Immunoassay for detecting TAU phosphorylated at serine 413

Abstract

Human tau protein phosphorylated at the amino acid, serine 413 (pS413 tau), can serve as a biomarker for tauopathies such as Alzheimer's disease. Detection and quantitation of pS413 tau in a biological sample such as cerebrospinal fluid can be useful in developing therapeutics for certain tauopathies. However, pS413 tau is present in biological samples at very low levels. Thus, the invention provides a highly sensitive assay for the detection and quantitation of pS413 tau in a biological sample comprising a series of steps as described herein.

Description

IMMUNOASSAY FOR DETECTING TAU PHOSPHORYLATED AT SERINE 413CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 587.537, filed October 3, 2023. the disclosure of which is incorporated herein by its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on August 30, 2024, is named 25834_WO_PCT_SL.XML and is 85,662 bytes in size.TECHNICAL FIELD

[0003] This disclosure relates generally to an ultrasensitive assay for quantitating human tau protein phosphorylated at serine 413 (pS413 tau) in a biological sample.BACKGROUND

[0004] Tauopathies are a group of progressive neurodegenerative disorders that are pathologically defined by the presence of tau protein aggregates in the brain. More than 26 different tauopathies have been identified. Sexton, C et al., Alzheimer s Dement, 2022 May; 18(5):988-1007. In primary tauopathies, tau is the major and prominent component of the pathology, such as PiD (Pick's disease), PSP (Progressive supranuclear palsy), CBD (corticobasal degeneration) and AGD (Argyrophilic grain disease). In some other tauopathies, tau aggregation is understood to be a response to other pathological proteins or events, like amyloid beta (A(3) in Alzheimer's disease (AD) and repetitive brain injury in chronic traumatic encephalopathy (CTE).

[0005] Alzheimer’s disease (AD) is the most common cause of dementia among older adults. It is a brain disorder involving slow destruction of memory and thinking skills, eventually leading to the loss of ability to carry out the simplest of tasks. AD is currently ranked as the seventh leading cause of death in the United States.

[0006] Histopathologically, brains of AD patients are characterized by the presence of two classes of microscopic abnormal structures: extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFTs). Miao J et al., Frontiers in Aging Neuroscience, March 2019, vol.11, Article 34. These structures comprise highly insoluble and densely packed filaments which are produced from soluble building blocks, amyloid-P (A|3) peptides for plaques and tau for neurofibrillary tangles. While amyloid-P peptides are produced from proteolytic fragmentation of the transmembrane amyloid precursor protein, tau is a brain-specific microtubule-associated protein enriched in axons. Bloom, GS. JAMA Neurology April 2014, 71(4). 505-508. Evidence indicates that the number of NFTs, but not the number of amyloid plaques correlates wi th the degree of dementia in AD patients. Arriagada, PV et al., Neurology, 42, 631-639. Evidence also indicates that soluble forms of Ap and tau work in coordination, independently of their accumulation into plaques and tangles, to drive healthy neurons into the diseased state, and that hallmark toxic properties of AP require tau. Bloom, GS, JAMA Neurology, April 2014, 71(4), 505-508.

[0007] The major function of tau is to promote microtubule assembly and maintain microtubule structure. This function is regulated by its degree of phosphorylation. Tau is abnormally hyperphosphorylated in AD brain, and neurofibrillary tangles are composed of hyperphosphorylated and aggregated tau. Abnormally hyperphosphorylated tau inhibits promotion of microtubule assembly. Alonso, AC et al., 1994, Proc. Natl. Acad. Sci. USA 91, 5562-5566. Evidence suggests that unlike normal tau. the hyperphosphorylated and oligomeric tau isolated from AD brain sequesters / captures normal tau and templates it into filaments in vitro in a prion-like manner. Id.

[0008] Six major tau isoforms, produced by alternative RNA splicing of exons 2, 3, andlO, are found in the human brain. These isoforms are: 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R. Among these, the 2N4R isoform is the longest and contains over 85 potential phosphorylation sites represented by either senne, threonine, or tyrosine residues. Goedert M. et al.. EMBO J. 1989;8(2):393-9.

[0009] In one study, using isotope labeled tau, the authors compared the post translation modification coverage (phosphorylation, acetylation, ubiquitinylation, and methylation) of tau enriched from Alzheimer’s brains across different disease stages and tau solubilities and found that soluble tau from AD and control brains carry similar phosphorylation, and that seeding competent tau from later disease stages, both oligomeric soluble and aggregated tau, has a higher extent of phosphorylation, and is acetylated and ubiquitinated in the microtubule-associating domain of tau. Hendrick W et al., Cell, 2020, 183. 1699-1713. Modifications on 43-55 different phosphorylation, 19 acetylation, 14-17 ubiquitination, and four methylation sites were identified.

[0010] In another study, based on immunoscreening of transgenic mice that dominantly express four-repeat human tau in adult age and exhibit abnormal tau phosphory lation, synapse loss, memory' impairment at 6 months, neurofibrillary' tangle formation and neuronal loss at 24 months, it was concluded that phosphory lation of tau at serine 413 occurs early and at a high level. Umeda et al., Annals of Clinical and Translational Neurology / 2015; 2(3): 241-255. Also, in this study, an anti-pSer413 antibody was shown to significantly improve memory. This cognitive improvement was found to parallel reduction in the levels of tau hyperphosphorylation, tau oligomer accumulation, synapse loss, tangle formation, and neuronal loss.

