Neurofilament light chain protein test kit

Abstract

This invention relates to kits for diagnosing biomarkers of neurodegenerative diseases, and more particularly to kits for detecting neurofilament light chain protein (NF-L), said kits being based on single-molecule detection technology for NF-L detection. Additionally, it relates to single-molecule detection systems for NF-L detection. This invention achieves extremely high sensitivity while enabling rapid detection, and the kits are highly stable, simple in design, and low in cost.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and in particular to a kit for detecting neurofilament light chain protein (NF-L) based on single-molecule counting, and a single-molecule detection system for detecting neurofilament light chain protein. Background Technology

[0002] Neurofilament light chain proteins (NF-Ls) are components of neurofilaments (NFs) and, along with glial filaments, are a major type of intermediate filaments (IFs) in the nervous system. Their physiological functions include maintaining axon diameter by preserving characteristic cell shape, regulating intracellular communication between axons and dendrites, and indirectly regulating nerve conduction velocity, thereby conferring resistance to mechanical stress. Recent studies have also shown that they are important for normal synaptic function. Axonal dysfunction and degeneration are crucial steps in the pathogenesis of neurodegenerative diseases (NDDs), occurring long before nerve cell death, typically prior to the deposition of detectable misfolded proteins. During these processes, neurofilament light chain proteins are released back into the extracellular space, thereby entering body fluids such as cerebrospinal fluid (CSF) and blood.

[0003] Neurofilament light chain protein (NSCLT) concentration is an important indicator of axonal degeneration. Serum / plasma NSCLT concentrations are closely correlated with cerebrospinal fluid NSCLT concentrations, directly reflecting neurodegenerative changes within the central nervous system. In various scenarios, NSCLT is currently the most promising candidate biomarker for the early identification of common neurodegenerative processes, supporting disease diagnosis, prognosis, and progression, as well as monitoring eventual disease improvement and treatment. Therefore, the research and development of NSCLT diagnostic kits has significant clinical value.

[0004] Currently known methods for NF-L detection mainly include enzyme-linked immunosorbent assay (see patent document 1), chemiluminescence assay (see patent documents 2-4), single-molecule detection method based on chemiluminescence (see patent document 5), immunochromatography (see patent document 6), and fluorescence immunoassay.

[0005] Specifically, Patent Document 1 discloses a kit and in vitro detection device for luminescent enzyme-linked immunosorbent assay (ELISA). The kit includes: an aqueous solution for stabilizing body fluid samples containing neuropathic protein markers; the solutes in the stabilizing solution include human serum, animal serum albumin, inorganic alkali metal salts, Tris base, protein denaturing agents, and nonionic surfactants; the pH of the aqueous solution is 6.7–7.6; and optionally, an antibody against the neuropathic protein markers. While this luminescent ELISA method achieves higher sensitivity than conventional ELISA, the operation is still cumbersome and time-consuming (1–2 hours, determined by the characteristics of the ELISA method itself), and the sensitivity is only around 20 pg / mL.

[0006] Patent document 2 discloses a magnetic microparticle chemiluminescence detection kit for determining the content of neurofilament light chain protein in humans, characterized by comprising: reagent R1, reagent R2, magnetic separation reagent, a series of calibrating solutions, and a chemiluminescent substrate solution; wherein, reagent R1 is a diluent for anti-neurofilament light chain protein monoclonal antibody labeled with fluorescein isothiocyanate, reagent R2 is a diluent for anti-neurofilament light chain protein antibody labeled with alkaline phosphatase, the magnetic separation reagent is a magnetic microparticle diluent coated with anti-fluorescein isothiocyanate monoclonal antibody, the series of calibrating solutions are antigen diluents containing different concentrations of neurofilament light chain protein, and the chemiluminescent substrate solution is a substrate solution catalyzed by alkaline phosphatase. The buffer solution used to prepare reagent R1 has a pH of 7.2–8.0 and includes Tris at a concentration of 12.0–12.3 g / L, sodium azide at a concentration of 1.98–1.99 g / L, sodium chloride at a concentration of 5.7–5.9 g / L, 1M magnesium chloride solution at a concentration of 0.8–1.2 mL / L, 0.1M zinc chloride solution at a concentration of 0.8–1.2 mL / L, fish skin gelatin at a concentration of 5–20 g / L, bovine serum albumin at a concentration of 2–5 g / L, and newborn calf serum at a concentration of 10–50 g / L. The remainder is deionized... The buffer solution used to prepare the magnetic separation reagent has a pH of 7.5–9.0 and consists of Tris at a concentration of 10.5–11.3 g / L, sodium azide at a concentration of 1.91–1.95 g / L, sodium chloride at a concentration of 5.5–5.7 g / L, 0.8–1.2 mL / L of 1M magnesium chloride solution, 0.8–1.2 mL / L of 0.1M zinc chloride solution, 4.7–4.9 g / L of bovine serum albumin, and 4.8–5.0 g / L of premium horse serum. The remaining component is deionized water. This method is based on the traditional chemiluminescence method, which is complex and involves numerous steps. The buffer solution also has many components, resulting in high cost. Furthermore, the sensitivity achieved by this method is 0.1 pg / mL, which, although improved compared to traditional ELISA kits (0.1 ng / mL), is still insufficient and cannot meet the needs for detecting trace amounts of NF-L. It should also be noted that although Table 2 states a sensitivity of 0.009 pg / mL (which is clearly contradictory to the content of paragraph 0033 in the instruction manual), those skilled in the art can determine from the fact that it only provides the concentrations at points A and B and the preparation of the standard curve that this is a clear typographical error, and it is impossible for it to achieve such a low detection sensitivity.

