Methods of immunoassays using neurofilament light chains

Abstract

Described herein are methods, kits, and compositions for detecting an analyte (typically an analyte associated with a neurodegenerative disease, such as a neurofilament light chain) in a sample using a chemiluminescent marker. Solid supports, reagents and compounds for use in these methods are also described. In general, the methods relate to specific forms of assays that provide the high resolution required for the detection of low concentrations of analytes in a sample, and can be used in a health care ecosystem at a location proximate to the patient. These methods, systems, and devices enable early detection and prediction of a variety of neurodegenerative diseases, such as Alzheimer's disease and multiple sclerosis.

Description

[0001] Cross-references to related applications This application claims priority and benefit to U.S. Application No. 63 / 592,693, filed October 24, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] open field This disclosure relates to high-resolution assay methods for detecting or measuring analytes that are typically present in biological samples at low concentrations, and to methods of use relating to neurological diseases, conditions, or states, such as early detection of disease. These assay methods typically involve the use of acridine monophosphate conjugated to an antibody fragment via a linker (such as polyethylene glycol) and a carrier protein (such as bovine serum albumin). The assays are suitable for indications of early neurodegenerative diseases.

[0003] background Detection of analytes of interest in certain biological samples is often hampered by the detection limits of immunoassays. Certain neurological diseases, disorders, and conditions are associated with biomarkers that could allow for improved diagnostic accuracy and prognostic assessment. For example, neurofilament light chains (NfL) are neuronal cytoplasmic proteins highly expressed in myelinated axons and are involved in amyotrophic lateral sclerosis (ALS), multiple sclerosis, Alzheimer's disease, and Huntington's disease. However, the concentration of NfL in the blood is limited, partly due to its necessity to cross the blood-brain barrier. Because of the extremely low concentrations of neurofilament light chains (NfL) in the blood (since this analyte must cross the blood-brain barrier), analytical methods lack the necessary resolution to identify its presence in the blood or to track changes in NfL concentration associated with disease progression.

[0004] Clinical laboratories typically define the lower limit of measurement for most assays as either the lower limit of quantification (LLOQ) or the limit of detection (LOD). The lower limit of quantification is the lowest value below which an accurate quantitative value (CV < 20%) can be reported, while the LOD is the value below which a measurement can distinguish the presence or absence of an analyte. Due to the inherently low limit of detection in typical immunoassays, some analytes with low concentrations, such as NfL, cannot be measured using most immunoassays.

[0005] Neurodegenerative diseases such as Alzheimer's disease, MS, and others are frequently diagnosed with delays, missed diagnoses, or misdiagnoses. Early diagnosis allows for more effective interventions, especially when cognitive decline and other aspects of the prodromal state can be slowed, or even preventative measures can be taken earlier. Once nerve damage has occurred, it is currently impossible to repair the damage.

[0006] There is a persistent need for assays that can provide highly sensitive analyte detection, particularly for analytes that are typically present in biological samples at low concentrations. Furthermore, there is a need for feasible and easily deployable methods suitable for large-scale population screening, routine physician clinic testing, or self-testing for indicative of neurodegenerative diseases such as Alzheimer's and MS, particularly at or near the point of care.

[0007] Overview Based on the foregoing and other objectives, this disclosure includes providing assays for the detection of highly sensitive analytes, particularly for analytes that are typically present in biological samples at low concentrations. Furthermore, this disclosure now provides feasible methods for large-scale population screening, routine physician clinic visits, or self-testing for indications of neurodegenerative diseases such as Alzheimer's disease and MS. In embodiments, indications of disease are characterized as early detection.

[0008] Based on the foregoing and other objectives, this disclosure includes embodiments that incorporate the use of NfL as a step in a combinatorial algorithm for early screening of individuals reporting cognitive problems (such as self-reported problems) and interested in assessing dementia risk, or in healthcare settings where such screening can become routine. Furthermore, due to its high sensitivity to neurodegenerative changes, minimally invasive acquisition method, and precise quantification, this disclosure is well-suited for monitoring the effectiveness of preventative or curative interventions.

[0009] Based on the foregoing and other objectives, this disclosure includes the feasible use of NfL for large-scale population screening for Alzheimer's disease, MS, or other neurodegenerative diseases. In embodiments, this disclosure includes providing NfL testing directly to consumers to assess their risk of developing such diseases or to monitor the impact of preventative measures such as exercise, good nutrition, optimized sleep patterns, cognitive training, and other interventions, including behavioral or pharmacological interventions.

[0010] Methods are provided for detecting or quantifying analytes in samples (e.g., biological samples such as blood, saliva, serum, or samples derived from biological samples, such as diluted biological samples). These methods may include: (a) Mixing a sample with a composition comprising a chemiluminescent label conjugated to a primary antibody or antibody fragment that binds to an analyte, wherein the chemiluminescent label binds to a carrier protein containing a linker, and the linker binds to an antibody or antibody fragment; (b) Add particles containing a secondary antibody or antibody fragment to the mixture, the secondary antibody or antibody fragment binding to the analyte and adhering to the particle surface; (c) Prepare mixtures to measure chemiluminescence (e.g., by separating particles having chemiluminescent acridinium adducted to the surface); (d) Triggering chemiluminescence from the preparation; and (e) The presence of the at least one analyte is detected or the concentration of the at least one analyte is calculated by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of emitted light with a known concentration of at least one of the multiple analytes.

[0011] In some embodiments, the analyte is a neurofilament, such as a neurofilament light chain (e.g., serum neurofilament light chain). In a particular embodiment, the sample is blood.

[0012] In various implementations, the method may include: a) Obtaining the first biological sample from the subject; b) Quantify the concentration of the first neurofilament (NfL) in the first biological sample; c) Obtain a second biological sample from the subject some time after the first biological sample is collected; d) Quantify the concentration of second neurofilaments (NfL) in the second biological sample; and e) Determine the rate of change of neurofilament concentration between the first concentration and the second concentration during the time period.

[0013] In various embodiments, the biological sample is a blood sample, and the method further includes correlating the rate of change in the biological sample with the rate of change in serum NfL. In some embodiments, the biological sample is independently a blood sample or a cerebrospinal fluid (CSF) sample. In some embodiments, the method further includes assessing the risk and / or progression of neurodegenerative diseases (e.g., by comparing the rate of change in blood NfL concentration and / or serum NfL concentration with one or more disease progression indicators based on said concentration). In some embodiments, the method further includes performing one or more cognitive tests on the subject. The first and second concentrations can (or may have been) obtained independently by: The first biological sample or biological blood sample is mixed with the composition, the composition comprising a chemiluminescent label conjugated to a primary antibody or antibody fragment that binds to a neurofilament, wherein the chemiluminescent label binds to a carrier protein comprising a linker, and the linker binds to the antibody or antibody fragment; Particles containing a secondary antibody or antibody fragment are added to the mixture, the secondary antibody or antibody fragment binding to the analyte and attaching to the particle surface; Mixtures are prepared to measure chemiluminescence (e.g., by separating particles having chemiluminescent acridine on the surface). Triggering chemiluminescence from the prepared product; and The presence of a neurofilament or the concentration of a neurofilament is detected by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of emitted light with a known concentration of the neurofilament.

[0014] If a risk of neurodegenerative disease is identified, the method may further include providing a treatment regimen to the subject. In some embodiments, the treatment regimen includes the administration of a therapeutic agent. In some embodiments, the treatment regimen includes guidance on preventative measures such as exercise, good nutrition, optimized sleep patterns, cognitive training, or behavioral training. In some cases, the first and / or second biological samples are obtained by the user. In some embodiments, the first and / or second biological samples are obtained by the user's primary care physician. Any step of the method disclosed herein, such as obtaining a biological sample, conducting cognitive tests (e.g., verbal tests), forming a binding complex, or measuring chemiluminescence output, can occur within the healthcare ecosystem, and particularly at the user's point of care and / or home. In some embodiments, the method further includes transmitting the concentration or measured chemiluminescence output to at least one or more servers.

[0015] A computer system that can be integrated with the methods and measurements of this disclosure is also provided. Typically, the computer system may include one or more servers configured collectively as follows: a) Receive data, including the concentration of neurofilaments in a user's biological sample; b) Compare the concentration with historical data on the progression of neurological disorders to assess the user's risk of developing the neurological disorder; and c) Risk assessment related to transmission and disease progression.

[0016] In some embodiments, the one or more servers are collectively configured to transmit performance tests (e.g., to a user's computing device), receive data related to the user's performance during the performance test, and use the user performance test-related data in risk assessments. In some embodiments, the performance test includes verbal analysis. In some embodiments, the one or more servers are collectively configured to: Receive data, which includes the concentration of neurofilaments in a second biological sample of the user collected at a time point following the collection of the user's biological sample. The change in neurofilament concentration over time was used in the second risk assessment; and Second risk assessment for transmission.

[0017] The computer system may further include an immunoassay device configured for quantifying the concentration of neurofilaments in biological samples, wherein the immunoassay device is capable of transmitting information via a network, and The one or more servers receive data from the immunoassay device via a network, the data including the concentration of neurofilaments and / or chemiluminescence output. For example, the immunoassay device may include a sequential array of reaction sites for measuring chemiluminescence from a biological sample (or a medium derived therefrom), wherein a mixture may be sequentially placed in each reaction site; the sequential array of reaction sites includes one or more of the following: Biological sample addition location: This is where biological samples are added to the reaction vessel. At the assay reagent site, the assay reagent (e.g., a solid-phase reagent comprising magnetizable particles on which molecules capable of forming binding complexes with neurofilament light chains or their binding partners are immobilized; chemiluminescent conjugates, such as acridinium compounds, including acridinium esters and acridinium sulfonamides capable of forming binding complexes with neurofilaments or molecules immobilized on magnetizable particles; or both) is added to the reaction vessel (e.g., a reaction vessel containing a biological sample). Incubation sites are used to bind analytes, chemiluminescent compounds, and magnetizable particles from biological samples; The isolation location magnetically isolates the magnetizable particles from the liquid medium from the biological sample and the assay reagents; Separation site, where liquid media (e.g., from biological samples, from assay reagents) are separated from magnetizable particles (e.g., isolated magnetizable particles); At the washing location, a washing buffer (e.g., a buffer solution that may contain one or more salts such as sodium chloride and sodium azide, detergents such as cationic detergents, buffers such as phosphates, blocking agents such as bovine serum albumin (BSA) or combinations thereof) is added to the magnetizable particles; and Wash buffer aspiration site, where the wash buffer is separated from the magnetizable particles to form a chemiluminescent sample; The first chemiluminescence reagent addition location is where the first chemiluminescence trigger reagent can be added to the chemiluminescent sample; The second chemiluminescent reagent addition site is where the second chemiluminescent triggering reagent is added to the chemiluminescent sample (or a portion thereof or a portion derived therefrom); and Chemiluminescence location, where chemiluminescence is collected by one or more photon detectors in order to measure the light output from the chemiluminescent sample; The amount of chemiluminescence is related to the concentration of NfL in the biological sample, and the immunoassay device transmits the amount of chemiluminescence and / or the concentration to the one or more servers.

[0018] Based on the foregoing and other objectives, this disclosure includes a method for detecting an analyte in a sample by using an acridine conjugate or a binding partner of the analyte, such as an antibody. These acridine conjugates are typically provided on a carrier protein, such as bovine serum albumin (e.g., in excess), which is linked to the analyte or its binding partner. These conjugates are typically capable of providing the desired chemiluminescent output over a range of standard concentrations in an assay (e.g., a sandwich assay) to achieve the required resolution for analytes that may be present in biological samples at low concentrations.

[0019] Furthermore, the preparation of conjugates can occur in a specific assay format suitable for characterization at the desired resolution. For example, the RLU slope factor (between 0 pg / mL and 660 pg / mL) associated with the conjugate and / or assay can be greater than (or up to 2000) 500 or greater than 600. This disclosure is partly based on the finding that it is possible to use the immunoassays described herein with respect to slope factors between 0 pg / mL and 660 pg / mL for immunoassays, and that such slope factors may be necessary for the clinical utility of detecting certain analytes. In some embodiments, the sample is blood, saliva, or serum. In some embodiments, the sample is derived from a biological sample, such as a diluted biological sample (e.g., as mixed with saline). In some embodiments, the analyte is an analyte that crosses the blood-brain barrier and enters the bloodstream (e.g., a neuronal analyte such as neurofilament light chain), and the sample is blood. In some embodiments, the analyte is a serum analyte (e.g., a serum neurofilament biomarker such as serum neurofilament light chain (sNfL)), and the biological sample is blood.

[0020] Methods can be used to characterize diseases, symptoms, or conditions. For example, if the analyte is a neuronal biomarker such as neurofilament light chains, then if the analyte level in the blood is above a certain concentration (e.g., between 10 pg / mL and 20 pg / mL), the subject from whom the sample was taken can be diagnosed with a neuronal disease (e.g., amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, and Huntington's disease). In some implementations, the method provides the resolution needed to track the progression of a disease, symptom, or condition. For example, the concentration of the analyte can be measured from a first sample taken from the subject. After a period of time, a second biological sample can be taken from the subject, and the concentration can be determined. Changes in biomarker levels can be correlated with disease progression. For example, the rate of change in Nfl concentration (e.g., [Nfl] / year) can be calculated and correlated with an estimate of symptom onset (see, for example...). Figure 7 ).

[0021] In some embodiments, the chemiluminescent label is conjugated to a first immunoglobulin antibody fragment (e.g., F(ab), F(c)). In a particular embodiment, the chemiluminescent label is conjugated to an F(ab) fragment, which can still bind to the antigen or analyte but is monovalent (and does not contain the F(c) portion). In some embodiments, the primary antibody or antibody fragment is a mouse monoclonal antibody or a fragment thereof (e.g., F(ab)). In some embodiments, the linker between the antibody or its fragment and the carrier protein comprises (or is) polyethylene glycol (PEG), which can be independently covalently linked to both portions. For example, the PEG linker may have 2-20 (e.g., 2-10, 2-5) ethylene glycol units. In some embodiments, the carrier protein is keyhole hemocyanin (KLH), bovine serum albumin (BSA), or cationic BSA. By utilizing the properties of the carrier protein, more labeling compounds can be present in the conjugate. For example, in some embodiments, the ratio of acridine to carrier protein is 50:1 to 1:1 by weight (e.g., 30:1 to 1:1, 25:1 to 5:1).

[0022] The assay disclosed herein is typically a two-step sandwich immunoassay using acridinium ester chemiluminescence technology. The assay can employ two anti-sNfL antibodies. The primary antibody in the luminescent reagent can be an acridinium ester-labeled mouse monoclonal anti-sNfL antibody. The secondary antibody can be a biotinylated mouse monoclonal anti-sNfL antibody that binds to paramagnetic microparticles coated with streptavidin in the solid phase. The desired sensitivity can be achieved by: 1) signal amplification (e.g., amplification using an acridinium ester conjugated to a carrier protein – particularly a specific excess of acridinium on the carrier protein), 2) reduction of nonspecific binding (e.g., reduction using an antibody fragment linked to the carrier protein), and 3) the timing of reagent addition in the analyzer (e.g., adding the luminescent reagent before adding the solid phase, the incubation time and intensity associated with each addition).

[0023] Chemiluminescent labels conjugated to primary antibodies or antibody fragments that bind to the analyte can be formed through the following: i) Reacting a chemiluminescent acridine trioxide compound containing reactive functional groups with a carrier protein to label the carrier protein; and ii) Reacting the linker compound (e.g., a compound containing a linker having a reactive functional group at each end), the primary antibody or antibody fragment, and the labeled carrier protein.

[0024] In the specific implementation scheme, the chemiluminescent acridinelon containing reactive functional groups is TSPAE-NHS: Or its salt.

[0025] The assays described herein may involve the presence of a solid phase conjugated with a secondary antibody or a fragment thereof. For example, the secondary antibody or antibody fragment may be a biotinylated antibody or antibody fragment (e.g., and the solid phase is coated with streptoacidin). Similar assays, systems, and methods can be found, for example, in PCT / US2024 / 026599, filed April 26, 2024, which is incorporated herein by reference in its entirety.

[0026] High-resolution assays can be performed using the conjugates and assay formats described herein. These assays can be performed on analytes in biological samples that are typically found at concentrations previously considered undetectable, such as those that cross the blood-brain barrier. Specific incubation and sequential steps as described herein can result in these assay formats with increased resolution. For example, the assay steps may be able to detect analyte concentration differences of less than 5 pg / mL (e.g., less than 4 pg / mL, 1 pg / mL to 5 pg / mL, 2–5 pg / mL, 2–4 pg / mL, 3–4 pg / mL, 2–3 pg / mL).

[0027] In some embodiments, the preparation steps may include separating particles (composite to the label) from the mixture, and triggering chemiluminescence by the particles or the separated mixture. In some embodiments, the method further includes incubating the mixture prior to the addition of the particles. In some embodiments, incubation prior to solid-phase addition includes heating the mixture for more than 30 minutes (e.g., 30 to 120 minutes, 30 to 60 minutes). In some embodiments, the method further includes incubating the mixture after the addition of the particles. In some embodiments, such incubation after particle addition may involve heating the mixture for less than 30 minutes (e.g., 10 to 30 minutes, 10 to 20 minutes). In some embodiments, the labeled conjugate is first added to a biological sample, optionally incubated, and then solid particles are added to the biological sample / labeled conjugate mixture, which optionally undergoes a second incubation.

[0028] Immunoassay compositions are also provided. These compositions typically comprise a chemiluminescent label conjugated to a primary antibody fragment that binds to an analyte (e.g., a neurofilament light chain), wherein the chemiluminescent label binds to a carrier protein comprising a linker (e.g., polyethylene glycol such as PEG2-PEG15, or PEG4), and the linker binds to the antibody fragment; and a carrier or excipient. In some embodiments, the composition further comprises a buffer. In various embodiments, the composition comprises one or more of a protease inhibitor, a surfactant, a preservative, an inhibitor, and / or a heterophile antibody inhibitor. In a particular embodiment, the carrier protein is bovine serum albumin. In some embodiments, the chemiluminescent acridine onionium is in a weight ratio of 50:1 to 1:1 (e.g., 30:1 to 1:1, 25:1 to 5:1) by weight. The reagent may comprise each detectable conjugate at a concentration of 10 to 30 ng / mL. The reagents of this disclosure comprise compositions containing the indicated components, and optionally excipients, carriers, or solvents. The reagents of this disclosure may include surfactants.

[0029] Kits for detecting analytes are also provided. The kit may contain the immunoassay reagent composition of this disclosure in a container adapted for use with an analyzer, and / or contain reagents associated with the immunoassay (e.g., luminescent reagents, solid-phase reagents) and / or sample collection devices (e.g., for obtaining biological samples from a user) and / or sample storage devices (e.g., devices for storing and / or transporting biological samples). The kit may also include instructions on accessing software or mobile device applications for installation and use to monitor and detect neurodegenerative diseases or their conditions. In some embodiments, the kit may include a sample collection device for collecting biological samples from a user, and instructions on how to use the sample collection device to collect biological samples and / or how to access the computer system of this disclosure.

[0030] Solid particles are also provided. These solid particles can be formed during the measurement process (and are typically used for subsequent analysis). The solid particles can be coated with streptavidin, which is optionally conjugated to a biotinylated mouse antibody (e.g., antineurofilament) via a linker (e.g., PEG, iodo-PEG), wherein the antineurofilament antibody binds to the analyte (e.g., neurofilament), and the bound analyte further binds to a monoclonal mouse antibody fragment linked to a carrier protein conjugated to one or more chemiluminescent acridine onions. In various embodiments, the monoclonal mouse antibody fragment is linked to the carrier protein via a linker (e.g., PEG).

[0031] After adding two sets of chemiluminescent labels, the sample is typically prepared in a manner that allows for the measurement and / or quantification of the analyte concentration to induce chemiluminescence. For example, the preparation steps may include: (e1) A solid support having molecules immobilized thereon, said molecules being capable of forming binding complexes with said at least one analyte and capable of forming binding complexes with chemiluminescent labels in a first and / or second set of chemiluminescent labels; and (e2) Separate the solid support from the mixture.

[0032] These and other aspects of the invention will be better understood by referring to the following detailed description, including the appended claims. Brief description of the attached diagram Figure 1 Detection methods that can be used to diagnose neurodegenerative diseases are provided. These methods include positron emission tomography (PET), single photon emission coupled tomography (SPECT), magnetic resonance imaging (MRI), and computed tomography (CT).

[0034] Figure 2 This disclosure showcases a healthcare-related ecosystem typically involved in the characterization and treatment of neurodegenerative diseases. The methods and systems disclosed provide greater detection and control in home care and physician offices (circled sections). It can be seen that by focusing on the home and primary care providers, this disclosure enables earlier and more efficient diagnosis of diseases such as Alzheimer's disease. These methods and systems cover key systems within the in vitro diagnostics (IVD) ecosystem, providing healthcare providers with the crucial information needed for accurate screening, diagnosis, and monitoring of patients. These methods and systems can provide customized solutions for better patient care.

