Component Assays for Alzheimer's Disease in Living Subjects

Abstract

Disclosed is a method for assaying for the presence of Alzheimer's Disease (AD) in a living human subject using a serum or plasma sample preparation from the human subject. In one embodiment, the presence of an approximately 90 kDa Filamin A (FLNA) polypeptide fragment in the sample preparation indicates that the sample donor likely had AD. More preferably, the ratio of the amount of the approximately 90 kDa FLNA polypeptide fragment to the amount of full-length (approximately 280 kDa) FLNA protein in the sample preparation is determined. If the ratio is between about 10 and about 2000, the donor likely has AD, and if the ratio is between about 0.005 and about 5, the donor likely does not have AD. Methods of determining the treatment prognosis of a living human subject suspected of having Alzheimer's Disease (AD), systems and kits for performing the assay are also contemplated.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 225,080, filed July 23, 2021, the disclosure of which is incorporated herein by reference. Government support The work reported herein was supported by the National Institute on Aging of the National Institutes of Health under Grant No. R44AG057329. The Government has certain rights in this invention. A blood-based assay for Alzheimer's disease in a living subject is disclosed. More specifically, contemplated assays contemplate using a blood component sample, such as plasma or serum, taken from a living subject to assay for the presence of Alzheimer's disease in that subject. [Background technology]

[0002] Alzheimer's disease (AD) represents one of the largest healthcare burdens, with an estimated 35 million people affected worldwide, and an estimated 115 million by 2050 [Wimo, Alzheimer's Disease International World Report 2010. The Global Economic Impact of Dementia, Alzheimer's Disease International (2010)]. AD is a severe form of dementia that initially presents with progressive memory loss and can later be accompanied by neuropsychiatric symptoms such as depression, delusions, agitation, and even aggression. Currently, available AD treatments are limited to nootropic drugs, which have limited and short-term efficacy. Previously, the diagnosis of AD could only be confirmed at autopsy by the presence of amyloid deposits and neurofibrillary tangles (NFTs) containing microtubule-associated tau protein. The current clinical diagnosis of AD meets the criteria for clinical confirmation of AD as outlined in the DSM-IV TR and the NINCDS-ADRDA Working Group by McKhann et al. [Neurology 34(7):939-944(1984)]. The initial diagnostic criteria presented by McKhann et al., which are based mainly on subjective assessment, require that the presence of cognitive impairment and a suspected cognitive syndrome be confirmed by neuropsychological testing for a clinical diagnosis of suspected or confirmed AD, but a definitive diagnosis requires postmortem histopathological confirmation (microscopic examination of brain tissue). The criteria define eight cognitive domains that may be impaired in AD, including memory, language, perceptual abilities, attention, constructional abilities, orientation, problem solving, and functional abilities. No motor, sensory, or coordination problems occur early in the disease. These criteria have good reliability and validity and are used herein as the basis for claims regarding the clinical diagnosis of AD. To date, the diagnosis has not been determined by laboratory assays. Such assays are primarily important in identifying possible causes other than dementia that must be ruled out before a diagnosis of Alzheimer's disease can be made with confidence. Neuropsychological testing can help confirm the diagnosis of dementia and evaluate the course and response to treatment. The criteria presented by McKhann et al. above are intended to be used as a guide for the clinical confirmation, clinical suspicion, and definitive diagnosis of Alzheimer's disease, and these criteria will likely be modified as more definitive information becomes available.

[0003] Diagnostic criteria have recently been refined to include a prodromal stage (early symptoms occurring before full-blown symptoms of the disease become apparent) called "Mild Cognitive Impairment with AD (MCI)". This new diagnosis reflects a desire to treat the disease earlier, as neuropathology is estimated to begin 10 years before symptoms appear [Trojanowski et al., Alzheimers Dement 6, 230-238 (2010)]. Clinical trials of potential disease-modifying therapies have been extremely disappointing, in part because even "early-stage" patients already have a large amyloid-β (Aβ) burden and substantial pathology with significant synaptic loss and inflammation in key brain regions responsible for cognition. According to Petersen et al. [Arch Neurol 56(3):303-308(1999)], the main difference between control and MCI subjects was in the memory domain, with other cognitive functions being comparable. However, when MCI subjects were compared with patients with the mildest form of AD, memory performance was similar, but the AD patients were more impaired in other cognitive domains. Long-term outcomes showed that MCI subjects declined more rapidly than controls, but less rapidly than patients with mild AD. Patients meeting criteria for MCI can be distinguished from healthy controls and from patients with minimal AD, and they appear to represent a characterizable clinical entity for therapeutic intervention.

[0004] Amyloid beta (Aβ) is a peptide of 39-42 amino acid residues in length that is generated in vivo by specific proteolytic cleavage of the amyloid precursor protein (APP) by beta-secretase and gamma-secretase. 42 contains residues 677–713 of the APP protein, itself a 770-residue transmembrane protein designated P05067 in the UniProtKB / Swiss-Prot system. Aβ, especially Aβ 42 Although it is generally believed to be the primary causative agent of AD, its mechanisms underlying the neuropathology of AD are debated. Until recently, the presence of Alzheimer's disease could only be diagnosed in living patients by symptom-based assays, as described in McKhann et al., supra. More recently, with the approval of Eli Lilly's Amyvid® in April 2012, followed by GE Healthcare's Vizamyl® and Piramal Imaging's Neuraceq®, PET scanning technology has been used to assay for AD in living humans. Radiolabeled positron-emitting compounds injected intravenously bind to Aβ in brain plaques. Although PET scans are accurate, they are inconvenient for patients because they require patients to place their head inside a relatively small space inside a scintillation detector, which limits their ability to move. PET scans are also expensive, especially when compared with regular blood tests, which require the withdrawal of a few milliliters of blood and perform as many as 40 assays (which unfortunately do not yet include an AD test commercially available).

[0005] Recent studies published by several groups have reported early AD immunoassay results based on the amount of tau protein phosphorylated at position 181 (p181 tau) present in serum and plasma. See, for example, Janelisze et al., Nat Med 26(3):379-386 (March02,2020) e-pub March02,2020; Karikari et al., Lancet Neurol 19(5):422-433 (May19,2020); Thijssen et al., Nat Med 26(3):387-397 (March02,2020) e-pub March02,2020; and Kasai et al., Mol Neurodegener 12(1):63 (September04,2017). Other groups have reported Ab-based serum-based assays. See Holtzman and Bateman, US Patent Publication 20140370619; Bateman et al., Alzheimer's Dement 16(Suppl5):e037518 (2020). Each of the above assays is used in combination with other assay techniques in determining patients with AD.

[0006] Discovered 45 years ago as the first non-muscle actin-binding protein [Hartwig et al., J Biol Chem 250:5696-5705(1975); Wang et al., Proc Natl Acad Sci USA 72:4483-4486(1975)], filamins [FLNs] are a family of cytoskeletal proteins (filamins A (FLNA) and B, but not C) expressed in non-muscle cells. Human FLNA has been given the identifier P21333 in the UniProtKB / Swiss-Prot database and has a sequence of 2647 amino acid residues (approximately 280 kDa). This protein is sometimes referred to in the art as actin-binding protein (ABP-280) [Gorlin et al., J Cell Biol 111:1089-1105(1990)]. FLNA proteins anchor various transmembrane proteins to the actin cytoskeleton and function as scaffolds for various cytoplasmic signaling proteins. Filamin is essential for mammalian cell locomotion and acts as an interface for protein-protein interactions [van der Flier et al., Biochim Biophys Acta 1538:99-117(2001)]. In addition to its role in cell motility, it has become increasingly clear that FLNA regulates cell signaling by interacting with various receptors and signaling molecules [Stossel et al., Nat Rev Mol Cell Biol 2:138-145(2001);Feng et al., Nat Cell Biol 6:1034-1038(2004)].

[0007] The FLNA protein consists of an N-terminal actin-binding domain (ABD) and a rod-shaped domain of 24 immunoglobulin-like repeat domains (IgFLNa), each approximately 96 amino acid residues long and numbered from the N-terminus, interrupted by two 30 amino acid residue flexible loops or hinges. The IgFLNa are numbered from 1 to 24, starting near the N-terminus and ending near the C-terminus. A loop called H1 is located between repeats 15 and 16, and a loop called H2 is located between repeats 23 and 24 [Gorlin et al., J Cell Biol 111:1089-1105 (1990); van der Flier et al., Biochim Biophys Acta 1538:99-117 (2001)]. H1 and H2 can be cleaved by calpains and caspases [Gorlin et al., J Cell Biol 111:1089-1105 (1990); Browne et al., J Biol Chem 275:39262-39266 (2000)]. Cleavage at H1 occurs between amino acid residues 1762 and 1764, generating an approximately 170 kDa fragment consisting of the ABD and repeats 1-15 (IgFLNa-1-15), and an approximately 110 kDa polypeptide fragment consisting of repeats 16-24 (IgFLNa-16-24). The UniProtKB / Swiss-Prot database lists the C-terminus of repeat 15 at amino acid residue 1740 and the N-terminus of repeat 16 at amino acid residue 1779. On the other hand, Gorlin et al., mentioned above, list the calpain cleavage site between residues 1762 and 1764, whereas Garcia et al. [Arch Biochem Biophys 446:140-150(2006)] list this site between residues 1761 and 1762. Similarly, Gorlin et al., mentioned above, state that the previous authors (Hartwig et al., J Cell Biol 87:841-848(1980)) reported the molecular weight of the full-length FLNA molecule to be 270 kDa on page 1089, and then reported a protein of 280 kDa on page 1093.

[0008] Garcia et al., supra, reported that calpain cleaves full-length FLNA into polypeptide fragments of 180, 100, 90, and 10 kDa, whereas Bedolla et al. [Clin Cancer Res 15(3):788-796(2009)] reported proteolytic fragments of 170 and 110 kDa, as well as a 90 kDa fragment cleaved from the 110 kDa fragment. Browne et al., supra, reported that granzyme B (grB), a protease of cytotoxic T lymphocytes, cleaves filamin in cooperation with the lytic protein perforin, and that filamin is also cleaved in a caspase-dependent manner after binding to the Fas receptor. Western blots (WB) of dying Jurkat cell lysates identified two caspase-cleaved polypeptides from the C-terminal region of FLNA with masses of approximately 110 and 95 kDa. Purified grB cleaved filamin into multiple polypeptides, including those with masses of approximately 205, 200, and 110 kDa. A polyclonal rabbit antibody raised against a fusion protein containing 476 amino acid residues from the C-terminal region of FLNA (positions 2172-2647) was used. Umeda et al. [J Biochem 130:535-542 (2001)] found somewhat similar results (C-terminal 135, 120, and 110 kDa polypeptide fragments) for proteolysis by caspase 3 in U937 monoblastic leukemia cells and Jurkat human T-lymphoblastoid cells.

[0009] It should be noted that Loy et al. [Proc Natl Acad Sci, USA, 100(8):4562-4567 (2003)] state that IgFLNa-16-24 has an apparent mass of approximately 110 kDa. The approximately 110 kDa polypeptide (IgFLNa-16-24) is further cleaved with H2 by calpain at long digestion times to yield an approximately 90 kDa fragment (IgFLNa-16-23) containing repeats 16-23 [Gorlin et al., J Cell Biol 111:1089-1105 (1990); van der Flier et al., Biochim Biophys Acta 1538:99-117 (2001)]. As described above, due to differences in residue positions and some molecular weights of full-length FLNA and its proteolytic fragments reported in the art, the full-length FLNA molecule and the smaller FLNA cleavage products are considered to have molecular weights of "approximately" 280 kDa and "approximately" 90 kDa, respectively.

[0010] FLNA promotes orthogonal branching of actin filaments and links them to membrane glycoproteins. Filamin A dimerizes through a carboxy-terminal repeat (repeat 24) near the transmembrane domain, resulting in an intracellular V-shaped structure that is important for its function. Each V-shaped FLNA dimer has two antiparallel self-binding domains24 that form the vertices of the "V", while the remaining domains are stretched out like beads on a string with their N-terminal ABD moieties each bound to an actin molecule. More recently, it has been reported that rod-shaped segment 1 (IgFLNa-1-15), which is the C-terminus of the ABD, forms an extended linear structure without any obvious interdomain interactions. Rod-shaped segment 2 (IgFLNa-16-23) adopts a compact structure due to multiple interdomain interactions in which domains 16-17, 18-19, and 20-21 form paired structures [Heikkinen et al., J Biol Chem, 284: 25450-25458 (2009); Lad et al., EMBO J, 26: 3993-4004 (2007)]. Proteolysis of FLNA is reported to be regulated in part by phosphorylation at Ser2152 (S2152) in repeat 20 (IgFLNa-20), which stabilizes the full-length protein and makes it resistant to cleavage [Gorlin et al., J Cell Biol 111:1089-1105 (1990); Garcia et al., Arch Biochem Biophys 446:140-150 (2006); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)].

[0011] Loy et al. [Proc Natl Acad Sci, USA, 100(8):4562-4567(2003)] reported that an H1 cleavage product of approximately 100 kDa, containing repeats 16-24, is localized in the nucleus together with the androgen receptor in prostate cancer cells. They noted that FLNA is generally regarded as a cytoplasmic architectural molecule, and stated that their finding that an approximately 100 kDa polypeptide generated by calpain cleavage additionally functions as a nuclear regulator of the androgen receptor was "completely unexpected" (p. 4565). The approximately 100 kDa FLNA fragment found in cell nuclei is not phosphorylated on pS2152, and in fact, phosphorylation on pS2152 has been reported to inhibit calpain-mediated cleavage of full-length FLNA in prostate cancer lines and platelets [Garcia et al., Arch Biochem Biophys 446:140-150 (2006); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)]. Wang et al. [Oncogene 26:6061-6070(2007)] showed that nuclear localization of FLNA correlates with hormone dependence in prostate cancer. A non-phosphorylated ≈90 kDa fragment (IgFLNa-16~23) translocates to the nucleus of hormone-sensitive cells in androgen-dependent prostate cancer. Conversely, in hormone-resistant androgen-independent prostate tumor cells, FLNA is phosphorylated, preventing its cleavage and nuclear translocation. The authors then showed that not only is prostate cancer metastasis correlated with the cytoplasmic localization of FLNA, but that cleavage and subsequent nuclear translocation of the phosphorylated protein can prevent metastasis [Bedolla et al.,Clin Cancer Res.15(3):788-796(2009)].

[0012] As a key regulator of the cytoskeletal network, FLNA interacts with many proteins involved in cancer metastasis [Yue et al., Cell & Biosci 3:7(2013)] and many other conditions, leading Nakamura et al. [Cell Adh Migr.5(2):160-169(2011)] to review the history of research on FLNA and note that the protein functions as a scaffold for over 90 binding partners, including channels, receptors, intracellular signaling molecules, and transcription factors. FLNA has also been implicated in tumor progression. FLNA knockout mice show reduced oncogenic activity of K-Ras, including downstream activation of ERK and Akt [Nallapalli et al., Mol Cancer 11:50(2012)]. FLNA expression is high in a variety of cancers, including colorectal and pancreatic cancers [Uhlen et al., Mol Cell Proteomics 4:1920-1932(2005)], and glioblastoma [Sun et al., Cancer Cell 9:287-300(2006)], in contrast to low expression in corresponding normal tissues. Inhibition of FLNA expression sensitizes cancer cells to both cisplatin and radiation [Sun et al., Cancer Cell 9:287-300(2006)], and FLNA deficiency in cancer cells also sensitizes them to chemotherapy [Yue et al., DNA Repair(Amst)11:192-200(2012)] and radiation [Yue et al., Cancer Res 69:7978-7985(2009); Yuan et al., J Biol Chem 276:48318-48324(2001)]. Meanwhile, Jiang et al. [Int.J.Biol.Sci.9:67-77(2013)] reported that inhibition of filamin A expression reduced metastasis in nude mice implanted with melanoma and breast cancer cells.