[0007] Patent document 5 describes a chemiluminescence-based single-molecule detection method. This method utilizes a unique single-molecule detection technology from Quanterix, Inc. (USA), dispersing the analyte into an extremely small (nano-level) chamber. This makes the operation cumbersome, requires extensive operational experience, and necessitates high-precision droplet generation equipment. Furthermore, the disclosed reagent kit is also complex in composition, with multiple components in the buffer system, further increasing costs. However, according to data published by Quanterix for NF-L detection, its optimal sensitivity is approximately 0.038 pg / mL, which is currently the best result achievable in the field.

[0008] Patent document 6 discloses a rapid detection method for Alzheimer's disease biomarkers. The detection method includes the following steps: (1) preparing a lateral flow test strip for detecting Alzheimer's disease biomarkers; (2) adding the sample solution to be tested onto the sample pad of the test strip; (3) after standing for 10-30 minutes, performing Raman spectroscopy analysis on the control line of the test strip using a Raman spectrometer: if the Raman spectrum of the obtained control line shows the characteristic peaks of all the Raman labels used, it indicates that the test strip detection result is valid; otherwise, a new test strip needs to be replaced and the detection repeated; if the Raman spectrum of the obtained control line shows the characteristic peaks of all the Raman labels used, then the Raman spectrum analysis on the detection line of the test strip is performed again using a Raman spectrometer to obtain the concentration of the corresponding Alzheimer's disease biomarker. This method is time-consuming (about half an hour) and does not disclose specific sensitivity data for NF-L. It is generally believed that the immunochromatographic method, as a semi-quantitative method, is difficult to achieve high sensitivity.

[0009] Single-molecule detection technology, characterized by counting, has significantly improved sensitivity compared to existing enzyme-linked immunosorbent assays (ELISA) and chemiluminescence immunoassays based on overall light intensity due to its breakthrough in principle. Therefore, its application for quantitative detection of low-content biomarkers is being researched (e.g., Patent Document 5). The inventors of this application have proposed a unique single-molecule detection method (see Patent Document 7), which uses in-situ signal-enhancing nanoparticles and magnetic beads, based on a double-antibody sandwich method, to perform single-molecule detection of cTnI protein, IL-6 protein, DNA, etc., achieving a low detection limit. However, it does not mention how to detect neurofilament light chain proteins, nor does it address the influence of buffer systems on the detection of neurofilament light chain proteins.

[0010] Existing technical documents

[0011] Patent Document 1: CN109696549A;

[0012] Patent Document 2: CN110531085A;

[0013] Patent document 3: CN115166229A;

[0014] Patent document 4: CN114184604A;

[0015] Patent Document 5: WO2019199871A1;

[0016] Patent document 6: CN111781376A;

[0017] Patent Document 7: WO2020156029A1 Summary of the Invention

[0018] The problem that the invention aims to solve

[0019] It is well known that longer detection times (especially incubation times) can improve detection sensitivity, and there is a trade-off between shortening detection time and improving detection sensitivity; it is difficult to simultaneously achieve both reduced detection time and increased sensitivity. Patent Document 7 does not mention how to detect neurofilament light chain proteins, nor does it address the impact of buffer systems on neurofilament light chain protein detection. The inventors of this application attempted to apply the single-molecule detection method of Patent Document 7 to the development of a neurofilament light chain protein kit. However, since neurofilament light chain proteins are completely different from the cTnI antigen, IL-6 antigen, DNA, etc., described in that document, directly applying the detection systems for cTnI antigen, IL-6 antigen, DNA, etc., described in its examples to the detection of neurofilament light chain proteins resulted in unsatisfactory sensitivity.

[0020] To address the aforementioned issues, the applicant repeatedly optimized the types of capture antibodies, detection antibodies, fluorescent dyes, and magnetic bead groups, but the sensitivity remained unsatisfactory. Regarding the buffer system, the inventors attempted to use buffers from existing NF-L kits, such as those described in Patent Documents 1-5, during the initial optimization process. However, they found that when these buffer systems were directly applied to single-molecule detection methods characterized by counting, even with extended incubation times, the optimal sensitivity was unsatisfactory and could not meet the requirements for detecting trace amounts of NF-L. Furthermore, these buffer systems had numerous components, resulting in high preparation costs. After careful analysis, the applicant concluded that this was because the buffers in existing NF-L kits were designed for use with their respective methods. When these buffer systems were directly used in single-molecule detection methods characterized by counting, they might exhibit adverse interactions with other components in the method (e.g., in-situ signal enhancement particles). Therefore, it is necessary to find a buffer that can appropriately complement fluorescent dye systems with specific particle sizes (180-450 nm) and magnetic beads in single-molecule detection methods characterized by counting.

[0021] The present invention was made in view of the above-mentioned problems, and aims to provide a neurofilament light chain protein kit that can achieve high sensitivity and good stability while enabling rapid detection.

[0022] Methods for solving problems

[0023] To address the aforementioned issues, the inventors of this application conducted repeated and in-depth research, resulting in the discovery of a neurofilament light chain protein reagent kit that can achieve high sensitivity and good stability while enabling rapid detection.

[0024] One technical solution of this application is as follows.

[0025] A kit for detecting neurofilament light chain proteins based on single-molecule counting, characterized in that it comprises magnetic beads coated with capture antibodies, detection antibodies labeled with fluorescent dyes, buffer 1 for the aforementioned magnetic beads coated with capture antibodies, and buffer 2 for the aforementioned detection antibodies labeled with fluorescent dyes.

[0026] The aforementioned capture antibody and detection antibody can bind to different sites on neurofilament light chain proteins.

[0027] The aforementioned fluorescent dyes contain fluorescent materials and carriers, and have a particle size of 180–450 nm.

[0028] The aforementioned buffer solution 1 has a pH of 5.5–6.5, and each 100 mL of buffer solution consists of 40 mM–60 mM MES (morpholinoethanesulfonic acid), 0.8 g–1.2 g of animal serum albumin, 0.8 mL–1.2 mL of nonionic surfactant, 500–700 mM of inorganic alkali metal salt, and sterile distilled water.

