosphorylation of tau, are thought to be a hallmark feature of AD.

[0070] 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 2.

[0071] The peripheral nervous system (PNS)-tau isoform (also referred to as “Big tau” or “peripheral tau”) is an isoform of tau preferentially expressed in peripheral tissues. Without being limited by any theory, Big tau is distinguishable from brain-derived isoforms of tau by the presence of a large peptide insert resulting from the transcription of an extra exon (exon 4a) of the MAPT gene. The amino acid sequence of Big Tau is listed in Table 1 where the amino acid sequence encoded by the 4a exon is underlined. The exon 4a insert breaks the junction between the domains encoded by exons 4 and 5, making it unique to Big Tau, as the domains expressed by exons 4 and 5 are continuous in the six brain derived tau isoforms. The glutamine (Q) and alanine (A) bolded refers to the connective portion between the amino acid sequence encoded by exon 4 and the amino acid sequence encoded by exon 5. This may also be referred to as the “exon 4-5 connective portion.”

[0072] The longest brain-derived tau isoform, 2N4R (441 amino acids in length) contains 2 aminoterminal inserts (2N) and 4 microtubule-binding repeats (4R). The two amino-terminal inserts are encoded by two alternatively spliced exons, E2 and E3, and encode 29 amino acids each. The other brain-derived tau isoforms arise from alternative splicing of exon 2, 3 and 10. These isoforms may differ in either 0, 1 or 2 inserts of the 29 amino acid amino-terminal part and three or four microtubule-binding repeats. Brain-derived tau isoforms differ from the PNS-tau isoform in that they lack the 4a exon. For example, as compared to 2N4R, the PNS-tau isoform includes an insertion between amino acids 124 of 2N4R and 125 of 2N4R. Non-limiting examples of other brain-derived tau isoforms include, the 0N3R isoform (352 amino acids in length), the 0N4R isoform (383 amino acids in length), the 1N3R isoform (383 amino acids in length), the 1N4R (412 amino acids in length), and the 2N3R isoform (410 amino acids in length).ii

[0073] Table 2: Exemplary Tau IsoformsSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKST PTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEE AG IGDTPS LEDEA A GHVTQEPES G KVVQEGFLREPG PPGLSHQLMSGMPGAPLLPEGPREATRQPSGTGPED TEGGRHAPELLKHQLLGDLHQEGPPLKGAGGKERP GSKEEVDEDRDVDESSPQDSPPSKASPAQDGRPPQT AAREATSIPGFPAEGAIPLPVDFLSKVSTEIPASEPDG PSVGRAKGQDAPLEFTFHVEITPNVQKEQAHSEEHL GRAAFPGAPGEGPEARGPSLGEDTKEADLPEPSEKQ PAAAPRGKPVSRVPQLKARMVSKS KDGTGS DDKK4 PNS-tau AKTSTRS S AKTLKNRPCLSPKHPTPGS SDPLIQPSS PA VCPEPPSSPKYVSSVTSRTGSSGAKEMKLKGADGKT KIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSG EPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPK KVA V VRTPPKSPS S AKSRLQTAPVPMPDLKN VKS KI GSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKD NIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHK PGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGG NKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDT SPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQ GL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKST PTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEE AGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDK KAKGADGKTKIATPRGAAPPGQKGQANATRIPAKT PPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTP5 SLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM tau-441 (2N4R)PDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSN VQS KCG S KDNIKH VPG G G S VQIV YKP VDL S KVTS K CGS LGNIHHKPGGGQ VEVKSEKLDFKDRVQSKIGSL DNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIV YKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGLSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQAR MVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPG QKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP6 tau-352 (0N3R)SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGG GKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVE VKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETH KLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSN VS STGSIDM VDSPQLATLADEVS AS LAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKST PTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKD GTGSDDKKAKGADGKTKIATPRGAAPPGQKGQAN ATRIP AKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGT7 PGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSR tau-381 (1N3R)LQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEK LDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFR ENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTG SIDMVDSPQLATLADEVSASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKST PTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEE AGIGDTPSLEDEA AGHVTQARM VSKS KDGTG S DDK KAKGADG KTKIATPRGAAPPGQKGQ AN ATRIP AKT PPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTP8 tau-410 (2N3R)SLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM PDLKNVKSKIGSTENLKHQPGGGKVQIVYKPVDLSK VTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQS KIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDH GAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQL ATLADEVSASLAKQGLSEQ ID NO Amino Acid Sequence Identifier MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKAEEAGIGDTPSLEDEAAGHVTQAR MVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPG QKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYS SPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSP9 SSAKSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGG tau-383 (0N4R) GKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEK LDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFR ENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTG S IDMVDSPQLATLADEVS ASLAKQGL MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQ DQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKST PTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKD GTGSDDKKAKGADGKTKIATPRGAAPPGQKGQAN ATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGT PGSRSRTPSLPTPPTREPKKVAVVRTPPKSPS S AKSR10 tau-412 (1N4R) LQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQII NKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPV DLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKD RVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKA KTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV D S PQL ATL A DE V S A S LAKQG L

[0074] Tau is thought to hold at least 80 potential serine, threonine, or tyrosine phosphorylation sites. If a tau isoform does not include phosphorylation, it may be referred to as nonphosphorylated tau or non-p-tau. Phosphorylated tau (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.

[0075] 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 iimodified 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.

[0076] 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.

[0077] 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.

[0078] 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). In particular, 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 ChimActa. 2022 Jun 1:531:100-111.

[0079] Amyloid-Related Imaging Abnormalities (ARIA) refer to brain abnormalities that can appear as side effects in patients receiving certain disease-modifying therapies (DMTs) for Alzheimer’s disease (AD), particularly those targeting amyloid-beta plaques. ARIA is typically categorized into two types: ARIA-E, which involves brain edema (swelling), and ARIA-H, which includes microhemorrhages (small brain bleeds) or superficial hemosiderosis (iron deposits from bleeding). These abnormalities are detectable by MRI and are believed to result from the inflammatory or vascular effects of amyloid-beta clearance, which may compromise blood-brain barrier integrity.

[0080] The APOE4 allele is a significant genetic factor influencing the risk of ARIA in patients undergoing amyloid-targeting therapies for AD. APOE4, especially in those carrying one (heterozygous) or two (homozygous) copies of the allele, is known to increase susceptibility to ARIA. Without being bound to any theory, this susceptibility may be due to the role of APOE4 in influencing blood-brain barrier integrity, amyloid clearance, and neuroinflammatory responses, all of which contribute to the development of these abnormalities. APOE4’s impact on these pathways may exacerbate amyloid plaque-related vascular damage and inflammation, key mechanisms in ARIA formation during anti-amyloid treatment. In the context of AD treatments, especially with monoclonal antibodies designed to reduce amyloid-beta accumulation in the brain, ARIA is a critical adverse event to monitor due to its potentially serious effects. This association has made ARIA management a key focus in clinical trials and therapeutic protocols, where patient selection, genotyping, and rigorous MRI monitoring are used to balance treatment benefits with the risk of ARIA

[0081] Clinical trials and studies on amyloid-targeting drugs, such as certain monoclonal antibodies, have demonstrated that APOE4 carriers face a higher incidence of ARIA, particularly in the first months of treatment. Patients homozygous for APOE4 are at the highest risk, with some data suggesting as much as a threefold increase in ARIA risk compared to non-carriers. Consequently, genetic testing for APOE4 status is often considered in clinical settings to guide decisions on monitoring and to weigh the risks and benefits of treatment for each patient. In light of these findings, clinical trials for AD therapies have adapted their protocols to include APOE4 status as a consideration in eligibility and to determine monitoring intensity.

[0082] For APOE4 carriers undergoing anti-amyloid therapy, close monitoring with MRI is recommended to detect and manage ARIA events, particularly within the first year of treatment when the risk is highest. Regular imaging enables early identification of ARIA, allowing adjustments to treatment or temporary pauses if significant ARIA is detected. This monitoring approach aims to improve patient safety by reducing the likelihood of severe ARIA-related complications while still offering APOE4 carriers access to potentially beneficial therapies.

[0083] By incorporating APOE4 genotyping into trial protocols and clinical practice, clinicians can determine the need for additional precautions, such as more frequent MRI monitoring, dose adjustments, or alternative therapeutic approaches for high-risk individuals. Theseif;countermeasures are essential to mitigate ARIA-related complications and enhance the safety and efficacy of amyloid-targeting therapies for Alzheimer’s disease.

[0084] High-throughput automated method for the investigation of ApoE isoforms, in particular the zygosity for the APOE s4 allele, the subsequent assessment of a subject’s Alzheimer’s Disease status based on p-tau concentrations are disclosed herein.II. Definitions

[0085] 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, TAPPT, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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 interferewith or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0091] 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 / ory" 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".

[0092] 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.

[0093] 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.

[0094] 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 liin all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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).

[0099] 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.

[0100] 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.

[0101] 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.

[0102] An “immunoassay analyzer” includes an automated analyzer that primarily relies on immune-based techniques (e.g. chemiluminescent or fluorescent immunoassays) to detectIIpresence 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.. An "immunoassay analyzer" can include an instrument on which immunoassays have been automated. Various immunoassay analyzer are commercially available including the Dxl® systems (Beckman Coulter, CA). In some aspects, the immunoassay analyzer is a high-throughput immunoassay analyzer.

[0103] 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.

[0104] 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.