[0013] Phosphorylation is recognized as a global regulator of cellular activity, and abnormal phosphorylation is implicated in many human diseases, especially cancer. Protein phosphorylation occurs when one or more hydroxyl groups of amino acid side chains of serine, threonine, or tyrosine residues are enzymatically converted to a phosphate group (-OPO3 -2 ) is included. Phosphorylation and its reverse reaction, dephosphorylation, occur through the action of two main classes of enzymes. Protein kinases phosphorylate proteins by transferring a phosphate group from a nucleotide triphosphate such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to their target proteins. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group. Thus, the amount of phosphate bound to a protein at a particular time is determined by the relative activity of that protein and the particular associated kinase and phosphatase enzymes specific for the particular amino acid residue(s) undergoing phosphorylation / dephosphorylation. If the phosphorylated protein is an enzyme, phosphorylation and dephosphorylation affect its enzymatic activity, essentially acting as a switch that can be turned on and off in a controlled manner. Phosphorylation can similarly regulate non-enzymatic protein-protein interactions by facilitating binding to partner proteins.

[0014] Protein phosphorylation can play an important role in intracellular signal transduction. Many of the proteins that make up signal transduction pathways, from cell surface tyrosine kinase receptors to downstream effector proteins, are kinases, many of which are serine / threonine kinases. FLNA is phosphorylated at many positions in its protein sequence in both normal and pathological cells, including cancer cells. For example, the enzyme PAK1 (EC:2.7.11.1) is a STE20 family protein kinase that controls cell motility and morphology. Phosphorylation of FLNA at position 2152 by PAK1 is required for PAK1-mediated actin cytoskeleton reorganization and PAK1-mediated membrane ruffling [Vadlamudi et al., Nat. Cell Biol. 4:681-690(2002); Woo et al., Mol Cell Biol. 24(7):3025-3035(2004)]. Cyclin B1 / Cdk1 (EC:2.7.11.22; EC:2.7.11.23) phosphorylates serine 1436 in vitro during FLNA-dependent actin remodeling [Cukier et al., FEBS Letters 581(8):1661-1672(2007)].

[0015] The UniProtKB / Swiss-Prot database entry for human FLNA (No. P21333) lists published reports of the following amino acid residue positions being phosphorylated under different circumstances: 11, 1081, 1084, 1089, 1286, 1338, 1459, 1533, 1630, 1734, 2053, 2152, 2158, 2284, 2327, 2336, 2414, and 2510. Additionally, polyclonal and monoclonal antibodies that immunoreact with FLNA phosphorylated at serine 1083, tyrosine 1046, serine 1458, serine 2152, and serine 2522 (phospho-FLNA) are commercially available from one or more of Abgent, Inc. (San Diego, CA), Abcam® Inc. (Beverly, MA), Bioss, Inc. (Woburn, MA), and GeneTex, Inc. (Irvine, CA). The 90 kDa FLNA fragment, which can localize to the nucleus and interact with transcription factors, contains a variant phosphorylated at serine 2152. However, the aforementioned ∼90 kDa FLNA fragment localized to the nucleus does not have phosphorylation at serine 2152. In fact, it has been reported that phosphorylation of FLNA at serine 2152 (pS2152 FLNA) protects FLNA from proteolysis forming the ∼90 kDa fragment [Garcia et al., Arch Biochem Biophys 446:140-150 (2006); Gorlin et al., J Cell Biol 111:1089-1105 (1990); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)].

[0016] In a publication underlying the allowed U.S. Patent Application Publication No. 16 / 030494 [Wang et al., J. Neurosci. 32(29):9773-9784 (July 18, 2012)], the present inventors and their co-workers reported that Alzheimer's disease-related ligand Aβ 42 We showed for the first time that FLNA activates TLR4 via CD14 and that this activation requires FLNA. The paper also showed that PTI-125 similarly reduces the association of FLNA with Toll-like receptor 4 (TLR4) and exerts an anti-inflammatory effect by preventing cytokine release [page 9774, top left paragraph]. Summary of the Invention

[0017] The present inventors have found that an approximately 90 kDa FLNA polypeptide fragment (IgFLNa-16-23) is present in the serum or plasma of living Alzheimer's disease (AD) patients but not in the serum or plasma of individuals without Alzheimer's disease. Early studies have shown that this 90 kDa FLNA polypeptide fragment can be detected by an antibody that immunoreacts with an epitope that includes the serine residue located at position 2152 of the human FLNA sequence, whether or not it is phosphorylated. This finding suggests that either or both the S2152 phosphorylated and S2152 non-phosphorylated polypeptides may be present, and that the approximately 90 kDa mass is a biomarker, regardless of the phosphorylation status of pS2152. It should be noted that the aforementioned references [e.g., Wang et al., Oncogene 26:6061-6070 (2007); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)] teach that this polypeptide is protected from phosphorylation compared to the full-length molecule, which is phosphorylated on the serine corresponding to serine 2152. Furthermore, this approximately 90 kDa fragment is not assayed for in the cell nucleus or cytoplasm [Loy et al., Proc Natl Acad Sci, USA, 100(8):4562-4567 (2003)], but is assayed in serum or plasma.

[0018] Thus, the present invention contemplates a method for assaying the presence of Alzheimer's disease (AD) in a living human subject using a serum or plasma sample from the human subject. Since AD ​​is a brain disease and there is little exchange of blood components with a molecular weight of about 1 kDa or more between the brain and the circulating blood, it is highly surprising that any accurate indicator of the presence of this brain disease has been found in the plasma or serum of the circulating bloodstream. It is even more surprising that this accurate marker of AD is a relatively high molecular weight, about 90 kDa FLNA polypeptide fragment that can be phosphorylated at the serine residue corresponding to position 2152 of full-length FLNA.

[0019] Contemplated methods include determining (detecting) the presence or absence of an approximately 90 kDa polypeptide fragment of FLNA (IgFLNa-16-23) among other proteinaceous material in a serum or plasma sample, typically using an aqueous serum or plasma preparation rather than serum or plasma per se, as described below. The approximately 90 kDa FLNA polypeptide fragment can be phosphorylated at FLNA serine 2152 (pS 2152 -90kDa FLNA or pS 2152 -IgFLNa-16~23). i) The presence of the 90 kDa FLNA polypeptide fragment (in an amount significantly above a predetermined background amount) indicates that the human subject from whom the blood sample was taken likely had AD at the time the sample was taken. ii) the absence of the 90 kDa FLNA polypeptide fragment (including amounts not significantly above a predetermined background amount) indicates that the human subject from whom the blood sample was taken was likely not suffering from AD at the time the sample was taken.

[0020] Preferably, the aqueous serum or plasma sample preparation is prepared using pS 2152 -90kDa and S 2152 The -90 kDa FLNA polypeptide is contacted with a paratope-containing receptor molecule that is immunoreactive with one or both of the -90 kDa FLNA polypeptides to form a reaction mixture. The reaction mixture comprises a mixture of the paratope-containing receptor molecule and the pS 2152 -90kDa and S 2152 The immunoreactant is maintained for a period of time sufficient for the -90 kDa FLNA polypeptide to form an immunoreactant, and the presence or absence of the phosphorylated or non-phosphorylated approximately 90 kDa polypeptide fragment of FLNA is detected in the immunoreactant. Preferably, the presence or absence of the 90 kDa FLNA polypeptide fragment is determined after separation of the proteinaceous material present in the sample. The proteinaceous material present in the aqueous serum or plasma sample preparation is separated into at least two portions prior to the contacting step described above, and the at least two portions are separated into at least two portions prior to the contacting step described above. 2152A first portion may contain the -90 kDa FLNA polypeptide fragment, and a second portion may contain the approximately 280 kDa FLNA protein. Proteins present in the sample may be separated by at least chromatography, such as size exclusion chromatography or affinity chromatography, isoelectric focusing, electrophoresis, and other methods as desired.

[0021] One preferred method of chromatographic separation is by affinity chromatography, in which a receptor, such as a paratope-containing receptor, is bound to a support, such as a polysaccharide resin, such as Sepharose® or Sephadex® resin, that has been activated with cyanogen bromide or other activating agent. Receptor-bound supports can be prepared as described in Scales et al., J Clin Microbiol 2(4):292-295 (1975), and references therein. Exemplary paratope-containing receptors for use in affinity binding include mouse monoclonal MAB1678 from Chemicon International, Inc., which binds to an epitope sequence near the N-terminus of FLNA outside the sequence of the 90 kDa C-terminal calpain cleavage fragment, and mouse monoclonal SC17749 IgG specific for an epitope mapping between amino acid residues 9-27 near the N-terminus of FLNA, available from Santa Cruz Biotechnology, Inc. 2a Using one of these exemplary receptors, the heavy approximately 280 kDa FLNA protein is attached to the support, and the approximately 90 kDa FLNA polypeptide fragment passes through with the eluate.

[0022] In another preferred embodiment, the presence or absence of about 90 kDa FLNA polypeptide is determined using reduced SDS-PAGE Western blot analysis using a monomercaptan such as 2-mercaptoethanol as a reducing agent to separate the proteinaceous portion of the sample. After separation, the presence or absence of the 90 kDa FLNA polypeptide can be illustratively determined using a receptor molecule that specifically binds to the 90 kDa FLNA polypeptide. Exemplary receptor molecules are described below. In a further preferred embodiment, the proteinaceous portion is separated by ultrafiltration, which separates material having a molecular weight of less than about 100 kDa from higher molecular weight proteinaceous material in a diluted aqueous serum or plasma sample preparation. The presence or absence of the 90 kDa FLNA polypeptide is determined using the filtrate (ultrafiltrate), while the higher molecular weight proteinaceous material is present in the retentate. Preferably, the proteinaceous portion of the separated sample is identified by contact with a detection reagent comprising an antibody or a portion thereof that specifically immunoreacts with an epitope that includes the serine residue present at position 2152 of the FLNA sequence and forms an immunoreactant, although other antibodies and portions thereof that immunoreact with other epitopes of IgFLNa-16-23 may also be used.

[0023] In another preferred embodiment, the presence or absence of the 90 kDa FLNA polypeptide is determined using a sandwich assay, such as an ELISA assay. In a preferred embodiment, receptors that bind to two different sites on the approximately 90 kDa FLNA polypeptide are utilized. Preferably, one of these receptors contains a paratope that immunoreacts with an epitope that contains a phosphorylated or non-phosphorylated serine residue present at FLNA sequence position 2152 to form an immunoreactant. In this assay, the proteinaceous material present in the sample may be separated before contact with the receptor, or the contact may be performed without prior separation of the proteinaceous material. In a more preferred embodiment, the ratio of the amount of the low molecular weight approx. 90 kDa FLNA polypeptide fragment (A) to the amount of a second proteinaceous substance (B) present in an aqueous serum or plasma sample preparation comprising a serum or plasma sample taken from a living human subject is determined. A particularly preferred second proteinaceous substance (B) can also be phosphorylated on serine at sequence position 2152 (pS 2152 -FLNA), or the approximately 280 kDa FLNA protein that cannot be so phosphorylated, but albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), or another protein present in the serum or plasma of the subject and thus in the aqueous serum or plasma sample preparation, can be used. From initial data, it appears that using the ratio allows different subject populations with different conditions to be closely correlated with each other, resulting in more reliable results.

[0024] When either A or B is present in a quantifiable amount (QAmt) and the other is not present in a quantifiable amount, assign an arbitrary predetermined fraction from about 0.1 to about 0.001 of the quantifiable amount to the other, such that zero is not used in the numerator or denominator [(0.1-0.001) x (QAmt)]. When both A and B are not present in quantifiable amounts, assign approximately the same arbitrary amount to both. If A is an approximately 90 kDa FLNA polypeptide fragment and B is an approximately 280 kDa FLNA protein, then if the A / B ratio is between about 10 and about 2000, the subject probably had AD at the time the sample was collected, and if the ratio is between about 0.005 and about 5, the subject probably did not have AD at the time the sample was collected. These two ratios are very different in magnitude, since the serum or plasma of subjects with AD contains high amounts of the 90 kDa FLNA polypeptide and very little, if any, the 280 kDa FLNA protein. In contrast, the serum or plasma of subjects without AD contains little or no low molecular weight 90 kDa FLNA polypeptide and high amounts of the full-length protein. Ratios made using amounts of proteinaceous material (B) other than the 280 kDa FLNA protein will differ from those made using filamin A itself, but the values ​​can be easily calculated. The determination of the above ratios can be performed in each of the assay formats disclosed herein. Although the mass or molar amounts of the two proteinaceous substances can be used, it is more convenient to utilize the relative amounts, usually determined by colorimetric, radioisotopic, fluorescent, or phosphorescent labeling techniques, as is well known.

[0025] Another embodiment of the present invention is an assay system for detecting whether or not the approximately 90 kDa FLNA polypeptide is present in a plasma or serum sample, thereby detecting the likelihood that the subject from whom the sample was taken had or did not have AD at the time the sample was taken. The contemplated system includes a solid support having an assay surface coated with a paratope-containing capture receptor molecule that immunoreacts with an epitope containing anti-FLNA (receptor molecule), and a container holding a first detection receptor molecule that, if present, binds and captures the approximately 90 kDa FLNA polypeptide fragment to form a capture complex, and a label indicating the presence of the capture complex, such that the presence or absence of the capture complex correlates with the presence or absence of the approximately 90 kDa FLNA polypeptide fragment in the sample. Preferably, the first detection receptor is a paratope-containing molecule (anti-pS or anti-S receptor) that immunoreacts with a phosphorylated or non-phosphorylated serine residue present at FLNA sequence position 2152-serine residue. In a preferred embodiment, an additional container is included that holds a second detector receptor molecule that reacts with a binding site present in full-length FLNA that is not present in the approximately 90 kDa FLNA polypeptide, and a label is also included that indicates the presence of that binding. In a preferred implementation, the system is a kit in which the listed elements are packaged together. Instructions for using these receptor molecules to bind to the approximately 90 kDa FLNA polypeptide to form an antibody-antigen complex are also preferably included in the kit. Exemplary solid supports include multi-well plates, individual test tubes, and particulate solids such as plastic beads and magnetic particles that allow for detection of the approximately 90 kDa FLNA polypeptide in multiple samples.

