Methods for detecting neurofilament light chains in plasma and cerebrospinal fluid - Patent Application 20070233633

Abstract

The present disclosure provides a method for detecting and optionally quantifying Nfl present in cerebrospinal fluid and blood, and the use of the method for detecting and optionally measuring the level of Nfl biomarkers indicative of neuronal damage.Anti-Nfl antibodies are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 147,833, filed February 10, 2021, U.S. Provisional Application No. 63 / 183,417, filed May 3, 2021, and U.S. Provisional Application No. 63 / 197,826, filed June 7, 2021, each of which is incorporated by reference herein in its entirety.

[0002] Government Rights This invention was made with government support under AG067559 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Reference to sequence listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on January 31, 2022 is named "716468_ST25.txt" and is 10,025 bytes in size.

[0004] Technology Areas The present disclosure encompasses methods for quantifying and analyzing various neurofilament light chain species and their use to measure neuronal damage, inform the diagnosis of neurodegenerative diseases, select patients for further diagnostic testing, and guide treatment decisions. [Background technology]

[0005] background Neurofilaments are major components of the neuronal cytoskeleton of myelinated axons and play a key role in neuronal conductance by enabling radial axonal growth. In the central nervous system, neurofilaments are composed of four proteins: neurofilament heavy chain (NfH), neurofilament medium chain (NfM), neurofilament light chain (NfL) and alpha-internexin. Of these four proteins, only NfL is well established as a marker of neuronal damage. It is elevated in Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease (PD), progressive supranuclear palsy (PSP), traumatic brain injury (TBI), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and other neurodegenerative disorders. This indicates that the biology and pathophysiology now known about NfL is useful for a variety of neurodegenerative diseases.

[0006] Currently, only one commercially available pair of anti-NfL antibodies is utilized to monitor clinical cohorts and clinical trials (anti-NF-L mAb 2:1 and anti-Nfl mAb 47:3, Umam). Although the use of commercially available assays has been successful in identifying neurodegenerative disease from healthy controls, the utility of the biomarker is currently limited by its nonspecific nature. The "noise" resulting from elevation of Nfl in multiple neurodegenerative and neuroinflammatory processes makes Nfl a less reliable marker of disease state and treatment response, limiting its clinical utility in treatment protocols. Nfl undergoes multiple post-translational modifications (PTMs), resulting in the possibility of many different Nfl isoforms in any given biological sample, but commercially available immunoassays bind to these PTMs. Also, significant gaps remain in our understanding of the extent to which, if any, Nfl species can be used to detect neurodegenerative disorders, stage subjects, and / or guide treatment decisions. Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there remains a need in the art for improved methods for detecting and quantitating Nfl isoforms present in cerebrospinal fluid and blood. [Means for solving the problem]

[0008] The application file contains at least one photograph executed in color. Copies of this application publication with the color photograph(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0009] [Figure 1] Figure 1 is a graph showing the amount of recombinant full-length Nfl (rec-Nfl) after immunoprecipitation with anti-Nfl antibodies and LC-MS analysis. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. See the first paragraph of Example 2 for an explanation of the nomenclature of the tryptic peptides. Each line is a different antibody (see legend in the figure). Data for individual antibodies are presented in Figures 4-27. [Figure 2A] Figure 2A is a graph showing the amount of Nfl immunoprecipitated from brain lysates using anti-Nfl antibodies as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. See the first paragraph of Example 2 for an explanation of the nomenclature of tryptic peptides. Each line is a different antibody (see legend in the figure). Data for individual antibodies are presented in Figures 4-21. Figure 2B is an expanded version of the graph shown in Figure 2A (note the difference in the scale of the y-axis between the two figures). [Figure 2B]Figure 2A is a graph showing the amount of Nfl immunoprecipitated from brain lysates using anti-Nfl antibodies as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. See the first paragraph of Example 2 for an explanation of the nomenclature of tryptic peptides. Each line is a different antibody (see legend in the figure). Data for individual antibodies are presented in Figures 4-21. Figure 2B is an expanded version of the graph shown in Figure 2A (note the difference in the scale of the y-axis between the two figures). [Diagram 3] Figure 3 is a graph showing the amount of Nfl immunoprecipitated from CSF using anti-Nfl antibodies as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. See the first paragraph of Example 2 for an explanation of the nomenclature of the tryptic peptides. Each line is a different antibody (see legend in the figure). Data for individual antibodies are presented in Figures 4-27. [Figure 4A] 4A and 4B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 4A) and CSF (FIG. 4B) using anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 4C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 4B]4A and 4B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 4A) and CSF (FIG. 4B) using anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 4C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 4C] 4A and 4B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 4A) and CSF (FIG. 4B) using anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 4C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 5A]5A and 5B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 5A) and CSF (FIG. 5B) using anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 5C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 5B] 5A and 5B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 5A) and CSF (FIG. 5B) using anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 5C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 5C]5A and 5B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 5A) and CSF (FIG. 5B) using anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 5C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 6A] 6A and 6B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 6A) and CSF (FIG. 6B) using anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 6C is a graph showing the amount of tryptic peptides of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 6B]6A and 6B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 6A) and CSF (FIG. 6B) using anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 6C is a graph showing the amount of tryptic peptides of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 6C] 6A and 6B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 6A) and CSF (FIG. 6B) using anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 6C is a graph showing the amount of tryptic peptides of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.3.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 7A]7A and 7B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 7A) and CSF (FIG. 7B) using anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 7C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 7B] 7A and 7B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 7A) and CSF (FIG. 7B) using anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 7C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 7C]7A and 7B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 7A) and CSF (FIG. 7B) using anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 7C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.4, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 8A] 8A and 8B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 8A) and CSF (FIG. 8B) using anti-Nfl antibody HJ30.3.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 8C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 8B]8A and 8B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 8A) and CSF (FIG. 8B) using anti-Nfl antibody HJ30.3.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 8C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 8C] 8A and 8B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 8A) and CSF (FIG. 8B) using anti-Nfl antibody HJ30.3.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 8C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.5, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 9A]9A and 9B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 9A) and CSF (FIG. 9B) using anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 9C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 9B] 9A and 9B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 9A) and CSF (FIG. 9B) using anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 9C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 9C]9A and 9B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 9A) and CSF (FIG. 9B) using anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 9C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.6, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 10A] 10A and 10B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 10A) and CSF (FIG. 10B) using anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 10C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 10B]10A and 10B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 10A) and CSF (FIG. 10B) using anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 10C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 10C] 10A and 10B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 10A) and CSF (FIG. 10B) using anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 10C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.7, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 11A]11A and 11B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 11A) and CSF (FIG. 11B) using anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 11C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 11B] 11A and 11B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 11A) and CSF (FIG. 11B) using anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 11C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 11C]11A and 11B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 11A) and CSF (FIG. 11B) using anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 11C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.8, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 12A] 12A and 12B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 12A) and CSF (FIG. 12B) using anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 12C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 12B]12A and 12B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 12A) and CSF (FIG. 12B) using anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 12C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 12C] 12A and 12B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 12A) and CSF (FIG. 12B) using anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 12C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.9, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 13A]13A and 13B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 13A) and CSF (FIG. 13B) using anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 13C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 13B] 13A and 13B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 13A) and CSF (FIG. 13B) using anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 13C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 13C]13A and 13B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 13A) and CSF (FIG. 13B) using anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 13C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.11, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 14A] 14A and 14B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 14A) and CSF (FIG. 14B) using anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 14C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 14B]14A and 14B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 14A) and CSF (FIG. 14B) using anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 14C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 14C] 14A and 14B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 14A) and CSF (FIG. 14B) using anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 14C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.12, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 15A]15A and 15B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 15A) and CSF (FIG. 15B) using anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 15C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 15B] 15A and 15B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 15A) and CSF (FIG. 15B) using anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 15C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 15C]15A and 15B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 15A) and CSF (FIG. 15B) using anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 15C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.13, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 16A] Figures 16A and 16B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (Figure 16A) and CSF (Figure 16B) using anti-Nfl antibody HJ30.14, as measured by LC-MS. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. Figure 16C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.14, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 16B] Figures 16A and 16B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (Figure 16A) and CSF (Figure 16B) using anti-Nfl antibody HJ30.14, as measured by LC-MS. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. Figure 16C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.14, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 16C]Figures 16A and 16B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (Figure 16A) and CSF (Figure 16B) using anti-Nfl antibody HJ30.14, as measured by LC-MS. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. Figure 16C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.14, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 17A] 17A and 17B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 17A) and CSF (FIG. 17B) using anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 17C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 17B]17A and 17B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 17A) and CSF (FIG. 17B) using anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 17C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 17C] 17A and 17B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 17A) and CSF (FIG. 17B) using anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 17C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.15, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 18A]18A and 18B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 18A) and CSF (FIG. 18B) using anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 18C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 18B] 18A and 18B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 18A) and CSF (FIG. 18B) using anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 18C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 18C]18A and 18B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 18A) and CSF (FIG. 18B) using anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 18C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.1, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 19A] 19A and 19B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 19A) and CSF (FIG. 19B) using anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 19C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 19B]19A and 19B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 19A) and CSF (FIG. 19B) using anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 19C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 19C] 19A and 19B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 19A) and CSF (FIG. 19B) using anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 19C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.16.2, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 20A]20A and 20B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 20A) and CSF (FIG. 20B) using anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 20C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 20B] 20A and 20B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 20A) and CSF (FIG. 20B) using anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 20C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 20C]20A and 20B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 20A) and CSF (FIG. 20B) using anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 20C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.17, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 21A] 21A and 21B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 21A) and CSF (FIG. 21B) using anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 21C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 21B]21A and 21B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 21A) and CSF (FIG. 21B) using anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 21C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 21C] 21A and 21B are graphs showing the amount of Nfl immunoprecipitated from brain lysates (FIG. 21A) and CSF (FIG. 21B) using anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. FIG. 21C is a graph showing the amount of rec-Nfl after immunoprecipitation with anti-Nfl antibody HJ30.18, as measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For a description of the nomenclature of tryptic peptides, see the first paragraph of Example 2. [Figure 22]Figure 22 summarizes the data from anti-Nfl antibody immunoprecipitation experiments and MS analysis (individual data are shown in Figures 4-21). The top figure shows the approximate Nfl isoforms enriched from CSF. The bottom figure shows the approximate Nfl isoforms enriched from brain lysates. Each figure includes a schematic of the full-length Nfl protein with amino acid numbering indicated along the bottom. In CSF, multiple Nfl isoforms were enriched, including isoforms with N-terminal truncations, isoforms with C-terminal truncations, and isoforms with N- and C-terminal truncations. The sizes of the isoforms are approximated from the MS data but are not specific at the residue level due to technical limitations of the methodology used. Thus, each line may represent multiple isoforms with different lengths at either end. Solid lines (dark blue) indicate higher confidence levels, and truncated isoforms contain the residues approximated by the solid line. Dashed lines (light blue) indicate lower confidence levels and represent possible variations. For example, the MS data suggest multiple N-terminally truncated isoforms containing amino acids 530-540 of SEQ ID NO:1 (represented by the four lower lines in the CSF diagram). The smallest of these isoforms is approximated to contain amino acids 462-543 of SEQ ID NO:1. This is only an approximation, however, the exact length cannot be determined using current techniques. It is therefore possible that the smallest isoform represented is multiple isoforms. At least two populations of Nfl isoforms were enriched in the brain lysates: a first population that is approximately full length, and a second population of N-terminally truncated isoforms that contain at least amino acids 530-540 of SEQ ID NO:1. The CSF diagram also includes approximate indications of the various regions that contain the epitopes to which anti-Nfl antibodies bind (shown above the schematic of the Nfl protein). For example, anti-Nfl antibodies HJ30.1, HJ30.2, HJ30.13 and HJ30.15 are shown to bind to an epitope within a region encompassing about amino acids 100 to about 225 of full-length Nfl, as determined from MS data.Note: this statement does not imply that these antibodies bind to the same epitope. [Figure 23] Figure 23 shows a schematic of the two-step enrichment process (left) and a graph showing the amount of tryptic peptide [323,330] immunoprecipitated with HJ30.4 at each step (y-axis, peak area). The red bar is 14N[323,330] and the blue bar is 15N[323,330]. See Example 2, first paragraph, for an explanation of the nomenclature of tryptic peptides. [Figure 24] Figure 24 depicts a schematic of the two-step enrichment process (left) and a graph showing the amount of tryptic peptide [529,539] immunoprecipitated with HJ30.11 at each step (y-axis, peak area). The red bar is 14N[529,539] and the blue bar is 15N[529,539]. See Example 2, first paragraph, for an explanation of the nomenclature of tryptic peptides. [Diagram 25] Figure 25 depicts a schematic of the two-step enrichment process (left) and a graph showing the amount of tryptic peptide [116,125] immunoprecipitated with HJ30.13 at each step (y-axis, peak area). The red bar is 14N[116,125] and the blue bar is 15N[116,125]. See Example 2, first paragraph, for an explanation of the nomenclature of tryptic peptides. [Figure 26] Figure 26 depicts two different three-step enrichment processes. Sample names are identified corresponding to the legends for Figures 27-28. [Figure 27] Figure 27 is a graph showing the amount of Nfl measured by LC-MS. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. For an explanation of the nomenclature of tryptic peptides, see the first paragraph of Example 2. Each color line is a different sample identified in the legend and in Figure 26. [Figure 28]Figure 28 is a graph showing the amount of Nfl measured by LC-MS. The y-axis is the 14N peak area for each tryptic identified peptide along the x-axis. See the first paragraph of Example 2 for an explanation of the nomenclature of tryptic peptides. Each color line is a different sample identified in the legend and in Figure 26. [Figure 29] Figure 29 depicts the three-step enrichment process. Sample names are identified corresponding to Figures 30-39 and 41. [Diagram 30] FIG. 30 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid positive with CDR=0.5. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Table 1] [Diagram 31] FIG. 31 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid positive with CDR=0.5. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Diagram 32]FIG. 32 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid positive with CDR=0.5. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Diagram 33] Figure 33 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid positive with CDR=0.5. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Diagram 34] FIG. 34 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Diagram 35]FIG. 35 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Diagram 36] FIG. 36 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Figure 37] FIG. 37 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Figure 38]FIG. 38 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Figure 39] FIG. 39 is a graph showing the amount of NF1 enriched from CSF obtained from a subject who was amyloid negative with CDR=0. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table A. The blue line is the "30.13" sample, the orange line is the "post30.13_30.4IP" sample, and the grey line is the "post30.13&4_30.11IP" sample. [Figure 40A] Figures 40A, 40B and 40C are graphs summarizing the individual data in Figures 31-40. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. The red line is the amyloid positive control and the blue line is the amyloid negative control. Figure 40D is a graph summarizing the individual data in Figures 31-40. The x-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the y-axis. [Figure 40B] Figures 40A, 40B and 40C are graphs summarizing the individual data in Figures 31-40. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. The red line is the amyloid positive control and the blue line is the amyloid negative control. Figure 40D is a graph summarizing the individual data in Figures 31-40. The x-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the y-axis. [Figure 40C] Figures 40A, 40B and 40C are graphs summarizing the individual data in Figures 31-40. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. The red line is the amyloid positive control and the blue line is the amyloid negative control. Figure 40D is a graph summarizing the individual data in Figures 31-40. The x-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the y-axis. [Figure 40D] Figures 40A, 40B and 40C are graphs summarizing the individual data in Figures 31-40. The y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the x-axis. The red line is the amyloid positive control and the blue line is the amyloid negative control. Figure 40D is a graph summarizing the individual data in Figures 31-40. The x-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide identified along the y-axis. [Figure 41A] Figures 41A and 41B are graphs showing the amount of Nfl enriched from CSF obtained from subjects who were amyloid positive ("AD") and CDR 0, CDR 0.5, CDR 1 or CDR 2, or subjects who were amyloid negative and CDR 0, CDR 0.5 or CDR 1. Subjects with CDR > 0.5 who were amyloid negative are considered "non-AD". Figure 41A shows the "post30.13_30.4 IP sample" and Figure 41B shows the "post30.13&4_30.11 IP sample". In each graph, the y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table B. [Figure 41B]Figures 41A and 41B are graphs showing the amount of Nfl enriched from CSF obtained from subjects who were amyloid positive ("AD") and CDR 0, CDR 0.5, CDR 1 or CDR 2, or subjects who were amyloid negative and CDR 0, CDR 0.5 or CDR 1. Subjects with CDR > 0.5 who were amyloid negative are considered "non-AD". Figure 41A shows the "post30.13_30.4 IP sample" and Figure 41B shows the "post30.13&4_30.11 IP sample". In each graph, the y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table B. [Figure 41C] Figures 41A and 41B are graphs showing the amount of Nfl enriched from CSF obtained from subjects who were amyloid positive ("AD") and CDR 0, CDR 0.5, CDR 1 or CDR 2, or subjects who were amyloid negative and CDR 0, CDR 0.5 or CDR 1. Subjects with CDR > 0.5 who were amyloid negative are considered "non-AD". Figure 41A shows the "post30.13_30.4 IP sample" and Figure 41B shows the "post30.13&4_30.11 IP sample". In each graph, the y-axis is the 14N / 15N ratio of the measured peak area for each tryptic peptide along the x-axis. The identity of the tryptic peptides numbered 1-24 along the x-axis is provided in Table B. [Table 2] [Figure 42A]Figures 42A and 42B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "30.13 sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 42A) or 14N amount (Figure 42B) of the measured peak area for each tryptic peptide along the x-axis. If no reliable MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Figure 42B] Figures 42A and 42B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "30.13 sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 42A) or 14N amount (Figure 42B) of the measured peak area for each tryptic peptide along the x-axis. If no reliable MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Figure 43A] Figures 43A and 43B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "post30.13_30.4IP sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 43A) or 14N amount (Figure 43B) of the measured peak area for each tryptic peptide along the x-axis. If no robust MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Figure 43B] Figures 43A and 43B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "post30.13_30.4IP sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 43A) or 14N amount (Figure 43B) of the measured peak area for each tryptic peptide along the x-axis. If no robust MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Figure 44A] Figures 44A and 44B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "post30.13&4_30.11 IP sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 44A) or 14N amount (Figure 44B) of the measured peak area for each tryptic peptide along the x-axis. If no robust MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Figure 44B] Figures 44A and 44B are graphs showing the amount of Nfl enriched from blood measured by LC-MS in the "post30.13&4_30.11 IP sample". In each graph, the y-axis is the 14N / 15N ratio (Figure 44A) or 14N amount (Figure 44B) of the measured peak area for each tryptic peptide along the x-axis. If no robust MS signal was detected for a given tryptic peptide of the ISTD, the data was not reported. Each line is a different blood sample. [Diagram 45] Figure 45 shows the immunoprecipitation of recombinant NfL. Each of the 23 NfL antibodies (HJ30.x) and one negative control antibody against amyloid beta (HJ5.1) were evaluated for their ability to immunoprecipitate full-length recombinant NfL. The relative amount of recombinant NfL recovery is shown (higher N14 / N15=higher N14 recovery). N15 was added after IP. [Figure 46] Figure 46 shows immunoprecipitation of native NfL from pooled CSF. Each of the 23 in-house NfL antibodies (HJ30.x) and one negative control antibody against amyloid beta (HJ5.1) were evaluated for their ability to immunoprecipitate NfL from CSF. Line colors correspond to individual antibodies, note figure legend. The antibodies appeared to target three distinct regions of NfL. [Figure 47] Figure 47 shows the linearity of the quantitative (6 peptide) MS method. Quantitation of all 6 peptides is linear within the N14 / N15 range tested. [Figure 48] FIG. 48 shows a map of neurofilament light chain species showing that CSF NfL exists as multiple fragment species. Antibodies targeting different domains of NfL used for immunoprecipitation along with mass spectrometry allowed the identification of multiple NfL species in CSF. Light dotted lines represent possible fragments in NfL species identification, while dark solid lines represent identified fragment species. NfL species were identified using 23 different custom antibodies and the data used to determine the NfL species are shown in FIGS. 49A-49B. [Figure 49A] Figures 49A, 49B and 49C show that while brain contains two major NfL species, CSF has at least three major NfL species. Experimental method for sequential IP-MS / MS assay to purify and identify at least three NfL fragment species. Figure 49A. Sequential NfL IP from pooled CSF shows three major NfL domains; a central domain region of NfL93-NfL224, another region of NfL324-NfL359 and the c-terminal region of NfL530. Figure 49B. and brain cortex lysate showing full length NfL from NfL2-NfL540 and the c-terminal peptide of NfL530. Figure 49C. The blue line represents peptides identified after the first IP with HJ30.13, the red line represents peptides identified during the second IP with HJ30.4, and the green line represents peptides identified during the third IP with HJ30.11. [Figure 49B]Figures 49A, 49B and 49C show that while brain contains two major NfL species, CSF has at least three major NfL species. Experimental method for sequential IP-MS / MS assay to purify and identify at least three NfL fragment species. Figure 49A. Sequential NfL IP from pooled CSF shows three major NfL domains; a central domain region of NfL93-NfL224, another region of NfL324-NfL359 and the c-terminal region of NfL530. Figure 49B. and brain cortex lysate showing full length NfL from NfL2-NfL540 and the c-terminal peptide of NfL530. Figure 49C. The blue line represents peptides identified after the first IP with HJ30.13, the red line represents peptides identified during the second IP with HJ30.4, and the green line represents peptides identified during the third IP with HJ30.11. [Figure 49C] Figures 49A, 49B and 49C show that while brain contains two major NfL species, CSF has at least three major NfL species. Experimental method for sequential IP-MS / MS assay to purify and identify at least three NfL fragment species. Figure 49A. Sequential NfL IP from pooled CSF shows three major NfL domains; a central domain region of NfL93-NfL224, another region of NfL324-NfL359 and the c-terminal region of NfL530. Figure 49B. and brain cortex lysate showing full length NfL from NfL2-NfL540 and the c-terminal peptide of NfL530. Figure 49C. The blue line represents peptides identified after the first IP with HJ30.13, the red line represents peptides identified during the second IP with HJ30.4, and the green line represents peptides identified during the third IP with HJ30.11. [Figure 50]Figure 50 shows that NfL species are increased in AD CSF compared to healthy controls. Sequential IP / MS of the three major CSF NfL species identifies increased NfL levels in Alzheimer's dementia (n=4) compared to controls (n=6) for each major species. The red line represents the relative abundance of NfL species for individuals with AD dementia and very mild dementia (CDR=0.5) as determined by the presence of amyloid plaques by PET, and the black line represents healthy age-matched controls (CDR=0). [Figure 51A] Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Figure 51B]Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Figure 51C] Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Fig. 51D]Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Figure 51E] Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Fig. 51F]Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Figure 51G] Figure 51A, Figure 51B, Figure 51C, Figure 51D, Figure 51E, Figure 51F and Figure 51G show that the validation cohort confirms increased NfL324 and NfL530 in AD compared to healthy controls. Schematic showing NfL map and peptide location in quantitative IP-MS method (Figure 51A). Comparison of NfL peptides between symptomatic AD participants (N=30; amyloid positive, CDR>0) and healthy controls (N=25; amyloid negative, CDR=0) for coil 1A and 1B regions. NfL101 (Figure 51B), NfL117 (Figure 51C) and NfL165 (Figure 51D) showed a non-significant trend towards increase in AD, no difference in the coil2B NfL283 region (Figure 51E), and highly significant increases in NfL323 (Figure 51F) and the C-terminal region NfL529 (Figure 51G). ** denotes statistical significance at p<0.01. [Figure 52A]Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Figure 52B] Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Figure 52C] Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Fig. 52D]Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Figure 52E] Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Fig. 52F] Figures 52A, 52B, 52C, 52D, 52E and 52F show the correlation between IP-MS and ELISA by Nfl species. The correlation between IP-MS and gold standard Uman Diagnostics ELISA results varies by NfL species: NfL101 (Figure 52A), NfL117 (Figure 52B), NfL165 (Figure 52C), NfL284 (Figure 52D), NfL324 (Figure 52E), NfL530 (Figure 52F). The highest correlation of these is observed between ELISA and NfL324. [Figure 53A]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Figure 53B]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Figure 53C]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Fig. 53D]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Figure 53E]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Fig. 53F]Figure 53A, Figure 53B, Figure 53C, Figure 53D, Figure 53E and Figure 53F show that NfL species correlate with the stage of AD dementia (CDR box sum). The amount of NfL species correlates minimally with the stage of dementia severity. The x-axis of each graph represents the CDR box sum, with the clinical scale of cognitive amount having a CDR-SB 0 being normal, 0.5-6 being mild dementia, and >6 being moderate clinical dementia. The relative amount of NfL species is shown on the y-axis as the N14 / N15 ratio in the NfL region. Spearman correlations and p-values ​​calculated for each group: NfL101: Amyloid+Spearman r=0.29 (ns, p=0.05), Amyloid-Spearman=0.18 (ns, p=0.30) (Figure 53A); NfL117: Amyloid+Spearman r=0.30 (p=0.04), Amyloid-Spearman r=0.18 (ns, p=0.31) (Figure 53B); NfL165: Amyloid+Spearman r=0.36 (p=0.01), Amyloid-Spearman r=0.19 (ns, p=0.28) (Figure 53C); NfL284: Amyloid+Spearman r=0.24 (ns, p=0.10), Amyloid-Spearman r=0.31 (ns, p=0.07) (Figure 53D); NfL324: amyloid+Spearman r=0.30 (p=0.04), amyloid-Spearman r=0.16 (ns, p=0.35) (Figure 53E); NfL530: amyloid+Spearman r=0.13 (ns, p=0.39), amyloid-Spearman r=0.25 (ns, p=0.14) (Figure 53F). Participants with amyloid plaques are shown in red circles and amyloid-negative participants in grey squares. NfL101, NfL117, NfL165, and NfL284 each have an outlier of n=1 that is not plotted on the graph but were included in the correlation calculation. [Figure 54A]Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Figure 54B] Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Fig. 54C]Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Fig. 54D] Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Figure 54E]Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Fig. 54F] Figure 54A, Figure 54B, Figure 54C, Figure 54D, Figure 54E and Figure 54F show log-transformed NfL concentrations by amyloid status and CDR. The amyloid-CDR 0 group was used as the reference group, and the other three groups were compared to the reference group using a two-sample t-test. P values ​​were corrected for multiple comparisons using the Benjamini-Hochberg method. NfL concentrations for each of the four groups (amyloid-negative CDR=0; amyloid-positive CDR=0, amyloid-positive CDR>0, amyloid-negative CDR>0) were compared for NfL101 (Figure 54A), NfL117 (Figure 54B), NfL165 (Figure 54C), NfL284 (Figure 54D), NfL324 (Figure 54E) and NfL530 (Figure 54F). [Figure 55A] Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Figure 55B]Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Figure 55C] Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Fig. 55D] Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Figure 55E] Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Fig. 55F]Figure 55A, Figure 55B, Figure 55C, Figure 55D, Figure 55E and Figure 55F show Log-transformed NfL concentrations by CDR global status. Differences between CDR 0 and CDR>0 groups were compared using two-sample t-tests for NfL101 (Figure 55A), NfL117 (Figure 55B), NfL165 (Figure 55C), NfL284 (Figure 55D), NfL324 (Figure 55E) and NfL530 (Figure 55F). [Figure 56] Figure 56 shows heatmaps for correlations between NfL species and clinical biomarkers of neurodegeneration, AD and tau. Maps represent Spearman correlations, with darker blue representing stronger correlations and white / light blue representing weaker correlations. The strongest correlations of CSF NfL regions were with each other and with NfL324 and NfL530, with the c-terminal region being least correlated with other NfL regions. There was a moderate correlation between NfL324 or NfL530 and CDR (Clinical Dementia Rating Scale), age, and p-tau and t-tau, while amyloid PET and MMSE measures had low correlations. Correlations between NfL and tau or ptau were higher for the c-terminal region of NfL (NfL324, NfL530) than for other peptides. [Figure 57] FIG. 57 depicts a schematic of the enrichment process (left) and sample types (right). [Figure 58A] Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Figure 58B]Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Figure 58C] Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Fig. 58D] Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Figure 58E] Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Fig. 58F]Figure 58A, Figure 58B, Figure 58C, Figure 58D, Figure 58E and Figure 58F show graphs showing differences in Nfl concentrations for ALS, spinal muscular atrophy (SMA), and controls. Figure 58F shows concentrations of NfL101. Figure 58E shows concentrations of NfL117. Figure 58C shows concentrations of NfL165. Figure 58D shows concentrations of NfL284. Figure 58B shows concentrations of NfL324. Figure 58A shows concentrations of NfL530. [Figure 59A] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Figure 59B] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Figure 59C] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Fig. 59D] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Figure 59E] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Fig. 59F] Figure 59A, Figure 59B, Figure 59C, Figure 59D, Figure 59E and Figure 59F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 59F shows the correlation of NfL101. Figure 59E shows the correlation of NfL117. Figure 59C shows the correlation of NfL165. Figure 59D shows the correlation of NfL284. Figure 59B shows the correlation of NfL324. Figure 59A shows the correlation of NfL530. [Figure 60A] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 60B] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 60C] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 60D] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 60E] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 60F] Figure 60A, Figure 60B, Figure 60C, Figure 60D, Figure 60E and Figure 60F are graphs showing the correlation between ALS progression and Nfl concentration. Figure 60F shows the correlation of NfL101. Figure 60E shows the correlation of NfL117. Figure 60C shows the correlation of NfL165. Figure 60D shows the correlation of NfL284. Figure 60B shows the correlation of NfL324. Figure 60A shows the correlation of NfL530. [Figure 61] FIG. 61 shows sequential IP / MS of CSF NfL species in ALS. [Figure 62] FIG. 62 shows sequential IP / MS of CSF NfL species in controls. [Figure 63] FIG. 63 shows sequential IP / MS of CSF NfL species in SMA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description Among various aspects of the present disclosure, the method for detecting and appropriately quantifying Nfl present in cerebrospinal fluid and blood, and the use of the method for detecting and appropriately measuring the level of Nfl biomarkers that indicate neuronal damage are provided.As described in more detail herein, it has been discovered that certain peptides or parts of Nfl are better indicators of neuronal damage than other peptides or parts of Nfl.Also disclosed herein are a number of anti-Nfl epitope binding agents and their use in the method of the present disclosure.