[0035] Figure 3 and Figure 4 This demonstrates the ecosystem changes that may occur when utilizing the methods and systems disclosed herein. Home screening using immunoassays (such as the Attellica CI immunoassay) within the healthcare ecosystem can bring diagnostic assessment closer to the patient and increase communication between physicians, clinics, reference laboratories, and patients. For example, as... Figure 4 As shown, the method disclosed herein can combine speech-based analysis (e.g., via a network) with home blood sampling testing.

[0036] Figure 5 The benefits associated with this disclosure are described.

[0037] Figure 6A schematic diagram is provided showing the effect of neurofilaments on nerve axons, resulting in CSF neurofilament concentration, the resulting blood concentration, and its significance in relation to the development of blood assays.

[0038] Figure 7 The association between NfL concentration and Alzheimer's disease progression has been demonstrated, as evidenced by changes in magnetic resonance imaging (MRI) in non-carriers, presymptomatic mutation carriers, and symptomatic mutation carriers, and by the estimated years to symptom onset (EYO) in these patients. It can be seen that NfL concentration can be correlated with disease progression and concentration (and the rate of change in NfL concentration). Changes in NfL predict disease progression in presymptomatic Alzheimer's disease.

[0039] Figure 8 Exemplary schematic diagrams of several steps of the sandwich assay protocol disclosed herein are provided. This assay protocol, and any methods of this disclosure, can be automated, for example, in point-of-care service equipment.

[0040] Figure 9 ( Figure 9A and Figure 9B ) provides arrays (or sample holders) and linear ( Figure 9A ) and ring ( Figure 9B () in the form of a stereoscopic diagram of the reaction vessel sequence therein.

[0041] Figure 10 An SDS-PAGE gel showing the digestion of neurofilament antibodies is displayed.

[0042] Figure 11A A schematic diagram of an exemplary binding complex formed by the sandwich assay of this disclosure is provided. Figure 11B An exemplary high-resolution measurement schematic diagram is provided.

[0043] Figure 12 RLU measurements at each standard concentration are provided for high-resolution determinations (slope factor greater than 600) for this disclosure.

[0044] Figure 13 It is a table that displays the measurement parameters and measurement outputs for various measurements with a slope factor less than 600.

[0045] Detailed description For convenience, certain terms used in this specification, including embodiments and appended claims, are summarized herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0046] Unless otherwise expressly defined, the following terms and phrases are expected to have the following meanings throughout this disclosure.

[0047] Unless otherwise stated, all percentages given herein refer to the weight percentage of a specific component relative to the entire composition (including the carrier). It should be understood that the sum of the weight percentages of all components within the composition does not exceed 100%.

[0048] As used herein, the term "an" or "a" means one or more. As used herein, the term "consistently composed of" is intended to limit the invention to the specified materials or steps, and those materials or steps that do not substantially affect the essential and novel features of the invention, as understood from reading this specification. The expression "comprising" includes both "consistently composed of" and "composed of".

[0049] Unless otherwise stated, the following definitions of various groups or substituents are used. The specific and general values ​​listed below regarding free radicals, substituents, and ranges are for illustrative purposes only; they do not exclude other defined values ​​or ranges for free radicals and substituents. Unless otherwise indicated, alkyl, alkenyl, alkynyl, alkoxy, etc., represent straight-chain, branched, and cyclic groups, and any combination thereof.

[0050] Throughout this application, the term "about" is used to indicate that a value includes inherent variation error of the composition / apparatus / device, error in the method used to determine the value, or variability among study subjects. For example, but not limited to, when using the term "about," a specified value may vary relative to a particular value by 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, provided that such variation is suitable for performing the disclosed method and as understood by one of ordinary skill in the art.

[0051] Antibody: The term “antibody” is used herein in the broadest sense and includes, for example, complete monoclonal and polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments and conjugates thereof that exhibit the biological activity of desired analyte binding (e.g., but not limited to Fab, Fab', F(ab')2, Fv, scFv, Fd, biantibodies, single-chain antibodies, and other antibody fragments and conjugates thereof that retain at least a portion of the variable region of a complete antibody), antibody substitute proteins or peptides (i.e., modified binding proteins / peptides), and combinations or derivatives thereof. The antibody may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody may bind, for example, neurofilaments, such as neurofilament light chains, neurofilament medium chains, or neurofilament heavy chains. For example, the antibody may be a monoclonal antibody such as mAB47:3 and mAb2:1, as included in the NF-Light assay from Uman Diagnostics (UmanDiagnostics, Umea, Sweden).

[0052] Biomarker: Consistent with its use in the art, the term "biomarker" or "biological marker" is used herein to refer to an entity whose presence, level, or form is associated with a particular biological event or state of interest, such that it is considered a "marker" of that event or state. To name just a few examples, in some embodiments, a biomarker may be or include a marker of a particular disease state, or a marker of the likelihood that a particular disease, symptom, or condition may develop, occur, or recur. In some embodiments, a biomarker may be or include a marker of a particular disease or treatment outcome or its likelihood. Thus, in some embodiments, a biomarker predicts an relevant biological event or state of interest, in some embodiments, a biomarker foreshadows a relevant biological event or state of interest, and in some embodiments, a biomarker diagnoses a relevant biological event or state of interest. In some embodiments, a biomarker is a possible biomarker of a relevant biological event or state of interest. A biomarker can be an entity of any chemical class. For example, in some embodiments, a biomarker may be or include nucleic acids, peptides, small molecules, or combinations thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is intracellular. In some embodiments, the biomarker is found extracellularly (e.g., secreted or otherwise generated or present in extracellular fluids such as blood, urine, tears, saliva, cerebrospinal fluid, etc.). An exemplary biomarker is the neurofilament light chain (NfL), as disclosed in Barro, C., et al. Ann Clin Transl Nerol 7.12 (2020):2508-2523, which is incorporated herein by reference in its entirety. Other biomarkers that may be used in this disclosure include those in Hansson, O., Nature Medicine 27 (2021): 954-963, which is also incorporated herein by reference in its entirety.

[0053] Diagnostic Test: As used herein, a “diagnostic test” is a step or series of steps performed or executed to obtain information that may be used to determine whether a patient has a disease, symptom, or condition and / or to classify the disease, symptom, or condition into a phenotypic category or to indicate the prognosis of the disease, symptom, or condition or the likely response to treatment (general treatment or any particular treatment) of the disease, symptom, or condition. Similarly, “diagnosis” refers to providing any type of diagnostic information, including but not limited to whether a subject is likely to have or develop a disease, symptom, or condition, such as the state, stage, or characteristics of the disease, symptom, or condition manifested in the subject, information relating to the nature or classification of a tumor, information relating to prognosis, and / or information useful in selecting appropriate treatment or additional diagnostic tests. Treatment options may include selecting a specific therapeutic agent or other treatment modality such as surgery, radiation, etc., options regarding whether to stop or deliver treatment, options related to the dosing regimen (e.g., the frequency or level of one or more doses of a specific therapeutic agent or combination of therapeutic agents), etc. The selection of additional diagnostic tests may include more specific tests for a given disease, symptom, or condition.

[0054] This document repeatedly refers to servers, services, interfaces, engines, modules, clients, peers, portals, platforms, or other systems formed by computing devices. It should be understood that the use of such terms is considered to refer to one or more computing devices having at least one processor (e.g., ASIC, FPGA, DSP, x86, ARM, ColdFire, GPU, multi-core processor) programmed to execute software instructions stored on computer-readable tangible non-transient media (e.g., hard disk drives, solid-state drives, RAM, flash memory, ROM). For example, a server may include one or more computers operating as web servers, database servers, or other types of computer servers to fulfill the described roles, responsibilities, or functions. It should be further understood that the disclosed computer-based algorithms, processes, methods, or other types of instruction sets may be embodied as computer program products containing non-transient tangible computer-readable media storing instructions that cause a processor to perform the disclosed steps. Various servers, systems, databases, or interfaces may exchange data using standardized protocols or algorithms, possibly based on HTTP, HTTPS, AES, public-private key exchange, web service APIs, known financial transaction protocols, or other electronic information exchange methods. Data exchange can be conducted through packet-switched networks, the Internet, LANs, WANs, VPNs, or other types of packet-switched networks.

[0055] As used herein, the term "substantially" means that the event or situation subsequently described occurs completely, or occurs to a large extent or in a measured sense. For example, when associated with a particular event or situation, the term "substantially" means that the event or situation subsequently described occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term "substantially adjacent" can mean that two items are 100% adjacent to each other, or that two items are very close to each other but not 100% adjacent, or that a portion of one of the two items is not 100% adjacent to the other item but is very close to the other item.

[0056] As used herein, in the context of the chemical section, the phrase “associated” includes both direct and indirect association between two parts. Non-limiting examples of association include: one part covalently bonded to another part by a direct bond or by a spacer group; one part non-covalently bonded to another part directly or by a specific binding pair member to said part; incorporating one part into another part (e.g., by dissolving one part in another part or by synthesis); and encapsulating one part onto another part.

[0057] As used herein, the term "patient" includes both humans and veterinary subjects. In some non-limiting embodiments, the patient is a mammal. In some other non-limiting embodiments, the patient is a human. For diagnostic / therapeutic purposes, the term "mammal" means any animal classified as a mammal, including humans, domesticated and farm animals, non-human primates, and zoo animals, sporting animals, or pet animals such as dogs, horses, cats, cattle, etc.

[0058] "Healthcare provider" or "healthcare decision-maker" includes any individual who is authorized to diagnose or treat patients, or assist in the diagnosis or treatment of patients.

[0059] "Point-of-care testing" refers to real-time diagnostic testing that can be performed within a rapid timeframe, making it faster than similar tests without such a system. Point-of-care testing can be rapid and on-site, such as in a physician's office, bedside, emergency lab, emergency room, or other such locations, especially where rapid and accurate results are required. Patients may be present, but are not required to be. Points of care include, but are not limited to: emergency rooms, operating rooms, hospital labs and other clinical laboratories, physician's offices, on-site, or any scenario where rapid and accurate results are desired.

[0060] As used herein, the term "immunoassay" refers to an assay that determines the presence of a diagnostic biomarker in a biological sample by reacting the sample with an antibody (or a fragment thereof) that specifically binds to the diagnostic biomarker, wherein the reaction is carried out for a period of time and under conditions that allow the formation of immune complexes between the antibody (or a fragment thereof) and the diagnostic biomarker. These immune complexes are then quantified.

[0061] A “threshold” can refer to a value (or values) used as a reference for obtaining information about a measurement result and / or classifying the measurement result, such as a measurement result obtained in an assay. The threshold can be determined based on one or more control samples. The threshold can be determined before, during, or after the measurement of interest. In some embodiments, the threshold can be a series of values. In some embodiments, the threshold can be a value (or a series of values) reported in a relevant domain (e.g., values ​​found in a standard table).

[0062] The term hydrocarbon can refer to a radical or group containing carbon and hydrogen atoms, which can be bonded at indicated positions (e.g., R, R', R'', R). N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7). Examples of hydrocarbon groups include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl, and any combination thereof (e.g., alkyl-aryl-alkyl). As used herein, unless otherwise indicated, hydrocarbons can be monovalent or polyvalent (e.g., divalent, trivalent) hydrocarbon groups. If the form is -(CH2) n A hydrocarbon group (including the methylene group, i.e., -CH2-) that does not have unsaturated bonds between carbon atoms is considered an alkyl group. Unless otherwise stated, all hydrocarbon groups (including substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl, aryl-alkyl, alkyl-aryl) may have 1-35 carbon atoms. In other embodiments, the hydrocarbon has 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Hydrocarbons may have 2 to 70 atoms, 4 to 40 atoms, or 4 to 20 atoms.

[0063] Substituted hydrocarbons may have one or more hydrocarbon groups, substituted hydrocarbon groups as substituents, or may contain one or more heteroatoms. Any hydrocarbon substituents disclosed herein (e.g., R, R', R'', R...)N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7 may optionally include 1-20 (e.g., 1-10, 1-5) heteroatoms. Examples of substituted hydrocarbon groups include, but are not limited to, heterocycles, such as heteroaryl groups. Unless otherwise stated, hydrocarbons substituted with one or more heteroatoms contain 1-20 heteroatoms. In other embodiments, hydrocarbons substituted with one or more heteroatoms contain 1-12, 1-8, 1-6, 1-4, 1-3, or 1-2 heteroatoms. Examples of heteroatoms include, but are not limited to, oxygen, nitrogen, sulfur, phosphorus, halogens (e.g., F, Cl, Br, I), boron, or silicon. In some embodiments, the heteroatom is selected from oxygen, nitrogen, sulfur, phosphorus, and halogens (e.g., F, Cl, Br, I). In some embodiments, the heteroatom may be selected from O, N, or S. In some embodiments, the heteroatom or group may replace carbon. In some embodiments, the heteroatom or group may replace hydrogen. In some embodiments, the substituted hydrocarbon may contain one or more heteroatoms (e.g., between two carbon atoms, as in "oxa") in the main chain or chain of the molecule. In some embodiments, the substituted hydrocarbon may contain one or more heteroatoms dangling from the main chain or chain of the molecule (e.g., covalently bonded to carbon atoms in the chain or main chain, as in "oxo").

[0064] When the indicated group is replaced by the indicated substituent, the specified group may be replaced by any and all of the one or more named substituents. For example, when the group, such as an alkyl or heteroaryl group, is replaced by an unsubstituted C1-C... 20 When alkyl or unsubstituted 2 to 20 heteroalkyl groups are substituted, the group may contain one or more unsubstituted C1-C groups. 20 Alkyl groups, and / or one or more unsubstituted 2- to 20-membered heteroalkyl groups. Furthermore, when partially substituted by an R substituent, the group may be referred to as "R-substituted". When partially R-substituted, the substitution is made by at least one R substituent, and each R substituent is optionally distinct. If the indicated group is used multiple times in the general chemical formula (e.g., R group), it should be understood that each group is chosen independently each time it appears.

[0065] Unless otherwise stated, any compound disclosed herein having one or more chiral centers may be in the form of a racemic mixture with respect to each chiral center, or may be pure or substantially pure with respect to each chiral center (e.g., greater than 98% ee). R or SEnantiomers exist, or can be used as a basis for understanding each chiral center. R or S A mixture of enantiomers is present, wherein the mixture comprises an excess of one or more enantiomers of another configuration, for example, more than 60%, or more than 70%, or more than 80%, or more than 90%, or more than 95%, or more than 98%, or more than 99% of the enantiomers in excess. R or S (The enantiomer is in excess.) In some implementations, any chiral center can be of the “S” or “R” configuration.

[0066] It should be understood that the description of compounds in this document is limited by the principles of chemical bonding. Therefore, when a group can be substituted by one or more of a number of substituents, such substituents are chosen in such a way as to conform to, for example, the principles of chemical bonding with respect to valence, and to give an inherently unstable compound. For example, consistent with the tetravalent electron configuration of carbon, any carbon atom is bonded to two, three, or four other atoms.

[0067] Substituent (group) prefix names can be derived from the parent hydride by: (i) replacing "ane" in the parent hydride with the suffix "yl", "diyl", "triyl", or "tetrayl"; or (ii) replacing "e" in the parent hydride with the suffix "yl", "diyl", "triyl", or "tetrayl" (here, when specified, atoms with free valences are given a lower number consistent with any established numbering of the parent hydride). Throughout this document, recognized abbreviations such as adamantyl, naphthyl, anthraceneyl, phenanthryl, furanyl, pyridyl, isoquinolinyl, quinolinyl, and piperidinyl, as well as common names such as vinyl, allyl, phenyl, and thiopheneyl, are also used.

[0068] Alkyl groups generally refer to saturated hydrocarbon chains, which can be straight or branched chains containing an indicated number of carbon atoms. For example, C1-C6 alkyl indicates that the group can have 1 to 6 carbon atoms (including the endpoints). Any atom can optionally be substituted, for example, by one or more substituents. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and tert-butyl. Any alkyl group mentioned herein (e.g., R, R', R'', R...) N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2cR3, R4, R5, R6, and R7 can have 1-35 carbon atoms. In other embodiments, the alkyl group has 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The alkyl group can be a lower alkyl group (e.g., C1-C4 alkyl).

[0069] A haloalkyl group is typically an alkyl group in which at least one hydrogen atom is replaced by a halogen. In some embodiments, more than one hydrogen atom (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) is replaced by a halogen. In these embodiments, the hydrogen atoms may each be replaced by the same halogen (e.g., fluorine), or the hydrogen atoms may be replaced by a combination of different halogens (e.g., fluorine and chlorine). A haloalkyl group may include an alkyl moiety in which all hydrogen atoms have been replaced by a halogen (sometimes referred to herein as a perhaloalkyl group, e.g., a perfluoroalkyl group, such as trifluoromethyl). The haloalkyl group may be optionally substituted.

[0070] Typically, alkoxy groups have the formula -O (alkyl). Alkoxy groups can be, for example, methoxy (-OCH3), ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, pentoxy, 2-pentoxy, 3-pentoxy, or hexoxy. Similarly, the term "thioalkoxy" refers to a group having the formula -S (alkyl). Finally, the terms "haloalkoxy" and "halothioalkoxy" refer to -O (haloalkyl) and -S (haloalkyl), respectively. The term "thiohydrothio" refers to -SH. As used herein, the term "hydroxyl," used alone or in combination with other terms, refers to a group having the formula -OH. Any alkoxy, thioalkoxy, or haloalkoxy groups mentioned herein (e.g., R, R', R'', R...) N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, and R7 can have 1-35 carbon atoms. In other embodiments, the alkoxy, thioalkoxy, or haloalkoxy groups have 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The alkoxy group can be a lower alkoxy group (e.g., C1-C4 alkoxy).

[0071] An aralkyl group generally refers to a group in which the alkyl hydrogen atom in the alkyl moiety is replaced by an aryl group. One of the carbon atoms in the alkyl moiety acts as the attachment point between the aralkyl group and the other moiety. Any ring or chain atom may optionally be substituted, for example, by one or more substituents. Non-limiting examples of aralkyl groups include benzyl, 2-phenylethyl, and 3-phenylpropyl groups.

[0072] The term alkenyl can refer to a straight-chain or branched hydrocarbon chain containing an indicated number of carbon atoms and having one or more carbon-carbon double bonds. Any atom may optionally be substituted, for example, by one or more substituents. Alkenyl groups may include, for example, vinyl, allyl, 1-butenyl, and 2-hexenyl. One of the double-bonded carbons may optionally be the attachment point for the alkenyl substituent. Any alkenyl group mentioned herein (e.g., R, R', R'', R...) N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7) can have 1-35 carbon atoms. In other embodiments, the alkenyl group has 1-20, 1-12, 1-8, 1-6, or 1-3 carbon atoms, including embodiments having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

[0073] The term alkynyl can refer to a straight-chain or branched hydrocarbon chain containing an indicated number of carbon atoms and having one or more carbon-carbon triple bonds. Alynyl groups (e.g., R, R', R'', R...) N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2c R3, R4, R5, R6, R7) may optionally be substituted by one or more substituents. The alkynyl group may include, for example, ethynyl, propynyl, and 3-hexynyl. One of the carbon atoms in the triple bond may optionally be the attachment site of the alkynyl substituent.

[0074] The term heterocyclic group generally refers to a fully saturated, partially saturated, or aromatic monocyclic, bicyclic, tricyclic, or other polycyclic ring system that has a ring independently selected from O and N (it should be understood that one or two other groups (e.g., R) may be present). N(This refers to the heteroatom or cyclic carbon atom that satisfies the valence requirement of nitrogen and / or forms a salt), or one or more constituent heteroatom ring atoms of S. The heteroatom or cyclic carbon can be the attachment point of the heterocyclic substituent to another moiety. Any atom may optionally be substituted, for example, by one or more substituents (e.g., heteroatom or substituent X). The heterocyclic group may include, for example, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidino), piperazine, morpholinyl (morpholino), pyrrolinyl, and pyrrolidinyl. For example, the phrase "a heterocyclic ring containing 5-6 ring atoms, wherein 1-2 ring atoms are independently selected from N, NH, N (C1-C6 alkyl), NC (O) (C1-C6 alkyl), O and S; and wherein the heterocyclic ring is optionally substituted by 1-3 independently selected R'', wherein R'' includes (but is not limited to) tetrahydrofuranyl, tetrahydropyranyl, piperidinyl (piperidino), piperazineyl, morpholinyl (morpholino), pyrrolinyl and pyrrolylalkyl.

[0075] The term heterocyclic alkenyl generally refers to a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group having one or more (e.g., 1-4) heteroatom ring atoms independently selected from O, N (it should be understood that one or two additional groups may be present to satisfy the nitrogen valence requirement and / or form a salt), or S. A cyclic carbon (e.g., saturated or unsaturated) or heteroatom can be the attachment point for a heterocyclic alkenyl substituent. Any atom may optionally be substituted, for example, by one or more substituents. Heterocyclic alkenyl groups may include, for example, dihydropyridyl, tetrahydropyridyl, dihydropyranyl, 4,5-dihydrooxazolyl, 4,5-dihydro-1H-imidazolyl, 1,2,5,6-tetrahydropyrimidinyl, and 5,6-dihydro-2H-[1,3]oxazinyl.

[0076] The cycloalkyl group can be a fully saturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. Any atom may optionally be substituted, for example, by one or more substituents. The ring carbon atom acts as the attachment point between the cycloalkyl group and another moiety. The cycloalkyl moiety may include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl (bicyclic [2.2.1]heptyl).

