Method for quantifying amyloid beta protofibril

Abstract

Disclosed herein are methods of measuring amyloid β protofibril levels in biological samples. Methods disclosed herein may detect amyloid β protofibril at femtomolar concentrations and selectively measure protofibril as compared to amyloid β monomers.

Description

[0001] Attorney Docket No.08061.0067-00304 METHOD FOR QUANTIFYING AMYLOID BETA PROTOFIBRIL RELATED APPLICATIONS The present application claims priority to U.S. Provisional Patent Application No. 63 / 613,407 filed December 21, 2023, the entire contents of which are hereby incorporated by reference for all purposes. SEQUENCE LISTING This application contains a Sequence Listing named 08061_6066-00000_SL.xml, which was created December 18, 2024, and is 31,432 bytes in size. The Sequence Listing has been filed electronically in XML format and is hereby incorporated by reference in its entirety. The present disclosure relates to methods of measuring amyloid β protofibril levels. Alzheimer’s disease (“AD”) is a progressive, neurodegenerative disorder of unknown etiology and the most common form of dementia among older people. In 2006, there were 26.6 million cases of AD in the world (range: 11.4-59.4 million) (Brookmeyer, R., et al., Forecasting the global burden of Alzheimer’s Disease. Alzheimer Dement.2007; 3:186- 91), while there were more than 5 million people in the United States reportedly living with AD (Alzheimer’s Association, Alzheimer’s Association report, 2010 Alzheimer’s disease facts and figures. Alzheimer Dement.2010; 6:158-94). By the year 2050, the worldwide prevalence of AD is predicted to grow to 106.8 million (range: 47.2-221.2 million), while in the United States alone the prevalence is estimated to be 11 to 16 million. (Brookmeyer, supra, and 2010 Alzheimer’s disease facts and figures, supra). The disease generally involves a global decline of cognitive function that progresses slowly and leaves end-stage subjects bedridden. AD subjects typically survive for only 3 to 10 years after symptom onset, although extremes of 2 and 20 years are known. (Hebert, L.E., et al., Alzheimer disease in the U.S. population: prevalence estimates using the 2000 census. Attorney Docket No.08061.0067-00304 Arch Neurol.2003; 60:1119-1122.) AD is the seventh leading cause of all deaths in the United States and the fifth leading cause of death in Americans older than the age of 65 years, despite the fact that mortality due to AD is greatly underestimated because death certificates rarely attribute the cause of death to AD. (Alzheimer’s Association. Alzheimer’s Association report. 2010 Alzheimer’s disease facts and figures. Alzheimer Dement.2010; 6:158-94.) AD represents a significant economic burden across industrialized countries with a substantial impact on healthcare systems and the public purse as well as on subjects and their families. In the United States alone, total payments for 2010 were estimated at $172 billion, including $123 billion for Medicare and Medicaid. Histologically, the disease is characterized by neuritic plaques, found primarily in the association cortex, limbic system, and basal ganglia. The major constituent of these plaques is amyloid beta (Aβ) peptide. Aβ exists in various conformational states - monomers, oligomers, protofibrils, and insoluble fibrils. Some details of the mechanistic relationship between onset of Alzheimer’s disease and Aβ production are unknown. The “amyloid hypothesis” proposes that amyloid β (Aβ) peptides play a central role in the pathogenesis of AD. Specifically, it is hypothesized that neurodegeneration in AD may be caused by deposition of Aβ plaques in brain tissue due to an imbalance between Aβ production and Aβ clearance, leading to formation of neurotoxic neurofibrillary tangles. Aβ peptides generally exist in a dynamic continuum of conformational states such that species tend to progress from monomeric Aβ, to soluble Aβ assemblies that include a range of low molecular weight oligomers to higher molecular weight protofibrils, and finally to insoluble fibrils (plaques). The protofibril form of Aβ is one of the largest soluble forms of Aβ and may play a leading role in AD. Development of therapies that selectively target Aβ protofibrils have led to the generation of humanized anti-Aβ protofibril antibodies, which are Attorney Docket No.08061.0067-00304 highly selective for the protofibril form of Aβ. As an example, Lecanemab, a recombinant humanized immunoglobulin gamma 1 (IgG1) monoclonal antibody directed against aggregated soluble and insoluble forms of amyloid beta, was recently approved for the treatment of Alzheimer’s disease. U.S. Patent No.8,025,878 (incorporated herein by reference in its entirety). Treatment with lecanemab may be initiated in patients with mild cognitive impairment or mild dementia stage of disease. Targeting soluble Aβ protofibrils, e.g., in these patients, may provide therapeutic benefit. A need remains, however, for measuring Aβ (particularly Aβ protofibrils) more accurately in patients. The detection and quantification of protofibrils in biological samples has been limited due to the need for a selective method of detection for the protofibril form of Aβ over other forms of Aβ (such as monomers) and the low concentration of protofibrils in biological samples. In some instances, the concentration of protofibrils is below the low limit of detection for an assay and may be in the femtomolar range. Provided herein is a method of quantifying Aβ protofibrils in biological samples. The assay may be sensitive to detect femtomolar concentrations of Aβ protofibrils. The assay may be selective for Aβ protofibrils compared to Aβ monomers. In some embodiments, the presence of a therapeutic anti-Aβ protofibril antibody (e.g., lecanemab or an antibody comprising its CDRs or variable regions, or an antibody binding the same epitope as or competing for binding with lecanemab) in a biological sample surprisingly does not interfere with the detection of Aβ protofibrils in an Aβ protofibril assay using the same antibody (e.g., the lecanemab as the anti-Aβ protofibril detection antibody as well as for clinical treatment). SUMMARY In some aspects, the present disclosure provides a method for quantifying amyloid β (Aβ) protofibrils in a biological sample from a human subject, comprising: contacting the Attorney Docket No.08061.0067-00304 biological sample with an anti-Aβ protofibril capture antibody comprising heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3) to form an immune complex; contacting the immune complex with a labeled anti-Aβ detection antibody; detecting a signal from the labeled detection antibody using a single molecule counting instrument; and quantifying Aβ protofibrils in the sample. In some embodiments, the anti-Aβ protofibril capture antibody comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8. In some embodiments, the anti-Aβ protofibril capture antibody comprises lecanemab. In some embodiments, the present disclosure provides a method wherein contacting the biological sample with the capture antibody forms a first immune complex and contacting the first immune complex with the detection antibody forms a second immune complex. In some embodiments, the present disclosure provides a method wherein the label on the anti-Aβ detection antibody comprises a tag, wherein the tag comprises a protein tag, a fluorescent tag, a quantum dot tag, an aptamer tag, an oligonucleotide tag, a SULFO-TAG, or a biotin tag. In some embodiments, the present disclosure provides a method wherein anti-Aβ detection antibody binds a region of Aβ that does not overlap a region bound by lecanemab, and / or wherein the anti-Aβ detection antibody does not compete for binding to Aβ protofibrils with lecanemab. In some embodiments, the anti-Aβ detection antibody binds to an N-terminus of an Aβ, e.g. Aβ1-5. In some embodiments, the anti-Aβ detection antibody comprises heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3) and light chain Attorney Docket No.08061.0067-00304 complementarity determining region (LCDR) sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO: 18 LCDR3. In some embodiments, the anti-Aβ detection antibody comprises a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20. In some embodiments, the anti-Aβ detection antibody comprises 3D6. In some embodiments, the present disclosure provides a method wherein the anti- Aβ detection antibody comprises lecanemab. In some embodiments, the present disclosure provides a method wherein the biological sample comprises a body fluid or tissue. In some embodiments, the present disclosure provides a method wherein the biological sample comprises cerebrospinal fluid, whole blood, plasma, serum, brain tissue homogenate, or brain tissue lysate. In some embodiments, the biological sample is a brain homogenate. In some embodiments, the biological sample is stored at -80°C prior to contacting the sample with the capture antibody. In some embodiments, the method does not comprise repetitive freeze-thaw cycles of the biological sample. In some embodiments, the method further comprises thawing the frozen biological sample and gently mixing and / or vortexing. In some embodiments, the biological sample is prepared and stored in low protein- binding tubes. In some embodiments, the present disclosure provides a method wherein the biological sample is at a concentration 0.1-10 mg of protein per mL of sample. In some embodiments, the biological sample is at a concentration of 1 mg of protein per mL of sample. In some embodiments, the present disclosure provides a method wherein the biological sample is diluted 1-fold to 4-fold. In some embodiments, the biological sample is diluted 1-fold, 2-fold, 3-fold, or 4-fold. In some embodiments, the biological sample is diluted 2-fold. In some embodiments, the biological sample is diluted in a sample buffer. Attorney Docket No.08061.0067-00304 In some embodiments, the present disclosure provides a method wherein the biological sample is a CSF sample and optionally the CSF sample is not diluted. In some embodiments, the CSF sample is diluted. In some embodiments, the CSF sample is diluted 1- fold to 4-fold. In some embodiments, the CSF sample is diluted 1-fold, 2-fold, 3-fold, or 4- fold. In some embodiments, the CSF sample is diluted 2-fold. In some embodiments, the CSF sample is diluted in a sample buffer. In some embodiments, the present disclosure provides a method wherein the biological sample is a blood sample and optionally the blood sample is not diluted. In some embodiments, the blood sample is diluted. In some embodiments, the blood sample is diluted 1-fold to 4-fold. In some embodiments, the blood sample is diluted 1-fold, 2-fold, 3-fold, or 4-fold. In some embodiments, the blood sample is diluted 2-fold. In some embodiments, the blood sample is diluted in a sample buffer. In some embodiments, the sample buffer for diluting the biological sample comprises a buffering agent, NaCl (e.g.150mM NaCl), a blocking agent (e.g. bovine serum albumin), and a detergent (e.g. non-ionic detergent, e.g. Tween 20). In some embodiments, the sample buffer for diluting the biological sample comprises a Good’s buffer, NaCl (e.g. 150mM NaCl), a blocking agent (e.g. bovine serum albumin), and a detergent (e.g. non-ionic detergent, e.g. Tween 20). In some embodiments, the sample buffer comprises a blocking agent (e.g. bovine serum albumin) and a detergent (e.g. non-ionic detergent, e.g. Tween 20) in buffered saline (e.g. phosphate-buffered saline (PBS), and tris-buffered saline (TBS)). In some embodiment, the sample buffer comprises 0.5% BSA and 0.05% Tween 20 in PBS. In some embodiments, the sample buffer is a commercially available dilution buffer or blocking buffer for immunoassay. In some embodiments, the sample buffer is Discovery Standard Diluent (Cat#02-0560-00. EMD Millipore). Attorney Docket No.08061.0067-00304 In some embodiments, the present disclosure provides a method wherein the anti- Aβ protofibril capture antibody is immobilized on a surface. In some embodiments, the surface is a plate, a bead, or a particle, optionally a magnetic bead or particle. In some embodiments, the particle is a tosylactivated magnetic bead. In some embodiments, the present disclosure provides a method wherein the capture antibody is conjugated to the bead at a concentration of 10-40 µg of antibody per mg of bead. In some embodiments, the present disclosure provides a method wherein the capture antibody is conjugated to the bead at a concentration of 20 µg of antibody per mg of bead. In some embodiments, the capture antibody is conjugated to the bead at a concentration of 20 µg antibody per mg of tosylactivated magnetic bead. In some embodiments, the present disclosure provides a method wherein the tag is a fluorescent tag, and wherein the signal from the fluorescent tag is measured by the single molecule counting instrument. In some aspects, the present disclosure provides a method for quantifying amyloid β (Aβ) protofibrils in a biological sample from a human subject, comprising: contacting the biological sample with an anti-Aβ protofibril capture antibody comprising heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3) immobilized on a surface to form an immune complex; contacting the sample with a fluorescently labeled anti-Aβ detection antibody to form an immune complex; dissociating the immune complex to release the fluorescently labeled anti- Aβ detection antibody; detecting a signal from the fluorescently labeled anti-Aβ detection antibody via a single molecule counting instrument; and quantifying Aβ protofibrils in the sample. Attorney Docket No.08061.0067-00304 In some embodiments, the present disclosure provides a method further comprising washing the sample to remove unbound materials. In some embodiments, the present disclosure provides a method wherein the capture antibody is conjugated to the bead at a concentration of 10-40 µg antibody per mg of bead. In some embodiments, the present disclosure provides a method, wherein the capture antibody is conjugated to the bead at a concentration of 20 µg antibody per mg of bead. In some embodiments, the present disclosure provides a method wherein the capture antibody is conjugated to the bead at a concentration of 20 µg antibody per mg of tosylactivated magnetic bead. In some embodiments, the present disclosure provides a method wherein the biological sample comprises Aβ protofibrils in an amount of at least about 0.01 picomolar, e.g., about 0.01 picomolar to about 2 picomolar. In some embodiments, the present disclosure provides a method wherein the method has a sensitivity for Aβ protofibrils of about 0.003 picomolar. In some embodiments, the present disclosure provides a method wherein the method has a lower limit of quantification of about 0.01 picomolar of Aβ protofibrils. In some embodiments, the present disclosure provides a method wherein the method provides a selectivity for an Aβ protofibril of 1,000,000-fold or greater than for an Aβ monomer. In some embodiments, the present disclosure provides a method wherein the quantity of Aβ protofibril is determined relative to a control. In some embodiments, the present disclosure provides a method wherein an Aβ protofibril standard curve is used to determine the Aβ protofibril quantity. Attorney Docket No.08061.0067-00304 In some embodiments, the present disclosure provides a method wherein the Aβ protofibril standard curve is prepared from Aβ1-42by size exclusion chromatography. In some embodiments, the present disclosure provides a method wherein the Aβ protofibril standard curve is prepared in low protein-binding tubes and stored at -80C. In some embodiments, the present disclosure provides a method wherein the method provides a S / N ratio greater than 100, e.g., at concentrations of Aβ protofibril of about 1 pM. In some embodiments, the protofibrils are measured in a sample from a human subject who has or is suspected of having Alzheimer’s disease (AD), preclinical AD, and / or mild cognitive impairment (MCI). In some embodiments, the human subject received treatment with lecanemab prior to or at the time of the biological sample collection. In some embodiments, the present disclosure provides a kit for carrying out the methods of quantifying amyloid β (Aβ) protofibrils in a biological sample from a human subject, wherein the kit comprises a capture antibody comprising lecanemab or an antigen- binding fragment thereof. In some embodiments, the detection antibody in the kit is fluorescently labeled. In some embodiments, the detection antibody is 3D6. In some embodiments, the kit further comprises a sample buffer, a washing buffer, and / or a dissociation buffer.