[0011] I. Definition In order to make the present invention easier to understand, certain terms are defined first.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention relates.Many methods and materials similar to, modified or equivalent to those described herein can be used in the implementation of the present invention without undue experimentation, and preferred materials and methods are described herein.In describing and claiming the present invention, the following technical terms are used according to the definitions set forth below.

[0012] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It should be understood that such range formats are used merely for convenience and brevity, and should be interpreted flexibly to include not only the numerical values ​​explicitly recited as range limits, but also to include all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. By way of illustration, a numerical range of "about 2 to about 50" should be interpreted to include not only the explicitly recited values ​​of 2 to 50, but also all individual values ​​or subranges within the stated range. Thus, this numerical range includes individual values ​​such as 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1, 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1, 38.0, 40, 44, 44.6, 45, 48, and subranges such as 1-3, 2-4, 5-10, 5-20, 5-25, 5-30, 5-35, 5-40, 5-50, 2-10, 2-20, 2-30, 2-40, 2-50, etc. This same principle applies to ranges in which only one numerical value is recited as the minimum or maximum. Moreover, such an interpretation shall apply regardless of the breadth of the range or feature being described.

[0013] The term "about" as used herein refers to the variation in quantity that may occur, for example, through typical measurement techniques and devices, for any quantifiable variable, including, but not limited to, mass, volume, time, distance and amount. Furthermore, given the solid and liquid handling procedures used in the real world, there are certain random errors and variations that may occur through differences in the manufacture, source or purity of the components used to make the composition or carry out the method. The term "about" also encompasses these variations, which may be up to ±5%, but may also be ±4%, 3%, 2%, 1%, etc. Whether modified by the term "about" or not, the claims include the equivalents of the amount.

[0014] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meanings ascribed to them in U.S. patent law and can mean "includes," "including," and the like, and are generally construed as open-ended terms. The terms "consisting of" or "consists of" are closed terms and include only those components, structures, steps, and the like specifically recited in conjunction with such terms, as well as those components, structures, steps, and the like, as per U.S. patent law. "consisting essentially of" or "consists essentially of" have the meanings generally ascribed to them by U.S. patent law. In particular, such terms are generally closed terms, except to the extent that they permit the inclusion of additional items, materials, components, steps, or elements that do not materially affect the basic and novel characteristics or functions of the items with which they are used. For example, trace elements present in the composition that do not affect the properties or characteristics of the composition would be permissible if present under the phrase "consists essentially of," even if they are not explicitly recited in the list of items following such terminology. It is understood that when open-ended terms such as "comprising" or "including" are used herein, direct support should be provided for the term "consisting essentially of," as well as the phrase "consisting of," as if expressly stated, and vice versa.

[0015] The term "Aβ" refers to a peptide derived from a region at the carboxy terminus of a large protein called amyloid precursor protein (APP). The gene encoding APP is located on chromosome 21. There are many forms of Aβ that can have toxic effects: Aβ peptides are generally 37-43 amino acid sequences long, but they can have truncations and modifications that change their overall size. They can be found in soluble and insoluble compartments, in monomeric, oligomeric and aggregated forms, inside or outside the cell, and may be complexed with other proteins or molecules. The harmful or toxic effects of Aβ can be due to any or all of the forms mentioned above, as well as others not specifically mentioned. For example, two such Aβ isoforms include Aβ40 and Aβ42, with the Aβ42 isoform being particularly fibrinogenic or insoluble and associated with disease states. The term "Aβ" typically refers to multiple Aβ species without distinguishing among the individual Aβ species. Specific Aβ species are identified by the size of the peptide, eg, Aβ42, Aβ40, Aβ38, etc.

[0016] As used herein, the term "Aβ42 / Aβ40 value" refers to the ratio of the amount of Aβ42 in a sample obtained from a subject compared to the amount of Aβ40 in the same sample.

[0017] "Aβ amyloidosis" is defined as clinically abnormal Aβ deposition in the brain. Subjects determined to have Aβ amyloidosis are referred to herein as "amyloid positive", while subjects determined not to have Aβ amyloidosis are referred to herein as "amyloid negative". There are indicators of Aβ amyloidosis that are accepted in the art. At the time of this disclosure, Aβ amyloidosis is measured directly by amyloid imaging (e.g., PiB PET, florbetapir, or other imaging methods known in the art) or indirectly by reduced cerebrospinal fluid (CSF) Aβ42 or reduced CSF Aβ42 / 40 ratio. [11C]PIB-PET imaging with a mean cortical binding potential (MCBP) score >0.18 is indicative of Aβ amyloidosis, as is a concentration of about 1 ng / ml of cerebrospinal fluid (CSF) Aβ42 measured by immunoprecipitation and mass spectrometry (IP / MS). Alternatively, the cut-off ratio for CSF Aβ42 / 40 that maximizes accuracy in predicting amyloid positivity as determined by PIB-PET can be used.These or other values ​​known in the art and / or values ​​such as those used in the examples can be used alone or in combination to clinically confirm Aβ amyloidosis.See, for example, Klunk WE et al. Ann Neurol 55(3) 2004, Fagan AM et al. Ann Neurol, 2006, 59(3), Patterson et al, Annals of Neurology, 2015, 78(3): 439-453, or Johnson et al., J. Nuc. Med., 2013, 54(7): 1011-1013, each of which is incorporated herein by reference in its entirety. A subject with Aβ amyloidosis may or may not be symptomatic, and a symptomatic subject may or may not meet clinical criteria for a disease associated with Aβ amyloidosis.Non-limiting examples of symptoms associated with Aβ amyloidosis may include impaired cognitive function, behavioral changes, abnormal language function, emotional dysregulation, seizures, dementia, and impaired nervous system structure or function.Diseases associated with Aβ amyloidosis include, but are not limited to, Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), Lewy body dementia, and inclusion body myositis.Subjects with Aβ amyloidosis are at increased risk of developing diseases associated with Aβ amyloidosis.

[0018] "Clinical signs of Aβ amyloidosis" refers to objective measures of Aβ deposition known in the art.Clinical signs of Aβ amyloidosis may include, but are not limited to, Aβ deposition identified by amyloid imaging (e.g., PiB PET, florbetapir, or other imaging methods known in the art) or by reduced cerebrospinal fluid (CSF) Aβ42 or Aβ42 / 40 ratio.See, for example, Klunk WE et al. Ann Neurol 55(3) 2004 and Fagan AM et al. Ann Neurol 59(3) 2006, each of which is incorporated herein by reference in its entirety. Clinical signs of Aβ amyloidosis may also include measurements of Aβ metabolism, particularly measurements of Aβ42 metabolism alone, or comparisons with measurements of the metabolism of other Aβ variants (e.g., Aβ37, Aβ38, Aβ39, Aβ40, and / or total Aβ), as described in U.S. patent application Ser. Nos. 14 / 366,831, 14 / 523,148, and 14 / 747,453, each of which is incorporated by reference in its entirety herein. Additional methods are described in Albert et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 170-179; McKhann et al., Alzheimer's & Dementia 2007 Vol. 7, pp. 263-269; and Sperling et al. Alzheimer's & Dementia 2007 Vol. 7, pp. 280-292, each of which is incorporated herein by reference in its entirety. Importantly, subjects with clinical signs of Aβ amyloidosis may or may not have symptoms associated with Aβ deposition. Nevertheless, subjects with clinical signs of Aβ amyloidosis are at increased risk of developing diseases associated with Aβ amyloidosis. Clinical indicia of Aβ amyloidosis may also include measurements of other soluble proteins in the CSF or blood, including, but not limited to, phosphorylated tau species (e.g., tau species phosphorylated at residues T217 and / or T181).

[0019] "Candidate for amyloid imaging" refers to a subject who has been identified by a clinician as an individual for whom amyloid imaging may be clinically justified. As a non-limiting example, a candidate for amyloid imaging may be a subject who has one or more clinical signs of Aβ amyloidosis, one or more Aβ plaque-related symptoms, one or more CAA-related symptoms, or a combination thereof. A clinician may recommend amyloid imaging for such a subject to direct the subject's clinical care. As another non-limiting example, a candidate for amyloid imaging may be a potential participant (either a control subject or a test subject) in a clinical trial for a disease associated with Aβ amyloidosis.

[0020] "Aβ plaque-associated condition" or "CAA-associated condition" refers to any condition caused by or associated with the formation of amyloid plaques or CAA, respectively, which are composed of regularly arranged fibrillar aggregates called amyloid fibrils. Exemplary Aβ plaque-associated symptoms may include, but are not limited to, neuronal degeneration, impaired cognitive function, impaired memory, behavioral changes, emotional dysregulation, seizures, impaired nervous system structure or function, and increased risk of developing or worsening Alzheimer's disease or CAA. Neuronal degeneration may include structural changes of neurons (including molecular changes such as intracellular accumulation of toxic proteins, protein aggregates, etc., and macro-level changes such as changes in the shape or length of axons or dendrites, changes in myelin sheath composition, myelin sheath loss, etc.), functional changes of neurons, loss of function of neurons, death of neurons, or any combination thereof. Impaired cognitive function may include, but is not limited to, difficulties with memory, attention, concentration, language, abstract thinking, creativity, executive function, planning, and organization. Behavioral changes may include, but are not limited to, physical or verbal aggression, impulsivity, decreased inhibition, apathy, decreased initiation, personality changes, alcohol, tobacco or drug abuse, and other addiction-related behaviors. Emotional dysregulation may include, but are not limited to, depression, anxiety, mania, irritability, and affective imbalance. Seizures may include, but are not limited to, generalized tonic-clonic seizures, complex partial seizures, and non-epileptic psychogenic seizures. Impaired nervous system structure or function may include, but is not limited to, hydrocephalus, Parkinson's disease, sleep disorders, psychosis, balance and coordination dysfunction. This may include motor dysfunction such as monoparesis, hemiparesis, quadriparesis, ataxia, ballism, and tremor. This may also include sensory loss or dysfunction, including the senses of smell, touch, taste, sight, and hearing. Furthermore, this may include autonomic nervous system dysfunction such as bowel and bladder dysfunction, sexual dysfunction, blood pressure and temperature dysregulation.Finally, this can include hormonal dysfunction resulting from hypothalamic and pituitary gland dysfunction, such as deficiencies and dysregulation of growth hormone, thyroid-stimulating hormone, luteinizing hormone, follicle-stimulating hormone, gonadotropin-releasing hormone, prolactin, and many other hormones and modulators.