[0077] The cycloalkenyl group can be a partially unsaturated monocyclic, bicyclic, tricyclic, or other polycyclic hydrocarbon group. The cyclic carbon (e.g., saturated or unsaturated) is the attachment point for the cycloalkenyl substituent. Any atom may optionally be substituted, for example, by one or more substituents. The cycloalkenyl moiety may include, for example, cyclohexenyl, cyclohexadienyl, or norbornyl.

[0078] The aryl group is often an aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (> three fused rings) hydrocarbon ring system. One or more ring atoms may optionally be substituted, for example, by one or more substituents. The aryl moiety includes, for example, phenyl and naphthyl groups.

[0079] The heteroaryl group is typically an aromatic monocyclic, bicyclic (two fused rings), tricyclic (three fused rings), or polycyclic (> three fused rings) hydrocarbon group, having one or more heteroatom ring atoms independently selected from O, N (it should be understood that one or two additional groups may be present to satisfy the valence requirement of nitrogen and / or form a salt), or S. One or more ring atoms may optionally be substituted, for example, by one or more substituents. Examples of heteroaryl groups include, but are not limited to, 2H-pyrrole, 3H-indolyl, 4H-quinolinazinyl, acridinel, benzo[b]thiophene, benzo[b]thiazolyl, β-carbolinyl, carbazolyl, coumarinyl, chromenyl, cenolinyl, dibenzo[b,d]furanyl, furazonyl, furyl, imidazole, imidizolyl, indazole, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthidyl, and others. Oxazolyl, perimidinyl, phenanthridine, phenanthrolinyl, phenarsazinyl, phenazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, pteridinyl, purine, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyridinyl, pyrimidinyl, pyrroleyl, quinazolinyl, quinolinyl, quinoxalolinyl, thiadiazolyl, thiaanthryl, thiazolyl, thiophene, triazolyl, and xanthonyl.

[0080] Generally speaking, when the definition of a particular variable includes both hydrogen and non-hydrogen (halogen, alkyl, aryl) possibilities, the term "substituents other than hydrogen" refers to the non-hydrogen possibilities of that particular variable, unless otherwise stated.

[0081] In general, any ranges described herein are within the scope of this invention and should be understood as the disclosed embodiments. Additionally, any half-integer values ​​within that range are also considered. For example, the range 0 to 4 explicitly discloses 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and any subset thereof (e.g., 1 to 2.5).

[0082] The term "substituent" can refer to a group that "substitutes" on a hydrocarbon (e.g., alkyl, haloalkyl, cycloalkyl, heterocyclic, heterocyclic alkenyl, cycloalkenyl, aryl, heteroaryl) group at any atom of that group, typically replacing one or more hydrogen atoms. In one aspect, groups (e.g., R, R', R'', R...) N ,Y,Y',Ω,L1,LC R L R C R1, R2, R 2a R 2b R 2c The substituents on R1, R2, R3, R4, R5, R6, R7 are independently any single or any combination of two or more of the permitted atoms or groups of atoms defined for that substituent. In another aspect, the substituent itself may be substituted by any of the aforementioned substituents. In some embodiments, the indicated substituent is not further substituted. Further, as used herein, the phrase "optionally substituted" means unsubstituted (e.g., substituted by H) or substituted. It should be understood that substitution at a given atom is restricted by valence state. Common substituents include halogens (e.g., F), C... 1-12 Straight-chain or branched alkyl groups, C 2-12 alkenyl, C 2-12 alkynyl group, C 3-12 cycloalkyl, C 6-12 Aryl, C 3-12 heteroaryl, C 3-12 Heterocyclic group, C 1-12 Alkylsulfonyl, nitro, cyano, -COOR, -C(O)NRR', -OR, -SR, -NRR', and oxo, for example, mono-, di-, or tri-substituted with moieties such as trifluoromethoxy, chlorine, bromine, fluorine, methyl, methoxy, pyridyl, furanyl, triazolyl, piperazine, pyrazolyl, imidazolyl, etc., each substituent optionally containing one or more heteroatoms, such as halogen, N, O, S, and P. R and R' are independently hydrogen, C 1-12 Alkyl, C 1-12 Haloalkyl, C 2-12 alkenyl, C 2-12 alkynyl group, C 3-12 cycloalkyl, C 4-24 cycloalkylalkyl, C 6-12 Aryl, C 7-24 Aryl alkyl, C 3-12 Heterocyclic group, C 3-24 Heterocyclic alkyl, C 3-12 heteroaryl, or C 4-24Heteroalkyl. Unless otherwise stated, all groups described herein optionally contain one or more common substituents, to the extent permitted by valence state. The term “substitution” generally means that a hydrogen atom and / or a carbon atom is removed and replaced by a substituent (e.g., a common substituent). The use of substituent (group) prefixes without the modifiers “optionally substituted” or “substituted”, such as alkyl, should be understood to mean that the particular substituent is unsubstituted. However, the use of “haloalkyl” without the modifiers “optionally substituted” or “substituted” should still be understood to mean an alkyl group in which at least one hydrogen atom is replaced by a halogen and any other relevant substitution if necessary. Any hydrocarbon described herein may be considered optionally substituted.

[0083] The present invention provides methods for diagnosing or predicting neurodegenerative diseases in subjects, identifying subjects at risk of neurodegenerative diseases, or prescribing treatment plans or predicting treatment benefits for subjects with neurodegenerative diseases.

[0084] In some embodiments, the method includes using the assays of this disclosure to determine whether a subject is at risk of or suffers from a neurological disease or condition based on a comparison of NfL levels in a second sample with NfL levels in a first sample [e.g., and a comparison of NfL levels in a third sample with NfL levels in the second sample (e.g., and optionally also with NfL levels in the first sample)] [e.g., compared to changes in NfL levels in a control subject over a similar time period (e.g., at a similar age (e.g., within three years)). In some embodiments, the method includes determining whether a subject is at risk of or suffers from a neurological disease or condition based on NfL levels in the second sample exceeding NfL levels in the first sample. In some embodiments, determining whether a subject is at risk of or suffers from a neurological disease or condition is further based on the subject's age. In some embodiments, determining whether a subject is at risk of or suffers from a neurological disease or condition is based on a threshold amount by which NfL levels in the second sample exceed NfL levels in the first sample. In some embodiments, determining that a subject is at risk of or has a neurological disorder is based on the NfL level in the second sample exceeding the NfL level in the first sample by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, or at least 1000% (e.g., said amount is a threshold amount), and said amount is determined using a assay of the present disclosure. In embodiments, the assay of the present disclosure is an IVD assay.

[0085] As provided herein, NfL includes gene products associated with NfL. For example, NfL may include, for example, proteins or nucleotides (e.g., RNA, such as mRNA). NfL also encompasses full-length proteins as well as fragments of NfL (e.g., characteristic fragments). In some embodiments, NfL includes fragments having an amino acid sequence having an adjacent span identical to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids of the amino acid sequences provided in Table 1. In some embodiments, NfL includes fragments having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences provided in Table 1. In some embodiments, NfL comprises a nucleic acid fragment having a nucleic acid sequence having an adjacency span identical to at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleic acids of the nucleic acid sequences provided in Table 2. In some embodiments, NfL comprises a fragment having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequences provided in Table 2. Variants or alternative forms of NfL include, for example, polypeptides encoded by any splice variant of a transcript encoding NfL.

[0086] Biomarkers considered herein also include truncated forms or polypeptide fragments of NfL as described herein. Truncated forms or polypeptide fragments of NfL may include forms with N-terminal deletions or truncations and forms with C-terminal deletions or truncations. Truncated forms or fragments of NfL may include fragments generated by any mechanism, such as, but not limited to, variable translation, exonuclease and / or endonuclease digestion and / or degradation, for example by physical, chemical and / or enzymatic digestion. Without limitation, biomarkers may include truncated forms or fragments of NfL that may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 8%, or at least 99% of the amino acid sequence of the NfL protein.

[0087] In some cases, the fragment is truncated by 1-20 amino acids from the N-terminus and / or C-terminus compared to the corresponding mature full-length NfL protein, for example, 1-15 amino acids, 1-10 amino acids, or 1-5 amino acids.

[0088] The NfL protein disclosed herein, such as the NfL protein or fragment thereof, may also encompass modified forms of NfL, such as those carrying post-expression modifications including but not limited to phosphorylation, glycosylation, lipidation, methylation, selenocysteine ​​modification, cysteine ​​modification, sulfonation, glutathioneization, acetylation, and / or oxidation of methionine to methionine sulfoxide or methionine sulfone.

[0089] In some implementations, NfL can be a nucleotide (also referred to herein as a nucleic acid or polynucleotide). In some implementations, the nucleotide can be RNA or DNA (e.g., cDNA). In some cases, the corresponding RNA or DNA may exhibit better distinguishing ability in diagnosis than the full-length protein.

[0090] Exemplary amino acid sequences of NfL are included in Table 1 below.

[0091] Table 1

[0092] Exemplary nucleic acid sequences for NfL are included in Table 2 below.

[0093] Table 2

[0094] As provided herein, an NfL can be a full-length protein or a fragment thereof, or a functional fragment thereof. In some embodiments, the fragment of an NfL is a characteristic protein fragment. In some embodiments, an NfL is a full-length protein. In some embodiments, for example, an NfL in a sample may include a subset of full-length NfL proteins and a subset of characteristic protein fragments of NfLs.

[0095] In some embodiments, NfL has a wild-type amino acid sequence. In some embodiments, NfL has a variant amino acid sequence, such as including one or more mutated amino acid sequences. In some embodiments, a subset of the NfL protein has a wild-type amino acid sequence, and a subset of the NfL protein has a variant amino acid sequence.

[0096] As provided herein, NfL can be a full-length nucleotide (e.g., DNA, cDNA, or RNA) encoding NfL or a fragment thereof. In some embodiments, the fragment of NfL is a characteristic nucleotide fragment. In some embodiments, NfL is a full-length nucleotide (e.g., DNA, cDNA, or RNA) encoding NfL. In some embodiments, for example, NfL in a sample may include a subset of full-length NfL nucleotides (e.g., DNA, cDNA, or RNA) and a subset of characteristic nucleotide fragments of NfL.

[0097] In some embodiments, NfL has a wild-type nucleic acid sequence. In some embodiments, NfL has a variant nucleic acid sequence, such as including one or more mutated nucleic acid sequences. In some embodiments, a subset of NfL comprises a wild-type nucleic acid sequence encoding NfL, and a subset includes variant nucleic acid sequences encoding NfL.

[0098] In some implementations, the neurodegenerative disease is selected from: Alzheimer's disease (AD), vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Lewy body dementia, tangles-predominant Alzheimer's disease, Pick's disease (PiD), aberrant granulomatosis, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson's-dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease. In various embodiments, the disease is selected from amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), frontotemporal dementia (FTD), Alzheimer's disease (AD), Creutzfeldt-Jakob disease (CJD), spinal muscular atrophy (SMA), Huntington's disease (HD), Parkinson's disease (PD), vascular dementia (VaD), Lewy body dementia (DLB), atypical Parkinson's syndrome (APS), progressive supranuclear palsy (PSP), primary lateral sclerosis (PLS), multiple system atrophy (MSA), fatal familial insomnia (FFI), corticobasal degeneration (CBD), fragile X-related tremor / ataxia syndrome (FXTAS), or hereditary spastic paraplegia (HSP).In some embodiments, the neurological disease, condition, or condition is selected from multiple sclerosis (e.g., relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS)), neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease (PD), Lewy body dementia (DLB), Huntington's disease (HD), multiple system atrophy (MSA), prions, acute neurological injuries (e.g., traumatic brain injury (TBI), stroke, spinal cord injury (SCI), cardiac arrest, hypoxic-ischemic encephalopathy (HIE), neurocritical care monitoring), and autoimmune diseases (e.g., neuromyelitis optica spectrum disorder (NMOSD), autoimmune encephalitis (AE), systemic lupus erythematosus). (SLE) (e.g., with central nervous system (CNS) involvement), primary Sjögren's syndrome (e.g., with CNS involvement), neurological complications (e.g., infectious diseases such as HIV, COVID-19), metabolic diseases such as type 2 diabetes, sleep disorders such as obstructive sleep apnea, substance use disorders such as alcohol use disorder, chronic kidney disease), mental health conditions (e.g., depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD)), brain function and health in certain environments (e.g., space medicine and astronaut health, military and combat-related trauma, environmental and occupational exposure, sports medicine / performance optimization), and age-related brain function and health (e.g., monitoring brain aging, predicting cognitive decline, assessing the impact of lifestyle factors, distinguishing normal aging from disease, assessing all-cause mortality risk). Typically, the disease, condition, or status can be assessed using a concentration cutoff value (or rate of change of concentration) determined based on a Z-score of the population mean. In some embodiments, the disease, condition, or status can be assessed based on a patient-specific baseline, such as a baseline established over a period of time and collected at intervals during risk assessment of the patient. In various implementations, the cutoff value is based on a percentage change relative to a baseline or in the form of a Z-score, which can be adjusted for various patient indicators such as age and body mass index.

[0099] In some embodiments, the present invention enables medical practitioners to diagnose or predict one or more neurodegenerative diseases in a subject. In other embodiments, the present invention enables medical practitioners to rule out or eliminate one or more neurodegenerative diseases as a diagnostic possibility. In still other embodiments, the present invention enables medical practitioners to identify subjects at risk of developing neurodegenerative diseases. In other embodiments, the present invention enables medical practitioners to predict whether a subject will later develop a neurodegenerative disease. In a further embodiment, the present invention enables medical practitioners to prescribe treatment plans or predict treatment benefits for subjects with neurodegenerative diseases.

[0100] The methods of this invention can be used in clinical settings to diagnose or predict neurodegenerative diseases in subjects, identify subjects at risk of neurodegenerative diseases, and / or to prescribe treatment regimens or predict treatment benefits for subjects with neurodegenerative diseases. Typically, the methods involve detecting biomarkers such as NfL and optionally monitoring the progression of that biomarker. In various embodiments, the methods involve correlating the concentrations of one or more biomarkers with population data and / or the rate of change of one of the biomarkers (e.g., concentration differences over one year) to assess disease progression and / or status.

[0101] Biomarker levels are typically determined in biological samples obtained from subjects who are suspected of having (e.g., determined by cognitive tests), have, or are at risk of having a neurodegenerative disease (e.g., Alzheimer's disease). In some embodiments, the biomarkers are neurofilaments (e.g., neurofilament light chains (NfL)), phosphorylated Tau, Aβ1-42, TDP-43, α-synuclein, SOD-1, FUS, FKBP51, IRS-1, phosphorylated IRS-1, CTSD, LAMP1, UBP, HSP70, NSE, NFL, CD9, CD63, CD81, CD171, or combinations thereof.

[0102] This disclosure relates to conjugates for the detection of analytes (e.g., NfL) in highly sensitive immunoassays. These assays can be utilized in point-of-care devices, enabling, for example, users and / or physicians to obtain samples and run the immunoassay device to quantify the analyte in the sample. Typically, these conjugates have a chemiluminescent acridine on a carrier protein conjugated to the carrier protein via a linker (e.g., PEG such as PEG2-PEG). 20 or PEG3PEG3-PEG 10The chemiluminescent acridine monophosphate compound is conjugated with an antibody or antibody fragment (e.g., F(ab)). In some embodiments, the conjugate comprises an excess of the acridine monophosphate moiety relative to the carrier protein (e.g., 2-40, 5-25 moieties). In some embodiments, the first reaction step occurs in a medium containing a buffer. In various implementations, the chemiluminescent acridine monophosphate compound is added in weight excess of the carrier protein (e.g., less than 100X, or less than 50X, or less than 40X, or less than 30X, or 5X to 25X excess).

[0103] In some embodiments, the chemiluminescent acridine onionium is conjugated to the protein by reacting the carrier protein with a compound containing reactive functional groups. For example, the compound may have the following structure: RFG is a reactive functional group used for protein conjugation. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: “ j "and" k "Independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 represents hydrogen, -R, or -X. b -R L -X b -L C -R、-L C -X b (For example, -L1-X) b -Z, -R L -Z、-L C -Z (e.g., -L1-Z), or -R L -L C -R L -Z (e.g., -R) L -L1-R L -Z); R2 and R3 are independently selected from hydrogen, -R, electron-donating groups, and -X each time they appear. c -R L -X c -L C -X c (For example, -L1-X) c-Z; wherein the two adjacent R2 or R3 groups can together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 can contain a bond with an imaging agent such as a fluorophore (e.g., rhodamine); L C divalent C atoms that are optionally substituted (e.g., having 1 to 20 heteroatoms or 1 to 20 substituents). 1-35 Alkyl, alkenyl, ynyl, aryl, or aralkyl groups; Z L It is a zwitterion junction group with the following structure: “ m "It is 0 (i.e., it is a key) or 1; “ n "and" p "Each occurrence is an independent integer from 0 (i.e., it is a key) to 10; Z represents a zwitterionic group that independently possesses the following structure each time it appears: “ q "and" l Independently 0 or 1; “ r "Independent integers from 0 to 10 (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; X c It is a protonated anionic group; L1 is independently -O-, -S-, -NH-, -N(R) each time it appears. N )-、-(CH2) 1-10 -、-S(=O) 1-2 -、-‍C=C-、-C=C-(CH2) 1-3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1-4 -、-(CH2) 1-4 -C(O)-, -‍C(O)-‍O-, -C(O)-N(R N -, -C(O)-NH-, -N(R) N )-C(O)-, -NH-C(O)-, -‍C(O)-‍N(R N )-‍(CH2) 1-3 -、-(CH2)1-3 -C(O)-N(R N )-、-(CH2) 1-3 -N(R N )-C(O)-、-NH-S(O) 1- ‍2-‍、-N(R N )-S(O) 1-2 -、-‍S(O) 1-2 -N(R N )-、-S(O) 1-2 -NH-, -(CH2) 1-3 -NH-S(O) 1-2 -‍、-(CH2) 1-3 -N(R N )-S(O) 1-2 -、-‍(CH2) 1-3 -S(O) 1-2 -N(R N )-、-(CH2) 1-3 -S(O) 1-2 -NH-, -O- (CH2) 1- ‍4-、-(CH2) 1-4 -O-、-S-(CH2) 1-4 -、-(CH2) 1-4 -S-, -NH-(CH2) 1-4 -、-‍N(R N )-‍(CH2) 1- ‍4-、-(CH2) 1-4 -N(R N )-、-‍(OCH2) 1-10 -、-(CH2O) 1-10 -、-(OCH2CH2) 1-10 -、or-‍(CH2CH2O) 1-10 -; R L Each time it appears, it is independently for C that optionally has one or more (e.g., 1-10, 1-5) substitution sites (e.g., 1-10 heteroatoms, 1-10 substituents). 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, aralkyl); R is independently hydrogen each time it appears, or optionally has one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., 1-20 heteroatoms, 1-20 substituents) of C. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, ynyl, or aralkyl) groups; R' and R'' are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, and carboxylates such as haloalkylcarboxylates and fluoroalkylcarboxylates). For example, chemiluminescent acridineonium containing reactive functional groups has the structure of formula (Ia): Where Ω represents O or N; Y is selected from -R or -R L -Z, or where Ω is 0, then Y does not exist; and Y' does not exist (i.e., Y' does not exist) ， It is a key, or selected from L1-, -R L -、-R L -L1-, -L1-L1-, -L1-R L -、-L1-R L -L1 and -R L -L1-R L - In some embodiments, the chemiluminescent acridine trioxide containing reactive functional groups has a structure of formula (Ia) or (Ib): R4-R7 are independently hydrogen, an electron-donating group, or C. 1-35 Alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and Y'' does not exist (i.e., it is a key), or it is -L. C -、-L1-、-R L -or-R L -L1-. In some embodiments, L is absent or is a C1-C5 alkylene group. In specific embodiments, the reactive functional group is an N-succinimide ester. In various implementations, at least one of R2 and R3 (e.g., at the C2 and / or C7 position of acridine) is independently -X. c -R L -X c -L C -X c (For example, -L1-X) cIn some embodiments, at least one of R2 and R3 is independently composed of -C(O)OH, -SO2OH, -OSO2OH, or -OP(O)(OR). P Alkoxy groups substituted with OH, -OH, or combinations thereof (e.g., C1-C4 alkoxy groups).

[0104] When a portion of the compounds disclosed herein is described in the context of molecular conjugates as comprising an analyte or its binding partner, a covalent bond can be formed between the analyte or its binding partner (e.g., using reactive functional groups that form covalent bonds) and the remaining portion of the conjugate. In such conjugates, the analyte or its binding partner may have hydrogen on the unconjugated analyte or its binding partner, said hydrogen forming a covalent bond with the indicator portion. The covalent bond on the analyte or its binding partner can be formed, for example, at a group on the analyte, its binding partner, or a derivative form of the analyte containing a group for forming the bond. The group can be, for example, an amine group, a thiol group, a carboxyl group, a maleimide group, or a carbohydrate group. For example, if a covalent bond is formed by a primary amine of the analyte or its binding partner, the compound can have the following structure: Where L is the linker (e.g., PEG), CP is the carrier protein, Ψ is acridine onionium, and the unconjugated analyte or binding partner A (e.g., antibody or antibody fragment) has the structure A'-NH2. Similarly, the conjugation of the linker to the carrier protein can also be operated similarly, such that the conjugate can have the following structure: Wherein L is independently a linker (e.g., PEG), Ψ is acridinium, A is an analyte or its binding conjugate (e.g., an antibody or antibody fragment), and the carrier protein has the structure H2N-CP' or H2N-CP''-NH2, and Ψ' is an acridinium ester Ψ having an amine-reactive functional group that reacts with an amine on the carrier protein. In some embodiments, the conjugate comprises an excess of the acridinium ester (e.g., 2 to 50) compared to the carrier protein.