[0002] Attorney Docket No.08061.0067-00304 BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several non-limiting embodiments of the invention and together with the description, serve to explain the principles of the disclosure. Fig.1. Detection of Aβ1-42 protofibril on SMCxPRO. Correlation between the concentration of Aβ1-42 protofibril and response measured by single molecule counting on an SMCxPRO instrument. Fig.2. Selectivity to PF, compared to monomer Aβ. Correlation between the response measurements on the SMCxPRO single molecule counting instrument and concentration of Aβ protofibril (closed circle), Aβ1-16 monomer (closed triangle) or Aβ1-40 monomer (open circle). Fig.3. Dilution linearity. Correlation between protofibril concentration and dilution factor of protofibril sample. Fig.4. BAN2401 interference. Concentration of protofibrils in samples with or without the addition of 500 ng / mL BAN2401. Fig.5. Measurement of brain homogenate. Protofibril concentrations in brain homogenates of patients with Alzheimer’s disease (AD; n=6 patients) compared to brain homogenates from controls (n=6 individuals). Figs.6A-B. CSF measurement. Fig.6(A) The concentration of Aβ protofibril in CSF samples (open circles) was determined relative to a standard curve (closed circles) of Aβ1-42 protofibril. Fig.6(B) The concentration of protofibril in CSF from patients with Alzheimer’s disease (AD; n=8) compared to controls (n=8). Fig.7. Evaluation of anti-Aβ detection antibodies by sandwich ELISA. Correlation between optical density (O.D.) at 450-650 nm relative to Aβ1-42 protofibril concentration for indicated detection antibodies in combination with BAN2401 capture antibody. Attorney Docket No.08061.0067-00304 Figs.8A-B. Comparison of antibody pairs BAN2401 / 3D6 and BAN2401 / BAN2401 on Meso Scale Discovery Electrochemiluminescence (MSD-ECL) platform. Fig.8(A) Correlation of the ECL signal (logarithmic scale) and PF standard. Fig. 8(B) Correlation of ECL signal (linear scale) and PF standard. Fig.9. Comparison of antibody pairs, BAN2401 / 3D6 and BAN2401 / BAN2401 on SMCxPro. The S / N ratio detected with BAN2401 as the capture antibody and 3D6 as the detection antibody compared to BAN2401 as the capture antibody and BAN2401 as the detection antibody for the detection of 0.1 pM PF or 1 pM PF. Figs.10A-B. Comparison of SMCxPRO to Simoa for detection of PF with BAN2401 / 3D6. Fig.10(A) Correlation of the S / N ratio (logarithmic scale) to PF concentration on SMCxPRO compared to Simoa. Fig.10(B) Correlation of the S / N ratio (linear scale) to PF concentration on SMCxPRO compared to Simoa. Fig.11. Measurement of Amyloid β protofibril levels on tosylactivated magnetic beads in CSF samples relative to blank samples on SMCxPRO. Fig.12(A) shows measurement of 0.003 and 0.01 pM of PF standard versus blanks after coating tosylactivated beads with 40 µg, 20 µg, or 10 µg of BAN2401. Fig.12(B) shows measurement of PFs in CSF following coating of 1 mg of tosylactivated beads with 10 µg, 20 µg, or 40 µg of BAN2401.