[0026] Also contemplated is a method for determining the prognosis of treatment of a living human subject suspected of having Alzheimer's disease (AD) with a therapeutic compound or a pharma- ceutically acceptable salt of the therapeutic compound. The method comprises determining the presence of a first amount of an approximately 90 kDa polypeptide fragment of Filamin A (FLNA) in a first aqueous serum or plasma sample preparation comprising a serum or plasma sample taken from the living human subject. The living human subject is treated with a therapeutic composition containing an anti-AD effective amount of a therapeutic compound or a pharma- ceutically acceptable salt of the compound. A second amount of the approximately 90 kDa polypeptide fragment of FLNA in a second aqueous serum or plasma sample preparation of the human subject is taken at a time point at least about one month after the start of treatment. The amount of the approximately 90 kDa polypeptide fragment of FLNA present in a serum or plasma sample preparation taken from the blood of the living patient before and after treatment is determined, and the later determined amount being significantly less than the earlier determined amount is consistent with a prediction of benefit from the use of the treatment for the patient from whom the sample was taken.

[0027] In a preferred embodiment, the amount of about 90 kDa polypeptide fragment of FLNA present in the first and second sample preparations is determined as a ratio to the amount of a second proteinaceous substance present in human serum or plasma, and the ratio of the two proteinaceous substances is compared before and after treatment, as mentioned. Again, the amount of the later determined ratio being significantly less than the amount of the earlier determined ratio is consistent with the prediction of benefit from the use of the treatment for the patient from whom the sample was taken. Exemplary second proteinaceous substances include albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and about 280 kDa FLNA protein. The use of about 280 kDa FLNA protein is particularly preferred for use in the ratio determination. Exemplary therapeutic compounds are aducanumab and simufilam, or a pharma- ceutically acceptable salt of simufilam. The use of simufilam or a pharma- ceutically acceptable salt thereof is particularly preferred.

[0028] The present invention has several benefits and advantages. Prominent among these benefits and advantages is the fact that accurate results regarding the presence or absence of AD can be obtained using relatively conservatively collected serum or plasma samples. A particular advantage of the present invention is that the determination of the presence or absence of AD is made in living humans, allowing treatment of AD or other diseases. A further advantage of the present invention is that undergoing the assay involves significantly less inconvenience for the patient compared to a PET scan. A further advantage of the present invention is that it poses minimal inconvenience to the laboratory performing the assay, in that the techniques required are common in the industry and the assay can be performed in a multiplexed and automated manner. Further benefits and advantages of the present invention will become apparent to those skilled in the art from the following discussion. The drawings which form a part of this disclosure are described below. [Brief description of the drawings]

[0029] [Figure 1]1 is a photographic image of a Western blot (WB) analysis of a 1.25 μL sample of human plasma from a subject, showing the full length FLNA of about 280 kDa and the FLNA polypeptide portion with a molecular weight of about 90 kDa (90 kDa FLNA). Separation was performed in the laboratory of the present inventor HY Wang at CUNY School of Medicine (Manhattan, NY) under denaturing conditions in 7.5% SDS-PAGE after boiling the diluted samples for 5 minutes in Laemmli's SDS-PAGE sample preparation buffer at pH 7.5 containing 2-mercaptoethanol as a reducing agent and cooling. After electrophoretic transfer of the separated samples to nitrocellulose, the proteinaceous material was first reacted with rabbit anti-human pS-2152-FLNA monoclonal antibody (Abcam® #75978) and then visualized by chemiluminescence visualization on X-ray film using HRP-conjugated anti-rabbit IgG, and the relative density value of each visualized protein was quantified. Albumin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were similarly visualized after binding a specific detection receptor for each protein (here, rabbit IgG antibody). The numbers under the dark bands of proteins labeled GAPDH are subject identification numbers. Protein density measurements were also performed for albumin and GAPDH present in the plasma of each subject indicated. Details of this method are described below. [Diagram 2]1 is a photographic image of a Western blot (WB) analysis of a 1.25 μL sample of human plasma from a subject, showing the full length FLNA of about 280 kDa and the FLNA polypeptide portion with a molecular weight of about 90 kDa (90 kDa FLNA). Separation was performed in the laboratory of the present inventor HY Wang at CUNY School of Medicine (Manhattan, NY) under denaturing conditions in 7.5% SDS-PAGE after boiling the diluted samples for 5 minutes in Laemmli's SDS-PAGE sample preparation buffer at pH 7.5 containing 2-mercaptoethanol as a reducing agent and cooling. After electrophoretic transfer of the separated samples to nitrocellulose, the proteinaceous material was first reacted with rabbit anti-human pS-2152-FLNA monoclonal antibody (Abcam® #75978) and then visualized by chemiluminescence visualization on X-ray film using HRP-conjugated anti-rabbit IgG, and the relative density value of each visualized protein was quantified. Albumin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were similarly visualized after binding a specific detection receptor for each protein (here, rabbit IgG antibody). The numbers under the dark bands of proteins labeled GAPDH are subject identification numbers. Protein density measurements were also performed for albumin and GAPDH present in the plasma of each subject indicated. Details of this method are described below. [Diagram 3]1 is a photographic image of a Western blot (WB) analysis of a 1.25 μL sample of human plasma from a subject, showing the full length FLNA of about 280 kDa and the FLNA polypeptide portion with a molecular weight of about 90 kDa (90 kDa FLNA). Separation was performed in the laboratory of the present inventor HY Wang at CUNY School of Medicine (Manhattan, NY) under denaturing conditions in 7.5% SDS-PAGE after boiling the diluted samples for 5 minutes in Laemmli's SDS-PAGE sample preparation buffer at pH 7.5 containing 2-mercaptoethanol as a reducing agent and cooling. After electrophoretic transfer of the separated samples to nitrocellulose, the proteinaceous material was first reacted with rabbit anti-human pS-2152-FLNA monoclonal antibody (Abcam® #75978) and then visualized by chemiluminescence visualization on X-ray film using HRP-conjugated anti-rabbit IgG, and the relative density value of each visualized protein was quantified. Albumin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were similarly visualized after binding a specific detection receptor for each protein (here, rabbit IgG antibody). The numbers under the dark bands of proteins labeled GAPDH are subject identification numbers. Protein density measurements were also performed for albumin and GAPDH present in the plasma of each subject indicated. Details of this method are described below. [Figure 4] Scatter plots of the visualized data from Figures 1, 2, and 3 arranged by pre-determined probable Alzheimer's disease status, where AD=AD patients (usually determined by Amyvid® assay), AMC=age-matched controls (no AD), YCI=young cognitively normal, MCI AD=moderately cognitively impaired AD patients, MCI NonAD=moderately cognitively impaired without AD. Data for the approximately 90 kDa FLNA protein only is shown. [Diagram 5] This is a scatter plot of the visualized data from Figures 1, 2, and 3 arranged according to pre-determined likelihood of Alzheimer's disease status, and shows data for full-length phosphorylated FLNA. [Figure 6]This is a scatter plot in which the visualized data of Figures 1, 2, and 3 are arranged according to the previously determined possibility of Alzheimer's disease status, and shows the ratio of approximately 90 kDa FLNA polypeptide / approximately 280 kDa FLNA protein (A / B). [Figure 7] 1 is a photograph of a Western blot analysis of a 1.0 μL sample of human plasma from a subject, showing full length FLNA of approximately 280 kDa and the FLNA polypeptide portion of approximately 90 kDa molecular weight, similar to FIGS. 1-3, except that protein density measurements of albumin and GAPDH present in the plasma were not performed. These studies were performed for the inventors at Altogen Labs (Austin, TX) using samples provided by Dr. Wang. [Figure 8] FIG. 8 is a graph of the 90 kDa FLNA / FLNA ratio obtained from density values ​​obtained by scanning the Western blot of FIG. 7. [Figure 9] 8 is a photograph of a Western blot analysis similar to that of FIG. 7, but using a different patient sample. [Figure 10] This is a graph of the ratio obtained from density values ​​obtained by scanning the Western blot of the approximately 90 kDa FLNA polypeptide / approximately 280 kDa FLNA protein of Figure 9. [Figure 11]1 is a graph showing the relative amounts of the FLNA polypeptide fragment of approximately 90 kDa and the full-length (approximately 280 kDa) FLNA protein in plasma from multiple subjects whose AD status was unknown at the time the assay was performed. Here, plasma samples were diluted 1:40 with Tris buffer, pH 7.4, and separated into two portions (unfiltered: light bars; filtered: dark bars). One portion was ultrafiltered using a Nanosep® 100K OMEGA™ (P / N OD100C33) centrifugal filter (PALL Corp., Ann Arbor, MI), and the resulting filtrate and unfiltered portion were separately contacted with the walls of a Reacti-Bind™ NeutrAvidin™ high-binding capacity coated 96-well plate coated with a biotinylated antibody specifically reacting with a rabbit monoclonal antibody against phosphorylated serine 2152 of FLNA as a capture molecule. The captured pS2152-approximately 90 kDa FLNA polypeptide fragment was detected using a mouse anti-phosphoserine monoclonal antibody [NeuroMab (UC Davis / NIH): Cat#: 73-292], and the captured pS2152-FLNA was detected using a mouse monoclonal IgG2a antibody (SC-17749, Santa Cruz Biotechnology, Inc.) specific for an epitope mapping between amino acid residues 9 to 27 near the N-terminus of FLNA. The relative amounts of each immunoreactant were determined by exciting the fluorescent label and measuring the relative fluorescence intensity after reaction with the respective FITC-labeled anti-mouse antibodies. [Figure 12A]12A and 12B are photographs of Western blot analysis of human plasma samples from the same subject using two different receptors that bind different epitopes. The receptor in FIG. 12A is a phospho-specific rabbit polyclonal antibody that binds to phosphorylated serine 2152 of FLNA, available from OriGene, Inc. under the designation TA313881. The receptor in FIG. 12B is a mouse monoclonal antibody raised against a 10-mer N-acetyl-terminal polypeptide corresponding in sequence to positions 2148 to 2157 of the FLNA sequence, including phosphorylated serine 2152, but which is not phospho-specific like the OriGene antibody. The letters in each column have the following meaning: AD=Alzheimer's disease patients, MCI-SNAP=MCI without suspected AD pathology, MCI-AD=MCI with AD pathology, EC=aged controls, YCI=cognitively normal young controls, and AD+=Amivid® positive AD patients. [Figure 12B] 12A and 12B are photographs of Western blot analysis of human plasma samples from the same subject using two different receptors that bind different epitopes. The receptor in FIG. 12A is a phospho-specific rabbit polyclonal antibody that binds to phosphorylated serine 2152 of FLNA, available from OriGene, Inc. under the designation TA313881. The receptor in FIG. 12B is a mouse monoclonal antibody raised against a 10-mer N-acetyl-terminal polypeptide corresponding in sequence to positions 2148 to 2157 of the FLNA sequence, including phosphorylated serine 2152, but which is not phospho-specific like the OriGene antibody. The letters in each column have the following meaning: AD=Alzheimer's disease patients, MCI-SNAP=MCI without suspected AD pathology, MCI-AD=MCI with AD pathology, EC=aged controls, YCI=cognitively normal young controls, and AD+=Amivid® positive AD patients. [Figure 13]13 is a graph showing the amount, in arbitrary units, of the approximately 90 kDa FLNA polypeptide fragment found in the plasma of 13 AD patients in a Phase 2a clinical trial who were treated with the drug twice daily for 28 days and whose plasma was assayed for the presence and amount of the approximately 90 kDa FLNA polypeptide fragment on days 0, 14, and 28. The data in the graph show that the amount of the approximately 90 kDa FLNA polypeptide fragment present was reduced in this patient group on day 14 versus day 0 and on day 28 versus day 14, indicating that the drug was effective in treating the disease. [Figure 14A] 14A and 14B are graphs of antibody binding data for peptides or recombinant approximately 90 kDa FLNA polypeptide fragments bound to solid supports in a microtiter plate well ELISA format. The UniProtKB / Swiss-Prot database P21233 sequence of bound human FLNA contained an additional C-terminal cysteine ​​amide that binds to the immunogenic carrier maleimide-activated keyhole limpet hemocyanin (KLH) of the immunogen. The serine residue at position 2152 of the polypeptide designated "pS2152" was phosphorylated. The bound antigens in each assay shown were, from right to left, the immunogenic polypeptide, the immunogenic polypeptide in which the phosphorylated serine residue was replaced with an alanine residue ("A2152"), and the 90 kDa FLNA polypeptide fragment phosphorylated at serine 2152 ("90 kDa FLNA"). Figure 14A shows data for mouse monoclonal antibody preparations diluted 1:1000 using a commercially available product (Origene) and two monoclonal antibodies (20H7 (IgG2) and 16E6 (IgM)) prepared by us at Aragen Biosciences, Inc. (Morgan Hill, Calif.). Figure 14B shows binding data using rabbit polyclonal antibodies against the same three targets using antibodies from the third bleed of numbered rabbits at a 1:200 dilution. [Figure 14B]14A and 14B are graphs of antibody binding data for peptides or recombinant approximately 90 kDa FLNA polypeptide fragments bound to solid supports in a microtiter plate well ELISA format. The UniProtKB / Swiss-Prot database P21233 sequence of bound human FLNA contained an additional C-terminal cysteine ​​amide that binds to the immunogenic carrier maleimide-activated keyhole limpet hemocyanin (KLH) of the immunogen. The serine residue at position 2152 of the polypeptide designated "pS2152" was phosphorylated. The bound antigens in each assay shown were, from right to left, the immunogenic polypeptide, the immunogenic polypeptide in which the phosphorylated serine residue was replaced with an alanine residue ("A2152"), and the 90 kDa FLNA polypeptide fragment phosphorylated at serine 2152 ("90 kDa FLNA"). Figure 14A shows data for mouse monoclonal antibody preparations diluted 1:1000 using a commercially available product (Origene) and two monoclonal antibodies (20H7 (IgG2) and 16E6 (IgM)) prepared by us at Aragen Biosciences, Inc. (Morgan Hill, Calif.). Figure 14B shows binding data using rabbit polyclonal antibodies against the same three targets using antibodies from the third bleed of numbered rabbits at a 1:200 dilution. [Figure 15] Scatter plots of the data in Table 1, plotting density values ​​for the FLNA 90 kDa polypeptide fragment, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the 280 kDa FLNA protein for serum samples from patients with previously determined Alzheimer's disease (AD) and age-matched controls known to be free of AD (AMC). Horizontal black bars indicate mean values ​​± standard error of the mean (SEM) paired with horizontal and vertical black lines connecting the values. [Figure 16A]Figures 16A, 16B, and 16C further replot the data from Table 1, showing the ratio of the density value of the approximately 90 kDa polypeptide fragment of FLNA divided by the density value of albumin (Figure 16A), or the ratio of the density value of GAPGH (Figure 16B), or the ratio of the density value of the approximately 280 kDa FLNA protein (Figure 16C) for multiple subjects, for AD subjects, age-matched control subjects (AMC), cognitively normal young subjects (YCI), subjects with mild cognitive impairment due to AD (MCI-AD), and subjects with mild cognitive impairment not due to AD (MCI-nonAD). [Figure 16B] Figures 16A, 16B, and 16C further replot the data from Table 1, showing the ratio of the density value of the approximately 90 kDa polypeptide fragment of FLNA divided by the density value of albumin (Figure 16A), or the ratio of the density value of GAPGH (Figure 16B), or the ratio of the density value of the approximately 280 kDa FLNA protein (Figure 16C) for multiple subjects, for AD subjects, age-matched control subjects (AMC), cognitively normal young subjects (YCI), subjects with mild cognitive impairment due to AD (MCI-AD), and subjects with mild cognitive impairment not due to AD (MCI-nonAD). [Figure 16C] Figures 16A, 16B, and 16C further replot the data from Table 1, showing the ratio of the density value of the approximately 90 kDa polypeptide fragment of FLNA divided by the density value of albumin (Figure 16A), or the ratio of the density value of GAPGH (Figure 16B), or the ratio of the density value of the approximately 280 kDa FLNA protein (Figure 16C) for multiple subjects, for AD subjects, age-matched control subjects (AMC), cognitively normal young subjects (YCI), subjects with mild cognitive impairment due to AD (MCI-AD), and subjects with mild cognitive impairment not due to AD (MCI-nonAD). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition In the context of this invention and the associated claims, the following terms have the following meanings. The articles "a" and "an" are used herein to 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.