[0021] As used herein, the term "blood sample" refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. A blood sample can be whole blood, plasma or serum, although plasma is typically preferred.

[0022] The term "isoform" as used herein refers to any of several different forms of the same protein that arise due to alternative splicing of the mRNA encoding the protein, post-translational modification of the protein, proteolytic processing of the protein that occurs in vivo, genetic mutation, and somatic recombination. The terms "isoform", "species" and "variant" are used interchangeably (e.g., the terms "Nfl isoform" and "Nfl species" can be used interchangeably).

[0023] Unless expressly stated otherwise, the term "neurofilament light chain" refers to "human neurofilament light chain" and includes all genetically encoded isoforms or variants, as well as in vivo C-terminally truncated, in vivo N-terminally truncated, in vivo N-terminally truncated and C-terminally truncated, in vivo post-translationally modified species thereof, or any combination thereof. The terms "neurofilament light chain", "neurofilament light chain polypeptide" and "Nfl" are used interchangeably herein. Full-length Nfl has the amino acid sequence of SEQ ID NO:1.

[0024] [Table 3]

[0025] The term "recombinant Nfl" refers to an Nfl encoded by a nucleic acid introduced into a system (e.g., a prokaryotic cell, a eukaryotic cell, or a cell-free expression system) that supports the expression of the nucleic acid and its translation into a protein. Methods for producing recombinant proteins are well known in the art, and the production of recombinant Nfl disclosed herein is not limited to a particular system.

[0026] The term "subject" refers to a human or a non-human animal that expresses human Nfl.

[0027] The terms "treat", "treating" or "treatment" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or diseases / disorders. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the disease, condition or disorder, as well as those prone to have the disease, condition or disorder, or those in whom the disease, condition or disorder is to be prevented.

[0028] The term "antibody", as used herein, is used in the broadest sense and encompasses a variety of antibodies and antibody-like structures, including, but not limited to, full-length monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), as well as heavy chain antibodies and antibody fragments, provided they exhibit the desired antigen-binding activity. The domain of an antibody involved in binding an antigen is referred to as the "variable region" or "variable domain" and is described in more detail below. A single variable domain may be sufficient to confer antigen-binding specificity. Preferably, but not necessarily, the antibodies useful in the present discovery are recombinantly produced. The antibodies may be glycosylated or non-glycosylated, although glycosylated antibodies may be preferred. An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibodies are purified to greater than 95% or 99% purity as determined by methods known in the art.

[0029] In addition to the antibodies described herein, it may be possible to design antibody mimetics or aptamers having substantially the same function as the antibodies of the present invention using methods known in the art. "Antibody mimetic" refers to a polypeptide or protein that can specifically bind to an antigen but is not structurally related to an antibody. Antibody mimetics have a mass of about 3 kDa to about 20 kDa. Non-limiting examples of antibody mimetics are affibody molecules, affilins, affimers, alphabodies, anticalins, avimers, DARPins, and monobodies. Aptamers are a class of small nucleic acid ligands composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Aptamers interact and bind to their targets by structural recognition, a process similar to antigen-antibody reactions. Aptamers have a lower molecular weight than antibodies, typically about 8 to 25 kDa.

[0030] The terms "full length antibody" and "intact antibody" may be used interchangeably and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein. The basic structural unit of a native antibody comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The light chains are classified as gamma, mu, alpha, and lambda. The heavy chains are classified as gamma, mu, alpha, delta, or epsilon, defining the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. The amino-terminal portion of each light and heavy chain contains a variable region (VL and VH, respectively) of about 100-110 or more amino acid sequences primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. Intact antibodies are suitably cross-linked through disulfide bonds, as is known in the art.

[0031] The variable domains of the heavy and light chains of antibodies generally have a similar structure, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. Kuby Immunology, 6 thed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0032] "Framework region" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the sequence of the HVRs and FRs generally appears in the following sequence: FR1-HVR1-FR2-HVR2-FR3-HVR3-FR4. The FR domains of the heavy and light chains may differ, as is known in the art.

[0033] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of the variable domain whose sequences are hypervariable (also commonly referred to as "complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, an antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). As used herein, "HVRs derived from a variable region" refer to HVRs that have no more than two amino acid substitutions compared to the corresponding HVR from the original variable region. Exemplary HVRs herein include, as defined below for various antibodies of the disclosure, (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD). (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol.262: 732-745 (1996);(d) CDR1-IMGT (positions 27-38), CDR2-IMGT (positions 56-65) and CDR3-IMGT regions (positions 105-116 or 105-117), which are based on the numbering unique to IMGT (Lefranc, "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains," The Immunologist, 1999, 7: 132-136;Lefranc et al., Nucleic Acids Research, 2009, 37(Database issue): D1006-D1012;Ehrenmann et al., "Chapter 2: Standardized Sequence and Structure Analysis of Antibody Using IMGT," in Antibody Engineering Volume 2, Eds. Roland E. Kontermann and Stefan Dubel, 2010, Springer-Verlag Berlin Heidelberg, doi: 10.1007 / 978-3-642-01147-4; www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html), and (e) a combination of (a), (b), (c) and / or (d). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) of a variable domain that are assigned a sequence identification number are numbered according to the unique numbering system of IMGT above.

[0034] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0035] A "variant Fc region" comprises an amino acid sequence that may differ from that of a native Fc region by one or more amino acid substitutions and / or an altered glycosylation pattern, as compared to a native Fc region or the Fc region of a parent polypeptide. In certain examples, a variant Fc region may have from about 1 to about 10 amino acid substitutions or from about 1 to about 5 amino acid substitutions in a native sequence Fc region or the Fc region of a parent polypeptide. A variant Fc region herein may have at least about 80% homology, at least about 90% homology, or at least about 95% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide.

[0036] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Non-limiting examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single chain forms of antibodies and higher variants thereof; single domain antibodies, and multispecific antibodies formed from antibody fragments.

[0037] Single chain forms of antibodies and their higher forms may include, but are not limited to, single domain antibodies, single chain variant fragments (scFv), bivalent scFv (di-scFv), trivalent scFv (tri-scFv), tetravalent scFv (tetra-scFv), diabodies, and triabodies, and tetrabodies. scFvs are composed of heavy and light chain variable regions connected by a linker. In most, but not all, cases, the linker may be a peptide. The linker peptide is preferably about 5-30 amino acids long, or about 10-25 amino acids long. Typically, the linker allows for stabilization of the variable domains without interfering with proper folding and creation of an active binding site. In a preferred embodiment, the linker peptide is rich in glycine, and serine or threonine. scFvs can be used to facilitate phage display, or for flow cytometry, immunohistochemistry, or as targeting domains. Methods for making and using scFvs are known in the art. The scFv may also be conjugated to a human constant domain (e.g., the heavy chain constant domain is derived from an IgG domain, e.g., IgG1, IgG2, IgG3 or IgG4, or the heavy chain constant domain is derived from IgA, IgM, or IgE). Diabodies, triabodies, and tetrabodies, as well as higher order variants, are typically created by varying the length of the linker peptide by zero to several amino acids. Alternatively, it is also well known in the art that multivalent binding antibody variants can be created using self-assembly units linked to variable domains.

[0038] A "single domain antibody" refers to an antibody fragment which consists of a single monomeric variable antibody domain.

[0039] Multispecific antibodies include bispecific antibodies, trispecific antibodies, or antibodies of four or more specificities. Multispecific antibodies may be created by combining the heavy and light chains of one antibody with the heavy and light chains of one or more other antibodies. These chains may be covalently linked.

[0040] "Monoclonal antibody" refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic or phage clone. "Monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be produced using hybridoma techniques well known in the art, as well as recombinant, phage display, synthetic, or combinations of such techniques, and other techniques readily known in the art. Furthermore, monoclonal antibodies can be labeled with a detectable label, immobilized on a solid phase, and / or conjugated with a heterologous compound (e.g., an enzyme or toxin) according to methods known in the art.

[0041] "Heavy chain antibody" refers to an antibody consisting of two heavy chains. The heavy chain antibody may be an IgG-like antibody from camel, llama, alpaca, shark, etc., or an IgNAR from cartilaginous fish.

[0042] "Humanized antibody" refers to a non-human antibody that has been modified to reduce the risk of the non-human antibody eliciting an immune response in humans after administration, but retains similar binding specificity and affinity as the starting non-human antibody. A humanized antibody binds to the same or similar epitope as a non-human antibody. The term "humanized antibody" includes antibodies that are partially or completely composed of amino acid sequences derived from human antibody germline by altering the sequence of an antibody with a non-human hypervariable region ("HVR"). The simplest such alteration may consist of simply replacing the mouse constant region with the constant region of a human antibody, thus resulting in a human / mouse chimera that may have sufficiently low immunogenicity to be acceptable for pharmaceutical use. Preferably, the variable region of the antibody is also humanized by techniques now well known in the art. For example, the framework regions of the variable region may be replaced by the corresponding human framework regions, but retaining one, some, or all six of the non-human HVRs. Some framework residues may be replaced with corresponding residues from a non-human VL or VH domain (e.g., the non-human antibody from which the HVR residues are derived) to, for example, restore or improve the specificity or affinity of the humanized antibody. Substantially, the human framework region has at least about 75% homology (i.e., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity) with a known human framework sequence. HVRs may also be randomly mutated to maintain or enhance the binding activity and affinity to the antigen in the context of fully human germline framework regions or substantially human framework regions. As noted above, for use in the method of discovery, it is sufficient to use antibody fragments. Furthermore, as used herein, the term "humanized antibody" refers to an antibody that comprises a substantially human framework region, at least one HVR from a non-human antibody, including where any constant region present is substantially human. A substantially human constant region has at least about 90% (ie, about 90%, about 95% or about 99% sequence identity) to a known human constant sequence.Therefore, all parts of a humanized antibody, except possibly the HVRs, are substantially identical to their corresponding counterparts in one or more germline human immunoglobulin sequences.

[0043] If desired, the design of humanized immunoglobulins may be performed as follows or using similar methods familiar to those skilled in the art (see, for example, Almagro, et al. Front. Biosci. 2008, 13(5):1619-33). The variable regions of the mouse antibody are aligned with the most similar human germline sequence (e.g., by using BLAST or a similar algorithm). The CDR residues from the mouse antibody sequence are grafted into the similar human "acceptor" germline. One or more positions near the CDR or within the framework (e.g., Vernier positions) may then be reverted back to the original mouse amino acid to achieve a humanized antibody with similar binding affinity as the original mouse antibody. Typically, several versions of the humanized antibody with different back mutations are generated and empirically tested for activity. The humanized antibody variant with the most similar properties to the parent mouse antibody and with the least reversion of the mouse framework is selected as the final humanized antibody candidate.

[0044] The term "epitope binder" refers to an antibody, aptamer, nucleic acid, peptide, protein, lipid, metabolite, small molecule, or fragment thereof that can recognize and specifically bind to a given epitope. An epitope may be a linear epitope or a conformational epitope. As used herein, the term "linear epitope" refers to an epitope that consists of a linear (or contiguous) sequence of amino acids. The term "conformational epitope" refers to an epitope that consists of non-contiguous amino acids on the surface of a protein aggregate that has a specific three-dimensional shape.

[0045] As used herein, the term "aptamer" refers to a polynucleotide, generally RNA or DNA, that has useful biological activity in terms of biochemical activity, molecular recognition or binding properties. Usually, aptamers have molecular activity such as binding to a target molecule at a specific epitope (region). It is generally accepted that aptamers that are specific in binding to a polypeptide can be synthesized and / or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art. See, for example, U.S. Patent No. 7,939,313, which is incorporated herein by reference in its entirety.

[0046] II. Anti-Nfl epitope binding agents "Anti-Nfl epitope binding agent" as used herein refers to an isolated epitope binding agent that binds to recombinant human neurofilament light chain polypeptide (Nfl) with an affinity constant or affinity of interaction (KD) of about 0.1 pM to about 10 μM, preferably about 0.1 pM to about 1 μM, more preferably about 0.1 pM to about 100 nM. Methods for determining the affinity of epitope binding agents for antigens are known in the art and are further described in the Examples. In some embodiments, the anti-Nfl epitope binding agent is a nucleic acid aptamer. In some embodiments, the anti-Nfl epitope binding agent is an antibody.

[0047] The anti-Nfl epitope binding agents disclosed herein can be described or defined in terms of the epitopes they recognize or bind. The portion of the target polypeptide that specifically interacts with the antigen-binding domain of the epitope binding agent is an "epitope". Nfl can contain any number of epitopes, depending on the source of the protein (e.g., recombinant, human), the location of the protein (e.g., intracellular, extracellular, brain, CSF, blood, etc.), conformational state, and isoform, etc. Furthermore, it should be noted that the "epitope" on Nfl can be a linear epitope or a conformational epitope, and in both cases, can include non-polypeptide elements, e.g., the epitope can include carbohydrate side chains, lipid side chains, phosphates, etc. The term "affinity" refers to a measure of the strength of binding of an individual epitope to the antigen-binding site of the epitope binding agent.

[0048] Anti-Nfl epitope binding agents of the present disclosure can be made by using recombinant full-length Nfl as an antigen, and anti-Nfl epitope binding agents useful for the methods of the present disclosure specifically bind to an epitope present on Nfl isolated from blood or plasma. A preferred anti-Nfl epitope binding agent of the present disclosure specifically binds to an epitope within amino acids 90-543 of SEQ ID NO:1. In various embodiments, an anti-Nfl epitope binding agent of the present disclosure specifically binds to an epitope within amino acids 90-300 of SEQ ID NO:1, or within amino acids 90-250 of SEQ ID NO:1. In other embodiments, an anti-Nfl epitope binding agent of the present disclosure specifically binds to an epitope within amino acids 125-300 of SEQ ID NO:1, within amino acids 125-250 of SEQ ID NO:1, or within amino acids 125-200 of SEQ ID NO:1. In other embodiments, anti-NFL epitope binding agents of the disclosure specifically bind to an epitope within amino acids 251-400 of SEQ ID NO:1, within amino acids 251-355 of SEQ ID NO:1, within amino acids 272-355 of SEQ ID NO:1, or within amino acids 272-350 of SEQ ID NO:1. In other embodiments, anti-NFL epitope binding agents of the disclosure specifically bind to an epitope within amino acids 350-543 of SEQ ID NO:1, within amino acids 397-543 of SEQ ID NO:1, within amino acids 400-543 of SEQ ID NO:1, within amino acids 397-540 of SEQ ID NO:1, or within amino acids 400-540 of SEQ ID NO:1. Methods for epitope mapping are well known in the art.

[0049] In one embodiment, the anti-Nfl epitope binding agent is HJ30.1, an antigen-binding fragment of HJ30.1, or an epitope binding agent that competitively inhibits the binding of HJ30.1 to full-length recombinant Nlf. HJ30.1 is a monoclonal antibody produced by hybridoma clone PTA-126966 deposited with the American Type Culture Collection (ATCC).

[0050] In one embodiment, the anti-Nfl epitope binding agent is HJ30.2, an antigen-binding fragment of HJ30.2, or an epitope binding agent that competitively inhibits the binding of HJ30.2 to full-length recombinant Nlf. HJ30.2 is a monoclonal antibody produced by hybridoma clone PTA-126967 deposited with the ATCC.

[0051] In one embodiment, the anti-Nfl epitope binding agent is HJ30.4, an antigen-binding fragment of HJ30.4, or an epitope binding agent that competitively inhibits the binding of HJ30.4 to full-length recombinant Nlf. HJ30.4 is a monoclonal antibody produced by hybridoma clone PTA-126968 deposited with the ATCC.

[0052] In one embodiment, the anti-Nfl epitope binding agent is HJ30.7, an antigen-binding fragment of HJ30.7, or an epitope binding agent that competitively inhibits the binding of HJ30.7 to full-length recombinant Nlf. HJ30.7 is a monoclonal antibody produced by hybridoma clone PTA-126969 deposited with the ATCC.

[0053] In one embodiment, the anti-Nfl epitope binding agent is HJ30.11, an antigen-binding fragment of HJ30.11, or an epitope binding agent that competitively inhibits the binding of HJ30.11 to full-length recombinant Nlf. HJ30.11 is a monoclonal antibody produced by hybridoma clone PTA-126970 deposited with the ATCC.

[0054] In one embodiment, the anti-Nfl epitope binding agent is HJ30.13, an antigen-binding fragment of HJ30.13, or an epitope binding agent that competitively inhibits the binding of HJ30.13 to full-length recombinant Nlf. HJ30.13 is a monoclonal antibody produced by hybridoma clone PTA-126971 deposited with the ATCC.

[0055] In embodiments where the epitope-binding agent is an antibody, the antibody may or may not have a variant Fc region. In some instances, the Fc region may be modified to increase or decrease affinity for Fc receptors on microglial cells and / or to change glycosylation patterns. In various embodiments, the anti-Nfl antibody may be a humanized antibody. For example, in some instances, the anti-Nfl antibody of the present disclosure is a humanized antibody derived from HJ30.1, HJ30.2, HJ30.4, HJ30.7, HJ30.11, or HJ30.13. The humanized anti-Nfl antibody may comprise one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity with known human framework sequences).

[0056] A test epitope binding agent is said to competitively inhibit the binding of a reference epitope binding agent (e.g., HJ30.1, HJ30.2, HJ30.4, HJ30.7, HJ30.11, HJ30.13, etc.) to a given epitope if the test epitope binding agent preferentially binds to that epitope to the extent that it blocks binding of the reference epitope binding agent to the epitope by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. Competitive inhibition can be determined by any method known in the art. In a specific example, competitive inhibition is determined by competitive inhibition ELISA comprising the steps of: coating a binding surface or support with a purified reference epitope binding agent to form an epitope binding agent-coated surface; combining a test sample containing a predetermined amount of purified labeled antigen and test epitope binding agent; adding an incubated mixture of labeled antigen and test epitope binding agent to the coated surface; incubating the coated surface with the combination of antigen and test epitope binding agent; and measuring the amount of antigen binding inhibition compared to conditions lacking test epitope binding agent.

[0057] The anti-Nfl epitope binding agents disclosed herein can also be described or defined in terms of their sequences.