[0003] Attorney Docket No.08061.0067-00304 DETAILED DESCRIPTION Definitions In order to better understand the disclosure, certain definitions are provided first. As used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. The phrase “and / or,” as used herein, means “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a non-limiting example, “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in some embodiments, to A only (optionally including elements other than B); in other embodiments, to B only (optionally including elements other than A); in yet other embodiments, to both A and B (optionally including other elements); etc. As used herein, “at least one” means one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. Attorney Docket No.08061.0067-00304 When a number is recited, either alone or as part of a numerical range, it should be understood that the numerical value can vary above and below the stated value by a variance of 10% of the stated value. When a range of values is listed herein, it is intended to encompass each value and sub-range within that range. For example, “2.5 mg / kg to 10 mg / kg” is intended to encompass, for example, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg, 10 mg / kg, 2.5 mg / kg to 3 mg / kg, 2.5 mg / kg to 4.5 mg / kg, 3 mg / kg to 4.5 mg / kg, 4.5 mg / kg to 8 mg / kg, 2.5 mg / kg to 9 mg / kg, and so forth. As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD) (or its inverse equilibrium association constant, KA). Affinity can be measured by common methods known in the art. See, for example, Pope M.E., Soste M.V., Eyford B.A., Anderson N.L., Pearson T.W., (2009) J. Immunol. Methods.341(1-2):86-96. As used herein, “competitive binding” refers to the relationship between two molecules which bind to the same site of a third molecule, for example two antibodies that bind to the same epitope of a protein. In such an instance, a first antibody or antigen-binding fragment may bind to an epitope of a protein (e.g., an epitope on an Aβ protofibril) in a manner sufficiently similar to the binding of a second antibody or antigen-binding fragment, such that the binding of the first antibody or antigen-binding fragment with its epitope is detectably decreased in the presence of the second antibody or antigen-binding fragment compared to the binding of the first antibody or antigen-binding fragment in the absence of the second antibody or antigen-binding fragment. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first Attorney Docket No.08061.0067-00304 antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. “Competitive binding” may be determined in an assay in which the immunoglobulin / antibody / binding fragment being evaluated inhibits specific binding of a reference antibody to a common antigen, such as Amyloid β (e.g., an Aβ sequence of Table 9), e.g., as measured by competitive radioimmunoassay (RIA), enzyme immunoassay (EIA), or a sandwich ELISA assay. In preferred embodiments, a sandwich ELISA is used. In some embodiments, an assay involves the use of purified antigen bound to a solid surface or expressed on a cell surface, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. In some embodiments, the test immunoglobulin is present in excess. In some embodiments, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody (e.g., radiolabeled antibody) to a common antigen by a detectable amount, e.g., at least 50% or more. As used herein “specifically binds” or “specific binding” in reference to an antibody means that the antibody binds to its target antigen or epitope with greater affinity than it does to structurally different antigen(s) or epitope(s). Specific binding refers, in some embodiments, to binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity, or by competition assay with a control molecule that shares similar binding affinity but is unlabeled. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. Binding kinetic properties may also be measured using surface plasmon resonance (SPR). For Attorney Docket No.08061.0067-00304 example, a molecule used in an assay disclosed herein may have a KD for the target of at least about 10−6M, alternatively at least about 10−7M, alternatively at least about 10−8M, alternatively at least about 10−9M, alternatively at least about 10−10M, alternatively at least about 10−11M, alternatively at least about 10−12M, alternatively at least about 10−13M, alternatively at least about 10−14M, or greater. As used herein, “Amyloid β peptide(s)” may be used interchangeably with “Aβ peptide(s)”, “Amyloid β monomer(s)” and “Aβ monomer(s)”, and refers to polypeptides, whether folded into a three-dimensional protein, unfolded and / or misfolded, resulting from the cleavage of the Amyloid Precursor Protein at various cleavage sites. Amyloid β 1-42 (Aβ1-42, Aβ(1-42), or Aβ1-42) refers to a 42 amino acid amyloid β monomer (Table 9, SEQ ID NO: 25). Amyloid β 1-40 (Aβ1-40 or Aβ1-40) refers to a 40 amino acid amyloid β monomer (Table 9, SEQ ID NO: 26). In some embodiments, an amyloid β monomer may be present intracellularly. In some embodiments, an amyloid β monomer may be extracellular, for example in the intracellular space or blood. In some embodiments, an amyloid β monomer may exist in biological samples, for example in the blood or CSF. As used herein, “Amyloid Precursor Protein (APP)” is a transmembrane protein comprising a 770 amino acid polypeptide (Table 9, SEQ ID NO: 29) with an extracellular domain, which may be cleaved by proteases to generate an Aβ peptide and / or protein. In some embodiments, the Aβ peptide comprises amino acids 1-42 (Table 9, SEQ ID NO: 25). In some embodiments, the Aβ peptide comprises amino acids 1-40 (Table 9, SEQ ID NO: 26). As used herein, “the Aβ aggregation process” refers to the dynamic continuum of Aβ three-dimensional conformational states such that species of Aβ tend to progress from monomeric Aβ, to soluble Aβ assemblies that include a range of low molecular weight oligomers to higher molecular weight protofibrils, and finally to insoluble fibrils and plaques. Attorney Docket No.08061.0067-00304 In some embodiments, the Aβ aggregates may take organized 3D conformational states (such as folded soluble oligomers or Aβ protofibrils), which may be associated with pathological processes. In some embodiments, during the Aβ aggregation process the Aβ polypeptide molecules may bind together in a disorganized state associated with misfolding or abnormal folding of Aβ polypeptides producing Aβ aggregates. In some embodiments, the Aβ aggregates may be the result of misfolding and exposure of hydrophobic residues. In some embodiments, the Aβ aggregates may undergo a nucleation step during which aggregation proceeds slowly until the Aβ aggregate reaches a size or number of Aβ monomers so that the addition of new Aβ monomers to the aggregate occurs more rapidly. As used herein, “Amyloid β oligomer” is used interchangeably with “oligomer” or “Aβ oligomer”. As used herein amyloid β oligomer refers to a molecule comprising two or more Aβ monomers. In some embodiments, an amyloid β oligomer may be intracellular. In some embodiments, an amyloid β oligomer may be extracellular, for example in the interstitium also known as the intracellular space. In some embodiments, an amyloid β oligomer may exist in biological samples, for example in the blood or CSF. As used herein, “Amyloid β protofibrils,” “Aβ protofibrils,” “Aβ protofibril,” “protofibril(s)” and “PF” are used interchangeably. Aβ protofibril refers to soluble Aβ aggregate species. In some embodiments, molecular weight of Aβ protofibril may be ≥75kDa, ≥80kDa, ≥100kDa or 75-5000 kDa. In some embodiments, protofibrils may be curved linear structures with a diameter of about 5 nm and a length of up to 200 nm (Englund et al. “Sensitive ELISA detection of amyloid-B protofibrils in biological samples” J of Neurochemistry 2007; 103, 334-345). In some embodiments, PF have a diameter of about 3 nm and range in length from 60 nm to 220 nm. (Dubnovitsky et al. “Amyloid B Protofibrils: Attorney Docket No.08061.0067-00304 Size Morphology and Synaptotoxicity of an Engineered Mimic" PLoS one 2013; 8(7): e66101) As used herein, “Amyloid β fibril” or “Aβ fibril” refers to an insoluble aggregation of Aβ molecules and is the major structural component of Amyloid β plaques. As used herein, “Amyloid β plaque” refers to a deposit of insoluble aggregated Aβ protein molecules. An Amyloid β plaque is comprised in large part of Amyloid β fibrils. The term “antibody” refers to an immunoglobulin, whether genetically engineered, natural, or wholly or partially synthetically or recombinantly produced. Intact antibodies typically comprise a heavy chain and a light chain, each comprised of a variable region forming the binding pocket for an antigen and a constant region that contributes to effector function. The antibody, by virtue of its chosen heavy chain, can be a member of any immunoglobulin class and subclass, including any of the human classes: IgG, IgM, IgA, IgD, and IgE, or a derivative or fragment thereof. Likewise, the light chain of the antibody may derive from any species, such as a human kappa (κ) or lambda (λ) light chain, determined based on the amino acid sequences of the constant region. The basic antibody structural unit typically comprises a tetramer. In various embodiments, the tetramer comprises two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino- terminal (also referred to as the N-terminus) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (also referred to as the C-terminus) portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more Attorney Docket No.08061.0067-00304 amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. See generally, Fundamental Immunology Ch 7. (Paul, W., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody typically has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids to each domain may be done in accordance with the IMGT numbering system. Alternative definitions are also known to know of ordinary skill in the art. See, e.g., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Clothia & Lesk J. Mol. Biol.196:901-917 (1987); Clothia et al. Nature 342:878-883 (1989). An “antibody fragment” or “antigen binding fragment” comprises a portion of a full- length antibody, generally at least the antigen binding portion / domain or the variable region thereof and preferably one retaining some or all of the antigen binding properties of the full- length antibody. The term antibody fragment is a subset of the term antibody discussed above. Examples of antibody fragments or antigen binding fragments include: Fab, Fab’, F(ab’)2, Fd, scFv, (scFv)2, scFv-Fc, Fv fragment, diabodies, single-chain antibody molecules, immunotoxins, and multi-specific antibodies formed from antibody fragments. In addition, antibody fragments may comprise single chain polypeptides having the characteristics of a VH chain capable of being able to assemble together with a VL chain to form a functional antigen binding pocket and thereby providing the property of binding to Aβ Attorney Docket No.08061.0067-00304 protofibril. The terms also comprise fragments that per se are not able to provide effector functions (e.g., Antibody-dependent cell-mediate cytotoxicity (“ADCC”) or complement dependent cytotoxicity (“CDC”) but provide this function after being combined with the appropriate antibody constant region(s). The term “identity” or “sequence identity” refers to the homology between at least two sequences (e.g., amino acid sequences), and may be quantified as a percentage. The percent identity between two sequences may be assessed using a mathematical algorithm. Suitable algorithms are known in the art. For example, the percent identity between two amino acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988 and Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; each of which is incorporated herein by reference in its entirety. Techniques for determining identity are codified in publicly available computer programs. Computer software to determine homology between two sequences include, but are not limited to, BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molecular Biol., 215, 403 (1990)). Preferably, BLAST alignment may be used unless specified otherwise. The term “chimeric antibody” refers to a monoclonal antibody comprising all or part of a variable region from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. In some embodiments, chimeric antibodies comprise a murine variable region and a human constant region. Such murine / human chimeric antibodies may be produced by expressing immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” may be those in which the class or subclass has been modified or changed from that of the original antibody. Such “chimeric” antibodies are also Attorney Docket No.08061.0067-00304 referred to as “class-switched antibodies.” Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos.5,202,238 and 5,204,244. The term “conjugated,” as used herein, refers to a bond or chemical moiety formed from a chemical reaction between a functional group of a first molecule (e.g., an antibody) with a functional group of a second molecule (e.g., a detectable signor or therapeutic agent or drug). Such bonds include, but are not limited to, covalent linkages and non-covalent linkages, while such chemical moieties include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. As used herein, the term “immune complex” refers to an antibody or other antigen binding fragment bound with its target antigen. In some embodiments, an immune complex comprises the target with the capture antibody and / or detection antibody. In some embodiments, the target bound to the capture antibody may be called the “first immune complex” and / or “immune complex I”. In some embodiments, an immune complex comprises the target bound to both the capture antibody and the detection antibody. In some embodiments, the target bound to both the capture and detection antibody may be called the “second immune complex” and / or “immune complex II”. The term “epitope” refers to a site on an antigen to which an immunoglobulin or antibody (or antigen binding fragment thereof) can specifically bind. Thus, an epitope on Aβ is the site on Aβ molecule where an immunoglobulin or antibody (or antigen binding fragment thereof) can specifically bind. An epitope, as the term is used herein, can be formed from either contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. An epitope may include additional stretches of amino acids anywhere in Attorney Docket No.08061.0067-00304 the antigen, e.g., 1, 2, 3, 4, 5, 10, 15, 20, or more amino acids on the N-terminus and / or C- terminus of a peptide, or at a region in the center of the peptide. When a polypeptide is considered in a linearized sequence, the sequence may be divided into sections. Amino acids are numbered within a protein beginning at the N-terminus identified by a terminal free amino group and proceeds to the C-terminus identified by a terminal free carboxylic acid group. An epitope may be located in a region closer to the N-terminus (e.g., N-terminal epitope) or the C-terminus (e.g., C-terminal epitope), or may be localized somewhere in the middle. Epitopes may be linear or conformational, i.e., the epitope bound by an antibody may form from the three-dimensional folding of an antigen and comprise more than one discrete section of the linear peptide. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, often in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, alanine scanning, x-ray crystallography, and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996). Exemplary methods are discussed and used herein for the disclosed antibodies. An epitope on Aβ, as discussed herein, is identified with reference to amino acid residues of a reference Aβ protein. For example, the epitope of 3D6 is Aβ1-5 (SEQ ID NO.: 28). One of skill in the art could readily identify corresponding amino acid residues on other Aβ isoforms and fragments, e.g., via alignment programs such as BLAST®. Unless otherwise indicated, a reference to a particular amino acid residue on Aβ and should be understood to encompass the corresponding positions on other Aβ isoforms and fragments. As used herein, the term “analyte” may be used to describe the molecule quantified by an assay. The terms “analyte” and “target” may be used interchangeably. In some Attorney Docket No.08061.0067-00304 embodiments, an analyte is a polypeptide or protein. In some embodiments, the analyte may be a biomarker. In some embodiments, the analyte levels may be determined as an exact concentration. In some embodiments, a concentration may be determined using linear regression from a standard curve. In some embodiments, the analyte levels may be determined as a relative concentration compared to other samples. In some embodiments, the analyte may be Aβ protofibrils. As used herein, a “biological sample” is any portion of tissue or fluid obtained or isolated from a living or once living being. In some embodiments, the biological sample is collected from a living or deceased human being. In some embodiments, biological samples include whole blood or blood-derived fluid, such as serum or plasma. In some embodiments, biological samples include cerebrospinal fluid (CSF) or brain tissue. In some embodiments, the biological sample is handled to protect the integrity of the sample and prevent degradation of the sample associated with removal of the sample from the living or deceased being and / or processing of the sample following isolation (e.g., avoid freeze thaw cycles). In some embodiments, the biological sample may contain analyte(s) and / or biomarker(s). As used herein, the term “blood sample” or “blood” refers to a sample of blood, including serum and / or blood plasma from a human subject. In some embodiments, the blood sample is serum from a human subject. In some embodiments, the blood sample is plasma from a human subject. In some embodiments, subjects are required to fast if possible before collection of the blood. In other embodiments and / or at other time points, subjects do not require fasting. As used herein, a “control sample” which may be used interchangeably with “control” refers to a biological sample that is distinct from samples being evaluated. In some embodiments, a control sample refers to a biological sample that is distinct from samples being evaluated following an intervention or treatment. In some embodiments, a control Attorney Docket No.08061.0067-00304 sample may be obtained from a healthy subject. In some embodiments, a control sample may be obtained from a healthy, cognitively unimpaired subject. In some embodiments, a control sample may be obtained from a healthy, cognitively normal subject. In some embodiments, a control sample may be obtained from a healthy age and / or gender matched subject. In some embodiments, a control sample may be obtained from a subject who does not have a diagnosis of Alzheimer’s disease. In some embodiments, a control may be obtained from a subject with co-morbidities that are not associated with AD. In some embodiments, a control sample may be obtained from a subject with a diagnosis along the spectrum of AD disease including, for example, preclinical AD or mild cognitive impairment. In some embodiments, a control sample may be a baseline sample collected from a subject prior to initiation of any treatment. In some embodiments a control may include a sample or multiple samples from a group of subjects. As used herein, the term “sandwich immunoassay” refers to a method of determining the concentration of an analyte comprising a capture antibody, e.g., one immobilized on a surface and a detection antibody conjugated to a tag that can be detected by an instrument. More particularly, a sandwich immunoassay may use two antibodies, which may bind to different sites on an analyte. The capture antibody, which is highly specific for the analyte, is typically attached to a solid surface. The analyte is then added, followed by the addition of a second antibody referred to as the detection antibody. The detection antibody may bind to the analyte at different site than the capture antibody. As a result, the antigen is “sandwiched” between the two antibodies. In some embodiments, the concentration of the analyte may be determined