[0011] These findings suggested that tau phosphorylated at serine 413 (pSer413 tau) is a promising target for intervention in the treatment of tauopathy. However, pSer413 tau is present at very low levels in biological fluids. Thus, a need exists for a highly sensitive assay to quantitate pSer413 tau in biological fluids, e.g., the CSF, of subjects having a tauopathy.SUMMARY OF THE INVENTION

[0012] In part, the present disclosure provides an immunoassay for detection and / or quantification of human tau protein phosphory lated at amino acid serine 413 (pS413 tau) in a biological sample. The immunoassay comprises the following steps:(a) contacting the sample with a capture antibody immobilized on a surface, wherein the capture antibody specifically binds to pS413 tau,(b) washing the surface to remove unbound sample,(c) adding a detection antibody to the surface after step (b), wherein the detection antibody specifically binds to tau bound to the capture antibody, thereby yielding a first complex, wherein a signaling tag is attached to the detection antibody,(d) washing to remove unbound detection antibody, and(e) detecting signal derived from the detection antibody, wherein the detection antibody comprises(i) a light chain variable region complementarity determining region 1 VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and(ii) a heavy chain variable region complementarity determining region 1 VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VH- CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0013] The immunoassay may further comprise between steps (d) and (e),(f) contacting the first complex with a signal enhancement reagent to form a second complex.(g) washing the second complex to remove unbound signal enhancement reagent,(h) contacting the second complex with a detection reagent to form a third complex,(i) washing the third complex to remove unbound detection reagent, and(j) contacting the third complex with a read reagent.

[0014] The capture antibody may comprise(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 11; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and(ii) a heavy chain variable region complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 14; a VEI-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0015] The biological sample is a bodily fluid, for example, cerebrospinal fluid.

[0016] Using this immunoassay, pS413 tau can be detected at a concentration as low as about 4.1 fM in human cerebrospinal fluid.

[0017] The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram showing the relative locations of the epitopes of the capture and detection antibodies used in the immunoassay for detecting human tau protein phosphorylated at serine 413 (pS413 tau).

[0019] Figure 2 is a graph showing detection of pS413 tau in CSF from normal subjects using the immunoassay disclosed herein.

[0020] Figure 3 is a graph showing detection of pS413 tau by ELISA in three CSF samples (1, 2, & 3) using antibodies HT7, Ta21-9, and Ta23-7 for capture, and the anti-pS413 tau antibody, Tai 505, for detection in each case.DETAILED DESCRIPTION OF THE INVENTION

[0021] The present disclosure is directed to a highly sensitive immunoassay for detecting human tau protein phosphorylated at the amino acid serine 413 (pS413 tau) in a biological sample such as a bodily fluid (e.g., CSF).

[0022] pS413 tau is present at very low levels in bodily fluids. In healthy subjects, pS413 tau is present at even lower levels than in subjects having a tauopathy. For therapies targeting pS413 tau, it is of interest to know the level of pS413 tau in the bodily fluid of choice at the start of the therapy. This information could be useful in selecting patients who could benefit from the therapy. It is of interest also to know the level of pS413 tau in the bodily fluid during or after the therapy. If the therapy is effective, the level of pS413 tau in the bodily fluid of the subject is likely to be further reduced. To assess if the agent used in the therapy, for example, an antibody, is engaging pS413 tau effectively, the assay should be able to measure this reduced level of pS413 tau. To date, no report exists of the detection of pS413 tau in the bodily fluids of either a healthy subject or a subject having a tauopathy. The immunoassay described herein overcomes this shortcoming.Definitions

[0023] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0025] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0026] As used herein, the term “about” means within a statistically meaningful range of a value, such as a stated concentration range, time frame, molecular weight, temperature, or pH. Such a range can be within an order of magnitude, typically within 10%, and more ty pically within 5% or within 1% of a given value or range (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides). Sometimes, such a range can be within the experimental error typical of standard methods used for the measurement and / or determination of a given value or range. The allowable variation encompassed by the term “about” will depend upon the system under study and can be readily appreciated by one of ordinary skill in the art. Whenever a range is recited w ithin this application, every whole number integer within the range is also contemplated as an embodiment of the disclosure.

[0027] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0028] In this disclosure, terms such as “comprises,” “comprised,” “comprising,” “contains,” “containing” and the like mean “includes,” “included,” “including” and the like. Such terms refer to the inclusion of particular ingredients or set of ingredients without excluding any other ingredients.

[0029] Terms such as “consisting essentially of’ and “consists essentially of’ allow for the inclusion of additional ingredients or steps that do not detract from the novel or basic characteristics of the disclosure, i.e., they exclude additional unrecited ingredients or steps that detract from the novel or basic characteristics of the disclosure. The terms “consists of’ and “consisting of’ are closed ended. Accordingly, these terms refer to the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.