[0105] The terms "treat", "treating", or "treatment" refer to administering to a subject a compound or pharmaceutical composition to partially or completely alleviate, inhibit, ameliorate, or relieve the condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a condition are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular condition refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by thecompounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a condition in the subject; arrest, inhibit, or slow the progression of the condition in the subject; and / or decrease the number, frequency, or severity of clinical symptoms of the condition in the subject. A “treatment protocol” is a protocol or regime developed regarding specific therapies (including pharmaceuticals or therapeutic interventions) for treatment. A “therapeutic intervention” refers to a clinical intervention intended to manage a disease, condition, disorder or injury and avoid further clinical interventions.

[0106] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and / or ameliorating a condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and / or substantially lessening the chances of a condition in a subject. The effective amount of the pharmaceutical composition may be administered orally or via intravenous injection. The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular composition, its mode of administration, its mode of activity, and the like.

[0107] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting 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. Assay Methods for Assessing a Subject’s Alzheimer’s Disease Status and Zygosity for the APOE ε4 Allele

[0108] The use of ApoE and tau to assess the pathology of neurodegenerative diseases, such as AD. has continued to evolve.

[0109] 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 differentiateAlzheimer’s disease from other neurodegenerative diseases, showing correlation to CSF tau and Amyloid PET in diagnosing AD.

[0110] The APOE (apolipoprotein) s4 genotype is also linked to AD risk. APOE s4 is associated with a significantly higher risk of developing AD. Having one copy is thought to increase a person’s AD risk, while having two copies further elevates the risk. At the same time, the APOE ε4 genotype is also linked to a higher risk of developing Amyloid-Related Imaging Abnormalities (ARIA) in patient’s receiving amyloid-targeting disease modifying therapies (DMT) for AD. These abnormalities typically appear on MRI scans and come in two primary forms: ARIA-E (Edema), which is characterized by brain swelling (edema) or an increase in fluid in the brain’s tissue, and ARIA-H (Hemosiderin), which involves small hemorrhages or bleeding in the brain, known as microhemorrhages. For example, Lecanemab, a monoclonal antibody targeting amyloid beta (Aβ) aggregates used for the treatment of AD in patients with mild cognitive impairment and confirmed amyloid pathology, has been found to be associated with ARIA-E and ARIA-H, with individuals with one or 2 copies APOE ε4 of having increased risk of such events. In some instances, candidates for a DMT having the APOE ε4 allele may require additional monitoring when receiving the DMT or may be precluded from receiving the DMT depending on other clinical factors.

[0111] Molecular testing, such as Polymerase Chain Reaction (PCR), is commonly used to determine the APOE genotype, including identifying the zygosity of the ε4 allele, which is associated with AD risk and treatment-related complications. By amplifying specific regions of the APOE gene, PCR enables the detection of key polymorphisms that define the ε2, ε3, and ε4 alleles. Subsequent analysis, such as restriction fragment length polymorphism (RFLP) or sequencing, differentiates these alleles, allowing precise determination of whether an individual carries zero, one, or two copies of the ε4 allele. This information provides critical insights into genetic predispositions related to AD.

[0112] In the context of DMTs for AD, APOE genotyping via PCR is utilized to assess a patient’s risk of developing ARIA. However, PCR-restriction fragment length polymorphism (RFLP) is labor intensive and prior to errors due to the number of steps. Assays, such as immunochemical assays and immunoassays, allow for the same genotyping as PCR, but have a much quicker runtime and are suitable for high-throughput analysis. Additionally, many of the diagnostic tools arelimited to ascribing a risk of late-onset AD and cannot differentiate between medium and high AD population groups.

[0113] Methods that rely solely on ApoE4 levels to assess AD risk are limited by high patient variability, caused in part by confounding pathological and physiological conditions. These factors, such as coexisting neurodegenerative processes, vascular conditions, liver conditions, and individual differences in amyloid-beta metabolism, can obscure the relationship between APOE4 and AD risk, thereby reducing the accuracy and reliability of such diagnostic approaches. The comparison of an ApoE isomer, such as ApoE4, with the total ApoE present in a subject provides a greater predictor of the potential development and / or progression of a neurodegenerative disease than the use an ApoE isomer alone. It has been found that ApoE levels in serum are higher in subjects with liver cirrhosis as compared to healthy controls and subjects carrying the E3 / E3 genotype are more likely to develop liver cirrhosis. It has also been found that subjects carrying the E4 / E4 genotype have a higher risk of heart disease than healthy controls. By predicting, based on a ApoE4 comparison value (i.e., ratio ApoE4 to total ApoE), whether the subject is heterozygous or homozygous for the APOE e4 allele, a clinician does not have to ascertain if there are confounding conditions which may skew the prediction if based solely on an ApoE genotype assessment.

[0114] The disclosed methods allow a user to rapidly determine a differentiation between ApoE4 heterozygous populations - which have roughly 3-fold increased risk of AD - from ApoE4 homozygous populations, which have roughly 12-fold increased risk of AD. In some aspects, the ApoE4 heterozygous populations carry an E3 / E4 or an E2 / E4 genotype (i.e., a single copy of each ApoE3 and ApoE4 or ApoE2 and ApoE4). In some aspects, the ApoE4 homozygous populations carry an E4 / E4 genotype (i.e., two copies of ApoE4). An additional advantage of the disclosed a methods, is that, unlike PCR, it allows for direct confirmation of the expression of the E4 isoform.

[0115] In some instances, to assess a subject’s Alzheimer’s Disease status and zygosity for the APOE e4 allele and assessing a degree of likelihood that the subject will be at risk of developing ARIA, the methods disclosed herein employ antibodies capable of binding to Apolipoprotein E (ApoE) an apolipoprotein E4 isoform (ApoE4) and p-tau 217.

[0116] 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.

[0117] As used herein, the term “antibody” or “antibodies” refers to a binding protein, immunoglobulin, or glycoprotein that maintains antigen-binding properties. An antibody often comprises a variable domain and a constant domain in each of a heavy chain and a light chain. Accordingly, most antibodies have a heavy chain variable domain (VH) and a light chain variable domain (VL) that together form the portion of the antibody that binds to the antigen. Within each variable domain are three complementarity determining regions (CDR) which form loops in the heavy chain variable domain (VH) and light chain variable domain (VL) that contact the surface of the antigen. Antibodies herein also include “antigen binding portion” or fragments of the antibody that are capable of binding to the antigen.

[0118] As used herein, the term “epitope” refers to a binding site recognized by an antibody. Epitopes may include any molecule or grouping thereof, including, but not limited to, amino acid side chains, sugars, and lipids, and can have a specific three-dimensional structure or conformation.

[0119] One aspect of the disclosure is an immunoassay method of assessing a subject’s Alzheimer’s Disease status and zygosity for the APOE e4 allele. In an embodiment, the method includes contacting a first portion of at least one biological sample from the subject with first and second apolipoprotein (ApoE) antibodies, wherein the second ApoE antibody is specific to apolipoprotein E4 isoform (ApoE4), generating a first reaction mixture. A detection reaction is conducted in the first reaction mixture, which generates a first signal, which is detected. A second portion of the at least one biological sample from the subject is then mixed with third and fourth ApoE antibodies capable of binding to multiple ApoE isoforms, wherein the third ApoE antibody and the first ApoE antibody bind to a same epitope of ApoE, generating a second reaction mixture. A detection reaction is conducted in the second reaction mixture, which generates a second signal, which is detected. A ratio of the first signal to the second signal is calculated and the subject is assigned an APOE ε4 genotype based on comparison of the ratio to a ratio established in reference population. A quantitative immunoassay is then performed on a third portion of the at least one sample from the subject to determine a concentration of tau phosphorylated at threonine 217 in thesample and the Alzheimer’s Disease status of the subject is assessed based on the comparison of the determined concentration to a concentration value established in a reference population.

[0120] Novel aspect of the methods disclosed are they employ a panel immunoassay, which is a laboratory technique used to simultaneously measure multiple analytes within a single sample. As disclosed herein, the methods allow for inclusion of several analytes to be assessed in parallel, enabling a comprehensive analysis of Alzheimer’s Disease status and zygosity for the APOE ε4 allele in one assay.

[0121] The disclosed methods provide distinct advantages to several methodologies used to diagnose AD, such as PCR combined with plasma p-tau assays, cerebrospinal fluid (CSF) analysis, and positron emission tomography (PET) scans. Each of these approaches presents distinct disadvantages that can impact their effectiveness and applicability in clinical settings.

[0122] PCR, while effective for genotyping, does not provide information on the actual expression levels of the APOE4 protein, potentially missing functional insights important for understanding disease risk. Biological variability, such as fluctuations in p-tau levels due to peripheral factors or other neurological conditions, can lead to inconsistent results. Moreover, there is a lack of standardization across different assays, which can affect the reliability and comparability of results between laboratories. These factors collectively reduce the accuracy of diagnosing AD solely based on PCR and plasma p-tau measurements.

[0123] The procedure to obtain CSF is invasive, requiring a lumbar puncture that can cause discomfort, pain, and carries risks such as headaches or infections. This invasiveness may limit the willingness of patients to undergo frequent testing, which is often necessary for monitoring disease progression or treatment efficacy. Additionally. CSF collection requires specialized medical settings and trained personnel, making it less accessible and more costly compared to blood-based assays. Variability in CSF biomarker levels due to factors like hydration status and diurnal fluctuations can also impact the consistency and reliability of results.