relative to a standard curve. In some embodiments, the concentration of the analyte may be determined relative to other samples. In some embodiments, the concentration of the analyte may be determined relative to samples designated “control(s)”. In some embodiments, a sandwich immunoassay may comprise an enzyme-linked immunosorbent Attorney Docket No.08061.0067-00304 assay (ELISA). In some embodiments, a sandwich immunoassay may comprise measuring the response from the detection antibody by single molecule counting. As used herein, the term “level(s)” may be used interchangeably with “value(s)” and “concentration(s)” and refers to the amount of an analyte or biomarker. In some embodiments, the level is an absolute concentration determined using linear regression and interpolating experimental values from a standard curve. In some embodiments, the level is a relative amount of something compared to other samples (e.g., a fold change relative to control samples). In some embodiments, the level may be a numerical value obtained as an output from an instrument capable of detecting fluorescence. As used herein, the term “response” used in a context of measurement, detection or quantification refers to the numerical output of single molecule counting instrument during the quantification of the signal from the detection antibody. In some embodiments, the response is a measure of the signal, for example a fluorescent signal, from the tag, for example a fluorescent tag, on the detection antibody. In some embodiments, there is a positive correlation between the response and the levels of signal from the detection antibody. In some embodiments, there is a positive correlation between the response and the levels of PF. In some embodiments, the response indicates the relationship between the signal of the detection antibody and the level of the analyte. In some embodiments, the response is a measure of the signal, for example a fluorescent signal from the tag on the detection antibody. The response may be generated mathematically in part through the integration of the intensity of a fluorescence signal into a numerical measurement or value. In some embodiments, the level of an analyte (e.g., Aβ PF) may be reported as a numerical value “response” generated in part through the integration of the intensity of a fluorescence signal by a digital ELISA instrument. In some embodiments, the level may be a “response” Attorney Docket No.08061.0067-00304 generated in part through the integration of the intensity of a fluorescence signal by a single molecule counting instrument (e.g., SMCxPRO). As used herein, the term “single molecule counting’ is a method of quantifying individual molecules in a sample. In some embodiments, individual molecules are counted digitally. In some embodiments, single molecule counting quantifies a signal from a tag on a detection antibody. In some embodiments, single molecule counting quantifies a fluorescent signal from a tag on a detection antibody. In some embodiments, the readout from single molecule counting is a response corresponding to a number. In some embodiments, the response is positively correlated with the analyte concentration such that the higher the number associated with a response the higher the concentration of the analyte. In some embodiments, the instrument used for single molecule counting is an SMCxPRO. In some preferred embodiments, protofibril concentrations are determined by single molecule counting on an SMCxPRO. As used herein, the term “lower limit of quantification (LLoQ)” may refer to the lowest concentration accurately measured by an assay. As used herein, the term “upper limit of quantification (ULoQ)” may refer to the highest concentration accurately measured by an assay. As used herein, the term “selectivity” maybe used interchangeably with “analytical selectivity” and refers to the ability of an assay to differentiate and quantify the target compared to other components in a sample. For example, a typical biological sample comprising cerebrospinal fluid may include different forms of amyloid β peptide, tau protein, or forms of phosphorylated tau protein. Selectivity is a measure of the ability of the bioanalytical method described herein to differentiate these and other components that may be present in the biological sample from the target biomarker of the method. Interference attributable to an extraneous biomarker can be investigated by spiking the sample with the Attorney Docket No.08061.0067-00304 extraneous biomarker and noting its effect on measurement. See, e.g., Examples 4 and 5 herein. In some embodiments, the selectivity of the protofibril assay may be determined using a standard curve of Aβ1-42PF compared to standard curves of Aβ1-40and Aβ1-16monomers. As used herein, the term “sensitivity” maybe used interchangeably with “analytical sensitivity” and refers to the smallest amount of substance in a sample that can be accurately measured. In some embodiments, the sensitivity of an assay may be determined by the thresholds set by the LLoQ. In some embodiments, the sensitivity of an assay may be determined by the thresholds set by the LLoQ and the ULoQ. As used herein, the term “signal-to-noise ratio” is used interchangeably with “S / N ratio”, “S / N”, or “SNR”. The S / N ratio is a measure that compares the desired signal level to the level of background noise, i.e., signal generated non-specifically. The terms “BAN2401” and “lecanemab” are interchangeably used and refer to the anti-Aβ protofibril antibody comprising the sequences of CDRs, variable regions and full chains shown in Tables 1-3. Unless otherwise specified by context, the terms “BAN2401” and “lecanemab” respectively encompass any antibodies having the same primary amino acid sequences of BAN2401 or lecanemab or functional variants thereof, e.g., any biosimilars thereof. “Biosimilar” is a biotherapeutic product that is similar in terms of quality, safety, and efficacy to an already licensed reference biotherapeutic product, for example, defined in WHO guidelines (Guidelines on evaluation of similar Biotherapeutic Products (SBPs), Annex 2, Technical Report Series No.977, 2009) or in FDA Biosimilars Guidances. Aβ Protofibril Assay Without being bound by theory, the detection and quantification of protofibrils in biological samples has been limited due to the requirement to have a selective method of detection for the protofibril form of Aβ over other forms of Aβ (such as monomers) and the Attorney Docket No.08061.0067-00304 low concentration of protofibrils in biological samples. In some instances, the concentration of protofibrils is below the low limit of detection for an assay and may be in the femtomolar range. Provided herein is a method of quantifying Aβ protofibrils in biological samples. The assay may be sensitive to detect femtomolar concentrations of Aβ protofibrils. The assay may be selective for Aβ protofibrils compared to Aβ monomers. In some embodiments, the assay comprises obtaining a sample from a patient (e.g., a CSF sample or a blood sample) and contacting it with a capture antibody that selectively binds Aβ protofibrils. In some embodiments, the capture antibody is directly immobilized on a solid surface. In other embodiments, the capture antibody is immobilized in a subsequent step (e.g., using a second anti-capture antibody on a solid surface, or by using biotin- streptavidin system with a biotin-conjugated capture antibody and streptavidin on the surface to which the capture antibody will be immobilized, or by similar methods). In some embodiments, the sample is contacted with a detection antibody. The detection and capture antibodies may be incubated at the same time, or they may be incubated in separate steps (e.g., with intervening purification, washing and / or transfer, steps, etc.). Capture Antibody In various embodiments of the protofibril assay, a capture antibody is used that selectively binds to the epitope of Aβ protofibrils. The capture antibody may be immobilized on a surface. The capture antibody may be used in an assay to determine the concentration of the target Aβ protofibrils. In some embodiments, immobilization of the capture antibody on a surface facilitates target retrieval and washing to promote assay sensitivity and selectivity. In some embodiments, the capture antibody is selective for Aβ protofibrils, in some preferred embodiments, the capture antibody may be BAN2401 (also referred to herein as lecanemab). Attorney Docket No.08061.0067-00304 In some embodiments, the assay comprises an anti-protofibril antibody for the capture antibody and an antibody that does not bind the same epitope as the capture antibody for the detection antibody. In some embodiments, the capture antibody binds the C-terminus of Aβ protofibrils and the detection antibody binds an N-terminus. In some embodiments, the capture antibody is BAN2401 or comprises the CDRs or variable regions from BAN2401, or binds the same epitope as BAN2401 (for CDR, variable region, and full chain sequences, see Tables 1-3). In some embodiments, the protofibril assay, e.g., using BAN2401 as the capture antibody is capable of accurate determination of the protofibril concentration to the femtomolar level. In some embodiments, the assay is capable of doing so in the presence of therapeutic BAN2401, e.g., in samples from patients being treated therapeutically with BAN2401. In various embodiments, quantification of protofibrils uses an immobilized capture antibody that preferentially binds protofibrils, such as anti-protofibril antibody. In some embodiments, the capture antibody is an anti-Aβ protofibril antibody that selectively binds Aβ protofibrils. In some embodiments, the capture antibody binds to an epitope of Aβ protofibrils with a greater affinity and lower dissociation constant (KD) in comparison to other proteins. In some embodiments, the anti-Aβ protofibril antibody binds the same epitope and / or competes for binding with BAN2401. In some embodiments, the capture antibody binds to an epitope of Aβ protofibrils with a greater affinity and lower dissociation constant (KD) than to other forms of Aβ (e.g., oligomers, monomers). In some embodiments, the capture antibody comprises the CDRs (Table 1; SEQ ID NOs.: 1, 2, 3, 4, 5, and 6) and / or variable regions of BAN2401 (Table 2; SEQ ID NOs.: 7 and 8). In some embodiments, the capture antibody competes for binding with, and / or binds the same or similar epitope (e.g., in the C-terminal region of Aβ) as BAN2401. In some embodiments, the capture antibody Attorney Docket No.08061.0067-00304 competes for binding with BAN2401. In some embodiments, the capture antibody comprises BAN2401. In some embodiments, the capture antibody comprises the complementarity determining regions of Table 1. In some embodiments, the capture antibody comprises heavy chain complementarity determining regions (CDRs) (i.e., HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NOs: 1, 2, and 3 and light chain CDRs (i.e., LCDR1, LCDR2 and LCDR3) comprising amino acid sequences of SEQ ID NOs: 4, 5, and 6. In some embodiments, the capture antibody may have HCDR1, HDCR2, HCDR3 and LCDR1, LCDR2, LCDR3 amino acid sequences that have 80% to 100% sequence identity to the sequences of Table 1. In some embodiments, the capture antibody may have HCDR1, HDCR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences that have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99% or 100% sequence identity to the sequences of Table 1. In some embodiments, the capture antibody comprises the heavy chain variable region and light chain variable region of Table 2. In some embodiments, the amino acid sequence of the heavy chain variable region of the capture antibody has 80-100% sequence identity to SEQ ID NO: 7. In some embodiments, the amino acid sequence of the heavy chain variable region of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 7. In some embodiments, the amino acid sequence of the light chain variable region of the capture antibody has 80-100% sequence identity to SEQ ID NO: 8. In some embodiments, the amino acid sequence of the light chain variable region of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the heavy chain variable region of the capture antibody comprises an amino Attorney Docket No.08061.0067-00304 acid sequence of SEQ ID NO: 7 and the light chain variable region of the capture antibody comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the capture antibody comprises the heavy chain and the light chain amino acid sequences of Table 3. In some embodiments, the amino acid sequence of the heavy chain of the capture antibody has 80-100% sequence identity to SEQ ID NO: 9. In some embodiments, the amino acid sequence of the heavy chain of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the amino acid sequence of the light chain of the capture antibody has 80- 100% sequence identity to SEQ ID NO: 10. In some embodiments, the amino acid sequence of the light chain of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the heavy chain of the capture antibody comprises an amino acid sequence of SEQ ID NO: 9 and the light chain of the capture antibody comprises an amino acid sequence of SEQ ID NO: 10. In some embodiments, the capture antibody comprises the heavy chain constant region and the light chain constant region amino acid sequences of Table 4. In some embodiments, the amino acid sequence of the heavy chain constant region of the capture antibody has 80-100% sequence identity to SEQ ID NO: 11. In some embodiments, the amino acid sequence of the heavy chain constant region of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the amino acid sequence of the light chain constant region of the capture antibody has 80-100% sequence identity to SEQ ID NO: 12. In some embodiments, the amino acid sequence of the light chain constant region of the capture antibody has 80%, 81%, 82%, 83%, 84%, 85%, Attorney Docket No.08061.0067-00304 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the heavy chain constant region of the capture antibody comprises an amino acid sequence of SEQ ID NO: 11 and the light chain constant region of the capture antibody comprises an amino acid sequence of SEQ ID NO: 12. Immobilization of Capture Antibody In various embodiments, the capture antibody is immobilized on a surface. The term “immobilized” indicates that the capture antibody may be bound (e.g., covalently) to the surface, which may be chemically modified to bind the capture antibody. As used herein, the term “surface” may refer to a structure upon which an antibody (e.g., a capture antibody) is immobilized, either covalently or non-covalently. In some embodiments, the surface is a plate. In some embodiments, the surface is a particle. In some embodiments, the surface is a bead. In some embodiments, the surface is a magnetic microparticle (MP) bead that can be isolated from a solution by a magnet. In some embodiments, the surface is chemically modified to bind and immobilize the capture antibody. In some embodiments, the surface is Protein A or Protein G modified to bind monoclonal or polyclonal antibodies. In some embodiments, the capture antibody is immobilized to a surface through the interaction of streptavidin and biotin. In some embodiments, the surface is modified to covalently bind primary amine and / or sulfhydryl residues. In some embodiments, the surface is chemically modified with tosyl groups to make it “tosylactivated”, such as with p-Toluene-sulfonyl groups. In some embodiments, tosyl groups on a surface bind primary amino and sulfhydryl groups to conjugate proteins to the surface of the magnetic particle. In some embodiments, when the capture antibody is immobilized on a surface, immobilization may occur before, at the same time as, or after the capture antibody is Attorney Docket No.08061.0067-00304 incubated with the analyte-containing sample. In some embodiments, the capture antibody is immobilized on a surface before the capture antibody is incubated with a biological sample. In some embodiments, the capture antibody is conjugated to the surface. In some embodiments, the capture antibody is immobilized on the surface of a plate. In some embodiments, the plate comprises for example 12-wells, 24-wells, or 96-wells. In some embodiments, the plate is clear plastic, such as polystyrene. In some embodiments, the plate is opaque or black. In some embodiments, when the capture antibody is immobilized to the surface of a particle, the particle may be magnetic. In some embodiments, the particle is a magnetic bead. In some embodiments, the magnetic bead diameter is about 1 µm to about 5 µm. In some embodiments the magnetic bead is a silica-coated iron oxide magnetic bead grafted with functional groups to facilitate the immobilization of the capture antibody. In some embodiments, the particle is a magnetic microparticle grafted with functional groups to facilitate the immobilization of the capture antibody. In some embodiments, the capture antibody is immobilized to the magnetic particle by a streptavidin-biotin linker. In some embodiments, the capture antibody is immobilized via a streptavidin-biotin linker to the surface using the Single Molecule Counting (SMC™) Capture Labeling Kit from Merck. In some embodiments, the surface is modified to covalently bind primary amine and / or sulfhydryl residues, for example, by modification with p-Toluene-sulfonyl groups, or “tosylactivated”. Tosyl-activated surfaces provide reactive sulfonyl esters that covalently link antibodies or other ligands that contain primary amine or sulfhydryl groups to the surface. In some embodiments, tosyl groups bind primary amino and sulfhydryl groups to conjugate proteins to the surface of a magnetic particle. Attorney Docket No.08061.0067-00304 In some embodiments, the capture antibody is immobilized to a tosylactivated particle. In some embodiments, the capture antibody is immobilized to the particle by a covalent bond formed between the capture antibody and the tosylactivated particle. In some embodiments, the tosylactivated beads are Dynabeads™ MyOne™ (Invitrogen). In some embodiments, the capture antibody may be immobilized on a tosylactivated surface according to principles known in the art for the conjugation of molecules to surfaces (see, e.g., Otieno BA et al., Chapter Seven – Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads. Methods in Enzymology Volume 571, Pages 135-150, 2016). In some embodiments, the capture antibody is conjugated to the surface at a concentration of 10-40 µg of the capture antibody per mg of bead. In some embodiments, the tosylactivated beads are washed in sodium borate buffer. In some embodiments, the capture antibody is immobilized to the tosylactivated beads by incubating for 24 hours at 37°C with continuous rotation of the reaction tube. In some embodiments, the capture antibody is conjugated to the surface at a concentration of 10, 15, 20, 25, 30, 35 or 45 µg of antibody per mg of bead. In some embodiments, the capture antibody is conjugated to the surface at a concentration of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37,38, 39, 40 µg of antibody per mg of bead. In some embodiments, the capture antibody is conjugated to surface at a preferred concentration of 20 µg of antibody per mg of bead. In some preferred embodiments, the capture antibody is conjugated to the surface at a concentration of 20 µg of capture antibody per mg of a bead such as a magnetic bead, e.g., a tosylactivated magnetic bead. In some embodiments, the supernatant (e.g., containing unbound capture antibody) is discarded and the capture antibody immobilized to the surface is retained for the assay. In some embodiments, the capture antibody immobilized to the surface is blocked with blocking buffer (e.g., PBS with 0.5% BSA and 0.05% Tween20). In some embodiments, the immobilized capture antibody was Attorney Docket No.08061.0067-00304 blocked for 1 hour to 24 hours at 20°C to 40°C. In some embodiments, blocking of the capture antibody immobilized to the beads is performed with continuous agitation (e.g., continuous rotation). In some embodiments, the capture antibody immobilized to the surface is blocked in PBS with 0.5% BSA and 0.05% Tween20 overnight (e.g., 18 to 24 hours) at 37°C. In some embodiments, the capture antibody immobilized to the beads is stored (e.g., at 4°C) until use.

