WO2026018204A1 - Method for the quantification of plasma amyloid-beta biomarkers in alzheimer's disease - Google Patents

Abstract

Provided herein is a method of detecting an amyloid peptide in a patient sample, including exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide; and analyzing the free amyloid peptide with a mass spectrometer.

Description

METHOD FOR THE QUANTIFICATION OF PLASMA AMYLOID-BETA BIOMARKERS IN ALZHEIMER’S DISEASE

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/672,952, filed July 18, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under AG083874, AG066468, and AG025204 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

[0003] Provided herein are methods for detection of biomarkers, including in nonlimiting embodiments amyloid peptides, in patient samples, for example in plasma samples.

Description of Related Art

[0004] Brain amyloid [3 (A|3) depositon is a pathological hallmark and diagnostic criterion of Alzheimer's disease (AD). Following the recent approval of anti-A|3 monoclonal anbody therapies by the United States Food and Drugs Administration, the importance of reliable yet accessible biomarkers in clinical settings has become increasingly essential. Currently, the most widely used biomarkers for assessing A|3 deposition are positron emission tomography (PET) imaging of A|3 plaques, and cerebrospinal fluid (CSF) measurements of A[342/40 peptide ratio via immunoassays. However, these methods are limited by their high costs, invasiveness, and lack of widespread availability, which restrict their use in routine clinical assessments and for drug development purposes.

[0005] Accordingly, there is a need in the art for new methods of detecting peptides in patient samples.

SUMMARY OF THE INVENTION

[0006] Provided herein is a method of detecting an amyloid peptide in a patient sample, including exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide; and analyzing the free amyloid peptide with a mass spectrometer.

[0007] Also provided herein is a binding assay buffer including tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D- maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

[0008] Also provided herein is a method of isolating an amyloid peptide in a patient sample, including exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; and eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide.

[0009] Further non-limiting embodiments are set forth in the following numbered clauses:

[0010] 1. A method of detecting an amyloid peptide in a patient sample, comprising: exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide; and analyzing the free amyloid peptide with a mass spectrometer.

[0011] 2. The method of clause 1 , wherein the assay binding buffer comprises tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D- maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

[0012] 3. The method of clause 1 or clause 2, wherein the surfactant is a non-ionic surfactant.

[0013] 4. The method of any of clauses 1 -3, wherein the non-ionic surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

[0014] 5. The method of any of clauses 1 -4, wherein the isothiazolone compound comprises isothiazolone chloride.

[0015] 6. The method of any of clauses 1 -5, wherein the binding reagent comprises an antibody for the amyloid peptide.

[0016] 7. The method of any of clauses 1 - 6, wherein the binding reagent further comprises a magnetic bead conjugated to the antibody. [0017] 8. The method of any of clauses 1 -7, wherein the antibody comprises an antibody for amyloid beta (A|3).

[0018] 9. The method of any of clauses 1 -8, wherein the washing step comprises washing the immunoprecipitated amyloid peptide with the assay binding buffer.

[0019] 10.The method of any of clauses 1 - 9, The method of claim 1 , wherein the washing step further comprises washing the immunoprecipitated amyloid peptide with phosphate-buffered saline.

[0020] 1 1. The method of any of clauses 1 -10, wherein the washed, immunoprecipitated amyloid peptide is eluted with a buffer comprising an alpha-cyano- hydroxycinnamic acid matrix and trifluoroacetic acid.

[0021] 12. The method of any of clauses 1 -1 1 , wherein the sample is a blood sample.

[0022] 13. The method of any of clauses 1 -12, wherein the sample is a plasma sample.

[0023] 14. The method of any of clauses 1 -13, wherein the amyloid peptide is A[3-

38, A|31 -39, Ap1 -40, Ap1 -41 , Ap1 -42, and/or APP669-71 1 .

[0024] 15. The method of any of clauses 1 -14, wherein the amyloid peptide is A|31 -

40 and Ap1-42.

[0025] 16. The method of any of clauses 1 -15, wherein the mass spectrometer is a matrix-assisted laser desorption/ionization-time of flight mass spectrometer.

[0026] 17. A binding assay buffer comprising tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D-maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

[0027] 18. The binding assay buffer of clause 17, wherein the surfactant is a nonionic surfactant.

[0028] 19. The binding assay buffer of clause 17 or clause 18, wherein the nonionic surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

[0029] 20. The binding assay buffer of any of clauses 17-19, wherein the isothiazolone compound comprises isothiazolone chloride.

[0030] 21 . The binding assay buffer of any of clauses 17-20, further comprising a binding reagent for an amyloid peptide.

[0031] 22. The binding assay buffer of any of clauses 17-21 , wherein the binding reagent further comprises a magnetic bead conjugated to the binding reagent. [0032] 23. The binding assay buffer of any of clauses 17-22, wherein the binding reagent is a binding reagent for amyloid beta (A|3).

[0033] 24. A kit comprising one or more containers holding tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D- maltoside, n-noyl-[3-D-thiomaltoside, 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol, and isothiazolone chloride.

[0034] 25. A method of isolating an amyloid peptide in a patient sample, comprising: exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; and eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows The PA[3 V2.0 assay protocol according to non-limiting embodiments described herein, including a simplified sample preparation procedure with only a single round of IP, saving time and resources.

[0036] FIG. 2 shows MALDI-TOF mass spectra of plasma A|3 peptides replicates utilizing the PA[3 V1 .0 assay procedure, comparing five different buffers or detergents using single IP procedure; 10% N4PE CSF diluent (PA[3 V2.0 assay), 10% SuperBlock, 10pg/ml TruBlock, 0.5% TritonWO, and 0.5% Tween20. Representative spectra from each experiment are presented. Interference peaks were consistently observed at 5771.1 m/z and 7746.8 m/z across all assays. Additionally, another interference peak at 6631 .0 m/z was consistently noted in all assays except the PA[3 V1 .0 assay. (B) Upon magnification in the range of 4000-4850 m/z, the theoretical m/z values of peptides were as follows: 4132.6 m/z for A|31— 38, 4144.7 m/z for A[33-40, 4231.8 m/z for A|31-39, 4330.9 m/z for Ap1-40, 4515.1 m/z for A[3 2, and 4689.4 m/z for APP669-71 1 . Ap 1 -38 IS at 4160.7 m/z, A|31 -40 IS at 4383.3 m/z, and Ap 1 - 42 IS at 4569.3 m/z were utilized as internal standards for the normalization of mass spectra. Notably, an interference peak was detected at 4153.4 m/z in samples processed using 10% SuperBlock, 10pg/ml TruBlock, 0.5% TritonWO, and 0.5% Tween20.

[0037] FIGS. 3A-3C show MALDI-TOF mass spectra of Ap peptides derived from plasma replicates utilizing the PA[3 V1.0 assay, 10% N4PE CSF diluent (PA[3 V2.0 assay) and PA[3 V1.0 assay with 11P. Representative spectra from each experiment are presented. Interference peaks were consistently observed at 5771.1 m/z and

7746.8 m/z across all assays. Additionally, another interference peak at 6631.0 m/z was consistently noted in all assay formats except the PA[3 V1.0 assay. Interference peaks at 3200 m/z to 3500 m/z and 6432.4 m/z were observed in PA[3 Vi.O assay with 1 1P only. Upon magnification to the range of 4000-4850 m/z, the theoretical m/z values of peptides are as follows: 4i32.6 m/z for A|31— 38, 4i44.7 m/z for A[33-40,

4231.8 m/z for Ap1-39, 4330.9 m/z for A|31-40, 45i5.i m/z for Ap1-42, and 4689.4 m/z for APP669-71 1 . Ap 1 -38 IS at 4160.7 m/z, A|31 -40 IS at 4383.3 m/z, and Ap 1 - 42 IS at 4569.3 m/z were utilized as internal standards for the normalization of mass spectra. Notably, an interference peak was detected at 4153.4 m/z in samples processed using PA[3 V1 .0 assay with 1 1P but not in the other assays. (B) S/N ratios were compared across three assays in triplicates, with asterisks indicating significant differences (*p < 0.05, **p < O.Oi) as determined by the Wilcoxon Rank Sum test. (C) The averages and standard deviations of the S/N ratios are listed.