[0124] PET scans are expensive and require access to specialized imaging facilities, limiting their availability to larger medical centers and increasing the overall cost of diagnosis. Additionally, PET scans involve exposure to radioactive tracers, raising concerns about radiation safety, especially with repeated scans needed for longitudinal studies or treatment monitoring. The procedure is time-consuming and may be uncomfortable for patients, particularly those withcognitive impairments or movement difficulties. Furthermore, interpreting PET scan results requires highly trained specialists, which can introduce variability and subjectivity in diagnosis.

[0125] These disadvantages highlight the need for a multimodal diagnostic approach to enhance accuracy and reliability in assessing Alzheimer’s Disease status and zygosity for the APOE ε4 allele and as well as assessing a degree of likelihood that the subject will be at risk of developing ARIA.

[0126] In an embodiment, a first portion of at least one biological sample from a subject is exposed to a first capture antibody capable of binding ApoE or capable of recognizing an epitope of ApoE, generating a first reaction mixture. A portion of the first reaction mixture is exposed to a first detector affinity molecule specific to apolipoprotein E4 isoform (ApoE4), generating a second reaction mixture. The first detector affinity molecule specific to ApoE4 may be specific to an epitope of ApoE4. A detection reaction is conducted in the second reaction mixture wherein the first detector affinity molecule generates a first detection signal, which is then recorded. In some aspects, the detection reaction is a chemiluminescent reaction, an electrochemiluminescence reaction, an electrogenerated chemiluminescence reaction, a photoluminescence reaction, or a bioluminescence reaction. In some embodiments, the chemiluminescent reaction is a dioxetane-based reaction, a 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 detector affinity molecule is conjugated to an enzyme, such alkaline phosphatase or horseradish peroxidase. In this embodiment, a substrate for the enzyme is added to the second reaction mixture triggering a chemiluminescent reaction.

[0127] In an embodiment, a second portion of the at least one biological sample from the subject is exposed to a second capture antibody capable of binding ApoE or capable of recognizing an epitope of ApoE, generating a third reaction mixture. In some embodiments, the first capture antibody and the second capture antibody bind to or recognize the same epitope of ApoE. A portion of the third reaction mixture is exposed to a second detector affinity molecule capable of binding to multiple isoforms of ApoE, generating a fourth reaction mixture. A detection reaction is conducted in the fourth reaction mixture wherein the second detector affinity molecule generates a second detection signal, which is then recorded. In some aspects, the detection reaction is a chemiluminescent reaction, an electrochemiluminescence reaction, an electrogenerated chemiluminescence reaction, a photoluminescence reaction, or a bioluminescence reaction. Insome 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 detector affinity molecule is conjugated to an enzyme, such alkaline phosphatase or horseradish peroxidase. In this embodiment, a substrate for the enzyme is added to the second reaction mixture triggering a chemiluminescent reaction.

[0128] A ratio of the recorded first detection signal to the recorded second detection signal is calculated to generate an ApoE4 comparison value. The ApoE4 comparison value (APOE4 relative to total APOE) is indicative of genotype as the APOE4 is expressed by e4 homozygous and heterozygous individuals. The ApoE4 comparison value is compared to a ratio established in a reference population and can be used to distinguish between e4 homozygous and heterozygous individual by, for example, determining if a significant amount of another isoform is being expressed or by controlling for another condition, such as coexisting neurodegenerative processes, vascular conditions, liver conditions, and individual differences in amyloid-beta metabolism.

[0129] Based on the comparison, a prediction is made whether the subject is heterozygous or homozygous for the APOE ε4 allele. In some embodiments, the method further comprises assessing a degree of likelihood that the subject will be at risk of developing ARIA based on the whether the subject is heterozygous or homozygous for the APOE ε4 allele. In some embodiments, the degree of likelihood that the subject will develop ARIA is low risk, medium risk, or high risk.

[0130] The presence of ApoE4 is also indictive of an increased risk of developing AD in a gene dose-dependent manner. Without being limited by any theory, it is thought that individuals with one copy of the ApoE4 gene have about three times greater the risk of developing AD as compared to individuals without ApoE4 present in their blood. While individuals with two copies of the ApoE4 gene have about a twelve-fold increased risk of developing AD, as compared to individuals without ApoE4 present in their blood.

[0131] In an embodiment, a third portion of the at least one biological sample from the subject is exposed to the third capture antibody capable of binding tau or capable of recognizing an epitope of tau, generating a fifth reaction mixture. A portion of the fifth reaction mixture is exposed to a third detector affinity molecule, wherein the third capture antibody or the third detector affinity molecule binds to a tau epitope comprising a residue subject to phosphorylation when the residueIBis phosphorylated, forming a sixth reaction mixture. In an aspect, the third detector affinity molecule only binds the tau epitope when the threonine 217 is phosphorylated (i.e., p-tau217).

[0132] 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.

[0133] In an embodiment, a detection reaction is conducted in the sixth reaction mixture wherein the third detector affinity molecule generates a third detection signal, which is then recorded. The third detection signal is then correlated to a concentration of a phosphorylated tau isoform and the subject’s Alzheimer’s Disease status is assessed based on the concentration.

[0134] In some aspects, assessing the subject’s Alzheimer’s Disease status comprises assessing a degree of likelihood that the subject will develop Alzheimer’s disease. For example, in nonlimiting aspects, the degree of likelihood that the subject will develop Alzheimer’s disease may be classified as low risk, medium risk, or high risk. In some aspects, the method may further include assessing the subject’s Alzheimer’s Disease status by analyzing a subject’s levels of amyloid protein detected through a PET scan of the brain and / or to the levels of tau and / or beta-amyloid proteins in CSF and correlating the analyzed levels to a reference population. In some embodiments, the correlations includes additional clinical factors such as a degree of cognitive decline or a level of cognitive impairment.

[0135] In some embodiments, if the concentration of the phosphorylated tau isoform is equal to or less than a reference range established in a reference population, the subject is diagnosed as being negative for Alzheimer’s disease. In other embodiments, if the concentration of the phosphorylated tau isoform is higher than a reference signal established in a reference population, the subject is diagnosed with Alzheimer’s disease. In some aspects, the methods may also be used to diagnose the subject with Alzheimer’s disease.

[0136] In some cases, for patients having AD, or at risk of progressing to AD, the levels of p-tau217 in blood may be in the AttoMole, Milli-international Unit (mIU), pg, or fg range. In a nonlimiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about 3 pg / mL, alternatively equal to or less than about 2 pg / mL, equal to or less than about 1 pg / mL. equal to or less than about 0.6 pg / mL and wherein the detected p-tau217 has a concentration above the LOQ. In a non-limiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about 100 pg / mL, less than about 90 pg / mL, less than about 80 pg / mL, less than about 70 pg / mL, less than about 60 pg / mL, less than about 50 pg / mL, less than about 40 pg / mL, less than about 30 pg / mL, less than about 20 pg / mL, or less than about 10 pg / mL. In a non-limiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about less than about 100 pg / mL, less than about 90 pg / mL, less than about 80 pg / mL, less than about 70 pg / mL, less than about 60 pg / mL, less than about 50 pg / mL, less than about 40 pg / mL, less than about 30 pg / mL, less than about 20 pg / mL, or less than about 10 pg / mL. In a non-limiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about 10 pg / mL, less than about 9 pg / mL, less than about 8 pg / mL, less than about 7 pg / mL, less than about 6 pg / mL, less than about 5 pg / mL, less than about 4 pg / mL, less than about 3 pg / mL, less than about 2 pg / mL, or less than about 1 pg / mL. In a nonlimiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about Ipg / mL, less than about 0.9pg / mL, less than about 0.8pg / mL, less than about 0.7pg / mL, less than about 0.6pg / mL, less than about 0.5pg / mL, less than about 0.4pg / mL, less than about 0.3pg / mL, less than about 0.2pg / mL, or less than about O.lpg / mL.

[0137] In a non-limiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about 100 fg / mL, less than about 90 fg / mL, less than about 80 fg / mL, less than about 70 fg / mL, less than about 60 fg / mL, less than about 50 fg / mL, less than about 40 fg / mL, less than about 30 fg / mL, less than about 20 fg / mL, or less than about 10 fg / mL. hi a non-limiting example, the quantitation detection limit (LOQ) for measuring p-tau217 is equal to or less than about 10 fg / mL, less than about 9 fg / mL, less than about 8 fg / mL, less than about 7 fg / mL, less than about 6 fg / mL, less than about 5 fg / mL, less than about 4 fg / mL, less than about 3 fg / mL, less than about 2 fg / mL, or less than about 1 fg / mL.

[0138] In another non-limiting example, the detected p-tau217 has a concentration that is at least IX greater than the LOQ, alternatively at least 2X greater than the LOQ, alternatively at least 3X lgreater than the LOQ, alternatively at least 4X greater than the LOQ, alternatively at least 5X greater than the LOQ, alternatively at least 10X greater than the LOQ, alternatively at least 100X greater than the LOQ, or alternatively at least 1OOX greater than the LOQ. In yet another nonlimiting example, the detected p-tau217 exhibits a coefficient of variation (CV) of 20% or less, 19% or less, 18% or less, 17% or less, 16% or less, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less. 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. In yet another non-limiting example, the detected p-tau217 exhibits a coefficient of variation (CV) of 20% or less. In yet another non-limiting example, the detected p-tau217 exhibits a coefficient of variation (CV) of 4% or less.