[0029] The pH of the aforementioned buffer 2 is 7.0–7.6. Each 100 mL of buffer consists of 20 mM–40 mM PBS (also known as phosphate buffer), 0.3 g–0.6 g of animal serum albumin, 100–200 mM of inorganic alkali metal salts, and sterile distilled water. It should be noted that the "inorganic alkali metal salts" mentioned here do not include NaCl, KCl, etc., found in PBS.

[0030] Preferably, the aforementioned nonionic surfactant is Tween-20, Tween-40, Tween-60, Tween-80, or a mixture of at least two of them, preferably Tween-20, and the aforementioned nonionic surfactant is 0.9 mL to 1.1 mL in every 100 mL of the aforementioned buffer solution 1.

[0031] Preferably, the aforementioned inorganic alkali metal salt is sodium chloride or potassium chloride, and more preferably, the inorganic alkali metal salt in buffer solutions 1 and 2 is sodium chloride.

[0032] Furthermore, in each 100 mL of the aforementioned buffer solution 1, the inorganic alkali metal salt concentration is 550–650 mM.

[0033] In each 100 mL of the aforementioned buffer solution 2, the inorganic alkali metal salt is 120–180 mM.

[0034] Preferably, the aforementioned animal serum albumin is bovine serum albumin (BSA), and its mass is 0.9g to 1.1g per 100mL of buffer 1 and 0.4g to 0.6g per 100mL of buffer 2.

[0035] Preferably, the aforementioned fluorescent material is an aggregation-induced emission material, a fluorescein-based luminescent material, a rhodamine-based luminescent material, or a quantum dot-based luminescent material, and the aforementioned carrier is silica, polyacrylamide, or polystyrene.

[0036] Another technical solution of this application is as follows.

[0037] A single-molecule detection system for detecting neurofilament light chain proteins includes the aforementioned reagent kit and optical imaging equipment.

[0038] The aforementioned optical imaging device includes a light source and an optical signal acquisition unit, and the aforementioned detection system does not include a total internal reflection microscope, a near-field microscope, or an Airy disk focusing detection device, nor does it include a microreactor with a volume of nanoliter, picoliter, or femtoliter.

[0039] Effects of the present invention

[0040] The kit for detecting neurofilament light chain protein (NF-L) of this invention achieves high sensitivity and good stability while enabling rapid detection. The inventors of this application have discovered that, in some embodiments for the detection of NF-L, the sensitivity can reach 0.035 pg / mL (superior to the best sensitivity for NF-L detection in the art), and in other embodiments, the sensitivity can reach 0.025 pg / mL. Furthermore, this application enables the detection of NF-L in a short time while maintaining high sensitivity; specifically, the incubation time can be reduced to approximately 5 minutes (approximately 5 minutes and 40 seconds from sample injection to result output), thereby significantly shortening the clinical testing time. In addition, the kit exhibits high stability, with both the relative error (RE) and coefficient of variation (CV) of the test results being less than 10% over a one-year period. Moreover, this invention allows the detection of neurofilament light chain protein using whole blood samples, serum samples, or plasma samples, eliminating the need for samples taken from cerebrospinal fluid, thus minimizing patient trauma and significantly reducing testing costs.

[0041] Compared to existing kits based on enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, chemiluminescence-based single-molecule detection, and immunochromatography (e.g., Patent Documents 1-6), the NF-L detection kit of the present invention, characterized by single-molecule counting based on a specific system, significantly shortens the detection time while improving sensitivity, and maintains comparable stability with a wide dynamic range. Since the detection kit of the present invention allows for a wider range of results, the amount of signal generated during measurement can be controlled, thereby reducing signal oversaturation and minimizing the hook effect. Furthermore, the buffer system of the detection antibody labeled with fluorescent dye and the magnetic beads coated with capture antibody in this application is simple, has few components, and uses inexpensive and readily available components, resulting in lower cost. For these reasons, the kit of the present invention has broad commercial application prospects. Attached Figure Description

[0042] Figure 1 The standard curve obtained in Example 1 is shown, where the vertical axis represents the number of single-molecule signals. Detailed Implementation

[0043] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but all such modifications or improvements are within the scope of the invention as long as they do not depart from the basic idea. Unless otherwise stated, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. In case of any conflict, this specification shall prevail. The description of numerical ranges in the specification explicitly includes each intermediate number within the range having the same precision. For example, for the range 40mM to 60mM, in addition to 40mM and 60mM, it also includes 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM and 60mM. For the range 5.5 to 6.5, it explicitly includes the values ​​5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5.

[0044] I. Reagent Kit

[0045] The first embodiment of this application relates to a kit for detecting neurofilament light chain proteins based on single-molecule counting, characterized in that it comprises magnetic beads coated with capture antibodies, detection antibodies labeled with fluorescent dyes, buffer 1 for the aforementioned magnetic beads coated with capture antibodies, and buffer 2 for the aforementioned detection antibodies labeled with fluorescent dyes.

[0046] The aforementioned capture antibody and detection antibody can bind to different sites on neurofilament light chain proteins.

[0047] The aforementioned fluorescent dyes contain fluorescent materials and carriers, and have a particle size of 180–450 nm.

[0048] The aforementioned buffer solution 1 has a pH of 5.5–6.5, and each 100 mL of buffer solution consists of 40 mM–60 mM MES, 0.8 g–1.2 g animal serum albumin, 0.8 mL–1.2 mL nonionic surfactant, 500–700 mM inorganic alkali metal salt, and sterile distilled water.

[0049] The pH of the aforementioned buffer 2 is 7.0–7.6, and each 100 mL of buffer consists of 20 mM–40 mM PBS, 0.3 g–0.6 g animal serum albumin, 100–200 mM inorganic alkali metal salt, and sterile distilled water.

[0050] The single-molecule counting used in this specification refers to determining the concentration of NF-L by counting individual molecules rather than measuring the overall fluorescence intensity of the solution for NF-L labeled with fluorescent dyes.