[0004] Attorney Docket No.08061.0067-00304 Detection Antibody In various embodiments of the assays disclosed herein, a detection antibody is used that is selective for the target, and which contains an epitope different from the capture antibody or is otherwise capable of binding to the target when bound by the capture antibody. In some embodiments, the detection antibody is conjugated to a detectable tag, e.g., to provide a readout of the presence and / or amount of the detection antibody present in a sample by an instrument capable of detecting the signal from the tag. In some embodiments, the detection antibody is an anti-Aβ antibody that selectively binds Aβ at an epitope distinct from that bound by the capture antibody. In some embodiments, the detection antibody binds to an epitope (e.g., in the N-terminal regions) of an Aβ protofibril with a greater affinity and lower dissociation constant (KD) than to other regions of Aβ or to other proteins. In some embodiments, the detection antibody binds to an epitope of Aβ protofibrils with a greater affinity and lower dissociation constant (KD) than to other forms of Aβ (e.g., oligomers, monomers). In some embodiments, the detection antibody binds to an epitope that becomes accessible for antibody binding when Aβ is organized into protofibrils. Table 16 contains exemplary anti-Aβ antibodies with predicted epitopes within the Aβ protein sequence. In some embodiments, the detection antibody binds the N-terminus of Aβ. In some embodiments, the detection antibody binds the first five amino acid residues of Aβ, Aβ1-5, of SEQ ID NO.: 28 or to an epitope comprising those first five amino acids. In some embodiments, the detection antibody binds the N-terminus of protofibrils, e.g., Aβ1-5(Table 9; SEQ ID NO.: 28). Exemplary detection antibodies with affinity to the N-terminus of Aβ include 3D6 (Table 6; SEQ ID NOs.: 19 and 20), 82E1 (Table 13; SEQ ID NOs.: 30 and 31) and 6E10 (Table 14; SEQ ID NOs.: 32 and 33) (Table 16). In some embodiments, 3D6 Attorney Docket No.08061.0067-00304 (Table 6; SEQ ID NOs.: 19 and 20) is an N-terminal binding antibody that may be used. In some embodiments, 3D6 may recognize an accessible epitope on Aβ protofibrils distinct from that bound by the capture antibody. In some embodiments, the detection antibody is 3D6 or comprises the CDRs and / or variable regions from 3D6 (Table 5, SEQ ID NOs.: 13, 14, 15, 16, 17, and 18; Table 6, SEQ ID NOs.: 19, 20). In some embodiments, antibodies with affinity to the C-terminus of Aβ are not used as detection antibodies, for example 21F12 (Table 15; SEQ ID NOs.: 34 and 35) and H31L21 (Table 16). In some embodiments, antibodies binding the C-terminus are used as detection antibodies. In some embodiments, lecanemab is used as the detection antibody, or an antibody comprising the CDRs and / or variable regions from lecanemab (Table 1, SEQ ID NOs.: 1, 2, 3, 4, 5, and 6; Table 2, SEQ ID NOs.: 7 and 8). In some embodiments, the detection antibody comprises the CDRs and / or variable regions from 82E1 (Table 13, SEQ ID NOs.: 30 and 31). In some embodiments, the detection antibody is 82E1. In some embodiments, the detection antibody binds amino acid residues 3-8 of Aβ, Aβ3-8. In some embodiments, the detection antibody comprises the CDRs and / or variable regions from 6E10 (Table 14, SEQ ID NOs.: 32 and 33). In some embodiments, the detection antibody is 6E10. In some embodiments, the detection antibody competes for binding or binds the same epitope as any one or more of antibodies 3D6 (Table 6, SEQ ID NOs.: 19 and 20), 82E1 (Table 13, SEQ ID NOs.: 30 and 31), and 6E10 (Table 14, SEQ ID NOs.: 32 and 33). In some embodiments, the detection antibody binds the same epitope or competes for binding with BAN2401. In some embodiments, the detection antibody comprises the CDRs and / or variable regions from BAN2401 (Table 1, SEQ ID NOs.: 1 (HCDR1), 2 (HCDR2), 3 (HCDR3), 4 (LCDR1), 5 (LCDR2), 6 (LCDR3); Table 2, SEQ ID NOs: 7 and 8). In some embodiments, the detection antibody is BAN2401. Attorney Docket No.08061.0067-00304 In some embodiments, the detection antibody comprises a set of six complementarity determining regions from the sets in Table 5. In some embodiments, the detection antibody comprises heavy chain complementarity determining regions (CDRs) (i.e., HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NOs.: 13 (HCDR1), 14 (HCDR2), and 15 (HCDR3) and light chain CDRs (i.e., LCDR1, LCDR2 and LCDR3) comprising amino acid sequences of SEQ ID NOs.: 16 (LCDR1), 17 (LCDR2), and 18 (LCDR3). In some embodiments, the detection antibody may have HCDR1, HDCR2, HCDR3 and LCDR1, LCDR2, LCDR3 amino acid sequences that have 80% to 100% sequence identity to the sequences of Table 5. In some embodiments, the detection antibody may have HCDR1, HDCR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences that have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to the sequences of Table 5. In some embodiments, the detection antibody comprises the heavy chain variable region and light chain variable region of Table 6. In some embodiments, the heavy chain variable region has 80-100% sequence identity to SEQ ID NO.: 19. In some embodiments, the heavy chain variable region has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO.: 19. In some embodiments, the light chain variable region has 80-100% sequence identity to SEQ ID NO.: 20. In some embodiments, the light chain variable region has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, 99% or 100% sequence identity to SEQ ID NO.: 20. In some embodiments, the heavy chain variable region of the detection antibody comprises an amino acid sequence of SEQ ID NO.: 19 and the light chain variable region of the detection antibody comprises an amino acid sequence of SEQ ID NO.: 20 Attorney Docket No.08061.0067-00304 Detectable Labels In some embodiments, the detection antibody (e.g., 3D6) is conjugated to a detectable label, e.g., a tag e.g., a fluorophore. In some embodiments, the detection antibody is tagged using an antibody labeling kit, such as the SMC™ Detection Antibody Labeling kit (EMD Millipore).In some embodiments, the concentration of the detection antibody may be determined by measuring the level of the signal from the tag, e.g., by measuring the absorbance of a fluorescently-tagged detection antibody, e.g., at 280 nm In some embodiments, the detection antibody conjugated to a fluorophore is diluted in a buffer, for example, Discovery Assay Buffer™ (EMD Millipore). In some embodiments, the detection antibody is diluted, e.g., to a concentration of 100 ng / mL, e.g., in Discovery Assay Buffer and filtered, e.g., through a 0.22 µm syringe filter (EMD Millipore). In some embodiments, a solution of detection antibody is stored at 4°C. As used herein, the term “tag” refers to a discrete unit that functions to detectably label a protein, i.e., is detectable and / or quantifiable by one or more means. In some embodiments, the tag is a detectable protein attached to an antibody disclosed herein. In some embodiments, the tag is a protein that may be incorporated into the 3’ or 5’ end of a DNA sequence of a protein coding gene in need of labeling (e.g., a detection antibody) to generate a fusion protein. In some embodiments, the tag is conjugated via chemical bonds, for example covalent chemical bonds. In some embodiments, the tag is conjugated to the labeled protein through the interaction of biotin and streptavidin. In some embodiments, the tag produces a fluorescent signal. In some embodiments, the tag is a quantum dot. In some embodiments, the tag is a fluorophore that absorbs photons of light and emits photons at a different wavelength. In some embodiments, the tag is a fluorescent protein, for example a protein that has inherent fluorescence, such as enhanced green fluorescent protein (EGFP) or yellow fluorescent protein (YFP). Attorney Docket No.08061.0067-00304 Generally, the tag is added to end of the protein in need of labeling. In some embodiments, the tag is conjugated to the detection antibody before, at the same time as, or after the detection antibody binds the analyte (e.g., forms the second immune complex). In some embodiments, the tag is conjugated to the detection antibody before binding the analyte and / or forming the second immune complex. In some embodiments, the tag is conjugated to the detection antibody through the interaction of biotin and streptavidin. In some embodiments, the tag is conjugated to streptavidin and labels biotinylated detection antibody. In some embodiments, the tag is conjugated to biotin and labels streptavidin conjugated detection antibody. In some embodiments, the tag is a quantum dot, with optical and electronic properties that can be detected. In some embodiments, the tag is modified to bind primary amine groups of proteins, as a method of conjugating the tag and the detection antibodies. In some embodiments, the tag is a SULFO-TAG, for example a SULFO-TAG NHS-Ester. In some embodiments, the tag is a SULFO-TAG comprising N-hydroxysuccinimide esters that can bind to primary amine groups of proteins, including detection antibodies. In some embodiments, the SULFO-TAG comprises a fluorescent tag. In some embodiments, the tag (e.g., the fluorescent tag), e.g., the quantum dot, is attached to 3D6 as the detection antibody. Biological Sample In various embodiments, protofibril levels are determined in one or more biological samples obtained from a subject, e.g., samples obtained over the course of treatment. In one embodiment, the biological sample is obtained from a subject treated with lecanemab. In some embodiments, a biological sample obtained from a human subject may Attorney Docket No.08061.0067-00304 comprise body fluid or tissue. In some embodiments, human subject is alive. In some embodiments, the subject is deceased. In some embodiments, the biological sample is tissue (e.g., brain tissue) obtained during a biopsy. In some embodiments, the biological sample is brain tissue obtained from a deceased individual. In some embodiments, the brain tissue is made into a brain tissue homogenate and / or tissue lysate. In some embodiments, the biological sample is a blood sample, including plasma and / or serum. In some embodiments, the biological sample is cerebrospinal fluid (CSF). In some embodiments, the biological sample contains no anti- Aβ antibody. In some embodiments, the biological sample contains an anti- Aβ antibody. In some embodiments, the biological sample contains lecanemab. In some embodiments, an Aβ protofibril level is measured in an undiluted biological sample, which may also be referred to as a “neat” sample. In some embodiments, the biological sample is diluted to a specific total protein concentration. In some embodiments, the biological sample is prepared at a concentration of 0.1 mg to 10 mg protein per ml of sample. In some embodiments, the biological sample is diluted at least 10-fold prior to the assay. In some embodiments, the sample is diluted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold. In some embodiments, the sample is diluted 1:1 with sample buffer. In some embodiments, the sample is diluted 1-fold to 4-fold. In some embodiments, the sample is diluted 1-fold, 2-fold, 3-fold, or 4-fold. In some embodiments the sample is diluted 1.5-fold. In some embodiments, the sample is diluted 2-fold. In some embodiments, the sample is diluted 3-fold. In some embodiments, the sample is diluted at least 100-fold prior to the assay. In some embodiments, the sample is diluted at least 1,000-fold prior to the assay. In some embodiments, the biological sample is treated to maintain the level of Aβ protofibrils. In some embodiments, the biological sample is diluted in a sample buffer to maintain the Aβ protofibril level. Attorney Docket No.08061.0067-00304 In some embodiments, the sample is diluted about 1.5-fold in a sample buffer, e.g., any of the sample buffers disclosed herein. In some embodiments, the CSF sample is diluted about 1.5-fold. In some embodiments, the CSF sample is diluted 1.5-fold. In some embodiments, the sample is diluted about 2-fold in a sample buffer, e.g., any of the sample buffers disclosed herein. In some embodiments, the CSF sample is diluted about 2-fold. In some embodiments, the CSF sample is diluted 2-fold. In some embodiments, the sample is diluted about 3-fold in a sample buffer, e.g., any of the sample buffers disclosed herein. In some embodiments, the CSF sample is diluted about 3-fold. In some embodiments, the CSF sample is diluted 3-fold. In some embodiments, the biological sample is prepared in low protein binding microcentrifuge tubes. In some embodiments, the low protein binding tubes reduce sample- to-surface binding and sample loss. In some embodiments, the low protein binding microcentrifuge tubes are made from plastic, such as polypropylene, and may be treated to reduce protein binding. In some embodiments, the low protein binding tubes are Protein LoBind® Tubes (Eppendorf). In some embodiments, the biological sample is stored frozen, e.g., at around -80°C. In some embodiments, the biological sample is thawed at room temperature. In some embodiments, the biological sample is thawed on wet ice. In some embodiments, the sample is gently mixed by pipetting or vortexing to avoid air entering the sample and bubble formation. In some embodiments, the biological samples are not freeze thawed. In some embodiments, the biological samples are not freeze- thawed more than once. In some embodiments, the biological samples are not freeze-thawed more than 2 times. In some embodiments, the biological samples are not freeze-thawed more than 3 times. In some embodiments, the biological samples are freeze-thawed less than 3 times. Attorney Docket No.08061.0067-00304 Assay Conditions In some embodiments, prior to incubating the immobilized capture antibody with a sample comprising the analyte (e.g., Aβ protofibrils), the antibody is washed to remove capture antibody that is not immobilized on a surface. In some embodiments a sample comprising analyte is contacted with capture antibody to form a first immune complex. In some embodiments, the first immune complex comprises the immobilized capture antibody bound to the analyte (e.g., Aβ protofibrils). In some embodiments, the first immune complex is washed to remove unbound material in the biological sample. In some embodiments, the sample and / or first immune complex is contacted with the detection antibody to form a second immune complex. In some embodiments, the second immune complex comprises the capture antibody (e.g., immobilized capture antibody), the analyte, and the detection antibody. In some embodiments, the second immune complex is washed to remove excess, unbound detection antibody. In some embodiments, the capture antibody is diluted, e.g., in Discovery Assay Buffer (EMD Millipore). In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody at room temperature. In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody at 27°C. In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody at 4°C. In some embodiments, the first immune complex is formed in a still solution, or solution only agitated intermittently (e.g., tapping periodically, e.g., every 20-30 minutes). In some embodiments, the first immune complex is formed in a continuously agitated solution (e.g., on a plate shaker, tube rotator). In some embodiments, the first immune complex formed in a solution incubated on a plate shaker set to 600-1000 rpm. In some embodiments, the first immune complex formed in Attorney Docket No.08061.0067-00304 a solution incubated on a plate shaker set to 700-900 rpm. In some preferred embodiments, the first immune complex formed in a solution incubated on a plate shaker set to 800 rpm. In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody for 0.5-5 hours. In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 hours. In some embodiments, the first immune complex is formed by incubating the biological sample with the capture antibody for 1-24 hours. In some embodiments, the first immune complex is formed by incubating the biological sample with the immobilized capture antibody for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the first immune complex is formed by incubating the biological sample with the immobilized capture antibody for 0.5-2 days. In some embodiments, the first immune complex is formed by incubating the biological sample with the immobilized capture antibody for 0.5, 1, 1.5, or 2 days. In some preferred embodiments, the first immune complex is formed by incubating the biological sample with the immobilized capture antibody for 2 hours with continuous agitation by a plate shaker set to 800 rpm at 27°C. In some embodiments, after an incubation period to form the first immune complex between the capture antibody and analyte, the first immune complex is washed, e.g., with a buffer, e.g., 10-fold diluted System Wash Buffer with Proclin™ (EMD Millipore) to remove unbound materials. In some embodiments, after the incubation period the first immune complex is washed using the SMC™ (EMD Millipore) washer. In some embodiments, a second immune complex is formed by incubating the sample containing the first immune complex with the detection antibody in a buffer, e.g., a Attorney Docket No.08061.0067-00304 dilution buffer. In some embodiments, the detection antibody is diluted with a buffer, such as Discovery Assay Buffer™ (EMD Millipore). In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody at room temperature. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody at 27°C. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody at 4°C. In some embodiments, the second immune complex is formed in a still solution, or solution only agitated intermittently (e.g., tapping periodically, e.g., every 5-30 minutes). In some embodiments, the second immune complex is formed in a continuously agitated solution (e.g., on a plate shaker or tube rotator). In some embodiments, the second immune complex is formed in a solution incubated on a plate shaker set to 600-1000 rpm. In some embodiments, the second immune complex formed in a solution incubated on a plate shaker set to 700-900 rpm. In some preferred embodiments, the second immune complex is formed in a solution incubated on a plate shaker set to 800 rpm. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody for 0.5-5 hours. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 hours. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody for 1-24 hours. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the second immune complex is formed by incubating the first immune complex with the detection antibody for 0.5-2 days. In some embodiments, the Attorney Docket No.08061.0067-00304 second immune complex is formed by incubating the first immune complex with the detection antibody for 0.5, 1, 1.5, or 2 days. In some embodiments, the second immune complex is formed by incubating the sample containing the first immune complex with the detection antibody for 1.5 hours with continuous agitation by a plate shaker set to 800 rpm at 27°C. In some embodiments, the second immune complex is washed with a wash buffer comprising Triton-X. In some embodiments, the buffer comprises 1-5% Triton-X-100, boric acid 0.1-1%, disodium tetraborate decahydrate 0.1-1% and 2-methyl-4-isothiazolin-3-one and ProClin (e.g., ProClin 300). In some embodiments, after the incubation period the second immune complex is washed with a buffer, such as 10-fold diluted SMC™ System Wash Buffer with ProClin™ (EMD Millipore). In some embodiments, after the incubation period the first immune complex is washed using the SMC (EMD Millipore) washer apparatus. In some embodiments, the Aβ protofibril level may be determined using an instrument capable of measuring a signal from the tag conjugated to the detection antibody. In some embodiments, the signal is measured using a plate reader. In some embodiments, the signal is measured using a fluorescence microscope. In some embodiments, the signal is measured using a digital ELISA. In some embodiments, the signal is measured using a digital single molecule counting instrument (e.g., SMCxPRO). In some embodiments, the instrument is a SMCxPRO. In some embodiments, the detection antibody in the second immune complex and / or the tag on the second antibody is dissociated to measure the signal from the tag of the detection antibody. In some preferred embodiments, the detection antibody comprises an antibody conjugated to a fluorescent tag. In some embodiments, the second immune complex is dissociated in an acidic elution buffer (e.g., Elution Buffer B™, EMD Millipore). In some embodiments, the second immune complex is incubated in the elution buffer for 1-20 Attorney Docket No.08061.0067-00304 minutes. In some embodiments, the second immune complex is incubated in the elution buffer for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes. In some embodiments, the second immune complex is incubated in the elution buffer at room temperature. In some embodiments, the second immune complex is incubated in the elution buffer at 27°C. In some embodiments, the second immune complex is incubated in the elution buffer at 4°C. In some embodiments, the second immune complex is incubated in the elution buffer without agitation and / or with intermittent mixing (e.g., at 1-2 minute intervals). In some embodiments, the second immune complex is incubated in the elution buffer with continuous agitation (e.g., plate shaker or tube rotator). In some highly preferred embodiments, the second immune is dissociated by incubation in the elution buffer for 10 minutes on a plate shaker at 800 rpm at 27°C. In some embodiments, the eluate resulting from incubation with the elution buffer and comprising the detection antibody and / or tag dissociated from the second immune complex is diluted with buffer that may neutralize the pH of the elution buffer (e.g., Buffer D™, EMD Millipore). In some embodiments, the signal from the dissociated detection antibody is measured by digital ELISA. In some embodiments, the signal from the dissociated detection antibody is measured by single molecule counting. In some embodiments, the signal from the dissociated detection antibody is measured using a Single Molecule Array (Simoa®) instrument (Quanterix). In some embodiments, the signal from the dissociated detection antibody is measured using a SMCxPRO® instrument (EMD Millipore). In some embodiments, the Aβ protofibril level of a biological sample may be determined relative to a control sample from a healthy subject. In some embodiments, the Aβ protofibril level of a biological sample may be determined relative to the mean Aβ protofibril level of two or more healthy subjects. In some embodiments, Aβ protofibril levels may be Attorney Docket No.08061.0067-00304 reported in a subject as a fold change relative to a baseline level of Aβ protofibril level in the same subject prior to treatment, e.g., with lecanemab. In some embodiments, a control sample is obtained from an age and / or gender matched subject. In some embodiments, a control sample is obtained from a subject with co-morbidities that are not associated with AD. In some embodiments, a control sample is obtained from a subject with mild cognitive impairment and / or early AD. In some embodiments, a control sample is obtained from a subject who does not have AD. In some embodiments, a control sample is obtained from the same subject as the test sample, but obtained prior to the start of treatment, e.g., to establish a baseline level of protofibril before treatment, e.g., treatment with lecanemab. In some embodiments, the Aβ protofibril level is a concentration determined using an Aβ1-42 protofibril standard curve. In some embodiments, the concentration of protofibrils in a biological sample may be determined by linear regression from a Aβ protofibril standard curve. In some embodiments, the Aβ protofibril standard is generated from known amounts of oligomerized Aβ monomers, for example Aβ1-42 monomers. In some embodiments the Aβ protofibril standard is generated by size exclusion chromatography (SEC). In some embodiments, the Aβ protofibril standard is generated by incubating Aβ1-42 monomers dissolved in 10 mM NaOH to form 100 µM and then diluted to 50 µM with 2X PBS. In some embodiments, the Aβ protofibril standard is generated by incubating 50 µM of dissolved Aβ1-42 monomers at 37°C for 90 min to form the protofibrils. In some embodiments, the Aβ protofibril standard is isolated by size exclusion chromatography with Superdex 7510 / 600 GL (GE). In some embodiments, the concentration of the Aβ1-42 protofibril standard is determined with V-PLEX Aβ1-42 Peptide (4G8) Kit (Meso Scale Diagnostics). In some embodiments, the Aβ protofibril standard is diluted to a stock concentration of 100 nM. In some embodiments, the Aβ protofibril standard is diluted to a stock concentration with PBS +0.6% Tween 20. In some embodiments, the protofibril standard is stored at -80°C. In some Attorney Docket No.08061.0067-00304 embodiments, the Aβ protofibril standard is diluted from 2 pM to 0.003 pM. In some embodiments, the Aβ protofibril standard is diluted in a diluent, for example Discovery Standard Diluent™ (EMD Millipore). In some embodiments, the Aβ protofibril standard is generated by a serial dilution (e.g., a 2-fold serial dilution). In some embodiments, the standard curve is generated from at least three and up to 10 concentrations to generate a range of points for the standard curve. In some embodiments, alternate assays may be used that are capable of detecting a signal from the anti-protofibril capture antibody, e.g., using alternate detection devices and / or labels such as horse radish peroxidase (HRP). In some embodiments, these assays may use lecanemab as the capture antibody and 3D6 as the detection antibody. In some embodiments, these assays may comprise plate-based sandwich ELISAs and / or the detection of a signal from the labeled detection antibody. In some embodiments, these protofibril assays may comprise detection by a Quanterix Simoa® instrument. In some embodiments, these protofibril assays may comprise single molecule counting assays, e.g., with detection by a SMCxPRO® instrument. In some embodiments, these assays may have a lower limit of quantification greater than 1 pM and detect concentrations of PF greater than 1 pM in samples. In some embodiments, these assays may be used to for the further identification of capture antibody and the detection antibody pairs. In some embodiments, these assays may quantify PF generated from recombinant Aβ peptides. In some embodiments, these assays quantify PF generated from recombinant Aβ peptides and diluted in body fluid (e.g., cerebrospinal fluid). Attorney Docket No.08061.0067-00304 Uses of the Aβ Protofibril Assay The PF assay methods discussed herein may be used to quantify Aβ Protofibril in a biological sample (e.g., a blood sample, a CSF sample, etc.). The disclosure and methods discussed herein may be used for determining an Aβ protofibril level in a biological sample (e.g., a blood sample, a CSF sample), e.g., by comparison to a control level. The biological sample may contain an anti-Aβ antibody (e.g., lecanemab). Kits In various embodiments, kits are disclosed herein, for detecting Aβ protofibrils in biological samples, comprising one or more of the capture antibodies or antigen binding fragments described herein and one or more of the detection antibodies or antigen binding fragments described herein. In certain embodiments, the kit comprises a capture antibody or antigen binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; and wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the capture antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the capture antibody Attorney Docket No.08061.0067-00304 comprises antibody BAN2401 or an antigen binding fragment thereof. In some embodiments, the kit comprises a capture antibody or antigen binding fragment immobilized to a surface, preferably a magnetic particle. In certain embodiments, the detection antibody or antigen binding fragment in the kit comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and the light chain variable region comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 15; and wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 18. In certain embodiments, the detection antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 19 and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the detection antibody comprises antibody 3D6 or an antigen binding fragment thereof. In some embodiments, the kit comprises a detection antibody or antigen binding fragment conjugated to a tag, preferably a fluorescent tag, such as a fluorescent tag detectable by a single molecule counting instrument (e.g., SMCxPRO). In various embodiments, the kit comprises: an immobilized capture antibody selective for Aβ protofibrils, a fluorescently tagged detection antibody capable of binding Aβ protofibrils already bound to the capture antibody, sample dilution buffer, washing buffer(s), and dissociation buffers. In another embodiment, any of the preceding kits may further Attorney Docket No.08061.0067-00304 comprise tubes treated to reduce protein binding (e.g., Protein LoBind® Eppendorf for example 1.5 ml tubes). In another embodiment, any of the preceding kits may further comprise instructions for using the one or more antibodies or antigen binding fragments to detect Aβ protofibrils in a sample from a subject according to the methods disclosed herein. The instructions may call for the sample to be cerebrospinal fluid or blood. The kits may further comprise additional components for use in sample collection and / or with a classic ELISA or a digital ELISA.