[0105] In some embodiments, any hydrocarbon or substituted hydrocarbon disclosed herein (e.g., R, R', R'', R) N ,Y,Y',Ω,L1,L C R L R C R1, R2, R 2a R 2b R 2cR3, R4, R5, R6, R7 can be substituted by one or more (e.g., 1-6, 1-4, 1-3, one, two, or three) substituents X, wherein X is independently selected each time it appears from one or more (e.g., 1-20) heteroatoms or one or more (e.g., 1-10) groups containing heteroatoms, or X is independently selected each time it appears from -F, -Cl, -Br, -I, -OH, -OR*, -NH2, -NHR*, -N(R*)2, -N(R*)3 + , -N(R*)-OH, -N(→O)(R*)2, -ON(R*)2, -N(R*)-OR*, -N(R*)-N(R*)2, -C=NR*, -N=C(R*)2, -C=NN(R*)2, -C(=NR*)(-N (R*)2), -C(H)(=N-OH), -SH, -SR*, -CN, -NC, -CHF2, -CCl3, -CF2Cl, -CFCl2, -C(=O)-R*, -CHO, -CO2H, -C(O)CH3, -CO2 - , -CO2R*, -C(=O)-SR*, -O-(C=O)-H, -O-(C=O)-R*, -SC(=O)-R*, -(C=O)-NH2, - C(=O)-N(R*)2, -C(=O)-NHNH2, -OC(=O)-NHNH2, -C(=S)-NH2, -(C=S)-N(R*)2, -N(R*)-CHO, -N(R*)-C(=O)-R*, -C(=NR)-OR*, -OC(=NR*)-R*, -SCN, -NCS, -NSO, -SSR*, -N(R*)-C(=O)-N(R*)2, -CH3, -CH2-CH3, -CH2-CH2-CH3, -C(H)(CH2) 2、 -C(CH3)3, -N(R*)-C(=S)-N(R*)2, -S(=O) 1-2 -R*, -OS(=O)2-R*, -S(=O)2-OR*, -N(R*)-S(=O)2-R*, -S(=O)2-N(R*)2, -O-SO3, -OS(=O)2-OR*, -OS(=O)-OR*, -OS(=O)-R*, -S(= O)-OR*, -S(=O)-R*, -NO, -NO2, -NO3, -O-NO, -O-NO2, -N3, -N2-R*, -N(C2H4), -Si(R*)3, -CF3, -O-CF3, -O-CHF2, -O-CH3, -O-(CH2) 1-6CH3, -OC(H)(CH2)2-OC(CH3)3, -PR*2, -OP(=O)(OR*)2, or -P (=O)(OR*)2; where R* can be H or C independently each time it appears. 1-10 Or C 1-8 Or C 1-6 Or C 1-4 Hydrocarbons, including but not limited to alkyl, alkenyl, alkynyl, aryl (e.g., phenyl), alkyl-aryl (e.g., benzyl), and aryl-alkyl (e.g., tolyl). In some embodiments, X may comprise a C1-C8, C1-C6, or C2-C4 perfluoroalkyl group. In some embodiments, X may be a C1-C8, C2-C6, or C3-C5 heterocycle (e.g., heteroaryl group). The term "halogenated" or "halogen" refers to any group of fluorine, chlorine, bromine, or iodine. In some embodiments, X is independently selected each time it appears from -OH, -SH, -NH2, -N(R*)2, -C(O)OR*, -C(O)NR*R*, -C(O)NR*R*, -C(O)OH, -C(O)NH2, F, or -Cl. In some embodiments, X is F. R and R* may be independently saturated or unsaturated alkyl groups (e.g., C1-C8 alkyl groups) each time they appear. In some embodiments, R and R* are independently selected from hydrogen, methyl, ethyl, propyl, or isopropyl. In some embodiments, R and R* are independently selected from hydrogen, methoxy, ethoxy, propoxy, or isopropoxy. In some embodiments, X is -CF3 or -O-CF3.

[0106] L C It can have the following structure: Where X1 is selected from =N-, -O-, -S-, or -NR. N -; X2-X4 are independently selected from -O-, -S-, and -NR. N -、-C(O)-、-NR N -C(O)-、-C(O)-NR N -、-OC(O)-、or -C(O)-O-、-SC(O)-、or -C(O)-S-; and R L Each time it appears, it is independently selected from -CH2-, -(CH2CH2O)-, or -‍(OCH2CH2)-. In each embodiment, L C Included between A and Ψ (or between A and Z) L At least one atom (or at least two atoms) in a chain between (between).

[0107] For example, anionic groups such as X aX b X c Each time it appears, it can independently be a carboxylate (-C(O)O) group. - ), sulfonate (-SO3) - ), sulfate (-OSO3) - ), phosphate (-OP (O)(OR) P )O - ), or oxygen anion (-O) - ), and R P C is hydrogen or optionally has one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents). 1-12 Hydrocarbons. The protonated forms of these groups (which can be protonated or in salt form to satisfy the charge neutrality requirement of the compound) include -C(O)OH, -SO2OH, -OSO2OH, -OP(O)(OR)OH. P )OH or -OH, and R P C is hydrogen or optionally has one or more (e.g., 1-10, 1-5) substitution sites (e.g., having 1-10 heteroatoms, having 1-10 substituents). 1-12 Hydrocarbons. For example, R1 may contain (or be) -R L -SO3 - (e.g., sulfopropyl). In some embodiments, R1 comprises (or is) sulfopropyl. In some embodiments, R1, R2, and / or R3 comprise (or are) sulfopropyl (which may be in zwitterionic form (e.g., R1) or neutral form (e.g., R2, R3)). In some embodiments, R1 is -S(O)2-NH-Z or -(CH2). 1-3 -S(O)2-NH-Z. In various realizations, R2 and R3 are independently hydrogen, alkyl, or optionally -C(O)OH, -SO2OH, -OSO2OH, -OP(O)(OR) each time they appear. P OH or -OH substituted alkoxy groups (e.g., lower alkoxy groups such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, R2 and R3 are each hydrogen. In other embodiments, one of R2 or R3 is hydrogen, and the other of R2 or R3 is an alkoxy group (e.g., lower alkoxy groups such as C1-C4 alkoxy, methoxy, ethoxy, propoxy, isopropoxy). In some embodiments, X a X b or X c Sulfonate (-SO3) - ), m R is 1 L It is propyl, and n andp Each is 3. For example, Z L It can have the following structure: .

[0108] Compounds can be used to detect the presence of materials in a sample, such as analytes (e.g., biomolecules). In some embodiments, the analyte is a neurofilament light chain (e.g., serum neurofilament light chains). In some embodiments, the biological sample is blood, and the analyte must cross the blood-brain barrier to enter the bloodstream (e.g., the analyte is a neurofilament light chain). In these embodiments, the high sensitivity provided by the assay even allows for the use of analytes at low blood concentration levels.

[0109] In some embodiments, the compounds of this disclosure may be zwitterionic and include one or more zwitterionic groups. For example, the R1 group attached to the positively charged nitrogen acridine nitrogen may optionally be substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, Br, F), and thus may combine with the positively charged acridine nitrogen atom to form a zwitterionic group. For example, the sulfopropyl or sulfobutyl group attached to the acridine nitrogen may form a zwitterionic pair. The R1 group may also be neutral (e.g., a lower alkyl group such as methyl) or zwitterionic on its own (e.g., R1 is -Z, -R). L -Z, -L8-Z, or -R L -L8-R M -Z. In some implementations, R1 has the following structure: .

[0110] When the acridineonium label is charged (e.g., R1 has a net neutral charge), the compound can be in its salt form and optionally include a counterion to balance the positively charged nitrogen on the acridineonium nucleus. The counterion can be selected from CH3SO4. - FSO3 - CF3SO4 - C4F9SO4 - CH3C6H4SO3 - Halogen anions (e.g., Cl-) - F - ,Br - ), CF3COO - CH3COO - Or NO3 - In some embodiments, R1 is methyl, ethyl, propyl, or isopropyl. In some embodiments, the acridine compound can become zwitterionic via covalent attachment to an anion. For example, R1 may contain -R L -X a And -Xa Sulfonate (-SO3) - In some implementations, R1 is -R. L -X a And -X a Sulfonate (-SO3) - In some implementations, R1 is -R. L -X or -L8-Z. In some implementations, L8 is -S(O)2-NH- or -(CH2). 1-3 -S(O)2-NH-. R1 can contain a sulfopropyl group (-(CH2)3-SO3). - In one specific implementation, R1 is sulfopropyl.

[0111] Substituents on chemiluminescent acridine esters can be modified to alter the rate and yield of light emission, thereby reducing nonspecific binding, increasing stability, or enhancing hydrophilicity. Typically, these modifications have minimal interference with the binding of the analyte and its binding partner. Examples of substituent variability are disclosed in U.S. Patent No. 7,309,615 (incorporated herein by reference), which describes high-quantum-yield acridine compounds containing, for example, an electron-donating group such as an alkoxy group (OR*) at C2 and / or C7, wherein R* is a moiety comprising a sulfopropyl or ethylene glycol moiety (e.g., -O(CH2CH2O)). 0-5 CH3) or a combination thereof. In some embodiments, R2 (e.g., R 2a R 2b R 2c R3 and / or R3 can be independently hydrogen, an electron-donating group such as an alkoxy group (e.g., OR such as -O(CH2CH2O)) each time they appear. 0-5 CH3 and / or OR*) or the OG group. Natrajan et al. also described hydrophilic chemiluminescent acridine esters with high quantum yields in International Publication No. WO2015 / 006174 (which is incorporated herein by reference in its entirety). These esters also possess certain electron-donating functional groups, such as those at the C2 and / or C7 positions. These electron-donating groups (-OG) can have the following structures: Among them, R9-R 14 Each time it appears, it is independently selected from a methyl group or the -(CH2CH2O) group. a CH3, where a is an integer from 1 to 5. G can be independently selected each time it appears, for example, from hydrogen, alkyl (e.g., C1-C4 alkyl), or -(CH2CH2O). 1-10-OCH3, for example -(CH2CH2O)2-OCH3 or -(CH2CH2O)5-OCH3.

[0112] Ψ can be included on the two flanking methyl groups of a phenolic ester to stabilize the bond, as Law et al. Journal of Bioluminescence and Chemiluminescence The references disclosed in 4:88-89 (1989) are incorporated herein by reference in their entirety. In some embodiments, the Ψ in the conjugate has the following structure: .

[0113] The reactive functional group can be an amine reactive group, a thiol reactive group, a carboxyl reactive group, a maleimide reactive group, or a carbohydrate reactive group. In some embodiments, the reactive functional group can react with the functional group of the analyte or its binding partner, such as a primary amine. The reactive functional group can comprise (or be) isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imide esters, carbodiimides, acid anhydrides, fluorophenyl esters, or combinations thereof. In various implementations, the reactive functional group labels the analyte or its binding partner by acylation or alkylation. For example, the bond can be formed by reactive functional groups (RFG) selected from: .

[0114] In some embodiments, the compound comprises a linker group having the following structures: -NH-C(O)- or -C(O)-NH-, -C(O)O- or -OC(O)-. In a preferred embodiment, the compound or a portion thereof (e.g., L...) C Ψ) contains at least one -NH-C(O)-, -C(O)-NH-, -C(O)O- or -OC(O)- linker group.

[0115] The covalent bond between RFG and Ψ (e.g., L) may contain (or be) a divalent C optionally substituted with up to 20 heteroatoms (e.g., N, O, S, P, Cl, F, Br). 1-20 Alkyl, alkenyl, alkynyl, aryl, or aralkyl groups. In some embodiments, L comprises a zwitterion connector. L may have the structure -L C -(Z L ) z -, where z is 0 or 1. L C It can have a structure: X1 is selected from -O-, -S-, and -NR. N -、-C(O)-、-NR N -C(O)-、-C(O)-NR N -, -OC(O)-, or -C(O)-O-, -SC(O)-, or -C(O)-S-, =N-, -O-, or -S-; X2-X4 are independently selected from -O-, -S-, and -NR. N -、-C(O)-、-NR N -C(O)-、-C(O)-NR N -、-OC(O)-、or -C(O)-O-、-SC(O)-、or -C(O)-S-; and R L Each time it appears, it is independently selected from -CH2-, -(CH2CH2O)-, or -(OCH2CH2)-; for example, where the premise is L C Included between A and Ψ (or between A and Z) L At least one atom (or at least two atoms) in a chain between (between).

[0116] In some embodiments, L and / or Ψ comprise -C(O)-NH-. In some embodiments, L C It has the following structure: .

[0117] The detectable label may contain a dimethyl acridine ester (DMAE) moiety and a zwitterion (which contains a zwitterion or a polyethylene glycol-derived connector) to improve the properties of the compound. When Ψ contains a zwitterion, a polyethylene glycol-derived connector, or dimethylphenyl ester, properties such as nonspecific binding, hydrophilicity, or compound stability can be improved. In some embodiments, Z L It has the following structure: .

[0118] In some embodiments, R' is hydrogen or a lower alkyl group (e.g., methyl, ethyl, propyl).

[0119] Chemiluminescence from multiple acridine esters can be measured using photomultiplier tubes (PMTs), each equipped with a filter that allows light from the acridine ester of interest to pass through while blocking unwanted light. An alternative detector is a charge-coupled device (CCD). Chemiluminescence can pass through a grating, allowing wavelength separation to occur along the detector (e.g., a CCD detector) and the image to be analyzed accordingly.

[0120] The acridine-onium markers in U.S. Patent No. 8,119,422 can be used as materials for forming a set of acridine-onium markers for wavelength separation and emission separation, the entire U.S. Patent being incorporated herein by reference.

[0121] The compounds disclosed herein can be characterized by their stability. Acridineonium labeling used in the assays disclosed herein, such as rapid acridineonium labeling and / or slow acridineonium labeling, can be characterized as stable. For example, a compound or conjugate can be considered stable if it exhibits a minimal loss of chemiluminescent activity, as measured by the loss of relative optical units (“RLU”), when stored in aqueous solutions typically in the pH range of 6–9. Compounds with increased instability may exhibit a greater loss of chemiluminescent activity compared to another compound. For example, the compounds disclosed herein can be characterized as having increased stability over 33 days at pH 6 and / or 7 and / or 8, at 4°C (common reagent storage temperature) and / or 37°C (acceleration temperature). In some embodiments, the compound can be characterized as having a change in chemiluminescent activity of less than (or 1% to) 40% (e.g., less than 30%, less than 20%, 10% to 40%, 10% to 30%, 10% to 20%) after being stored at 37°C and pH 7 and / or pH 8 for 33 days.

[0122] Compounds can be prepared using standard synthetic methods (other than those described herein) from commercially available raw materials, compounds known in the literature, or readily prepared intermediates. Standard synthetic methods and procedures for the preparation of organic molecules, as well as functional group transformations and manipulations, are readily available from relevant scientific literature or standard textbooks in the field. It should be understood that other process conditions may also be used given typical or preferred process conditions (e.g., reaction temperature, time, molar ratio of reactants, solvent, pressure), unless otherwise stated. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art through conventional optimization procedures. Those skilled in the art of organic synthesis will recognize that the nature and order of the presented synthetic steps can be modified for the purpose of optimizing the formation of the compounds described herein.

[0123] Synthetic chemical transformations (including protecting group methods) that can be used to synthesize the compounds described herein are known in the art and include, for example, those described below: RC Larock, Comprehensive Organic Transformations 2nd edition, Wiley-VCH Publishers (1999); PGM Wuts and TW Greene, Protective Groups in Organic Synthesis4th edition, John Wiley and Sons (2007); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis John Wiley and Sons (1994); and L. Paquette, eds. Encyclopedia of Reagents for Organic Synthesis John Wiley and Sons (1995), and subsequent editions thereof, each of which is incorporated herein by reference in its entirety.

[0124] The process described herein can be monitored using any suitable method known in the art. For example, product formation can be monitored using spectroscopic methods, such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C) Infrared spectroscopy (FT-IR), spectrophotometry (e.g., UV-Vis), or mass spectrometry (MS), or chromatography such as high-performance liquid chromatography (HPLC) or thin-layer chromatography (TLC).

[0125] The preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by those skilled in the art. The chemistry of protecting groups can be, for example, as described by Greene et al. Protective Groups in Organic Synthesis The reference is found in the 2nd edition, Wiley & Sons, 1991, and is incorporated herein by reference in its entirety.

[0126] The reactions described herein can be carried out in a suitable solvent, which can be readily selected by those skilled in the art of organic synthesis. At the temperature under which the reaction takes place (i.e., a temperature ranging from the freezing point to the boiling point of the solvent), the suitable solvent can be substantially non-reactive with the raw materials (reactants), intermediates, or products. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the specific reaction step, a suitable solvent can be selected for that particular reaction step.

[0127] The resolution of racemic mixtures of compounds can be performed using any of the numerous methods known in the art. For example, the absolute configuration of stereoisomers can be determined using 1D and 2D NMR techniques such as COSY, NOESY, HMBC, and HSQC. Specific implementations of these NMR techniques can be found in Hauptmann, H. et al. Bioconjugate Chem . 11 (2000):239-252 or Bowler, J. SteroidsThe references are found in 54 / 1 (1989): 71-99, each of which is incorporated herein by reference in its entirety. Another example method involves the preparation of Mosher esters or amides of the respective alcohols or amines. The absolute configuration of the ester or amide is then determined by proton and / or... 19 The determination is made by F NMR spectroscopy. An example method includes fractional recrystallization using a "chiral resolving acid," which is an optically active salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids such as D- and L-type tartaric acid, diacetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be performed by elution on a column packed with an optically active resolving agent (e.g., N-(3,5-dinitrobenzoyl)phenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0128] Generally, as described in U.S. Patent Nos. 6,664,043, 7,309,615, 9,575,062, or 9,487,480 to Natrajan et al., each of which is incorporated herein by reference in its entirety, and particularly with respect to the zwitterionic acridine esters described therein and their synthesis, zwitterionic acridine esters (“ZAE”) containing reactive functional groups for forming covalent bonds can be used to synthesize the compounds disclosed herein. For example, zwitterionic acridine ester raw materials may include an N-sulfopropyl (“NSP”) group in the zwitterionic moiety and / or include a charged nitrogen atom (“DIZAE”) attached to a charged acridineonium nucleus and / or include a sterically stable dimethyl acridine ester (“DMAE”) and / or include an isopropoxy-functionalized acridineonium nucleus (“ISO”) and / or include a zwitterionic (“Z”) and / or a hexa(ethylene) glycol-derived (“HEG”) and / or a glutarate-derived (e.g., -C(O)-(CH2)3-C(O)-) linker between the acridine ester and the reactive functional group. The reactive functional group may be NH2 or N-hydroxysuccinimide ester (“NHS”). For example, compounds (e.g., compounds for conjugation with analytes or analyte coupling pairs such as peptides, proteins, or macromolecules including antibodies) may have the structure of formula (IV): RFG is a reactive functional group used for conjugation with the analyte or its binding partner. L does not exist (i.e., it is a key) or is a connector, and Ψ represents a chemiluminescent acridine. The chemiluminescent conjugate or the compound used to form it can also be synthesized using acridine sulfonamide reactants. For example, the acridine sulfonamide disclosed in U.S. Patent No. 5,543,524 to Mattingly et al. (which is incorporated herein by reference in its entirety) is a useful starting material for preparing the chemiluminescent compounds disclosed herein.

[0129] The particles in the solid phase and how they are prepared during the assay process can be important for achieving the desired assay resolution. The particles may have an average diameter of at least about 0.02 micrometers and no more than about 100 micrometers. In some embodiments, the particles have an average diameter of about 0.05 micrometers to about 20 micrometers, or about 0.3 micrometers to about 10 micrometers. The particles may be organic or inorganic, expandable or non-expandable, and porous or non-porous. In a particular (but non-limiting) embodiment, the particles have a density close to that of water, generally about 0.7 g / mL to about 1.5 g / mL, and are composed of materials that may be transparent, partially transparent, or opaque. The particles may be composed of organic and inorganic polymers, latex particles, magnetic particles, or non-magnetic particles, etc. In some non-limiting examples, the particles are chromium particles or latex particles.

[0130] Polymer particles can be formed from addition polymers or condensation polymers. Particles can also be derived from naturally occurring materials, synthetically modified naturally occurring materials, and synthetic materials. Of particular interest are organic polymers containing polysaccharides, especially cross-linked polysaccharides, such as (but not limited to) agarose, which is available as Sepharose; dextran, which is available as Sephadex and Sephacryl; cellulose; starch; etc.; addition polymers, such as homopolymers and copolymers of polystyrene, polyvinyl alcohol, acrylates, and methacrylate derivatives, especially (but not limited to) esters and amides having free hydroxyl functional groups, etc.