[0030] The term “detecting” is used in the broadest sense to include both qualitative and quantitative measurements of a target molecule. In one aspect, the detecting method as described herein is used to identify the mere presence of human tau protein phosphorylated at the amino acid serine 413 (pS413 tau) in a biological sample. In another aspect, the method is used to test whether pS413 tau in the sample is at a detectable level. In yet another aspect, the method can be used to quantify the amount of pS413 tau in the sample and further to compare the pS413 tau levels from different samples.

[0031] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature. 256:495 (1975), or may be made by recombinant DNA methods (see. e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.Immunoassay for detecting pS413 tan

[0032] The immunoassay developed by the inventors for detecting and quantitating pS413 tau is based on the S-PLEX technology of MESO SCALE DISCOVERY (MESO SCALE DIAGNOSTICS. Rockville, Maryland). S-PLEX technology requires detection of electrochemiluminescence (ECL) signal. The immunoassay described requires the use of two antibodies: (1) a capture antibody that specifically recognizes an epitope on human tau that includes phosphoserine 413 and (2) the detection antibody that recognizes an epitope on tau close to serine 413. for example, Ta21-9 which recognizes an epitope comprising the peptide region comprising amino acids 365 to 388. The capture antibody is immobilized on a surface, for example, the capture antibody is biotinylated which allows its attachment to a plate having streptavidin coated wells. The detection antibody is labeled with a Meso Scale Discovery' (MSD) proprietary reagent TURBO-BOOST™, which in combination with another MSD proprietary' reagent TURBO-TAG™ generates enhanced ECL signal.

[0033] For the assay perform at a high level of sensitivity, several parameters had to be optimized. For example, to improve reproducibility, a tau peptide phosphorylated at serine 413(SEQ ID NO: 26) was used as a calibrator instead of recombinant tau protein phosphorylated at serine 413. With this change, inconsistency in results arising due to the low (<5%) and variable in vitro phosphorylation of recombinant tau protein from one batch to another could be avoided. To eliminate heterophilic interference, a heterophilic blocking reagent (HBR) was used. Heterophilic interference refers to problem caused by antibodies present in a biological sample such as plasma which bind to capture and detection antibodies used in immunoassay, often resulting in false positive interference, and sometimes also false negative interference. A HBR may contain immunoglobulins of murine origin with specific binders that by attaching to the heterophilic antibodies render the heterophilic antibodies incapable of cross linking the capture and the detection antibodies in the immunoassay (e.g., HBR 1, Scantibodies Laboratory, Inc., Santee, CA, Catalog No. 3KC534-075). Other parameters that were varied until optimum results were obtained include capture antibody concentration, detection antibody concentration, various incubation time, and sample volume.Capture and detection antibodies

[0034] US 2015 / 0183854 discloses several antibodies (Tal5 series) that were produced to recognize human tau protein (SEQ ID NO: 22; see Table 1 below) phosphorylated at serine 413. Each of these antibodies, namely Tal501, 1502. 1505, 1506, 1507, 1508, and 1509 may be used as the capture antibody in the immunoassay of the invention.

[0035] These antibodies were produced by immunizing mice with the synthetic peptide, pSer413(Im) (SEQ ID NO: 21; see Table 1 below), which corresponds to the peptide region from 403rdamino acid (Thr) to the 423rdamino acid (Pro), The peptide has GlyCysSerGly (SEQ ID NO: 88) attached at the N-terminus and is phosphorylated on serine at position 413. The specificity of these antibodies was determined by testing for binding to a battery of peptides phosphorylated at different serine and / or threonine residues and to a control peptide referred to as non-pS413 (corresponding to the region from the 405thamino acid (Pro) to the 421stamino acid (Asp) (SEQ ID NO: 24; see Table 1 below). These antibodies did not bind to any of the peptides except pS413 (same as peptide non-pS413, but with phosphorylation at serine 413 (SEQ ID NO: 23; see Table 1 below) and to a much smaller extent or not at all to non-pS413.

[0036] For detection, the assay used the antibody, Ta21-9, which was produced by immunizing mice with the synthetic peptide GCGGGNKKIETHKLTFRENAKAKTDH (ImPep 365-388; SEQ ID NO: 25), which corresponds to the region from 365thamino acid (Gly) to the 388thamino acid (His) of human tau protein (SEQ ID NO: 22) with GlyCys attached at the N- terminus.

[0037] The amino acid sequences of various regions of Tal505, Tal501, Tal502, Tal506- Tal509, and Ta21-9 are shown in Table 1 below.Table 1In SEQ ID NOs: 21, 23, and 26, phosphorylated serine is indicated by bold letter and underlining.

[0038] Like Tal505, Ta21-9 recognizes an epitope near the C-terminus of tau (see Fig. 1). The epitope of Ta21-9 is proximal to the epitope of Tal505 and relatively shifted toward the N- terminus of tau. Such proximal positioning of the epitope of Ta21-9 vis-a-vis that of Tal505 issignificant for the following reason. Tau is subject to proteolysis across its entire length (see Quinn JP et al., Journal of Alzheimer ’s Disease 63 (2018) 13-33). Thus, in immunoassay for tau based on a capture and a detection antibody that bind the protein simultaneously, the farther away the epitope of one antibody of the pair is from that of the other antibody, the greater the likelihood of a drop in signal intensity due to the possibility of proteolysis at one more site in the region separating the two epitopes. In addition, the two antibodies should be such that one does not sterically hinder the other.Kits

[0039] As a matter of convenience, the immunoassay method of this invention can be provided in the form of a kit. Such a kit is a packaged combination including the basic elements of:(a) a capture antibody that specifically recognizes an epitope of tau that includes phosphorylated serine 413. e.g., Tal505 (which may be biotinylated by the user); and(b) detection reagents comprising the detection antibody Ta21-9 (labeled or with Turboboost®).