[0038] FIGS. 4A-4B show the calibration curves were generated using Api— 40 concentrations of 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml, and Opg/ml, and Api— 42 concentrations of 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml, 6.25pg/ml, 3.125pg/ml, and Opg/ml, normalized with Api — 38 IS. (B) The matrix effect recovery was assessed across three different concentrations, each with three replicates, utilizing Api— 38 IS normalization. (C) The IP recovery was measured through the SIMOA assay. (D) The relationship between plasma dilution and normalized intensity of the PA[3 V1.0 and PA[3 V2.0 assays. Three replicates were performed for each volume. Both Api — 40 and Api— 42 were normalized by Api— 38 IS. (E) The S/N ratios of plasma samples with various volumes were compared between PA[3 V1 .0 and PA[3 V2.0 assays for Api — 40 and A i — 42.

[0039] FIG. 5 shows the calibration curves were generated using Api— 40 concentrations of 400pg/ml, 200pg/ml, 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml, and Opg/ml, and Api— 42 concentrations of 100pg/ml, 50pg/ml, 25pg/ml, 12.5pg/ml, 6.25pg/ml, 3.125pg/ml, and Opg/ml, normalized with Api— 40 IS and Api— 42 IS. (B) The matrix effect recovery was assessed across three different concentrations, each with three replicates, utilizing Api — 40 IS and Api — 42 IS normalization.

[0040] FIGS. 6A-6C show box and whisker plot categorizes the ADRC cohort into clinically assessed probable AD and normal control groups, analyzed using the Wilcoxon Rank Sum test, with p-values indicated. N represents the sample size. (B) Box and whisker plot shows the AGUEDA cohort split into A|3 PET positive and PET negative groups, analyzing three assay formats: PA[3 V1 .0 assay A|31 — 42/A|31 — 40, PA[3 V2.0 assay A|31 — 42/A|31 — 40, and PA[3 V2.0 assay A|31 — 42/A|31 — 40 normalized with A|31 — 40 IS and A|31 — 42 IS. Differences between groups were evaluated using the Wilcoxon Rank Sum test, with p-values provided. (C) Box and whisker plot dividing the AGUEDA cohort into CL positive, CL transition, and CL negative groups, with differences assessed using the Kruskal-Wallis test and p-values noted.

[0041] FIGS. 7A-7D show the correlation between the PA[3 V1 .0 assay and PA[3 V2.0 assay, normalized using A|31 — 38 IS, was illustrated for the ADRC (A) and AGUEDA (B) cohorts. Spearman correlation was employed to evaluate the strength of the correlation between A|3 peptide measurements across the two assays. Additionally, A|31 — 40 and A|31 — 42, normalized using A|31 — 40 IS and A|31 — 42 IS in the PA[3 V2.0 assay, were further assessed for correlation with their respective A|3 peptides in the PA[3 V1.0 assay, normalized using A|31 — 38 IS.

[0042] FIG. 8 shows demographic data for participants. Mean and Standard Deviation are reported for continuous variables. Frequencies and percentages are shown for categorical variables. § AD diagnosis was assessed by clinical diagnosis for ADRC cohort, and A|3 PET neuroimaging for AGUEDA cohort. * P-values were calculated using Wilcoxon Rank Sum or Kruskal-Wallis test for a continuous variable and Fisher’s exact test for a categorical variable, respectively. c[ Two participants had missing data for APOE alleles. The percentage was calculated based on a sample size of 75. Abbreviations: APOE, apolipoprotein E; CDR, Clinical Dementia Rating; MMSE, Mini Mental State Examination; MoCA, Montreal Cognitive Assessment; CL, Centiloid.

DESCRIPTION OF THE INVENTION

[0043] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word "about". In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein, "a" and "an" refer to one or more. [0044] As used herein, the term "comprising" is open-ended and may be synonymous with 'including', 'containing', or 'characterized by'. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps, and those that do not materially affect basic and novel characteristic(s). The term "consisting of" excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments "comprising" one or more stated elements or steps also include but are not limited to embodiments "consisting essentially of" and "consisting of" these stated elements or steps.

[0045] As used herein, the term "patient" or "subject" refers to members of the animal kingdom including but not limited to human beings, and "mammal" refers to all mammals, including, but not limited to human beings.

[0046] Provided herein are methods of analyzing patient samples for biomarkers (e.g., peptides or proteins) of interest, in non-limiting embodiments biomarkers relating to Alzheimer's Disease. The methods disclosed herein provide for improved analysis in terms of access to the sample, cost and simplicity of the assay, and improved signal- to-noise ratio.

[0047] Accordingly, in non-limiting embodiments, method of detecting an amyloid peptide in a patient sample is provided. In non-limiting embodiments, the method may include an immunoprecipitation step. In non-limiting embodiments, the immunoprecipitation step may include exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide. In non-limiting embodiments, only a single immunoprecipitation step is conducted. In non-limiting embodiments, the binding reagent may be an antibody for the amyloid peptide, or a fragment thereof (e.g., an ScFV fragment).

[0048] An antigen binding reagent or complexes thereof may be, for example and without limitation, a monoclonal antibody, including fragments, derivatives, or analogs thereof, or complexes thereof, including without limitation: Fab, Fab’, Fv fragments, single chain Fv (scFv) fragments, disulfide stabilized Fv (dsFv) fragments, Fab1 fragments, F(ab’)2 fragments, single domain antibodies, camelized (camelid) antibodies and antibody fragments, humanized antibodies and antibody fragments, and multivalent versions of the foregoing; multivalent binding reagents including without limitation: monospecific or bispecific antibodies, such as dsFv fragments, scFv tandems ((ScFv)2 fragments), diabodies, triabodies, tetrabodies, which typically are covalently linked or otherwise stabilized (e.g., leucine zipper or helix stabilized) scFv fragments, bi-specific T-cell engager (BITE, e.g., a DbTE), di-scFv (dimeric singlechain variable fragment), single-domain antibody (sdAb), or antibody binding domain fragments. Antibody fragments also include miniaturized antibodies or other engineered binding reagents that exploit the modular nature of antibody structure, comprising, often as a single chain, one or more antigen-binding or epitope-binding sequences (e.g., paratope) and, at a minimum, any other amino acid sequences needed to ensure appropriate specificity, delivery, and stability of the composition. [0049] scFv molecules may be manufactured using any suitable technology. Typically, recombinant cells comprising genes for expressing scFv-containing polypeptides are engineered, e.g., according to decades-old methods using any of a variety of publicly- and commercially-available expression systems. Huston J. S., M. Mudgett-Hunter, M. S. Tai et al., "Protein engineering of single-chain Fv analogs and fusion proteins," Methods in Enzymology, vol. 203, pp. 46-88, 1991 ; Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250; G?ciarz A, Ruddock LW. Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv. PLoS One. 2017 Dec 18;12(12):e0189964; Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int J Mol Sci. 2020 Aug 31 ;21 (17):6324; Petrus MLC, Kiefer LA, Puri P, Heemskerk E, Seaman MS, Barouch DH, Arias S, van Wezel GP, Havenga M. A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli. Appl Microbiol Biotechnol. 2019 Nov;103(21 -22):8875- 8888; and Toleikis L, Frenzel A. Cloning single-chain antibody fragments (ScFv) from hybridoma cells. Methods Mol Biol. 2012;907:59-71 ; see, also, www.kbdna.com/cloning-scfv.