[0131] The particles are typically readily dispersible in an aqueous medium and can be adsorbent or functionalized to allow direct or indirect conjugation to a monoclonal antibody (or fragment thereof) via a linker group. When a linker group is used (e.g., within the particle or between the antibody fragment and the carrier protein), in some non-limiting embodiments, the linker group may comprise about 2 to about 50 atoms, or 4 to about 30 atoms, excluding hydrogen atoms, and may comprise a chain of 2 to about 30 atoms, or 3 to about 20 atoms, each of which is independently selected from the group typically consisting of carbon, oxygen, sulfur, nitrogen, and phosphorus. In some instances, the linker group contains an oxime functional group.

[0132] The number of heteroatoms in the linking group can range from 0 to about 20, or 1 to about 15, or about 2 to about 10. The linking group can be aliphatic or aromatic. When heteroatoms are present, oxygen is usually present as an oxo or oxygen group bonded to carbon, sulfur, nitrogen, or phosphorus; nitrogen is usually present as a nitro, nitroso, or amino group bonded to carbon, oxygen, sulfur, or phosphorus; sulfur is similar to oxygen; and phosphorus is usually present as a phosphonate and a mono- or diester of phosphate, bonded to carbon, sulfur, oxygen, or nitrogen. Common functional groups that form covalent bonds between the linking group and the molecule to be conjugated are alkylamines, amidines, thioamides, ethers, ureas, thioureas, guanidines, azo compounds, thioethers, as well as carboxylic acid esters, sulfonates, and phosphate esters, amides, and thioesters.

[0133] In most cases, when the linking group has a linking functional group (a functional group that partially reacts with a crosslinking functional group), such as a nonoxocarbonyl group including nitrogen and sulfur analogs, a phosphate group, an amino group, an alkylating agent such as a halogenated or p-toluenesulfonylalkyl group, an oxygen group (hydroxyl or sulfur analogs, mercapto), an oxocarbonyl group (e.g., an aldehyde or ketone), or an active olefin (e.g., a vinyl sulfone or an α-, β-unsaturated ester), these functional groups are linked to an amine group, a carboxyl group, an active olefin, or an alkylating agent (e.g., a bromoacetyl group). When an amine is linked to a carboxylic acid or its nitrogen derivative or a phosphate, amides, amidines, and phosphoramides are formed. When a thiol is linked to an active olefin, a thioether is formed. When a thiol is linked to an alkylating agent, a thioether is formed. When an aldehyde and an amine are linked under reducing conditions, an alkylamine is formed. When a ketone or aldehyde is linked to a hydroxylamine (including its derivatives in which a substituent replaces the hydrogen of a hydroxyl group), an oxime functional group (=NO-) is formed. When a carboxylic acid or phosphate is linked to an alcohol, an ester is formed. Various linking groups are provided, for example, in Cautrecasas, J. Biol. Chem. (1970): 245:3059, which is incorporated herein by reference in its entirety, and particularly in the portion relating to linking groups.

[0134] Various forms of immunoassays, including, for example, competitive and non-competitive immunoassays, antigen / analyte capture assays, and double-antibody sandwich assays, can be used according to the reagent cartridges, kits, and methods described herein. For example, an assay can be a competitive immunoassay, which typically involves the detection of macromolecules, also known as macromolecular analytes, using binding molecules such as antibodies. Antibodies are immobilized or attached to a solid phase, such as particles, beads, membranes, microtiter plates, or any other solid surface.

[0135] In examples of competitive heterogeneous assays, a support having an antibody against an analyte (e.g., bovine monoclonal antibody, mouse monoclonal antibody, antibody fragment such as bovine antibody fragment, mouse antibody fragment) bound to it is contacted with a medium containing a sample suspected of containing the analyte and the chemiluminescent conjugate (or “labeled analog”) described herein. The analyte from the sample can compete with the labeled analog for binding to the analyte antibody. After separation of the support and the medium, the labeling activity of the support or medium is determined using conventional techniques and is related to the amount of analyte in the sample. In variations of the competitive heterogeneous assay described above, the support contains an analyte analog that competes with the analyte in the sample for binding to the antibody reagent, according to the principles described herein. The labeled analyte analog can be covalently attached to a chemiluminescent or fluorescent molecule, often referred to as a label or tracer.

[0136] When a solid phase containing immobilized antibodies is mixed with a sample containing analytes and labeled analytes, binding complexes typically form between the analytes or labeled analytes. Because a solid phase is involved, this type of assay is often referred to as a heterogeneous assay. The chemiluminescent signal associated with the binding complex can then be measured, and the presence or absence of the analyte in the sample can be inferred. Typically, the binding complex is separated from the remainder of the binding reaction components, such as excess labeled analyte, before signal generation. For example, if the binding complex is bound to magnetic beads, a magnet can be used to separate the bead-bound binding complex from the bulk solution.

[0137] In examples employing a sandwich assay using two antibodies (or fragments thereof), a solid support having an immobilized primary antibody or fragment thereof against an analyte is mixed with a sample containing the analyte and a labeled conjugate containing a secondary antibody or fragment thereof. A binding complex is formed between the solid particles and the labeled conjugate via the analyte in the sample. A signal associated with the binding complex can be measured, and the presence or absence, or amount, of the analyte can be inferred. Typically, the binding complex is separated from the remainder of the binding reaction components, such as excess labeled analyte, before a signal is generated. For example, if the binding complex is bound to magnetic beads, a magnet can be used to separate the bead-bound binding complex from the bulk solution. In some embodiments, the immobilized primary antibody is a biotinylated mouse monoclonal antibody that binds to coated (e.g., streptoacidin-coated) optionally paramagnetic particles. In some embodiments, the secondary antibody is a fragment of a mouse monoclonal antibody labeled with acridine (e.g., acridine ester).

[0138] By using a series of "standards," i.e., analytes at known concentrations, dose-response curves can be generated for known labeled analytes. These dose-response curves can be identified individually for any acridineonium label, or based on a combination of acridineonium labels used in the assay. Therefore, the dose-response curve correlates a certain amount of measured signal with a specific concentration of analyte. In competitive assays, as the concentration of the analyte increases, the amount of signal decreases if chemiluminescence from the binding complex is measured. The concentration of the analyte in an unknown sample can then be calculated by comparing the signal generated via the unknown sample containing the macromolecular analyte with the dose-response curve.

[0139] Methods for attaching binding molecules, such as antibodies, to a solid phase typically involve mixing the necessary components to induce attachment. For example, antibodies can be covalently attached to particles containing amines on their surface by using cross-linking molecules such as glutaraldehyde. Attachment can also be non-covalent and can involve simple adsorption of the binding molecule onto the surface of a solid phase, such as polystyrene beads or microtiter plates. Labeling of binding molecules, such as antibodies, with other binding proteins is also well known in the art and is commonly referred to as conjugation reactions, with labeled antibodies often called conjugates. Typically, the reactive amine portion of the label reacts with the amine on the antibody to form an amide bond. Other bonds between the antibody and the label can also be used, such as thioethers, esters, carbamates, etc.

[0140] In another aspect of the invention, a reagent for detecting an analyte may be provided, comprising a chemiluminescent acridine compound bound to the analyte or a binding pair. The reagent may comprise 0.1 to 100 ng / mL of the chemiluminescent acridine compound, or 1 to 50 ng / mL of the chemiluminescent acridine compound, or 5 to 30 ng / mL of the chemiluminescent acridine compound. In some embodiments, the compound is provided in a reagent further comprising a buffer solution.

[0141] The determination and methods for measuring the amount of neurofilaments in samples that can be used in this disclosure include those described in U.S. Provisional Patent Application No. 63 / 588,465 (titled “High-Resolution Immunoassay”) and U.S. Patent Publication No. 2022 / 0229073 (titled “Quantification of Neurofilament Light Chains in Physiological Samples”, U.S. Application No. 17 / 046,122), both of which are incorporated herein by reference in their entirety, and particularly with respect to the description of the quantitative determination of neurofilaments.

[0142] In some embodiments, the sample is derived from a mammal (e.g., a human). In some embodiments, the sample comprises saliva and / or blood and / or serum. In some embodiments, the sample is saliva and / or blood and / or serum.

[0143] In some assays, the sample to be analyzed is pretreated to release the analyte from endogenously bound substances (e.g., plasma or serum proteins that bind the analyte). Release of the analyte from endogenously bound substances can be achieved, for example, by adding a digestive agent or a releasing agent, or a combination of digestive and releasing agents used sequentially. The digestive agent is a reagent that breaks down the endogenously bound substances so that they can no longer bind the analyte.

[0144] Conditions for measuring a portion of a sample according to the principles described herein may include measurement in an aqueous buffer medium of moderate pH, typically the pH that provides optimal assay sensitivity. The aqueous medium may be water only or may include 0.1% to 40% by volume a co-solvent. The pH of the medium may range from 4 to 11, or 5 to 10, or 6.5 to 9.5, or 7 to 8. Typically, the pH of the solution is a trade-off between optimal binding of any specific binding pair, optimal pH of other reagents in the assay such as members of the signal generation system, etc. Various buffers can be used to achieve the desired pH and maintain that pH during the assay. Exemplary buffers include, for example, borates, phosphates, carbonates, TRIS, barbiturates, PIPES, HEPES, MES, ACES, MOPS, and BICINE.

[0145] Various auxiliary materials can be used in the assay method. For example, in addition to buffer solutions, the composition, reagents, or reaction media may also contain stabilizers for the media and the reagents used. In some embodiments, the media may contain proteins (e.g., albumin), organic solvents (e.g., formamide), quaternary ammonium salts, polyanionic compounds (e.g., dextran sulfate), binding enhancers (e.g., polyalkylene glycols), polysaccharides (e.g., dextran, trehalose), blocking agents (e.g., blocking antibodies to prevent false positives), and combinations thereof.

[0146] The chemiluminescence of analogues can be triggered by adding a chemiluminescence triggering agent. The chemiluminescence triggering agent can be acidic or basic. Multiple chemiluminescence triggering agents can be added sequentially. For example, an acidic solution can be added first, followed by a basic solution. In some embodiments, the chemiluminescence triggering agent comprises hydrogen peroxide, hydrogen peroxide salts, nitric acid, nitrates, sodium hydroxide, ammonium salts, or combinations thereof.

[0147] The kits according to this disclosure may relate to immunoassay reagent compositions comprising an antibody (or a fragment thereof) conjugated to a carrier protein. The kit may contain auxiliary components such as buffers, blocking reagents, ions such as divalent or monovalent cations, calibration proteins, secondary antibodies, detection reagents such as detection dyes, and any other suitable compounds or liquids required for performing analyte detection. Additionally, the kit may include instructions for use and / or information that can provide relevance to the results obtained (or such information may be obtained, for example, via the server of this disclosure) and / or additional tests that may be prescribed for further risk assessment. In some embodiments, the kit further comprises a solid-phase reagent. In some embodiments, the kit further comprises a chemiluminescent triggering reagent.

[0148] This disclosure relates to the assessment of the risk of neurological diseases in subjects. Users identified as having an increased risk of neurodegenerative diseases (such as Alzheimer's disease) or being in their early stages may receive one or more treatment options. For example, the present invention provides a method of treating a neurodegenerative condition in a subject, comprising administering to the subject an effective amount of a composition to treat or delay the progression of the neurodegenerative disease or condition. The composition may comprise one or more pharmaceutically acceptable excipients and active substances, such as acampolic acid, ambesentan, aminocaproic acid, amlodipine, amobarbital, apralinidine, argatroban, baclofen, benidipine, carbamazepine, carbamazine, carbexolone, cefotaxime, cefotetan, ciclopirox, cilostazol, cinacalcet, cinnarizine, clopidogrel, dihydroxypropyltheophylline, enprofen Theophylline, eplerenone, eprosartan, erythritol tetranitrate, etomidate, fenodopan, leflunomide, lecanidipine, levosimendan, adenomyine, methimazole, methylchlorothiazide, miglitol, moxifloxacin, oxtriphylline, methylethyldione, phenformin, prilocaine, rifabutin, risedronate, sulfamethoxazole, sulodexixil, tadalafil, terbinafine, torasemide, and zonisamide. In some embodiments, the pharmaceutical composition may comprise one or more of the following: an AMPK modulator (e.g., phenformin) and a sodium channel SCN1A inhibitor and a BK channel activator (e.g., zonisamide) or a BK channel modulator (e.g., methaqualizine); an AMPK modulator (e.g., phenformin) and a GABAergic and glutamatergic receptor activity modulator (preferably selected from acampolic acid, etomidate, and apralinidine); an AMPK modulator (e.g., phenformin) and an EDNRA endothelin receptor antagonist (e.g., sulfamethoxazole); a sodium channel SCN1A inhibitor and a BK channel activator (e.g., zonisamide) or a BK channel modulator (e.g., methaqualizine) and an RYR3 rennetine receptor modulator (e.g., prilocaine); or a GABBR2 receptor. Modulators (e.g., baclofen) and RHOA modulators (preferably selected from terbinafine and risedronate), GABBR2 receptor modulators (e.g., baclofen) and EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole), GABBR2 receptor modulators (e.g., baclofen) and sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., meclothiazide), GABBR2 receptor modulators (e.g., baclofen) and HAS1-3 hyaluronic acid synthase modulators (e.g., leflunomide), sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., meclothiazide) and adenosine receptor ADORA1 / 2 / 3 modulators (e.g., dihydroxypropyltheophylline).Sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., mechlorothiazide) and EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole), RHOA modulators (preferably selected from terbinafine and risedronate) and EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole), RHOA modulators (preferably selected from terbinafine and risedronate) and phospholipase PLA1A and PLA2 inhibitors (e.g., adenomyine), RHOA modulators (preferably selected from terbinafine and risedronate) and GABAergic and glutamatergic receptor activity modulators (preferably selected from acampolic acid, etomidate, and apralinidine), RHOA modulators (preferably selected from terbinafine and Rifampin and its chemical chaperones (e.g., rifabutin), AMPK modulators (e.g., phenformin) and PDE11A, PDE4A, PDE5A phosphodiesterase inhibitors (e.g., tadalafil, enprophylline, choline), sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., meclothiazide) and thrombin receptor F2R signaling modulators (e.g., argatroban, cefotaxime), AMPK modulators (e.g., phenformin) and purinergic receptor P2RY1 and P2RY12 modulators (e.g., clopidogrel), GABAergic and glutamatergic receptor activity modulators (e.g., acampate, etomidate, and apralindone) and CASR modulators ( Examples include cinacalcet, EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole) and CASR modulators (e.g., cinacalcet), RHOA modulators (preferably terbinafine and risedronate) and thrombin receptor F2R signaling modulators (preferably argatroban and cefotaxime), GABBR2 receptor modulators (e.g., baclofen) and purinergic receptor P2RY1 and P2RY12 modulators (e.g., clopidogrel), RHOA modulators (preferably terbinafine and risedronate) and purinergic receptor P2RY1 and P2RY12 modulators (e.g., clopidogrel), sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., ... Examples of active agents include: methylchlorothiazide and voltage-gated calcium CACNA channel antagonists (preferably selected from cinnarizine, benidipine, methylethyldione, and amlodipine); GABAergic and glutamatergic receptor activity modulators (preferably selected from acampate, etomidate, and apralindone); sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., methylchlorothiazide) and HIF1A signaling modulators (e.g., ciclopirox ol), GABAergic and glutamatergic receptor modulators (e.g., acampate, etomidate, and apralindone) and HIF1A signaling modulators (e.g., ciclopirox ol).EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole) and oxidative phosphorylation modulators (preferably selected from amobarbital and methimazole), sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., chlorothiazide) and oxidative phosphorylation modulators (e.g., amobarbital, methimazole), EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole) and vitamin K metabolism modulators (e.g., cefotetan), sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., chlorothiazide) and vitamin K metabolism modulators (e.g., cefotetan), GABAergic and glutamatergic receptor activity modulators (e.g., acampolic acid, etomidate, apralinidine) and PRKG1 modulators (e.g., erythritol). Sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., chlorothiazide) and PRKG1 modulators (e.g., erythritol tetranitrate), EDNRA endothelin receptor antagonists (e.g., sulfamethoxazole) and PRKG1 modulators (e.g., erythritol tetranitrate), KCNJ11 modulators (e.g., miglitol, levosimendan) and sodium channel SCN1A inhibitors and BK channel activators (e.g., zonisamide) or BK channel modulators (e.g., chlorothiazide), KCNJ11 modulators (e.g., miglitol, levosimendan) and RHOA modulators (e.g., terbinafine, risedronate).

[0149] Different aspects of this invention can be used in and developed for healthcare systems. Examples of such systems include point-of-care systems, healthcare operating systems, or combinations thereof. For example, a user can use a device to test a blood sample and can provide information from the test to a system or method of this invention, which in turn sends additional information to the user.

[0150] In one implementation, the healthcare system is part of an integrated infrastructure built around a point-of-care blood monitoring device. Within this integrated infrastructure, information can be transmitted (e.g., wirelessly) from the measuring device to a database that integrates data from the device with stored data from various databases (patient records, genetic or genomic information, data from clinical trials) into a central database. The system can then allow for the automatic application of mathematics to the database within the pathophysiological context of a given neurological condition.

[0151] In one embodiment of the invention, the healthcare system can be used for home or physician monitoring of biological samples (e.g., blood). Systems of this disclosure that include point-of-care devices communicating with the healthcare system can eliminate the need to transfer samples, such as transporting them to another laboratory, thereby improving sample integrity. Detection of analytes in the systems or methods of the invention can be provided at the point of care. Such detection, integrated with a central healthcare database system, can allow the creation of complex longitudinal data sequences acquired by immunoassay devices.

[0152] The systems and methods disclosed herein can be used to provide early diagnosis of a variety of neurodegenerative diseases. For example, diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease can begin 10-20 years before clinical manifestation. Multiple sclerosis (MS) can have a pronounced prodromal period, such as 5 years, during which signs and symptoms appear, but the more typical symptoms of the disease do not manifest. Furthermore, diseases such as MS have highly variable rates and types of progression. For these diseases, early and accurate diagnosis (as provided in this disclosure) may be particularly important for intervention and treatment. For example, these identifications can aid in treatment decisions or help identify candidates for clinical trials. In addition, this disclosure provides measurements that can optimize treatment decisions and / or enhance current identification strategies, such as imaging techniques like magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Figure 1 As shown. Early detection modalities for various neurodegenerative diseases are provided in Stoessl Translational Neurodegeneration 15 (2012), Sheinerman et al., Cell Cycle 12.11 (2013): 1-2, and Li, Yanxiang et al., AmJ Biomed Sci&Res 11.3 (2020): 216-218, Wijnands, Jose et al., Mult. Scler 28.5 (2019): 1092-1101, each of which is incorporated herein by reference in its entirety. Typically, all neurodegenerative diseases exhibit some heterogeneity and are prone to misdiagnosis. However, the detection of many neurodegenerative biomarkers, such as NfL assays, has previously relied on study-based assays. This disclosure provides more reliable markers for the detection of both prediction and treatment response, typically involving fully automated platforms.

[0153] The systems and methods disclosed herein can bring the measurement of specific biomarkers in a healthcare ecosystem closer to the patient and provide more easily accessible data. Furthermore, the systems disclosed herein can enable more efficient flow of patient information to specific groups within the ecosystem, thereby improving the efficiency of networks involving the transmission of confidential information. Exemplary ecosystems in... Figure 2 As shown in the diagram. The methods and systems disclosed herein can be used at home or in a doctor's office (circled section) to allow for the diagnosis and detection of neurological disorders that are frequently misdiagnosed.

[0154] like Figure 3 and Figure 4 As shown, measurements can occur at home or in a doctor's office, thereby communicating via a wireless network. In some embodiments, sample collection and measurement can be performed in a doctor's office while cognitive assessments can be performed at home. Cognitive assessments, such as home speech analysis (e.g., provided by one or more servers as disclosed herein), can be performed before or after the measurement results are obtained. These assessments can identify the risk of neurodegenerative diseases, thereby referring patients to clinics for further cognitive studies. In some embodiments, sample collection can be performed at home and samples can be sent to a reference laboratory for analysis. High-risk individuals can be referred to a major hospital system for further diagnosis and testing, such as imaging and CSF testing. These individuals can access early behavioral interventions, as well as opportunities for treatment and testing, particularly due to early disease identification. Figure 5 As shown, the methods and systems disclosed herein provide laboratories with an easy assay, enabling clinicians to know what measurements should be performed and to easily interpret the results.

[0155] Figure 6 A schematic diagram of the mechanism of NfL serum concentration, which can be measured using the protocols described herein, is provided. NfL is particularly abundant in large-diameter axons projecting deep into the brain and spinal cord. Damage to neural axons (e.g., damage caused by neurodegenerative diseases) dissociates NfL, leading to increased NfL concentrations in cerebrospinal fluid (CSF). Concentrations in CSF can be significantly variable, making typical ELISA measurements potentially too sensitive to detect appropriate changes for early prediction. Some NfL may cross the blood-brain barrier, thus blood NfL concentrations may be 40–200 times lower than CSF concentrations, and are typically measured using electrochemiluminescence (ECL) immunoassays. This disclosure provides systems and methods, generally involving acridineonium-based chemiluminescence, which can be automated to detect NfL concentrations in these biological samples in potentially automated settings, thereby providing more detection opportunities within the healthcare ecosystem.