[0031] "Blood serum" or "serum" is the clear liquid that separates from blood when the blood has completely clotted. Thus, fibrinogen has been removed from the plasma during the blood clotting process [Dorland's Illustrated Medical Dictionary, 29th ed., WB Saunders Co. Philadelphia, PA, p. 1629 (2000)]. "Blood plasma" or "plasma" is the clear, pale yellow liquid portion remaining after removal of cellular components such as red blood cells, white blood cells, and platelets from unclotted blood. Plasma is the largest component of human blood, accounting for about 55%, and contains water, salts, enzymes, antibodies, and other proteins [The Practice of Medicinal Chemistry, C. Wermuth ed., Academic Press, New York, p. 46 (1996)]. A "serum or plasma sample" is an aliquot of serum or plasma obtained from a subject after minimal processing as described above. Serum or plasma samples are typically too viscous for use in the assays described herein and are therefore typically diluted with water or an aqueous buffer or other aqueous composition to form a "serum or plasma sample preparation." Exemplary dilutions are typically from about 10 to about 100-fold, more typically from about 20 to about 50-fold. The dilution composition may contain one or more organic solvents that are compatible with the proteinaceous components of the serum or plasma sample.

[0032] The FLNA molecules of interest in the present invention have apparent molecular weights (masses) determined by electrophoresis to be "about 90 kDa" and "about 280 kDa". These two FLNA molecules are generally referred to herein as "about 90 kDa FLNA polypeptide fragment" and "about 280 kDa FLNA protein". For ease of expression in the present specification, the word "about" may be omitted, as well as one or more of the words "FLNA polypeptide fragment" and "FLNA protein", on the assumption that those skilled in the art will understand that such masses are approximate values ​​depending on the method of their determination.

[0033] As mentioned above, FLNA contains a sequence of 2647 amino acid residues, with a molecular weight of approximately 280 kDa, according to the accession number P21333 in the UniProtKB / Swiss-Prot database. Each of these amino acid residues has a specific position number, counting from the amino terminus of the protein. When a protein is cleaved by an enzyme, the relative positions of the residues in the resulting polypeptide chain do not change. Therefore, it is convenient to refer to specific residues by their position numbers in the UniProtKB / Swiss-Prot database, whether they are in the intact (full-length) protein or in the polypeptide portion of the protein. Thus, the serine at position 2152 of the intact FLNA molecule corresponds to residue 2152 of the approximately 90 kDa FLNA polypeptide portion of the FLNA molecule (i.e., S 2152 When this serine residue is phosphorylated and present in the 90 kDa polypeptide, it is called pS 2152 When phosphorylated and present in full-length FLNA, it is called pS 2152 -It is called FLNA. It should further be noted that the terms "full length" and "90 kDa polypeptide portion" or "90 kDa polypeptide fragment" refer to those proteinaceous substances obtained from the body sample to be analyzed, and are therefore intended to include sample substances whose respective molecular weights are altered by processing during or after collection. It should also be understood that full-length FLNA and the 90 kDa FLNA polypeptide fragment are present in serum or plasma by themselves without lysis of cells such as lymphocytes that may also be present in blood, as may occur during collection of blood, plasma, or serum.

[0034] As used herein, the term "receptor" is used broadly to refer to an entity to which another entity, a ligand, specifically binds. Receptors are generally macromolecules, while ligands are generally small, low molecular weight molecules, although this distinction is not necessary. Receptor molecules contemplated herein include whole antibodies and antibody binding site portions (paratopes) that immunoreact with a specific epitope of the ligand, as well as proteins such as Staphylococcus aureus proteins A and G that bind Fab and Fc antibody portions. Biotin and avidin (streptavidin) can also be considered as ligand-receptor pairs, with either molecule being the receptor or the ligand, as can aptamers. The receptor molecule of the present invention may be an antibody, a substantially intact antibody in a substantially purified form as found in the ascites fluid or serum of an immunized animal, or an idiotype-containing polypeptide portion of an antibody, such as the Fab and F(ab')2 antibody portions described below. Antibody receptor molecules may be monoclonal and polyclonal antibody receptors, and may be referred to herein as "monoclonal receptors" or "polyclonal receptors." The biological activity of a receptor molecule is evidenced by the receptor specifically binding to its ligand when mixed in an aqueous medium at at least physiological pH values ​​and ionic strengths. Preferably, the receptor also binds to the ligand within pH values ​​of about 5 to about 9 and ionic strengths ranging from distilled water to about 1 molar sodium chloride.

[0035] The terms "binding" and "binding" are used herein as shorthand for specific binding, such as in an immune reaction between an antibody and its antigen, the binding of an enzyme to its substrate, or the binding of biotin to avidin, which is contrasted with nonspecific binding, such as the indiscriminate attachment of proteins in solution to the plastic or glass walls of a microtiter plate by hydrophobic, ionic, or other nonspecific means. The paratope-containing molecule or polypeptide portion of an antibody (antibody binding site) is the portion of the antibody molecule that specifically binds to the epitope of a ligand and may include the Fab, Fab', and F(ab')2 portions of the antibody. The Fab and F(ab')2 portions of antibodies are well known in the art and are prepared by reacting papain and pepsin, respectively, on a substantially intact antibody by well-known methods. See, for example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon. The Fab' portion of an antibody is also well known and is prepared by reducing the F(ab')2 disulfide bond, such as by mixing with mercaptoethanol, followed by alkylating the reduced cysteine ​​residues with a reagent such as iodoacetamide. Intact antibodies are the preferred receptors and are utilized as exemplary of the receptor molecules of the present invention.

[0036] Suitable monoclonal antibody receptors (usually whole antibodies) can be prepared using hybridoma techniques as described by Niman et al., Proc. Natl. Sci., USA, 80:4949-4953 (1983), which is incorporated herein by reference. Briefly, to form a hybridoma from which the monoclonal receptor is produced, a myeloma or other autologous growing cell line is fused with lymphocytes taken from the spleen of a mammal hyperimmunized with a selected proteinaceous immunogen. The myeloma cell line is preferably from the same species as the lymphocytes. +Mice of this strain are the preferred mammal. Mouse myelomas suitable for use in the present invention include the hypoxanthine-aminopterin-thymidine sensitive (HAT) cell lines P3X63-Ag8.653 (ATCC CRL1580) and Sp2 / 0-Ag14 (ATCC CRL1581). Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by sensitivity to HAT medium. Hybridomas producing useful receptor molecules can be identified using enzyme-linked immunosorbent assays (ELISA).

[0037] Exemplary monoclonal receptors include mouse anti-human FLNA IgG1 monoclonal (MAB1678) and mouse anti-human FLNA IgG1 monoclonal (MAB1680) commercially available from Chemicon International, Inc. Mouse monoclonal MAB1680 binds to an epitope sequence within the approximately 90 kDa C-terminal calpain cleavage fragment of the FLNA polypeptide, and mouse monoclonal MAB1678 binds to an epitope sequence near the N-terminus of FLNA, outside the sequence of the 90 kDa C-terminal calpain cleavage fragment. Other monoclonal receptors useful herein include mouse anti-phosphoserine NeuroMab clone N259 / 48 available from the UC Davis / NIH NeuroMab Facility at UC Davis, Davis, Calif., and rabbit monoclonal anti-pS 2152 Filamin A receptor [EP2310AY] Abcam® (cat#: ab75978). The monoclonal receptor need not be obtained from the hybridoma supernatant, but can generally be obtained in a more concentrated form from the ascites fluid of a mammal injected with the desired hybridoma. The production of monoclonal antibodies using ascites fluid is also well known and will not be discussed further herein. Both MAB1678 and MAB1680 are produced in ascites fluid.

[0038] A "polyclonal receptor" (pAb) is a receptor produced by clones of different antibody-producing cells that produce antibodies against multiple epitopes of an immune molecule. Exemplary polyclonal antibodies useful herein include rabbit pAb sc28284, whose epitope is said to be at the C-terminal amino acid residues 2348-2647 of FLNA, rabbit pAb sc130190, whose epitope is said to include phosphorylated serine 2151 of FLNA, and goat pAb SC7565, whose epitope is said to map near the N-terminus of FLNA from Santa Cruz Biotechnology, Inc. In addition to the above-mentioned mice, rabbits, and goats, non-human warm-blooded animals that can be used in the present invention as hosts for producing monoclonal or polyclonal receptors can include poultry (such as chickens or pigeons), ratites (such as emus, ostriches, cassowaries, or moas), or mammals (such as dogs, cats, monkeys, pigs, cows, horses, guinea pigs, or rats). Preferably, the host animal is a rabbit or a mouse.

[0039] It should be noted that depending on the assay format, one or two receptor molecules can be used. The first, as used in solid-phase assays such as ELISA, is called the capture receptor molecule, or more simply the capture molecule. The capture molecule is usually itself bound to a solid support and is composed of FLNA or a phosphorylated proteinaceous portion of FLNA (pS 2152 The second type of receptor molecule is referred to herein as the detection receptor molecule or analytical molecule. In its bound form, the second receptor binds to the analyte ligand present in the liquid phase, such as the fluorophore ...

[0040] The detection molecule is utilized in conjunction with an indicator labeling means or "indicating group" or "label". The indicator group or label is used in conjunction with an analytical molecule as a means to confirm that a particular ligand has bound to the capture molecule. Exemplary are labeled paratope-containing molecules that bind to the Fc portion of a genus-specific antibody, such as FITC-labeled goat anti-mouse antibody (Sigma-Aldrich or Abcam®) and HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology or GE Lifesciences). The terms "indicating labeling means", "indicating group", or "label" are used interchangeably herein to include single atoms and molecules that are attached to a receptor or used alone, whether the atoms or molecules are used alone or in combination with additional reagents. Such indicator groups or labels are well known in immunochemistry. The indicator labeling means may be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorescent dye (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorescent dyes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), dimethylaminonaphthalene-S-sulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), Lissamine rhodamine B200 sulfonyl chloride (RB200SC), and the like. A description of immunofluorescence analysis techniques is provided by DeLuca in "Immunofluorescence Analysis," Antibody As A Tool, Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1985), which is incorporated herein by reference.

[0041] The indicator labeling means may be directly bound to the analyte molecule or may comprise a separate molecule. It is particularly preferred that the indicator means is a separate molecule, such as an antibody that binds to the receptor of the invention. Staphylococcus aureus protein A (sometimes referred to herein as protein A) can also be used as an indicator or labeling means for separate molecules when utilizing an intact or substantially intact antibody receptor. In such uses, protein A itself contains a label, such as a radioactive element or a fluorescent dye. The indicator group can also be a biologically active enzyme such as horseradish peroxidase (HRP) or glucose oxidase. When the primary indicator group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the formation of the receptor-ligand complex. Such additional reagents for HRP include hydrogen peroxide and an oxidative dye precursor such as diaminobenzidine. Another additional reagent useful for glucose oxidase is 2,2'azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

[0042] More modern techniques utilize luminol as an oxidation precursor that is chemiluminescent when oxidized, similar to the HRP hydrogen peroxide reaction product. Exemplary chemiluminescent reagents for use with HRP include those sold under the tradename SuperSignal® ELISA Pico Chemiluminescent Substrate by ThermoFisher Scientific (Waltham, Massachusetts), westernSure® ECL Substrate by LI-COR Biosciences (Lincoln, Nev.), and PicoMax™ Sensitive Chemiluminescent HRP Substrate by Rockland Immunochemicals, Inc., (Limerick, Pennsylvania). It is understood that chemiluminescent assays are more accurate and precise than colorimetric assays, and that sandwich ELISA format assays provide increased signal-to-noise and improved sensitivity. See, e.g., Hatch et al., J. Med. Biol Sci 3(1):1-6 (2009) and Wang et al., J Bioprocess Biotech 3(2):136 (2013). HRP-conjugated goat anti-rabbit IgG polyclonal antibodies useful as indicator groups are available from several sources, exemplary materials include EMD Millipore product 12-348, Abcam® ab6721, ThermoFisher Scientific product 65-6120, and Santa Cruz Biotechnology product sc-2030.

[0043] Radioactive elements provide another class of labels. Exemplary radiolabeling agents that can be utilized in the present invention are radioactive elements that produce gamma radiation. 124 I, 125 I, 128 I, 131 I, 132 I, and 51 Elements that themselves emit gamma rays, such as Cr, represent one class of radioactive element indicator groups that produce gamma radiation. Particularly preferred are 125I. Another class of useful indicator groups are those which themselves emit positrons. 11 C. 18 F, 15 O, and 13 Elements such as N. The positrons thus emitted produce gamma rays when they encounter electrons present in the analytical medium. 111 Beta emitters such as indium are also useful. Radioactive receptor molecules can be produced, as is well known, by culturing receptor-producing cells in a medium containing radioactive amino acids and, after isolating the receptor, labeling it with one of the radioactive elements mentioned above. Radioactive labeling of proteins is well known in the art and will not be described further herein.

[0044] The receptor molecule and separate indicator means of any of the diagnostic kits described herein can be provided in solution, as a dispersion, or as a substantially dry powder, for example in lyophilized form. When the indicator means is a molecule separate from the analyte receptor, it is preferred that the indicator means is packaged separately. When the indicator means is an enzyme, the enzyme substrate can also be provided in a separate package in the kit. A solid support such as a microtiter plate, one or more buffers, and other desired reagents can also be included as separately packaged elements in the diagnostic assay kit. The terms "system" and "kit" are used herein with slightly different meanings. A "system" includes the recited elements, and a "kit" may include the same elements. The difference between the two is that a kit packages the elements together, whereas a system simply uses the elements regardless of their source. The packaging described herein with respect to diagnostic kits is that conventionally utilized in diagnostic systems, and includes glass and plastic (e.g., polyethylene, polypropylene, polystyrene, and polycarbonate) bottles, vials, plastic, and plastic-foil laminated envelopes.

[0045] As used herein, phrases such as "significantly different" and "significantly different" mean that when a difference is observed between two or more findings, the compared results differ by more than one standard deviation of either measurement, preferably more than two standard deviations, and more preferably three standard deviations, when the assay is repeated a sufficient number of times to obtain statistically reliable results. Each of the patents, patent applications, and articles cited herein is incorporated by reference. Use of the article "a" or "an" is intended to include one or more.