[0050] The antigen binding molecules described herein, may include, at their core paratopes formed from VL and VH polypeptides, that are defined by three CDR's (typically loops), CDR1 , CDR2, and CDR3, which for VH peptides may be termed HCDR1 , HCDR2, and HCDR3, respectively, and which for VL peptides may be termed LCDR1 , LCDR2, and LCDR3, respectively, each of which are flanked by, and separated by framework (e.g., joining or scaffold) amino acid sequences that space apart and support the CDRs, and which may differ from antibody-to-antibody, and which may be "humanized" to minimize antigenicity when administered to a human patient. In nature, HCDR3 and LCDR3 are typically the most variable of the CDRs, contributing significantly to antibody specificity. Various methods may be used to identify the precise limits of each CDR, but the sequences provided herein can be evaluated by any suitable method to determine the CDRs.

[0051] Binding reagents for biomarkers (e.g., peptides or proteins) of interest, such as, prion proteins, tau proteins (including total tau and/or phosphorylated tau), synuclein proteins (such as alpha-synuclein), amyloid proteins (such as amyloid beta (A|3)) and/or the like are known in the art, for example as described in van Dyck, Anti- Amyloid-p Monoclonal Antibodies for Alzheimer’s Disease: Pitfalls and Promise, Biol. Psych 2017 83(4): 31 1 -319) and Wu et al., The FDA-approved anti-amyloid-[3 monoclonal antibodies for the treatment of Alzheimer’s disease: a systematic review and meta-analysis of randomized controlled trials, Eur. J. Med. Res. 2023, 28: 544 (the contents of which are incorporated herein by reference in their entirety), and/or are commercially available from, for example and without limitation, Cell Signaling Technology, Abeam, NovusBio, ACROBiosystems, and ThermoFisher Scientific.

[0052] In non-limiting embodiments the binding reagent (e.g., antibody or fragment thereof) may be conjugated to a substrate that may, for example, increase the ability to isolate the bound protein. In non-limiting embodiments, the substrate may be a bead, for example an agarose bead or a magnetic bead. Suitable substrates may be modified as is known in the art, for example with coatings that properly configure the binding reagent, that improve signaling during various blotting procedures, and/or that improve adhesion.

[0053] In non-limiting embodiments, the assay binding buffer may include one or more of tris(hydroxymethyl)aminomethane hydrochloride (e.g, Tris-HCI), sodium chloride (e.g., in a solution, such as saline), n-dodecyl-[3-D-maltoside, n-noyl-[3-D- thiomaltoside, a surfactant, and/or an isothiazolone compound.

[0054] Isothiazolones (also referred to as isothiazolinones) are a class of chemicals that include a heterocyclic five-membered ring. In non-limiting embodiments the isothiazolone compound has the formula (CH)2SN(H)CO. In non-limiting embodiments, the isothiazolone compound is isothiazolinone chloride, which is also referred to as 3(2H)-lsothiazolone, 5-chloro-2-methyl-, mixt. with 2-methyl-3(2H)- isothiazolone; 3(2H)-lsothiazolone, 2-methyl-, mixt. contg.; Kathon 886MW; and Kathon 886. [0055] In non-limiting embodiments, the surfactant is a non-ionic surfactant. In nonlimiting embodiments, the surfactant is TritonX-100 (e.g., 2-[4-(2,4,4-trimethylpentan- 2-yl)phenoxy]ethanol). Those of skill in the art will appreciate that other non-ionic surfactants are commercially available and may be used.

[0056] In non-limiting embodiments the isothiazolone compound may be part of a composition added to the assay binding buffer, that may include water, a surfactant (e.g., Triton X-100), and the isothiazolone compound, for example in amounts of, by percent of the composition, 66-90% water, 0.01 -0.1 % surfactant, and 0.005-0.05% isothiazolone compound, all values and subranges therebetween inclusive. In nonlimiting embodiments, the composition including the isothiazolone compound may be included in the assay binding buffer in an amount of 1 % to 20%, 5% to 15%, about 10%, and/or 10%, all values and subranges therebetween inclusive.

[0057] In non-limiting embodiments, the method further includes washing the immunoprecipitated amyloid peptide. Washing may be performed with any suitable solvent and/or buffer. In non-limiting embodiments, the washing is conducted with saline, for example a buffered saline such as phosphate-buffered saline (PBS).

[0058] In non-limiting embodiments, following immunoprecipitation, the immunoprecipitated peptide (and substrate, e.g., beads) may be re-suspended in the assay binding buffer. In non-limiting embodiments, following this resuspension, the immunoprecipitated peptide (and substrate, e.g., beads) may be washed with binding buffer. In non-limiting embodiments, the immunoprecipitated, washed peptide (and substrate, e.g., beads), may be washed with a buffer, such as PBS, for example 1 , 2, 3, 4, 5, or more times. In non-limiting embodiments, following this washing, the immunoprecipitated peptide may be washed with water, for example HPLC-grade water.

[0059] In non-limiting embodiments, the method further includes eluting the washed, immunoprecipitated amyloid peptide (And substrate, e.g., beads), thereby generating free amyloid peptide (e.g., free of the beads). In non-limiting embodiments, the elution may be conducted with one or more solutions, for example, a glycine- containing buffer (e.g., a glycine elution buffer) and/or a-cyano-4-hydroxycinnamic acid matrix (for example, dissolved in acetonitrile, e.g., 50% acetonitrile, and trifluoroacetic acid, (e.g., TA50). In non-limiting embodiments, a multi-step elution is performed. In non-limiting embodiments, a single elution step is performed, and, in non-limiting embodiments, the single elution step is elution with a-cyano-4- hydroxycinnamic acid matrix dissolved in TA50. Suitable elution reagents are commercially available from, for example, Sigma-Aldrich, Bruker, and ThermoFisher Scientific, and may also include (in addition to those identified above, an SDS- containing buffer and/or a urea-containing buffer, which are also commercially available).

[0060] In non-limiting embodiments, the method further includes analyzing the free amyloid peptide, for example with a mass spectrometer. Those of skill in the art will appreciate that various mass spectrometers are commercially available, and that instructions for use for each are available, such that no one mass spectrometer is necessarily required with the methods disclosed herein. In non-limiting embodiments, the mass spectrometer is a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer.

[0061] In non-limiting embodiments, the patient sample is a patient blood sample. In non-limiting embodiments, the sample is a plasma sample.

[0062] In non-limiting embodiments the amyloid peptide is one or more of A[3-38, A|31 -39, A|31 -40, A|31 -41 , A|31 -42, and/or APP669-71 1 . In non-limiting embodiments, the amyloid peptide is A|31 -40 and/or A|31 -42. In non-limiting embodiments, data obtained from the mass spectrometer is analyzed for a ratio of one or more biomarkers of interest relative to one or more other biomarkers of interest. In non-limiting embodiments, the ratio used for the analysis is an amount of A[31 -42 to an amount of Api -40. Amounts of the biomarker of interest may be normalized against another biomarker, for example a standardized biomarker, prior to, during, or after the analysis. [0063] Also provided herein is a binding assay buffer useful for a method described herein. In non-limiting embodiments, the assay binding buffer includes tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride (e.g., in a solution, e.g. saline), n-dodecyl-[3-D-maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

[0064] In non-limiting embodiments, the isothiazolone compound is isothiazolinone chloride, which is also referred to as 3(2H)-lsothiazolone, 5-chloro-2-methyl-, mixt. with 2-methyl-3(2H)-isothiazolone; 3(2H)-lsothiazolone, 2-methyl-, mixt. contg.; Kathon 886MW; and Kathon 886.

