Neurodegeneration marker detection reagent and detection method

Abstract

This application relates to a reagent and method for detecting neurodegenerative markers. The reagent comprises an oxygen-generating agent, a catalyst, and an immunoassay reagent capable of specifically binding to the target neurodegenerative marker in the sample. The oxygen-generating agent reacts under the action of the catalyst to generate oxygen, which increases the oxygen content in the reaction system. The immunoassay reagent utilizes reactive oxygen species in the reaction solution to generate a light signal, and the target molecule is selected from neurodegenerative markers. The method provided in this application can increase the light signal value even at low concentrations of neurodegenerative markers, thus improving the detection capability of neurodegenerative markers.

Description

Technical Field

[0001] This application relates to the field of biological reagent technology, and in particular to a reagent and method for detecting neurodegenerative markers. Background Technology

[0002] Axonal injury is the pathological basis for permanent disability in various neurological diseases. Reliable quantification and longitudinal follow-up of this injury are crucial for assessing disease activity, monitoring treatment response, facilitating treatment development, and determining prognosis. Currently, most clinical examination methods for neurological diseases rely on clinical assessment, brain imaging, and laboratory tests. Magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid analysis have limitations in clinical and research use due to their invasiveness, complexity, and high cost. In the treatment of neurological diseases, reliable biomarkers are urgently needed to improve the accuracy of identification and prognostic assessment.

[0003] Neurofilament light chain (NfL) is a sensitive but non-specific biomarker of axonal injury, highly expressed in the cytoplasm of neurons with large-diameter myelinated axons. In various neurological diseases, including inflammatory, neurodegenerative, traumatic, and cerebrovascular diseases, the levels of NfL in cerebrospinal fluid and blood increase proportionally to the degree of axonal injury. Over the past two decades, the role of NfL as a biomarker in multiple sclerosis (MS), Alzheimer's disease (AD), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), atypical Parkinson's disease (APD), and traumatic brain injury (TBI) has been extensively reported. Complementing other neurological assessments and more disease-specific biomarkers and brain imaging results, NfL can assist clinicians in predicting various neurological diseases, monitoring disease progression, and assessing treatment efficacy and prognosis, thus possessing significant clinical value.

[0004] NfL is typically measured from cerebrospinal fluid, serum, or plasma. Early NfL assays primarily utilized enzyme-linked immunosorbent assay (ELISA) or fluorescence immunoassay. These methods suffer from drawbacks such as low sensitivity, narrow linear range, and unstable results, making them inaccurate in measuring the low concentrations of NfL present in the blood of most individuals. Therefore, there is an urgent need for a method that can improve precision and sensitivity while remaining simple to operate. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a reagent and method for detecting neurodegenerative markers, which can increase the light signal value when the concentration level of neurodegenerative markers is low, thereby improving the detection capability of neurodegenerative markers.

[0006] The first aspect of this application provides a neurodegenerative marker detection reagent, comprising an oxygen-generating agent, a catalyst, and an immunoassay reagent capable of specifically binding to the neurodegenerative marker to be tested in the sample.

[0007] The oxygen-generating agent reacts under the action of the catalyst to generate oxygen, and the generated oxygen is used to increase the oxygen content of the reaction system; the immunoassay reagent can generate a light signal using reactive oxygen species in the reaction solution, and the target molecule to be tested is selected from neurodegenerative markers.

[0008] The detection reagents described above, wherein the oxygen-generating agent comprises a metal-peroxide salt and / or a hydrogen peroxide complex;

[0009] Preferably, the oxygen-generating agent includes at least one of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxide.

[0010] The detection reagent described above, wherein the catalyst comprises at least one of an enzyme compound, a halide, and a metal oxide;

[0011] Preferably, the catalyst comprises at least one selected from catalase, iodide, manganese dioxide, iron(III), silver and dichromate.

[0012] The detection reagent described above has an oxygen-generating agent concentration of 10 mg / mL to 100 mg / mL, and / or

[0013] The concentration of the catalyst is 0.5 mg / mL to 100 mg / mL.