[0005] Attorney Docket No.08061.0067-00304 EXAMPLES The present disclosure is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference in their entirety for all purposes. Example 1. Aβ and biological fluid preparation Aβ1-42Protofibril Aβ1-42Protofibril (PF) was produced by the method below. Aβ1-42 Protofibril was made from Aβ1-42 aggregates by Size Exclusion Chromatography (SEC) purification. In brief, Aβ1-42peptide (AnaSpec, USA, Cat#64129) was dissolved in 10 mM NaOH to form 100 µM and then diluted to 50 µM with 2X PBS.50 µM solution was incubated at 37°C for 90 min to form the protofibrils. After the incubation, 10% Tween20 was added to form 0.6% Tween20 in solution and the protofibril solution was centrifuged at 16,000 g for 5 min at room temperature. The supernatant was collected for further purification by size exclusion chromatography to separate PF from small oligomer and monomer. Superdex 7510 / 600 GL (GE, Cat#1004-90) was used for the purification. Monitoring 214 nm, first peak which was eluted in void volume was collected as Aβ1-42 PF. 6.5 µM Aβ1-42PF solution was aliquoted in Protein LoBind tube 1.5 mL (Eppendorf, Germany) and stored at -80°C before use. Concentration of Aβ1-42 PF was determined using V-PLEX Aβ42 Peptide (4G8) Kit (Meso Scale Diagnostics, USA, Cat#K150-SLE) by the measurement of PF solution treated with 50% formic acid followed by neutralization of pH. 6.5 µM PF solution was further diluted to 100 nM with PBS+0.6% Tween20 and aliquoted as the working stock. Working stock was stored at -80°C until use. Attorney Docket No.08061.0067-00304 Aβ1-40, Aβ1-16peptide Lyophilized Aβ1-40peptide (Cat#4307-v) and Aβ1-16peptide (Cat#4359-v) were purchased from Peptide Institute, Inc. (Osaka, Japan). They were dissolved in 10 mM NaOH to make 100 µM solution. They were used for the evaluation of assay selectivity after appropriately diluted with Discovery Standard Diluent (EMD Millipore, USA, Cat#02-0560-00). Human brain homogenate Human brain tissues were purchased from TRANS-HIT BIOMARKERS (Canada).0.5 g of brain tissue was frozen with liquid nitrogen and crushed by MULTI-BEADS SHOCKER MB601(S) (Yasui Kikai, Japan). To the crushed tissue, buffer solution (1mM NaHCO3, 1mM MgCl2, 0.5mM CaCl2, 10% Sucrose, protease inhibitor cocktail (Nakalai Tesque, Japan, Cat#03969-21) and phosphatase inhibitor cocktail (Nakalai Tesque, Japan, Cat#07575-51) was added to make 200 mg tissue weight / mL homogenate. According to the protein concentration determined using Pierce 660 nm Protein Assay Kit (Thermofisher, USA, Cat#), each homogenate was diluted to 3 mg / mL protein concentration. The homogenate diluted to 3 mg / mL protein concentration was aliquoted and stored at -80°C before use.