[0058] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.1, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.1. The HVRs from the VL of HJ30.1 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.1 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.1 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.1. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.1, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.1. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0059] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.2, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.2. The HVRs from the VL of HJ30.2 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.2 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.2 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.2. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.2, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.2. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0060] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.4, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.4. The HVRs from the VL of HJ30.4 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.4 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.4 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.4. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.4, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.4. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0061] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.7, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.7. The HVRs from the VL of HJ30.7 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.7 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.7 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.7. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.7, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.7. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0062] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.11, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.11. The HVRs from the VL of HJ30.11 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.11 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.11 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.11. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.11, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.11. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0063] In one embodiment, the anti-Nfl antibody comprises a light chain variable region (VL) having one or more HVRs from HJ30.17, and / or a heavy chain variable region (VH) having one or more HVRs from HJ30.17. The HVRs from the VL of HJ30.17 may be L1, L2, L3, or any combination thereof. The HVRs from the VH of HJ30.17 may be H1, H2, H3, or any combination thereof. The antibody comprising one or more HVRs from the VH of HJ30.17 may further comprise a VL comprising the L1, L2, L3, or any combination thereof of the VL of HJ30.17. In various embodiments above, the antibody may be a humanized antibody, or the antibody may have a VL with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.17, and / or a VH with 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.17. In each of the above embodiments, the anti-Nfl antibody may optionally include one or more constant regions or portions of constant regions that are substantially human (i.e., at least 90%, 95%, or 99% sequence identity to known human framework sequences). The present disclosure also encompasses corresponding nucleic acid sequences, which may be readily determined by those skilled in the art, and may be incorporated into vectors or other large DNA molecules, e.g., chromosomes, to express the antibodies of the present disclosure.

[0064] Anti-Nfl epitope binding agents disclosed herein can also be described or defined in terms of their cross-reactivity. The term "cross-reactivity" refers to the ability of an epitope binding agent specific for one antigen to react with a second antigen, and refers to a measure of relatedness between two different antigenic substances. Thus, an epitope binding agent is cross-reactive if it binds to an epitope other than the one that induces its formation. A cross-reactive epitope generally contains many of the same complementary structural features as the induced epitope, and in some cases may actually fit better than the original. For example, a certain antibody has a degree of cross-reactivity in that it binds to related but non-identical epitopes, such as epitopes that have at least about 85%, at least about 90%, or at least about 95% identity (calculated using methods known in the art) to a reference epitope. An antibody or other epitope-binding agent can be said to have little or no cross-reactivity if it does not bind to an epitope having less than about 95%, less than about 90%, or less than about 85% identity to a reference epitope. An antibody or other epitope-binding agent can be considered to be "highly specific" for a particular epitope if it does not bind to any other analogs, orthologs, or homologs.

[0065] III. Methods for detecting NF1 in biological samples In another embodiment, the present disclosure provides a method for detecting Nfl in a biological sample. The method includes preparing a biological sample, enriching one or more Nfl isoforms in the biological sample, and detecting the previously enriched one or more Nfl isoforms. As used herein, the terms "multiple Nfl isoforms" and "population of Nfl isoforms" can be used interchangeably.

[0066] (a) preparing a biological sample Suitable biological samples include blood samples or cerebrospinal fluid (CSF) samples obtained from a subject. Blood and CSF contain multiple Nfl isoforms, as detailed in the Examples.

[0067] The size of the biological sample used may vary depending on the type of sample, the health status of the subject from whom the sample was obtained, and the analyte added to the Nfl to be analyzed (e.g., Aβ, tau, ApoE, α-synuclein, etc.). The volume of the CSF sample may be about 0.01 mL to about 5 mL, or about 0.05 mL to about 5 mL. In a specific example, the size of the sample may be about 0.05 mL to about 1 mL of CSF. The volume of the plasma sample may be about 0.01 mL to about 20 mL, or about 0.1 mL to about 20 mL. In a specific example, the size of the sample may be about 1 mL to about 20 mL of blood.

[0068] In some embodiments, the subject is a human.The human subject may be waiting for medical treatment or treatment, may be under medical treatment or treatment, or may be undergoing medical treatment or treatment.In various embodiments, the human subject may be a healthy subject, a subject at risk of developing neurodegenerative disease, a subject with signs and / or symptoms of neurological damage and / or neurodegenerative disease, or a subject diagnosed with neurological damage and / or neurodegenerative disease. Neurodegenerative diseases include amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease, dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease, Huntington's disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury (TBI), Guam amyotrophic lateral sclerosis / parkinsonism-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, and frontotetemporal dementia. dementia), frontotemporal dementia linked to chromosome 17 with parkinsonism, Hallervorden-Spatz disease, Lewy body dementia (LBD), multiple sclerosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, Palido-Ponto-nigral degeneration, Parkinson's disease, Pick's disease, progressive subcortical gliosis, postencephalitic parkinsonism, PART (primary age-related tauopathy), progressive supranuclear palsy , subacute sclerosing panencephalitis, subacute sclerosing panencephalopathy, tangle-only dementia (or tangle-predominant dementia), tangle-predominant dementia, white matter tauopathy with globular glial inclusions, mild cognitive impairment (MCI), glaucoma, familial british dementia, familial danish dementia, Guadeloupe parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-associated mental retardation, HIV-associated dementia, senile cardiac amyloidosis. Healthy subjects, which may be referred to as "control subjects" or "healthy controls," minimally have no clinical signs or symptoms of cognitive impairment and may be "negative" for other clinical signs or symptoms of neurological injury, neurodegenerative disease, and / or traumatic brain injury.

[0069] In other embodiments, the subject is an experimental animal. In further embodiments, the subject is an experimental animal genetically engineered to express human Nfl.

[0070] CSF may be obtained by lumbar puncture with or without an indwelling CSF catheter. Blood may be collected by venipuncture with or without a venous catheter, or by finger prick (or the equivalent). Multiple blood or CSF samples collected from a subject at the same time may be pooled to create a "sample". Once collected, blood or CSF samples may be processed according to methods known in the art (e.g., centrifugation to remove whole cells and cell debris; use of additives designed to stabilize and preserve specimens prior to analytical testing, etc.). Blood or CSF samples may be used immediately or may be frozen and stored indefinitely.

[0071] Prior to use in the methods disclosed herein, the biological sample may also be modified, as necessary or desired, to include protease inhibitors, internal standards, detergents, and chaotropic agents, to be depleted of other analytes (e.g., proteins, peptides, metabolites, etc.), or any combination thereof.

[0072] The term "deplete" means to reduce the amount or number. Thus, a protein-depleted sample may have any amount of protein that is measurably less than the amount in the original sample, or may not contain any amount of protein. As a non-limiting example, proteins may be depleted from a sample by ultrafiltration, or protein precipitation using acids, organic solvents, or salts. Generally speaking, these methods are used to ensure that high abundance and high molecular weight proteins are reduced, which in turn enriches for low molecular weight and / or low abundance proteins and peptides (e.g., tau, Aβ, Nfl, etc.). In a specific example, proteins may be depleted from a sample by precipitation. Briefly, precipitation involves adding a precipitating agent to a sample and mixing thoroughly, incubating the sample with the precipitating agent to precipitate proteins, and separating the precipitated proteins by centrifugation or filtration. The resulting supernatant may then be used in downstream applications. The amount of reagent required may be determined experimentally by methods known in the art. Suitable precipitating agents include perchloric acid, trichloroacetic acid, acetonitrile, methanol, and the like. In an exemplary embodiment, proteins are depleted from the sample by acid precipitation. In a further embodiment, proteins are depleted from the sample by acid precipitation using perchloric acid.

[0073] In a further example, proteins may be depleted from a sample by acid precipitation using perchloric acid. As used herein, "perchloric acid" refers to 70% perchloric acid unless otherwise indicated. In some embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 10% v / v. In other embodiments, perchloric acid is added to a final concentration of about 1% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 15% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 10% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of 3.5% v / v to about 15% v / v, 3.5% v / v to about 10% v / v, or 3.5% v / v to about 5% v / v. In other embodiments, perchloric acid is added to a final concentration of about 3.5% v / v. After addition of perchloric acid, the sample is thoroughly mixed (e.g., by vortex mixing) and typically maintained at low temperature for about 10 minutes or more to promote precipitation. For example, the sample may be maintained for about 10 minutes to about 60 minutes, about 20 minutes to about 60 minutes, or about 30 minutes to about 60 minutes. In other examples, the sample may be maintained for about 15 minutes to about 45 minutes, or about 30 minutes to about 45 minutes. In other examples, the sample may be maintained for about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes. In other examples, the sample is maintained for about 30 minutes. The sample is then centrifuged at low temperature to pellet precipitated proteins, e.g., the supernatant containing soluble tau (i.e., the acid-soluble fraction) is transferred to a new container. As used in the above context, "low temperature" refers to a temperature of 10°C or lower. For example, low temperature may be about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C. In some embodiments, a narrower temperature range may be preferred, e.g., about 3°C ​​to about 5°C, or even about 4°C. In certain embodiments, low temperature may be achieved by placing the sample on ice.

[0074] Alternatively or in addition, proteins, peptides, and / or metabolites may be depleted from a sample by methods that specifically target the biomolecules of interest, for example, by affinity depletion, solid phase extraction, or other methods known in the art. Targeted depletion of a protein / peptide or multiple proteins / peptides may be used in situations where downstream analysis of the protein / peptide is desired (e.g., identification, quantification, analysis of post-translational modifications, etc.). Typically, at least 50% (e.g., 50%, 60%, 70%, 80%, 90%, or more) of the targeted protein in the starting material is depleted. In some embodiments, about 70% or more, about 80% or more, or about 90% or more of the targeted protein in the starting material is depleted. Affinity depletion refers to a method of depleting a protein of interest from a sample by its specific binding to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support, for example, a bead, a resin, a tissue culture plate, etc. Immobilization of the ligand to the solid support may also occur after ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope binding agents. For example, Aβ peptides may be identified and quantified by methods known in the art after affinity depletion of Aβ with a suitable epitope binding agent. Tau may be identified and quantified in a similar manner. The molecule may also be a polymer or other material that selectively absorbs the protein of interest. As a non-limiting example, polyhydroxymethylene substituted with fatty oxethylized alcohol (e.g., PHM-L LIPOSORB, Sigma Aldrich) may be used to selectively absorb lipoproteins (including ApoE) from serum. Identification of ApoE isoforms ("ApoE status") and / or quantification of ApoE may then be performed by methods known in the art.Targeted depletion may also be used to isolate other proteins, including, but not limited to, apolipoprotein J, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukins, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, prion protein, pNFH, and DJ-1, for subsequent analysis.

[0075] (b) enriching one or more Nfl isoforms in the biological sample; The term "enrich" refers to an increase in amount or number. Blood and CSF contain multiple Nfl isoforms. Thus, "enriching one or more Nfl isoforms in a biological sample" refers to measurably increasing the amount of an Nfl isoform or multiple Nfl isoforms per volume of sample compared to the starting sample (i.e., biological sample). In some examples, the enrichment may be at least about 5-fold. In some examples, the enrichment may be about 5-fold to about 1000-fold. For example, the enrichment may be at least about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, or more.

[0076] In some embodiments, the method of the present disclosure includes enriching one or more Nfl isoforms in a biological sample, where the Nfl isoforms are about 350 amino acids or less in length. For example, the Nfl isoforms may be about 300 amino acids or less, about 250 amino acids or less, about 200 amino acids or less, about 150 amino acids or less, about 100 amino acids or less, or even about 50 amino acids or less in length. The Nfl isoforms about 350 amino acids or less in length may contain an N-terminal truncation, a C-terminal truncation, or an N-terminal and a C-terminal truncation of the full-length Nfl (having the amino acid sequence of SEQ ID NO: 1). In a further example, the method of the present disclosure may include enriching one or more Nfl isoforms having an amino acid sequence that includes amino acids 92-100, amino acids 101-107, amino acids 108-116, amino acids 117-126, amino acids 137-144, amino acids 148-157, amino acids 158-164, amino acids 165-172, amino acids 178-185, amino acids 192-196, or amino acids 198-206 of SEQ ID NO: 1, or any combination thereof. In another example, the method of the present disclosure may include enriching one or more Nfl isoforms having an amino acid sequence that includes amino acids 282-292, amino acids 323-330, amino acids 331-338, or amino acids 339-353 of SEQ ID NO: 1, or any combination thereof. In yet another example, the method of the present disclosure may include enriching for one or more Nfl isoforms having an amino acid sequence including amino acids 422-437, amino acids 438-462, and / or amino acids 530-540 of SEQ ID NO: 1. In yet another example, the method of the present disclosure may include enriching for a first population of Nfl isoforms, and then subsequently enriching for a second, third, fourth, or more populations of Nfl isoforms, in each case using a previously depleted sample. In yet another example, the method of the present disclosure may include enriching for one or more isoforms described in FIG.

[0077] The disclosed method may enrich for a truncated Nfl isoform or multiple truncated Nfl isoforms by isolating the Nfl isoform from a biological sample (e.g., affinity purification, solid-phase extraction, etc.) and / or removing other Nfl isoforms from a biological sample (e.g., affinity depletion, solid-phase extraction, etc.). Affinity depletion is described above. Affinity purification refers to a method of enriching a protein of interest by its specific binding to a molecule. Typically, the molecule is a ligand (referred to as an immobilized ligand) attached to a solid support, such as a bead, a resin, a tissue culture plate, etc. Immobilization of the ligand to the solid support may also occur after the ligand-protein interaction has occurred. Suitable ligands include antibodies, aptamers, and other epitope binding agents. Purifying Nfl by affinity purification includes contacting a sample containing Nfl with a suitable immobilized ligand, one or more washing steps, and elution of the Nfl from the immobilized ligand. Reagents for affinity purification and / or affinity depletion of Nfl can be made by methods known in the art (e.g., using full-length recombinant Nfl to make epitope binding agents, and then selecting epitope binding agents that bind to a given epitope, using recombinant Nfl peptides to make epitope binding agents for a certain fragment of Nfl, etc.). Commercially available epitope binding agents, such as ABIN6025698 (Abbexa), ABIN4339158 (Novus Biologicals), 13-0400 (Invitrogen Antibodies), UD1 or UD2 (Uman Diagnostics), etc., can also be used. Suitable epitope binding agents are also described in Section II.

[0078] Alternatively, enrichment of an Nfl isoform may not be performed directly, but rather amplified detection of an Nfl isoform or multiple Nfl isoforms may be performed indirectly. For example, a proximity ligation assay [e.g., Duo-Link (Sigma Aldrich)] may be used to detect the N- and C-terminal regions of an Nfl isoform using a reagent that can generate an amplified signal when the reagent binds to the N- and C-terminal regions, respectively. Typically, the reagent is an epitope binding agent. Suitable epitope binding agents may include those described in Section II and / or commercially available antibodies. Amplified detection of an Nfl isoform or multiple Nfl isoforms, such as by a proximity ligation assay, may also be performed after an enrichment or depletion step (e.g., after a single enrichment step using an epitope binding agent that enriches for isoforms that include amino acids 530-540 of SEQ ID NO:1).

[0079] In a specific embodiment of the above, the step of enriching one or more NF1 isoforms in the biological sample comprises contacting the biological sample with an epitope binding agent that specifically binds to a first population of NF1 isoforms and isolating the first population of NF1 isoforms, wherein the epitope binding agent is selected from the group consisting of: (i) an epitope binding agent that specifically binds to an epitope within amino acids 90-250 of SEQ ID NO:1; (ii) an epitope binding agent that specifically binds to an epitope within amino acids 116-184 of SEQ ID NO:1; (iii) an epitope binding agent that specifically binds to an epitope within amino acids 250-400 of SEQ ID NO:1; (iv) an epitope binding agent that specifically binds to an epitope within amino acids 283-338 of SEQ ID NO:1; and (v) an epitope binding agent that specifically binds to an epitope within amino acids 400-543 of SEQ ID NO:1. epitope binding agents; (vi) epitope binding agents that specifically bind to an epitope within amino acids 437 to 543 of SEQ ID NO:1; (vii) an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl; (viii) HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; and (ix) HJ30.11, an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.11 to full-length recombinant Nfl.

[0080] In another specific embodiment of the above, the step of enriching one or more Nfl isoforms in a biological sample may include: (a) contacting the biological sample with a first epitope binding agent that specifically binds to a first population of Nfl isoforms, and isolating the first population of Nfl isoforms; and (b) contacting the sample depleted of the first population of Nfl isoforms with a second epitope binding agent that specifically binds to a second population of Nfl isoforms, and isolating the second population of Nfl isoforms. Alternatively, the biological sample may be contacted with the first epitope binding agent and the second epitope binding agent simultaneously, and the first population of Nfl isoforms and the second population of Nfl isoforms may be isolated sequentially or simultaneously. In a preferred embodiment, the second epitope binding agent binds to an epitope downstream of the epitope of the first epitope binding agent. In one example, the first epitope binding agent specifically binds to a first epitope within amino acids 1-450 of SEQ ID NO:1; the second epitope binding agent specifically binds to an epitope within amino acids 400-543 of SEQ ID NO:1. In another example, the first epitope binding agent specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1 or within amino acids 200-400 of SEQ ID NO:1; the second epitope binding agent specifically binds to an epitope within amino acids 400-543 of SEQ ID NO:1. In another example, the first epitope binding agent specifically binds to a first epitope within amino acids 90-250 of SEQ ID NO:1 or within amino acids 250-400 of SEQ ID NO:1; the second epitope is within amino acids 400-543 of SEQ ID NO:1 or within amino acids 430-540 of SEQ ID NO:1. Suitable epitope binding agents include those described in Section II and / or commercially available antibodies. In a specific embodiment, the first epitope binding agent may be HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits the binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl.In another specific embodiment, the first epitope binding agent may be HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits the binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl. In another specific embodiment, the second epitope binding agent may be HJ30.11, an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits the binding of HJ30.11 to full-length recombinant Nfl. In another specific embodiment, the second epitope binding agent may be HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits the binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl.

[0081] In another specific embodiment of the above, the step of enriching one or more Nfl isoforms in a biological sample may include: (a) contacting the biological sample with a first epitope binding agent that specifically binds to a first population of Nfl isoforms, and isolating the first population of Nfl isoforms; (b) contacting the sample depleted of the first population of Nfl isoforms with a second epitope binding agent that specifically binds to a second population of Nfl isoforms, and isolating the second population of Nfl isoforms; and (c) contacting the sample depleted of the first and second populations of Nfl isoforms with a third epitope binding agent that specifically binds to a third population of Nfl isoforms, and isolating the third population of Nfl isoforms. Alternatively, the biological sample may be contacted with the first epitope binding agent, the second epitope binding agent, and the third epitope binding agent simultaneously, and the first group of Nfl isoforms, the second group of Nfl isoforms, and the third group of Nfl isoforms may be isolated sequentially or in any combination.In a preferred embodiment, the second epitope binding agent may bind to the downstream epitope of the first epitope binding agent, and the third epitope binding agent may bind to the downstream epitope of the second epitope binding agent.Alternatively, the second epitope binding agent may bind to the upstream epitope of the first epitope binding agent. In one example, a first epitope binding agent specifically binds to a first epitope within amino acids 1-400 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 90-450 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 200-400 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope.In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-250 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope downstream from the first epitope within amino acids 250-400 of SEQ ID NO:1; and a third epitope binding agent specifically binds to a third epitope downstream from the second epitope within amino acids 400-543 of SEQ ID NO:1. Suitable epitope binding agents include those described in Section II and / or commercially available antibodies. In specific embodiments, (i) the first epitope-binding agent is HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl; (ii) the second epitope-binding agent is HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; (iii) the third epitope-binding agent is HJ30.11, an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.11 to full-length recombinant Nfl; or (iv) any combination thereof.