[0139] The complex and specific information provided by the disclosed assay methods may become a critical feature in individualized therapeutics. As such, a clinician may use this information to better inform their treatment, intervention, and / or monitoring efforts of a subject.

[0140] In some aspects, assessing a subject’s Alzheimer’s Disease status includes analyzing a subject’s p-tau levels. In some aspects, the analyzed levels are correlated to a reference population. In some aspects, the method may also include assessing additional clinical factors such as a degree of cognitive decline or a level of cognitive impairment or the presence of the APOE4 genotype. In some embodiments, the assessment is used to identify patients having AD, or at risk of progressing to AD; detect abnormal amyloid pathology; monitor the treatment responses to clinical and / or pharmaceutical interventions; and / or monitor the progression of AD. In some embodiments, the assessment is used to identify patients who have a low risk, medium risk, or high risk of developing or progressing to AD.

[0141] In some aspects, a clinician may use the disclosed method to determine a treatment selection and prognosis. If the subject is diagnosed with Alzheimer’s disease, the subject may recommended for further treatment. The further treatment may include qualifying the subject for a clinical trial, assessing the risks associated with the subject entering the clinical trial, and / or increasing safeguards for the subject in clinical trial.

[0142] In some aspects, further treatment may include assessing a subject’s eligibility for a disease-modifying therapy (DMT), administering a DMT and / or providing monitoring or additional treatments in conjunction with the DMT. Rather than just treating the symptoms, DMTs for Alzheimer's disease aim to slow down the progression of the disease. Non-limiting examples iiof DMT include anti-amyloid therapies, which include monoclonal antibody therapies such as Aducanumab, Lecanemab, and Donanemab; anti-tau-targeted therapies, which are being developed to prevent the spread of tau tangles and disrupt neuronal function in Alzheimer's; immunotherapies such as vaccines; beta-site amyloid precursor protein cleaving enzyme (BACE) inhibitors, which aim to reduce the production of amyloid-beta; anti-inflammatory drugs, including those that target glial cells (which become overactive in Alzheimer's) or drugs that reduce general neuroinflammation; and neurotrophic drugs, which include Brain-Derived Neurotrophic Factor (BDNF) mimetics, Nerve Growth Factor (NGF) analogs, Glial-Derived Neurotrophic Factor (GDNF) agents, and neuroprotective drugs.

[0143] In some aspects, the method includes assessing a degree of likelihood that the subject will be at risk of developing ARIA after the administration of or treatment with certain DMTs (such as anti-amyloid therapies) and includes analyzing a subject’s ApoE4 comparison value and determining whether the subject is heterozygous or homozygous for the APOE s4 allele. In some aspects, the comparison value is correlated to a reference population.

[0144] In some aspects, if an ApoE4 comparison value is between about 0 and about 0.5. the subject likely has an ApoE2 / ApoE2, ApoE2 / ApoE3, or ApoE3 / ApoE3 genotype.

[0145] In some aspects, if an ApoE4 comparison value is between about 0.5 and about 1.0, the subject likely has an ApoE2 / ApoE4 or ApoE3 / ApoE4 genotype.

[0146] In some aspects, if an ApoE4 comparison value ratio is between about 1.0 or greater, the subject likely has an ApoE4 / ApoE4 genotype.

[0147] In an embodiment, if degree of likelihood that the subject will develop ARIA is medium risk or high risk and the subject is diagnosed with Alzheimer’s disease, and the subject is recommended for further treatment, wherein the further treatment may include administration of a DMT with increased monitoring. In some embodiments, the further treatment may include administration of an anti-amyloid therapy with increased monitoring as long as the subject is also not taking anti-coagulants.

[0148] In some aspects, a clinician may use the disclosed methods to inform decisions regarding the recommendation or administration of clinical or therapeutic interventions. In a non-limiting aspect, if a subject is assigned a medium or high risk of developing or progressing to AD, a clinician may recommend multimodal interventions including, but not limited to, improvednutrition, increased physical activity, cognitive engagement, and management of comorbidities or improved cognitive functioning.

[0149] In some aspects, a clinician may use the disclosed assays to monitor the clinical progression of AD. In this embodiment, the disclosed assay may be used on a biological sample from a subject taken at different time points. In an aspect, at least a first sample and a second sample may be collected at least several days, alternatively at least several months, alternatively at least several years apart. However, it is appreciated that any time point or number of samples may be analyzed.

[0150] A novel aspect of the disclosure is that two APOE sandwich immunoassays performed as the steps of the APOE4 genotyping assay can be combined into a single workflow and can use the same capture reagent. In some cases, calibration of the APOE immunoassays is not necessary because they are performed as part of the same workflow and leverage the same capture antibody. The signals produced by each immunoassay can be compared to generate a ratio indicative of APOE genotype and without being converted to concentration values using a calibration curve. In one embodiment the capture antibodies and detection antibodies are combined into a single reagent pack. A “reagent pack” may include any suitable container that can store a reagent. An example of a reagent pack can include a generally rectangular elongated body formed to include multiple reagent vessels including one or more large reagent vessels, and one or more relatively smaller reagent vessels, as well as features to facilitate handling and automation. US Patent No.9,519,000, which is incorporated by reference in its entirety herein, discloses non-limiting examples of a reagent pack that may be used in an aspect of the invention.

[0151] In some instances, using separate APOE and APOE4 assays that are not combined into a single workflow with a shared antibody may introduce variance into the analysis relative to the present assay (e.g., lot-to-lot variability) which may affect the specificity, sensitivity, and accuracy of the measurement. It also increases the run time, lowering the throughput of the assay. In addition to overcoming the problems with separate assays, this combined assay is robust, highly accurate, inexpensive, does not require a calibrator, and requires less run time than conventional ApoE4 or p-tau assays, allowing a user to rapidly analyze a biological sample.

[0152] For example, in conventional assays methods, a calibration curve is employed to quantify the analyte present in the sample. In some instances, to prepare a calibration curve, a one or more preparations (e.g., calibrator levels) containing known concentrations of the antigen to bemeasured are required. The signal produced by each preparation is plotted against the known concentration of antigen in each preparation, and a curve is generated from the data points using a math model. When the assay is performed on a sample having an ApoE4 analyte, the calibration curve can be used to correlate the signal produced by the analyte to a concentration. The calibration curve must be constructed before running the assay. In some instances, a calibration curve for an particular assay must be reconstructed after a certain time period has elapsed or amount of assays have been performed. The disclosed combined assay methods do not require a calibrator because they are not measuring the concentration of an analyte, but ratio of signals produced by each analyte. An advantage of the disclosed assay methods includes the elimination of calibrant interference, while providing a faster, more precise, and reproducible method for determining an ApoE isoform: total ApoE ratio. In exemplary embodiments, an ApoE4: total ApoE ratio can be used to distinguish between ApoE4 heterozygous populations and ApoE4 homozygous populations. In these embodiments, the total ApoE antibody serves as an internal control, reducing the potential ApoE4 detection variability.

[0153] A “reagent vessel” may refer to a vessel, unit, fluid container, well, or the like that is configured to store reagents. In some aspects, the reagent pack comprises enough reagent vessels to perform a combined assay. 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 embodiments, each reagent vessel is configured to store a volume of reagent required for at least one instance of the assays, wherein at least one reagent comprises the first capture antibody, at least one reagent comprises the second capture antibody, at least one reagent comprises the third capture antibody, at least one reagent comprises the first detector affinity molecule, at least one reagent comprises the second detector affinity molecule, and at least one reagent comprises the third detector affinity molecule.

[0154] 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.Bl

[0155] 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.

[0156] In an embodiment, the reagent vessels include an elastomeric self-sealing membrane. An elastomeric self-sealing membrane may be a polymer, such as polypropylene, which is able to regain its original shape when pierced. For some embodiments, the elastomeric membrane can be a thermoplastic elastomer with hardness of 30-40 durometer (Shore) A. In other embodiments, the hardness can be 20-50 (Shore) A, or about 30 (Shore) A. Elastomers deform sufficiently to form a tight seal with the vessel base. Thermoplastic elastomers are advantageous because of their compatibility with plastics injection molding processes.

[0157] The elastomeric membrane can be large enough to provide adequate compression without bottoming on the sealing portion of the vessel. The hardness and dimensions can cooperate to allow the elastomeric membrane to the sealing portion with reasonable sealing force. In some embodiments, the elastomeric membrane diameter is small enough so that, when compressed by engagement of the pipettor tip, it conforms to the sealing portion without contacting the wall of the pipettor tip. This advantageously concentrates sealing force to the sealing portion of vessel and distributes sealing force evenly to prevent leaks. In some embodiments, the sealing force is about 44 newtons (about 9.9 lbs.) and produces a pressure on the sealing surface of about 300 (about 43.5 pounds per square inch) to about 1000 kPa (145.0 pounds per square inch).