[0006] Attorney Docket No.08061.0067-00304 Human cerebrospinal fluid Human cerebrospinal fluid (CSF) was purchased from PrecisionMed, LLC. (USA) and stored at -80°C before use, and avoided freeze-thaw cycling. In the case of sample preparation, spike recovery and dilution test, Protein LoBind 1.5 mL tube (Eppendorf, Germany) was used. CSF was thawed in room temperature and mixed gently by vortex mixer on the day of use. BAN2401 BAN2401103 mg / mL solution was aliquoted and stored at -80°C until use. Example 2. Conjugation and labeling of antibodies Preparation of BAN2401 immobilized magnetic particles BAN2401 was immobilized to Dynabeads MyOne Tosylactivated (Invitrogen, USA, Cat#65501) following to instructions of the beads, and used for the assay on SMCxPRO (EMD Millipore, USA). Briefly, Tosylactivated beads were washed by 0.1M sodium borate buffer (pH 9.5) several times. After initial wash, BAN2401 and 0.1M sodium borate buffer (pH 9.5) and 3M ammonium sulphate were added to the beads and then reaction mixture was incubated for 24 hour at 37°C with continuous rotation of the reaction tube, 20 µg of BAN2401 was added per 1 mg of beads. After the generation of covalent link between BAN2401 and the beads, supernatant was removed and blocking buffer (PBS with 0.5% BSA and 0.05% Tween20) was added to the remaining beads, and then reaction tube was further incubated for overnight at 37°C with continuous rotation. After blocking, the beads were washed several times with wash / storage buffer (PBS with 0.1% BSA and 0.05% Tween20) Attorney Docket No.08061.0067-00304 and then beads solution was prepared for 20 mg / mL.20 mg / mL BAN2401 immobilized beads solution was used as stock solution and stored at 4°C until use. Preparation of dye-labeled 3D6 Anti Aβ N-terminal antibody clone 3D6 was purchased from ADx Neurosciences NV (Gent, Belgium). Labeling with fluorescent dye was conducted using SMC Detection Antibody Labeling Kit (EMD Millipore, USA, Cat# 03-0076-02) following the kit instruction. Concentration of the labeled antibody was determined from the absorbance at 280 nm. The dye labeled 3D6 was stored at 4°C until use. Example 3. Detection of Aβ1-42PF BAN2401 was covalently immobilized to the magnetic particles (MP), Dynabeads MyOne Tosylactivated (Invitrogen, USA) according to the instruction manual of the beads. In 96 well 500μL V-bottom plate (Axygen, USA, Cat# P-96-450V-C), BAN2401-bound MP solution which was diluted to 100 µg / mL in Discovery Assay Buffer (EMD Millipore, USA, Cat#02-0474-00) was added at 50 µL to 150 µl of Aβ1-42 PF which was diluted from 2 to 0.003 pM in Discovery Standard Diluent (EMD Millipore, USA, Cat#02-0560-00) followed by incubation for 2 h at 27°C with 800 rpm shaking on the plate shaker (BIOSAN LTD., Latvia, Cat# PST-60HL-4). After washing with 10-fold diluted System / Wash Buffer w / Proclin (EMD Millipore, USA, Cat#02-0111-03) by the washer for SMC (EMD Millipore, USA, Cat#95-0004-05), Fluorescent-labeled detection antibody 3D6 was diluted with Discovery Assay Buffer to a final concentration of 1000 ng / mL and filtered through a 0.22 µm syringe filter (EMD Millipore, USA, Cat#SLGPR33RS) and the diluted antibody was added at 20 µL / well. The 96-well was incubated for 1.5 hour at 27°C with 800 rpm shaking. After washing step, buffer was aspirated from well and 11.5 µL / well of Elution Buffer B Attorney Docket No.08061.0067-00304 (EMD Millipore, USA, Cat#02-0297-00) was added, followed by 10 min incubation at 27°C with 800 rpm shaking. After the 10 min incubation, 10 µL of eluate was collected and transferred to 384 glass bottom plate (Aurora, USA, Cat#ABB2-00160A) which was filled with 10 µl / well of Buffer D (EMD Millipore, USA, Cat#02-0368-00). The plate was sealed with Nunc sealing tape for multiwell plates (Thermofisher Scientific, USA, Cat#276014) and then it was read by the SMCxPRO instrument. Response (readout of SMCxPRO) was obtained depending on Aβ1-42PF concentration even at very low level as tens of fM order (Fig.1). LLOQ (Lower Limit Of Quantification) and ULOQ (Upper Limit Of Quantification) of this assay were 0.01 pM and 2 pM, respectively. Example 4. Selectivity to PF, compared to monomer Aβ Aβ1-42 PF, Aβ1-40, and Aβ1-16 were measured on SMCxPRO. Each Aβ species was diluted with Discovery Standard Diluent (EMD, USA, Cat#02-0560-00) in Protein LoBind 1.5 mL tube (Eppendorf, Germany). Selectivity was estimated by dividing the interpolated values by the theoretical values based on an Aβ1-42PF standard curve at comparable protein concentrations. Aβ1-16 was used to represent complete monomer which includes epitopes of both BAN2401 and 3D6 (Englund et al. “Sensitive ELISA detection of amyloid-β protofibrils in biological samples”, Journal of Neurochemistry, 2007, 103, 334–345). In the preparation of Aβ1-40 solution, formation of oligomerized Aβ species were not avoidable even amount of them was very small. Aβ1-16was therefore more precisely reflect cross-reactivity to monomer of this PF assay rather than Aβ1-40. Calculated selectivity to PF was 3.2 or 2 million-fold greater than that to Aβ1-16. This assay was over 1,000,000-fold selective to PF compared to Aβ monomer (Fig.2). Attorney Docket No.08061.0067-00304 Example 5. Dilution linearity, spike recovery 100 nM PF stock was diluted with Discovery Standard Diluent to make 10 pM and 5 pM PF solution. Dilution linearity: 6.2 µL of 10 pM Aβ1-42PF was spiked to 613.8 µL CSF and 2-fold serially diluted. The samples were measured on SMCxPRO. Spike Recovery: 6.2 µL of 5 pM Aβ1-42PF or Discovery Standard Diluent was spiked to 303.8 µL CSF (Spiked in CSF and neat CSF).6.2 µL of 5 pM Aβ1-42 PF was spiked to 303.8 µL Discovery Standard Diluent and measured. Measured value of the sample was PF spiked (Spiked in buffer). From neat to 8-fold dilution, good dilution linearity was obtained (Fig.3). Spike recovery was also good (Table 10). It was confirmed that this assay was not interfered from sample matrix. Example 6. BAN2401 interference 103 mg / mL BAN2401 was diluted with Discovery Standard Diluent (EMD Millipore, USA, Cat#02-0560-00) to the concentration of 25 µg / mL.6.2 µL of 25 µg / mL BAN2401 or Discovery Standard Diluent was spiked to 303.8 µL of CSF to make samples containing 0 or 500 ng / mL BAN2401. Samples were prepared using Protein LoBind 1.5 mL tube (Eppendorf, Germany). They were used for the assay on SMCxPRO and their measured values were compared. Addition of BAN2401 to CSF did not interfere the assay result (105% compared to BAN24010 ng / mL) (Fig.4). It was confirmed that this assay was not affected by the Attorney Docket No.08061.0067-00304 presence of 500 ng / mL BAN2401 in CSF. This indicates that the assay can be used to quantify Aβ PF in a sample from a subject administered lecanemab. Example 7. Measurement of brain homogenate Brain homogenate (N=12, 6 AD and 6 Control) was measured on SMCxPRO. Before measurement, each homogenate was centrifuged at 13,000 g for 10min and then supernatant was 5000-fold diluted with Discovery standard diluent (EMD Millipore, USA, Cat#02-0560- 00). The diluted supernatant was used for the assay on SMCxPRO. Calibration curve was generated using Aβ1-42 PF. It was not statistically significant but brain homogenate from AD showed higher trend compared to that from control (Fig.5). Example 8. CSF measurement AD and Control CSF specimens purchased from PrecisionMed were measured on SMCxPRO. Annotations (AD or Control) were assigned by the vender based on symptoms. CSF were stored at -80°C before use. The samples were thawed and mixed on the day of use. CSF were used as neat for the measurement. All CSF specimens could be detected in the measurement range (0.01 pM-2 pM). Significant difference was not observed between AD and Control groups (Fig.6(A) and Fig. 6(B)). Example 9. Evaluation of anti-Aβ detection antibodies by sandwich ELISA In combination with BAN2401 as a capture antibody, detection antibodies were evaluated for Aβ1-42 PF detection by sandwich ELISA. Three anti Aβ N-terminal antibodies and two anti-Aβ 1-42 C-terminal antibodies were evaluated (Table 16). The N-terminal Attorney Docket No.08061.0067-00304 antibodies are 3D6 (ADx Neurosciences, Belgium), 82E1 (IBL, Japan) and 6E10 (Biolegend, USA). The C-terminal antibodies are 21F12 (ADx Neurosciences, Belgium) and H31L21 (Thermofisher Scientific, USA, Cat#700254). The detection antibodies were biotinylated using Simoa®Homebrew Assay Development Kit (Quanterix, USA, Cat#101354) according to the kit instructions. BAN2401 was adjusted with 50 mM Tris / HCl (pH 7.5) to a concentration of 2 µg / mL, and then injected at 100 µl / well in a Nunc Maxisorp 96 well plate (Thermofisher Scientific, USA). The plate was sealed and stood for overnight at 4 ̊C. After the antibody solution was removed by aspiration, SuperBlock Blocking Buffer (Thermofisher Scientific, USA, Cat#37515) was injected at 250 µl / well, followed by the incubation at room temperature for 30 min. The plate was washed three times with wash buffer (PBS with 0.05% Tween20) and then series of Aβ1-42protofibril solution were added at 50 µL / well and incubated for 1.5 hour at room temperature. After washing for three times, 1 µg / mL of the biotinylated detection antibodies were added at 100 µL / well and incubated for 1.5 hour at room temperature. After washing and 30 min incubation with Streptavidin-HRP (R&Dsystems, USA), 3,3',5,5'-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA (KPL, SeraCare Life Sciences) was injected at 100 µl / well and reacted at room temperature for 20 minutes. Reaction was terminated by the addition of 100 µL / well 1M H2SO4and Optical Density (450 to 650 nm) was then measured by a spectrophotometer, SpectraMAX190 (Molecular Devices, USA). Aβ1-42PF, the biotinylated detection antibodies and Streptavidin-HRP were diluted in PBS with 0.5% BSA and 0.05% Tween20. The N-terminal antibodies showed higher signal compared to the C-terminal antibodies (Fig.7). Among the N-terminal antibodies, 3D6 was selected because it showed the highest signal in combination with BAN2401 as a capture antibody. Attorney Docket No.08061.0067-00304 Example 10. Comparison of antibody pairs, BAN2401 / 3D6 and BAN2401 / BAN2401 (MSD-ECL) Comparison of antibody pairs, BAN2401 / 3D6 and BAN2401 / BAN2401 was conducted on Meso Scale Discovery Electrochemiluminescence (MSD-ECL) platform (Fig. 8(A) and Fig.8(B)). Biotinylated capture antibody and SULFO-TAG labeled detection antibody were used for MSD-ECL platform. Biotinylated BAN2401 was prepared using EZ- Link™ NHS-PEG4-Biotin (Thermo Fisher Scientific, USA, Cat# A39259) and SULFO-TAG labeled 3D6 and BAN2401 were prepared using MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack (Meso Scale Discovery, USA, Cat#R31AA). The biotinylated capture antibody, the SULFO-TAG labeled detection antibody and Aβ1-42 protofibril were diluted with PBS + 0.5% BSA + 0.05% Tween20 and used.150 µL of blocking solution (1% BSA in PBS) was added to wells of biotinylated microplate, MSD GOLD 96-well Small Spot Streptavidin SECTOR Plate (Meso Scale Discovery, USA, Cat#L45SA) and the plate was incubated for 1 hour at room temperature. After 1 time wash with PBS+0.05% Tween20, 25 µL of 1 µg / mL biotinylated BAN2401 solution was added to the wells and the plate was incubated for 1 hour at room temperature. The plate was washed 3 times with PBS+0.05% Tween20 and 25 µL of series of diluted Aβ protofibril standard were added to appropriate wells. After 1 hour incubation at room temperature and 3 times wash with PBS+0.05% Tween20, 25 µL of 1 µg / mL SULFO-TAG labeled 3D6 or BAN2401 were added and incubated for 1 hour at room temperature. The plate was washed 3 times with PBS + 0.05% Tween20 and 150 µL of 1:1 mixture of MSD Read Buffer T (4x) (Meso Scale Discovery, USA, Cat#R92TC) and milli-Q water was added to the wells. Electrochemiluminescence signal was read by MESO SECTOR S 600 (Meso Scale Discovery, USA, Cat# IC0AA-0). Attorney Docket No.08061.0067-00304 BAN2401 / 3D6 showed higher ECL signal compared to BAN2401 / BAN2401. BAN2401 / 3D6 pair is more optimal for MSD-ECL platform. Example 11. Comparison of antibody pairs, BAN2401 / 3D6 and BAN2401 / BAN2401 (SMCxPRO) Comparison of antibody pairs, BAN2401 / 3D6 and BAN2401 / BAN2401 was conducted on SMCxPRO platform (Fig.9). Assay procedure was following to the method described in elsewhere (Example 3).1000 ng / mL of fluorescent-labeled 3D6 or BAN2401 were used as detection antibody. Aβ1-42 PF was diluted with Discovery Standard Diluent (EMD Millipore, USA, Cat#02-0560-00) to make 0.1 pM and 1 pM solution. BAN2401 / 3D6 effectively worked for the detection of Aβ1-42 PF, compared to BAN2401 / BAN2401. BAN2401 / 3D6 showed 30 times (1 pM) and 12 times (0.1 pM) larger S / N ratio than BAN2401 / BAN2401. Example 12. Comparison of platform, BAN2401 / 3D6 on SMCxPRO®and Simoa®Comparison of platform, SMCxPRO and Simoa was conducted using the same antibody pair, BAN2401 / 3D6. For SMCxPRO assay, BAN2401 coated magnetic beads and fluorescent dye labeled 3D6 were prepared following the procedure described elsewhere (Example 2) and measurement was conducted following the method previously described (Example 3). For Simoa assay, BAN2401 coated beads and biotinylated 3D6 were prepared using Simoa® Homebrew Assay Development Kit (101354, Quanterix, Billerica, MA, USA) according to the manufacturer’s instructions. BAN2401 coated beads was diluted with Beads Diluent Buffer (Quanterix, USA, Cat#101362), biotinylated 3D6 and Aβ1-42 protofibril were diluted with Homebrew Detector / Sample Diluent (Quanterix, USA, Cat#101359). The 25 µL of BAN2401 beads solution and 100 µL of diluted Aβ1-42 protofibril were added to the Attorney Docket No.08061.0067-00304 microplate, Simoa® 96-well Assay Plate (Quanterix, USA, Cat#101457) and incubated for 30 min at 30°C with 800 rpm shaking. After incubation, the plate was washed with Wash Buffer A (Quanterix, USA, Cat#103078) twice on the plate washer (BioTek, USA, Cat#405TSRVS) following the washing procedure installed by Quanterix. After washing, 100 µL of 0.3µg / mL biotinylated 3D6 was added to wells and incubated for 10 min at 30°C with 800 rpm shaking. After second reaction, the plate was washed with Wash Buffer A twice on the plate washer. After second wash, 100 µL of 150 pM SBG solution (SBG concentrate was diluted with SBG Diluent) (101361; Simoa Enzyme and Substrate Kit, Quanterix, Billerica, MA, USA) was added to the plate and incubated for 10 min at 30°C with 800 rpm shaking. After SBG incubation, the plate was washed with Wash Buffer A twice on the plate washer, and then the plate was washed with Wash Buffer B (Quanterix, USA, Cat#103079) twice on the plate washer. The plate after wash was set on Simoa SR-X analyzer (Quanterix, Billerica, MA, USA, Cat#102917) to read AEB (signal of Simoa platform). Compared to Simoa platform, SMCxPRO showed better S / N ratio for the measurement of Aβ1-42 protofibril (Fig.10(A) and Fig.10(B)). Example 13. Selection of magnetic beads for sensitive detection of Aβ1-42 PF on SMCxPRO Dynabeads MyOne Tosylactivated (Invitrogen, USA, Cat#65501) was compared to the beads supplied as default beads for SMCxPRO platform by the vendor. Coating of BAN2401 to the default beads for SMCxPRO (SMC_BAN2401) was conducted using SMC™ Capture Labeling Kit (EMD Millipore, USA, Cat# 03-0077-02). Coating of BAN2401 to Dynabeads MyOne Tosylactivated (TA_BAN2401) was conducted by following the procedure described in elsewhere (Example 2). Human IgG1 (Sigma, USA, Cat#I5154) was also used to immobilize on SMC default beads (SMC_IgG) and Dynabeads MyOne Tosylactivated Attorney Docket No.08061.0067-00304 (TA_IgG), conjugation method was the same for BAN2401 beads. BAN2401 beads and IgG1 beads were used for human CSF measurement on SMCxPRO. Compared to SMC default beads, Dynabeads MyOne Tosylactivated beads showed higher signal in human CSF measurement without increasement of non-specific signal from IgG1 beads (Fig.11 and Table 11). Example 14. Optimization of antibody amount on beads coating procedure (SMCxPRO) To optimize antibody immobilization procedure, antibody amount during coating reaction was investigated. Coating procedures were same for 3 reaction conditions except antibody / beads ratio in the immobilization reaction. Evaluated conditions were 40 µg BAN2401 / mg magnetic beads, 20 µg BAN2401 / mg magnetic beads and 10 µg BAN2401 / mg magnetic beads. Coating procedure was the same as described elsewhere (Example 2). Aβ1-42 protofibril and human CSF were measured with each BAN2401 coated beads according to the method described in elsewhere (Example 3). Discovery Standard Diluent was measured as buffer blank. 20 µg BAN2401 per 1 mg magnetic beads was selected for the optimal preparation procedure judging from S / N ratio in PF standard and CSF measurement (Fig.12(A) and Fig. 12(B) and Table 12).