[0040] The kit may further comprise a solid support on which the capture antibody may be immobilized.Embodiments

[0041] The invention is further described below in the following embodiments.

[0042] In one embodiment, the invention provides an immunoassay for detection and / or quantification of human tau protein phosphorylated at amino acid serine 413 (pS413 tau) in a biological sample. The immunoassay comprises(a) contacting the sample with a capture antibody immobilized on a surface, wherein the capture antibody specifically binds to pS413 tau,(b) washing the surface to remove unbound sample,(c) adding a detection antibody to the surface after step (b). wherein the detection antibody specifically binds to tau bound to the capture antibody, thereby yielding a first complex, wherein a signaling tag is attached to the detection antibody,(d) washing to remove unbound detection antibody, and(e) detecting signal derived from the detection antibody, wherein the detection antibody comprises(i) a light chain variable region complementarity determining region 1 VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and(ii) a heavy chain variable region complementarity determining region 1 VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VH- CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VH- CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0043] In some embodiments, the immunoassay further comprises between steps (d) and (e),(f) contacting the first complex with a signal enhancement reagent to form a second complex,(g) washing the second complex to remove unbound signal enhancement reagent,(h) contacting the second complex with a detection reagent to form a third complex,(i) washing the third complex to remove unbound detection reagent, and(j) contacting the third complex with a read reagent.

[0044] In some embodiments, the signal enhancement reagent comprises S-PLEX El, S-PLEX E2, and S-PLEX E3 (MESO SCALE DIAGNOSTICS).

[0045] In some embodiments, the detection reagent comprises S-PLEX DI and S-PLEX D2 (MESO SCALE DIAGNOSTICS, LLC ).

[0046] In some embodiments, the read reagent comprises MSD Gold Read Buffer A or MSD Gold Read Buffer B (MESO SCALE DIAGNOSTICS, LLC ).

[0047] In some embodiments, the signal is an electrochemiluminescence signal.

[0048] In some embodiments, the surface is a well of a 96-well or a 384-well plate.

[0049] In some embodiments, the well is coated with streptavidin.

[0050] In some embodiments, the well comprises an electrode, allowing detection of electrochemiluminescence signals.

[0051] In some embodiments, the signaling tag is TURBO-BOOST® (MESO SCALE DIAGNOSTICS, LLC ).

[0052] In some embodiments, the capture antibody is biotinylated.

[0053] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 11; a VL-CDR2 comprising the amino acidsequence of SEQ ID NO: 12; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 13;(ii) a heavy chain variable region complementarity' determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 14; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

[0054] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity' to the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0055] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 17 and a VH having the amino acid sequence of SEQ ID NO: 18.

[0056] In some embodiments, the capture antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 19 and a heavy chain having the amino acid sequence of SEQ ID NO: 20.

[0057] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 28; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 29; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 30;(ii) a heavy chain variable region complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 31; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33.

[0058] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 34 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity7to the amino acid sequence of SEQ ID NO: 35.

[0059] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 34 and a VH having the amino acid sequence of SEQ ID NO: 35.

[0060] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 38; a VL-CDR2 comprising the amino acidsequence of SEQ ID NO: 39; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 40;(ii) a heavy chain variable region complementarity' determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 41; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

[0061] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity' to the amino acid sequence of SEQ ID NO: 44 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 45.

[0062] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 44 and a VH having the amino acid sequence of SEQ ID NO: 45.

[0063] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 48; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 49; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 50;(ii) a heavy chain variable region complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 51; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

[0064] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 54 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity' to the amino acid sequence of SEQ ID NO: 55.

[0065] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 54 and a VH having the amino acid sequence of SEQ ID NO: 55.

[0066] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity' determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 58; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 60;(ii) a heavy chain variable region complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 61; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 62; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 63.

[0067] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 64 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 65.

[0068] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 64 and a VH having the amino acid sequence of SEQ ID NO: 65.

[0069] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 68; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 70;(ii) a heavy chain variable region complementarity' determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 71; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

[0070] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity7to the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 75.

[0071] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 74 and a VH having the amino acid sequence of SEQ ID NO: 75.

[0072] In some embodiments, the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence of SEQ ID NO: 78; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 80;(ii) a heavy chain variable region complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence of SEQ ID NO: 81; a VH-CDR2 comprising the amino acidsequence of SEQ ID NO: 82; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 83.

[0073] In some embodiments, the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 84 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 85.

[0074] In some embodiments, the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 84 and a VH having the amino acid sequence of SEQ ID NO: 85.

[0075] In some embodiments, the detection antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 7 and heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0076] In some embodiments, the detection antibody comprises a VL having the amino acid sequence of SEQ ID NO: 7 and a VH the amino acid sequence of SEQ ID NO: 8.

[0077] In some embodiments, the detection antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO: 10.

[0078] In some embodiments, the sample comprises human cerebrospinal fluid (CSF).