[0158] In an aspect, the capture antibody is conjugated to a magnetic bead or a magnetic particle. In some instances, magnetic beads (also known as magnetic particles, paramagnetic particles, or superparamagnetic particles) consist of a polystyrene core surrounded by a thin layer of small iron oxide particles (-20-30 nm), such as magnetite. On the surface, the magnetic beads are encapsulated by, for example, a polymer, protein A, protein G, protein L, a secondary antibody, or an epoxy. Surface modification of the coating minimizes any non-specific protein binding. Antibodies targeting the analyte of interest or capture antibody can be covalently coupled to the surface of the magnetic bead. In some embodiments, there is about 2 pg or antibody per mg of magnetic bead. In some embodiments, there is about 3 pg or antibody per mg of magnetic bead. In some embodiments, there is about 4 pg or antibody per mg of magnetic bead. In some embodiments, there is about 5 pg or antibody per mg of magnetic bead. In some embodiments,ithere is about 6 pg or antibody per mg of magnetic bead. In some embodiments, there is about 7 pg or antibody per mg of magnetic bead. In some embodiments, there is about 8 pg or antibody per mg of magnetic bead. In some embodiments, there is about 9 pg or antibody per mg of magnetic bead. In some embodiments, there is about 10 pg or antibody per mg of magnetic bead. In some embodiments, there is about 12 pg or antibody per mg of magnetic bead. In some embodiments, there is about 13 pg or antibody per mg of magnetic bead. In some embodiments, there is about 14 pg or antibody per mg of magnetic bead. In some embodiments, there is about 15 pg or antibody per mg of magnetic bead.

[0159] In some aspects, the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity molecule is conjugated to an enzyme, such as alkaline phosphatase. In certain embodiments, the affinity molecule is an enzyme-conjugated antibody, an enzyme-conjugated antigen, an alkaline phosphatase (ALP)-conjugated antibody, or a labeled antibody. In some aspects, the first detector affinity molecule is anti- ApoE4- ALP. In some aspects, the second detector affinity molecule is anti- ApoE- ALP. In some aspects, the third detector affinity molecule is anti-p-tau217-ALP.

[0160] In an aspect, the first detection signal, the second detection signal, and the third detection signal are generated via a detection reaction between the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity and a substrate formulation. Depending on the analysis desired, a first substrate formulation, may be added to the second reaction mixture, a second substrate formulation, may be added to the fourth reaction mixture, and a third substrate formulation may be added to the sixth reaction mixture. In some aspects, the first substrate formulation, the second substrate formulation, and the third substrate formulation are the same.

[0161] In an aspect, the detection signal may be detected using a detector, such as a luminometer, an electrochemiluminescence (ECL) detector, a photomultiplier Tube (PMT) detector, photometer, a fluorometer, or a bioluminescence detector.

[0162] In an aspect, the substrate formulation is configured to produce a colorimetric response. These substrates produce a visible color based on a chemical reaction between an analyte and a reagent. At operation, this substrate is added to a vessel with a reaction mixture and the light generated is measured with a photometer.. s

[0163] In an aspect, the substrate formulation is configured to produce chemiluminescence. These substrates can produce light and thereby provide detection corresponding to a quantity of analytes captured. The term “chemiluminescent compound” refers to a compound that produces chemiluminescence in the presence of a phosphatase enzyme and oxygen under appropriate conditions as provided herein. Chemiluminescent compounds useful in the present formulations are capable of generating chemiluminescence when contacted with an alkaline phosphatase.

[0164] In a non-limiting example, the substrate formulation includes a chemiluminescent compound of formula I or a salt thereof:

[0166] wherein

[0167] A is Ci-ehaloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ris, CN or NO2 substituents;

[0168] Ri is selected from the group consisting of Cg-uaryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;

[0169] R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or Rn-R12 or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;

[0170] R15 is C1-6 alkyl;

[0171] 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;

[0172] Zis O or S; and

[0173] n is 0, 1, or 2;

[0174] a cationic aromatic compound (CAC);

[0175] a background reducing agent; and

[0176] an ether-linked nonionic surfactant or a hydrophilic polymer.

[0177] 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. 6,45,727, 6,90,571, 6,139,782, 6,218,137, 6,270,695, 6,296,787, and 10,703,971 each of which is incorporated by reference herein.

[0178] At operation, this substrate is added to a vessel with a reaction mixture and light generated by the reaction is measured with a luminometer.

[0179] 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.

[0180] In an exemplary method of producing light from the reaction of the chemiluminescent substrate with a phosphatase enzyme (e.g.. detection antibody), the reaction is performed at a temperature between 5° C and 50° C, preferably between 20° C and 40° C in an aqueous buffer solution at a pH between 7 and 12, 8 and 11, or preferably between 8.5 and 10. The enzyme is preferably an alkaline phosphatase or an alkaline phosphatase conjugate.

[0181] In one aspect, the substrate formulation comprises 0.01 mM-50 mM compound 1. 0.01-200 pM cationic aromatic compound, 1 pM -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.

[0182] In one aspect, the substrate formulation comprises 0.05 mM-10 mM compound I, 0.05-50 pM cationic aromatic compound, 10 uM-1000 pM 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.

[0183] In one aspect, the substrate formulation comprises 0.1 mM-5 mM compound I, 0.1-25 pM cationic aromatic compound, 50 to 500 pM background reducing agent, 0.2 to 5 g / L ether-linkednon-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.

[0184] In one aspect, the compound I has the formula□ OIV. Automated Analyzer

[0185] In an aspect, an automated analyzer disclosed herein includes the following basic structural and functional modules: a sample presentation unit, an analytic unit, an incubator station, a washing station, a read station, and reagent storage. In addition, the automated analyzer may include a pipettor arrangement with at least one sample pipettor and at least one reagent pipettor, and at least one transport device. In an aspect, the transport device includes mechanisms, such as pick-and-place grippers, which are used to transport sample and reaction vessels among the various modules of the automated analyzer.

[0186] The automated analyzer may include a container carriage device which is configured to hold and carry the containers at various locations in the instrument so that the analytic unit, incubator station, wash station, and read station can use the containers in various manners. Examples of container carriage devices include vessel racks (e.g., a sample rack, a reagent rack, and a diluent rack), the sample presentation unit, vessel carriage units (e.g., a sample carriage unit, a reaction vessel carriage unit, and a reagent carriage unit), vessel transfer units (e.g., a sample transfer unit, a reagent transfer unit, an incubator transfer unit, and an reaction vessel transfer unit), and vessel holding plates or wheels (e.g., a sample wheel, an incubator, and a wash wheel), which are described herein.

[0187] The read station may include a detector arrangement. In an aspect, the detector arrangement may include a detector that is configured to detect colorimetric response, light or luminescence. The detector may be a photometer, luminescence detector, a chemiluminescence detector, aluminometer, a photomultiplier-based detection instrument, a electrochemiluminescence (ECL) detector, a fluorometer, or a bioluminescence detector.

[0188] In an embodiment, the detector includes a photometer (or photometry unit) configured to emit light of a predetermined wavelength toward a reaction vessel and receive the light that transmits through the reaction mixture. In an embodiment, the detector includes a light detector configured to sense photons emitted from assay reactions over a period of time, an analog circuit configured to provide an analog signal based on the photons emitted from the assay reactions over the period of time, and a counter circuit configured to provide a photon count based on the photons emitted from the assay reactions over the period of time. U. S. Patent Nos. 5,014,216 and 11,604,146, which are incorporated by reference in its entirety herein, discloses non-limiting examples of a detector that may be used in an aspect of the invention.

[0189] In an aspect, the analytic unit is configured to receive and analyze samples, hi an aspect, the analytic unit configured to perform an assay, such as an immunochemical assay or immunoassay. In certain embodiments, the analytic unit includes the pipettor arrangement. The pipettor arrangement may be configured to aliquot, aspirate, and dispense fluidic substances into various vessels, including, but not limited to sample vessels, diluent vessels, reagent vessels, and reaction vessels. Fluidic substances are substances that have fluidic characteristics. In some embodiments, the fluidic substance is a single fluidic substance. In other embodiments, the fluidic substance is a mixture of a plurality of substances.

[0190] 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 uL.it

[0191] 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.

[0192] The disclosed simultaneous and / or selective operation of the pipettors allows for a high-throughput analysis. In some embodiments, the method is configured to analyze at least about 200 biological samples / hr., alternatively at least about 300 biological samples / hr., alternatively at least about 400 biological samples / hr., alternatively at least about 440 biological samples / hr., or alternatively at least 500 biological samples / hr.

[0193] At operation, a biological sample and a reagent comprising capture antibody configured to bind to total ApoE are dispensed into a reaction vessel and mixed. The mixing can be performed with a stirrer in direct contact with the fluidic substances, an ultrasonic probe in direct or indirect contact with the fluidic substances, or any other suitable mixing apparatus. In some aspects, the immunoassay analyzer includes an ultrasonic mixing module. For instance, a reagent pipettor may be outfitted with a tip that allows it to perform ultrasonic mixing of a reagent in a reagent pack before aspirating it for transport to a reaction vessel, thereby ensuring that the aspirated reagent would not be impacted by any settling that may have taken place in the reagent pack. Sample pipettors may similarly be specialized.

[0194] The mixture is then transferred to an incubator. The transfer unit transfers the reaction vessels to and from the incubator station which includes an incubator. In some embodiments, the transfer unit transfers one or more of the pipetted reaction vessels from the reagent carriage unit to the incubator. Further, the transfer unit can transfer one or more reaction vessels from the incubator to the reagent carriage unit. The transfer unit can also remove from the reaction vessels that have been read or completed the incubator. The incubator is thermally controlled to maintain a predetermined temperature. In some embodiments, the incubator is maintained about 30 °C to 40 °C. In other embodiments, the incubator is maintained about 37 °C to ensure immunological reaction and enzyme reaction, for example. By way of example, the incubator performs assay incubation.ii

[0195] During the incubation, the biological sample and the capture antibody interact. The resulting “first reaction mixture” is a result of the incubation between the sample and the reagent.

[0196] 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.