[0065] In non-limiting embodiments, the surfactant is a non-ionic surfactant. In nonlimiting embodiments, the surfactant is TritonX-100 (e.g., 2-[4-(2,4,4-trimethylpentan- 2-yl)phenoxy]ethanol). Those of skill in the art will appreciate that other non-ionic surfactants are commercially available and may be used.

[0066] In non-limiting embodiments, the buffer may include a binding reagent, for example one or more of those described herein. In non-limiting embodiments, the binding reagent is an antibody for an amyloid peptide of interest. In non-limiting embodiments, the binding reagent further includes a bead, such as a magnetic bead, conjugated to the binding reagent. In non-limiting embodiments, the antibody is an antibody for amyloid beta (A|3).

[0067] Also provided herein are kits including one or more containers, holding a binding assay buffer or one or more components thereof (e.g., in non-limiting embodiments, tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n- dodecyl-[3-D-maltoside, n-noyl-[3-D-thiomaltoside, 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol, and isothiazolone chloride). In non-limiting embodiments, the kit may further include a binding reagent for a biomarker of interest, such as an amyloid peptide. In non-limiting embodiments, a kit may include instructions for immunoprecipitating, washing, and/or eluting a biomarker of interest, such as an amyloid peptide.

Example

Methods

[0068] This study included plasma samples from two cohorts. For the first cohort, we enrolled participants from the University of Pittsburgh Alzheimer’s Disease Research Center (ADRC) in Pittsburgh, Pennsylvania, USA. The participants in this ongoing study undergo annual clinical evaluation to assess their longitudinal brain health and potential development of cognitive impairment and dementia. Annual evaluations include neuroimaging, cognitive testing, and blood collection for use in plasma biomarker analysis outside of the clinical assessment. Neuropsychological evaluation and diagnoses were established through clinical assessments. The battery of cognitive tests included the Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), and the Clinical Dementia Rating (CDR) scale. The current investigation was a prospective, blinded sub-study where participants were enrolled based on their order of clinical attendance and their informed consent to participate. This involved agreeing to provide an additional tube of blood for the project. The ADRC study was approved by the University of Pittsburgh Institutional Review Board (MODI 9110245-023). [0069] The second cohort was sourced from the Active Gains in Brain Using Exercise During Aging (AGUEDA) project (NCT05186090). Participants were recruited from Granada, Spain, based on their classification as physically inactive and cognitively normal, assessed by the Spanish Telephone Interview for Cognitive Status modified (STICS-M), MMSE, and MoCA. As an outcome, A|3 PET was performed using the [18F] Florbetaben tracer, quantified using standardized uptake value ratio (SUVR) values and the Centiloid (CL) scale. Detailed information on eligibility criteria, participant selection methods, and recruitment procedures, as well as details about the study setting, locations, and data collection, can be found in a comprehensive description provided in the AGUEDA protocol. Prior to enrollment in the AGUEDA trial, participants provided informed consent, and the trial was conducted in accordance with the approval of the Research Ethics Board of the Andalusian Health Service (CEIM/CEI Provincial de Granada; #2317-1X1-19). In this cross-sectional analysis, we focused on the baseline data.

[0070] Researchers were blinded to all participant information until the completion of data acquisition.

[0071] Blood collection and processing procedures

[0072] At the University of Pittsburgh ADRC, blood samples were collected via venipuncture by nurses with extensive clinical experience and trained in ADRC procedures. Blood collection was performed between 9:00 am and 2:00 pm, with the time of last meal recorded. For the AGUEDA cohort, blood samples were collected at 08:00-10:00 am following longer than 8 hours of fasting, at the Virgen de las Nieves University Hospital, Spain.

[0073] Briefly, a 10 and 4 ml Lavender top ethylenediaminetetraacetic acid (EDTA) tube was used to collect whole blood from each participant in the ADRC and AGUEDA cohort, respectively. Following each blood draw, the tubes were promptly inverted 8 to 10 times and subsequently centrifuged at 2000 xg for 10 minutes for the AGUEDA cohort and 15 minutes for the ADRC cohort at 4°C to effectively separate the plasma. The resulting plasma samples were aliquoted into cryovials and frozen at -80 °C until use, following standard guidelines.

[0074] Immunoaffinity enrichment

[0075] Pittsburgh plasma A|3 assay (PA[3) V1 .0

[0076] The PA[3 V1 .0 assay was developed at the University of Pittsburgh based on the method originally described by Nakamura et al. (High performance plasma amyloid-fi biomarkers for Alzheimer’s disease. Nature, 2018. 554(7691 ): p. 249-254). For each sample, 250 pl of binding buffer (100 mM Tris-HCI pH 7.4 [Sigma #T2788- 1 L], 300 mM NaCI [Sigma #S7653-250G], 0.2% w/v n-dodecyl-[3-D-maltoside [DDM; Sigma #D4641-1 G], 0.2% w/v n-nonyl-[3-D-thiomaltoside [NTM; Anatrace #148565- 55-3]) containing 62.4 pg/ml of A|31 — 38 internal standard (IS) (Anaspec #AS-65220), was added to a 1.5 ml Eppendorf Protein LoBind Tube (ThermoFisher #13-698-794), followed by the addition of 250 pl plasma sample. To facilitate direct comparison with the PA[3 V2.0 assay, 100 pg/ml A|31 — 40 IS (Rpeptide #A-1 101-2) and 30 pg/ml A|31 — 42 IS (Rpeptide #A-1102-1 ) were also added to the binding buffer for the evaluation of analytical performance.

[0077] The samples were immunoprecipitated with 10 pl of 50 mg/ml Dynabeads (M-270 Epoxy; ThermoFisher #14301 ) coupled with 5 pg 6E10 anti-A|3 antibody (BioLegend #803003) for 1 hour at 4°C with rotation. The beads were coupled with the antibody following the protocol recommended by the manufacturer. After the IP, the supernatant was discarded, and the beads washed once with 0.5 ml of cold phosphate-buffered saline (PBS, Gibco #2537136). The washed beads were then transferred to a fresh Eppendorf tube using 0.5 ml of cold PBS and eluted with 25 pl of glycine elution buffer (50 mM glycine [pH 2.8, Sigma #G2879-100G], 0.1 % DDM) after removing all liquid. The eluates were collected and transferred to fresh tubes containing 0.5 ml of the binding buffer (without any A|3 ISs) for a second round of IP. Following one hour of rotation at 4 °C, the beads were washed twice with 0.5 ml of cold HPLC-grade H2O (Fisher #7732-18-5) and transferred to a fresh Eppendorf tube by resuspending in 0.2 ml H2O. After complete removal of all liquid through vacuum aspiration, the beads were eluted using 6 pl of 3 mg/ml a-cyano-4-hydroxycinnamic acid matrix (Bruker #8201344) dissolved in TA50 (50% Acetonitrile [Fisher #75-05-8], 0.1 % Trifluoroacetic acid [Alfa Aesar #UN2699], 1 mM ammonium dihydrogen phosphate [Sigma #204005]). The eluate was spotted four times with 1 pl each onto the MALDI target plate (Bruker #8280823) for MS analysis. A schematic illustration of the workflow for this assay is shown in FIG. 1 .

[0078] The PA[3 V1 .0 assay protocol (A) entails two rounds of immunoprecipitation. In contrast, the PA[3 V2.0 assay protocol (B) includes a simplified sample preparation procedure with only a single round of IP, saving time and resources.