[0046] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention contemplates a method for assaying serum or plasma samples from a living human subject for the possible presence of Alzheimer's disease (AD) in that human subject using the relatively non-invasive technique of drawing blood. In another aspect, the contemplated assays can provide prognosis for treatment in living putative AD patients, as well as be used as the basis for objective prognostic and biomarker assays to track disease progression and treatment efficacy. The plasma of AD patients contains a 90 kDa pS 2152 -FLNA fragment, 280 kDa pS 2152 We found that the 90 kDa pS 2152 We hypothesize that the -FLNA fragment originates from degenerated and ruptured neurons in the brain. To evaluate this hypothesis, we investigated the mechanism by which long-term Aβ 42 Using ex vivo organotypic brain slice cultures that showed AD-like pathology and neurodegeneration after exposure [Wang et al., Biol Psychiatry 67(6):522-530(2010)]. Two brain regions known to be vulnerable to AD damage were used to prepare 200 μm slices of the prefrontal cortex and hippocampus from 10-week-old rats. These brain slices were cultured for 4 days as previously described (Wang et al., supra) to allow for recovery from slice-induced damage. The slices were then serum-deprived and incubated with 100 μM Aβ. 1-42 (Aβ 42 ) was incubated with 90 kDa pS in the medium2152 -FLNA fragment and pS 2152 -FLNA protein, as well as other biomarkers reflecting neuronal destruction, nitrated tau (nY 29 The presence of Aβ-tau, and glial damage [truncated glial fibrillary acidic protein (GFAP)] was assayed by Western blot. 42 The specificity of Aβ is due to the intrareversal peptide Aβ 42-1 It is defined by: 90kDa pS 2152 -FLNA fragment is Aβ 42 It appeared in the medium 4 hours after addition and steadily increased with time. 2152 -FLNA protein is not. 29 -Similar to tau progression, with a pS of 90 kDa 2152 This suggests that the -FLNA fragment originates from damaged nerve cells (axons). 1-42 Full-length GFAP appears early after exposure to GFAP, followed by its truncated form.

[0047] These data suggest that glial damage occurs before neuronal destruction. Furthermore, axonal / neuronal damage was more prominent than glial damage in hippocampal tissue compared with prefrontal cortex. These data suggest that Aβ 42 have shown that it destroys both neurons and glia. FLNA is expressed in both neurons and glia, but is a 90 kDa pS 2152 The time course of -FLNA appearance suggests that degenerating neurons are the major source. 2152 -FLNA protein was absent, and in addition, the 280 kDa pS 2152 -FLNA protein levels are elevated, indicating that the 90 kDa pS 2152 -FLNA fragments are mainly derived from destroyed neurons and are expressed as pS of 280 kDa before being released into plasma. 2152 These data suggest that the Aβ-FLNA protein is the result of protease digestion.42 This also supports the view that may be a causative agent of AD pathology.

[0048] Brain slice data showed that full-length pS 2152 -FLNA are likely to be of peripheral origin. Platelets are known to have large amounts of FLNA and may therefore be a source. Thus, the approximately 90 kDa FLNA polypeptide fragment, or the approximately 90 kDa FLNA polypeptide fragment phosphorylated on serine at FLNA residue 2152 (pS 2152 It has been found that subject plasma or serum containing the approximately 90 kDa FLNA polypeptide fragment positively correlates with the subject having AD. Conversely, the absence of the approximately 90 kDa FLNA polypeptide fragment in a subject's serum or plasma sample correlates with the subject not having AD. Although these correlations are generally accurate, the FLNA of approximately 90 kDa in serum or plasma (pS 2152 -90kDa or S 2152 Based solely on the presence or absence of the -90 kDa FLNA protein fragment (polypeptide) or the approximately 280 kDa FLNA protein, inaccurate conclusions may be obtained, particularly in elderly subjects, e.g., subjects over about 45 years of age, and particularly subjects over about 60 years of age.

[0049] Examination of Figures 4 and 5 shows the variability of the data points obtained by plotting the individual density values ​​obtained from the data in Figures 1, 2, and 3. However, when the ratios of the density values ​​of the FLNA polypeptide fragment at approximately 90 kDa divided by the value of the FLNA protein at approximately 280 kDa are examined in subject sample preparations, as shown in Figure 6, the data become less variable, but there is still no overlap between subjects with AD (AF and MCI AD) and those without AD (AMC and MCI NonAD). Therefore, as described below, in order to more accurately evaluate the presence or absence of AD in the subjects at the time of blood collection, it is preferable to use both the amounts of low molecular weight [about 90 kDa FLNA] and high molecular weight [about 280 kDa; presumed intact FLNA], both of which are phosphorylated or not at serine residue at position 2152, in the ratio. Although data from normal young control (YCI) subjects are somewhat variable, these subjects rarely have AD. Although the serum of AD patients contains about 90 kDa FLNA fragments, it is not known whether all of the 90 kDa FLNA fragments present in serum or plasma are derived from AD brain, other tissues, or both. In a preferred embodiment, phosphorylated or unphosphorylated S 2152 -90kDa FLNA and / or S 2152 The presence or absence of -FLNA is determined using reducing SDS-PAGE Western blot separation and analysis. A monomercaptan such as 2-mercaptoethanol is used as a sample reducing agent prior to separating the proteinaceous portion of the sample. Exemplary Western blot analyses of several human serum or plasma sample preparations prepared from serum or plasma samples are shown in the visualized separations shown in Figures 1-3, 7, and 9.

[0050] The baseline data in Table 1, described in the Results section below, show that the upper limit for cognitively normal young (YCI) subjects and age-matched controls (AMC) approaches but does not overlap with the lower limit for AD patients. The data in Table 1 for the (approximately 90 kDa polypeptide fragment) / (approximately 280 kDa FLNA protein) ratio and the scatter plot in FIG. 6 show that there is no overlap when the ratio of the amounts of the two FLNA-associated phosphorylated proteinaceous substances is used as described herein. This separation is particularly relevant for the AMC population. Similar results are obtained using a receptor that does not immunoreact with the unphosphorylated serine 2152 residue. It was unexpected that the approximately 90 kDa FLNA polypeptide fragment was found in the plasma or serum of a suspected AD subject. The present inventors have found that the phosphorylated approximately 90 kDa FLNA polypeptide fragment (pS 2152We are not aware of any reports regarding the existence and / or detection of approximately 90 kDa FLNA (-90 kDa FLNA) or any approximately 90 kDa FLNA polypeptide fragment, which, as mentioned above, has previously only been reported to occur in the cell nucleus.

[0051] Furthermore, the species shown in Figures 1 to 6 were identified using an antibody that specifically binds to an epitope containing phosphorylated serine at FLNA position 2152, and it was found that the approximately 90 kDa FLNA polypeptide fragment in these figures is phosphorylated at serine at protein position 2152 and possibly also at other positions that are currently not defined. Not only is this blood-derived approximately 90 kDa FLNA polypeptide fragment species phosphorylated, but it is also phosphorylated at a serine residue corresponding to serine 2152 in the full-length approximately 280 kDa FLNA protein molecule, as has been shown by other methods in the art [Garcia et al., Arch Biochem Biophys 446:140-150 (2006); Gorlin et al., J Cell Biol 111:1089-1105 (1990)], and inhibition of proteolytic processing of FLNA results in the presence of full-length phosphorylated (pS2152) FLNA and the approximately 90 kDa FLNA polypeptide fragment that is not phosphorylated at serine 2152 in the nuclei of prostate cancer cells. The approximately 90 kDa FLNA polypeptide fragment species can generally be readily distinguished from full-length FLNA by Western blot analysis, regardless of whether or not those molecules are phosphorylated at serine 2152. As a result, receptor molecules that immunoreact with epitopes that include or do not include phosphorylated serine 2152 can be utilized in this assay.

[0052] Phosphorylated FLNA (pS) of approximately 90 kDa present in plasma or serum samples 2152It was further unexpected that the amount of the approximately 90 kDa FLNA polypeptide, or the approximately 90 kDa FLNA polypeptide not phosphorylated at the serine 2152 species, together with the amount of another protein, could be used to accurately diagnose the AD status of subjects from whom plasma or serum samples were taken. Thus, the data in Figures 16A, 16B, and 16C, detailed below, show that the ratio of the amount (density) of the approximately 90 kDa FLNA polypeptide to the amount (density) of another proteinaceous material present in the same aqueous plasma or serum sample preparation can be used to reduce the variability in the amount of the approximately 90 kDa FLNA polypeptide alone to create a viable assay. Exemplary other sample preparation proteinaceous species are albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the approximately 280 kDa FLNA protein. Also surprisingly, the approximately 280 kDa FLNA protein has proven to be the most useful counterpart ever discovered. It is preferred to use a ratio of the approximately 90 kDa FLNA polypeptide fragment or the approximately 280 kDa FLNA protein to another proteinaceous substance from serum or plasma of the same subject, but it is understood that the use of either of these substances alone will also result in an assay that will yield the correct answer. Thus, although the assays described herein are generally described in terms of the use of ratios, these individual materials can also be used separately. Contemplated assays can also be used in conjunction with methods of treating AD to indicate progression, stagnation, or regression of the disease. Thus, an increase in the ratio of either the approximately 90 kDa FLNA polypeptide fragment / the approximately 280 kDa FLNA protein, or their 2152 phosphorylated counterparts, indicates disease progression (worsening), whereas a decrease in the ratio indicates a reduction in disease progression, and no change in the ratio indicates no change in the disease state.

[0053] In one embodiment, the contemplated assay is preferably performed in a Western blot format after SDS-PAGE separation under reducing conditions, as exemplified and explained below: Monomercaptan reducing agents are used for SDS-PAGE separation, which denatures proteins under reducing conditions. 2-mercaptoethanol is the preferred and commonly used reducing agent, but thioglycolic acid and thioglycolic acid derivatives can be used, such as glyceryl thioglycolate, ethylene glycol monothioglycolate, thioglycolamide (2-mercaptoacetamide), or C1-C6-hydrocarbyl thioglycolates. The use of reducing agents with two or more mercapto (-SH) groups (polymercapto compounds), such as dithiothreitol (DTT) or dithioerythritol (DTE), has been found to interfere with the assay, and the reducing composition used in this assay does not contain such polymercapto reducing agent compounds.

[0054] After electrophoretic separation, the separated protein portions are transferred to a membrane, such as nitrocellulose or polyvinylidene difluoride (PVDF), and conjugated or reacted with an identifying reagent, as described below and as is well known in the art. Continuing with the steps of the method, a detection reagent is contacted with the first separated FLNA fraction (e.g., about 90 kDa FLNA polypeptide fragment) to form a first detection reagent-bound FLNA fraction. If the low molecular weight polypeptide is present, the subject may have had AD at the time of sample collection. The diluted aqueous serum or plasma preparation is ultrafiltered using a suitable ultrafilter to obtain about 90 kDa FLNA polypeptide fragment in the ultrafiltrate (filtrate), as also shown herein. More preferably, the detection reagent is contacted with a second high molecular weight separated FLNA fraction (e.g., about 280 kDa FLNA protein) to allow the detection reagent to bind to the second separated FLNA fraction. When about 90 kDa FLNA polypeptide fragment and about 280 kDa FLNA protein are present, respectively, a first and a second detection reagent-bound FLNA fraction are formed. It is preferable to use the same detection reagent to contact both phosphorylated FLNA species. By using the same detection reagent for both phosphorylated FLNA species, the binding and affinity difference between two different receptor molecules can be eliminated, which simplifies calculations.

[0055] The amount of the first fraction of detection reagent-bound FLNA (A) and the second fraction of detection reagent-bound FLNA (B) are quantified. The quantification step is usually performed by densitometry, by fluorimetry, or, if a radioactive tracer is used, by radioactive decay using commercially available equipment specifically designed for Western blot analysis. The amount of detection reagent-binding substance present may be determined by the actual amount in grams or moles. More commonly, the amount present is determined relative to an internal standard or as an amount above background. Methods for determining these relative amounts are well known in the art. Therefore, one preferred assay utilizes the ratio of the amounts of the above low and high molecular weight polypeptides and proteins, respectively.Currently, it is preferred to use the ratio of the amount of low molecular weight polypeptides to the amount of high molecular weight proteins.In one embodiment, this determination is also carried out as a Western blot assay after SDS-PAGE protein separation, preferably using monomercaptan as reducing agent.

[0056] The ratio of the amounts of two phosphorylated proteins is preferred for at least two reasons. First, almost everyone whose serum or plasma is tested, except for "young cognitively normal" (YCI) subjects who are unlikely to be tested in the proposed assay, will have small amounts of one or both of the polypeptides and / or proteins. Second, determining the ratio using two assayable proteinaceous substances allows the assay to be controlled in that if too much or too little sample is used in the assay, or if not enough receptor molecules are used, the use of two values ​​for the ratio can minimize errors. In contrast to such common practice, in the assay of the present invention, one or both of the FLNA-related molecules being assayed may be absent or absent in quantifiable amounts. When either the amount of low molecular weight polypeptide (A) or the amount of high molecular weight (B) protein is present in a quantifiable amount (QAmt) and the other is absent in a quantifiable amount, any predetermined fraction of about 0.1 to about 0.001 of the quantifiable amount is assigned to the amount of the other that is not quantifiable [(0.1 to 0.001) x (QAmt)] to avoid using zero in the numerator or denominator. When both A and B are absent in quantifiable amounts, approximately the same arbitrary amount is assigned to both. When one of A and B is present in a quantifiable amount and the other is not, its abundance is usually in a relative unit, such as a few thousand density units, and any amount between about 30 to about 70, preferably about 50 or 60, can be used for the non-quantifiable amount.

[0057] In a preferred assay, using a ratio of polypeptide / protein amount, with the amount of low molecular weight protein as the numerator and the amount of high molecular weight protein as the denominator, a ratio value of about 10 to about 2000, more typically about 120 to about 400, indicates that the subject from whom the sample was taken probably had AD at the time of taking the sample, whereas a ratio value of about 0.005 to about 15, typically about 0.01 to about 1.2, more typically about 0.015 to about 0.5, indicates that the subject from whom the sample was taken probably did not have AD at the time of taking the sample. It should be understood that the ratio value will vary depending on the pair of proteins selected for comparison. Thus, if albumin is used as the second proteinaceous substance (B) and the FLNA polypeptide fragment of about 90 kDa is (A), an A / B ratio of about 0.35 to about 1.5 indicates that the subject had AD at the time of sample collection, whereas an A / B ratio of about 0.003 to about 0.25 indicates that the subject did not have AD at the time of sample collection. If (A) is the same as above and GADPH is the second proteinaceous substance (B), an A / B ratio of about 0.6 to about 2.1 indicates that the subject had AD at the time of sample collection, whereas an A / B ratio of about 0.005 to about 0.35 indicates that the subject did not have AD at the time of sample collection. Similar ratios can be made using the approximately 280 kDa FLNA protein as the (A) substance and another proteinaceous substance present in the plasma or serum used to determine the ratio. One or more such reference lists of ratios can be made from a large number of subjects known to have or not have AD, based on one or the other of the approximately 90 kDa FLNA polypeptide fragment and the approximately 280 kDa FLNA protein, and a second proteinaceous substance present in the serum or plasma used for comparison. The use of the approximately 90 kDa FLNA polypeptide fragment and the approximately 280 kDa FLNA protein is a particularly preferred ratio at present.