[0079] In some embodiments, pS413 tau is detected at a concentration as low as about 4. 1 fM.EXEMPLIFICATION

[0080] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention and are not intended to limit the invention.Example 1: Production of the monoclonal antibodies Tal505 and Ta21-9

[0081] As noted in the previous section, Tai 505 is one of several antibodies disclosed in US 2015 / 0183854 that were produced to recognize human tau protein phosphory lated at serine 413. TA1505 binds specifically to tau phosphorylated at serine 413 (pS413 tau).

[0082] The antibody Ta21-9 was produced by immunizing mice with the synthetic peptide GCGGGNKKIETHKLTFRENAKAKTDH (ImPep 365-388; SEQ ID NO: 25), which corresponds to amino acids 365 (Gly) to 388 (His) of human tau protein (SEQ ID NO: 22) withGlyCys attached at the N-terminus. Following conventional procedures, in the initial immunization, the peptide was used as a KLH conjugate in Freund's complete adjuvant. Booster immunizations were carried out by intraperitoneal injection of the peptide with Freund's incomplete adjuvant. Specificity of Ta21-9 was evaluated by testing its binding to nonphosphorylated tau, phosphorylated tau, hyperphosphorylated tau, and MAP2, a microtubule- associated protein that is highly homologous to tau.

[0083] Ta21-9 did not bind to MAP2, bound roughly equally well to phosphory lated tau and hyper phosphorylated tau, and relatively less well to non-phosphorylated Tau. It should be noted that like Tal505, Ta21-9 also recognizes an epitope near the C-terminus of tau. but compared to Tal505, its epitope is shifted toward the N-terminus.Example 2: Selection of an antibody that can pair with Tai 505 in an immunoassay for the detection of pS413 tau

[0084] As noted above. TA1505 binds specifically to tau phosphorylated at serine 413. Also as noted above, Tau is subject to proteolysis across its entire length. As such, for an antibody to work effectively with TA1505 (as a capture and detection antibody pair) in an immunoassay for the detection of pS413 tau, the antibody should recognize an epitope of tau proximal to the TA1505 epitope, but not sterically hinder or be hindered by TA1505.

[0085] In this regard, three antibodies were tested, namely Ta21-9, Ta2001, and 6B12. As described in Example 1, Ta21-9 recognizes an epitope in the amino acid region 365-388 of tau. Ta2001 recognizes epitope in the peptide region 379-408 of tau in which serine residues 396, 400, and 404 are phosphorylated. The epitope of 6B12 is unknown. As Table 2 below shows, among these antibodies, Ta21-9 was found to yield the highest signal and the highest signal / blank ratio. Note that in this immunoassay, Tal505, which recognizes tau phosphorylated at serine 413, was used as the capture antibody.Table 2

[0086] The results show that in the immunoassay for detecting pS413 tau in which Tai 505 was one of the antibodies of the antibody pair, Ta21-9 performed the best among the three anti-tau antibodies tested. Ta2001 did not perform as well even though it recognizes an epitope in the amino acid region 379-408, which, compared to the epitope of Ta21-9, is closer to the epitope of Tai 505.

[0087] In another study, two additional anti-tau antibodies were examined for their ability to be paired with Tal505 for detecting pS413 tau. These antibodies are HT7 and Ta23-7. HT7 is a commercially available anti-tau mouse monoclonal antibody (INNOTEST, Fujirebio) that recognizes the amino acid region 159-163 in normal and phosphorylated tau protein. Chai X et al., 2011, J. Biol. Chem. vol. 286, issue 39, pp. 34457-34467. Ta23-7 is a monoclonal anti-tau antibody which was produced by the inventors of the this application by immunizing mice with a peptide having the sequence GCSPQLATLADEVSASLAKQGL (SEQ ID NO: 27), which corresponds to amino acids 422-441 of tau with a GlyCys tag at the N-terminus (ImPep 422-441; SEQ ID NO: 27). In the initial immunization, the peptide was used as a KLH conjugate in Freund's complete adjuvant. Booster immunizations were carried out by intraperitoneal injection of the peptide with Freund's incomplete adjuvant. Specificity of Ta23 was evaluated by testing its binding to non-phosphorylated tau, phosphorylated tau, hyperphosphorylated tau, and MAP2. Ta23 bound almost equally well all to three forms of tau, but not to MAP2.

[0088] A sandwich ELISA (Enzyme-Linked Immunosorbent Assay) was performed with three antibody pairs: each of HT7, Ta21-9, and Ta23-7 as the capture antibody paired with Tal505 as the detection antibody. CSF was used as the sample. Results showed that pairing Ta21-9 with Tal505 led to highest assay sensitivity. See Fig. 3. With Ta23-7, the assay sensitivity was about 60% as good as with Ta21-9. However, sensitivity was poor when HT7 was paired with Tal505. It should be noted that the epitope of HT7, amino acid region 159-163, is farther from the epitope of Tal505 than the epitope of Ta21-9, amino acid region 365-388, and therefore more likely to have been lost due to proteolytic cleavage of tau.Example 3: Immunoassay for the detection and quantitation of nS413 tau in CSF

[0089] An immunoassay for the detection and quantitation of pS413 tau in CSF was developed using an ultra-sensitive immunoassay platform called S-PLEX (S-PLEX Development Pack, Catalog #K15291S-4 purchased from MESO SCALE DISCOVERY (MESO SCALE DIAGNOSTICS. Rockville, Maryland). The assay was performed following manufacturer's instructions with modifications and is described below.