[0014] The test reagents described above, wherein the sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma; and / or,

[0015] The target molecules to be tested are selected from neurofilament light chains, glial fibrous acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

[0016] The detection reagent described above, wherein the immunoassay reagent is a photochemiluminescence detection reagent, and the photochemiluminescence detection reagent comprises:

[0017] A luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles are capable of reacting with reactive oxygen species to generate a detectable light signal;

[0018] The labeling reagent includes a tag-labeled second biomolecule; wherein both the first biomolecule and the second biomolecule are capable of specifically binding to the target molecule in the test sample.

[0019] Preferably, it also includes a donor reagent, which can convert ground-state oxygen into reactive oxygen under laser irradiation of a certain wavelength;

[0020] More preferably, the donor reagent comprises photosensitive microparticles filled with a photosensitive substance.

[0021] A second aspect of this application provides a method for detecting neurodegenerative markers as described above, wherein the method includes:

[0022] After the sample to be tested and the immunoassay reagent are mixed and reacted, the oxygen-generating agent and catalyst are added, and the amount of emitted photons is measured to obtain the light signal value.

[0023] The detection method described above includes the following specific steps:

[0024] S1. Mix the sample to be tested with the immunoassay reagent, and incubate for the first time to obtain the first reaction solution;

[0025] S2. The oxygen-generating agent and catalyst are added to the first reaction solution, and the second reaction solution is obtained after a second incubation.

[0026] S3. The second reaction solution is irradiated with excitation light, and the light signal is detected.

[0027] In the detection method described above, the oxygen-generating agent and catalyst are premixed and then added to the first reaction solution;

[0028] Preferably, the concentration ratio of the oxygen-generating agent and the catalyst premix is ​​(1-10):(1-20); more preferably, the concentration ratio of the premix is ​​10:1.

[0029] Preferably, the volume ratio of the oxygen-generating agent to the catalyst premix is ​​1:1;

[0030] Preferably, the premixing time of the oxygen-generating agent and the catalyst is 1 to 10 minutes; more preferably, the premixing time is 3 minutes.

[0031] The detection method described above, wherein,

[0032] The incubation temperature is the same for each incubation; preferably, the incubation temperature is 37-42°C; more preferably, it is 37°C.

[0033] The technical solution provided in this application may include the following beneficial effects:

[0034] The neurodegenerative marker detection reagent provided in this application includes an oxygen-generating agent and a catalyst. The oxygen-generating agent reacts under the action of the catalyst to release oxygen. This oxygen can be activated into reactive oxygen species, increasing the reactive oxygen species content in the detection system, thereby enhancing the light signal intensity of photo-induced chemiluminescence. This improves the stability, sensitivity, and precision when applied to the photo-induced chemiluminescence detection of neurodegenerative markers, and helps solve the problems of low luminescence signal values, significant interference from instrument noise, and poor precision in the detection of low-level neurodegenerative markers.

[0035] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0036] To facilitate understanding of this application, it will be described in detail below. However, before describing this application in detail, it should be understood that this application is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive.

[0037] Where a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered within this application. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered within this application, subject to any explicitly excluded limits within the specified range. Where the specified range includes one or two limits, the range excluding any or both of those included limits is also included within this application.

[0038] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While the methods and materials described herein, or any equivalent methods and materials, may also be used in the implementation or testing of this application, preferred methods and materials are now described.

[0039] Terminology Explanation

[0040] The term "oxygen-generating agent" as used in this application refers to a chemical substance capable of producing oxygen, which undergoes a decomposition reaction to release oxygen. In some specific embodiments of this application, the oxygen-generating agent may be, for example, compounds such as hydrogen peroxide, sodium peroxide, and urea peroxide. Other oxygen-generating agents known to those skilled in the art may also be used in this application.