[0156] Figure 7Exemplary NfL measurements for Alzheimer's disease are provided, which correlate serum NfL change rate with imaging changes (left panel, Figure a) and serum NfL change rate with predicted years to symptom onset (EYO) in non-carrier or mutation carriers (such as amyloid precursor protein (App), presenilin 1, or presenilin 2 mutations) patients (right panel, Figure b). It can be seen that serum NfL change rate (e.g., detected using the systems and methods of this disclosure) can be used as a predictor of disease status and provide early identification of individuals at moderate or high risk of symptom development.

[0157] Figure 8 Exemplary schematic diagrams of the sandwich assay scheme of this disclosure are provided, which can be automated in the systems described herein. An acridine-tagged antibody (e.g., acridine ester (AE)) can be added to a biological sample (e.g., blood, CSF) and then incubated to allow NfL to bind to the antibody. Paramagnetic particles coated with a capture antibody (which are generally also capable of binding to NfL bound to acridine via a first antibody) can be added to the mixture. After an optional second incubation period, the solid complex containing solid particles conjugated to acridine (via NfL) can be separated from the unbound AE (e.g., by using a magnetic field and / or washing), and chemiluminescence from the solid complex can be induced by adding one or more triggering agents. In some embodiments, the chemiluminescent sample can be further washed once or multiple times with a washing buffer, as shown in U.S. Patent No. 6,143,578, which is incorporated herein by reference in its entirety, and particularly with respect to the reaction sites relating to sample preparation, washing, and resuspension washing. The measured RLU output is correlated with the NfL concentration of the original biological sample, which can then be used for risk assessment of the individual providing the biological sample.

[0158] Now refer to Figure 9 ( Figure 9A and Figure 9B The diagram provides an exemplary array that can be used to automate portions of the methods disclosed herein. A position AP can be considered a reaction location for performing the indicated steps described herein. A reaction vessel can be inserted into a slot (e.g., a slot appearing at each location) and translated along the track of the array. The determination may involve lateral translation of the sample (e.g., by moving along the track to the respective locations), which allows the indicated reaction location or condition to be achieved. In some embodiments, subsequent reaction locations may not involve lateral translation of the sample. For example, in... Figure 9AIn the diagram, reaction positions AP indicate a series of locations along a linear track where the sample can be manipulated. At reaction position A, a biological sample and assay reagents (including chemiluminescent conjugates and magnetizable particles with analytes or analyte-binding pairs immobilized thereon) are added to the reaction vessel. The reaction vessel can then be moved along the track to position B, where the magnetizable particles begin to be isolated by a magnet, for example, as shown in the diagram. Figure 8 As shown. Translation at position BE may each involve the isolation of the magnetizable particles. At some positions, such as position F, the liquid medium can be removed, and at position G, a wash buffer can be added. Position H includes a magnet 42 further separated from the reaction vessel, which can allow the magnetizable particles to disperse and be resuspended or redispersed in the wash buffer. Position IN may involve an additional washing step (e.g., at position M). At position N, the wash buffer can be aspirated from the reaction vessel. At position O, a first chemiluminescent reagent (e.g., a triggering reagent such as an acid) can be added to the reaction vessel. At position P, the isolation of the magnetizable particles may be affected by magnets 44 and / or 46 (e.g., depending on their position relative to the reaction vessel, depending on their field strength, or a combination thereof). The reaction vessel at position P is movable (e.g., with a translation arm) into the luminometer, where a second chemiluminescent reagent is added and chemiluminescence is measured. In various embodiments, the liquid medium is aspirated from the reaction vessel at position P and added to a new reaction vessel that has been placed or will be placed in the luminometer for chemiluminescence measurement. Figure 9B Similar embodiments are provided, using potential reaction location tracks in the form of circular turntables or toroidals, wherein the sample is exposed to magnets 50, 52, 54, and 56 as it moves around the toroidal ring. After the reaction vessel has moved past location AP, chemiluminescence can be ultimately induced by adding a second chemiluminescent reagent to a suitable medium. Addition can occur at a location close to a photomultiplier tube used for light collection (e.g., in a luminometer). The steps described herein can be generated by multiple tracks, such as adjacent turntables, each providing any indicated reaction location. In various embodiments, the system includes a shunt to move the reaction vessel between adjacent turntables. Removal of the liquid phase may result in a portion of the liquid phase remaining in the reaction vessel or at any reaction location. For example, if the reaction vessel is aspirated to remove the liquid phase, as understood regarding aspiration, some portion of the liquid phase may remain in the reaction vessel. In various embodiments, aspiration can be performed without taking further steps to dry any indicated reaction location. In various implementations, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% of the liquid phase can be removed during the removal of the liquid phase (e.g., aspiration).

[0159] Additionally, a non-transitory computer-readable medium containing executable instructions is provided, which, when executed, cause a processor to perform operations including the methods described herein. For example, the non-transitory computer-readable medium contains executable instructions that, when executed, cause a processor to perform operations including the methods described herein. In embodiments, the non-transitory computer-readable medium includes media for storing data such as hard disks, external hard disks, optical disks, CDs, and DVDs. In embodiments, software disposed within a physical medium is applicable herein.

[0160] In some embodiments, a non-transitory computer-readable medium containing executable instructions, when executed, causes a processor to perform operations including methods for determining the presence, severity, and / or susceptibility to neurodegenerative symptoms or neurological disorders (such as Alzheimer's disease and MS) in an individual, the methods comprising the steps of: incubating the assay components of this disclosure together with a biological sample, and determining the amount of one or more biomarkers in the sample.

[0161] Advances in analyzing speech, gait, internet use, gameplay, and eye-tracking patterns have provided very early detection of individual cognitive changes, but lack the specificity and sensitivity necessary for diagnosing Alzheimer's disease and other neurodegenerative symptoms. In embodiments, the methods disclosed herein include one or more components that are practical and provide excellent diagnostic and therapeutic opportunities: • Use easily distributed and usable detection methods, such as smartphone-based speech sample collection and advanced speech analysis, to identify individuals with characteristic speech patterns associated with cognitive decline, and in particular, with a risk of Alzheimer's disease.

[0162] • Use sample collection devices, such as self-collection devices for capillary blood, like finger prick (BD) or shoulder patches with microneedles (Tasso), which allow individuals to collect samples at home (DTC sold) or through a primary care physician (professional sold).

[0163] • Use NfL and / or other peripheral biomarkers of neurodegenerative diseases from blood (or also saliva, urine, feces, etc.) to confirm that the initial results are likely due to the neurodegenerative process rather than other causes (i.e., mood disorders).

[0164] • After initial test results are obtained, whether directly by the individual or by self-reporting cognitive problems to a healthcare professional, individuals identified as more suspected of having Alzheimer's disease or other neurodegenerative diseases may be referred for a neurological consultation, which will lead to more comprehensive cognitive testing, brain imaging, CSF testing, and other means to confirm the diagnosis.

[0165] In its implementation, this disclosure includes a speech-based screening database for neurodegenerative diseases such as Alzheimer's disease and dementia. For example, speech data (at home) is used for risk assessment, and intermediate-risk and high-risk individuals are marked for further investigation according to a protocol (e.g., through a physician such as a primary care physician) → laboratory + speech + cognitive scoring (in a clinic) → patient is flagged for imaging / CSF-based diagnosis → treatment recommendations → patient monitoring (laboratory + speech + cognitive scoring).

[0166] In embodiments, this disclosure includes kits and methods applicable to use according to this disclosure, including the detection of available AD-specific blood-based biomarkers (pTau, NfL, GFAP, ApoE) using an immunoassay platform as disclosed herein. In embodiments, blood samples can be collected via venipuncture and / or finger-prick blood collection. In embodiments, immunoassay results from venous blood samples and finger-prick blood samples are compared to investigate home-based self-collection for analyzing patterns of AD-specific blood-based biomarkers.

[0167] The methods disclosed herein may involve communication across one or more networks to aid in an individual's cognitive assessment (typically used in conjunction with biomarker assays such as neurofilament assays). The system may include a server that can communicate with one or more client devices via a network (e.g., the Internet). The server may be one or more computing devices including at least one processor, memory, and communication interface, located in one or more locations, capable of storing data and communicating data with other components of the system. The server may include a database storing multiple performance tests.

[0168] Performance testing may include computer-executable instructions that enable the system to administer performance tests to a user, acquire performance information captured by one or more sensors (such as a camera or microphone), and then enable a server (or a processor connected to the server) to analyze the performance of the test and determine whether a certain condition exists.

[0169] Performance tests in the database can be associated with specific types of tests (e.g., psychological, cognitive, emotional, motor skills). Furthermore, performance tests can be associated with one or more conditions.

[0170] One example of a performance test is a cognitive test in which a patient is instructed to sequentially touch their eyes, nose, and jaw. This performance test can be designed to assess a patient's cognitive abilities and evaluate the likelihood of impairment. The test is typically scored based on the patient's ability to identify each facial point they are asked to touch and subsequently perform them correctly in sequence. In this case, the condition that may be determined is the level of impairment. If the patient is able to successfully perform the test by touching the indicated points and in the correct order, the test indicates no impairment. If the patient can identify one or more points but misses one or more points or performs the test in a different order than the correct order, it is assessed as "some impairment." If the patient cannot identify any points, it is assessed as "impaired." In some variations of this test, a percentage of impairment may be provided based on the amount of the test the patient performs correctly.

[0171] The database can store executable instructions that enable the presentation of instructions (e.g., via a virtual agent, which could be a video of someone performing a test), capture of the patient performing the test (e.g., via a camera on a computing device), analysis of the test to determine the condition (e.g., level of damage), and transmission of the test to the appropriate party (the patient themselves, a healthcare provider such as a primary care physician). The instructions can further enable the presentation of the results of biomarker immunoassays (e.g., NfL immunoassays) to the patient and / or healthcare provider.

[0172] In some implementations, a cognitive performance test may require users to name as many animals as possible that begin with a certain letter within a specific time period. Based on their ability to recall animal names, the test can determine whether there is a certain amount of impairment. As in the example above, the database stores executable instructions that enable the system to present the performance test, capture the performance, and evaluate the performance to determine whether there is any impairment (and combine or present the results of the performance test with biomarker assay results).

[0173] The results of cognitive tests can lead to the determination (diagnosis, confirmation, or probability assessment) of one or more degenerative cognitive conditions (such as Parkinson's disease, dementia, Alzheimer's disease, ALS, or MLS). This determination is typically associated with biomarker assay results.

[0174] Performance tests and their scoring can include those found in tests such as the Montreal Cognitive Assessment and the Mini-Mental State Examination.

[0175] The method may include a client computing device configured to interact with one or more servers (e.g., to receive cognitive tests or to transmit cognitive test results). The client computing device may access the functionality of this disclosure in various ways, such as via a downloadable application or via a web portal accessible through a browser. The client computing device typically includes at least one processor, at least one non-transitory computer-readable storage medium, and I / O interfaces (e.g., monitor, touchscreen, speaker, mouse, keyboard, camera) that allow the user to receive data from and interact with the computing device. The client computing device may also have a communication interface (e.g., Wi-Fi, wired internet connection, cellular network) that enables the device to exchange data over a network. Examples of suitable computing devices may include desktop computers, laptops, tablets, smartphones, and video game consoles.

[0176] To administer the test and enable further interaction with the patient, the client computing device can execute a virtual agent. This virtual agent can be installed on the client computing device and / or executed by a server and presented on the client computing device only via a web browser or other user-facing portal.

[0177] Before the cognitive test, users can log in to their accounts via a web browser or application installed on their device to access the system's functions.

[0178] To initiate a cognitive test, the server can retrieve one or more performance tests to be presented to the user. The retrieval of performance tests, including the selection of one or more performance tests, can be based on prior advice or instructions, such as those given by the user's doctor or other medical professional. For example, if a doctor recommends performance tests focusing on motor skills / motor degeneration, the server can select one or more performance tests designed to measure the user's motor skills and / or determine the extent of motor degeneration. Other criteria for selecting performance tests may include age, sex, ethnicity, family history, medical history, and occupation. As an illustrative example, the server could select a test, based on a healthcare provider's request, in which the user must touch their eyes, nose, and jaw in sequence.

[0179] In some cases, a server may have a default set of performance tests for administration, which may cover multiple areas of a patient's health. For example, the set may include one or more performance tests designed to measure a person's cognitive abilities, or one or more tests designed to measure a person's motor skills. These performance tests may be transmitted to the user via a virtual agent. The activation of the virtual agent may be in response to a server that recognizes the need to administer one or more performance tests to that particular user, for example, based on biomarker readings, healthcare provider instructions, or population information related to any disease being evaluated. In an implementation, the virtual agent may be activated based on a user logging into their account and accessing the tests. The server may execute the tests, such that the tests are administered via the virtual agent. The administration of the tests via the virtual agent may include presenting instructions for the tests, which may include visual and / or audio instructions presented via the virtual agent that explain the tests to the user. The visual components of the instructions may include text, still images, and / or video images.

[0180] For example, a virtual agent could present a video showing a user the actions to be performed to demonstrate a test. The video might show a person sequentially touching their eyes, nose, and jaw. The video could also include audio instructions describing the test as the video is shown to the user. The instructions could also include framing instructions to ensure the user is at the appropriate distance from the camera for capture, and any additional instructions. The instructions could include a textual explanation of the test and a start prompt.

[0181] In another instance, a performance test could ask a user to repeat a series of words. For this test, a virtual agent could then present the words in an audible manner (via the computing device's speakers) and / or display them on a screen for the user to read. This could also include a prompt to begin saying the words. In yet another instance, a performance test could be one that asks a user to name as many animals as possible within a specific timeframe (e.g., 10 seconds). In this instance, the test could be administered by simply instructing the user to begin the test after the steps are explained, followed by starting a timer.

[0182] To begin the test, the virtual agent may prompt the user to click a "Ready" button or utter a word indicating their readiness. In implementations, the virtual agent may have a countdown timer or other indication that the test will begin shortly after the steps are described, allowing the user to enter the test without any interaction with the system. The computing device may capture the user's test performance via one or more sensors integrated into or connected to the computing device. Depending on the nature of the test, the sensor may be one or more of a camera, microphone, touchscreen, keyboard, or mouse. For tests requiring the user to perform actions, the sensor may be a camera or other device capable of capturing the user's actions during test execution. For example, in an "eye, nose, jaw" touch test, the user's computing device camera may capture the user attempting to perform the steps of touching their eyes, nose, and jaw in sequence. For tests requiring the user to perform the test verbally (e.g., uttering certain words or phrases or reciting terms from memory), the sensor may be a microphone or a camera with a built-in microphone. Sensor data captured during test execution can be transmitted from the computing device to a server. The server or a processor connected to it can analyze the performance of the test based on the sensor data captured by the sensors. In an implementation scheme, the analysis may be a comparison of the performance test with established metrics for that particular test, wherein the execution or non-execution of certain aspects of the test is flagged or scored.

[0183] To perform the analysis, the server can employ image recognition technology (e.g., capable of accurately determining the position of a user's hands relative to their face), speech recognition technology (e.g., capable of identifying the words spoken and subsequently determining aspects of speech such as fluency, volume, stuttering, etc.), pattern recognition technology, and other technologies that enable the server to "understand" the user's test performance. The analysis can utilize artificial intelligence algorithms to help detect certain parameters associated with the sensor data transmitted to the server. For example, the server can apply image recognition technology to correctly identify which areas of the face are "eyes," "nose," and "mouth," and the "hands" moving relative to the face, and based on this, determine whether the user touched their eyes, nose, and jaw, and / or whether the actions were performed sequentially, based on captured video. The server can compare the test performance to one or more condition indicators representing a specific condition. Generally, the comparison may be a comparison of the test performance captured in the sensor data to a baseline or standard performance for the test. The baseline may be derived from a set of sensor data performed by multiple individuals considered "healthy" (i.e., not suffering from the condition being tested). In some implementations, the baseline can be derived from the patient and established by measuring NfL concentrations (particularly blood NfL concentrations) at intervals (e.g., monthly, annually) over a period of time (e.g., 1-10 or 1-5 years). The systems and methods of this disclosure, particularly when located close to the point of care within a healthcare ecosystem, are better able to establish these patient-specific baselines through, for example, more measurement opportunities. Baselines and subsequent cutoff values ​​for further diagnosis, testing, and / or treatment can be derived from patient-specific models based on the level of statistically or functionally significant change relative to the baseline (e.g., percentage change relative to baseline, Z-scores optionally adjusted for patient indicators such as age and body mass index (BMI)). The condition can be a degenerative condition or a neurological condition that can generally be assessed using biomarker detection.

[0184] The server can determine how many of the eyes, nose, and jawline the user was able to touch, and whether they were touched in the correct order. If the server determines that the test was performed correctly—each of the eyes, nose, and jawline was touched in the correct order—the server can assess the condition as "undamaged." If the server determines that some (but not all) touches were performed and / or not in the correct order, the server can assess the condition as "somewhat damaged." If the server determines that no touches were performed correctly, the server can assess the condition as "damaged." As discussed above, in an implementation, the server can determine more granular conditions based on the percentage of tests performed correctly and in the correct order.

[0185] Performance tests can be timed or require recording specific items within a defined time limit. The server can access a range of databases to evaluate the results of the performance test. For example, a performance test might involve listing multiple specific items within a specific time frame. For instance, if a user is asked to name as many animals as possible that begin with a specific letter within one minute, and the user does so, the server can analyze the test performance by first converting the speech to text (e.g., via speech recognition technology), then comparing the text to an existing animal name ontology / database, and scoring the results based on the matches. In this example, the analysis could further include tracking the time taken to name the animals and the comprehensibility / fluency of the responses.

[0186] In another instance, performance testing could involve a server (via a virtual agent) transmitting information (such as a series of numbers) to a user and asking the user to repeat them. Therefore, the analysis in the steps could involve speech recognition of the stated numbers and comparing them to numbers provided by the virtual agent.

[0187] Scoring algorithms can be used to score / measure user performance in tests. These algorithms can be constructed using classifiers (logistic regression or random forest), taking information such as the accuracy of spoken words or the percentage accuracy of touching specific body parts as input, and outputting a score or decision. Additional details regarding the training of the classifier and suitable examples of this embodiment of the subject matter can be found in Ramanarayanan, Vikram. Journal of Speech, Language, and Hearing Research (2024): 1-13, Hecker, Pascal et al., Frontiers in Digital Health 4 (2022): 842301, and Bowden, Molly et al., NPJdigital medicine 6.1 (2023): 228, all of which are incorporated herein by reference in their entirety, and particularly concerning speech recognition algorithms for disease detection.

[0188] Once the condition (and optionally its severity) is determined, the server can send the results to one or more recipients. One recipient could be the user themselves. Other recipients could be healthcare providers or pharmacies.

[0189] In implementations, multiple tests can be administered in a single session (e.g., the examples given herein), and the results of each test can be combined by the server to form an overall score of cognitive function. In some implementations, the server can be programmed to select a second performance test from a database based on the results of a first performance test. Furthermore, one or more performance tests can be assigned based on the results of previous tests. For example, if a user's performance on a particular performance test and / or biomarker analysis leads to the identification of a condition, the server can retrieve performance tests applicable to the condition itself (e.g., the likelihood of Alzheimer's disease) or applicable to the type of condition (e.g., a neurological condition). The results of the second performance test can be used to confirm the identification of the initial condition and / or determine the severity of the condition following the first performance test.

[0190] In implementations, the server can be programmed to account for poor test performance due to reasons other than the test itself. In these implementations, the server can be programmed (via a virtual agent) to execute a new performance test similar to the previous one. For example, if analysis shows poor performance on a spoken language test, the poor performance might be due to a person's knowledge rather than their mental ability / sensitivity. This could happen, for example, if a person is asked to memorize words from a topic unfamiliar to them. If a person is asked to name cities that begin with "S" but is unfamiliar with those cities, the performance test result might be low, but this doesn't reflect a situation related to mental acuity. The server can retrieve a new test, such as asking the person to name a four-legged animal. If the result of the second test still indicates a certain situation, that result can be reported. In variations, additional tests can be selected and administered. In one variation of this implementation, if English is not the user's primary language, the language of the performance test can be changed, and the same test can then be administered in the user's primary language.

[0191] Similarly, poor performance on cognitive tests based on a patient's execution of a single action or series of actions may be due to mobility difficulties rather than cognitive reasons. For example, in a test instructing a person to touch their eyes, nose, and jaw in sequence, a person might accidentally touch their forehead, cheek, and lips. In this case, the person clearly understood the test instructions and remembered the steps, but a motor skill condition prevented them from performing the test correctly. Using image recognition technology, a server can identify that the person attempted to perform the steps in the correct order by determining that their hands were close to (i.e., within a specific distance and sufficiently far from other body parts) the correct body parts. In this implementation, the server can obtain performance tests related to motor skills and dexterity and administer these tests via a virtual agent to determine whether a motor skill condition actually exists and, if so, its severity.