[0197] At operation, a first portion of the first reaction mixture and a reagent comprising a first enzyme-conjugated affinity molecule configured a single isoform of ApoE at a different binding site than the capture antibody are dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, the sample and the first enzyme-conjugated affinity molecule interact. The resulting “second reaction mixture” is a result of the incubation between the first enzyme-conjugated affinity molecule and the reagent.

[0198] At operation, a second portion of the first reaction mixture and a reagent comprising a second enzyme-conjugated affinity molecule configured to bind total ApoE at a different binding site than the capture antibody are dispensed into a reaction vessel and mixed. The mixture is then transferred to the incubator. During the incubation, the sample and the first enzyme-conjugated affinity molecule interact. The resulting “third reaction mixture” is a result of the incubation between the second enzyme-conjugated affinity molecule and the reagent.

[0199] In a non-limiting example, the incubation time of the first, second, and / or third reaction mixtures is at least about 30 minutes, alternatively at least about 40 minutes, alternatively at least about 50 minutes, alternatively at least about 55 minutes, or alternatively at least about 60 minutes.

[0200] The wash station receives and supports reaction vessels thereon such that various aspects of diagnostic process are performed with the automated analyzer. In an embodiment, the wash station is configured to wash away at least some of the unreacted components. Unreacted components may include unreacted reagents (e.g., free antigens, antibodies, unbound reactants, particles, and / or fluid, etc.) and unreacted sample. The wash station may be configured to perform a set number of wash actions depending on the assay. The wash station may also be configured toperform a set number of washes within a predetermined sequence. In certain embodiments, the wash station is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions. In some embodiments, the wash station is a thermally controlled device to separate bound or free analytes from particles after incubation. In some embodiments, the wash unit is maintained about 30 °C to 40 °C. In other embodiments, the wash unit is maintained about 37 °C to ensure enzyme reaction, for example, U. S. Patent Publication No. 2022 / 0357352, which is incorporated by reference in its entirety herein, discloses configurable wash processes according to an aspect of the invention.

[0201] 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).

[0202] In an embodiment, the capture antibody is conjugated to at least one magnetic bead and the first 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.

[0203] At operation, the vessel containing the first reaction mixture is moved near one or more magnets. The one or more magnets attract the magnetic bead(s) or magnetic particle(s) to one or more sides of the reaction vessel. The reaction vessel is then subject to a wash process in which a cleaning dispense nozzle dispenses a rinsing fluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the 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.

[0204] At operation, a substrate is dispensed into the second reaction mixture. After mixing and 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.

[0205] At operation, a substrate is dispensed into the third reaction mixture. After mixing, 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 rinsingIIfluid and a cleaning aspiration nozzle aspirates the unreacted components. The aspiration nozzle may be washed with a probe washer before and / or after the aspiration. The reaction vessel may undergo a series of wash 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.

[0206] Assays including features and / or characteristics described herein may benefit from one or more additional number(s) of wash actions.

[0207] In certain embodiments, the transport device includes three pick-and-place grippers, where a first pick-and-place gripper may be used to transport sample containers among the onload section, the transfer station, and reagent pipetting stations. A second pick-and-place gripper may be used to transport reaction vessels between the reagent pipetting stations and the incubator station or read station. A third pick-and-place gripper may be used to transport reaction vessels between the incubator station and the wash station or read station. A detailed description of the configurations and functions of one embodiment of the vessel pick-and-place grippers is provided in U. S. Patent No. 7128874 and is incorporated herein in its entirety by reference. However, it should be understood that other pick-and-place mechanism that are capable of transporting sample and reaction vessels among the various modules of the automated analyzer is also contemplated for the purpose of the present invention.

[0208] In an aspect, the automated analyzer includes a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the automated analyzer. In some embodiments, instrument functionalities may include optical sensors, pressure sensors and thermistors. In some embodiments, the assay functionalities may include sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring. The machine vision apparatus operates to evaluate the preparation of samples for subsequent analysis, In some embodiments, the machine vision apparatus utilizes one or more image capture units to determine whether samples have been appropriately prepared for analysis. As described herein, the machine vision apparatus provides direct and simple measurements of volume or integrity of a sample to determine whether theitsample 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] The dispense tip image capture unit operates to capture images of dispense tips in one or more locations. In some embodiments, the dispense tip image capture unit is fixed at a particular location in the instrument. In other embodiments, the dispense tip image capture unit is movably disposed in the instrument, which can move either independently from other components of the instrument or together with one or more components of the instrument. Some embodiments of the instrument include a plurality of dispense tip image capture units. As described herein, the dispense tip image capture unit can include a camera unit.

[0213] 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 theimmunoassay 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.

[0214] 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.

[0215] All of the units of the automated analyzer are connected to a controller, which can perform block control of all of the analyzer functions by using, for example, a microcomputer. The controller may contain subunits such as a data processing unit, a communication interface, 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.

[0216] The data processor may include any suitable data computation device or combination of such devices. An exemplary data processor may comprise one or more microprocessors working together to accomplish a desired function. The data processor may include a CPU that comprises at least one high-speed data processor adequate to execute program components for executing user and / or system-generated requests. The CPU may be a microprocessor such as AMD’s Athlon, Duron and / or Opteron; IBM and / or Motorola’s PowerPC; IBM’s and Sony’s Cell processor; Intel’s Celeron, Itanium, Pentium, Xeon, and / or XScale; Apple Ml, and / or the like processor(s).

[0217] 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.

[0218] The computer-readable medium may comprise code, executable by the data processor to perform any suitable method. For example, the computer-readable medium may comprise code, executable by the processor, to cause the controller to operate on a pre-determined schedule. In some embodiments of the presently claimed technology, the pre-determined schedule is a constituent test.

[0219] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0220] EXAMPLES

[0221] Example 1: Exemplary panel immunoassay for APOE genotype and ptau217

[0222] The assay is performed using an exemplary immunoassay analyzer having (i) four reagent pipettors and one sample pipettor; (ii) two reagent packs configured to store volumes of reagents for at least 50 instances of each assay; (iii) an ultrasonic mixer; (iv) the capability to perform over 5 wash cycles per reaction vessel; and (v) a luminometer.

[0223] The reagent packs include several reagent vessels. The APOE reagent pack includes a first reagent vessel with capture antibodies capable of binding to all major of isoforms of ApoE conjugated to paramagnetic particles. The second reagent vessel incudes includes ApoE4 antibodies conjugated to alkaline phosphatase (“ALP”). The third reagent vessel incudes includes ApoE antibodies - capable of binding to all major isoforms of ApoE - conjugated to alkaline phosphatase (“ALP”). The remaining reagent vessel includes buffers required for the assay. The ptau217 reagent pack includes a fourth reagent vessel including capture antibodies specific to ptau217. The sixth reagent vessel includes an N-terminal tau antibody conjugated to ALP. The remaining reagent vessel includes buffers required for the assay.

[0224] The paramagnetic particle conjugated with capture antibodies from the first reagent vessel (ApoE capture antibodies) were pipetted into a reaction vessel using one of the four reagentpipettors along with a buffer. A first sample aliquot was pipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated. A magnetic field was applied to the reaction vessel and the first incubated mixture was washed using a wash buffer to remove any unreacted components.

[0225] A secondary antibody capable of binding ApoE4 conjugated to ALP was added to the reaction vessel containing the incubated mixture. The reaction vessel was mixed ultrasonically and incubated, generating a first reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0226] The paramagnetic particle conjugated with capture antibodies from the first reagent vessel (ApoE capture antibodies) were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A second 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 second incubated mixture. A magnetic field was applied to the reaction vessel and the second incubated mixture was washed using a wash buffer to remove any unreacted components.

[0227] . A secondary antibody capable of binding multiple isoforms of ApoE conjugated to alkaline phosphatase ALP was added to the reaction vessel containing the second mixture. The reaction vessel was mixed ultrasonically and incubated, generating a second reaction mixture. A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0228] Test results were determined automatically by the system software. The signal generated by the assay was measured in relative light units (RLUs). The resulting signals were compared to generate an ApoE4 comparison value. The subject was then assigned a zygosity for the APOE e4 allele based on the comparison value.

[0229] The paramagnetic particle conjugated with capture antibodies from the ptau217 reagent pack including the fourth reagent vessel (ptau217 capture antibodies) were pipetted into a reaction vessel using one of the four reagent pipettors along with a buffer. A third sample aliquot waspipetted into the reaction vessel using a sample pipettor. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a third incubation mixture. A magnetic field was applied to the reaction vessel and the third incubation mixture was washed using a wash buffer to remove any unreacted components.

[0230] A secondary antibody capable of binding the N-terminal of tau conjugated to alkaline phosphatase ALP was added to the reaction vessel containing the third incubation mixture. The reaction vessel was mixed ultrasonically and incubated, generating a third reaction mixture. A magnetic field was applied to the reaction vessel and the third mixture was washed five times using a wash buffer to remove any unreacted components. A substrate was added to the reaction vessel, allowed to incubate, and the signal generated from the resulting reaction was read using a luminometer.

[0231] Test results were determined automatically by the system software. The signal generated by the assay was measured in relative light units (RLUs). The resulting signals were compared to reference to generate a concentration of a phosphorylated tau isoform. The subject’s Alzheimer’s Disease status was then assessed based on the concentration.

[0232] Example 2: ApoE4 Assay Clinical Performance

[0233] An exemplary ApoE4 / total ApoE combination assay was assessed for clinical performance including the ability to distinguish between APOE4+ / +(ApoE4 homozygous) populations, APOE4" / +(ApoE4 heterozygous) populations, and APOE4 / _populations.