[0058] The results for samples from AD patients (AD) diagnosed by techniques such as PET scans and age-matched controls (AMC) are shown in Table 1 and Figures 1-11 below. As can be seen from the data in Table 1 and Figures 1-11, subjects who had AD at the time of sample collection had abundant amounts of the .about.90 kDa FLNA polypeptide fragment and virtually no FLNA (.about.280 kDa FLNA) in their serum or plasma samples. These results were reversed in age-matched cognitively normal controls; most subjects had no quantifiable .about.90 kDa FLNA polypeptide fragment and abundant amounts of .about.280 kDa FLNA protein. The graphs in these figures show that, although the magnitudes vary widely, the results obtained using the A / B ratios are similar with little variability. These probable and non-probable AD results are easily distinguishable from each other. As a result of the contemplated assay, it may occur that neither A nor B is present in quantifiable amounts. If both the approximately 90 kDa FLNA polypeptide fragment and the approximately 280 kDa FLNA protein are not present in quantifiable amounts (absent), approximately the same arbitrary amount is assigned to both. For example, when both A and B are absent or cannot be quantified, an arbitrary assignment of 50 and 60 is used herein for the amounts of A and B. Thus, the A / B ratio is 0.83 or 1.2 (50 / 60 or 60 / 50), or about 1. This result is typically obtained in samples from cognitively normal young adult (YCI) subjects (preferably under 45 years old) without AD symptoms. The results obtained from assays of samples from such subjects are shown in Table 1 below.

[0059] As is well understood in science, the presence and absence of a substance does not necessarily equate to a yes or no answer. Rather, as science advances, a substance that was undetectable (or unquantifiable) yesterday may be detectable today, tomorrow, or in 20 years' time. Due to advances in science and technology, the absence of the aforementioned low or high molecular weight polypeptides / proteins is defined herein as the inability to detect or quantitate the substance using published methods utilizing Western blot techniques and reagents commercially available in the United States in 2021 and densitometric scanning with a GS-800™ calibrated densitometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The presence of either or both substances is determined as well. Reagents and equipment useful for such analysis are described below.

[0060] In another preferred embodiment, the proteinaceous portion is separated by ultrafiltration, which removes material with a molecular weight of more than about 100 kDa from the sample, and the presence or absence of the about 90 kDa FLNA polypeptide fragment (phosphorylated or non-phosphorylated) is determined using an ultrafiltrate with a proteinaceous material mass of less than about 100 kDa. The full-length FLNA (about 280 kDa FLNA protein) phosphorylated at serine 2152 and non-phosphorylated are among the separated proteinaceous molecules with a molecular weight of more than about 100 kDa that are excluded from the ultrafiltrate and present in the retentate. The full-length FLNA has a molecular weight of about 280 kDa, and is often referred to herein as pS for FLNA, about 280 kDa FLNA, and the 2152 serine phosphorylated molecule. 2152-It is called FLNA. Therefore, pS 2152 Assays can be utilized that simply determine whether -90 kDa and / or approximately 90 kDa FLNA polypeptides are present in the ultrafiltrate, and more preferably, assays are performed for both the lighter mass (approximately 90 kDa) FLNA polypeptide species and the heavier mass (approximately 280 kDa) FLNA protein.

[0061] The results of such an assay are shown in Figure 11. Plasma samples were collected and diluted 40-fold in volume with Tris buffer, pH 7.4. The resulting sample was split into two portions. One portion was ultrafiltered using a Nanosep® 100K OMEGA™ (P / N OD100C33) from Pall Life Sciences (Ann Arbor, MI) to remove proteinaceous material with a molecular weight above about 100 kDa. An amount of ultrafiltrate thus prepared, equivalent to approximately 0.25 or 0.5 □L of plasma, was added to a well of a 96-well assay ELISA plate (filtration). The same amount of unfiltered plasma sample preparation was added to another well. The well walls of the ELISA plate were pre-coated with streptavidin, and then FLNA phosphorylated serine 2152 [rabbit monoclonal anti-pS 2152 The wells were coated with a biotinylated rabbit monoclonal antibody raised against an epitope containing the filamin A receptor [EP2310AY] Abcam® (cat#: ab75978). Filtered and unfiltered samples were added to each well, and the proteins were bound to anti-pS 2152 After paratope binding, the wells were rinsed again. A mouse monoclonal antibody against the epitope mapping between residues 9 and 27 of the N-terminus of FLNA (E3; SC-17749, Santa Cruz Biotechnology, Inc.) was used to identify pS 2152 - The presence of FLNA was detected and pS was analyzed using a mouse anti-phosphoserine monoclonal antibody [NeuroMab (UC Davis / NIH): Cat#: 73-292]. 2152-90kDa FLNA polypeptide was detected. After the existing proteins were allowed to bind to the detection receptor, the wells were rinsed and then contacted with FITC-labeled anti-rabbit IgG to immunoreact with the mouse antibody-bound phosphorylated protein. After rinsing, the fluorescence of the wells was measured, and the results are shown in the graph in Figure 11. As can be seen from a comparison of the diagnoses shown in FIG. 11 with those shown in Table 1 for the same subjects, the two assays were in perfect agreement.

[0062] Assay Systems and Kits FLNA is a protein found abundantly in the peripheral circulation and is processed following platelet activation [Buitrago et al., bioRxiv307397]. found that FLNA is cleaved into 100kDa and 90kDa fragments upon the onset of platelet activation. Because FLNA is abundant in platelets and FLNA cleavage products are likely to be released into the blood upon platelet activation, methods must be used to minimize platelet activation. These methods include the use of coagulation inhibitors such as sodium citrate / citric acid buffer, EDTA and similar chelating agents, avoidance of vigorous centrifugation, and minimizing transit and storage times.

[0063] Yet another embodiment of the present invention relates to a method for determining whether a serum or plasma sample from a living subject is capable of detecting approximately pS 2152 A system for detecting a -90 kDa polypeptide and / or an approximately 90 kDa FLNA polypeptide (non-phosphorylated at serine 2152), thereby detecting the possibility that Alzheimer's disease (AD) was present in the living subject at the time the sample was taken for analysis. One aspect of this embodiment is a peptide having a molecular weight of about 90 kDa and / or pS 2152 -90 kDa FLNA polypeptides and any pS that may be present in the assay sample. 2152The contemplated system includes a kit-form containing a container holding a capture receptor molecule, such as an antibody or a paratope-containing portion of an antibody, that forms a capture complex when separated from the approximately 90 kDa and / or approximately pS FLNA protein in the sample. 2152 The formation of a capture complex is shown to correlate with the presence or absence of one or both of the -90 kDa FLNA polypeptides and therefore the presence or absence of AD in the living subject whose blood sample was used.

[0064] In another embodiment, the system contemplated is a kit in which the recited elements are packaged together. Thus, for example, the capture receptor molecule is present in a separate package or container, such as a vial. The above-mentioned label may also be present in a separate package. Also preferably, such a kit includes a capture receptor molecule having a molecular weight of about 90 kDa and / or about pS 2152 Instructions are included for the use of these receptor molecules to bind to one or both of the -90 kDa FLNA polypeptides to form a capture complex. Exemplary containers include those containing about 90 kDa and / or about pS in multiple samples. 2152 There are glass or plastic vials and multi-well plates that can detect either or both of the -90kDa FLNA polypeptides. The contemplated kits may include suitable labels as described above, as well as other packaged reagents and materials (e.g., washing buffers, etc.), depending on the particular assay used. Standard immunoassays as described above can be performed using these kits.

[0065] A particularly preferred assay kit comprises a packaged solid support having an assay surface coated with a paratope-containing capture receptor molecule that immunoreacts with an epitope common to both the low and high molecular weight proteinaceous FLNA materials described above. A specific example of such a capture antibody binds to an antigen (epitope) that contains phosphorylated serine 2152 of the low and high molecular weight FLNA of interest in the present invention (anti-pS 2152 receptor molecule), forming a capture complex. The contemplated kit also has a container that holds a first detection receptor molecule that selectively binds to the captured phosphorylated and / or non-phosphorylated approximately 90 kDa FLNA polypeptide to form a capture complex. A label that detects the presence of the captured low molecular weight FLNA-containing complex may be bound or bindable to the first detection receptor. There may also be another container that holds a second receptor that selectively binds to any captured phosphorylated and / or non-phosphorylated approximately 280 kDa FLNA protein molecule from the calpain H1 cleavage site (approximately position 1761) toward the N-terminus, along with a bound or bindable label that detects binding to high molecular weight FLNA protein molecules. The two labels utilized emit different signals so that the presence of both masses of filamin can be detected.

[0066] In a further contemplated system, another particularly preferred assay kit comprises two or more solid supports having an assay surface coated with a paratope-containing capture receptor molecule that immunoreacts with an epitope common to both the low molecular weight (about 90 kDa) and high molecular weight (about 280 kDa) proteinaceous FLNA materials described above, together with a further container as described below, and further comprising instructions for use. The paratope-containing capture receptor molecules on the surface of the two or more solid supports of the kit are the same, i.e., have the same binding characteristics for FLNA molecules of both molecular weights. One of the containers of the kit contains a first detection receptor molecule that specifically binds both low molecular weight (about 90 kDa) and high molecular weight (about 280 kDa) proteinaceous FLNA material when present in an aliquot of the sample preparation. The detection receptor binds to a different region than the capture receptor molecule coated on the solid support. Labels bound or capable of binding to the detection receptor emit a signal indicative of the total amount of both FLNA species present. If bound to the first detection receptor molecule, the labels are typically co-contained with their receptors. If capable of binding, the label molecule is typically in a separate container of the kit.

[0067] The kit further comprises a second container with a second detection receptor that specifically binds only high molecular weight FLNA molecules, and another label as described above. When the second solid support described above is used with a second sample preparation aliquot, the same amount of low molecular weight and high molecular weight FLNA binds to the support. The second detection receptor and the labels are reacted and the amount present is determined, and the amount of high molecular weight FLNA molecules is obtained. This amount can be subtracted from the total amount obtained as described above to obtain the amount of low molecular weight FLNA molecules. To avoid having to handle different reactions of different labels, it is preferable that the labels used to assay the total amount of high molecular weight and low molecular weight FLNA molecules that may be present are the same. Alternatively, the above kit further comprises a second container with a second detection receptor that specifically binds only low molecular weight FLNA molecules, and another label as described above.When the above-mentioned second solid support is used with the second sample preparation aliquot, the same amount of low molecular weight and high molecular weight FLNA binds to this support.The second receptor and the label are reacted, and the amount of existence is determined, and the amount of low molecular weight FLNA molecules is obtained.This amount is subtracted from the total amount to obtain the amount of high molecular weight FLNA molecules.

[0068] Exemplary second detection receptors that immunoreact with high molecular weight FLNA molecules have been generated against portions of the molecule that are not typically present in low molecular weight polypeptides. Thus, oligopeptides of the IgFLNa-1-15 or ABD regions of the molecule bound to a carrier molecule, such as Hepatitis B core antigen (HBsAg) or maleimide-activated keyhole limpet hemocyanin (KLH), can be used to induce the production of antibodies that recognize the peptide sequence and detect only the approximately 280 kDa FLNA protein molecule. The amino and carboxy termini of the approximately 90 kDa FLNA polypeptide each present a neo-epitope that is not present in the high molecular weight FLNA molecule and can be similarly bound to a carrier and further used to induce antibodies that bind only the approximately 90 kDa polypeptide. See, e.g., U.S. Patent No. 6,942,866 to Birkett and references therein. In comparable systems, the solid support is provided separately and can be prepared by the user.Exemplary solid supports include multi-well plates, such as the AcroPrep™ Advance filter plate available from Pall Life Sciences or the microtiter plate available from Thermo Fisher Scientific, that allow the detection of the FLNA polypeptide of about 90 kDa in multiple samples, individual test tubes, and particulate solids, such as plastic beads, such as those manufactured by Spherotech, Inc. (Lake Forest, Illinois), Firefly® particles from Abcam® Plc, and magnetic particles, such as superparamagnetic polystyrene (SPP) particles sold as Dynabeads® M-280 Strepavidin from Thermo Fisher Scientific.

[0069] Contemplated kits may also include a label or indicator means that signals the presence of an immunological reaction between the detection receptor and the reactive protein in the capture complex. The indicator means allows for detection of the reaction product capture complex and, as previously described, is packaged separately from the detection receptor if it is not directly bound to the receptor. More specifically, the kits may include one or more of the receptor molecules described herein, such as those used to detect pS2152-phosphorylated or pS2152-nonphosphorylated anti-FLNA paratope-containing molecules, biotinylated paratope-containing molecules that bind to receptors of another species, etc. Labeled receptor molecules that immunoreact with Fc portions illustratively include detection molecules such as FITC-labeled anti-mouse Fc antibodies and HRP-conjugated anti-rabbit IgG, or other receptors familiar to those of skill in the art that bind to receptors of non-self species. Other optional components of the kit may include buffers such as 50 mM Tris at pH 7.4 for use in diluting plasma or serum samples, buffers for use in carrying out the various described immune reactions, and buffers useful for carrying out separations or denaturing such as in SDS-PAGE analysis. The various components of the kit may be contained in separate containers, and certain compatible components may be prepackaged in a single container if desired. In addition to the above components, the kit may further include instructions for carrying out the above methods. These instructions may be present in the kit in various forms, one or more of which may be present in or on the kit. One form of these instructions may be as information printed on a suitable medium or substrate, such as one or more pieces of paper, in or on the kit packaging, in a package insert, etc. As a further means, the information may be present on a computer readable medium, such as a diskette, CD, etc. Yet another means that may be present is a website address that can be used to access the information at the removed site via the Internet. Any convenient means may be present in the kit.

[0070] In a preferred embodiment, a contemplated kit comprises an assay surface, e.g., well walls and bottom, comprising a paratope-containing molecule, e.g., rabbit monoclonal anti-pS, that immunoreacts with an epitope comprising phosphorylated or non-phosphorylated serine 2152 of FLNA as a capture receptor. 2152 Filamin A receptor [EP2310AY] Abcam® (cat#: ab75978), and anti-pS designated TA313881 and TA325463 from Origene Technologies, Inc. 2152 ELISA solid supports include microtiter plates coated with Filamin A rabbit polyclonal antibody. A particularly preferred multi-well plate is the AcroPrep™ Advance 96-well filter plate with an Omega™ 100 kDa molecular weight cutoff filter, available from Pall Life Sciences (Ann Arbor, Mich.). Contemplated kits also include a container of a detector receptor molecule that reacts with the capture complex, if present. One preferred such detector receptor molecule is a paratope-containing molecule that immunoreacts with mouse monoclonal anti-phosphorylated serine residue (anti-pS) receptors, such as NeuroMab clone N259 / 48; UC Davis / NIH NeuroMab Facility (Cat#:73-292).