[0082] In another specific embodiment of the above, the step of enriching one or more Nfl isoforms in a biological sample may include (a) contacting the biological sample with two or more epitope binding agents simultaneously (but not sequentially), where each epitope binding agent specifically binds to a different epitope of Nfl; and (b) detecting and, where appropriate, quantifying the one or more Nfl isoforms enriched in step (a). In one example, a first epitope binding agent specifically binds to a first epitope within amino acids 1-400 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 90-450 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 200-400 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-250 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 250-400 of SEQ ID NO:1; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1. Suitable epitope binding agents include those described in Section II and / or commercially available antibodies.In specific embodiments, the two or more epitope binding agents are selected from the group consisting of HJ30.1, an antigen-binding fragment of HJ30.1, HJ30.2, an antigen-binding fragment of HJ30.2, HJ30.13, an antigen-binding fragment of HJ30.13, HJ30.4, an antigen-binding fragment of HJ30.4, HJ30.7, an antigen-binding fragment of HJ30.7, HJ30.11, an antigen-binding fragment of HJ30.11, or an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2, HJ30.4, HJ30.7, HJ30.11, and HJ30.13 to full-length recombinant Nfl. In another specific embodiment, one epitope binding agent is selected from group (i), (ii), or (iii) and at least one additional epitope binding agent is selected from a different group (i), (ii), or (iii), where group (i), (ii), or (iii) is selected from (i) HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or HJ30.14 or an antigen-binding fragment thereof, (ii) an epitope binding agent that competitively inhibits the binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; (iii) an epitope binding agent that competitively inhibits the binding of HJ30.11, an antigen-binding fragment thereof, or HJ30.11 to full-length recombinant Nfl.In another specific embodiment, the at least one epitope binding agent is selected from each of groups (i), (ii), and (iii), where group (i), (ii), or (iii) is (i) an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or full-length recombinant Nfl; (ii) an epitope binding agent that competitively inhibits binding of HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or HJ30.4 or HJ30.7 to full-length recombinant Nfl; or (iii) an epitope binding agent that competitively inhibits binding of HJ30.11, an antigen-binding fragment thereof, or HJ30.11 to full-length recombinant Nfl.

[0083] An internal standard (abbreviated as "ISTD" herein) may be used to account for variations during enrichment and calculate absolute concentrations accordingly. In general, an internal standard is added prior to processing of the sample of interest, which may be added more than once if necessary. One or more full-length Nfl isoforms may be used. Alternatively or additionally, isoforms of Nfl and / or peptide fragments of Nfl with post-translational modifications may also be used. For example, in embodiments using sequential isolation of multiple isoforms, it may be advantageous to use a number of internal standards equal to the number of isolation steps, where each internal standard is a different peptide fragment of Nfl, such that each isolation step isolates only a single internal standard. In some embodiments, each internal standard may be a different AQUA peptide (i.e., a stable isotope-labeled peptide corresponding to the peptide of interest detected by MS). Alternatively, a single internal standard may be used when the Nfl isoform of the final isolation step is the only one of interest, where the internal standard is a peptide fragment that is not isolated / enriched until the final step. In some embodiments, the internal standard may be an AQUA peptide. Typically, the internal standard is detectably labeled to distinguish the Nfl standard from endogenous Nfl analytes without affecting the chemical properties on which the separation is based. In some embodiments, the internal standard is an isotopically labeled internal Nfl standard. A suitable isotopically labeled internal Nfl standard has a heavy isotope label incorporated into at least one amino acid residue. Generally speaking, the incorporated labeled amino acid residue should increase the mass of the peptide without affecting its chemical properties, and the mass shift resulting from the presence of the isotope label should be sufficient to allow the mass spectrometry method to distinguish the internal standard (IS) from the signal of the endogenous Nfl analyte. As shown herein, suitable heavy isotope labels include, but are not limited to: 2 H, 13 C and 15 In one example, the internal standard is Lys, Arg. 13 C and 15The internal standard may be N-labeled recombinant full-length Nfl or a peptide fragment of Nfl. Typically, about 0.1 to 10 ng of the internal standard is sufficiently diluted in an appropriate buffer (e.g., TBEAC, about 0.01 to 2% albumin, etc.). In an exemplary embodiment, it is about 1 ng to about 2.5 ng in about 1% human serum albumin (HSA).

[0084] (c) detecting one or more previously enriched Nfl isoforms; Nfl isoforms may be detected and appropriately quantified in enriched samples by mass spectrometry as further detailed below or in the Examples, or by other methods known in the art, including but not limited to immunoassays, multiplex assays (such as xMAP technology by Luminex), single molecule array assays (such as Simoa® bead technology), proximity ligation assays (such as DuoLink® by Sigma Aldrich), etc.

[0085] In some embodiments, Nfl isoforms are detected and appropriately quantified in enriched samples by mass spectrometry. Briefly, detection by mass spectrometry includes cleaving enriched Nfl isoforms with protease, and appropriately desalting the obtained cleavage products by solid phase extraction to obtain a sample containing proteolytic peptides of Nfl; and performing liquid chromatography-mass spectrometry (LC / MS) of the sample containing proteolytic peptides of Nfl to detect at least one proteolytic peptide of Nfl. In any of the methods disclosed herein, the amount of any proteolytic peptide of Nfl can also be quantified (e.g., from the peak height or integral of MS analysis corresponding to appropriate proteolytic peptide). Thus, in practice, one or more proteolytic peptides of Nfl are used to detect and measure the amount of Nfl protein present in biological samples. Because blood and CSF contain multiple Nfl isoforms, and a subset of the isoforms share sequence similarity (see Figure 22), measurement of Nfl proteolytic peptides can describe the levels of multiple Nfl isoforms in a biological sample containing the measured proteolytic peptides, as long as the enrichment method is specific for a given isoform.

[0086] In embodiments where trypsin is the protease, suitable Nfl proteolytic peptides indicative of the presence of Nfl can be as described in the Examples. For example, tryptic peptides [370,378], [379,389], and [391,398] are not unique to Nfl and are therefore not preferred when the objective is to quantify Nfl in a biological sample. In some embodiments, suitable Nfl proteolytic peptides indicative of the presence of Nfl can be selected from the peptides listed in Table C. Exemplary tryptic peptides include tryptic peptides having amino acids 108-116 of SEQ ID NO:1, amino acids 117-126 of SEQ ID NO:1, amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, amino acids 324-331 of SEQ ID NO:1, amino acids 400-421 of SEQ ID NO:1, amino acids 422-437 of SEQ ID NO:1, amino acids 438-462 of SEQ ID NO:1, and amino acids 530-540 of SEQ ID NO:1. When using alternative enzymes for digestion, the resulting proteolytic peptides may be slightly different, but can be easily determined by those skilled in the art. Without wishing to be bound by theory, it is believed that the variation in the amount of tryptic peptides between two biological samples of the same type (e.g., two blood samples) reflects the difference in the Nfl isoforms that constitute these biological samples. As disclosed herein, the amount of a certain Nfl proteolytic peptide and the ratio of a certain Nfl proteolytic peptide can provide clinically significant information for diagnosing neuronal damage, informing the diagnosis of neurodegenerative disease, selecting patients for further diagnostic testing, guiding treatment decisions, or any combination thereof. Thus, a method that allows detection and quantification of Nfl tryptic peptides has utility in the diagnosis, prognosis, and treatment of many diseases.

[0087] [Table 4]

[0088] The proteolytic peptides of Nfl may be separated by a liquid chromatography system coupled to a high-resolution mass spectrometer. A suitable LC-MS system may include a column with an ID of <1.0 mm and use a flow rate of less than about 100 μl / min. In a preferred embodiment, a nanoflow LC-MS system is used (e.g., a column with an ID of about 50-100 μm and a flow rate of <1 μL / min, preferably about 100-800 nL / min, more preferably about 200-600 nL / min). In an exemplary embodiment, the LC-MS system may include a column with an ID of 0.05 mm and use a flow rate of about 400 nL / min.

[0089] As known in the art, tandem mass spectrometry can be used to improve resolution, or the technique can be improved to achieve the resolution of tandem mass spectrometry using a single mass analyzer.Suitable mass analyzer types are known in the art.These include, but are not limited to, quadrupole, time-of-flight, ion trap and Orbitrap, and hybrid mass analyzers that combine different types of mass analyzers into one configuration (e.g. Orbitrap Fusion™ Tribrid™ mass analyzer, Orbitrap Fusion™ Lumos™ mass analyzer, Orbitrap Tribrid™ Eclipse™ mass analyzer, Q Exactive mass analyzer, each ThermoFisher Scientific). In an exemplary embodiment, the LC-MS system may include a mass analyzer selected from an Orbitrap Fusion™ Tribrid™ mass analyzer, an Orbitrap Fusion™ Lumos™ mass analyzer, an Orbitrap Tribrid™ Eclipse™ mass analyzer, or a mass analyzer with similar or improved ion focusing and ion transmission in the quadrupole. A suitable mass analysis protocol may be developed by optimizing the number of ions collected prior to analysis (e.g., AGC settings using an Orbitrap) and / or injection time. In an exemplary embodiment, the mass analysis protocol outlined in the examples is used.

[0090] The proteolytic peptides analyzed by MS can be quantified by methods known in the art. Generally speaking, a known amount of internal standard is added to the sample. The sample is then digested and analyzed by LC-MS. Extracted ion chromatograms are generated for native peptides and internal standards. Peak ratios (e.g., 14N / 15N) are used to calculate the amount of native peptides.

[0091] IV. METHODS FOR DETECTING BIOMARKERS AND USES THEREOF In another aspect, the present disclosure provides a method for detecting and optionally quantifying protein biomarkers in a sample obtained from a subject.The method comprises detecting and optionally quantifying Nfl according to the method of section III, where the biological sample is a sample obtained from a subject with or at risk of having neuronal injury, and the biomarker is enriched Nfl isoform, the ratio of a first Nfl isoform to a second Nfl isoform, an enriched population of Nfl isoforms, or the ratio of a first population of enriched Nfl isoforms and a second population of enriched isoforms.In some embodiments, the neuronal injury can include axonal injury.In some embodiments, the neuronal injury is axonal injury.The type of neuronal injury is not limited and can be caused by acute or chronic injury (such as traumatic brain injury), neuroinflammation, and / or neurodegenerative disease.Non-limiting examples of neurodegenerative diseases include amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, chronic traumatic encephalopathy (CTE), Creutzfeldt-Jakob disease, dementia pugilistica, Down's syndrome, Gerstmann-Straussler-Scheinker disease, Huntington's disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, Guam amyotrophic lateral sclerosis / parkinsonism-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, diffuse neurofibrillary tangle disease with calcifications, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 and parkinsonism, Hallervorden-Spatz disease, and Lewy body dementia (LBD). These include multiple sclerosis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, Palido-Ponto-nigral degeneration, Parkinson's disease, Pick's disease, progressive subcortical gliosis, postencephalitic parkinsonism, PART (primary age-related tauopathy), progressive supranuclear palsy, subacute sclerosing panencephalitis, subacute sclerosing panencephalopathy, tangle-only dementia (or tangle-predominant dementia), tangle-predominant dementia, white matter tauopathy with globular glial inclusions, mild cognitive impairment (MCI), glaucoma, familial British dementia, familial Danish dementia, Guadeloupe parkinsonism, neurodegeneration with brain iron accumulation, SLC9A6-related mental retardation, HIV-associated dementia, and senile cardiac amyloidosis. Healthy subjects, which may be referred to as "control subjects" or "healthy controls," minimally have no clinical signs or symptoms of cognitive impairment and may be "negative" for other clinical signs or symptoms of neurological injury, neurodegenerative disease, and / or traumatic brain injury.

[0092] In some embodiments, the biomarker may be a population of Nfl isoforms selected from the group consisting of isoforms having an amino acid sequence consisting essentially of the rod domain of Nfl, isoforms having an amino acid sequence consisting essentially of a portion of the rod domain of Nfl, isoforms having an amino acid sequence consisting of a portion of the rod domain of Nfl with no amino acids outside this region, or any combination thereof. The rod domain is typically described as comprising amino acids 90-396 of SEQ ID NO: 1. For example, the biomarker may comprise one or more Nfl isoforms comprising amino acids 90-396 of SEQ ID NO: 1 having a length of up to about 330 amino acids (e.g., about 330, about 325, about 320, about 315, about 310 amino acids). Alternatively or additionally, the biomarker may comprise one or more Nfl isoforms that are fragments of amino acids 90-396 of SEQ ID NO: 1. For example, the biomarker may be less than 310 amino acids in length (about 305, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 25, etc.) and may include one or more Nfl isoforms that include amino acids 164-171 of SEQ ID NO:1, amino acids 197-205 of SEQ ID NO:1, amino acids 323-330 of SEQ ID NO:1, or any combination thereof. In some examples, the biomarker may be less than about 200 amino acids in length and may include one or more Nfl isoforms that include amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, or any combination thereof. Alternatively or in addition, the biomarker may include one or more Nfl isoforms that are fragments of amino acids 125-396 of SEQ ID NO:1. For example, the biomarker may include one or more Nfl isoforms that are about 275 amino acids in length or less and include amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, amino acids 324-331 of SEQ ID NO:1, or any combination thereof.

[0093] In some embodiments, the biomarker may be a population of Nfl isoforms selected from the group consisting of isoforms consisting essentially of the tail region of Nfl, isoforms having an amino acid sequence consisting of a portion of the tail region of Nfl, or combinations thereof. The tail region is typically set forth as amino acids 397-543 of SEQ ID NO: 1. For example, the biomarker may include one or more Nfl isoforms that include amino acids 397-543 of SEQ ID NO: 1 that are 147 amino acids long to about 200 amino acids long. Alternatively or in addition, the biomarker may include one or more Nfl isoforms that are fragments of amino acids 397-543 of SEQ ID NO: 1. For example, the biomarker may include one or more Nfl isoforms that are less than 147 amino acids long and include amino acids 422-437 of SEQ ID NO: 1, amino acids 438-462 of SEQ ID NO: 1, amino acids 530-540 of SEQ ID NO: 1, or any combination thereof. In another embodiment, the biomarker may include one or more Nfl isoforms that are less than about 100 amino acids in length and include amino acids 422-437 of SEQ ID NO:1, amino acids 438-462 of SEQ ID NO:1, amino acids 530-540 of SEQ ID NO:1, or any combination thereof.

[0094] In further embodiments, the biomarker may be the ratio of two biomarkers described above. As a non-limiting example, the biomarker may be the ratio of two biomarkers from rod region, or the ratio of two biomarkers from tail region, or more preferably, the ratio of one biomarker from rod region and one biomarker from tail region. In yet further embodiments, the biomarker may be the ratio of the biomarker to the total amount of Nfl. Other mathematical operations and the use of more than two biomarkers are also contemplated. For example, the rate of change of the biomarker may be used when a first and a second biological sample are analyzed, where the second sample is obtained at a period (e.g., days, weeks, months, years) after the first.

[0095] In some embodiments, the biomarker may be a population of Nfl isoforms affinity purified using an epitope binding agent selected from: (i) HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl; (ii) HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; or (iii) HJ30.11, an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.11 to full-length recombinant Nfl.

[0096] In some embodiments, the biomarkers may be: (a) a population of Nfl isoforms comprising amino acids 165-172 of SEQ ID NO:1 and / or amino acids 198-206 of SEQ ID NO:1, wherein the population of Nfl isoforms has been affinity purified using an epitope binding agent selected from (i) HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl; (b) a population of Nfl isoforms comprising amino acids 324 to 331 of SEQ ID NO:1, wherein the population of Nfl isoforms has been affinity purified using an epitope binding agent selected from HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; (c) a population of Nfl isoforms comprising amino acids 530-540 of SEQ ID NO:1, wherein the population of Nfl isoforms has been affinity purified using an epitope binding agent selected from HJ30.11, an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.11 to full-length recombinant Nfl; (d) the ratio of (a) to (b); (e) the ratio of (b) to (c); or (f) The ratio of (a) to (c).

[0097] In the above embodiment, each population of Nfl isoforms may be isolated from a biological sample, or may be isolated from a sample that has previously been depleted of other Nfl isoforms. As a non-limiting example, the population of Nfl isoforms in (c) may be isolated from a sample that has been depleted of (a), depleted of (b), or depleted of (a) and (b).

[0098] In one example, the method includes the steps of: (a) providing a biological sample obtained from a subject, the biological sample being a blood sample or a CSF sample; (b) enriching one or more Nfl isoforms in the biological sample, the enriching step comprising: (i) contacting the biological sample with a first epitope binding agent that specifically binds to a first population of Nfl isoforms to isolate the first population of Nfl isoforms; (ii) contacting the sample depleted of the first population of Nfl isoforms with a second epitope binding agent that specifically binds to a second population of Nfl isoforms to isolate the second population of Nfl isoforms; and (iii) conjugating the sample to a second epitope binding agent that specifically binds to a second population of Nfl isoforms. The method comprises the steps of: (a) contacting a sample depleted of the first and second populations of Nfl isoforms with a third epitope-binding agent that specifically binds to a third population of Nfl isoforms, and isolating the third population of Nfl isoforms; and (c) detecting and optionally quantifying one or more Nfl isoforms in the first population, the second population, the third population, or any combination thereof, wherein the biomarker is the Nfl isoform or population of Nfl isoforms detected in (c), or a ratio between the Nfl isoform or population of Nfl isoforms detected in (c). In a preferred embodiment, the second epitope-binding agent may bind to an epitope downstream of the epitope of the first epitope-binding agent, and the third epitope-binding agent may bind to an epitope downstream of the epitope of the second epitope-binding agent. Alternatively, the second epitope binding agent may bind to an epitope upstream of the first epitope binding agent. In one example, the first epitope binding agent specifically binds to a first epitope within amino acids 1-400 of SEQ ID NO:1; the second epitope binding agent specifically binds to a second epitope within amino acids 90-450 of SEQ ID NO:1; and the third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1.In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 200-400 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-250 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 250-400 of SEQ ID NO:1; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1. Suitable epitope binding agents include those described in Section II and / or commercially available antibodies. In specific embodiments, (i) the first epitope-binding agent is HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full-length recombinant Nfl; (ii) the second epitope-binding agent is HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; (iii) the third epitope-binding agent is HJ30.11, an antigen-binding fragment thereof, or an epitope-binding agent that competitively inhibits binding of HJ30.11 to full-length recombinant Nfl; or (iv) any combination thereof.

[0099] In some embodiments, the method includes: (a) preparing a biological sample obtained from a subject, where the biological sample is a blood sample or a CSF sample; (b) enriching multiple Nfl isoforms by contacting the biological sample with two or more epitope binding agents, where each epitope binding agent specifically binds to a different epitope of Nfl; and (c) detecting and, where appropriate, quantifying one to more Nfl isoforms enriched in step (b), where the biomarker is an Nfl isoform or population of Nfl isoforms detected in (c), or a ratio between an Nfl isoform or population of Nfl isoforms detected in (c). In one example, a first epitope binding agent specifically binds to a first epitope within amino acids 1-400 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 90-450 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1; a second epitope binding agent specifically binds to a second epitope within amino acids 200-400 of SEQ ID NO:1 downstream of the first epitope; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO:1 downstream of the second epitope. In another example, a first epitope binding agent specifically binds to a first epitope within amino acids 90-250 of SEQ ID NO: 1; a second epitope binding agent specifically binds to a second epitope within amino acids 250-400 of SEQ ID NO: 1; and a third epitope binding agent specifically binds to a third epitope within amino acids 400-543 of SEQ ID NO: 1. Suitable epitope binding agents include those described in Section II and / or commercially available antibodies.In specific embodiments, the two or more epitope binding agents are selected from the group consisting of HJ30.1, an antigen-binding fragment of HJ30.1, HJ30.2, an antigen-binding fragment of HJ30.2, HJ30.13, an antigen-binding fragment of HJ30.13, HJ30.4, an antigen-binding fragment of HJ30.4, HJ30.7, an antigen-binding fragment of HJ30.7, HJ30.11, an antigen-binding fragment of HJ30.11, or an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2, HJ30.4, HJ30.7, HJ30.11, and HJ30.13 to full-length recombinant Nfl. In another specific embodiment, one epitope binding agent is selected from group (i), (ii), or (iii) and at least one additional epitope binding agent is selected from a different group (i), (ii), or (iii), where group (i), (ii), or (iii) is selected from (i) HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or HJ30.14 or an antigen-binding fragment thereof, (ii) an epitope binding agent that competitively inhibits the binding of HJ30.4 or HJ30.7 to full-length recombinant Nfl; (iii) an epitope binding agent that competitively inhibits the binding of HJ30.11, an antigen-binding fragment thereof, or HJ30.11 to full-length recombinant Nfl.In another specific embodiment, the at least one epitope binding agent is selected from each of groups (i), (ii), and (iii), where group (i), (ii), or (iii) is (i) an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or full-length recombinant Nfl; (ii) an epitope binding agent that competitively inhibits binding of HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or HJ30.4 or HJ30.7 to full-length recombinant Nfl; or (iii) an epitope binding agent that competitively inhibits binding of HJ30.11, an antigen-binding fragment thereof, or HJ30.11 to full-length recombinant Nfl.