[0041] The term "catalyst" as used in this application refers to a substance capable of altering the chemical reaction rate of an oxygen-generating agent, while maintaining essentially unchanged mass and chemical properties before and after the reaction. In some specific embodiments of this application, the catalyst may be, for example, platinum, palladium, manganese dioxide, ferric oxide, enzymes, etc. Other catalysts known to those skilled in the art may also be used in this application.

[0042] The term "sample to be tested" as used in this application refers to a mixture containing or suspected of containing the target molecule. Samples to be tested that can be used in this application include bodily fluids, such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, cerebrospinal fluid, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells derived from prokaryotes. Samples to be tested can be diluted with a diluent as needed before use. For example, to avoid the hook effect, the sample can be diluted with a diluent before being tested on the instrument.

[0043] The term "neurodegenerative markers" as used in this application refers to a class of indicators that can reflect the occurrence and development of neurodegenerative diseases, and assist in disease diagnosis, disease monitoring, and prognosis assessment. Neurodegenerative markers that can be used in this application include neurofilament light chains, glial fibrillary acidic proteins, ubiquitin C-terminal hydrolases, or laminin, etc.

[0044] The term "luminescent microparticle" as used in this application refers to particles containing compounds capable of reacting with reactive oxygen species to generate a detectable signal. In some specific embodiments of this application, the luminescent microparticles comprise a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0045] The term "luminescent microparticle and first biomolecule bound thereto" as used in this application refers to binding (coating) a first biomolecule onto the surface of the luminescent microparticle. The first biomolecule is an organic molecule that can specifically bind to the target molecule in the sample to be tested. For example, the first biomolecule can be an antibody, enzyme, receptor, nucleic acid aptamer, etc.

[0046] The term "tagged second biomolecule" as used in this application refers to a biomolecule labeled with a marker, such as biotin, fluorescent, or radioactive markers. A second biomolecule is an organic molecule capable of specifically binding to a target molecule in a test sample; for example, a second biomolecule can be an antibody, enzyme, receptor, or nucleic acid aptamer.

[0047] The term "bonding" as used in this application refers to the direct union between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0048] The term "specific binding" as used in this application refers to a mutually distinguishable and selective binding reaction between two substances, which, from a stereostructural perspective, refers to the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in this application, detection methods for specific binding reactions include, but are not limited to: double-antibody sandwich assays, competitive assays, neutralization competitive assays, indirect assays, or capture assays.

[0049] The term "donor reagent" as used in this application refers to a reagent containing a sensitizer that, upon activation by energy, can generate an active intermediate, such as reactive oxygen species, that reacts with the luminescent microparticles. Donor reagents include photosensitive microparticles filled with a photosensitive substance. In some specific embodiments of this application, the photosensitive microparticles are polymeric microparticles filled with a photosensitizer, and the photosensitive substance is a photosensitizer. The photosensitizer can be a photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with singlet oxygen, such as compounds like methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, as well as derivatives of these compounds having 1-50 substituents, the substituents being used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. Other examples of photosensitizers known to those skilled in the art may also be used in this application.

[0050] The first aspect of this application provides a neurodegenerative marker detection reagent, comprising an oxygen-generating agent, a catalyst, and an immunoassay reagent capable of specifically binding to neurodegenerative markers in a test sample; wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen, and the generated oxygen is used to increase the oxygen content in the reaction system; the immunoassay reagent can utilize reactive oxygen species in the reaction solution to generate a light signal, and the target molecule to be tested is selected from neurodegenerative markers.

[0051] The immunoassay reagent in this application's neurodegenerative marker detection reagent can specifically bind to neurodegenerative markers and emit a light signal under photo-induced chemiluminescence (PET). The oxygen-generating agent in this application can react under the action of a catalyst to release oxygen. This oxygen can be activated into reactive oxygen species in the PET detection system, thereby increasing the reactive oxygen species content in the detection system and enhancing the light signal intensity of the PET. This improves the stability, sensitivity, and precision of the detection results when applied to the PET detection of neurodegenerative markers, effectively addressing the problems of low luminescence signal values, significant interference from instrument noise, and poor precision in the detection of low-level neurodegenerative markers.