[0051] Neurofilament light chain protein (NF-L) is a neuron-specific cytoskeletal protein with a molecular weight of 68 kDa. It is the first substructure of neurofilament proteins to be expressed, particularly in large-diameter myelinated axons. It can be actively (e.g., via exosomes) or passively released from neurons, secondary to axonal injury and loss of neuronal membrane integrity, into the interstitial space, where it freely exchanges with cerebrospinal fluid (CSF).

[0052] A kit is a container used to hold chemical reagents for detecting chemical components, drug residues, virus types, etc. In this instruction manual, it specifically refers to a kit containing multiple reagents for detecting NF-L concentration in a sample. The kit also includes accompanying instructions, reaction cups, and waste cups, details of which are omitted here.

[0053] As used in this specification, "antibody" refers to monoclonal antibodies, monospecific antibodies (e.g., antibodies produced by methods other than those using ordinary germ cells), multispecific antibodies, human antibodies, humanized antibodies (fully or partially humanized antibodies), animal antibodies (e.g., but not limited to: birds (e.g., ducks or geese), sharks, whales, and mammals, including non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.) or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, and single-chain Fv ("scFv"). Antibodies can be monoclonal or polyclonal, including single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, etc., preferably monoclonal / polyclonal mouse / rabbit antibodies. Additionally, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing analyte binding sites. Immunoglobulin molecules can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of protein molecules, preferably IgG. For simplicity, antibodies against NF-L are generally referred to as NF-L antibodies in this specification.

[0054] This invention is also a kit based on a double-antibody sandwich assay, in which the capture antibody and the detection antibody bind to different epitopes of the antigen (NF-L). Ideally, the binding of the capture antibody to the NF-L epitope does not interfere with the binding of the detection antibody to the NF-L epitope. Both monoclonal and polyclonal antibodies can be used as the capture and detection antibodies in the sandwich immunoassay. Capture antibodies are classified according to their antibody specificity characteristics as one or both of polyclonal and monoclonal antibodies, and according to their source as one or more of mouse, rabbit, sheep, and alpaca antibodies. Detection antibodies are classified according to their antibody specificity characteristics as one or both of polyclonal and monoclonal antibodies, and according to their source as one or more of mouse, rabbit, sheep, and alpaca antibodies.

[0055] Magnetic beads can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluidic. Typical ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, and Fe3O4 (FeO·Fe2O3). Magnetic beads can be a magnetic solid core surrounded by one or more non-magnetic layers. The magnetic portion can also be a layer surrounding the non-magnetic core. The surface of magnetic beads is modified with active functional groups capable of covalently coupling with antibodies, such as hydroxyl, carboxyl, amino, succinimidyl, sulfonyl (e.g., toluenesulfonyl) and one or more of their derivatives.

[0056] The term "capture antibody-coated magnetic beads" refers to a complex obtained by combining a capture antibody with magnetic beads whose surface is modified with active functional groups capable of covalently coupling with the capture antibody. The preparation process may include steps such as magnetic bead washing, activation treatment, coupling of the magnetic beads with the capture antibody, post-coupling washing, and blocking. Regarding the activator used in the activation treatment, for example, when the modified group on the magnetic bead surface is a carboxyl group, the activator can be one or more of N-hydroxysuccinimide (NHS), SulfoNHS, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), etc.; when the modified group is an amino group, the activator can be succinimide 4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), Sulfo-SMCC, etc.

[0057] The preparation process of the aforementioned "magnetic beads coated with capture antibodies" may include the following steps: magnetic bead washing, magnetic bead activation, magnetic bead antibody conjugation, post-conjugation washing, magnetic bead blocking, and storage of the magnetic bead working solution. In the storage of the magnetic bead working solution after magnetic bead blocking, the aforementioned "buffer solution 1 for magnetic beads coated with capture antibodies" is added; therefore, it can also be called a magnetic bead preservation solution. In the art, it is generally believed that the magnetic bead preservation solution has little impact on the detection system and will not cause significant fluctuations in sensitivity. However, the applicant has unexpectedly discovered that in a single-molecule detection system based on a specific particle size, as described in this invention, the composition and concentration of the magnetic bead preservation solution significantly affect the sensitivity when detecting the specific protein NF-L. The inventors of this application, focusing on magnetic bead preservation solutions that have not received much attention in the past, have discovered, through repeated experiments, a buffer system that can be used well with the following "buffer solution 2 for detecting antibodies labeled with fluorescent dyes" to significantly improve the detection sensitivity of NF-L. Specifically, the pH is 5.5 to 6.5, and each 100 mL of buffer solution consists of 40 mM to 60 mM MES, 0.8 g to 1.2 g animal serum albumin (preferably BSA), 0.8 mL to 1.2 mL nonionic surfactant (preferably Tween-20), 500 to 700 mM inorganic alkali metal salt (preferably NaCl), and sterile distilled water.

[0058] The term "detection antibody labeled with fluorescent dye" refers to a complex obtained by directly or indirectly binding a fluorescent dye to a detection antibody. Direct binding means the detection antibody is directly adsorbed or coupled to the fluorescent dye through physical adsorption or chemical modification. Indirect binding refers to binding via an anti-detection antibody (i.e., a secondary antibody) or a biotin-streptavidin system, specifically labeling the detection antibody with the fluorescent dye. The preparation process of this fluorescently labeled detection antibody may include the following steps:

[0059] (1) Preparation of diluent for fluorescent dye

[0060] Dilute the fluorescent dye to the specified concentration using buffer a (such as carbonate buffer, phosphate buffer, PBS, or borate buffer);

[0061] (2) Preparation of labeling incubation solution

[0062] The activator was dissolved in PBS buffer to prepare a buffer solution containing the activator. This solution was then added to diluted fluorescent dye, mixed, centrifuged, and the buffer solution was added to prepare the labeling incubation solution for later use.

[0063] (3) Preparation of labeling working solution

[0064] Add the original antibody solution to the above-mentioned labeling incubation solution, mix well, and incubate for 1 hour.