[0071] Detection (anti-pS 2152 The anti-pS receptor is induced in an animal genus (genus 2; i.e., mouse) other than the one used to induce the capture receptor (rabbit). 2152 The receptor itself may be labeled, or a labeled anti-genus 2 receptor may be used. Receptor molecules that specifically immunoreact with the Fc portion of antibodies raised in other genera are commercially available, and their use is described herein. Particularly preferred kits utilize receptor molecules that specifically immunoreact with antigenic (epitope) sites present in full-length FLNA and that are absent in either phosphorylated or unphosphorylated 90 kDa FLNA polypeptide, and these receptors are preferably contained in their own container. One such contemplated receptor immunoreacts with the N-terminal FLNA sequence, and thus binds to the portion of the FLNA molecule that is between the N-terminus of repeat 16, which begins at position 1779 of the FLNA sequence in the UniProtKB / Swiss-Prot database, accession number P21333. These receptor molecules are used to detect capture complexes that contain either phosphorylated or non-phosphorylated approximately 280 kDa FLNA protein species.

[0072] One exemplary detection receptor is a mouse monoclonal antibody sold by Santa Cruz Biotechnology, Inc. as Filamin 1(E-3):sc-177749, which is reported in the product literature to be specific for an epitope mapping near the N-terminus of full-length FLNA between amino acid residues 9-27. Another exemplary detection molecule is FITC-conjugated naloxone (naloxone fluorescein #7315, Setareh Biotech, LLC, Eugene, OR), which binds to a site in repeat 24 near the C-terminus of full-length FLNA. As mentioned above, these paratope-containing detector molecules (receptors) are preferably derived in an animal genus (genus 2; i.e., mouse) other than the one used to induce the capture receptor (rabbit). 2152 (Anti-pS 2152 ) The FLNA receptor itself may be labeled, or a labeled anti-genus 2 receptor may be used. Exemplary mouse monoclonal receptor molecules that immunoreact with the N-terminal FLNA sequence are described elsewhere herein and can be used in the kits. Such antibodies can have a conjugated label or can be used as a label-binding paratope-containing molecule, such as FITC-conjugated anti-mouse IgG.

[0073] Assay In one embodiment, serum or plasma samples are collected from a living subject and preferably maintained at a temperature of about 4 to about 10° C. prior to use in the assay. Protease and phosphatase inhibitors are added to the sample. Exemplary commercially available protease and phosphatase inhibitors include Complete EDTA-Free Protease Inhibitor Cocktail (Millipore Sigma, Catalog No. 4693159001) and PhosSTOP® (Millipore Sigma, Catalog No. 4906845001). Dissolve one tablet of protease inhibitor and one tablet of phosphatase inhibitor in 500 μL of double distilled water, vortex, and add approximately 53 μL to each mL of plasma or serum. The sample is then preferably diluted to form a sample preparation and separated into proteinaceous constituents. Exemplary separation methods include size and charge. When separation by charge is performed, the proteinaceous materials in the serum sample are separated according to their isoelectric point (pI) (the pH value at which the net charge of a particular molecule is zero). Preferably, separation by size is performed, in which case the proteinaceous materials are preferably separated by electrophoresis, ultrafiltration, or other means.

[0074] After separation, the proteinaceous material is preferably immobilized and then contacted and reacted with the analyte receptor. In this assay, only one receptor molecule that binds the desired proteinaceous ligand is required. For example, an antibody that binds phosphorylated FLNA serine 2152 may be used, as described above and used illustratively in some examples herein. The analyte receptor may be bound to a labeling means, or may be labeled separately, as is well known with fluorescently labeled antibodies that bind to the Fc portion of anti-phosphorylated FLNA serine 2152. Most biochemistry laboratories have equipment to perform electrophoretic tests. Although such equipment is useful, standard gel electrophoresis equipment can be subject to user error and subjective bias. More recent equipment, such as those sold under the names WES™, Peggy Sue™, Sally Sue™, and Simon™ by the ProteinSimple division of Bio-Techne Corp. (Minneapolis, Minnesota), allows for assays that are faster, more accurate, and less subjective to user subjectivity.

[0075] A second embodiment is suitable for an ELISA type format and utilises two different capture receptor molecules, preferably two microtiter plate sample wells or similar structures per assay. Preferably, two aliquots are taken from the diluted plasma or serum sample preparation. The desired FLNA proteinaceous material of the first aliquot from the sample preparation is bound to a first capture molecule that is itself immobilized on a first solid support. Exemplary solid supports are well known and include the inner surface of a well of a microtiter plate, magnetic beads, and non-magnetic beads. As exemplified herein, using a preferred microtiter plate well, the aliquots are placed into a first well, the surface of which is coated with a first capture molecule. Illustratively, these first capture receptor molecules bind to a region of FLNA that is N-terminal to the calpain H1 cleavage site that occurs between repeats 15 and 16 of FLNA. As a result, the first capture receptor binds to full-length FLNA, but not to the approximately 90 kDa phosphorylated or non-phosphorylated polypeptide fragment. After a suitable maintenance time to allow binding of the capture molecules with the FLNA species present in the sample, the unbound sample is removed. Typically, the binding moieties (if present) are rinsed off. Unbound areas of the surface are often treated with an aqueous dispersion of bovine serum albumin or skim milk powder to prevent non-specific binding, as is well known. The amount of bound FLNA moieties is determined using an analytical molecule as described below. Exemplary first capture molecules include mouse monoclonal MAB1678 and goat pAb sc7565. Exemplary analytical (detection) receptor molecules include rabbit pAb75978 and rabbit pAb sc130190, both of which bind to sequences containing phosphorylated serine 2152 (or 2151). After removing any unbound analytical receptor molecules, the receptor containing the indicator means is reacted with an exemplary bound rabbit antibody, any excess is removed, and the amount of full length FLNA is determined.

[0076] A second well, the surface of which is coated with a second, different capture receptor molecule, is utilized to determine the amount, if any, of the approximately 90 kDa FLNA fragment phosphorylated at serine 2152 or unphosphorylated in the sample. An exemplary second capture receptor molecule is rabbit pAb sc28284 (Santa Cruz), which binds near the C-terminus of FLNA. This capture receptor binds both full length and phosphorylated approximately 90 kDa FLNA molecules. The total amount of FLNA full length and approx. 90 kDa fragment molecules is determined using an analytical molecule such as rabbit pAb75978 (abcam Inc., Cambridge, Mass.) or rabbit pAb sc130190 (Santa Cruz), which binds to an epitope containing phosphorylated or non-phosphorylated serine 2152, as described above. Preferably, the same analytical receptor molecule is used in both parts of the assay (full length FLNA, and full length and approx. 90 kDa polypeptide fragments) to minimize possible errors due to differences in binding activity. The amount of full-length FLNA determined in the first well is subtracted from the total amount of FLNA species determined in the second well to obtain the amount of approximately 90 kDa FLNA polypeptide fragment present. If desired, the ratio of the approximately 90 kDa FLNA polypeptide fragment species to the amount of full-length FLNA is determined. In this second embodiment, it is preferred to use equal volumes of plasma or serum sample in each well. If unequal amounts of sample are used, the amounts of the numerator or denominator of the ratio are adjusted appropriately so that the resulting ratio is the ratio that would have been determined if equal volumes had been used.

[0077] result Western blotting: An exemplary study was performed in the laboratory of Dr. Haou-Yan Wang at the CUNY School of Medicine (Manhattan, NY). Frozen plasma (1 mL) collected from subjects was removed from a -80°C freezer and placed on ice until completely thawed. The thawed plasma was then divided into ten 100 μL aliquots. 5 μL of plasma was diluted 20-fold with 50 mM Tris-HCl (pH 7.5) and then mixed with 100 μL of SDS-PAGE sample buffer containing 2-mercaptoethanol as a reducing agent. The resulting solution was boiled for 5 min and then cooled to room temperature for SDS-PAGE. 25 μL of solubilized plasma was loaded onto a 7.5% SDS-PAGE, usually in the first left lane, along with a molecular weight marker (5 μL, protein ladder, ThermoFisher Scientific, Corp., Waltham, MA, USA). Protein samples were then size-fractionated under denaturing conditions according to the manufacturer's specifications. Well-separated proteins were electrophoretically transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) [300 mA, 2 h].

[0078] The resulting nitrocellulose membrane was then washed three times (2 min each) with phosphate buffered saline (PBS, pH 7.2) containing 0.1% Tween® 20, followed by blocking for 1 h at room temperature with 10% nonfat milk in PBS containing 0.1% Tween® 20. The blot was washed three times (2 min each) with PBS containing 0.1% Tween® 20 and blocked with a 1:1000 dilution of rabbit monoclonal anti-human pS 2152The membrane was incubated with FLNA antibody [EP2310AY] (ab75978, Abcam® plc, Cambridge, UK) overnight (approximately 18 hours) at 4° C. The membrane was then washed three times (2 minutes each) with PBS containing 0.1% Tween® 20, and then incubated with 1:7500 HRP-conjugated anti-rabbit IgG (pre-adsorbed, Santa Cruz Biotechnology, Inc., Dallas, TX, USA and GE Lifesciences, Chicago, IL, USA, 1:1) at room temperature for 1 hour. The membrane was again washed three times (1 min each) with PBS containing 0.1% Tween® 20, then once (1 min) with distilled water. Immunoreactivity was detected by chemiluminescence (SuperSignal™ chemiluminescence reagent, Pierce / Thermo) and immediately visualized by exposure to X-ray film for 10–30 s (depending on the intensity of the signal). Specific protein bands were quantified by densitometry scanning (GS-800™ calibrated densitometer, Bio-Rad Laboratories). It should be understood that the density values ​​thus obtained are in arbitrary units and are not absolute, since a longer exposure time may result in more proteins being observed.

[0079] Blood samples were coded and assays were performed blinded to the operators performing them. Diagnoses of AD and mild cognitive impairment (MCI) were made individually and independently using PET scanning techniques such as Amyvid®, Vizamyl®, Neuraceq®, and fluorodeoxyglucose assays in conjunction with clinical criteria. The results of these assays are shown in Table 1 below. [Table 1] TIFF2024527899000002.tif177147 *Subject ID = identification number or letters assigned to the subject at the time of sample collection, Age = subject's age at the time of collection, -- = unknown age, DX = independent diagnosis, AD = Alzheimer's disease, AMC = age-matched controls, MCI = mild cognitive impairment, YCI = cognitively normal young; 90kD = density measurement of the phosphorylated band at approximately 90kD, 280kD = density measurement of the phosphorylated band at approximately 280kD, 90 / 280 = ratio of density measurements.

[0080] Similar density scans were performed for albumin and GAPDH present in the serum of each subject and are shown in the exemplary images in Figures 1, 2, and 3 and in the two right-hand columns of Table 1. The respective density values ​​of the approximately 90 kDa FLNA polypeptide fragment, albumin, GAPDH, and the approximately 280 kDa FLNA protein from serum samples of AD subjects, as well as the density values ​​of the same proteinaceous material from the serum of age-matched controls, are shown in Figure 15. As can be seen, the albumin and GAPDH levels are substantially similar in the AD subjects and the age-matched non-AD controls. In contrast, the respective levels of the approximately 90 kDa FLNA polypeptide fragment and the approximately 280 kDa FLNA protein differ between previously diagnosed AD and previously non-AD subjects. Thus, the density of the ∼90 kDa FLNA polypeptide fragment differs by a factor of ∼10 between subjects with AD and age-matched controls. The density values ​​for the ∼280 kDa FLNA protein appear to differ by several thousand-fold, but this is because the ∼280 kDa FLNA protein is not found in AD subjects and an artificial value of 50 has been used for these data.

[0081] Research is currently underway to determine whether the approximately 280 kDa FLNA protein is truly absent or whether its presence cannot be confirmed by identifying antibodies by some currently unknown means. If the approximately 280 kDa FLNA protein is in fact absent from a subject's serum or plasma, its absence could also be a biomarker that indicates that the subject has Alzheimer's disease. The density ratios of albumin, GAPDH, and the FLNA polypeptide fragment of about 90 kDa to the FLNA protein of about 280 kDa are shown in Figures 16A, 16B, and 16C, respectively. These ratios of density values ​​shown in both Figures 16A and 16B were indicative of the Alzheimer's disease status of AD subjects. However, as can be seen, the ratio values ​​are relatively similar for all subject types, making the distinction between each relatively difficult but possible. In contrast, in Figure 16C, it can be seen that the difference between AD subjects (AD and MCI-AD) and non-AD subjects (AMC and MCI-nonAD) is more than about 100-fold, making it easy to distinguish between AD and non-AD subjects. The data for cognitively normal young (YCI) subjects was widely variable, calling into question the accuracy of the diagnosis. Fortunately, these subjects are unlikely to have AD.

[0082] Altogen Labs (Austin, TX, USA) Western Blot (WB) Procedure: One microliter of plasma from the old sample was diluted with 50 mM Tris (pH 7.5) and then mixed with Laemmli SDS-PAGE sample buffer containing 2-mercaptoethanol as a reducing agent as described previously. The resulting solution was boiled for 5 min and then cooled to room temperature for SDS-PAGE. Samples were loaded onto a 7% SDS-PAGE, usually in the first left lane, along with a molecular weight marker (10 mL, protein ladder, ThermoFisher Scientific). Protein samples were then size-fractionated under denaturing conditions according to the manufacturer's specifications. Well-separated proteins were electrophoretically transferred to a nitrocellulose membrane [300 mA, 2 h]. The resulting membranes were then washed three times (5 min each) with phosphate-buffered saline (PBS, pH 7.2) containing 0.1% Tween® 20, followed by blocking with 5% nonfat milk in PBS containing 0.1% Tween® 20 for 1:15 h at room temperature. Blots were washed three times (5 min each) with PBS containing 0.1% Tween® 20, followed by incubation with anti-pS2152FLNA antibody [EP2310AY] (ab75978, Abcam®) at 1:1000 dilution or pan-FLNA 1:2500 (Santa Cruz) at 4°C overnight (approximately 18 h). The membranes were then washed three times (5 min each) with PBS containing 0.1% Tween® 20, followed by incubation with 1:7500 HRP-conjugated anti-rabbit IgG (pre-adsorbed, Santa Cruz Biotechnology and GE Lifesciences, 1:1) for 1 h at room temperature. The membrane was again washed three times (5 min each) with PBS containing 0.1% Tween® 20, then once (1 min) with distilled water. Immunoreactivity was detected by chemiluminescence (SuperSignal™ chemiluminescence reagent, Pierce / Thermo) and immediately visualized by exposure to X-ray film for 30 s–2 min (depending on the intensity of the signal). Specific protein bands were quantified by densitometric scanning (GS-800 calibrated densitometer, Bio-Rad Laboratories).

[0083] The details of the reagents are shown in the table below: [Table 2]

[0084] Using the protocol described above, Altogen Labs assayed four previous samples and four additional samples. The Alzheimer's disease status of each subject had been previously determined (Indept.DX) and was blinded to the people performing the assay. More limited assay results are shown below in Table 2. [Table 3] *Indept.DX = independent diagnosis, Assay DX = diagnosis by this assay

[0085] A comparison of the results from each group is shown below in Table 3 for the four replicates. [Table 4] Although the density values ​​obtained for the two phosphorylated proteins differ between the two assays, it is easy to see that similar values ​​were obtained using the ratio between the Western blot densities of the two phosphorylated proteins, leading to the same conclusions about the Alzheimer's disease status of the subjects from whom the samples were taken. This similarity of results indicates the robust nature of this assay, which uses the density ratio of two phosphorylated FLNA-associated protein species, rather than the densities of the individual phosphorylations.