[0100] The detection and quantification of biomarkers can be used for several purposes.Non-limiting examples include diagnosing nerve damage, diagnosing diseases or conditions characterized by nerve damage (e.g., traumatic brain injury, neurodegenerative diseases, etc.), monitoring / measuring the occurrence or progression of nerve damage and / or disease conditions characterized by nerve damage, treating subjects with nerve damage, determining / measuring the effectiveness of a given treatment, etc.

[0101] Thus, in another aspect, the method comprises detecting and quantifying the level of a biomarker as described in any of the above embodiments, and determining whether the level is reduced compared to the level in a cognitively normal control subject. In another aspect, the method comprises detecting and quantifying the level of a biomarker as described in any of the above embodiments, and determining whether the level is reduced compared to the level in a cognitively normal control subject who is also negative for one or more additional clinical signs or symptoms of neurodegenerative disease or traumatic brain injury. In another aspect, the method comprises detecting and quantifying the level of a biomarker as described in any of the above embodiments, and determining whether the level is reduced compared to the level in a cognitively normal control subject who is also negative for pathological levels of tau deposition in the brain. Clinical tests for assessing cognitive impairment, including dementia, are known in the art. As a non-limiting example, the Clinical Dementia Scale (CDR) test may be used. The use of tau or Aβ PET tracers to assess pathogenic protein deposits in the brain is also well known in the art.

[0102] In some embodiments, a subject may be diagnosed as having neurological damage if the level of biomarker significantly deviates from the mean in control subjects. "Significantly deviates from the mean" refers to a value that is at least 1 standard deviation, preferably at least 1.3 standard deviation, more preferably at least 1.5 standard deviation, or even more preferably at least 2 standard deviations above or below the mean (e.g., 1σ, 1.1σ, 1.2σ, ​​1.3σ, 1.4σ, 1.5σ, etc., where σ is the standard deviation defined by normal distribution measured in a control population). In addition to using a threshold value (e.g., at least 1 standard deviation above or below the mean), in some embodiments, the degree of change above or below the mean may be used to diagnose a subject.

[0103] In another aspect, the method comprises detecting and quantifying levels of biomarkers in a first biological sample obtained from a subject and a second biological sample obtained from the subject, wherein the first biological sample and the second biological sample are both blood samples or both CSF samples, and the second biological sample was obtained after the first biological sample, as described in any of the above embodiments.

[0104] In another aspect, the method comprises detecting and quantifying the level of biomarkers in a first biological sample obtained from a subject and a second biological sample obtained from the subject as described in any of the above embodiments, wherein the first biological sample and the second biological sample are both blood samples or both CSF samples, and the second biological sample is obtained after the first biological sample.The increase in the level of biomarkers in the second sample compared to the first sample indicates an increase in neurological damage.In some embodiments, the rate of change in the level of biomarkers between the first and subsequent samples is used to determine the stage of disease and / or the extent of neurological damage.Therefore, such methods may be used to monitor subjects who have or are at risk of having neurological damage.

[0105] In some embodiments, one or more of the above methods may be used in combination with one or more disease biomarkers known in the art to diagnose, stage, and / or treat specific neurodegenerative diseases.For example, data show that the pathophysiological cascade of events in AD progression begins with changes in the plasma Aβ42 / Aβ40 ratio in CSF and blood, followed by an increase in amyloid plaques measured by amyloid PET, which is associated with increased phosphorylation of specific CSF tau species (e.g., p-tau217, p-tau231, p-tau181, p-tau153, p-tau111), before increases in p-tau205, NfL, total tau concentration, metabolic decline, and atrophy. Finally, some species of MTBR-tau (e.g., MTBR-tau299, MTBR-tau243, MTBR-tau354, MTBR-tau3R) increase before or with the onset of tau aggregation and clinical symptoms by tau PET, and correlate with long-term tau aggregation and clinical progression. These findings suggest that measures of CSF and blood NF1, combined with one or more measures of Aβ, p-tau, and MTBR-tau, are highly accurate biomarkers of brain amyloidosis, tauopathy, and neurodegeneration, and can accurately identify stages of preclinical and clinical AD and predict the efficacy of treatments targeting certain disease processes. Use with other disease biomarkers for the diagnosis, staging, and treatment of neurodegenerative diseases other than AD is also contemplated.

[0106] In some embodiments, one or more of the above methods may be used in addition to or as an alternative to more invasive diagnostic methods such as PET or lumbar puncture.Thus, in any of the above methods, the subject in some aspects has not previously undergone diagnostic examination such as PET imaging.

[0107] In some embodiments, one or more of the above methods can be used to determine whether a subject should undergo additional diagnostic tests, which can be, for example, more invasive diagnostic methods.For example, if Nfl level or Nfl level combined with another clinical sign suggests Alzheimer's disease, the subject can be selected to undergo amyloid-based PET or tau-based PET.Alternatively or in addition, Nfl level can be used to select the subject to undergo cognitive tests, or further cognitive tests when the first test is performed, for example, to further confirm the presence of disease or a certain disease stage. Alternatively or additionally, Nfl levels may be used to select patients for additional biomarker testing, such as tests to determine total levels or levels of specific isoforms such as apolipoprotein J, huntingtin protein, synuclein, soluble amyloid precursor protein, alpha-2 macroglobulin, S100B, myelin basic protein, interleukins, superoxide dismutase, TNF, TREM-2, TDP-43, YKL-40, VILIP-1, prion protein, pNFH, DJ-1, etc.

[0108] In some embodiments, one or more of the above methods can also be used to perform the prognosis of cognitive status or cognitive decline.Therefore, in some embodiments, the present disclosure relates to the above method for use in predicting cognitive status or predicting cognitive decline in human subjects with neurological damage, including neurological damage caused by neurodegenerative disease, neuroinflammation, or traumatic brain injury.Therefore, in further embodiments, the present disclosure relates to the above method for use in predicting the progression of neurodegenerative disease.

[0109] In another aspect, the method comprises: (a) detecting and quantifying the level of a biomarker in a first biological sample obtained from a subject, the biomarker being a biomarker described herein, as described in any of the above embodiments; (b) administering a treatment to the subject; and (c) detecting and quantifying the biomarker quantified in step (a) in a second biological sample obtained from the subject after the treatment, where the first biological sample and the second biological sample are both blood samples or both CSF samples. Either the absence of a change in the level of the biomarker or a decrease in the level of the biomarker in the second sample compared to the first sample indicates a positive treatment response. In addition, an increase in the level of the biomarker in the second sample compared to the first sample can also indicate a positive treatment response if the increase is less than the increase that occurs in a control group of subjects that have nerve damage but have not received treatment. Preferably, the control subjects have nerve damage resulting from the same disease process or type of injury. Thus, such methods are used to measure treatment response in subjects that have or are at risk of having nerve damage.

[0110] In another embodiment, the disclosure includes treating a subject diagnosed with or at risk of having neurological damage. The method includes the steps of: (a) quantifying a biomarker described herein in a sample obtained from the subject; and (b) administering to the subject a pharmaceutical composition that reduces or stabilizes the amount of the biomarker measured in step (a).

[0111] V. Composition In another embodiment, the disclosure also provides a composition comprising a biomarker of Section IV and an internal standard.

[0112] In another aspect, the disclosure provides a composition comprising an internal standard and a plurality of Nfl isoforms selected from the group consisting of an isoform having an amino acid sequence consisting essentially of the rod domain of Nfl (typically set forth as amino acids 90-396 of SEQ ID NO:1), an isoform having an amino acid sequence consisting essentially of a portion of the rod domain of Nfl, an isoform having an amino acid sequence consisting of a portion of the rod domain of Nfl with no amino acids outside this region, or any combination thereof. For example, the composition may comprise one or more Nfl isoforms comprising amino acids 90-396 of SEQ ID NO:1 having a length of up to about 330 amino acids. Alternatively or in addition, the composition may comprise one or more Nfl isoforms that are fragments of amino acids 90-396 of SEQ ID NO:1. For example, the composition may include one or more Nfl isoforms that are less than 310 amino acids in length (such as about 305, about 300, about 275, about 250, about 225, about 200, about 175, about 150, about 125, about 100, about 75, about 50, about 25, etc.) and include amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, amino acids 324-331 of SEQ ID NO:1, or any combination thereof. In some examples, the composition may include one or more Nfl isoforms that are less than about 200 amino acids in length and include amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, or any combination thereof. Alternatively or in addition, the composition may include one or more Nfl isoforms that are fragments of amino acids 125-396 of SEQ ID NO:1. For example, the composition may include one or more Nfl isoforms that are about 275 amino acids in length or less and include amino acids 165-172 of SEQ ID NO:1, amino acids 198-206 of SEQ ID NO:1, amino acids 324-331 of SEQ ID NO:1, or any combination thereof.

[0113] In another aspect, the disclosure provides a composition comprising an internal standard and a plurality of Nfl isoforms selected from the group consisting of an isoform consisting essentially of the tail region of Nfl, an isoform having an amino acid sequence consisting of a portion of the tail region of Nfl, or a combination thereof. The tail region is typically set forth as amino acids 397-543 of SEQ ID NO: 1. For example, the composition may comprise one or more Nfl isoforms comprising amino acids 397-543 of SEQ ID NO: 1 that are 147 amino acids in length to about 200 amino acids in length. Alternatively or in addition, the composition may comprise one or more Nfl isoforms that are fragments of amino acids 397-543 of SEQ ID NO: 1. For example, the composition may comprise one or more Nfl isoforms that are less than 147 amino acids in length and comprise amino acids 422-437 of SEQ ID NO: 1, amino acids 438-462 of SEQ ID NO: 1, amino acids 530-540 of SEQ ID NO: 1, or any combination thereof. In another embodiment, the composition may include one or more Nfl isoforms less than about 100 amino acids in length and including amino acids 422-437 of SEQ ID NO:1, amino acids 438-462 of SEQ ID NO:1, amino acids 530-540 of SEQ ID NO:1, or any combination thereof.

[0114] In another aspect, the present disclosure provides a composition comprising an internal standard and a peptide selected from the group consisting of a peptide consisting of amino acids 165-172 of SEQ ID NO: 1, a peptide consisting of amino acids 198-206 of SEQ ID NO: 1, a peptide consisting of amino acids 324-331 of SEQ ID NO: 1, and a peptide consisting of amino acids 530-540 of SEQ ID NO: 1. In another aspect, the present disclosure provides a composition comprising an internal standard and a peptide consisting of amino acids 530-540 of SEQ ID NO: 1, wherein the composition contains negligible amounts of the peptide consisting of amino acids 165-172 of SEQ ID NO: 1, the peptide consisting of amino acids 198-206 of SEQ ID NO: 1, and the peptide consisting of amino acids 324-331 of SEQ ID NO: 1.

[0115] In some embodiments of the above compositions, the internal standard is a recombinant Nfl, or a fragment thereof, which is detectably labeled. In some embodiments, the internal standard is 2 H, 13 C and 15 N detectably labeled with a heavy isotope label selected from. In some embodiments, the internal standard is an AQUA peptide. In certain embodiments, the internal standard detectably labeled with a heavy isotope label and the biomarker or a plurality of Nfl isoforms or peptides may be in a ratio of more than 0.01 to 1, and less than 1 to 100, or may be in a ratio of about 0.1 to 1 to about 10 to 1, respectively.

[0116] The amount of peptide, or the amount of biomarker, or the amount of multiple Nfl isoforms is at least 0.1 pg. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 0.1 pg to about 10,000 ng, or about 0.1 pg to about 5,000 ng, or about 0.1 pg to about 1,000 ng. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 1 pg to about 10,000 ng, or about 1 pg to about 5,000 ng, or about 1 pg to about 1,000 ng. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 10 pg to about 10,000 ng, or about 10 pg to about 5,000 ng, or about 10 pg to about 1,000 ng. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 100 pg to about 10,000 ng, or about 100 pg to about 5,000 ng, or about 100 pg to about 1,000 ng. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 500 pg to about 10,000 ng, or about 500 pg to about 5,000 ng, or about 500 pg to about 1,000 ng. In some embodiments, the amount of peptide, biomarker, or multiple Nfl isoforms is about 1,000 pg to about 10,000 ng, or about 1,000 pg to about 5,000 ng, or about 1,000 pg to about 1,000 ng. In some embodiments, the amount of the peptide, biomarker, or multiple Nfl isoforms is about 5 ng to about 10,000 ng, or about 5 ng to about 5,000 ng, or about 5 ng to about 1,000 ng. In some embodiments, the amount of the peptide, biomarker, or multiple Nfl isoforms is about 10 ng to about 10,000 ng, or about 10 ng to about 5,000 ng, or about 10 ng to about 1,000 ng.

[0117] In each of the above embodiments, the composition may further comprise one or more anti-Nfl epitope binding agents, as described in Section II and incorporated by reference herein. EXAMPLES

[0118] The following examples are included to demonstrate preferred embodiments of the present invention. It should be recognized by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results, and therefore, all matters described or shown in the accompanying examples and drawings should be interpreted as illustrative and not limiting. [Example 1]

[0119] This example describes an immunization protocol for the production of NfL monoclonal antibodies.

[0120] Antigen: recombinant protein [neurofilament light chain-human produced and purified in E. Coli]. NfL 1-543aa was produced and purified. 100 micrograms of recombinant NfL protein was injected intraperitoneally (IP) into B6 / C3 mice. The first injection consisted of 100 micrograms of recombinant NfL 1-543aa, IP per mouse in 200 microliters of PBS + 200 microliters of Freund's complete adjuvant. The second injection 14 days later consisted of 100 micrograms of recombinant NfL 1-543aa, IP per mouse in 200 microliters of PBS and 200 microliters of Freund's incomplete adjuvant. After 21 days, 30 microliters of blood was taken from each mouse and serum was screened by direct ELISA against recombinant NfL. Mice received a third injection of 100 micrograms of recombinant NfL 1-543aa in 200 μl of PBS and 200 microliters of Freund's incomplete adjuvant IP 28 days later. Thirty microliters of blood was taken from the mice 35 days later (second bleed) and serum was again assessed by direct ELISA against recombinant NfL. Once the mouse titers were above 1:10,000, fusions were then performed. Mice were then given a final boost of 50 micrograms of recombinant NfL 1-543aa in PBS IP injected 3 days prior to fusion.

[0121] The antibodies produced from each clone were then screened by coating the plate with recombinant NfL 1-543aa. To do this, NfL 1-543aa was plated and then the plate was blocked with 1% milk. The plate was then washed with PBS and cell culture medium was added from each clone at a dilution of 1:10 in 0.5% BSA and mouse anti-IgG HRP in PBS buffer. TMB was then added and read by absorbance at 650, which was compared to cell culture medium alone as blank.

[0122] Twenty-three antibodies were selected for further characterization (see Example 2). Antibodies HJ30.1, HJ30.2, HJ30.4, HJ30.7, HJ30.11, and HJ30.13 were deposited with the ATCC.

[0123] [Table 5] [Example 2]

[0124] In this example, Nfl was immunoprecipitated using one of the 23 antibodies described in Example 1, proteolytically digested, and then the proteolytic peptides of Nfl were detected and quantified by mass spectrometry (MS). In all MS experiments described in this and other examples, if no reliable MS signal was detected for a tryptic peptide of a given ISTD, no data was reported for either the ISTD (15N) or the experimental Nfl (14N). See, for example, tryptic peptides [55,83] in Figure 1. Tryptic peptides are identified throughout the specification, including the examples herein, using a specific shorthand notation - "[X,Y]", where X is the first amino acid of the tryptic peptide, Y is the last amino acid of the tryptic peptide, and X+1 or Y+1 identifies the amino acid of the full-length Nfl (SEQ ID NO: 1). For example, the nomenclature "[55,83]" is shorthand for the tryptic peptide SYSSSSGSLMPSLENLDLSQVAAISNDLK (SEQ ID NO: 5), which consists of amino acids 56 to 84 of full-length Nfl (SEQ ID NO: 1).

[0125] To facilitate recovery of immunoprecipitated Nfl, individual antibodies were cross-linked to Dynabeads® at a concentration of 25 μg antibody per mg beads according to the manufacturer's instructions. HJ5.1, an anti-Aβ antibody, was used as a negative control. Full-length recombinant Nfl (rec-Nfl) and biologically produced Nfl isolated from brain lysates or CSF were evaluated.

[0126] For full-length recombinant Nfl analysis (rec-Nfl), 5ng / uL rec-Nfl in 10uL 1% HSA was added to 40uL 100mM TEABC. Nfl was immunoprecipitated by adding 30uL of 30% (i.e., 3mg / mL) slurry of antibody-conjugated bead preparation and rotating the sample for 120 minutes at room temperature. Antibody-conjugated beads were magnetically separated and post-IP supernatant was removed. Beads were washed 3 times with 1mL 25mM TEABC (per wash).

[0127] NfL was also affinity purified from brain lysates and CSF. The brain samples used were previously lysed samples and stored at -80°C during assay development. Patient demographics for the subjects were not obtained (e.g., age, sex, clinical signs or symptoms of neurological disease). Frozen lysates were thawed and an aliquot was diluted 1:1000 in 1% HSA. 450 μl of thawed brain lysate was transferred to a new 1.6 mL tube. Then, 25 uL of master mix containing detergent (1% NP-40), chaotropic agent (5 mM guanidine), and protease inhibitor (Roche complete Protease Inhibitor Cocktail) was added, as well as 20 uL of 0.5 ng / ml Nfl internal standard (ISTD) in 50 mM triethylammonium bicarbonate buffer (TEABC). Lys, Arg 13 C and 15 N-labeled recombinant full-length Nfl was used as ISTD. Nfl was immunoprecipitated by adding 30uL of 30% (i.e., 3mg / mL) slurry of antibody-conjugated bead preparation and rotating the sample for 120 minutes at room temperature. Antibody-conjugated beads were magnetically separated and post-IP supernatant was removed. Beads were washed 3 times with 1mL of 25mM TEABC (per wash).

[0128] The CSF samples used were previously obtained from human subjects for assay development and stored at -80°C. Patient demographics for the subjects were not known (e.g., age, sex, clinical signs or symptoms of neurological disease). Frozen CSF samples were thawed at room temperature and 500 μl of thawed CSF was transferred to a new tube. Then, 25 uL of master mix containing detergent (1% NP-40), chaotropic agent (5 mM guanidine), and protease inhibitor (Roche complete Protease Inhibitor Cocktail) was added, as well as 20 uL of 0.5 ng / ml Nfl internal standard (ISTD) in 50 mM triethylammonium bicarbonate buffer (TEABC). Lys, Arg 13 C and 15 N-labeled recombinant full-length Nfl was used as ISTD. Nfl was immunoprecipitated by adding 30uL of 30% (i.e., 3mg / mL) slurry of antibody-conjugated bead preparation and rotating the sample for 120 minutes at room temperature. Antibody-conjugated beads were magnetically separated and post-IP supernatant was removed. Beads were washed 3 times with 1mL of 25mM TEABC (per wash).