[0065] (4) Seal the labeling working solution

[0066] Add the blocking solution to the above-mentioned labeling working solution, mix well, and incubate for 1 hour.

[0067] (5) Washing and labeling working solution

[0068] (6) Preserve the labeling working solution

[0069] In step (6), the aforementioned "buffer solution 2 for the detection antibody labeled with fluorescent dye" was used. Specifically, this buffer solution was added to a centrifuge tube containing washed labeled working solution, and the labeled working solution was stored for later use. Therefore, this buffer solution can also be called a labeled storage solution. The applicant analyzed that by using the buffer solution of the specific composition and concentration of the present invention to store the specific labeled working solution (i.e., the liquid containing the detection antibody labeled with a specific fluorescent dye), the specific particle size of the fluorescent dye used in the present invention can be stably maintained without excessive aggregation, avoiding fluorescence quenching, thereby achieving high sensitivity. In addition, it is speculated that the buffer solution of the specific composition and concentration of the present invention has good compatibility with the detection antibody against NF-L, ensuring the stability of the system, which in turn helps to improve detection sensitivity and storage stability. The pH of buffer 2 for detecting antibodies labeled with fluorescent dyes is 7.0–7.6. Each 100 mL buffer consists of 20 mM–40 mM PBS, 0.3 g–0.6 g animal serum albumin, 100–200 mM inorganic alkali metal salt, and sterile distilled water.

[0070] The detection sensitivity used in this specification, also known as the limit of detection (LoD), refers to the lowest concentration of the analyte (i.e., the quantity to be measured) that can be detected at a specific confidence level. The confidence level is typically 95%, and the probability of a false negative measurement is 5%. Sensitivity is the lowest analyte concentration that can be reliably distinguished from the blank limit (LoB) and detected.

[0071] The fluorescent dye of the present invention is a material that enhances the fluorescence signal in situ to a level that can be detected by conventional optical imaging equipment, and it contains two parts: a fluorescent material and a carrier.

[0072] In the fluorescent dye, the carrier plays a crucial role. For example, it can bind more fluorescent material, resulting in a stronger luminescence signal; it provides sites for functionalization modification, enabling the binding of a large number of antibodies and improving reactivity; and it makes single-molecule detection possible using conventional fluorescence microscopy, which is impossible without a carrier. The carrier, classified by material, can be one or more of silica, polystyrene, polyacrylamide, poly(meth)acrylate, dextran, agarose, and inorganic metal compounds. Classified by structure, the carrier can be one or more of hollow, core-shell, porous, alloy, and hydrogel structures. From the viewpoint of ensuring uniform distribution and high brightness of the fluorescent material, silica, polyacrylamide, polystyrene, and dextran are preferred carriers.

[0073] The fluorescent material in the fluorescent dye is also essential for achieving single-molecule detection. The fluorescent material can be one or more of the following: fluorescent dye molecules, rare earth elements, rare earth chelates, fluorescent proteins, quantum dots, aggregation-induced emission materials, and upconversion nanoparticles. Preferably, the fluorescent material is a fluorescein (e.g., fluorescein isothiocyanate), a rhodamine (e.g., rhodamine green, rhodamine B, etc.), a coumarin, a quantum dot (e.g., CdS, CdSe, CdTe, ZnSe), a rare earth element (e.g., Eu, Ce), or its complexes. The fluorescent material is adsorbed or encapsulated on or inside the carrier surface through one or more of the following mechanisms: covalent modification, chelation, spatial encapsulation, hydrophobic interaction, and electrostatic adsorption. It should be noted that, from the viewpoint of facilitating optical imaging recognition and improving sensitivity, it is preferable that the fluorescent material is uniformly encapsulated inside the carrier.

[0074] In this application, the fluorescent dye is preferably a fluorescent particle formed by encapsulating fluorescent dye molecules (such as fluorescein) with silica, a fluorescent particle formed by encapsulating fluorescent dye molecules (such as fluorescein) with polyacrylamide, a fluorescent particle formed by encapsulating quantum dots with polystyrene, a fluorescent particle formed by encapsulating rare earth elements or rare earth chelates with polystyrene, a fluorescent particle formed by encapsulating fluorescent proteins with dextran, or a fluorescent particle formed by encapsulating quantum dots with cross-linked agarose.

[0075] In this invention, the particle size of the fluorescent dye needs to be controlled within the range of 180–450 nm, for example, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, and 440 nm. If the particle size of the fluorescent dye is less than 180 nm, for example, 150 nm, no signal can be detected in conventional optical imaging equipment. If the particle size is greater than 450 nm, for example, 460 nm, the detection sensitivity is low and it is difficult to achieve the sensitivity required in clinical practice. It should be noted that the particle size can be a primary particle size or a secondary particle size. The secondary particle size refers to the particle size formed after the primary and secondary particles agglomerate.

[0076] In addition, items required but not provided in the kit include reaction cups, washing solution, waste cup box, and flow-through cell care solution.

[0077] The kit of this invention can be used for the rapid diagnosis of traumatic brain injury or primary brain injury, and is especially suitable for the rapid diagnosis of neurodegenerative diseases (Alzheimer's disease, vascular dementia, Parkinson's disease, Huntington's disease, amyotrophic axonal sclerosis, etc.).

[0078] II. <Single-molecule detection system for detecting neurofilament light chain proteins>

[0079] The detection system includes the aforementioned reagent kit and optical imaging equipment. The optical imaging equipment includes a light source and an optical signal acquisition unit. The detection system does not include a total internal reflection microscope, a near-field microscope, or an Airy disk focusing detection device, nor does it include a microreactor with a volume in the nanoliter, picoliter, or femtoliter range. It also does not require expensive detection equipment based on nanopores or nanowells as used in document CN110249226A.