[0079] Single IP procedure for detergents and blocking buffer tests [0080] Similar to the first IP step of the PA[3 V1 .0 assay, we prepared 250 pl of the same assay binding buffer, either used as is or supplemented with one of the following detergents or blocking buffers: 10% v/v SuperBlock (Thermo #37535), 10 pg/ml TruBlock (Meridian #A66803H), 0.5% v/v TritonWO (Millipore #648462), 0.5% v/v Tween20 (BioRad #1610781 ), or 10% Quanterix Neurology Plex 4E CSF sample diluent (N4PE CSF diluent [Quanterix #103727]) for different tests.

[0081] This mixture was transferred to a 1 .5 ml Eppendorf Protein LoBind tube with 62.4 pg/ml of A|31— 38 IS, 100 pg/ml of A|31— 40 IS, and 30 pg/ml of A|31— 42. Subsequently, 250 pl of human plasma sample was added to the mixture. The sample was immunoprecipitated with 5 pl of 50 mg/ml Dynabeads coupled with 1.25 pg 6E10 A|3 antibody (BioLegend #803003) for 1 hour at 4°C with rotation. After IP, the supernatant was discarded, and the beads resuspended in 0.5 ml of the assay binding buffer with the corresponding supplement added as appropriate and transferred to a new tube. The beads underwent an additional wash with 0.5 ml of the binding buffer with corresponding supplement, two washes with 0.5 ml of PBS and one wash with 0.5 ml of HPLC-grade H2O. Finally, the beads were transferred to a fresh Eppendorf tube using 0.2 ml of H2O. After removal of all liquid through vacuum aspiration, the beads were eluted using 6 pl of 3 mg/ml a-cyano-4-hydroxycinnamic acid matrix dissolved in TA50. The eluate was spotted four times with 1 pl each onto the MALDI target plate for analysis.

[0082] Screening of buffers and blockers for the PA[3 V2.0 assay

[0083] We evaluated the effects of several buffer systems and heterophilic blocking agents for the PA[3 V2.0. These included the 10% N4PE CSF diluent from Quanterix, the 10% v/v SuperBlock, 10 pg/ml TruBlock, 0.5% v/v TritonWO and 0.5% v/v Tween20. The results from the PA[3 V2.0 assay were compared to those obtained using the PA[3 V1 .0 assay.

[0084] MALDI-TOF MS

[0085] After sample spotting, the MALDI target plate was air dried and then loaded into a benchtop MALDI- TOF mass spectrometer, Microflex LT (Bruker Daltonics), equipped with a 337 nm nitrogen laser to acquire mass spectra. The Microflex LT operated in linear mode with a pulsed positive ion extraction setting, utilizing an attenuator offset of 12%, an attenuator range of 30%, and 63% laser power. An external mass calibration was performed using a peptide calibration mixture consisting of two calibration standards (Bruker #8222570, #8206355). The auto scan function was utilized, acquiring one spectrum for each spot through the combination of ion signals from 2,500 laser shots, resulting in four spectra per sample. A|31 — 38 IS was employed to ensure spectrum quality in the auto scan function. Only spectrum, generated from every 50 shots, with A|31 — 38 IS S/N ratios greater than three were collected. After acquisition, the spectra underwent smoothing using the SavitzkyGolay algorithm with a width of 0.1 mass-to-charge (m/z) and baseline subtraction using the TopHat algorithm. The peak intensity and S/N ratios were measured using FlexControl (v3.4, Bruker Daltonics). Subsequently, ClinPro Tools Software (v2.1 , Bruker Daltonics) was employed for m/z alignment, peak detection, and peak area calculation. [0086] Analytical assessment

[0087] Linearity analysis was conducted using a two-fold serial dilution of an A|3 peptide mixture, starting with concentrations of 400 pg/ml for A|31 — 40 (Anaspec, #AS- 24235) and 10 pg/ml for A|31— 42 (Anaspec, #AS-20276), in 6% bovine serum albumin (BSA)/PBS, diluting up to 64x. The analysis involved six replicates for each dilution, totaling 36 samples, which were evenly processed across two batches. The lower limit of quantification (LLOQ) was established as the lowest concentration measurable with a coefficient of variation (CV) under 20%. The working range was defined as the range from the LLOQ to the highest concentration tested. To evaluate the plasma matrix effect, we assessed the recovery by comparing the results in plasma to those in 6% BSA/PBS at three different concentration levels. Both media were spiked with equal amounts of A|31 — 40 and A|31 — 42 prior to the IP procedures. Recovery was calculated using the formula:

% Reroverv - . x (^P-^sP^iM plasma - P_plas a)

Recovery - x _z p.spiked BSA where P represents the normalized peak area.

[0088] Intra- and inter-assay variability were determined by analyzing samples at three A|3 concentrations levels across five batches, each containing six replicates per concentration.

[0089] The linearity, LLOQ, working range, matrix effect recovery and precision of A|31 — 40 and A|31 — 42 were normalized using either common IS (A|31 — 38 IS) or analyte specific IS (A|31 — 40 IS and A|31 — 42 IS), respectively.

[0090] Plasma dilution linearity [0091] The effect of plasma dilution on normalized intensity for both the PA[3 Vi.O and PA[3 V2.0 assay formats were investigated by testing five separate amounts of a pooled plasma sample (50 pl to 250 pl), with three replicates each. All samples in this test were diluted to 250 pl prior to processing, and A|31 — 40 and A|31 — 42 levels were normalized using the A|31 — 38 IS only.

[0092] Simoa assay for IP recovery assessment

[0093] To quantify the proportion of A|3 peptides retained after the IP procedures, Single Molecule Array (Simoa) assays were utilized. These assays were performed using the Simoa Human Neurology 4-Plex E assay (N4PE) kit from Quanterix (#i03670) on an HD-X analyzer (Quanterix, Billerica, MA, USA). To monitor assay performance, quality control samples at three different concentrations were analyzed at the beginning and end of each assay run. The average %CV for the quality controls was below 5%.

[0094] Mass spectrometric and immunoassay experiments were performed in the Mass Spectrometry facility at the Biofluid Biomarker Laboratory, Department of Psychiatry, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

[0095] Clinical Performance Assessment

[0096] We compared three different A|3 biomarkers: A|31 — 42/A|31 — 40 using the PA[3 Vi.O assay, A|31 — 42/A|31 — 40 using the PA[3 V2.0 assay, and A|31 — 42/A|31 — 40 normalized with the A|31 — 42 IS and the A|31 — 40 IS correspondingly using the PA[3 V2.0 assay. The evaluation of biomarker performance was conducted across the PITT- ADRC based on the clinical assessments for cognitive status (ADRC cohort), and the AGUEDA cohort based on the A|3 PET imaging results (AGUEDA cohort) using CL scales (AGUEDA cohort).

[0097] The assay performance over multiple batches was evaluated using pooled quality control plasma samples at two concentration levels by measuring the A|31 — 40 and A|31 — 42. In both assays, normalization of A|31— 40 and A|31— 42 was conducted using the A|31 — 38 IS. The intra- and inter-assay %CV were determined to be less than i5% for both cohorts.

[0098] Correlation Analysis

[0099] The correlation between the PA[3 Vi.O and PA[3 V2.0 assay formats was evaluated using the normalized peak areas of multiple A|3 biomarkers, including A|31 - 42, A 1-4O, A 1-39, A[33-40, Ap1 -38, and APP669-7ii, across the PITT-ADRC and AGUEDA cohorts. All analytes were normalized using A|31 — 38 as the IS. Additionally, A|31 — 42 and A|31— 40 signals in the PA[3 V2.0 assay format were further normalized using their respective IS; A|31 — 42 IS and A|31 — 40 IS.