[0129] Regardless of the sample, antibody-bound Nfls were digested on-bead with 400 ng of MS-grade trypsin or Lys-C in 25 mM TEABC for 16 h at 37°C. Protease concentrations, incubation times, and buffers can be adjusted according to methods known in the art. Digestions were loaded onto a TopTip C18 conditioned according to the manufacturer's instructions, desalted by washing twice with 0.1% formic acid (FA), and eluted with 60% acetonitrile (ACN) and 0.1% FA. Eluted peptides were dried by vacuum centrifugation and either stored at -80°C or immediately resuspended.

[0130] For MS analysis, the eluted peptides were reconstituted with 25 uL of 0.1% FA / 0% CAN. A 4.5 uL aliquot of each digest was then injected into a nano-Acquity LC for MS analysis. The nano-Acquity LC (Waters Corporation, Milford, MA, USA) was adapted to an HSS T3 75 μm×100 μm, 1.8 μm column and a flow rate of 0.5 μL / min. Peptides were separated with a gradient of 2% to 95% solvent B. Solution A consisted of 0.1% formic acid in MS grade water, and solution B consisted of 0.1% formic acid in acetonitrile. Samples were analyzed in positive ion mode with a spray voltage of 2,200 V and an ion transfer tube temperature of 275 °C. Data were analyzed using intrinsic (N14) and isotopic labeling (C14) targeting 2+ or 3+ charge states. 13 N 15 ) peptides were collected by parallel reaction monitoring (PRM). HCD collision energies ranged from 18-27% and were optimized for each peptide. Maximum injection times ranged from 24-118 ms and were optimized for each peptide. The normalized AGC target was set at 400%. Data were extracted using Skyline software (McCoss laboratory) and analyzed in Skyline and Microsoft Excel.

[0131] Figure 1 provides a summary of Nfl recovery / IP efficiency for rec-Nfl. Figure 2 provides a summary of Nfl recovery / IP efficiency from brain lysates. Figure 3 provides a summary of Nfl recovery / IP efficiency from CSF. Figures 4-21 show data for individual antibodies. In each graph, the y-axis is the 14N / 15N ratio and each point on the x-axis is a tryptic peptide of Nfl.

[0132] In Figure 1, the 14N / 15N ratios were consistent along the sequence, which was expected given that both the ISTD and experimental Nfls (rec-Nfl) were full-length proteins. Notably, HJ13.3.2, HJ30.19, HJ30.19-40, and HJ30-.19-77 did not show rec-Nfl recovery.

[0133] In brain lysates, the Nfl detected was also nearly full-length (Figure 2). Analysis indicated that the N-terminal methionine was cleaved and the second amino acid (serine) was acetylated (data not shown). The high signal seen with tryptic peptides [370,378], [379,389], and [391,398] was determined to be due to contamination (data not shown).

[0134] The contaminations seen in the analysis of brain lysate samples were also seen in the analysis of CSF samples (Figs. 3-21). Tryptic peptides [370,378], [379,389], and [391,398] are not unique to Nfls, so they should not be used for detection and quantification of Nfls in biological samples. Acetylation of [2,14] was also seen in CSF Nfls (Figs. 3-21). Unlike the 14N / 15N ratios seen in rec-Nfls and brain lysate-derived Nfls, CSF-derived Nfls showed inconsistent 14N / 15N ratios along the sequence, indicating the number of truncated Nfl species in CSF (Fig. 3). Comparing the profiles of individual antibodies (Figs. 4-21), it was evident that certain antibodies were more effective at immunoprecipitating CSF Nfls than others, and that CSF Nfls contain multiple Nfl isoforms of variable length with truncations at the N-terminus, C-terminus, or N- and C-terminus.

[0135] A summary of some of the possible isoforms enriched from brain lysates and CSF by the methods described herein is illustrated in FIG. 22 with approximate sizes provided. [Example 3]

[0136] In this example, Nfl were immunoprecipitated from CSF using one of HJ30.4, HJ30.11, or HJ30.13. The CSF samples used were previously obtained from human subjects for assay development and stored at -80°C. Patient demographics for the subjects were not known (e.g., age, sex, clinical signs or symptoms of neurological disease). Immunoprecipitation and LC-MS analysis were generally as described in Example 2, except that the supernatants collected after immunoprecipitation were subjected to a second round of immunoprecipitation with the same antibodies. Data for 14N (red bars) and 15N (blue bars) are shown separately (Figures 23-25). The first two bars in each figure correspond to the first enrichment step, and the second two bars correspond to the second enrichment step.

[0137] Most of the internal standards (ISTDs, blue bars) are recovered from the samples by the first enrichment step. This suggests that it is necessary to re-add the full-length internal standards if subsequent rounds of enrichment with the same or different epitope-binding agents are performed. Similarly, HJ30.4 and H30.13 efficiently enriched Nfl isoforms from CSF in a single step (Figures 23-24). In contrast, HJ30.11 did not immunoprecipitate all Nfl isoforms to which it specifically binds in a single step (Figure 25). [Example 4]

[0138] Sequential immunoprecipitation of Nfl from CSF with multiple antibodies was also performed. The CSF samples used were previously obtained from human subjects for assay development and stored at -80°C. Patient demographics for the subjects were not known (e.g., age, sex, clinical signs or symptoms of neurological disease). Immunoprecipitation and LC-MS analysis were generally as described in Example 2, except that the supernatant collected after the first immunoprecipitation was subjected to a second round of immunoprecipitation with a second antibody, and then the supernatant collected after the second immunoprecipitation was subjected to a third round of immunoprecipitation with a third antibody. In one experiment, HJ30.2, HJ30.7, and then HJ30.11 were used sequentially for immunoprecipitation. In the second experiment, HJ30.11, HJ30.7, and HJ30.2 were used sequentially for immunoprecipitation. In these experiments, internal standards were only added to the first CSF sample. Each experiment was performed in duplicate. A schematic of the experimental design is shown in Figure 26. An alternative approach using an internal standard was also added to each post-IP supernatant prior to the subsequent round of enrichment shown in Figure 29.

[0139] Data from both experiments are shown together in Figures 27-28. In Figure 27, the y-axis is the 14N / 15N ratio and each point on the x-axis is a tryptic peptide of Nfl. In Figure 28, the y-axis is the 14N signal and each point on the x-axis is a tryptic peptide of Nfl. Each color line is a different sample, as specified in the legend and in Figure 26. If no reliable MS signal was detected for a given tryptic peptide of ISTD, the data was not reported. Nfl isoforms containing the C-terminus were more abundant after sequential enrichment using HJ30.11 at the end of the process compared to the beginning of the process (compare "30.11" or "30.11_duplicate" with "post 30.2&7_30.11IP" or "post 30.2&7_30.11IP_duplicate" in Figure 28). Detection of these C-terminal isoforms was negligible in other samples analyzed. These data indicate that the sequential use of different epitope binding agents enriches populations of different Nfl isoforms. [Example 5]

[0140] In this example, the Nfl isoforms from CSF samples obtained from human subjects with neurological injury were compared with those from CSF samples obtained from control human subjects. Immunoprecipitation and LC-MS analysis were generally as described in Example 2, except that the supernatant collected after the first immunoprecipitation with HJ30.13 was subjected to a second round of immunoprecipitation with HJ30.4, and then the supernatant collected after the second immunoprecipitation was subjected to a third round of immunoprecipitation with HJ30.11. An internal standard was also added to each post-IP supernatant before the subsequent round of enrichment. A schematic diagram of the experimental design is shown in Figure 29.

[0141] The data presented in Figures 30-39 are from experiments using CSF samples obtained from human subjects with one or more biomarkers for Alzheimer's Disease (AD) or from control human subjects. More specifically, subjects identified as having AD were amyloid positive as assessed by PIB-PET imaging or CSF Aβ42 levels (Figures 30-33). All AD subjects in this cohort also had a Clinical Dementia Scale (CDR) score of ≧0.5. Control subjects had a CDR score of zero and were amyloid negative (Figures 34-39). Each figure is an individual subject, and in each figure, the blue line is the Nfl isoform immunoprecipitated by HJ30.13, the orange line is the Nfl isoform immunoprecipitated by HJ30.4, and the gray line is the Nfl isoform immunoprecipitated by HJ30.11, where the y-axis is the 14N / 15N ratio, and each point on the x-axis is the tryptic peptide of Nfl. If no reliable MS signal was detected for a given tryptic peptide of ISTD, the data was not reported. As summarized in Figure 40, some parts of Nfl seem to be able to better distinguish AD subjects from control subjects.

[0142] The data shown in Figure 41 are from experiments using additional CSF samples obtained from human subjects with one or more biomarkers for AD (i.e., at least amyloid positive, as assessed by PIB-PET imaging or CSF Aβ42 levels) grouped by CDR score (0, 0.5, 1, or 2), additional samples from control human subjects (amyloid negative and CDR=0), and samples obtained from human subjects with very mild dementia (CDR 0.5) or mild dementia (CDR 1) but who were amyloid negative. The amyloid negative status with very mild to mild dementia may indicate other neurological disease processes (i.e., neurological diseases affecting cognition other than AD). Due to technical errors, no Nfl data were obtained from the first enrichment step. However, data from the second and third enrichment steps appeared similar to the initial analysis of AD vs. controls (amyloid negative, CDR 0). In particular, the data again show that the Nfl isoform comprising the C-terminal amino acids 530-540 of SEQ ID NO: 1 distinguishes AD subjects from control subjects. In addition, data from amyloid-negative controls with mild dementia (CDR 1) suggest that the detected Nfl isoforms may be useful as markers of neuronal damage due to other causes. [Example 6]

[0143] In this example, Nfl isoforms from blood samples obtained from human subjects were analyzed. Two different samples of pooled blood were used, each of which consisted of blood previously obtained from multiple human subjects for assay development, pooled, and stored at -80°C. Immunoprecipitation and LC-MS analysis were generally as described in Example 5, except that 0.5 mL of blood was used instead of CSF. As shown in Figures 42-44, Nfl concentrations in blood are lower than in CSF, but are detectable. Furthermore, blood is also composed of multiple Nfl isoforms, similar to CSF. Also, there are several truncated Nfl isoforms in blood that are composed mainly of the C-terminus, similar to CSF. [Example 7]

[0144] This example expands on the previous example by utilizing the developed antibodies that bind to different regions of NfL and characterizing the NfL domains retrieved by these antibodies in brain tissue and CSF using immunoprecipitation mass spectrometry (IP-MS). This example shows that brain NfL primarily constitutes the full-length protein, whereas CSF NfL consists of a mixture of different protein fragment species. The newly identified NfL fragment species in control and AD discovery cohorts were then tested and further validated in validation cohorts.

[0145] Pooled CSF samples used for assay development were previously obtained from human subjects and stored at -80°C. At the time of initial collection, CSF was centrifuged at 1000 x g for 10 min to remove cellular debris and immediately frozen at -80°C. Brain samples included previously lysed samples that were stored at -80°C during assay development. All AD and control CSF samples were collected during a previous study, aliquoted, and stored at -80°C. The validation cohort included CSF samples from 30 symptomatic amyloid-positive participants, 16 asymptomatic amyloid-positive participants, 10 symptomatic amyloid-negative participants, and 25 negative controls. Participant demographics are shown in Table 2.

[0146] [Table 6]

[0147] Both recombinant and native NfL were immunoprecipitated by adding 30uL of 30% (i.e., 3mg / mL) slurry of antibody-conjugated bead preparation and rotating the samples for 120 minutes at room temperature. Antibody-conjugated beads were magnetically separated and post-IP supernatant was removed. Beads were washed 3 times with 1mL of 25mM TEABC (per wash). Bound NfL was digested on-bead with 400ng MS-grade trypsin / Lys-C (Promega) for 16 hours at 37°C. Digestion was loaded onto a TopTip C18 (Glygen, TT2C18.96), desalted, and eluted. Eluates were dried in vacuum without heating and stored at -80°C until analysis by liquid chromatography tandem mass spectrometry (LC-MS / MS) (see LC-MS / MS method below). Sixteen antibodies recovered full-length recombinant protein (Figure 45). Based on peptide profiles from native NfL immunoprecipitated from pooled CSF, it was determined that the antibodies had epitopes to the amino-terminal portion of the rod domain, the carboxy-terminal portion of the rod domain, or the carboxy-terminus of NfL (Figure 46). Antibodies with high recovery and NfL specificity were selected for each of these NfL domains and used in qualitative and quantitative IP-MS assays. The custom antibodies did not recognize the N-terminus of NfL.

[0148] A three-step sequential immunoprecipitation was used to characterize NfL in brain lysates and CSF. Antibodies targeting the rod domain coil 1A / 1B (HJ30.13), rod domain coil 2B (HJ30.4), and tail region (HJ30.11) were used. Frozen brain lysates were thawed and 450 μL aliquots of frozen brain lysates were diluted 1:1000 in 1% HSA. Frozen CSF samples were thawed at room temperature and 450 μL of thawed CSF was transferred to a new 1.6 mL tube for immunoprecipitation.

[0149] Both brain and CSF samples were immunoprecipitated as described above for native CSF using 30 μL of 30% (i.e., 3 mg / mL) slurry of antibody-conjugated bead preparation of HJ30.13 (coil 1A / 1B antibody). The washed beads were stored on ice until all samples were ready for on-bead digestion. In the second step, a second immunoprecipitation was performed by adding 20 μL of 0.5 ng / ml NfL ISTD in 50 mM TEABC and adding NfL to 30 μL of 30% (i.e., 3 mg / mL) slurry of antibody-conjugated bead preparation of HJ30.4 (coil 2B antibody). The remaining steps were identical to the first immunoprecipitation. 10 ng ISTD in 50 mM TEABC was added again to the post-IP supernatant before the third sequential immunoprecipitation performed with HJ30.11 (tail antibody). Bound NfL was digested on-beads with 400 ng MS-grade trypsin / Lys-C (Promega) for 16 hours at 37° C., and samples were extracted as described above.

[0150] To eliminate the need for sequential addition of ISTDs, antibodies targeting rod domain coil 1A / 1B (HJ30.13), rod domain coil 2B (HJ30.4), and tail region (HJ30.11) were mixed 1:1:1 to create an antibody slurry with a final concentration of each antibody at 10% (i.e., 1 mg / mL). 25 μL of master mix containing detergent (1% NP-40), chaotropic agent (5 mM guanidine), and protease inhibitor (Roche Complete Protease Inhibitor Cocktail) was added to a 96-well plate. 5 μL of ISTD (0.1 ng / μL in 1% HSA; ISTD solvent and amount optimized for quantitative recovery and dynamic range of the assay) was then added, followed by 450 μL of thawed CSF and 30 μL of antibody slurry. Immunoprecipitation and on-bead digestion were performed as described above.

[0151] Pooled CSF was screened to identify pools with low and high concentrations of NfL. CSF pools with the lowest (NfL-L1) and highest (NfL-L2) NFL concentrations were selected and used to determine the linear range of the assay. NfL-L2 CSF was serially diluted with NfL-L1 CSF to generate 8-point curves at 100%, 50%, 25%, 12.5%, 6.25%, 3.13%, and 1.56% NfL-L2. IP was performed as described above for NfL in triplicate for each concentration. N14 / N15 ratios were determined for each of the six peptides in a quantitative manner, and the mean N14 / N15 ratios of the replicates were plotted against NfL-L2% and linear regression was performed. All six peptides showed good linearity across the tested NfL concentrations with R2≧0.988 (FIG. 47). The average CV% for each peptide over the linear range was 8-12% (Table 3).

[0152] [Table 7]

[0153] The extracted digests were reconstituted with 25 μL of 0.1% formic acid / 0% ACN. A 4.5 uL aliquot of each digest was then injected into a nano-Acquity LC for MS analysis. The nano-Acquity LC (Waters Corporation, Milford, MA, USA) was fitted with an HSS T3 75 μm×100 μm, 1.8 μm column, and peptides were separated using a 0.5 μL / min flow rate of a gradient of solutions A and B. Solution A consisted of 0.1% formic acid in MS-grade water, and solution B consisted of 0.1% formic acid in acetonitrile. Samples were analyzed in positive ion mode with a spray voltage of 2,200 V and an ion transfer tube temperature of 275° C. Data were collected by parallel reaction monitoring (PRM) for endogenous (N14) and isotopically labeled (Lys, Arg:13C15N) peptides. Tryptic peptides specific to NfL were identified via Blast searches, and those with good ionization were included in a qualitative PRM designed to optimize sequence coverage. Quantitative methods were optimized for assay precision, and multiplexing was reduced to the analysis of six NfL peptides across various NfL domains and their corresponding ISTDs (Table 4).

[0154] [Table 8]

[0155] The validation cohort consisted of 81 previously collected CSF samples from individuals with AD dementia (amyloid positive, CDR 0–2), non-AD dementia (amyloid negative, CDR 0.5–1), and healthy controls (amyloid negative, CDR 0). Amyloid positivity was previously determined by the Aβ42 / Aβ40 ratio (Patterson et al., 2015). For each CSF sample, six NfL peptides corresponding to four distinct domains of NfL (coil 1A, coil 1B, coil 2B, and tail) were measured using the quantitative IP-MS method described above. NfL was also measured via a commercially available ELISA kit (UMAN Diagnostics) according to the manufacturer's instructions. Briefly, for ELISA measurements, CSF samples were thawed on wet ice and vortexed. Samples were then diluted 2-fold with the provided sample diluent in a 96-well preplate and transferred to the assay plate after mixing.

[0156] To determine the relationship of soluble NfL species to AD clinical, cognitive, imaging and biomarker measures, correlation analyses were performed between each NfL region (IP-MS) and previously obtained biomarker data. The following measures were assessed: age, CDR global and CDR box sum (CDR-SB), Mini-Mental State Examination (MMSE), amyloid plaque imaging (PET PiB), CSF Aβ42 / Aβ40, CSF total tau (t-tau), CSF ptau181 and ptau181 / tau181, CSF ptau205 and ptau205 / tau205, CSF ptau217 and ptau217 / tau217.

[0157] Twenty-three monoclonal antibodies were generated against NfL and assessed for their ability to immunoprecipitate full-length ref-NfL, brain lysate-derived NfL, and pooled CSF-derived NfL. Antibodies were characterized by the NfL domain they target, their IP efficiency, and their specificity. Representative antibodies for each NfL domain were selected and used for further assay development. Using antibodies targeting various NfL domains, we determined that multiple NfL species are present in CSF (Figure 48). To better elucidate the NfL species in CSF, pooled CSF samples were sequentially immunoprecipitated starting with an antibody targeting coil1A / 1B (approximately aa93-252, HJ30.13), followed by an antibody targeting coil2B (approximately aa272-396, HJ30.4), and finally an antibody targeting the C-terminus of the tail region (aa520-550, HJ30.11). Based on these protein profiles, we identified three major NfL fragment species at a minimum in CSF, although multiple versions of these species may exist. These include two different N- and C-terminal truncations containing the rod domain enriched by HJ30.13 (aa92 to at least aa224, possibly a version extending to aa360) and HJ30.4 (aa324 to aa360), as well as a C-terminal fragment containing the tail of NfL (enriched by HJ30.11, containing aa530 to at least aa540). No N-terminal fragments were recovered, and full-length NfL was not present in quantifiable concentrations in CSF (Figures 49A-49B).

[0158] In contrast to the highly fragmented protein in CSF, brain tissue homogenates contained nearly full-length NfL. To determine whether any truncated isoforms were also present in brain, the same sequential immunoprecipitations on human brain tissue were performed. Most brain NfL appeared to be full-length, but a C-terminal fragment of tail subdomain B containing at least amino acids 530-540 was also observed, similar to the fragment identified in CSF (Figure 49A, 49C). A fragment containing aa165-224 appears to be enriched by HJ30.13. No additional NfL fragments were enriched in brain during the second IP (HJ30.4, coil 2B of rod domain).