[0080] In this invention, the optical imaging device mainly includes the following components: an excitation light source, an objective lens, a filter, a photosensitive element, a data acquisition module, a data processing module, and a dichroic mirror (if it is an upright microscope, the dichroic mirror may be omitted). The excitation light source is an optical emitting device used to excite optical signals from the reacted sample. The objective lens is used for signal acquisition and amplification of the sample. The dichroic mirror is used for reflection of the excitation light path and acquisition of the sample's optical signals. The filter is used for filtering the excitation light band and filtering the sample's emitted light signals. The photosensitive element is used for acquiring the sample's optical signals. The data acquisition module is configured to receive the optical signals captured by the photosensitive element and convert them into digital signals. The data processing module is configured for digital signal conversion and the formation and processing of optical images.

[0081] In some embodiments of this device, the excitation source includes one or more of a gas laser, a solid-state laser, a semiconductor laser, a liquid laser, and a free-electron laser. In some embodiments of this device, the objectives are classified according to magnification, including one or more of 1X, 2X, 4X, 5X, 10X, 20X, 40X, 50X, and 100X; the objectives are classified according to field curvature correction, including plane objectives and curved objectives. In some embodiments of this device, the photosensitive element includes one or both of a CCD (Charge Coupled Device) and a CMOS (Complementary Metal-Oxide Semiconductor).

[0082] The optical imaging device of the present invention can be a conventional optical imaging device (i.e., an optical imaging device that does not break the optical diffraction limit), without the need for expensive imaging devices that break the optical diffraction limit, such as total internal reflection fluorescence microscopes, epifluorescence microscopes, scanning near-field optical microscopes, confocal fluorescence microscopes, and wide-field fluorescence microscopes.

[0083] Example

[0084] The present invention will be further described in detail below with examples and comparative examples, but the present invention is not limited thereto. For methods of determining the particle size of fluorescent dyes, single-molecule imaging, and standard curve plotting, please refer to the examples section of Patent Document 7 filed by the applicant, as detailed below.

[0085] 1. Determination of particle size of in-situ signal-enhanced nanoparticles

[0086] Taking silica fluorescent nanoparticles as an example, the silica fluorescent nanoparticles obtained in each example and comparative example were diluted with water by 1000 times, and then 100 μL was dropped onto the surface of a clean silicon wafer, dried, and 5 nm of platinum was sputtered onto its surface using a small sputtering instrument. The particle size was determined by imaging analysis using SEM (SU3900 manufactured by Hitachi High Technology Co., Ltd., Japan).

[0087] Taking polyacrylamide fluorescent nanoparticles as an example, the obtained polyacrylamide fluorescent nanoparticles were diluted 1000 times with pure water, and the particle size was determined using a Malvern particle size analyzer (Zetasizer Nano S90).

[0088] 2. Single-molecule imaging

[0089] Single-molecule imaging can be performed using conventional fluorescence microscopes such as the Nikon Eclipse Ti-U fluorescence microscope or the Olympus fluorescence microscope. Alternatively, other fluorescence microscopes from the Nikon Eclipse Ti series or the Leica DMi8 fluorescence microscope can also be used.

[0090] 3. Method for drawing standard curves

[0091] In this application, the combined use of single-molecule counting mode and fluorescence intensity integration mode can significantly improve the dynamic detection range of the standard curve for detecting biomarkers. The specific implementation method is as follows:

[0092] When the concentration of the analyte is low, the number of magnetic beads is greater than the number of analyte molecules bound to the magnetic beads. Therefore, a standard curve is plotted for analyte samples of different concentrations using a single-molecule counting mode.

[0093] When the concentration of the analyte exceeds a certain threshold, more than one analyte may be bound to the surface of a magnetic bead. The signals of a single molecule are easily superimposed, which can lead to deviations in the detection results. Therefore, the fluorescence intensity integration mode is more suitable.

[0094] Specifically, when the number of single molecules in an image does not exceed a set threshold, the single molecule counting mode is used to plot the standard curve; when the number of single molecules in an image exceeds the set threshold, the fluorescence intensity integration mode is used, and the total fluorescence intensity area is divided by the average fluorescence intensity area of ​​each molecule to convert it into an "approximate single molecule number" for plotting the standard curve.

[0095] Finally, the standard curve obtained using the single-molecule counting mode and the standard curve obtained using the fluorescence intensity integral mode are combined, and the curve is fitted using the fitting formula to draw the complete standard curve.

[0096] 4. Preparation of Buffer 1 Example 1(Preparation of Buffer 1)

[0097] Prepare 50 mL of sterile distilled water, add 40 mM MES (purchased from Sigma-Aldrich), 0.8 g bovine serum albumin (purchased from Sigma-Aldrich), 500 mM sodium chloride (purchased from Beijing Innocare Technology), and 0.8 mL of Tween-20 (purchased from Sigma-Aldrich), and finally add the remaining sterile distilled water, mix well, and bring the volume to 100 mL. The pH should be around 6.

[0098] 5. Preparation of Buffer 1 (Examples 2-8) (Preparation of buffer solutions 2-8)

[0099] Except for changing the amounts of the above-mentioned components, the same procedure as in Preparation Example 1 was followed to obtain buffer solutions 2 to 8. The contents of each component in these buffer solutions are shown in Table 1 below.

[0100] Table 1

[0101] MES BSA NaCl Tween-20 Buffer 2 50mM 1g 600mM 1mL Buffer 3 50mM 1g 600mM 1.2mL Buffer 4 60mM 0.8g 600mM 1mL Buffer 5 60mM 1.2g 600mM 1mL Buffer 6 40mM 0.8g 700mM 0.8mL Buffer 7 40mM 1g 700mM 0.8mL Buffer 8 40mM 1g 500mM 0.8mL

[0102] 6. Comparative Preparation Examples of Buffer 1 (Examples 1-8) (Preparation of Comparative Buffers 1-8)

[0103] Except for changing the amounts of the above components, the same procedure as in Preparation Example 1 was followed to obtain Comparison Buffers 1 to 8. The contents of each component in these buffers are shown in Table 2 below.