[0086] Ultrafiltration An exemplary study was performed in the laboratory of Dr. Haou-Yan Wang at the CUNY School of Medicine (Manhattan, NY). The plasma samples were diluted 1:40 with Tris buffer, pH 7.4, and separated into two portions. One portion was ultrafiltered with a Nanosep® 100K OMEGA™ (P / N OD100C33) centrifugal filter (PALL Corp., Ann Arbor, MI). This ultrafiltration device allows the passage of molecules in the filtrate with a molecular weight of approximately 100 kDa or less. The resulting filtrate and the unfiltered portion (retentate) were separately contacted with the walls of a Reacti-Bind™ NeutrAvidin™ high-binding capacity coated 96-well plate coated with a biotinylated antibody that specifically reacts with an avidin-labeled rabbit monoclonal antibody against phosphorylated serine 2152 of FLNA as a capture molecule. The captured pS 2152 The approximately 90 kDa FLNA polypeptide was detected using a mouse anti-phosphoserine monoclonal antibody [NeuroMab (UC Davis / NIH): Cat#: 73-292] and was isolated from the captured pS 2152-FLNA is a mouse monoclonal IgG specific for an epitope mapping between amino acid residues 9-27 near the N-terminus of the FLNA protein. 2a The antibodies were detected using the antibody (SC-17749, Santa Cruz Biotechnology, Inc.). The relative amounts of each immunoreactant were determined by exciting the fluorescent label after reaction with the respective FITC-labeled anti-mouse antibodies, and measuring and plotting the relative fluorescence intensity. The results of this test are shown in Figure 11.

[0087] Non-phosphorylated specific receptor A study was performed using a commercially available phospho-specific polyclonal rabbit antibody (TA325463) (OriGene, Inc.) that immunoreacts with an epitope containing the phosphorylated serine residue at position 2152 of FLNA. A second study used a mouse monoclonal antibody (Antibody A) raised against an immunogenic polypeptide containing an amino acid residue sequence containing phosphorylated serine at position 2152 of FLNA and native FLNA sequence on either side of that residue. A 10-mer peptide corresponding sequentially to positions 2148 to 2157 of the human FLNA UniProtKB / Swiss-Prot database P21233 sequence was used as the immunogenic sequence. The serine residue at position 2152 of the peptide was phosphorylated. A cysteine ​​amide residue was added to the C-terminus of the peptide and used for conjugation with maleimide-activated keyhole limpet hemocyanin (KLH) as an immunogenic carrier for the immunogen. From the immunization, two mouse hybridomas and two different monoclonal antibodies (A and B) were prepared. The precise specificities of these molecules have not yet been identified. The first monoclonal [and hybridoma] (A) was used in the studies described below.

[0088] Monoclonal antibody A is of the IgG2 type. These antibodies specifically immunoreact with their immunizing peptide and the approximately 90 kDa FLNA polypeptide fragment in Western blot (WB) assays. They are not immunoreactive with the phosphorylated approximately 280 kDa full-length FLNA protein bound by OriGene rabbit polyclonal phosphospecific antibodies TA313881 and TA325463 in either Western blot or ELISA assay formats. Monoclonal antibody B is of the IgM type. These antibodies specifically immunoreact with their immunizing peptide and the approximately 90 kDa FLNA polypeptide fragment in Western blot assays. They are not immunoreactive with the phosphorylated approximately 280 kDa full-length FLNA protein in Western blot format, but they do specifically bind to the full-length 2152 phosphorylated FLNA protein in ELISA format. We believe that these antibodies have unique properties that distinguish them from all other antibodies against the neighboring region of FLNA S2152 in that they only recognize the FLNA polypeptide fragment of approximately 90 kDa and not the full-length FLNA protein in WB assays, which is unpredictable since the epitope is present in both FLNA proteoforms, the FLNA fragment of approximately 90 kDa and the full-length FLNA protein of 280 kDa.

[0089] Samples were numbered and assayed with each antibody. Sample #88 from Table 1, taken from a known AD patient, was included as a positive control sample in each assay. Each antibody was used to identify the presence of the approximately 90 kDa FLNA polypeptide fragment and / or full-length FLNA after SDS gel separation and visualization as described above. The results of this study are shown in Figures 12A and 12B. As can be seen, both antibodies bound to the 90 kDa FLNA polypeptide fragment, but only the phospho-specific rabbit polyclonal antibody bound to both the 90 kDa FLNA polypeptide fragment and the full-length 280 kDa FLNA protein. Both Antibody A and Antibody B accurately identified AD patients and clearly distinguished them from cognitively normal young (YCI) and elderly control (EC) subjects.

[0090] Prognostic Assays The contemplated assays may also be used to prognostically assay the efficacy of treatment in patients presumed to have Alzheimer's Disease (AD). Drugs currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of AD include the monoclonal antibody aducanumab, marketed as Aduhelm™. A small molecule drug currently in clinical trials that has recently shown some efficacy in small-scale clinical trials is a patented compound formerly known as PTI-125, now known as simufilam, whose structural formula is shown below: [ka]

[0091] In accordance with contemplated prognostic methods, a living human patient is treated with a therapeutic composition containing an anti-AD effective amount of a drug (therapeutic compound such as simufilam or aducanumab) or a pharma- ceutically acceptable salt of the drug. Exemplary salts useful for contemplated compounds include, but are not limited to, sulfate, bisulfate, hydrochloride, hydrobromide, acetate, adipate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanesulf ... Salts of the carboxylate group include sodium, potassium, magnesium, calcium, aluminum, ammonium, and many substituted ammonium salts. For a list of commonly used pharma-ceutically acceptable acids and bases that form pharma-ceutically acceptable salts with pharmaceutical compounds, see Berge, J. Pharm. Sci. 1977 68(1):1-19.

[0092] The amount of the about 90 kDa FLNA polypeptide fragment present in a serum or plasma sample preparation taken from the blood of a living patient before and after treatment is determined, and the amount of the about 90 kDa FLNA polypeptide fragment determined later is significantly less than the amount of the about 90 kDa FLNA polypeptide fragment determined earlier, indicating that the drug is binding to its target and is effective in ameliorating the patient's condition. A small Phase 2a clinical trial utilizing the proposed prognostic assay, along with other methods, was first reported in the fall of 2019. Results of this trial were subsequently reported at Clinical Trials on Alzheimer's Disease (CTAD), held December 4-7, 2019, San Diego, CA, and published by Wang et al. in the February 7, 2020 issue of J Prev Alzheimers Dis. Use of the assay disclosed herein is not shown or discussed in that paper. Briefly, the study included 13 patients with mild to moderate Alzheimer's disease aged 50-85 years, Mini-Mental State Examination score ≥16 and ≤24, and cerebrospinal fluid total tau / Aβ42 ratio ≥0.30. Patients were administered a pharmaceutical composition in tablet form for oral administration containing 100 mg of simufilam (PTI-125) twice daily for 28 consecutive days. The study was conducted at five sites in the United States under an Investigational New Drug (IND) application.

[0093] Consistent with the drug's mechanism of action and preclinical data, simufilam (PTI-125) reduced cerebrospinal fluid biomarkers of Alzheimer's disease pathology, neurodegeneration, and neuroinflammation from baseline to day 28. All patients demonstrated biomarker responses to simufilam. Total tau, neurogranin, and neurofilament light chain were reduced by 20%, 32%, and 22%, respectively. Phosphorylated tau (pT181) was reduced by 34%, indicating that this compound inhibits Aβ expression via the α7 nicotinic acetylcholine receptor. 42 We demonstrated that cerebrospinal fluid biomarkers of neuroinflammation (YKL-40 and inflammatory cytokines) were reduced by 5-14%. Biomarker effects were similar in plasma. Aβ 42 was slightly increased. Low Aβ 42 This is a desirable result, since α7 is indicative of Alzheimer's disease. This increase indicates that the compound is blocking Aβ receptors at the α7 nicotinic acetylcholine receptor. 42This is consistent with a 1,000-fold decrease in the femtomolar binding affinity of

[0094] Biomarker reductions were at least p ≤ 0.001 by paired t-test. Target binding was indicated by conformational change of Filamin A from abnormal to native in lymphocytes, from 93% abnormal on day 1 to 40% on day 28. Results showed that Filamin A binds to α7 nicotinic acetylcholine receptors and Toll-like receptor 4, and Aβ 42 The α7 nicotinic acetylcholine receptor and its complex with CD14 were all significantly decreased by treatment. The drug was safe and well tolerated in all patients. The plasma half-life was 4.5 hours, and drug accumulation was approximately 30% on day 28 compared to day 1. The presence and amount of the approximately 90 kDa FLNA polypeptide fragment in the plasma of each patient was also determined separately on days 0, 14, and 28 of the study. The assay results for the group of 13 patients showed a decrease in the amount of the 90 kDa FLNA polypeptide fragment in plasma on days 14 and 28 of the study, respectively, with P<0.01. These results, shown graphically in Figure 13, indicate that the treatment appears to be effective and are consistent with more standard assays.

[0095] The drug (therapeutic compound) is usually administered multiple times over the course of a month and may continue for the rest of the patient's life. The treatment regimen illustratively consists of a tablet containing 100 mg of simufilam (PTI-125) administered twice daily for 28 days. Comparison of the presence and amount of 90 kDa FLNA polypeptide is typically performed multiple times each month for the first month to about 12 months, and may be performed less frequently after an effective dosage and administration regimen has been determined. The foregoing description and examples are intended to be illustrative and not limiting. Further variations within the spirit and scope of the invention are possible and will readily occur to those skilled in the art.

Claims

**Claim 1** A method for assisting in the determination of whether a living human subject has Alzheimer's disease (AD), the method comprising detecting the presence of an approximately 90 kDa polypeptide fragment of filamin A (FLNA) protein in an aqueous serum or plasma sample preparation comprising a serum or plasma sample collected from the living human subject, wherein the serum or plasma sample collected from the living human subject may contain the FLNA protein, the FLNA protein having a molecular weight of approximately 280 kDa and comprising an amino-terminal actin-binding portion bound to 24 immunoglobulin-like repeat domains (IgFLNa-1 to IgFLNa-24) called repeats 1 to 24 from the amino terminus to the carboxy terminus, the approximately 90 kDa FLNA polypeptide fragment comprising the amino acid residue sequence of FLNA containing repeats IgFLNa-16 to IgFLNa-23, and the presence of the approximately 90 kDa FLNA polypeptide fragment in the sample assisting in the determination of whether the living human subject has AD. **Claim 2** A method for assisting in the determination of whether a living human subject has Alzheimer's disease (AD), the method comprising determining the ratio of an approximately 90 kDa polypeptide fragment of filamin A (FLNA) to a second proteinaceous substance present in an aqueous serum or plasma sample preparation comprising a serum or plasma sample collected from the living human subject, the method comprising the following steps: a) separating the proteinaceous substances present in the aqueous serum or plasma sample preparation, if present, into at least two portions: a first portion (A) containing the approximately 90 kDa FLNA polypeptide and a second portion (B) containing the second proteinaceous substance; b) determining the relative amounts of the approximately 90 kDa FLNA polypeptide and the second proteinaceous substance to determine the ratio of the two amounts, and assigning an arbitrary value of one-hundredth of a quantifiable amount to the denominator or numerator when either amount of A and / or B is a small, non-quantifiable amount such that zero does not exist in the denominator or numerator; and c) determining whether the obtained ratio is within a range of values of a predetermined A / B ratio for the approximately 90 kDa FLNA polypeptide and the second proteinaceous substance taken from subjects known to have AD and subjects known not to have AD, thereby assisting in determining whether the human subject had AD at the time of blood collection; A method comprising. **Claim 3** A method for assisting in determining that a living human subject has Alzheimer's disease (AD), the method comprising determining a ratio of an approximately 90 kDa polypeptide fragment of filamin A (FLNA) to filamin A (FLNA) in an aqueous serum or plasma sample preparation comprising a serum or plasma sample taken from the living human subject, the method comprising the following steps: a) separating the proteinaceous substances present in the aqueous serum or plasma sample preparation, if present, into at least two portions, a first portion comprising the approximately 90 kDa FLNA polypeptide, if present, and a second portion comprising the approximately 280 kDa FLNA, if present; b) contacting the separated serum or plasma sample preparation portions with paratope-containing receptor molecules that immunoreact with the approximately 90 kDa FLNA polypeptide and the approximately 280 kDa FLNA, or both if present, to form a reaction mixture; c) maintaining the reaction mixture for a time sufficient for the paratope-containing receptor molecules and, if present, the approximately 90 kDa FLNA polypeptide and / or, if present, the approximately 280 kDa filamin A, FLNA to form immunoreaction product conjugates A and B, respectively; d) detecting and quantifying, if any, the relative amounts of the immunoreaction product conjugates A and B; and e) determining the A / B ratio by assigning an arbitrary value of approximately 1 / 100 of a quantifiable amount such that zero does not exist in the denominator or numerator for a small, non-quantifiable amount of either A and / or B, and if the A / B ratio is from approximately 10 to approximately 2000, this subsidiarily indicates that the living human subject was probably AD at the time of sample collection, and if the A / B ratio is from approximately 0.005 to approximately 5, this subsidiarily indicates that the living human subject was probably not AD at the time of sample collection; A method comprising. **Claim 4** A system for assaying the possibility of the presence of Alzheimer's disease (AD) in a living subject using an aqueous serum or plasma sample preparation containing a serum or plasma sample collected from the living subject, comprising: a) A solid support having an assay surface coated with a paratope-containing capture receptor molecule that immunoreacts with an epitope present in a polypeptide fragment of filamin A (FLNA) of about 90 kDa to form an immune complex, wherein the FLNA protein has a molecular weight of about 280 kDa and contains an amino-terminal actin-binding portion bound to 24 immunoglobulin-like repeat domains (IgFLNa) called repeats 1 to 24 (IgFLNa-1 to IgFLNa-24) in the direction from the amino terminus to the carboxy terminus, and the about 90 kDa FLNA polypeptide fragment contains the amino acid residue sequence of FLNA containing repeats IgFLNa-16 to IgFLNa-23; b) A container holding a first detection receptor molecule that binds to the captured 90 kDa FLNA polypeptide fragment to form a capture complex; and c) A label for detecting the presence of the capture complex. A system comprising the above.

5. A method for assisting in determining the prognosis of treatment of a living human subject presumed to have Alzheimer's disease (AD) with a therapeutic compound or a pharmaceutically acceptable salt of the therapeutic compound, comprising the following steps: a) Determining a first amount of a polypeptide fragment of filamin A (FLNA) of about 90 kDa present in a first aqueous serum or plasma sample preparation containing a serum or plasma sample collected from the living human subject; c) At a time point of at least about one month after the start of the treatment, determining a second amount of a polypeptide fragment of FLNA of about 90 kDa in a second aqueous serum or plasma sample preparation from the human subject; and d) Comparing the amounts of the polypeptide fragment of about 90 kDa of FLNA present in the sample preparations of serum or plasma collected from the blood of the living patient before and after the treatment, wherein a significantly lower amount determined later than the amount determined earlier assists in determining the prediction of benefit from the use of the treatment for the patient from whom the sample was collected. A method comprising the above steps.

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