[0090] S-PLEX uses electrochemiluminescence (ECL) technology7. The improved sensitivity of S-PLEX is due, in part, to the use of the so-called TURBO-BOOST® and TURBO-TAG® proprietary reagents. The detection antibody is labeled with TURBO-BOOST® which is enhanced in an additional step and then bound by an electrochemiluminescence TURBO-TAG® molecule. Additional S-PLEX reagents reduce non-specific signals. With the combination of TURBO-TAG and TURBO-BOOST®, more signal is generated when compared to other ECL formats that use MSD SULFO-TAG™ as the detection label. The first step of an S-PLEX assay is to assemble a sandwich complex containing capture antibody immobilized on a solid surface, pS413 Tau analyte, and detection antibody labeled with TURBO-BOOST® in an assay plate. The next step involves adding certain MSD proprietary reagents that aid in the generation and enhancement of signal. The third step is to simply add a buffer called MSD GOLD Read Buffer to measure the assay signals on an MSD instrument. The Buffer allows the voltage applied to the plate to cause the captured TURBO-TAG labels to emit light.

[0091] As noted above, Tai 505 and Ta21-9 were used as the capture and detection antibodies, respectively. S-PLEX 96-well SECTOR microplate was washed three times in phosphate buffer saline (PBS) containing 0.05% TWEEN 20. Next, the microplate was coated with biotinylated Tal505 in the MSD S-PLEX coating reagent Cl solution at room temperature with shaking for 1.5 hours. The microplate was washed three times with PBS containing 0.05% TWEEN 20.Next 25 JJ.1 of MSD S- PLEX Blocker S I in the MSD Diluent 100 solution was added to each well, followed by 75 JJ.1 of a standard sample (phospho-tau peptide; SEQ ID NO: 26), or a 2-fold diluted CSF sample. The mixture was incubated at 4 °C overnight with shaking. The microplate was washed three times with PBS containing 0.05% TWEEN 20. Then 50 pl of MSD S- PLEX TURBO-BOOST™ conjugated Ta21-9 (detection antibody) in MSD Diluent 101 were added to each well. The microplate was incubated at room temperature with shaking for 1.5 hours followed by three washes with PBS containing 0.05% TWEEN 20. Next, 50 pl of MSD S- PLEX Enhancement solution containing S- PLEX Enhancer El, E2, and E3 was added to each well. The microplate was incubated at room temperature with shaking for 30 minutes. Next, the microplate was w ashed three times with PBS containing 0.05% TWEEN 20 followed by addition into each well 50 pl of MSD S-PLEX Detection solution containing S-PLEX Detect DI and D2. The microplate w as incubated at room temperature with shaking for 1.5 hours. The plate w as then washed three times with PBS containing 0.05% TWEEN 20. Lastly, 150 pl of MSD Gold read buffer A (MSD Catalog No. R92TG) was added to the plate and the plate was read on the detection instrument MESO SECTOR® S 600MM.

[0092] Details of the equipment, material, and reagents used for the immunoassay, as w ell as the various steps of the assay are described below. Several parameters of the immunoassay w ere varied to improve the sensitivity of the assay including concentrations of the biotinylated capture antibody and the TURBO-BOOST® conjugated detection antibody, CSF sample volume, various incubation times, introduction of a heterophilic blocking reagent, and use of a phosphopeptide calibrator / standard.

[0093] EQUIPMENTMSD Sector Imager (Sl-600) and MSD Workbench Software

[0094] REAGENTS1. Peptide standard (SEQ ID NO: 26)2. Capture antibody Tal505. Tal505 was biotinylated. 100X stocks of the biotinylated Tal505 in Diluent 100 (MSD Catalog No. R50AA-4). containing 0.05% sodium azide were prepared and stored at 4 °C. The concentration of the 100X biotinylated Tal505 stock was 50 pg / mL. Diluent 100 contains protein, blockers, and preservatives.3. S-PLEX Development Pack including El, E2, E3, DI, D2, coating reagent, and blocking reagent. MSD4. Detection antibody Ta21-9. Ta21-9was conjugated with TURBO-BOOST®. 100X stocks of the TURBO-BOOST® conjugated Ta21-9 in Diluent 100 containing 1 mM EDTA and0.05% sodium azide were prepared and stored at 4 °C. The concentration of the 100X conjugated Ta21-9 stocks was 10 pg / mL.5. HyPure Cell Culture Grade Water, Hy clone Cat. No SH30529.036. 10X DPBS with TWEEN 20 pH 7.4, TEKNOVA Cat. No P02977. MSD GOLD Read Buffer (IX), MSD Cat. No R92TG-48. Diluent 100; MSD; Catalog # R50AA-49. Diluent 101; MSD; Catalog # R51AD-310. Heterophilic Blocking Reagent 1 (HBR-1); Scantibodies Laboratory; Catalog#: 3KC533