[0234] 298 samples were evaluated on Beckman Coulter’s Dxl 9000 analyzer having (i) four reagent pipettors and one sample pipettor configured to aspirate and dispense less than about 10 pL; (ii) a reagent pack with 5 reagent vessels where each reagent vessel is configured to store volumes of reagents for at least 50 instances of assays; (iii) an ultrasonic mixer; and (iv) the capability to perform over 5 wash cycles per reaction vessel.

[0235] Step 1: An aliquot from a vessel containing paramagnetic particles conjugated with ApoE capture antibodies was pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot was pipetted into the reaction vessel using a sample pipettor and an aliquot from a reagent vessel containing assay buffers was pipetted into the reaction vessel using one of four reagentitpipettors. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a first reaction mixture.

[0236] Step 2: A magnetic field was applied to the reaction vessel and the first reaction mixture was washed three times using a wash buffer to remove any unreacted components.

[0237] Step 3: An aliquot from the first reaction mixture was pipetted into a different reaction vessel using one of four reagent pipettors. An aliquot from a reagent vessel containing ApoE4 monoclonal antibodies conjugated to ALP was pipetted into the reaction vessel containing the first aliquot, which was then mixed ultrasonically and incubated, generating a second reaction mixture.

[0238] Step 4: A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components.

[0239] Step 5: A substrate according to the present disclosure was added to the reaction vessel containing the second reaction mixture, and the signal generated from the resulting reaction was read using a luminometer.

[0240] Step 6: An aliquot from a vessel containing paramagnetic conjugated with ApoE capture antibodies was pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot was pipetted into the reaction vessel using a sample pipettor and an aliquot from a reagent vessel containing assay buffers was pipetted into the reaction vessel using one of four reagent pipettors. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a third reaction mixture.

[0241] Step 7: A magnetic field was applied to the reaction vessel and the third reaction mixture was washed three times using a wash buffer to remove any unreacted components.

[0242] Step 8: An aliquot from the third reaction mixture was pipetted into a different reaction vessel using one of four reagent pipettors. An aliquot from the fourth reagent vessel containing antibodies capable of binding multiple isoforms of ApoE conjugated to alkaline phosphatase ALP was pipetted into the reaction vessel containing the second aliquot, which was then mixed ultrasonically and incubated, generating a fourth reaction mixture.

[0243] Step 9: A magnetic field was applied to the reaction vessel and the fourth reaction mixture was washed five times using a wash buffer to remove any unreacted components.ii

[0244] Step 10: A substrate according to the present disclosure was added to the reaction vessel containing the fourth reaction mixture, and the signal generated from the resulting reaction was read using a luminometer.

[0245] All samples were analyzed in duplicate.

[0246] For each sample a ratio of the signal produced in Step 5 (ApoE4) to the signal produced in Step 10 (Total ApoE) was calculated. The calculated ratios were used to identify the sample as APOE4 / _, APOE4 / +(patients who are thought to have roughly a 3-fold increased risk of developing AD) or APOE4+ / +(patients who are thought to have roughly a 12-fold increased risk of AD). The results are depicted in FIG.2.

[0247] Table 3 lists the minimum, maximum, mean and SD ratios for the patient samples categorized by the predicted genotype. The SD separation between the sample populations, APOE4+ / + / APOE4 / _and APOE4_ / + / APOE4+ / +, is 5.2 for both, indicating that the patient samples for each genotype are well- separated and the difference between them is statistically significant.

[0248] Table 3: Results Summary for Patient SamplesPredicted GenotypeAPOE4' APOE4 / +APOE4+ / +Minimum Ratio 0.003 5.372 1.156Max Ratio 0.007 7.806 3.786Mean Ratio 0.004 6.726 2.404SD Ratio 0.001 0.684 0.462

[0249] The predicted genotype results were also compared with PCR results. As shown in Table 4, the ApoE4 / total ApoE combination assay has > 99% concordance to PCR genotype prediction. Concordance generally refers to the agreement or consistency between diagnostic tests or methods. The high concordance indicates that the ApoE4 / total ApoE combination assay is a reliable and accurate tool for predicting APOE genotypes.

[0250] Table 4: Concordance ComparisonApoE4 / total ApoEPCR Genotype Combination Assay ConcordancePredictioniiAPOE4'7181 181 100%APOE4 / +12 12 100.0%APOE4+ / +107 105 98.1%All 300 298 99.3%

[0251] Example 3: p-Tau217 Immunoassay Clinical Performance

[0252] An exemplary p-Tau217 immunoassay using an exemplary antibody pair was assessed for clinical performance including the ability to distinguish between Alzheimer’s Disease (AD) confirmed patient samples, other neurodegenerative disease patient samples, and normal patient samples.

[0253] The samples included 45 samples from patients with an Alzheimer’s diagnosis, 20 samples from patients with other neurodegenerative diseases (including frontotemporal dementia and Lewy body disease), and 25 aged match control patient samples. The patients with an Alzheimer’s diagnosis were characterized for their amyloid status by CSF Ab42 / Ab40 and / or amyloid PET.

[0254] The p-Tau217 assay as described herein was performed on a Beckman Coulter’s Dxl 9000 analyzer having (i) four reagent pipettors and one sample pipettor configured to aspirate and dispense less than about 10 pL; (ii) a reagent pack with 4 reagent vessels, where each reagent vessel is 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.

[0255] Step 1: An aliquot from the vessel containing paramagnetic particle conjugated with tau capture antibodies was pipetted into a reaction vessel using one of four reagent pipettors. A sample aliquot was pipetted into the reaction vessel using a sample pipettor and an aliquot from the reagent vessel containing ancillary buffers was pipetted into the reaction vessel using one of four reagent pipettors. Then the reaction vessel was mixed using the ultrasonic mixer and incubated, generating a first reaction mixture.

[0256] Step 2: A magnetic field was applied to the reaction vessel and the first reaction mixture was washed three times using a wash buffer to remove any unreacted components.

[0257] Step 3: An aliquot of the first reaction mixture was pipetted into a different reaction vessel using one of the four reagent pipettor. An aliquot from the vessel containing ALP conjugated p-tau217 antibodies was pipetted into the reaction vessel using one of four reagent pipettors. The reaction vessel was mixed ultrasonically and incubated, generating a second reaction mixture.

[0258] Step 4: A magnetic field was applied to the reaction vessel and the second reaction mixture was washed five times using a wash buffer to remove any unreacted components.

[0259] Step 5: 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.

[0260] All samples were analyzed in duplicate. As shown in FIG. 3, the exemplary p-Tau217 immunoassay was able discriminate between amyloid positive and negative patients.

[0261] The sensitivity versus 1-Specifity for the resulting data was plotted, generating a receiver operating characteristic (ROC) curve. The area under the curve (AUC), which is an effective and combined measure of sensitivity and specificity that describes the inherent validity of diagnostic tests, was then calculated. The ROC curve shows an AUC = 0.93 with 95% confidence interval 0.858 to 0.994 for the detection of Alzheimer’s disease (43 AD patient samples and 48 Non-AD patient samples) and an AUC = 0.95 with 95% confidence interval 0.892 to 1.004 for the prediction of amyloid status (48 amyloid(+) patient samples and 40 amyloid(-) patient samples) indicating an excellent diagnostic performance of the exemplary p-Tau217 assay.

[0262] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0263] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. An immunoassay method of assessing a subject’s Alzheimer’s Disease status and zygosity for the APOE e4 allele, the method comprising:contacting a first portion of at least one biological sample from the subject with first and second apolipoprotein (ApoE) antibodies, wherein the second ApoE antibody is specific to apolipoprotein E4 isoform (ApoE4), generating a first reaction mixture;conducting a detection reaction in the first reaction mixture generating a first signal; detecting the first signal;contacting a second portion of the at least one biological sample from the subject with third and fourth ApoE antibodies capable of binding to multiple ApoE isoforms, wherein the third or fourth ApoE antibody and the first ApoE antibody bind to a same epitope of ApoE, generating a second reaction mixture;conducting a detection reaction in the second reaction mixture generating a second signal: detecting the second signal;calculating a ratio of the first signal to the second signal;assigning the subject an APOE e4 genotype based on comparison of the ratio to a ratio established in a reference population;performing a quantitative immunoassay on a third portion of the at least one sample from the subject to determine a concentration of phosphorylated tau isoform in the sample;and assessing the Alzheimer’s Disease status of the subject based on the comparison of the determined concentration to a concentration value established in a reference population.

2. An immunoassay method of assessing a subject’s Alzheimer’s Disease status and zygosity for the APOE e4 allele, the method comprising:exposing a first portion of at least one biological sample from the subject to a first capture antibody capable of binding to Apolipoprotein E (ApoE) and a first detector affinity molecule specific to apolipoprotein E4 isoform (ApoE4), generating a first reaction mixture:conducting a detection reaction in the first reaction mixture wherein the first detector affinity molecule generates a first detection signal;recording the first detection signal;exposing a second portion of the at least one biological sample from the subject to a second capture antibody capable of binding to ApoE and a second detector affinity molecule capable of binding to multiple isoforms of ApoE, generating a second reaction mixture, wherein the first capture antibody and the second capture antibody bind to the same epitope of ApoE; conducting a detection reaction in the second reaction mixture wherein the second detector affinity molecule generates a second detection signal;recording the second detection signal;calculating a ratio of the recorded first detection signal to the recorded second detection signal to generate an ApoE4 comparison value and,predicting, based on the ApoE4 comparison value, whether the subject is heterozygous or homozygous for the APOE e4 allele;exposing a third portion of the at least one biological sample from the subject to a third capture antibody capable of binding to tau, generating a third reaction mixture;conducting a detection reaction in the third reaction mixture;quantitatively determining an amount of a phosphorylated tau isoform in the plasma based on the presence of the reaction in the third reaction mixture;correlating the third detection signal to a concentration of a phosphorylated tau isoform; andassessing the subject’s Alzheimer’s Disease status based on the concentration of the phosphorylated tau isoform.