[0104] Table 2

[0105] MES BSA NaCl Tween-20 Comparison Buffer 1 35mM 1g 600mM 1mL Comparison Buffer 2 65mM 1g 600mM 1mL Comparison Buffer 3 50mM 0.7g 600mM 1mL Comparison Buffer 4 50mM 1.3g 600mM 1mL Comparison Buffer 5 50mM 1g 450mM 1mL Comparison Buffer 6 50mM 1g 750mM 1mL Comparison Buffer 7 50mM 1g 600mM 0.7mL Comparison Buffer 8 50mM 1g 600mM 1.3mL

[0106] 7. Preparation of Buffer 2 (Example 1) (Preparation of buffer 1')

[0107] Prepare 50 mL of sterile distilled water, add 30 mM PBS (Thermo Fisher Scientific), 0.5 g bovine serum albumin (Sigma-Aldrich), and 150 mM sodium chloride (Beijing Innocare Technology), and finally add the remaining sterile distilled water, mix well, and bring the volume to 100 mL. The pH should be around 7.4.

[0108] 8. Preparation of Buffer 2: Examples 2' to 7' (Preparation of buffer solution 2' to 7')

[0109] Except for changing the amounts of the above components, the same procedure as in Preparation Example 1 was followed to obtain buffer solutions 2' to 7'. The contents of each component in these buffer solutions are shown in Table 3 below.

[0110] Table 3

[0111] PBS BSA NaCl Buffer 2' 20mM 0.5g 150mM Buffer 3' 60mM 0.5g 150mM Buffer 4' 30mM 0.3g 150mM Buffer 5' 30mM 0.6g 150mM Buffer 6' 30mM 0.5g 100mM Buffer 7' 30mM 0.5g 200mM

[0112] 9. Comparative Preparation Examples of Buffer 2: 1' to 6' (Preparation of Comparative Buffers 1' to 6')

[0113] Except for changing the amounts of the above components, the same procedure as in Preparation Example 1' was followed to obtain Comparison Buffers 1' to 6', and the contents of each component in these buffers are shown in Table 4 below.

[0114] Table 4

[0115] PBS BSA NaCl Comparison Buffer 1' 18mM 0.5g 150mM Comparison buffer 2' 64mM 0.5g 150mM Compare buffer 3' 30mM 0.2g 150mM Comparison buffer 4' 30mM 0.7g 150mM Compare buffer 5' 30mM 0.5g 96mM Compare buffer 6' 30mM 0.5g 210mM

[0116] 10. Example of reagent kit preparation

[0117] (1) Experimental components

[0118] Carboxyl-activated magnetic beads (purchased from Merck), NF-L capture antibody (self-developed), NF-L detection antibody (self-developed), silane coupling agent (APTES), silica microspheres coated with fluorescein isothiocyanate (FITC), polyacrylamide coated with fluorescent dye, serum sample to be tested, PBS buffer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), microsphere preservation solution, sample dilution solution, PBS washing solution, labeling blocking solution (0.01% NaCl, 0.5% BSA dissolved in 10mM PBS, pH=7.4), labeling dispersion solution (0.01% NaCl, 0.1% BSA dissolved in 10mM PBS). PBS (pH=7.4), labeling and preservation solutions (buffers 1' to 7' obtained in preparation examples 1' to 7' above, with a pH of approximately 7.4; and comparison buffers 1' to 6' obtained in comparative preparation examples 1' to 6'), magnetic bead washing solution (PBST solution), crosslinking agent dissolving solution (DMSO), magnetic bead coupling solution (TRIS buffer), magnetic bead blocking solution (0.01% NaCl, 0.2% BSA and 0.02% PC300 dissolved in 10mM PBS, pH=7.4), and magnetic bead preservation solutions (buffers 1 to 8 obtained in preparation examples 1 to 8 above, with a pH of approximately 6.0; and comparison buffers 1 to 8 obtained in comparative preparation examples 1 to 8).

[0119] (2) Preparation of magnetic bead solution coated with NF-L capture antibody

[0120] a. Take 100 μL of carboxyl-modified magnetic beads (purchased from Merck), wash 5 times with 15 mM PBS buffer, and remove the buffer.

[0121] b. Take 70 μg of NF-L capture antibody and add it to the magnetic beads in (1). Mix well and incubate at 20°C on a roller incubator for 1.5 h at a speed of 80 rpm / min.

[0122] c. Add magnetic bead cleaning solution for cleaning, mix well, and place on a magnetic rack for magnetic separation.

[0123] d. Add magnetic bead blocking solution to the coated tube and incubate on a roller incubator for 40 min at a speed of 80 rpm / min. Remove the supernatant after magnetic separation.

[0124] e. Take buffer 1 to 8 (pH around 6.0) obtained in Preparation Examples 1 to 8 above and comparison buffer 1 to 8 obtained in Comparative Preparation Examples 1 to 8 and add them to a centrifuge tube. Mix well to obtain a magnetic bead solution coated with NF-L capture antibody.

[0125] (3) Preparation of NF-L detection antibody solution labeled with fluorescent dye

[0126] a. Preparation of diluent for fluorescent dyes

[0127] Dilute 20 μL of fluorescent dye (fiTC-coated silica microspheres with a particle size of 250 nm) with 20 μL of PBS buffer.

[0128] b. Preparation of labeling incubation solution

[0129] Dissolve 0.004 g of activator EDC in 20 μL of PBS buffer to prepare a buffer solution containing the activator. Add this solution to diluted fluorescent dye, mix well, centrifuge, and add the buffer solution to prepare the labeling incubation solution for later use.

[0130] c. Preparation of labeling working solution

[0131] Add 25 μg of NF-L detection antibody stock solution to the above labeling incubation solution, mix well and incubate for 1 hour.

[0132] d. Seal the labeling working solution

[0133] Add 25 μL of the labeling blocking solution to the above labeling working solution, mix well, and incubate for 1 hour.