[00100] Statistical Analysis

[00101] For participant demographic characteristics, continuous variables were summarized using means and standard deviations, while categorical variables were reported as numbers and percentages. Differences across cohorts for continuous variables were examined using the Wilcoxon Rank Sum test or Kruskal-Wallis test, depending on the number of groups involved. Categorical variables were analyzed using Fisher’s exact tests. For S/N ratio comparison between different assays, Wilcoxon Rank Sum test was used. For clinical assessments, box and whisker plots were generated using clinical assessments, A|3 PET imaging results, and CL scales over the cohorts. Wilcoxon Rank Sum test was used to assess the disease discriminating performance of biomarkers across cohorts based on the clinical assessments or the A|3 PET imaging results. The Kruskal-Wallis test was used to evaluate the difference among the CL scale groups. The Cohen’s d was calculated for multiple assay biomarkers to evaluate the standardized difference between different diagnostic groups. For correlation study, Spearman correlation analysis was conducted to evaluate the strength of the association between A|3 peptide measurements from the two different assays. For all the tests, a p-value less than 0.05 was considered statistically significant. All analyses were performed using R statistical software (version 4.2.1 , R Foundation for Statistical Computing, Vienna, Austria), available at [http://www.r-project.org/]. Results

[00102] Effectiveness of detergents and blocking buffers in reducing IP-MS background

[00103] To streamline the PA[3 V1 .0 assay into a single IP step, we experimented with various supplements in the IP binding buffer to reduce background interference. These included 10% N4PE CSF diluent, 10% SuperBlock, 10 pg/ml TruBlock, 0.5% Triton O, and 0.5% Tween20, were all tested following the Single IP protocol (see Methods section).

[00104] Among the supplements tested, the N4PE CSF diluent demonstrated the best performance, effectively eliminating interference peaks while maintaining the highest S/N ratio. Consistent with the PA[3 V1 .0 assay, the interference peak at 4450 m/z, which often obscures the A|31— 38 and A[33-40 signals, was significantly eliminated with the use of the N4PE CSF diluent but not with the other supplements. Notably, SuperBlock and TruBlock resulted in significantly lower S/N ratios when compared with the PA[3 V1.0 assay. The detergents, on the other hand, showed the lowest S/N ratios for all A|3 peptides. (FIG. 2).

[00105] As a confirmation comparison, the original PA[3 V1 .0 assay and the single IP assay that used the N4PE CSF diluent were compared with the original PA[3 V1 .0 assay configuration with one IP step and no binding buffer supplementation. As shown in the representative spectra (FIG. 3A), supplementing the IP binding buffer with the N4PE CSF diluent resulted in the cleanest spectra. Similar to the PA[3 V1 .0 assay, the interference peaks observed in the PA[3 V1 .0 assay with a single round of IP at 3200 m/z to 3500 m/z, and at 6400 m/z and 6600 m/z, were reduced by using the N4PE CSF diluent. Furthermore, the single round of IP procedure using N4PE CSF diluent achieved a significantly higher S/N ratio, with means of 143.9 for A|31 — 40 and 9.5 for A|31— 42, compared with 72.4 and 5.5 respectively in the PA[3 V1.0 assay, and 23.9 and 1 .6 in the PA[3 V1 .0 assay with one IP (FIGS. 3B-3C). Similar improvements were also observed for other A|3 peptides. In the PAB V1 .0 assay, the S/N ratios were 16.0, 7.8, 4.9, and 3.6 in PA[3 V1.0 assay and 5.5, 0, 1.8, and 2.4 in the PA[3 V1.0 assay with a single IP for A|31— 38, A[33-40, A|31— 39, and APP669-71 1 , respectively. Conversely, in the single IP with N4PE CSF diluent, these ratios improved to 29.8, 13.7, 9.1 , and 7.1 for the same peptides (FIGS. 3B-3C).

[00106] Due to the optimal performance, we selected the single IP with N4PE CSF diluent-supplemented binding buffer as the Pittsburgh assay PA[3 V2.0.

[00107] Analytical assessment

[00108] We proceeded to compare the analytical performance of the PA[3 V1.0 assay with the PA[3 V2.0 assay.

[00109] Linearity test, LLOQ, ULOQ and Assay range

[00110] To assess linearity, we constructed standard curves using two-fold serial dilutions of a mixture of A|31 — 40 and A|31 — 42 in 6% BSA/PBS. A total of seven samples containing varying concentrations of A|31— 40 (0.00 pg/ml, 12.5 pg/ml, 25.0 pg/ml, 50pg/ml 100 pg/ml, 200 pg/ml, 400 pg/ml) and A|31 — 42 (0.00 pg/ml, 3.125 pg/ml, 6.25 pg/ml, 12.5 pg/ml, 25.0 pg/ml, 50 pg/ml and 100 pg/ml) were included in the linearity test. The measured A|31 — 40 and A|31 — 42 peak areas were normalized using the A|31 — 38 IS (FIG. 4A) or the analyte specific IS (A|31 — 40 IS and A|31 — 42 IS) (FIG. 5, panel A). Both the PA[3 V1.0 and the PA[3 V2.0 assay formats exhibited robust linearity across the tested concentration range, with r2 values for the linear regression lines exceeding 0.99 for both A|31 — 40 and A|31 — 42.

[00111] The inter-assay CV for both A|31 — 40 and A|31— 42 were below 20% in the sample with the lowest non-zero concentrations. Thus, we set the LLOQs for both assays at 12.5 pg/ml for A|31— 40 and at 3.125 pg/ml for A|31— 42. Additionally, since the linearity extended to the sample with the highest concentrations, we set the upper limits of quantification (ULOQs) for the assays at 400 pg/ml for A|31 -40 and 100 pg/ml for A 1-42.

[00112] Matrix effect assessment

[00113] To assess plasma matrix effect, we compared signals of A|3 peptides in plasma samples relative to BSA/PBS at three concentration levels (1 18.2pg/ml, 53.6pg/ml, and 21.4pg/ml for A|31— 40, 47.2pg/ml, 23.0pg/ml and 10.8pg/ml for A|31 — 42) and calculated the matrix effect recovery following the formula outlined in the Methods section. Both the PA[3 V1.0 and PA[3 V2.0 assay formats demonstrated similar matrix effects (Table 1 , FIG. 4A). The detailed results are listed in Table 1 (below).

Table 1 [00114] Interestingly, we observed overall better recovery when using analyte specific IS to normalize peak area (Table 1 , FIG. 5, panel B). These results suggest that different A|3 peptides might exhibit varying matrix effects, and analyte specific IS might be more robust in normalizing the matrix effect of corresponding analytes.

[00115] Assay precision

[00116] Assay precision was evaluated at three concentration levels (37.5 pg/ml, 146.4 pg/ml and 382.5 pg/ml for A|31 — 40, 82.8 pg/ml, pg/ml and 13.9 pg/ml for A|31 — 42) using normalized peak areas for both intra- and inter-assay assessments. The detailed results are listed in Table 2.

Table 2

V2.0 assay (%)

V1 .0 assay (%)

[00117] Similar %CVs were observed across both assays and normalization techniques, indicating strong reproducibility (%CV < 10%) for both PA[3 V1 .0 and PA[3 V2.0 assays.

[00118] Relationship between plasma dilution and normalized intensity

[00119] The relationship between plasma dilution and normalized intensity for both the PA[3 V1 .0 and PA[3 V2.0 assays was linear (r2 > 0.99), except for the A|31 — 42 of the PA[3 V1.0 assay for which r2 was 0.758. This deviation can be attributed to the inaccuracy introduced by low S/N ratio at low concentration level (FIG. 4B).