[0007] Attorney Docket No.08061.0067-00304 TABLES Table 1. Amino acid sequences of monoclonal antibody (mAb) CDRs Table 2. Amino acid sequences of mAb variable regions (CDRs shown in bold)

[0008] Attorney Docket No.08061.0067-00304 Table 3. Amino acid sequences of mAb heavy and light chains

[0009] Attorney Docket No.08061.0067-00304 Table 4. Amino acid sequences of mAb constant regions

[0010] Attorney Docket No.08061.0067-00304 Table 5. Amino acid sequences of mAb CDRs Table 6. Amino acid sequences of mAb variable regions (CDRs shown in bold)

[0011] Attorney Docket No.08061.0067-00304 Table 10. Spike recovery Attorney Docket No.08061.0067-00304 Table 11. S / N ratio Table 12. S / N ratio for 3 different beads in PF standard and human CSF measurement

[0012] Attorney Docket No.08061.0067-00304 Table 13. Amino acid sequences of mAb variable regions (CDRs shown in bold) Table 14. Amino acid sequences of mAb variable regions (CDRs in bold) Table 15. Amino acid sequences of mAb variable regions (CDRs shown in bold) Attorney Docket No.08061.0067-00304 Table 16. Epitopes of anti-Aβ antibodies

[0013] Attorney Docket No.08061.0067-00304 Abbreviations Aβ amyloid beta Aβ(1-42) amyloid beta monomer from amino acid 1 to 42 AD Alzheimer’s disease CDR Complementarity Determining Region LC-MS / MS liquid chromatography – tandem mass spectrometry

Claims

AMENDED CLAIMS received by the International Bureau on 20 May 2025 (20.05.2025)1. A method for quantifying amyloid p (Ap) protofibrils in a biological sample from a human subject, comprising: contacting the biological sample with an anti-Ap protofibril capture antibody comprising heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), andSEQ ID NO: 3 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5(LCDR2), and SEQ ID NO: 6 (LCDR3) to form an immune complex; contacting the immune complex with a labeled anti-Ap detection antibody; dissociating the immune complex to release the labeled anti-Ap detection antibody; detecting a signal from the labeled anti-Ap detection antibody using a single molecule counting instrument; and quantifying Ap protofibrils in the sample.

2. The method of claim 1, wherein contacting the biological sample with the capture antibody forms a first immune complex and contacting the first immune complex with the detection antibody forms a second immune complex.

3. The method of claim 1 or 2, wherein the anti-Ap protofibril capture antibody comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.AMENDED SHEET (ARTICLE 19)4. The method of any one of claims 1 to 3, wherein the anti-Ap protofibril capture antibody comprises lecanemab.

5. The method of any one of claims 1 to 4, wherein the label on the anti-Ap detection antibody comprises a tag, wherein the tag comprises a protein tag, a fluorescent tag, a quantum dot tag, an aptamer tag, an oligonucleotide tag, a SULFO-TAG, or a biotin tag.

6. The method of any one of claims 1 to 5, wherein the anti-Ap detection antibody binds a region of Ap that does not overlap a region bound by lecanemab, and / or wherein the anti-Ap detection antibody does not compete for binding to Ap protofibrils with lecanemab.

7. The method of any one of claims 1 to 6, wherein the anti-Ap detection antibody binds to an N-terminus of an Ap, e.g. AP1-5.

8. The method of any one of claims 1 to 7, wherein the anti-Ap detection antibody comprises heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 13 (HCDR1), SEQ ID NO: 14 (HCDR2), and SEQID NO: 15 (HCDR3) and light chain complementarity determining region(LCDR) sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), andSEQ ID NO: 18 LCDR3.

9. The method of any one of claims 1 to 8, wherein the anti-Ap detection antibody comprises a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20.AMENDED SHEET (ARTICLE 19)10. The method of any one of claims 1 to 9, wherein the anti-Ap detection antibody comprises 3D6.

11. The method of any one of claims 1 to 5, wherein the anti-Ap detection antibody comprises lecanemab.

12. The method of any one of claims 1 to 11 , wherein the biological sample comprises a body fluid or tissue.

13. The method of claim 12, wherein the biological sample comprises cerebrospinal fluid, whole blood, plasma, serum, brain tissue homogenate, or brain tissue lysate.

14. The method of any one of claims 1 to 13, wherein the biological sample is stored at -80°C prior to contacting the sample with the capture antibody.

15. The method of any one of claims 1 to 14, wherein the method does not comprise repetitive freeze-thaw cycles of the biological sample.

16. The method of any one of claims 14 or 15, further comprising thawing the frozen biological sample and gently mixing and / or vortexing.

17. The method of any one of claims 1 to 16, wherein the biological sample is prepared and stored in low protein-binding tubes.AMENDED SHEET (ARTICLE 19)18. The method of any one of claims 1 to 17, wherein the biological sample is a brain homogenate.

19. The method of any one of claims 1 to 18, wherein the biological sample is at a concentration 0.1-10 mg of protein per mb of sample.

20. The method of any one of claims 1 to 19, wherein the biological sample is at a concentration of 1 mg of protein per mb of sample.

21. The method of any one of claims 1 to 20, wherein the biological sample is diluted 2-fold, optionally in a sample buffer.

22. The method of any one of claims 1 to 17, wherein the biological sample is aCSF sample and optionally the CSF sample is not diluted.

23. The method of any one of claims 1 to 17, wherein the biological sample is aCSF sample diluted 2-fold, optionally in a sample buffer.

24. The method of any one of claims 1 to 17, wherein the biological sample is a blood sample and optionally the blood sample is not diluted.

25. The method of any one of claims 1 to 17, wherein the biological sample is a blood sample diluted 2-fold, optionally in a sample buffer.AMENDED SHEET (ARTICbE 19)26. The method of any one of claims 1 to 25, wherein the anti-Ap protofibril capture antibody is immobilized on a surface.

27. The method of claim 26, wherein the surface is a plate, a bead, or a particle, optionally a magnetic bead or particle.

28. The method of claim 27, wherein the particle is a tosylactivated magnetic bead.

29. The method of claim 27 or claim 28, wherein the capture antibody is conjugated to the bead at a concentration of 10-40 pg of antibody per mg of bead.

30. The method of claim 29, wherein the capture antibody is conjugated to the bead at a concentration of 20 pg of antibody per mg of bead.

31. The method of claim 29, wherein the capture antibody is conjugated to the bead at a concentration of 20 pg antibody per mg of tosylactivated magnetic bead.

32. The method of any one of claims 5 to 31, wherein the tag is a fluorescent tag, and wherein the signal from the fluorescent tag is measured by the single molecule counting instrument.

33. A method for quantifying amyloid p (Ap) protofibrils in a biological sample from a human subject, comprising:AMENDED SHEET (ARTICLE 19)contacting the biological sample with an anti-Ap protofibril capture antibody comprising heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 1 (HCDR1), SEQ ID NO: 2 (HCDR2), andSEQ ID NO: 3 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO: 4 (LCDR1), SEQ ID NO: 5(LCDR2), and SEQ ID NO: 6 (LCDR3) immobilized on a surface to form an immune complex; contacting the sample with a fluorescently labeled anti-Ap detection antibody to form an immune complex; dissociating the immune complex to release the fluorescently labeled anti-Ap detection antibody; detecting a signal from the fluorescently labeled anti-Ap detection antibody via a single molecule counting instrument; and quantifying Ap protofibrils in the sample.

34. The method of claim 33, further comprising washing the sample to remove unbound materials.

35. The method of claim 33 or claim 34, wherein the anti-Ap protofibril capture antibody comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.

36. The method of any one of claims 33 to 35, wherein the anti-Ap protofibril capture antibody is lecanemab.AMENDED SHEET (ARTICLE 19)37. The method any one of claims 33 to 36, wherein the fluorescently labeled anti-Ap detection antibody binds to the N-terminus of an Ap, e.g. AP1-5.

38. The method of any one of claims 33 to 37, wherein the fluorescently labeled anti-Ap detection antibody comprises heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 13 (HCDR1), SEQ IDNO: 14 (HCDR2), and SEQ ID NO: 15 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO: 16(LCDR1), SEQ ID NO: 17 (LCDR2), and SEQ ID NO:18 (LCDR3).

39. The method of any one of claims 33 to 38, wherein the fluorescently labeled anti-Ap detection antibody comprises a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQID NO: 20.

40. The method of any one of claims 33 to 39, wherein the fluorescently labeled anti-Ap detection antibody comprises 3D6.

41. The method of any one of claims 33 to 40, wherein the biological sample comprises a body fluid or tissue.

42. The method of claim 41 , wherein the biological sample comprises cerebrospinal fluid (CSF), whole blood, plasma, serum, brain tissue homogenate, or brain tissue lysate.AMENDED SHEET (ARTICLE 19)43. The method of claim 41 or claim 42, wherein the biological sample is stored at-80°C prior to contacting the sample with the capture antibody.

44. The method of any one of claims 41 to 43, wherein the method does not comprise repetitive freeze-thaw cycles of the biological sample.

45. The method of claim 43 or claim 44, further comprising thawing the frozen biological sample and gently mixing and / or vortexing.

46. The method of any one of claims 41 to 45, wherein the biological sample is prepared and stored in low protein-binding tubes.

47. The method of any one of claims 41 to 46, wherein the biological sample is at a concentration 0.1-10 mg of protein per mb of sample.

48. The method of any one of claims 41 to 47, wherein the biological sample is at a concentration of 1 mg of protein per mb of sample.

49. The method of any one of claims 41 to 48, wherein the biological sample is diluted 2-fold, optionally in a sample buffer.

50. The method of any one of claims 41 to 48, wherein the biological sample is a brain homogenate.

51. The method of any one of claims 41 to 48, wherein the biological sample is aCSF sample and optionally the CSF sample is not diluted.AMENDED SHEET (ARTICLE 19)52. The method of any one of claims 41 to 48, wherein the biological sample is aCSF sample diluted 2-fold, optionally in a sample buffer.

53. The method of any one of claims 41 to 48, wherein the biological sample is a blood sample, e.g. serum or plasma, and optionally the blood sample is not diluted.

54. The method of any one of claims 41 to 48, wherein the biological sample is a blood sample, e.g. serum or plasma, diluted 2-fold, optionally in a sample buffer.

55. The method of any one of claims 33 to 54, wherein the surface is a plate, a bead, or a particle, optionally a magnetic bead or particle.

56. The method of claim 55, wherein the particle is a tosylactivated magnetic bead.

57. The method of claim 55 or claim 56, wherein the capture antibody is conjugated to the bead at a concentration of 10-40 pg antibody per mg of bead.

58. The method of claim 57, wherein the capture antibody is conjugated to the bead at a concentration of 20 pg antibody per mg of bead.AMENDED SHEET (ARTICLE 19)59. The method of claim 57, wherein the capture antibody is conjugated to the bead at a concentration of 20 pg antibody per mg of tosylactivated magnetic bead.

60. The method of any one of claims 1 to 59, wherein the biological sample comprises Ap protofibrils in an amount of at least about 0.01 picomolar, e.g., about 0.01 picomolar to about 2 picomolar.

61. The method of any one of claims 1 to 59, wherein the method has a sensitivity for Ap protofibrils of about 0.003 picomolar.

62. The method of any one of claims 1 to 61 , wherein the method has a lower limit of quantification of about 0.01 picomolar of Ap protofibrils.

63. The method of any one of claims 1 to 62, wherein the method provides a selectivity for an Ap protofibril of 1 ,000,000-fold or greater than for an Ap monomer.

64. The method of any one of claims 1 to 63, wherein the quantity of Ap protofibril is determined relative to a control.

65. The method of any one of claims 1 to 64, wherein an Ap protofibril standard curve is used to determine the Ap protofibril quantity.

66. The method of claim 65, wherein the Ap protofibril standard curve is prepared from AP1-42 by size exclusion chromatography.AMENDED SHEET (ARTICLE 19)67. The method of claim 65 or claim 66, wherein the Ap protofibril standard curve is prepared in low protein-binding tubes and stored at -80C.

68. The method of any one of claims 1 to 67, wherein the method provides a S / N ratio greater than 100, e.g., at concentrations of Ap protofibril of about 1 pM.

69. A kit for detecting Ap protofibrils in biological samples, comprising: an anti-Ap protofibril capture antibody comprising heavy chain complementarity determining region (HCDR) sequences of SEQ ID NO: 1(HCDR1), SEQ ID NO: 2 (HCDR2), and SEQ ID NO: 3 (HCDR3) and light chain complementarity determining region (LCDR) sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO: 5 (LCDR2), and SEQ ID NO: 6 (LCDR3), optionally immobilized on a solid surface, e.g. a magnetic bead; a labeled anti-Ap detection antibody; a dissociation buffer; and instructions for using the kit with a single molecule counting instrument.

70. The kit of claim 69, wherein the capture antibody is lecanemab.

71. The kit of claim 69 or 70, wherein the detection antibody is fluorescently labeled.

72. The kit of any one of claims 69 to 71, wherein the detection antibody is 3D6.AMENDED SHEET (ARTICLE 19)73. The kit of any one of claims 69 to 72, further comprising a sample buffer and / or a washing buffer.95AMENDED SHEET (ARTICLE 19)

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