[0095] REAGENTS PREPARATIONWash Buffer200 ml of 10X PBST (PBS containing 0.05% TWEEN 20)1800 ml of milliQ filter waterBiotinylated capture antibody (Tal505) solution6 ml of Diluent 100120 pl of biotinylated Tal505 antibody30 pl of 200X S-PLEX Coating Reagent Cl, MSD Catalog No. C20H0-3 Assay Buffer: HBR-1 in Diluent 100 (3 pg HBR-1 per 25 pl of cerebrospinal fluid)70 pl of HBR-1 at 20.7 mg / ml was added into 12 ml of Diluent 100 and mixed thoroughly.Blocking Solution3.5 ml of Diluent 10035 pl of Blocker SI, MSD Catalog No. R93AG-1 Preparation of Standard Dilutions of the peptide standard (SEP ID NO: 26)Peptide standard (STD 1) peptide stock at 169.2 fMSTD 2 was prepared by transferring 200 pl of STD 1 to 200 pl of Assay Buffer and mixing well by pipetting up and down several times. Without changing the pipette tip, the 2-fold serial dilution was repeated six additional times to generate STD 3-STD 8. See Table 3 below.Table 3Assay Buffer = Diluent 100 + HBR-1Preparation of TURBO-BOOST® conjugated detection antibody (Ta21-9) solution120 pl of TURBO-BOOST® conjugated Ta21-9 antibody was added to 6 mb of Diluent 101. This was sufficient for one plate. The antibody solution was prepared 5-10 minutes before use.S-PLEX Enhancement Solution3ml of Molecular Biology7Grade Water1.5 ml of 4X S- PLEX Enhance El1.5 ml of 4X S- PLEX Enhance E230 pl of 200X S- PLEX Enhance E3The S- PLEX Enhancement Solution was prepared 5-10 minutes before use.S- PLEX Detection Solution4.5 ml of Molecular Biology Grade Water1.5 ml of 4X S-Plex Detect DI30 pl of 200X S-Plex Detect D2The S- PLEX Detection Solution was prepared 5-10 minutes before use.

[0096] ASSAY PROCEDUREEach test / assay point was performed in duplicate.Plate washThe assay plate was washed 3 times with wash buffer using program 3XS-PLEX. The plate was gently tapped upside down on paper towels.Addition of biotinylated capture antibody50 pl of biotinylated capture antibody solution w as added to each well of the plate. The plate was sealed with plate sealing tape and incubated at room temperature with shaking at 700 rpm for 1.5 hours.Washing and addition of SamplesThe plate was w ashed 3 times with wash buffer and gently tapped upside down on paper towels.Next 25 pl of Blocking solution was added to each wellEach STD or sample w as mixed by pipetting. Next, 75 pl of the w orking solution of STDs or samples was added per well. The plate was sealed with plate sealer and incubated overnight at 4 °C with shaking at 700 rpmWashing and addition of Turbo-Boost detection antibody solutionThe plate was washed 3 times with wash buffer and gently tapped upside down on paper towels.50 pl of Turbo-boost detection antibody solution w as added to each well. The plate was sealed with sealing tape and incubated at room temperature with shaking at 700 rpm for 1.5 hours.Washing and addition of S-PLEX Enhancement SolutionThe plate was w ashed 3 times with wash buffer and gently tapped upside down on paper towels.50 pl of Enhancement Solution was added to each well. The plate was sealed with sealing tape and incubated at room temperature with shaking at 700rpm for 30 min Washing and addition of S-Plex Detection SolutionThe plate was w ashed 3 times with wash buffer and gently tapped upside down on paper towels.50 pl of detection antibody solution was added to each well. The plate was sealed with plate tape and incubated at room temperature with shaking at 700rpm for 1.5 hour Washing and reading the plateThe plate was washed 3 times with wash buffer and gently tapped upside down on paper tow els.150 pl of MSD Gold Read Buffer was added to each well and the plate read on MSD Sector Imager (SI-600). Raw data was analyzed. The regression analysis used was 4PL with a l / Y2weighting factor.Example 4: Establishment of lower limit of quantification (LLOQ)

[0097] Assay sensitivity, i.e., LLOQ (lower limit of quantification), was determined by precise determination of the concentration of pS413 Tau in eight human CSF samples using the assay described in Example 3. The lowest level of pS413 tau level detected in a sample with interassay coefficient of variation (CV) of <25% was set as the limit of assay sensitivity. Table 4 below shows that human CSF_2 met this criterion. Thus, the assay sensitivity was determined to be 4. 1 fM in human CSF.Table 4Incorporation by reference

[0098] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.Equivalents

[0099] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Various structural elements of the different embodiments and various disclosed method steps may be utilized in various combinations and permutations, and all such variants are to be considered forms of the invention. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. An immunoassay for detection and / or quantification of human tau protein phosphorylated at amino acid serine 413 (pS413 tau) in a biological sample, comprising(a) contacting the sample with a capture antibody immobilized on a surface, wherein the capture antibody specifically binds to pS413 tau,(b) washing the surface to remove unbound sample,(c) adding a detection antibody to the surface after step (b), wherein the detection antibody specifically binds to tau bound to the capture antibody, thereby yielding a first complex, wherein a signaling tag is attached to the detection antibody,(d) washing to remove unbound detection antibody, and(e) detecting signal derived from the detection antibody, wherein the detection antibody comprises(i) a light chain variable region complementarity determining region 1 VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and(ii) a heavy chain variable region complementarity determining region 1 VH- CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

2. The immunoassay of claim 1, further comprising between steps (d) and (e).(f) contacting the first complex with a signal enhancement reagent to form a second complex,(g) washing the second complex to remove unbound signal enhancement reagent,(h) contacting the second complex with a detection reagent to form a third complex,(i) washing the third complex to remove unbound detection reagent, and(j) contacting the third complex with a read reagent.