[00120] Plasma volume requirement

[00121 ] The PA[3 V1 .0 assay was designed around the use of 250 pl plasma sample for each measurement. To test whether the PA[3 V2.0 assay could enable measurement of A|3 peptides at lower plasma volumes, we examined both assays using varying amounts of plasma, ranging from 50 pl to 250 pl in increments of 50 pl, with three replicates for each sample volume. The results showed the PA[3 V2.0 assays provided 178.0-22.7% higher S/N of A|31— 40 and 87.6-26.1% higher S/N of A|31 — 42 from 50 pl to 250 pl (FIG. 4B).

[00122] Using a S/N ratio cutoff of 3, the PA[3 V1.0 assay required a minimum of 100 pl to achieve quality measurement of A|31— 40 and A|31— 42, respectively, compared with 50-100 pl for the PA[3 V2.0 assay (FIG. 4B).

[00123] IP recovery

[00124] To evaluate the proportion of A|3 peptides that were retained after the IP procedures, we utilized Simoa immunoassay to provide absolute quantification of A|3 peptides before and after IP IP recovery was evaluated at three concentration levels of low, medium, and high (27.4 pg/ml, 51.4 pg/ml, and 99.2 pg/ml for A|31— 40; 7.0 pg/ml, 13.2 pg/ml, and 27.4 pg/ml for A|31— 42) in triplicates. The result demonstrated that the PA[3 V2.0 assay retained a higher proportion of A|3 peptides after IP (FIG. 4A). [00125] Clinical assessment [00126] Participant characteristics

[00127] In the PITT-ADRC cohort, the mean age was 75.6 years (SD 7.8), with 16 (53.3%) females. Nine participants (30.0%) carried the APOE s4 genotype, and eight (26.7%) were diagnosed with probable AD. In terms of cognitive performance, the mean MMSE and MocA scores were 24.7 (SD 6.3) and 22.9 (SD 7.3), respectively. Regarding CDR scores, nine participants (30.0%) had a score of “disease absent” (CDR = 0), sixteen participants (53.3%) had a score of “questionable” (CDR = 0.5), three participants (10.0%) had a score of “disease present but mild” (CDR = 1 ), and two participants (6.7%) were categorized as “moderate” (CDR = 2). Comparing these metrics between the probable AD and normal control groups indicated significant differences in MoCA, MMSE and CDR scores. (FIG. 8).

[00128] In the AGUEDA cohort, the mean age was 71.4 years (SD 3.9), with 44 (57.1 %) females. Twelve participants (16.0%) were APOE s4 genotype carriers, eighteen (23.4%) were A[3-PET positive and the averaged CL level was 7.5 (SD 25.2). The mean MMSE was 28.9 (SD 1.1 ), and MoCA score was 25.8 (SD 2.2). The average years of education was 1 1 .7 (SD 4.8). Comparing these metrics between the A[3-PET- positive and A[3-PET-negative groups revealed no significant differences. The AGUEDA A[3-PET data was also classified into three categories according to the CL scales: CL < 12 (A[3-PET negative), 12 < CL < 24 (transition zone), and CL > 24 (A[3- PET positive). Comparing the metrics among the categories revealed significant differences in APOE s4 genotype carriers. (FIG. 8)

[00129] Clinical performance assessment

[00130] As mentioned in the Method section, we compared three different A|3 biomarkers: A|31 — 42/A|31 — 40 using the PAB V1.0 assay, A|31 — 42/A|31 — 40 using the PA[3 V2.0 assay, and A|31 — 42/A|31 — 40 normalized with A|31— 42 and A|31— 40 IS correspondingly using the PA[3 V2.0 assay.

[00131] In the ADRC cohort, the PA[3 V1 .0, PA[3 V2.0, and PA[3 V2.0 with analytespecific IS assays all showed equivalent performance in the A|31 — 42/A|31 — 40 ratio when comparing clinically assessed probable AD and normal control groups, with p- values of 0.13, 0.05, and 0.14, respectively (FIG. 6A). The effect sizes were consistent with those observed in the ADRC cohort, measuring 0.18, 0.20, and 0.30 for the PA[3 V1.0, PA[3 V2.0, and PA[3 V2.0 with analyte specific IS assays, respectively.

[00132] In the AGUEDA cohort, significantly lower levels in the A|31 — 42/A|31 — 40 ratio were observed in A[3-PET-positive versus A[3-PET-negative groups using both PA[3 V1 .0 and PA[3 V2.0 assays, with p-values of 0.031 and 0.019, respectively (FIG. 6B). Implementing analyte specific IS in the PA[3 V2.0 assay revealed a similar performance, yielding a p-value of 0.0013. The effect sizes for the A[3-PET-positive versus negative groups were 0.53, 0.56, and 0.73 for the PA[3 V1.0, PA[3 V2.0, and PA[3 V2.0 with analyte specific IS assays, respectively.

[00133] The AGUEDA A[3-PET data was also assessed according to the CL scales into three categories. In all assays, lower A|31— 42/1 — 40 levels were observed in the A[3-PET positive CL group, with p-values of 0.031 for PA[3 V1.0, 0.061 for PA[3 V2.0, and 0.0046 for PA[3 V2.0 with analyte specific IS assays, respectively (FIG. 6C). The effect sizes for A[3-PET-positive versus negative groups were 0.61 , 0.59, and 0.76 for the PA[3 V1 .0, PA[3 V2.0, and PA[3 V2.0 with analyte specific IS assays, respectively. [00134] These results indicate that the PA[3 V2.0 assay with common IS normalization and analyte specific IS normalization performed comparably to the PA[3 V1.0 assay in the clinical predictive performance.

[00135] Correlation between A|3 peptides measured in the PA p V1 .0 and the PA[3 V2.0 assays

[00136] To assess the measurement consistency between the PA[3 V1 .0 and PA[3 V2.0 assays for A|3 peptides, we evaluated their correlation across two cohorts for A 1-42, APMO, A 1-39, Ap3— 40, Ap1-38, and APP669-71 1. For both assays, A|31— 39, A[33-40, A|31— 38, and APP669-71 1 were normalized using the A|31— 38 IS. A|31 — 42 and A|31— 40 were normalized using both A|31— 38 IS and analyte specific IS for the PA[3 V2.0 assay, and A|31 — 38 IS only for the PA[3 V1 .0 assay. The correlation strength interpretation was based on previous publication.

[00137] In the ADRC cohort, we observed strong correlations for A|31 — 38, A|31 — 39 and A|31— 40 when normalized using A|31 — 38 IS (r > 8). The remaining A|3 peptides also exhibited strong correlations of 0.8 > r > 0.6 (FIGS. 7A-7B).

[00138] Similar results were obtained in the AGUEDA cohort, where there were strong correlations (r > 0.8) between A|31 — 40 measures when normalized using A|31 — 38 IS or A|31 — 40 IS. The correlation of A|31 — 42 (normalized using A|31 — 38 IS or A|31 — 42 IS) and A|31 — 38 was strong (0.8 > r > 0.6). Additionally, the correlation of the other A|3 peptides was moderate (0.6 > r > 0.5) (FIGS. 7C-7D), all indicating good agreement between the peptide levels measured in the different assay formats.

Discussion [00139] Mounting evidence indicate that plasma A|3 ratio has utility to measure brain A|3 pathology, and target engagement in therapeutic programs targeting brain A|3 aggregates. It is critical for healthcare systems to utilize cost-effective and minimally invasive methods for clinical diagnosis and patient selection and monitoring for the recently approved immunotherapies. Additionally, A|3 is an early biomarker showing changes in patients with incipient disease including in cognitively normal older adults compared to non-diseased individuals, highlighting its critical role in the pre- clinical diagnosis of AD. Early detection provides an opportunity for intervention and potentially altering the disease course. Furthermore, implementing a blood-based biomarker test for patient triaging could potentially reduce the current 50-month wait for treatment access to just 12 months, as projected by specialist referral models for cognitive impairment and dementia patients. These factors highlight the necessity of plasma-based IP-MS A|3 assay as a tool for early diagnosis.