[0192] In embodiments of the subject matter of this invention, the server can adjust the application of one or more performance tests based on conditions other than the test itself. For example, considering a decrease or lack of system resources, or environmental conditions. In some of these embodiments, the server can modify or otherwise adjust the application of some or all of the performance tests (e.g., one or more descriptions, the application of the test itself, etc.) based on unstable or unreliable network connectivity and / or a lack of sufficient computing resources. In these embodiments, the performance test may have one or more required resource metrics necessary for the test to function properly. Resource metrics may include hardware requirement metrics (e.g., camera, microphone), network bandwidth requirement metrics (e.g., the minimum bandwidth required to render video commands in real-time or near real-time and / or return results from computing devices in real-time or near real-time), and computing hardware requirement metrics (e.g., the computing device has specific capabilities to execute virtual agents and render video at a certain resolution, capture video). The server can perform checks to determine whether the resource metric requirements of the test are met. This may include checking the necessary hardware (camera, microphone, etc.), testing available network bandwidth, and testing computing devices to determine available resources. If the metrics obtained by the server do not meet the requirements of the performance test, the server can adjust the application of the test using one or more of the following adjustments.

[0193] The server can enable the virtual agent to change the mode of the test being applied (e.g., from video to plain audio, or from plain audio to text). For example, if some or all of the test is applied via video, the virtual agent can instead render only audio instructions (thus saving bandwidth compared to transmitting video).

[0194] The server can be adapted by transferring some or all of the instructions (code) for administering the test from the server to the computing device, enabling the computing device to perform the performance test locally. In these implementations, the computing device can then store the results of the performance test locally until conditions improve and the computing device can transfer the results to the server for analysis. In a variation of these implementations, the instructions transferred to the computing device may include instructions that enable the computing device to perform some or all of the analysis.

[0195] The server can measure latency (e.g., latency caused by network delays) that affects the real-time or near-real-time nature of the interaction between the virtual agent and the user. Latency can be the delay between the server executing a part of the test process and the actual presentation of that part through the virtual agent, the delay between the virtual agent and the user's questions and responses, or the delay in capturing the user's test performance and forwarding it to the server in real time. The server can then account for latency by buffering more parts of the test process (e.g., video or instruction steps) on the client's computing device. The server can also buffer information captured via sensors so that it can be sent as a continuous stream once network conditions improve. For tests that require back-and-forth interaction between the server (via the virtual agent) and the user, buffering can be done in such a way that the presented information is smooth and uninterrupted, allowing the user to respond immediately in a natural manner.

[0196] The server can analyze one or more resource metrics for a specific performance test. If one or more resource metrics fail to meet the requirements of the performance test, the server can select a second performance test where the available resource metrics satisfy the requirements. The second performance test will therefore be a test for a similar purpose to the first performance test (e.g., testing specific aspects of cognitive or motor function) but with lower resource intensity. For example, the second performance test might only require capturing audio, instead of the video and audio in the first test, thus placing lower hardware and bandwidth requirements on the user's computing device. The server then acquires the second performance test and continues to present it via a virtual agent. Example

[0197] The following examples illustrate the synthesis of a representative number of compounds, the characterization of parameters involved in their development, and their use in measuring samples in heterogeneous competitive assays. Therefore, the examples are intended to be illustrative and not limiting of this disclosure. Other compounds not specifically exemplified may be synthesized using conventional methods in conjunction with the methods described herein.

[0198] Example 1: Generation of antibody fragments To produce immunoglobulin fragments, the antibody is digested with fig protease to obtain two F(ab) fragments (each with an approximate weight of 50 kDa) and one Fc fragment.

[0199] To produce the fragment, mouse monoclonal antibody (Mab) was heated at 37°C for 1 hour. Figinase F6008-100UN / 086K770 was dissolved at 10 mg / ml, pH 7, and then added to the Mab at a mass ratio of 1:30 (270 mg Mab / 30) ÷ 10 mg / ml = 0.9 ml. L-cysteine ​​(121.6) (Aldrich 168149 / BCBW3951) was then dissolved at 10 mg / ml, pH 7, and incorporated into the mixture to 1 mM. N-ethylmaleimide (NEM, Sigma E3871 / SLBW6111) was then added to the mixture at 0 / 1 M, pH 7, and the mixture was stirred at room temperature (21°C) for 30 minutes. The mixture was then incubated overnight at 4°C to produce the digestion mixture.

[0200] The digestion mixture was diluted 1:1 (v / v) with 1.5 M glycine-2 M NaCl, pH 8.6 and separated using equilibrated 1×18 Protein A Millipore ProSep-vA 113115827 / R7AA65871 separation medium. The flow-through was captured by washing with 40 mL of the medium at pH 8.6. The flow-through (F / T) was concentrated at 30 K and purified to 0.1 M NaPO4-150 mM NaCl-5 mM EDTA, pH 7.4 (PBSE 7.4) using a 26 / 70 Superdex-200 column. SDS-PAGE analysis was performed. Figure 10 Provided by China.

[0201] Example 2: Formation of acridine-BSA-PEG-F(ab) conjugate Bovine serum albumin was labeled with an excess of 5 to 25 acridinium ester, said acridinium ester containing a reactive functional group having a structure such as TSPAE-NHS: The reaction mixture was purified through a 2.3 / 18cm G-25 fine filter medium to 0.1 M NaPO4- 150 mM NaCl- 0.1% Tween-20, pH 7.4 (PBST7.4), and concentrated to the desired amount of protein.

[0202] The carrier protein / acridonium complex then reacts to form a specific antibody / antibody fragment conjugated protein / acridonium complex. Both are linked using a linker molecule such as a polyethylene molecule (e.g., RFG-(OCH2CH2)). n -RFG, where n is, for example, 1-15, and RFG is independently a reactive functional group used to form a conjugate with the protein, is coupled. The complex is then purified using a chromatography procedure.

[0203] Specific conjugates formed using this procedure include neurofilament light chain antibody fragments (Nfl Mab Fab') and bovine serum albumin carrier protein (NflMab Fab'-PEG4-mBSA-AE) conjugated to TSPAE (AE) via polyethylene glycol (PEG, e.g., PEG4 (n=4)). Conjugates having a 25X excess of acridine ester / carrier protein and an antibody fragment targeting the UD2 clone of the neurofilament light chain are referred to herein as 25X UD2-Fab'-PEG4-m-BSA-AE.

[0204] Example 3: Immunoassay Measurement A fully automated two-step sandwich assay was performed using a Siemens ATELLICA® immunoassay and the conjugates disclosed herein. The assay employed two anti-sNfl antibodies. The primary antibody in the luminescent reagent was 25X-UD2-Fab-PEG4-m-BSA-AE. The secondary antibody was a biotinylated mouse monoclonal anti-sNfl antibody (UD1) that bound to paramagnetic microparticles coated with streptavidin in the solid phase (5XUD1-iodo-PEG2-biotin). Exemplary assay formats used in the experiments and schematic diagrams are shown in… Figure 11A and 11B middle.

[0205] The luminescent reagent immunoassay compositions used are provided in Tables 3 and 4 (Table 3 provides the sNfL luminescent reagent buffer formulation).

[0206] Table 3 Components concentration Function sNfL luminescent reagent buffer 1 L / L buffer <![CDATA[25X UD2-Fab'-PEG4-m-BSA-AE]]> 0.35 mg / L Luminescent reagent concentrate

[0207] Table 4

[0208] The solid-phase immunoassay compositions used are provided in Tables 4 and 6 (Table 6 provides the components of the sNfL solid-phase buffer).

[0209] Table 5 Components concentration Function sNfL solid-phase buffer 1 L / L buffer <![CDATA[5X UD1-Iodo-PEG2-Biotin]]> 0.02 mg / mg granules solid-phase (captured) conjugates Dynabeads MyOne Streptoacid T1 0.5 mg particles / mL solid particles

[0210] Table 6

[0211] The measurement was performed as follows: a) Add 100 μL of sample (or standard) to a cuvette; b) Add 100 μL of the luminescent reagent to the sample and incubate for the required time and temperature (e.g., 32 minutes at 37°C); c) Add 100 μL of solid phase to the incubation mixture, and specify the time and temperature required for the incubation mixture to be incubated again (e.g., 17 minutes at 37°C); d) The mixture containing the solid phase after washing and incubation; e) Add a chemiluminescence triggering reagent (300 μL acid / 300 μL base) to induce chemiluminescence; and f) Measure and report chemiluminescence.

[0212] Figure 11B An example of this order of addition (including the first and second reagent dispensings and washes) is described in detail, where A shows the addition of 100 μL sample, B indicates the first reagent dispensing, C indicates the first magnetic separation (without washing), D indicates the second reagent dispensing, and E indicates the second magnetic separation.

[0213] Typically, chemiluminescence reporting is performed by identifying the relative light units (RLU) generated after chemiluminescence triggering. Table 7 provides the RLU measurements for a range of standards with identified doses of neurofilament light chains present, using this assay procedure, solid phases, and conjugates.

[0214] Table 7 sample Dosage (pg / mL) RLU STD01 0 1901 STD02 10.6 12965 STD03 28.6 30168 STD04 64.5 66649 STD05 137 130988 STD06 318 298172 STD07 660 553451

[0215] Figure 12 The graph shows the RLU measured at each NfL concentration, including the best-fit linear fit (with the function RLU = 840.46 ([NfL]) + 10003 shown as a dashed line). These results have a low-end slope factor of 836 (RLU(S07-S01) / dose(S07-S01) - (553451-1901) / (660-0) = 836).

[0216] Other assay architectures cannot produce slope factors greater than 600, including those lacking antibody fragments, carrier proteins, and / or the linker between the carrier protein and the antibody fragment. Some highly sensitive assays require high slope factors to produce the high-resolution assays needed to measure certain analytes (e.g., neurofilaments). Table 8 provides the RLUs and assay formats that result in slope factors less than 600, leading to lower-resolution assays obtained using various arrangement of assay architectures. These measurements were performed using the same conjugate (25X-UD2-Fab-PEG4-m-BSA-AE). Figure 13 A complete dataset is provided, including RLU measurements for each assay type under each standard.

[0217] Table 8 Differences in measurement methods Slope factor (between 0 and 660 pg / mL) Incubate for ~50 minutes: SP (SP then LR) 250 Incubate for 18 minutes: DP (SP then LR) 296 Incubate for ~50 minutes: DP (SP then LR) 417 Incubate for 18 minutes: DP (LR then SP) 258 Incubate for ~50 minutes: DP (LR then SP) 381 Incubate for ~50 minutes: SP (SP then LR) 246 Warm-up ~18 minutes: SP 276 Resuspended in 200 μL LR 272

[0218] Example 4: Specific monitoring of healthy patients Blood samples are collected annually from healthy patients during their physical examinations. Blood is measured using assays of this disclosure (e.g., those described in Example 3), and a baseline NfL concentration is established. In years 2 through 5, no change in NfL concentration is observed (e.g., NfL concentration measurements fall within the noise level of the assay, and NfL concentration measurements are not statistically significant relative to previous measurements). In year 6, an increase in blood NfL concentration is observed, exceeding a certain percentage from baseline (e.g., exceeding 1%, exceeding 5%, exceeding 10%, 1-20%, 5%-20%, 10%-20%). Repeated samples are collected and measured, showing a statistically significant change relative to a patient-specific baseline (e.g., determined by Z-scores and adjusted for patient indicators such as age and body mass index (BMI)). This triggers a clinical decision to identify the healthy patient as having an increased risk of neurological disease, condition, or status. The patient begins further testing, diagnosis, and treatment at an early stage of the investigation.

[0219] Example 5: Specific monitoring of confirmed patients Blood samples are collected monthly from patients diagnosed with a neurological disorder and undergoing treatment. Blood is measured using assays of this disclosure (e.g., those described in Example 3), and a baseline for NfL concentration is established. In years 2 through 4 after treatment initiation, the rate of change in NfL concentration relative to the previous month's measurement remains unchanged (e.g., the NfL concentration change rate measurement falls outside the noise level of the assay, or the NfL concentration change rate measurement shows no statistical significance relative to previous measurements). In year 5, the rate of change in blood NfL concentration decreases relative to the baseline established in years 1–4, exceeding a certain percentage of the baseline (e.g., exceeding 1%, exceeding 5%, exceeding 10%, 1–20%, 5%–20%, 10%–20%). Repeated samples are collected and measured, showing a statistically significant change relative to a patient-specific baseline (e.g., determined by Z-scores and adjusted for patient indicators such as age and body mass index (BMI)). This triggers a clinical decision to identify the therapy as potentially beneficial. The rate of change in serum concentration levels continues to decrease, prompting a clinical decision to modify the patient's treatment regimen.

[0220] Non-restrictive illustrative implementation plan The following provides non-limiting illustrative embodiments, each of which should be considered part of the disclosure of this application. These embodiments can be applied to any of the embodiments described herein.

[0221] Illustrative Embodiment 1. A method for detecting or quantifying analytes, such as neurological biomarkers, in a sample (e.g., a biological sample such as blood, saliva, serum, or a sample derived from a biological sample, such as a diluted biological sample), comprising: (a) Mixing a sample with a composition comprising a chemiluminescent label conjugated to a primary antibody or antibody fragment that binds to an analyte, wherein the chemiluminescent label binds to a carrier protein containing a linker, and the linker binds to an antibody or antibody fragment; (b) Add particles containing a secondary antibody or antibody fragment to the mixture, the secondary antibody or antibody fragment binding to the analyte and adhering to the particle surface; (c) Prepare mixtures to measure chemiluminescence (e.g., by separating particles having chemiluminescent acridinium adducted to the surface); (d) Triggering chemiluminescence from the preparation; and (e) The presence of the at least one analyte is detected or the concentration of the at least one analyte is calculated by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of emitted light with a known concentration of at least one of the multiple analytes.

[0222] Illustrative Implementation Scheme 2. According to the method of Illustrative Implementation Scheme 1, the analyte is a nerve filament.

[0223] Illustrative Implementation Scheme 3. The method according to Illustrative Implementation Scheme 1 or 2, wherein the sample is blood.

[0224] Illustrative Embodiment 4. The method according to any one of Illustrative Embodiments 1-3, wherein the chemiluminescent label is conjugated to a primary antibody fragment (e.g., F(ab)).

[0225] Illustrative Embodiment 5. The method according to any one of Illustrative Embodiments 1-4, wherein the primary antibody or antibody fragment is a mouse monoclonal antibody or a fragment thereof (e.g., F(ab)).

[0226] Illustrative Embodiment 6. The method according to any one of Illustrative Embodiments 1-5, wherein the connector comprises (or is) polyethylene glycol (PEG).

[0227] Illustrative Embodiment 7. The method according to Illustrative Embodiment 6, wherein the polyethylene glycol is 2-20 (e.g., 2-10, 2-5) ethylene glycol units.

[0228] Illustrative Embodiment 8. The method according to any one of Illustrative Embodiments 1-7, wherein the carrier protein is keyhole hemocyanin (KLH), bovine serum albumin (BSA), or cationic BSA.

[0229] Illustrative Embodiment 9. The method according to any one of Illustrative Embodiments 1-8, wherein the ratio of acridine to carrier protein is 50:1 to 1:1 by weight (e.g., 30:1 to 1:1, 25:1 to 5:1).

[0230] Illustrative Embodiment 10. According to the method of any one of Illustrative Embodiments 1-9, the chemiluminescent label conjugated to the primary antibody or antibody fragment binding to the analyte is formed by: i) Reacting a chemiluminescent acridine trioxide compound containing reactive functional groups with a carrier protein to label the carrier protein; and ii) Reacting the linker compound (e.g., a compound containing a linker having a reactive functional group at each end), the primary antibody or antibody fragment, and the labeled carrier protein.

[0231] Illustrative Embodiment 11. The method according to Illustrative Embodiment 10, wherein the first reaction step occurs in a medium containing a buffer solution.

[0232] Illustrative Embodiment 12. The method according to Illustrative Embodiment 10 or 11, wherein the chemiluminescent acridine compound is added in excess of the carrier protein by weight (e.g., less than 100X excess, or less than 50X excess, or less than 40X excess, or less than 30X excess, or 5X to 25X excess).

[0233] Illustrative Embodiment 13. According to the method of any one of Illustrative Embodiments 10-12, the chemiluminescent acridine trioxide compound containing reactive functional groups has the following structure: RFG is a reactive functional group used for protein conjugation. L is absent (i.e., it is a bond) or optionally contains the group L. C or Z L The connector, and Ψ is a chemiluminescent acridine trioxide containing the following structure: “ j "and" k "Independently 0 (e.g., all R2 groups are hydrogen, all R3 groups are hydrogen), 1, 2, 3 or 4; R1 represents hydrogen, -R, or -X. b -R L -X b -L C -R、-L C -X b (For example, -L1-X) b -Z, -R L -Z、-L C -Z (e.g., -L1-Z), or -R L -L C -R L -Z (e.g., -R) L -L1-R L -Z); R2 and R3 are independently selected from hydrogen, -R, electron-donating groups, and -X each time they appear. c -R L -X c -L C -X c (For example, -L1-X) c -Z; wherein the two adjacent R2 or R3 groups can together form a fused cyclic group (e.g., a 5-7 fused aryl or heteroaryl group, a 5-7 fused heterocyclic group), and wherein R2 or R3 can contain a bond with an imaging agent such as a fluorophore (e.g., rhodamine); LC divalent C atoms that are optionally substituted (e.g., having 1 to 20 heteroatoms or 1 to 20 substituents). 1-35 Alkyl, alkenyl, ynyl, aryl, or aralkyl groups; Z L It is a zwitterion junction group with the following structure: “ m "It is 0 (i.e., it is a key) or 1; “ n "and" p "Each occurrence is an independent integer from 0 (i.e., it is a key) to 10; Z represents a zwitterionic group that independently possesses the following structure each time it appears: “ q "and" l Independently 0 or 1; “ r "Independent integers from 0 to 10 (e.g., 1 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); X a and X b It is an anionic group each time it appears; X c It is a protonated anionic group; L1 is independently -O-, -S-, -NH-, -N(R) each time it appears. N )-、-(CH2) 1-10 -、-S(=O) 1-2 -、-‍C=C-、-C=C-(CH2) 1-3 -, -C(O)-, -OC(O)-, -C(O)-(CH2) 1-4 -、-(CH2) 1-4 -C(O)-, -‍C(O)-‍O-, -C(O)-N(R N -, -C(O)-NH-, -N(R) N )-C(O)-, -NH-C(O)-, -‍C(O)-‍N(R N )-‍(CH2) 1-3 -、-(CH2) 1-3 -C(O)-N(R N )-、-(CH2) 1-3 -N(R N )-C(O)-、-NH-S(O) 1- ‍2-‍、-N(RN )-S(O) 1-2 -、-‍S(O) 1-2 -N(R N )-、-S(O) 1-2 -NH-, -(CH2) 1-3 -NH-S(O) 1-2 -‍、-(CH2) 1-3 -N(R N )-S(O) 1-2 -、-‍(CH2) 1-3 -S(O) 1-2 -N(R N )-、-(CH2) 1-3 -S(O) 1-2 -NH-, -O- (CH2) 1- ‍4-、-(CH2) 1-4 -O-、-S-(CH2) 1-4 -、-(CH2) 1-4 -S-, -NH-(CH2) 1-4 -、-‍N(R N )-‍(CH2) 1- ‍4-、-(CH2) 1-4 -N(R N )-、-‍(OCH2) 1-10 -、-(CH2O) 1-10 -、-(OCH2CH2) 1-10 -、or-‍(CH2CH2O) 1-10 -; R L Each time it appears, it is independently for C that optionally has one or more (e.g., 1-10, 1-5) substitution sites (e.g., 1-10 heteroatoms, 1-10 substituents). 1-20 Divalent hydrocarbon groups (e.g., alkyl, alkenyl, aryl, phenyl, monoalkyl-substituted phenyl, dialkyl-substituted phenyl, alkynyl, aralkyl); R is independently hydrogen each time it appears, or optionally has one or more (e.g., 1-20, 1-10, 1-5) substitution sites (e.g., 1-20 heteroatoms, 1-20 substituents) of C. 1-35 Hydrocarbon (e.g., alkyl, alkenyl, ynyl, or aralkyl) groups; R' and R'' are independently hydrogen or C each time they appear. 1-10 alkyl; R N Each time it appears, it originates independently from either hydrogen or C. 1-5 Alkyl groups (e.g., methyl, ethyl, propyl); and R' is hydrogen or C 1-10 Alkyl; or Its salts (e.g., halide salts such as chloride salts, sulfonates such as halosulfonates, haloalkylsulfonates, fluoroalkylsulfonates, carboxylates such as haloalkylcarboxylates, fluoroalkylcarboxylates).

[0234] Illustrative Embodiment 14. According to the method of any one of Illustrative Embodiments 10-13, the chemiluminescent acridineonium containing the reactive functional group has the structure of formula (Ia): Where Ω represents O or N; Y is selected from -R or -R L -Z, or where Ω is 0, then Y does not exist; and Y' does not exist (i.e., Y' does not exist) ， It is a key, or selected from L1-, -R L -、-R L -L1-, -L1-L1-, -L1-R L -、-L1-R L -L1 and -R L -L1-R L -

[0235] Illustrative Embodiment 15. According to the method of any one of illustrative embodiments 10-14, the chemiluminescent acridine trioxide containing the reactive functional group has a structure of formula (Ia) or (Ib): R4-R7 are independently hydrogen, electron-donating groups, or C. 1-35 Alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, or amino; and "Y" does not exist (i.e., it is a key), or it is -L. C -、-L1-、-R L -or-R L -L1-.