[0052] In some embodiments, the oxygen-generating agent includes a metal-peroxide salt and / or a hydrogen peroxide complex; preferably, the oxygen-generating agent includes at least one or more combinations of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxides. Of course, the oxygen-generating agent can also be other metal-peroxide salts or hydrogen peroxide complexes; this is merely illustrative and not intended to be limiting. The above-mentioned oxygen-generating agents have high oxygen production, high stability, good solubility, and high environmental friendliness. When applied to the detection of neurodegenerative markers, they can efficiently generate ground-state oxygen while avoiding side reactions between the oxygen-generating agent and immunoassay reagents, as well as environmental pollution, thereby improving the stability, sensitivity, and precision of the detection.

[0053] In some embodiments, the catalyst includes at least one selected from enzyme compounds, halides, and metal oxides; preferably, the catalyst includes at least one or more combinations of catalase, iodides, manganese dioxide, iron(III), silver, and dichromates. Of course, the catalyst can also be other enzyme compounds, halides, or metal oxides; this is merely illustrative and not limiting. The above-mentioned catalysts have advantages such as high catalytic activity, high stability, high environmental friendliness, and low cost. They can efficiently and stably catalyze oxygen production from oxygen-generating agents and do not interfere with photo-induced chemiluminescence detection, thus improving the stability, sensitivity, and precision of the detection.

[0054] In some embodiments, the concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL. For example, the concentration of the oxygen-generating agent can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL, etc. This is only an example and is not a limitation.

[0055] In some embodiments, the concentration of the catalyst is 0.5 mg / mL to 100 mg / mL. For example, the concentration of the catalyst can be 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL, etc. These are only examples and are not intended to be limiting.

[0056] It should be noted that the concentrations of the oxygen-generating agent and the catalyst mentioned above in this application are the independent configuration concentrations of the oxygen-generating agent and the catalyst, and their concentrations when stored separately, i.e., their individual concentrations before being mixed and used together. When the concentrations of the oxygen-generating agent and the catalyst are within the aforementioned ranges, the oxygen-generating agent can fully and efficiently release oxygen under the action of the catalyst, increasing the reactive oxygen species content in the detection of neurodegenerative markers, reducing raw material waste caused by excessive oxygen-generating agent or catalyst, improving the stability, sensitivity, and precision of the detection, and saving costs.

[0057] In some implementations, the sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma. When cerebrospinal fluid, whole blood, serum, or plasma is selected as the sample to be tested, the target molecules in the sample are stably present, and the sample is easy to collect, which is beneficial for the widespread application of photochemiluminescence detection methods in clinical medicine.

[0058] In some embodiments, the target molecule to be tested is selected from neurodegenerative markers; preferably, neurodegenerative markers may be selected from neurofilament light chains, glial fibrillary acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

[0059] In some embodiments, the immunoassay reagent is a photochemiluminescence assay reagent, which includes a luminescent reagent and a labeling reagent. The luminescent reagent includes luminescent microparticles and a first biomolecule bound to them; the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal. The labeling reagent includes a tag-labeled second biomolecule. Both the first and second biomolecules can specifically bind to the target molecule in the test sample.

[0060] Specifically, the luminescent reagent of this application includes luminescent microparticles and a first biomolecule bound thereto. The luminescent microparticles can react with reactive oxygen species to generate a detectable light signal, and the first biomolecule can specifically bind to the target molecule in the sample to be tested. This application does not limit the specific selection of the luminescent microparticles, which can be selected according to actual needs. This application does not limit the specific selection of the first biomolecule, as long as the first biomolecule can specifically bind to the target molecule in the sample to be tested. For example, when the target molecule in the sample to be tested is an antigen, the first biomolecule can be an antibody that can specifically bind to the antigen. For example, if the target molecule is a neurofilament light chain, the first biomolecule in the luminescent reagent should be a neurofilament light chain antibody; for example, if the target molecule is a glial fibrillary acidic protein, the first biomolecule in the luminescent reagent should be a glial fibrillary acidic protein antibody.