3. The immunoassay of claim 1 or claim 2, wherein the signal enhancement reagent comprises S-PLEX El, S-PLEX E2, and S-PLEX E3 (MESO SCALE DIAGNOSTICS).

4. The immunoassay of any one of claims 1-3, wherein the detection reagent comprises S-PLEX DI and S-PLEX D2 (MESO SCALE DIAGNOSTICS, LLC ).

5. The immunoassay of any one of claims 1-4, wherein the read reagent comprises MSD Gold Read Buffer A or MSD Gold Read Buffer B (MESO SCALE DIAGNOSTICS. LLC ).

6. The immunoassay of any one of claims 1-5, wherein the signal is an electrochemiluminescence signal.

7. The immunoassay of any one of claims 1-6, wherein the surface is a well of a 96- well or a 384-well plate.

8. The immunoassay of any one of claims 1-7, wherein the well is coated with streptavidin.

9. The immunoassay of any one of claims 1-8, wherein the well comprises an electrode, allowing detection of electrochemiluminescence signals.

10. The immunoassay of any of any one of claims 1-9, wherein the signaling tag is TURBO-BOOST® (MESO SCALE DIAGNOSTICS, LLC ).

11. The immunoassay of any one of claims 1-10, wherein the capture antibody is biotinylated.

12. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 11; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 13;(ii) a heavy chain variable region complementarity determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 14; a VH-CDR2 comprisingthe amino acid sequence of SEQ ID NO: 15; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 16.

13. The immunoassay of claim 12, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 18.

14. The immunoassay of claim 12, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 17 and a VH having the amino acid sequence of SEQ ID NO: 18.

15. The immunoassay of any of claims 12, wherein the capture antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 19 and a heavy chain having the amino acid sequence of SEQ ID NO: 20.

16. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 28; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 29; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 30;(li) a heavy chain variable region complementarity determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 31; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33.

17. The immunoassay of claim 16, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 34 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 35.

18. The immunoassay of claim 16, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 34 and a VH having the amino acid sequence of SEQ ID NO: 35.

19. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 38; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 39; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 40;(ii) a heavy chain variable region complementarity' determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 41; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43.

20. The immunoassay of claim 19, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 44 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 45.

21. The immunoassay of claim 19, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 44 and a VH having the amino acid sequence of SEQ ID NO: 45.

22. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 48; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 49; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 50;(ii) a heavy chain variable region complementarity determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 51; a VH-CDR2 comprisingthe amino acid sequence of SEQ ID NO: 52; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53.

23. The immunoassay of claim 22, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 54 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 55.

24. The immunoassay of claim 22, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 54 and a VH having the amino acid sequence of SEQ ID NO: 55.

25. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 58; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 59; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 60;(ii) a heavy chain variable region complementarity determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 61; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 62; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 63.

26. The immunoassay of claim 25, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 64 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 65.

27. The immunoassay of claim 25, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 64 and a VH having the amino acid sequence of SEQ ID NO: 65.

28. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity' determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 68; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 69; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 70;(ii) a heavy chain variable region complementarity' determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 71; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 73.

29. The immunoassay of claim 28, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 74 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 75.

30. The immunoassay of claim 28, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 74 and a VH having the amino acid sequence of SEQ ID NO: 75.

31. The immunoassay of any one of claims 1-11, wherein the capture antibody comprises(i) a light chain variable region complementarity determining region 1 (VL- CDR1) comprising the amino acid sequence of SEQ ID NO: 78; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 79; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 80;(ii) a heavy chain variable region complementarity determining region 1 (VH- CDR1) comprising the amino acid sequence of SEQ ID NO: 81; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 83.

32. The immunoassay of claim 31, wherein the capture antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO: 84 and a heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 85.

33. The immunoassay of claim 31, wherein the capture antibody comprises a VL having the amino acid sequence of SEQ ID NO: 84 and a VH having the amino acid sequence of SEQ ID NO: 8534. The immunoassay of any of claims 1-33, wherein the detection antibody comprises a light chain variable region (VL) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 7 and heavy chain variable region (VH) having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 8.

35. The immunoassay of any of claims 34, wherein the detection antibody comprises a VL having the amino acid sequence of SEQ ID NO: 7 and a VH the amino acid sequence of SEQ ID NO: 8.

36. The immunoassay of any of claims 1-35, wherein the detection antibody comprises a light chain having the amino acid sequence of SEQ ID NO: 9 and a heavy chain having the amino acid sequence of SEQ ID NO: 10.

37. The immunoassay of any of claims 1-36, wherein the sample comprises human cerebrospinal fluid (CSF).

38. The immunoassay of claim 37, wherein pS413 tau is detected at a concentration as low as about 4. 1 fM.

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