[0236] Illustrative Embodiment 16. The method according to any one of Illustrative Embodiments 13-15, wherein L is a C1-C5 alkylene group.

[0237] Illustrative Embodiment 17. The method according to any one of Illustrative Embodiments 10-16, wherein the chemiluminescent acridine onion containing the reactive functional group is an N-succinimide ester.

[0238] Illustrative Implementation Scheme 18. The method according to any one of Illustrative Implementation Schemes 13-17, wherein R2 and R3 are independently -X c -R L -X c -L C -X c (For example, -L1-X) c ).

[0239] Illustrative Implementation Scheme 19. The method according to any one of Illustrative Implementation Schemes 13-17, wherein R2 and R3 are independently derived from -C(O)OH, -SO2OH), -OSO2OH), -OP(O)(OR P Alkoxy groups substituted with OH, -OH, or combinations thereof (e.g., C1-C4 alkoxy groups).

[0240] Illustrative Embodiment 20. The method according to any one of Illustrative Embodiments 10-19, wherein the chemiluminescent acridineonium containing reactive functional groups is TSPAE-NHS: Or its salt.

[0241] Illustrative Embodiment 21. The method according to any one of Illustrative Embodiments 1-20, wherein the secondary antibody or antibody fragment is a biotinylated antibody or antibody fragment.

[0242] Illustrative Embodiment 22. The method according to any one of Illustrative Embodiments 1-21, wherein the particles are coated with streptavidin.

[0243] Illustrative Implementation Scheme 23. The method according to any one of Illustrative Implementation Schemes 1-22, wherein the detection step can detect concentration differences of less than 5 pg / mL (e.g., less than 4 pg / mL, 1 pg / mL to 5 pg / mL, 2-5 pg / mL, 2-4 pg / mL, 3-4 pg / mL, 2-3 pg / mL).

[0244] Illustrative Embodiment 24. The method according to any one of Illustrative Embodiments 1-23, wherein the preparation step includes separating particles from the mixture and triggering chemiluminescence by the particles or the separated mixture.

[0245] Illustrative Embodiment 25. The method according to any one of Illustrative Embodiments 1-24, wherein the method further comprises incubating the mixture prior to adding the particles.

[0246] Illustrative Embodiment 26. The method according to Illustrative Embodiment 25, wherein the incubation includes heating the mixture for more than 30 minutes (e.g., 30 to 120 minutes, 30 to 60 minutes).

[0247] Illustrative Embodiment 27. The method according to any one of Illustrative Embodiments 1-26, wherein the method further includes incubating the mixture after adding the particles.

[0248] Illustrative Embodiment 28. The method according to Illustrative Embodiment 27, wherein the incubation includes heating the mixture for less than 30 minutes (e.g., 10 to 30 minutes, 10 to 20 minutes).

[0249] Illustrative Embodiment 29. An immunoassay composition comprising a chemiluminescent label conjugated to a primary antibody fragment binding to a neurofilament, wherein the chemiluminescent label comprises a polyethylene glycol linker (e.g., PEG2-PEG). 15 The linker binds to a carrier protein such as PEG4, and the linker binds to an antibody fragment; and a carrier or excipient.

[0250] Illustrative Embodiment 30. An immunoassay composition according to Illustrative Embodiment 29, wherein the composition further comprises a buffer solution.

[0251] Illustrative Embodiment 31. An immunoassay composition according to Illustrative Embodiment 29 or 30, wherein the carrier protein is bovine serum albumin.

[0252] Illustrative Embodiment 32. An immunoassay composition according to any one of Illustrative Embodiments 29-31, wherein the weight ratio of the chemiluminescent acridine to the carrier protein is 50:1 to 1:1 by weight (e.g., 30:1 to 1:1, 25:1 to 5:1).

[0253] Illustrative Embodiment 33. A solid particle coated with streptavidin, said streptavidin optionally being conjugated to a biotinylated mouse antineurofilament antibody via a linker (e.g., PEG, iodo-PEG), said antineurofilament antibody binding to neurofilaments, and The bound neurofilaments further bind to a monoclonal mouse antibody fragment linked to a carrier protein, which is conjugated with one or more chemiluminescent acridine onions.

[0254] Illustrative Embodiment 34. Solid particles according to Illustrative Embodiment 33, wherein the monoclonal mouse antibody fragment is linked to a carrier protein via a linker (e.g., PEG).

[0255] Illustrative Implementation Scheme 37. A method comprising: a) Obtaining the first biological sample from the subject; b) Quantify the concentration of the first neurofilament (NfL) in the first biological sample; c) Optionally, a second biological sample may be obtained from the subject some time after the first biological sample collection; d) Optionally quantify the concentration of second neurofilaments (NfL) in a second biological sample; e) Optionally determine the rate of change of neurofilament concentration between the first and second concentrations during the said time period; and f) Associate the rate of change in the first concentration and / or neurofilaments with the risk associated with the subject having a neurological disease, condition, or status.

[0256] Illustrative Embodiment 38. The method according to Illustrative Embodiment 37, wherein the biological sample is a blood sample, and the method further includes correlating the rate of change in the biological sample with the rate of change in serum NfL.

[0257] Illustrative Embodiment 39. The method according to Illustrative Embodiment 37 or 38, wherein the method further includes assessing the risk and / or progression of neurodegenerative diseases (e.g., by comparing the rate of change of blood NfL concentration and / or serum NfL concentration with one or more disease progression indicators based on said concentration).

[0258] Illustrative Implementation Scheme 40. The method according to any one of Illustrative Implementation Schemes 37-39 further includes conducting one or more cognitive tests on the subject.

[0259] Illustrative Embodiment 41. The method according to any one of Illustrative Embodiments 37-40, wherein the first concentration and the second concentration are (or were) obtained independently by: The first biological sample or biological blood sample is mixed with the composition, the composition comprising a chemiluminescent label conjugated to a first antibody or antibody fragment that binds to a neurofilament, wherein the chemiluminescent label binds to a carrier protein comprising a linker, and the linker binds to the antibody or antibody fragment; Add particles to the mixture, the particles having a second antibody or antibody fragment attached to the particle surface that binds to the analyte; The mixture is prepared to measure chemiluminescence (e.g., by separating particles with chemiluminescent acridine on their surface). Triggering chemiluminescence from the prepared material; and The presence of a neurofilament or the concentration of a neurofilament can be detected or calculated by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of emitted light with a known concentration of the neurofilament. By any of the methods in illustrative implementation schemes 1-28.

[0260] Illustrative Implementation Scheme 42. The method according to any one of Illustrative Implementation Schemes 37-41 further includes providing the subject with a treatment plan if a risk of neurodegenerative disease is found.

[0261] Illustrative Embodiment 43. The method according to Illustrative Embodiment 42, wherein the treatment regimen includes the administration of a therapeutic agent.

[0262] Illustrative Implementation Scheme 44. The method according to Illustrative Implementation Scheme 42 or 43, wherein the treatment scheme includes guidance on preventive measures, such as exercise, good nutrition, optimized sleep patterns, cognitive training or behavioral training.

[0263] Illustrative Implementation Scheme 45. The method according to any one of Illustrative Implementation Schemes 37-44, wherein the first biological sample and / or the second biological sample is obtained by a user.

[0264] Illustrative Implementation Scheme 46. The method according to any one of Illustrative Implementation Schemes 37-45, wherein the first biological sample and / or the second biological sample is obtained by the user's primary healthcare physician.

[0265] Illustrative Implementation Scheme 47. The method according to any one of Illustrative Implementation Schemes 37-46 is performed at the user's point of care and / or home.

[0266] Illustrative Embodiment 48. The method according to any one of Illustrative Embodiments 37-47, wherein the method further comprises transmitting the concentration or measured chemiluminescence output to at least one or more servers.

[0267] Illustrative Implementation Scheme 49. The method according to any one of Illustrative Implementation Schemes 37-48, wherein the neurological disease, condition, or condition is selected from multiple sclerosis (e.g., relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS)), neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease (PD), Lewy body dementia (DLB), Huntington's disease (HD), multiple system atrophy (MSA), prions, acute neurological injuries (e.g., traumatic brain injury (TBI), stroke, spinal cord injury (SCI), cardiac arrest, hypoxic-ischemic encephalopathy (HIE), neurocritical care monitoring), and autoimmune diseases (e.g., neuromyelitis optica spectrum disorder (NMOSD), autoimmune encephalitis (AE)). Systemic lupus erythematosus (SLE) (e.g., with central nervous system (CNS) involvement), primary Sjögren's syndrome (e.g., with CNS involvement)), neurological complications (e.g., infectious diseases such as HIV, COVID-19), metabolic diseases such as type 2 diabetes, sleep disorders such as obstructive sleep apnea, substance use disorders such as alcohol use disorder, chronic kidney disease), mental health conditions (e.g., depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD)), brain function and health in certain settings (e.g., space medicine and astronaut health, military and combat-related trauma, environmental and occupational exposure, sports medicine / performance optimization), and age-related brain function and health (e.g., monitoring brain aging, predicting cognitive decline, assessing the impact of lifestyle factors, distinguishing normal aging from disease, assessing all-cause mortality risk).

[0268] Illustrative Embodiment 50. A computer system comprising one or more servers, said servers being collectively configured to: a) Receive data, including the concentration of neurofilaments in a user's biological sample; b) Compare the concentration with historical data on the progression of neurological disorders to assess the user's risk of developing the neurological disorder; and c) Risk assessment related to transmission and disease progression.

[0269] Illustrative Embodiment 51. A computer system according to Illustrative Embodiment 50, wherein the one or more servers are collectively configured to transmit performance tests (e.g., to a user's computing device), receive data related to the user's performance during the performance test, and use the data related to the user's performance test in a risk assessment.

[0270] Illustrative Implementation Scheme 52. A computer system according to Illustrative Implementation Scheme 51, wherein the performance test includes verbal analysis.

[0271] Illustrative Implementation Scheme 53. A computer system according to any one of Illustrative Implementation Schemes 50-52, wherein the one or more servers are collectively configured as follows: Receive data, including the concentration of neurofilaments in a second biological sample of the user, which is collected at a time point after the user's biological sample is collected. The change in neurofilament concentration over time was used in the second risk assessment; and Second risk assessment for transmission.

[0272] Illustrative Embodiment 54. A computer system according to any one of Illustrative Embodiments 50-53, wherein the computer system further includes an immunoassay device configured for quantifying the concentration of neurofilaments in a biological sample, wherein the immunoassay device is capable of transmitting information via a network, and The one or more servers receive data, including neurofilament concentrations, from the immunoassay device via a network.

[0273] Illustrative Embodiment 55. A computer system according to Illustrative Embodiment 54, wherein the immunoassay device includes a sequential array of reaction sites for measuring chemiluminescence from a biological sample (or a medium derived therefrom), wherein a mixture can be sequentially placed in each reaction site; the sequential array of reaction sites includes one or more of the following: Biological sample addition location: This is where biological samples are added to the reaction vessel. At the assay reagent site, the assay reagent (e.g., a solid-phase reagent comprising magnetizable particles on which molecules capable of forming binding complexes with neurofilament light chains or their binding partners are immobilized; chemiluminescent conjugates, such as acridinium compounds, including acridinium esters and acridinium sulfonamides capable of forming binding complexes with neurofilaments or molecules immobilized on magnetizable particles; or both) is added to the reaction vessel (e.g., a reaction vessel containing a biological sample). Incubation sites are used to bind analytes, chemiluminescent compounds, and magnetizable particles from biological samples; The isolation location magnetically isolates the magnetizable particles from the liquid medium from the biological sample and the assay reagents; Separation site, where liquid media (e.g., from biological samples, from assay reagents) are separated from magnetizable particles (e.g., isolated magnetizable particles); At the washing location, a washing buffer (e.g., a buffer solution that may contain one or more salts such as sodium chloride and sodium azide, detergents such as cationic detergents, buffers such as phosphates, blocking agents such as bovine serum albumin (BSA) or combinations thereof) is added to the magnetizable particles; and Wash buffer aspiration site, where the wash buffer is separated from the magnetizable particles to form a chemiluminescent sample; The first chemiluminescence reagent addition location is where the first chemiluminescence trigger reagent can be added to the chemiluminescent sample; The second chemiluminescent reagent addition site is where the second chemiluminescent triggering reagent is added to the chemiluminescent sample (or a portion thereof or a portion derived therefrom); and Chemiluminescence location, where chemiluminescence is collected by one or more photon detectors in order to measure the light output from the chemiluminescent sample; The amount of chemiluminescence is related to the concentration of NfL in the biological sample, and the immunoassay device transmits the amount of chemiluminescence and / or the concentration to the one or more servers.

[0274] Illustrative Embodiment 56. A kit comprising a specimen collection device for collecting biological samples from a user and instructions on how to use the specimen collection device to collect biological samples and / or how to access a computer system according to any one of Illustrative Embodiments 50-55.

[0275] Illustrative Embodiment 56. Illustrative embodiments 1-55 are configured for large-scale population screening, routine physician office use, or self-testing methods suitable for and feasible indicative of neurodegenerative diseases such as Alzheimer's disease and MS. In these embodiments, indications for the disease are characterized as early detection. In these embodiments, a kit is provided containing components that allow the execution of embodiment 56.

[0276] Illustrative Embodiment 57. Illustrative embodiments 1-55 are configured to use NfL as a key step in a combined algorithm for early screening of individuals reporting cognitive problems (such as self-reported problems) and interested in assessing dementia risk, or for use in such screening as can become routine in a healthcare setting. Furthermore, due to its high sensitivity to neurodegenerative changes, minimally invasive acquisition method, and precise quantification, this disclosure is well-suited for monitoring the effectiveness of preventative or curative interventions.

[0277] Illustrative Embodiment 58. Illustrative Embodiments 1-55 are configured to use NfL for large-scale population screening for Alzheimer's disease, MS, or other neurodegenerative diseases in a practical manner. In these embodiments, this disclosure includes providing NfL testing directly to consumers to assess their risk of developing such diseases or to monitor the impact of preventative measures such as exercise, good nutrition, optimized sleep patterns, cognitive training, and other interventions, including behavioral or pharmacological interventions.

[0278] All references cited herein, including patent applications and publications, are incorporated herein by reference and for all purposes to the same extent that each individual publication or patent or patent application is specifically and individually indicated by reference as incorporated herein by reference in its entirety for all purposes. Many modifications and variations of the invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The embodiments described herein are provided by way of example only, and the invention is limited only by the terms of the appended claims, together with the full scope of equivalents conferred by such claims.

Claims

1. A method comprising: g) Obtain the first biological sample from the subject; h) Quantify the concentration of the first neurofilament (NfL) in the first biological sample; i) Optionally, a second biological sample may be obtained from the subject some time after the first biological sample has been collected; j) Optionally quantify the concentration of the second neurofilament (NfL) in the second biological sample; k) Optionally determine the rate of change of neurofilament concentration between the first concentration and the second concentration during the said time period; and l) Associate the rate of change of the first concentration and / or the neurofilament with the risk associated with the subject having a neurological disease, condition, or status.

2. The method of claim 1, wherein the biological sample is a blood sample, and the method further comprises correlating the rate of change in the biological sample with the rate of change in serum NfL.

3. The method according to claim 1 or 2, wherein the method further comprises assessing the risk and / or progression of neurodegenerative diseases.

4. The method according to any one of claims 1-3, further comprising performing one or more cognitive tests on the subject.

5. The method according to any one of claims 1-4, wherein the first concentration and the second concentration are, or were, obtained independently by: The first biological sample or biological blood sample is mixed with the composition, the composition comprising a chemiluminescent marker conjugated to a first antibody or antibody fragment that binds to a neurofilament, wherein the chemiluminescent marker binds to a carrier protein comprising a linker, and the linker binds to the antibody or antibody fragment; Add particles to the mixture, the particles having a second antibody or antibody fragment attached to the particle surface that binds to the analyte; The mixture was prepared to measure chemiluminescence; Trigger chemiluminescence from the prepared material; as well as The presence of a neurofilament or the concentration of a neurofilament is detected by comparing the amount of chemiluminescence with a standard dose-response curve that correlates the amount of emitted light with a known concentration of the neurofilament.

6. The method according to any one of claims 1-5, further comprising providing a treatment plan to the subject if a risk of neurodegenerative disease is detected.

7. The method of claim 6, wherein the treatment regimen includes the administration of a therapeutic agent.

8. The method of claim 6 or 7, wherein the treatment regimen includes guidance on preventive measures, such as exercise, good nutrition, optimized sleep patterns, cognitive training, or behavioral training.

9. The method according to any one of claims 1-8, wherein the first biological sample and / or the second biological sample is obtained by a user.

10. The method according to any one of claims 1-9, wherein the first biological sample and / or the second biological sample is obtained by the user's primary care physician.

11. The method according to any one of claims 1-10, wherein the method is performed at the user's point of care and / or home.

12. The method according to any one of claims 1-11, wherein the method further comprises transmitting the concentration or measured chemiluminescence output to at least one or more servers.

13. The method according to any one of claims 1-12, wherein the neurological disease, condition, or condition is selected from multiple sclerosis (e.g., relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS), secondary progressive multiple sclerosis (SPMS)), neurodegenerative diseases (e.g., amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), frontotemporal dementia (FTD), Parkinson's disease (PD), Lewy body dementia (DLB), Huntington's disease (HD), multiple system atrophy (MSA), prions, acute neurological injuries (e.g., traumatic brain injury (TBI), stroke, spinal cord injury (SCI), cardiac arrest, hypoxic-ischemic encephalopathy (HIE), neurocritical care monitoring), and autoimmune diseases (e.g., neuromyelitis optica spectrum disorder (NMOSD), autoimmune encephalitis (AE)). Systemic lupus erythematosus (SLE) (e.g., with central nervous system (CNS) involvement), primary Sjögren's syndrome (e.g., with CNS involvement)), neurological complications (e.g., infectious diseases such as HIV, COVID-19), metabolic diseases such as type 2 diabetes, sleep disorders such as obstructive sleep apnea, substance use disorders such as alcohol use disorder, chronic kidney disease), mental health conditions (e.g., depression, bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD)), brain function and health in certain settings (e.g., space medicine and astronaut health, military and combat-related trauma, environmental and occupational exposure, sports medicine / performance optimization), and age-related brain function and health (e.g., monitoring brain aging, predicting cognitive decline, assessing the impact of lifestyle factors, distinguishing normal aging from disease, assessing all-cause mortality risk).

14. A computer system comprising one or more servers, said servers being collectively configured as follows: a) Receive data, including the concentration of neurofilaments in a user's biological sample; b) Compare the concentration with historical data on the progression of neurological disorders to assess the user's risk of developing the neurological disorder; and c) Risk assessment related to transmission and disease progression.

15. The computer system of claim 14, wherein the one or more servers are collectively configured to transmit performance tests, receive data related to a user's performance during a performance test, and use the data related to the user's performance test in a risk assessment.

16. The computer system of claim 15, wherein the performance test includes verbal analysis.

17. The computer system according to any one of claims 14-16, wherein the one or more servers are collectively configured as follows: Receive data, including the concentration of neurofilaments in a second biological sample of the user, which is collected at a time point after the user's biological sample is collected. The change in neurofilament concentration over time was used in the second risk assessment; as well as Second risk assessment for transmission.

18. The computer system according to any one of claims 14-17, wherein the computer system further comprises an immunoassay device configured for quantifying the concentration of neurofilaments in a biological sample, wherein the immunoassay device is capable of transmitting information via a network, and The one or more servers receive data, including neurofilament concentrations, from the immunoassay device via a network.

19. The computer system of claim 18, wherein the immunoassay device comprises a sequential array of reaction sites for measuring chemiluminescence from a biological sample or a medium derived therefrom, wherein a mixture may be sequentially placed in each reaction site; the sequential array of reaction sites comprises one or more of the following: Biological sample addition location: This is where biological samples are added to the reaction vessel. The reagent is located here; add the reagent to the reaction vessel. Incubation sites are used to bind analytes, chemiluminescent compounds, and magnetizable particles from biological samples; The isolation location magnetically isolates the magnetizable particles from the liquid medium from the biological sample and the assay reagents; Separation point, where the liquid medium is separated from the magnetizable particles; Washing location, where washing buffer is added to the magnetizable particles; as well as Wash buffer aspiration site, where the wash buffer is separated from the magnetizable particles to form a chemiluminescent sample; The first chemiluminescence reagent addition location is where the first chemiluminescence trigger reagent can be added to the chemiluminescent sample; The second chemiluminescent reagent addition site is where the second chemiluminescent triggering reagent is added to the chemiluminescent sample; as well as Chemiluminescence location, where chemiluminescence is collected by one or more photon detectors in order to measure the light output from the chemiluminescent sample; The amount of chemiluminescence is correlated with the concentration of NfL in the biological sample, and the immunoassay device transmits the amount of chemiluminescence and / or the concentration to the one or more servers.

20. A kit comprising a specimen collection device for collecting biological samples from a user and instructions on how to use the specimen collection device to collect biological samples and / or how to access a computer system according to any one of claims 14-19.

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