[00140] Among the various blood-based A|3 assays, IP-MS methods such as the assay from Nakamura et al. stands out for its performance but has limitations needing improvement. We adopted and enhanced this assay, resulting in the PA[3 V2.0 assay with several enhancements. Firstly, our assay streamlined sample preparation time and preanalytical processing. Secondly, our new assay demonstrated a substantially stronger signal to noise ratio. Thirdly, the PA[3 V2.0 and PA[3 V1.0 assays exhibited similar clinical performance and analytical performance across multiple cohorts. To our knowledge, this is the first time that such significant enhancements have been achieved in refining the landmark Nakamura et al. plasma A|3 method.

[00141] The PA[3 V2.0 assay successfully streamlined the IP steps using a commercially available buffer - the N4PE CSF diluent. The high detergent, high salt content and the interference blocker mixture in the buffer helped reduce the background noise. This buffer was selected after comparing its performance against several detergents and blocking buffers. While all other tested reagents exhibited lower S/N ratios compared with PA[3 V1.0 assay, the N4PE diluent demonstrated higher S/N ratio, supporting its selection for further use as PA[3 V2.0 assay.

[00142] The PA[3 V2.0 assay maintained comparable analytical performance with a higher recovery rate compared to the PA[3 V1.0 assay. This result was verified by SIMOA, an immunoassay with a different measurement mechanism than MS. We also tested the S/N of A|31-40 and A|31 -42 in the PA[3 V2.0 assay, utilizing a diluted sample volume of pooled plasma. The results demonstrated a higher S/N ratio and suggested the potential feasibility of decreasing the sample volume to 50-100 pl for the PA[3 V2.0 assay. However, further investigation is warranted, including comparisons to A|3 PET imaging and/or CSF analysis, to assess the clinical utility and determine the feasibility of utilizing reduced sample volumes. Additionally, the PA[3 V2.0 assay preserved similar clinical performance, with peptide concentrations showing strong correlation with those in the PA[3 V1 .0 assay.

[00143] We further tested the performance of using analyte specific IS (A|31 — 40 IS and A|31— 42 IS) comparing to the common IS (A|31— 38 IS) for normalization. Our results indicated that using analyte specific IS for normalization can slightly improve the matrix effect recovery of plasma A|3 peptides. However, the use of the analyte specific IS did not significantly change the analytical performance of the A|3 biomarkers. In the clinical performance analysis, regardless of whether the analyte specific IS was used in the PA[3 V2.0 assay or not, the results did not show a significant improvement compared to the PA[3 V1 .0 assay. Our findings supported Nakamura et al.’s approach, confirming that using a common IS in the MALDI-TOF based IP-MS A|3 assay did not significantly alter clinical performance.

[00144] The MS instrument we utilized for our assays was a Bruker Microflex LT MALDI-TOF, widely adopted across numerous clinical facilities. Notably, it has received FDA approval for clinical microbiology diagnosis in humans, and is widely available in many laboratories. In comparison to other MS instruments utilized in alternative IP-MS plasma A|3 assays, the Microflex is distinguished by its affordability and simplicity. Furthermore, it offers practical advantages, such as direct compatibility with a standard 110V outlet, without necessitating the use of any special electrical modifications or voltage converter. Moreover, its user-friendly interface facilitates straightforward operation, enabling general laboratory technicians to operate the instrument proficiently without requiring specialized training in mass spectrometry.

[00145] Our study has several notable strengths. Firstly, we describe in detail the technical development, analytical and clinical validation of an improved plasma A|3 assay by IP-MS. Secondly, our study included two different cohorts. This diverse representation enhances the generalizability and practical relevance of our findings. Thirdly, the cohorts had been characterized for biological evidence of disease using brain A|3 PET and neuropsychologically using established evaluation instruments such as the MMSE, MoCA, and CDR. [00146] In conclusion, we report successful development of a more resourceefficient and cost-effective IP-MS plasma A|3 assay. Compared with the in-house reproduced Nakamura et al. assay, the new assay demonstrated comparable clinical and analytical performance. The cost, time, and reagent savings, coupled with the utilization of a more affordable and widely available instrument, will enable research laboratories to conduct IP-MS analysis of A|3 in blood more effectively.

[00147] Having described this invention above, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. Any document incorporated herein by reference is only done so to the extent of its technical disclosure and to the extent it is consistent with the present document and the disclosure provided herein.

Claims

THE INVENTION CLAIMED IS

1 . A method of detecting an amyloid peptide in a patient sample, comprising: exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide; and analyzing the free amyloid peptide with a mass spectrometer.

2. The method of claim 1 , wherein the assay binding buffer comprises tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n- dodecyl-[3-D-maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

3. The method of claim 2, wherein the surfactant is a non-ionic surfactant.

4. The method of claim 3, wherein the non-ionic surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

5. The method of claim 2, wherein the isothiazolone compound comprises isothiazolone chloride.

6. The method of claim 1 , wherein the binding reagent comprises an antibody for the amyloid peptide.

7. The method of claim 6, wherein the binding reagent further comprises a magnetic bead conjugated to the antibody.

8. The method of claim 6, wherein the antibody comprises an antibody for amyloid beta (A|3).

9. The method of claim 1 , wherein the washing step comprises washing the immunoprecipitated amyloid peptide with the assay binding buffer.

10. The method of claim 9, The method of claim 1 , wherein the washing step further comprises washing the immunoprecipitated amyloid peptide with phosphate-buffered saline.

11 . The method of claim 1 , wherein the washed, immunoprecipitated amyloid peptide is eluted with a buffer comprising an alpha-cyano-hydroxycinnamic acid matrix and trifluoroacetic acid.

12. The method of claim 1 , wherein the sample is a blood sample.

13. The method of claim 1 , wherein the sample is a plasma sample.

14. The method of claim 1 , wherein the amyloid peptide is A[3-38, A 1 -39, Ap 1 -40, Ap 1 -41 , Ap 1 -42, and/or APP669-711 .

15. The method of claim 1 , wherein the amyloid peptide is Api -40 and A 1-42.

16. The method of claim 1 , wherein the mass spectrometer is a matrix-assisted laser desorption/ionization-time of flight mass spectrometer.

17. A binding assay buffer comprising tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D- maltoside, n-noyl-[3-D-thiomaltoside, a surfactant, and an isothiazolone compound.

18. The binding assay buffer of claim 17, wherein the surfactant is a non-ionic surfactant.

19. The binding assay buffer of claim 18, wherein the non-ionic surfactant comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol.

20. The binding assay buffer of claim 17, wherein the isothiazolone compound comprises isothiazolone chloride.

21 . The binding assay buffer of claim 17, further comprising a binding reagent for an amyloid peptide.

22. The binding assay buffer of claim 21 , wherein the binding reagent further comprises a magnetic bead conjugated to the binding reagent.

23. The binding assay buffer of claim 21 , wherein the binding reagent is a binding reagent for amyloid beta ( A|3) .

24. A kit comprising one or more containers holding tris(hydroxymethyl)aminomethane hydrochloride, sodium chloride, n-dodecyl-[3-D- maltoside, n-noyl-[3-D-thiomaltoside, 2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol, and isothiazolone chloride.

25. A method of detecting an amyloid peptide in a patient sample, comprising: exposing the patient sample to a binding reagent in the presence of an assay binding buffer, thereby immunoprecipitating the amyloid peptide; washing the immunoprecipitated amyloid peptide; and eluting the washed, immunoprecipitated amyloid peptide, thereby generating free amyloid peptide.