[0134] e. Washing and labeling working solution

[0135] f. Preservation of the labeling working solution

[0136] Add the labeled dispersion to a centrifuge tube and mix well. After mixing, add the labeled preservation solution (i.e., buffer 1' to 7' obtained in preparation examples 1' to 7' above; and comparison buffer 1' to 6' obtained in comparison preparation examples 1' to 6') and store in a refrigerator at 2 to 8°C.

[0137] (4) Combine the magnetic bead solution coated with NF-L capture antibody, the NF-L detection antibody solution labeled with fluorescent dye, and the NF-L quality control and calibrators prepared in the above steps to form kits 1-8 and comparison kits 1-10. The combinations of magnetic bead preservation solution and labeling preservation solution used in each kit are shown in Tables 5 and 6 below.

[0138] Table 5

[0139]

[0140] Table 6

[0141]

[0142] Example 1 The concentration of NF-L was determined using kit 1 based on single-molecule detection technology.

[0143] (1) The concentration of NF-L was diluted to 0, 0.034, 0.103, 0.31, 0.93, 2.78, 8.33, 33.33, 66.67 and 100 pg / mL, respectively.

[0144] (2) Load the samples and reagents into the designated positions in sequence according to the requirements of the AST-Dx90 fully automated fluorescence immunoassay analyzer (a self-developed device of Suzhou Yuce Biotechnology). After preparation, start the test. The device will automatically send the sample into the loading position and load the reaction cup into the incubation tray. The sampling needle will draw 20 μL of the sample obtained in (1) from the sample tube and add it to the reaction cup. The reagent needle will draw 25 μL of magnetic bead solution coated with NF-L capture antibody (reagent 1) from reagent kit 1 and add it to the reaction cup. Mix and incubate for 3 min. The magnetic beads modified with surface-specific antibodies in reagent 1 can recognize and capture target molecules with extremely low content in the sample.

[0145] (3) Take 15 μL of NF-L detection antibody labeled with fluorescent dye (reagent 2) from reagent kit 1 and add it to the reaction cup. Mix well and incubate for 2 min. Reagent 2 contains a single-molecule signal marker that modifies the detection antibody, which can convert the target molecule into a single-molecule signal.

[0146] (4) The detection probe transfers the reaction system to the flow cell. Magnetic separation is used to attract the magnetic beads to the bottom of the flow cell and spread them evenly on the surface of the detection well. Other components are washed away, and single-molecule imaging is performed using a standard fluorescence microscope (purchased from Olympus). The single-molecule counting mode and fluorescence intensity integration mode are used in combination to complete the subsequent single-molecule counting statistics and analysis. It should be noted that the incubation time is 5 minutes, and the time from sample injection to result output is 5 minutes and 40 seconds.

[0147] (5) Complete a series of concentration tests, repeating each concentration point 6 times, and draw a standard curve based on the test results.

[0148] Test results as follows Figure 1 As shown, in this embodiment, the detection range of NF-L is 0.025 pg / mL to 100 pg / mL. Within this range, the number of single molecules shows a good linear relationship with the sample concentration (R0). 2 It is close to 1), with a detection limit of 0.025 pg / mL, and the time from sample injection to result output is only 5 minutes and 40 seconds. In addition, the CV value is also less than 10%.

[0149] Furthermore, the results of six independent experiments conducted two months apart show that the NF-L kit of the present invention has highly stable detection results. Specifically, the relative errors calculated from the results of the six independent experiments are all less than 10%.

[0150] Examples 2-12 and Comparative Examples 1-16 (using kits 2-12 and 1-16 as described above, respectively)

[0151] For the above-mentioned kits 2-12 and comparative kits 1-16, the same operation as in Example 1 was performed (the incubation time was 5 minutes and the time from sample injection to output result was 5 minutes and 40 seconds) to obtain the detection limit and CV value. The results of the detection limit and CV value are shown in Table 7 and Table 8, respectively.

[0152] Table 7

[0153]

[0154] Table 8

[0155]

Claims

1. A kit for detecting neurofilament light chain proteins based on single-molecule counting, characterized in that, The buffer comprises magnetic beads coated with capture antibodies, detection antibodies labeled with fluorescent dyes, buffer 1 for the magnetic beads coated with capture antibodies, and buffer 2 for the detection antibodies labeled with fluorescent dyes. The capture antibody and the detection antibody can bind to different sites on neurofilament light chain proteins, respectively. The fluorescent dye contains fluorescent materials and a carrier, and has a particle size of 180~450nm. The pH of buffer 1 is 5.5-6.5, and each 100 mL of buffer consists of 40 mM-60 mM MES, 0.9 g-1.1 g bovine serum albumin, 0.9 mL-1.1 mL Tween-20, 550-650 mM sodium chloride, and sterile distilled water. The pH of buffer 2 is 7.0-7.6, and each 100 mL buffer consists of 20 mM-40 mM PBS, 0.4 g-0.6 g bovine serum albumin, 120-180 mM sodium chloride, and sterile distilled water.

2. The kit for detecting neurofilament light chain proteins as described in claim 1, wherein, The kit also includes neurofilament light chain protein calibrators and neurofilament light chain protein controls.

3. The kit for detecting neurofilament light chain proteins as described in claim 1 or 2, wherein, The fluorescent material is a fluorescein-based luminescent material, a rhodamine-based luminescent material, an aggregation-induced luminescent material, or a quantum dot-based luminescent material, and the carrier is silica, polyacrylamide, or polystyrene.

4. A single-molecule detection system for detecting neurofilament light chain proteins, comprising the kit and optical imaging device as described in any one of claims 1 to 3. The optical imaging device includes a light source and an optical signal acquisition unit, and the detection system does not include a total internal reflection microscope, a near-field microscope, or an Airy disk focusing detection device, nor does it include a microreactor with a volume of nanoliter, picoliter, or femtoliter.

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