3. The method of claim 2, wherein the first capture antibody and the second capture antibody are the same.

4. The method of claim 2 or claim 3, wherein the third capture antibody capable of binding to tau binds to a tau epitope comprising an isoform subject to phosphorylation when the isoform is phosphorylated.

5. The method of any one of claims 2 to 4, wherein the method further comprises exposing the third portion of the at least one biological sample to a third detector affinity molecule prior to conducting the detection reaction.

6. The method of any one of claims 1 to 5, wherein the phosphorylated tau isoform is threonine 217.

7. The method of any one of claims 1 to 6, wherein assessing the subject’s Alzheimer’s Disease status comprises comparing the concentration of the phosphorylated tau isoform to concentrations of phosphorylated tau established in a reference population of plasma samples, wherein each of the reference population samples have been classified as positive or negative for Alzheimer’s disease based on a correlation to tau levels measured via positron emission tomography (PET) or cerebrospinal fluid (CSF) analysis.

8. The method of any one of claims 1 to 7, wherein the subject has mild cognitive impairment and / or is 55 years or older.

9. The method of any one of claims 1 to 8, wherein the assessing the subject’s Alzheimer’s Disease status comprises diagnosing the subject with Alzheimer’s disease.

10. The method of claim 9, wherein if the concentration of the phosphorylated tau isoform is equal to or less than a reference range established in the reference population, the subject is determined to not have Alzheimer’ s disease.

11. The method of claim 9, wherein if the concentration of the phosphorylated tau isoform is higher than a reference signal established in the reference population, the subject is determined to have Alzheimer’s disease.

12. The method of any one of claims 1 to 11, wherein the quantitation detection limit (LOQ) for measuring the phosphorylated tau isoform is equal to or less than about 3 pg / mL,lalternatively equal to or less than about 2 pg / mL, equal to or less than about 1 pg / mL, equal to or less than about 0.6 pg / mL, and wherein the detected phosphorylated tau isoform has a concentration above the LOQ.

13. The method of claim 12, wherein the detected phosphorylated tau isoform has a concentration that is at least IX greater than the LOQ; alternatively at least 2X greater than the LOQ; or alternatively at least 3X greater than the LOQ.

14. The method of any one of claims 1 to 13, wherein the detected phosphorylated tau isoform exhibits a coefficient of variation (CV) of 20% or less, alternatively 15% or less, alternatively 10% or less, alternatively 5% or less, or alternatively 4% or less.

15. The method of any one of claims 1 to 14, wherein the method further comprises assessing a degree of likelihood that the subject will be at risk of developing Amyloid-Related Imaging Abnormalities (ARIA) based on the whether the subject is heterozygous or homozygous for the APOE e4 allele.

16. The method of claim 15, wherein the degree of likelihood that the subject will develop ARIA is low risk, medium risk, or high risk.

17. The method of any one of claims 1 to 16, wherein the subject is recommend further treatment.

18. The method of claim 17, wherein the further treatment comprises qualifying the subject for a clinical trial, assessing the risks associated with the subject entering the clinical trial, and / or increasing safeguards for the subject in clinical trial.

19. The method of claim 17, wherein the further treatment comprises assessing a subject’s eligibility for a disease-modifying therapy (DMT), administering a DMT and / or providing monitoring or additional treatments in conjunction with the DMT.

20. The method of claim 19, wherein the DMT is selected from the group consisting of antiamyloid therapies, anti-tau therapies, immunotherapies, vaccines, BACE inhibitors, antiinflammatory drugs, neurotrophic drugs, and combinations thereof.

21. The method of any one of claims 1 to 20, wherein the detector affinity molecule is an antibody, a monoclonal antibody, a polyclonal antibody, an antibody fragment, a synthetic antibody mimic, an aptamer, an affimer, DARPins, oligonucleotide, peptide, or antigen.

22. The method of any one of claims 1 to 21, wherein the capture antibody, the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity molecule is conjugated to at least one magnetic bead.

23. The method of any one of claims 1 to 22, wherein the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity molecule is conjugated to an enzyme.

24. The method of claim 23, wherein the enzyme comprises horseradish peroxidase or alkaline phosphatase.

25. The method of any one of claims 1 to 24, wherein the method is performed using an 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.

26. The method of claim 25, wherein the detector arrangement comprises a luminometer, an electrochemiluminescence (ECL) detector, a photomultiplier tube (PMT) detector, a photometer, a fluorometer, or a bioluminescence detector.

27. The method of any one of claims 2 to 26, wherein the first detection signal, the second detection signal, and / or the third detection signal are luminescent signals, eletrochemiluminescent signals, or chemiluminescent signals.

28. The method of any one of claims 2 to 27, wherein the first detection signal, the second detection signal, and / or the third detection signal are generated via a detection reaction between the first detector affinity molecule, the second detector affinity molecule, and / or the third detector affinity and a substrate formulation, wherein the substrate formulation comprises:a chemiluminescent compound of the formula A or a salt thereof:whereinA is Ci-ehaloalkyl, naphthyl, phenyl, substituted phenyl, or heteroaryl, wherein substituted phenyl comprises from 1 to 3 halo, Ci-6 alkyl, Ci-6 alkoxy, Ci-6 haloalkyl, C(O)Ri5, CN or NO2 substituents;Ri is selected from the group consisting of Cs-uaryl, C1-6 alkyl, C1-6 haloalkyl, and C5-14 aralkyl groups;R7-R14 are independently H, C1-6 alkoxy, halo, C1-4 alkyl, or R7 or R8-R9 or R9-R10 or Rn-R^or R12-R13 or R13-R14, can be joined together as a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring;R15 is C1-6 alkyl;each M is independently selected from the group consisting of H, an alkali metal, alkaline earth metal, transition metal, ammonium, phosphonium, organic amine salt, and an amino acid salt; Z is O or S; andn is 0, 1, or 2;a cationic aromatic compound (CAC);a background reducing agent; andan ether-linked nonionic surfactant or a hydrophilic polymer.ill29. The method of any one of claims 25 to 28, wherein the immunoassay analyzer further comprises at least one reagent pack configured to hold a plurality of reagent vessels, wherein each reagent vessel is configured to store a volume of reagent required for at least one instance of the assays.

30. The method of claim 29, wherein the immunoassay analyzer further comprises at least two reagent packs configured to hold a plurality of reagent vessels, whereinthe first reagent pack comprises a reagent vessel comprising the first capture antibody, a reagent vessel comprising the first detector affinity molecule, and a reagent vessel comprising the second detector affinity molecule, andthe second reagent pack comprises a reagent vessel comprising the third capture antibody and a reagent comprising the third detector affinity molecule.

31. The method of any one of claims 25 to 30, wherein the immunoassay analyzer further comprises an ultrasonic mixing module.

32. The method of any one of claims 25 to 31, wherein the immunoassay analyzer further comprises:a machine vision apparatus comprising an image capture device and an image interpretation device configured to monitor instrument and / or assay functionalities of the immunoassay analyzer.

33. The method of claim 32, wherein the instrument functionalities are selected from the group consisting of optical sensors, pressure sensors and thermistors.

34. The method of claim 33, wherein the assay functionalities are selected from the group consisting of sample volume monitoring, total reagent volume monitoring, residual volume monitoring, and particle retention monitoring.

35. The method of any one of claims 25 to 34, wherein the pipettor arrangement comprises at least a first reagent pipettor, a second reagent pipettor, and a third reagent pipettor.

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

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

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

39. The method of any one of claims 25 to 38, wherein the first reaction mixture, the second reaction mixture, and / or the third reaction mixture comprise unreacted components, and the immunoassay analyzer further comprises a washing arrangement,wherein the washing arrangement is configured to perform at least one wash action to wash away at least a portion of unreacted components, alternatively configured to perform at least two wash actions, alternatively configured to perform at least three wash actions, alternatively configured to perform at least four wash actions, alternatively configured to perform at least five wash actions, alternatively configured to perform at least six wash actions, alternatively configured to perform at least seven wash actions, alternatively configured to perform at least eight wash actions, alternatively configured to perform at least nine wash actions, or alternatively configured to perform at least ten wash actions.

40. The method of claim 39, wherein the first reaction mixture, the second reaction mixture, and / or the third reaction mixture is subjected to a magnetic field prior to performing the at least one wash action.

41. The method of any one of claims 1 to 40, wherein the biological sample is serum, whole blood, plasma, and / or cerebral spinal fluid.

42. The method of any one of claims 1 to 41, wherein the biological sample volume is less than about 10 µL, alternatively between about 2 µL to about 9.9 µL.

43. The method of any one of claims 1 to 42, wherein the method is configured to analyze at least about 200 biological samples / hr; alternatively at least about 300 biological samples / hr; or alternatively at least about 400 biological samples / hr.

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