[0159] The sequential IP-MS method was first tested in an experiment, which was repeated in CSF samples from a discovery cohort of Alzheimer's dementia (AD, N=4) and healthy controls (N=6) that was done in pooled CSF. The peptides observed in both clinical groups were similar to those observed in pooled CSF, but there was an increased abundance of the three major NfL species in AD compared to controls (Figure 50). In addition, some peptides appeared to be better than others at discriminating between AD and control samples. The most striking differences were observed for NfL530 (tryptic peptide VEGAGEEQAAK (SEQ ID NO:29), aa530-540) in the C-terminal tail and tryptic peptide GADEAALAR (SEQ ID NO:16) within coil 1B of the rod domain.

[0160] To better compare CSF NfL species in AD, non-AD dementia, and healthy controls, we developed a quantitative NfL assay to reliably measure select regions across multiple NfL species. To improve accuracy, we reduced the multiplexing of our quantitative method to measure six peptides and their corresponding internal standards across various NfL domains. The IP-MS quantitative method was then applied to measure specific CSF NfL species in a validation cohort of 81 AD and control samples (30 amyloid positive, CDR>0; 16 amyloid positive, CDR=0; 10 amyloid negative, CDR=0; 25 amyloid negative, CDR=0, Table 2). CSF NfL concentrations were also quantified using the gold standard Uman NfL immunoassay for comparison.

[0161] Consistent with the results of sequential IP of the discovery cohort (Figure 51), increased NfL concentrations were observed in a validation cohort of symptomatic amyloid-positive individuals (N=30) compared to amyloid-negative healthy controls (N=25) (Figure 52). Interestingly, the differences between groups were greater for some regions than others, with the largest differences observed in coil 2B of the rod domain [NfL324;GMNEALEK (SEQ ID NO:20), aa324-331] and the C-terminus of the tail [NfL530;VEGAGEEQAAK (SEQ ID NO:29), aa530-540]. NfL324 was increased 1.5-fold in AD compared to controls (P=0.008, Figure 52F) and NfL530 was increased 1.7-fold (P=0.002, Figure 52G).

[0162] The NfL immunoassay was increased 1.4-fold in AD compared to controls. It had the strongest correlation with NfL324 peptide concentrations (R2 = 0.84), suggesting that the Uman immunoassay targets CSF NfL fragments containing the coil 2B region. Importantly, correlations between the immunoassay and the other investigated peptides were lower for NfL101, 117, 165 and 530 (R2 ranging from 0.34 to 0.49), and no correlation was found with NfL284 (Figure 52).

[0163] Since NfL is a marker of general neurodegeneration and not specific for AD, we hypothesized that NfL was increased regardless of the presence of amyloid plaques in those with clinical dementia and neurodegeneration. The correlation between CDR-SB (a clinical measure of dementia severity) and NfL species was evaluated for amyloid-positive and amyloid-negative samples (Figure 53). Although the correlation was slightly higher for some NfL species in the amyloid-positive group than in the amyloid-negative group (NfL101, NfL117, NfL165 and NfL324), the correlation was marginal or low for all of the NfL species in both groups. The correlation between NfL50 and CDR-SB was not significantly non-zero for either group.

[0164] Spearman correlation analyses were also performed between each of the six quantified NfL peptides, and additional previously measured biomarkers, and clinical measures including general markers of clinical dementia (CDR-SB, MMSE), biomarkers of amyloid plaques (PET PiB, CSF Aβ42 / Aβ40) and tau biomarkers (CSF total tau, CSF phospho-tau immunoassay, and mass spectrometry measurements of CSF ptau 181, 205 and 217 occupancy); Figure 54, Table 5. The goal of this analysis was to form hypotheses about the biology of different NfL species in general neurodegeneration compared to disease-specific neurodegeneration. The strongest correlations were observed between peptides within coil 1A (NfL101 and NfL117, r=0.99) and coil 1B (NfL101 and NfL165, r=0.98; NfL117 and NfL165 r=0.98) of the rod domain. Peptides in coil 2B of the rod domain have similar but slightly lower correlations with coil 1A peptides (NfL101 and NfL284, r = 0.89; NfL101 and NfL324, r = 0.87; NfL117 and NfL284, r = 0.90; NfL117 and NfL324, r = 0.88). The correlation between C-terminal tail peptides and coil 1A is the lowest among the NfL peptides investigated (NfL101 and NfL530, r = 0.75; NfL117 and NfL530, r = 0.76). Interestingly, most of the measured c-terminal peptides (NfL324 and NfL530) have the highest correlations between disease biomarkers and NfL. Moderate correlations between NfL324 or NfL530 and ptau181, 205 or 217 ranged from r=0.45 to 0.49. Correlations between the same NfL peptides and tTau ranged from r=0.42 to 0.43. Correlations with CSF Aβ42 / Aβ40 and CSF NfL530 were lower, at -0.37 (Figure 53, Table 4).

[0165] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0166] This example establishes that there are at least three major truncated species of NfL in CSF and that there is a variable degree of increase in AD. Furthermore, brain NfL is full-length with a newly identified c-terminal fragment. The major CSF NfL species have different relationships with each other and with other AD measures. This would indicate that the truncated species of NfL are secreted differently for NfL biology and neurodegeneration, some of which may be more relevant as biomarkers than others. NfL peptide levels from the coil 1 domain of NfL correlated with each other and behaved slightly differently from peptides measured from the coil 2 and C-terminal regions. Significant increases in NfL peptides 324 and 530 were found in symptomatic AD CSF, supporting that these domains may be more relevant as biomarkers. The higher correlation observed between NfL324 and NfL immunoassays, combined with the similar fold increase between AD and control (1.5-fold) and ELISA assays (1.4-fold) for NfL325, would suggest that the antibodies used by this Uman-proprietary assay may be selected to target these best performing NfL isoforms. There was moderate correlation with CDR, age, and phosphorylated and total tau, while amyloid PET and MMSE measures had low correlations. [Example 8]

[0167] In this example, Nfl isoforms from CSF samples obtained from control, ALS and SMA human subjects were analyzed. CSF samples from patients with sporadic amyotrophic lateral sclerosis (ALS, n=12), familial ALS (n=6), spinal muscular atrophy (SMA, n=10), and normal neuronal controls were analyzed for differences in NfL species utilizing the same method described. Briefly, 0.5 ng of 15N13C internal standard NfL was added to 0.5 ml of CSF followed by immunoprecipitation with a 1:1:1 mixture of antibodies HJ30.13, HJ30.4 and HJ30.11. Processed samples were analyzed by LC-MS / MS to quantify the amount of NfL species in each region shown in Figures 57-63. These findings revealed an increase in sporadic and familial ALS symptomatic cases over asymptomatic cases, SMA, or controls. The amount of increase in NfL species was highly correlated with the rate of decrease measured by the ALS FRS scale measured in monthly decreases. The ALS profile of NfL species showed an increased amount of amino acid positions 37 to 352, then and another increase in 437 to 539 (c-terminal peptide). However, the profile was different in controls, where the amount of c-terminal peptide and other regions were less prominent. Finally, the SMA profile showed very little central domain region, but the region from 339 to 461 was relatively increased. This indicates that there may be specific profiles for each disease state that can be quantified and used for different diagnoses.

Claims

1. 1. A method for detecting neurofilament light chain (Nfl) in a biological sample, comprising: (a) providing a biological sample selected from a blood sample or a CSF sample; (b) enriching one or more Nfl isoforms in the biological sample; and (c) detecting one or more Nfl isoforms enriched in step (b). The method includes:

2. Step (b) (i) contacting the biological sample with a first epitope-binding agent that specifically binds to a first population of Nfl isoforms and isolating the first population of Nfl isoforms, thereby generating a sample that is depleted of the first population of Nfl isoforms; (ii) contacting the sample depleted of the first population of Nfl isoforms with a second epitope-binding agent that specifically binds to a second population of Nfl isoforms and isolating the second population of Nfl isoforms, thereby generating a sample depleted of the first and second populations of Nfl isoforms; and (iii) contacting the sample depleted of the first and second populations of Nfl isoforms with a third epitope binding agent that specifically binds to a third population of Nfl isoforms, and isolating the third population of Nfl isoforms. Including, step (c) comprises detecting one to more Nfl isoforms in a first population of Nfl isoforms, one to more Nfl isoforms in a second population of Nfl isoforms, one to more Nfl isoforms in a third population of Nfl isoforms, or any combination thereof; The method of claim 1.

3. a first epitope binding agent that specifically binds to a first epitope within amino acids 90-300 of SEQ ID NO:1, optionally within amino acids 90-250 of SEQ ID NO:1; a second epitope binding agent that specifically binds to a second epitope within amino acids 200-450 of SEQ ID NO:1 downstream of the first epitope; and the third epitope binding agent specifically binds to a third epitope within amino acids 397-543 of SEQ ID NO:1 downstream of the second epitope; The method of claim 2.

4. Step (b) (i) contacting the biological sample with a first epitope-binding agent that specifically binds to a first population of Nfl isoforms and isolating the first population of Nfl isoforms, thereby generating a sample that is depleted of the first population of Nfl isoforms; (ii) contacting the sample depleted of the first population of Nfl isoforms with a second epitope binding agent that specifically binds to a second population of Nfl isoforms, and isolating the second population of Nfl isoforms; Including, step (c) comprises detecting one to more Nfl isoforms in a first population of Nfl isoforms, one to more Nfl isoforms in a second population of Nfl isoforms, or any combination thereof; The method of claim 1.

5. the first epitope binding agent specifically binds to a first epitope within amino acids 90-450 of SEQ ID NO:1; and / or the second epitope binding agent specifically binds to an epitope within amino acids 396-543 of SEQ ID NO:1; Optionally, the first epitope is within amino acids 250-400 of SEQ ID NO:1 and the second epitope is within amino acids 400-543 of SEQ ID NO:

1. The method according to claim 4.

6. Step (b) comprises contacting the biological sample with an epitope binding agent that specifically binds to a first population of Nfl isoforms and isolating the first population of Nfl isoforms, the epitope binding agent comprising: (i) an epitope binding agent that specifically binds to an epitope within amino acids 90-250 of SEQ ID NO:1 or amino acids 125-250 of SEQ ID NO:1; (ii) an epitope binding agent that specifically binds to an epitope within amino acids 116-184 of SEQ ID NO:1; (iii) an epitope binding agent that specifically binds to an epitope within amino acids 250-400 of SEQ ID NO:1; (iv) an epitope binding agent that specifically binds to an epitope within amino acids 283-338 of SEQ ID NO:1; (v) an epitope binding agent that specifically binds to an epitope within amino acids 400-543 of SEQ ID NO:1; (vi) an epitope binding agent that specifically binds to an epitope within amino acids 437-543 of SEQ ID NO:1; (vii) HJ30.1 or an antigen-binding fragment thereof, HJ30.2 or an antigen-binding fragment thereof, HJ30.13 or an antigen-binding fragment thereof, or an epitope binding agent that competitively inhibits binding of HJ30.1, HJ30.2 or HJ30.13 to full length recombinant Nfl; (viii) an epitope binding agent that competitively inhibits binding of HJ30.4 or an antigen-binding fragment thereof, HJ30.7 or an antigen-binding fragment thereof, or HJ30.4 or HJ30.7 to full-length recombinant Nfl; and (ix) an epitope-binding agent that competitively inhibits the binding of HJ30.11 to HJ30.11, an antigen-binding fragment thereof, or full-length recombinant Nfl. Selected from the group consisting of: The method of claim 1.

7. Step (b) (i) amino acids 92-100 of SEQ ID NO:1, amino acids 101-107 of SEQ ID NO:1, amino acids 108-116 of SEQ ID NO:1, amino acids 117-126 of SEQ ID NO:1, amino acids 137-144 of SEQ ID NO:1, amino acids 148-157 of SEQ ID NO:1, amino acids 158-164 of SEQ ID NO:1, amino acids 165-172 of SEQ ID NO:1, amino acids 178-185 of SEQ ID NO:1, amino acids 192-196 of SEQ ID NO:1, or amino acids 198-206 of SEQ ID NO:1; (ii) amino acids 283-293 of SEQ ID NO:1, amino acids 324-331 of SEQ ID NO:1, amino acids 332-339 of SEQ ID NO:1, or amino acids 340-354 of SEQ ID NO:1; (iii) amino acids 438-462 of SEQ ID NO:1 or amino acids 530-540 of SEQ ID NO:1; or (iv) amino acids 438 to 462 of SEQ ID NO:1 or amino acids 530 to 540 of SEQ ID NO:1 The method of any one of claims 1 to 6, comprising enriching one to more Nfl isoforms having an amino acid sequence comprising:

8. Step (b) enriches for one or more Nfl isoforms comprising N-terminal truncations, the N-terminal truncations for each Nfl isoform being (1) amino acids 1 to 90 of SEQ ID NO:1; (2) amino acids 1 to 92 of SEQ ID NO:1; (3) amino acids 1 to 124 of SEQ ID NO:1; (4) amino acids 1 to 135 of SEQ ID NO:1; (5) amino acids 1 to 137 of SEQ ID NO:1; (6) amino acids 1 to 146 of SEQ ID NO:1; (7) amino acids 1 to 162 of SEQ ID NO:1; (8) amino acids 1 to 222 of SEQ ID NO:1; (9) amino acids 1 to 223 of SEQ ID NO:1; (10) amino acids 1 to 234 of SEQ ID NO:1; (11) amino acids 1 to 252 of SEQ ID NO:1; (12) amino acids 1 to 271 of SEQ ID NO:1; (13) amino acids 1 to 281 of SEQ ID NO:1; (14) amino acids 1 to 323 of SEQ ID NO:1; (15) amino acids 1 to 339 of SEQ ID NO:1; (16) amino acids 1 to 359 of SEQ ID NO:1; (17) amino acids 1 to 396 of SEQ ID NO:1; (18) amino acids 1 to 399 of SEQ ID NO:1; (19) amino acids 1 to 420 of SEQ ID NO:1; (20) amino acids 1 to 436 of SEQ ID NO:1; (21) amino acids 1 to 437 of SEQ ID NO:1; and (22) Amino acids 1 to 528 of SEQ ID NO:1 and Optionally, step (b) enriches for a single isoform. The method according to any one of claims 1 to 7.

9. A method for detecting a biomarker in a sample obtained from a subject, comprising detecting neurofilament light chain according to the method of any one of claims 1 to 14, wherein the biological sample is a sample obtained from a subject having or at risk of having neuronal damage, and the biomarker is one to more Nfl isoforms detected in step (c).

10. 10. The method of claim 9, comprising quantifying a first and a second population of Nfl isoforms, and optionally the biomarker is a ratio of the two populations.

11. 1. A method for measuring treatment response in a subject having or at risk of having neurological injury, comprising: (a) quantifying a biomarker according to claim 9 or 10 in a first biological sample obtained from a subject; (b) administering the treatment to the subject; and (c) quantifying the biomarkers quantified in step (a) in a second biological sample obtained from the subject after treatment. Including, the first biological sample and the second biological sample are both blood samples or both CSF samples; An absence or decrease in the amount of the biomarker in the second sample compared to the first sample indicates a positive treatment response, or the amount of the biomarker increases in the second sample compared to the first sample, but the change is less than that which occurs in a control group of subjects with neurological injury but who were not administered the treatment. method. (a) an epitope-binding agent that competitively inhibits binding of HJ30.1 to HJ30.1, a humanized version of HJ30.1, or full-length recombinant Nfl; (b) an epitope-binding agent that competitively inhibits binding of HJ30.2 to HJ30.2, a humanized version of HJ30.2, or full-length recombinant Nfl; (c) an epitope-binding agent that competitively inhibits binding of HJ30.4, a humanized version of HJ30.4, or HJ30.7 to full-length recombinant Nfl; (d) an epitope-binding agent that competitively inhibits binding of HJ30.7 to HJ30.7, a humanized version of HJ30.7, or full-length recombinant Nfl; (e) an epitope binding agent that competitively inhibits binding of HJ30.11 to HJ30.11, a humanized version of HJ30.11, or full-length recombinant Nfl; or (f) an epitope-binding agent that competitively inhibits binding of HJ30.13 to HJ30.13, a humanized version of HJ30.13, or full-length recombinant Nfl.

2. An isolated anti-neurofilament light chain (Nfl) epitope binding agent selected from: Claim 13: (a) PTA-126966; (b) PTA-126967; (c) PTA-126968; (d) PTA-126969; (e) PTA-126970; or (f) PTA-126971 An isolated antibody produced by a hybridoma deposited with the American Type Culture Collection having an ATCC designation selected from: (a) a light chain variable region of HJ30.1 (ATCC No. PTA-126966), and / or a heavy chain variable region of HJ30.1 (ATCC No. PTA-126966); (b) the light chain variable region of HJ30.2 (ATCC No. PTA-126967), and / or the heavy chain variable region of HJ30.2 (ATCC No. PTA-126967); (c) the light chain variable region of HJ30.4 (ATCC No. PTA-126968), and / or the heavy chain variable region of HJ30.4 (ATCC No. PTA-126968); (d) the light chain variable region of HJ30.7 (ATCC No. PTA-126969), and / or the heavy chain variable region of HJ30.7 (ATCC No. PTA-126969); (e) the light chain variable region of HJ30.11 (ATCC No. PTA-126970), and / or the heavy chain variable region of HJ30.11 (ATCC No. PTA-126970); or (f) the light chain variable region of HJ30.13 (ATCC No. PTA-126971); and / or the heavy chain variable region of HJ30.13 (ATCC No. PTA-126971) 1. An isolated anti-neurofilament light chain (Nfl) antibody or an antigen-binding fragment thereof, comprising: (a) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.1 (ATCC No. PTA-126966), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.1 (ATCC No. PTA-126966); (b) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.2 (ATCC No. PTA-126967), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.2 (ATCC No. PTA-126967); (c) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.4 (ATCC No. PTA-126968), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.4 (ATCC No. PTA-126968); (d) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.7 (ATCC No. PTA-126969), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.7 (ATCC No. PTA-126969); (e) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.11 (ATCC No. PTA-126970), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.11 (ATCC No. PTA-126970); or (f) a light chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VL of HJ30.13 (ATCC No. PTA-126971), and / or a heavy chain variable region having 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the VH of HJ30.13 (ATCC No. PTA-126971).

1. An isolated anti-neurofilament light chain (Nfl) antibody or an antigen-binding fragment thereof, comprising:

16. Use of an isolated anti-Nfl epitope binding agent described in claim 11, an isolated antibody described in claim 12, or an isolated anti-Nfl antibody or antigen-binding fragment thereof described in claim 13 or 14, for detecting and / or quantifying Nfl in a biological sample, optionally wherein the biological sample is a blood sample or a cerebrospinal fluid sample, and optionally wherein the epitope binding agent, or the antibody or antigen-binding fragment thereof is attached to a detectable label.

17. (a) (i) an epitope-binding agent that competitively inhibits binding of HJ30.1 to HJ30.1, a humanized version of HJ30.1, or full-length recombinant Nfl; (ii) an epitope-binding agent that competitively inhibits the binding of HJ30.2, a humanized version of HJ30.2, or HJ30.2 to full-length recombinant Nfl; (iii) an epitope binding agent that competitively inhibits the binding of HJ30.4, a humanized version of HJ30.4, or HJ30.4 to full-length recombinant Nfl; (iv) an epitope-binding agent that competitively inhibits the binding of HJ30.7, a humanized version of HJ30.7, or HJ30.7 to full-length recombinant Nfl. (v) an epitope binding agent that competitively inhibits the binding of HJ30.11, a humanized version of HJ30.11, or HJ30.11 to full-length recombinant Nfl; or (vi) an epitope-binding agent that competitively inhibits the binding of HJ30.13, a humanized version of HJ30.13, or HJ30.13 to full-length recombinant Nfl. one or more antibodies selected from: (b) (i) detecting a biomarker in a sample obtained from a subject according to claim 9 or 10; or (ii) measuring the treatment response in a subject having or at risk of having a neurological injury according to claim 11. Instructions for Including the kit.

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