[0061] Specifically, the labeling reagent of this application includes a tag-labeled second biomolecule capable of specifically binding to the target molecule in the test sample. This application does not limit the specific choice of the tag; it can be selected according to actual needs, such as biotin. This application also does not limit the specific choice of the second biomolecule, as long as it can specifically bind to the target molecule in the test sample. For example, when the target molecule in the test sample is an antigen, the second biomolecule can be an antibody capable of specifically binding to the antigen. For example, if the target molecule is neurofilament light chain, the second biomolecule in the labeling reagent should be a neurofilament light chain antibody; for example, if the target molecule is glial fibrillary acidic protein, the second biomolecule in the labeling reagent should be a glial fibrillary acidic protein antibody. For the same target molecule, the aforementioned first and second biomolecules can be the same antibody or different antibodies.

[0062] In some embodiments, the immunoassay reagent of this application may further include a donor reagent, which can convert ground-state oxygen into reactive oxygen species under laser irradiation of a certain wavelength. Preferably, the donor reagent is also called a photosensitive reagent, which includes photosensitive microparticles filled with a photosensitive substance. The surface of the photosensitive microparticles is bonded with a substance that can specifically bind to the label of the labeled reagent, such as avidin, which facilitates binding with biotin, preferably streptavidin. This application does not limit the specific selection of photosensitive microparticles, which can be selected according to actual needs, as long as they can convert ground-state oxygen generated by the oxygen-generating agent into reactive oxygen species under laser irradiation of a certain wavelength. This application does not limit the specific selection of photosensitive substance, as long as it can receive a laser signal of a certain wavelength.

[0063] A second aspect of this application provides a method for detecting the aforementioned neurodegenerative markers, comprising:

[0064] After the sample to be tested and the immunoassay reagent are mixed and reacted, an oxygen-generating agent and a catalyst are added, and the amount of emitted photons is measured to obtain the light signal value.

[0065] Specifically, the sample to be tested is mixed with an immunoassay reagent to obtain a test mixture. Then, an oxygen-generating agent and a catalyst are added to the test mixture. The test mixture is then irradiated with a laser, and finally, the amount of emitted photons is measured to obtain the light signal value.

[0066] In this process, the oxygen-generating agent reacts under the action of a catalyst to release oxygen. This oxygen can be activated into reactive oxygen species, thereby increasing the amount of reactive oxygen species in photochemiluminescence, which in turn improves the luminescence efficiency and enhances the light signal intensity of photochemiluminescence.

[0067] In some embodiments, the specific steps of the detection method for neurodegenerative markers of this application include:

[0068] S1. Mix the sample to be tested with the immunoassay reagent, and incubate for the first time to obtain the first reaction solution.

[0069] The immunoassay reagents include the luminescent reagent, labeling reagent, and donor reagent mentioned above, which will not be elaborated further here. For example, the sample to be tested can be first mixed with the luminescent reagent and labeling reagent and incubated to form a complex of "luminescent microparticle-first biomolecule-target molecule-second biomolecule-biotin"; then the donor reagent can be added and mixed and incubated to form a sandwich immunoassay complex of "luminescent microparticle-first biomolecule-target molecule-second biomolecule-biotin-avidin-photosensitive microparticle".

[0070] Among them, the target molecules in the test samples are neurodegenerative markers.

[0071] In some implementations, the incubation time for the first stage is 25 minutes. For example, the first stage incubation is 15 minutes, and the second stage incubation is 10 minutes. The incubation temperature for this step can be 37°C to 42°C.

[0072] S2. Add the oxygen-generating agent and catalyst to the first reaction solution, and after a second incubation, obtain the second reaction solution.

[0073] In some embodiments, the oxygen-generating agent and catalyst are premixed before being added to the first reaction solution. In the detection method of this application, the oxygen-generating agent and catalyst are stored separately before sample testing to avoid premature oxygen release due to decomposition of the oxygen-generating agent upon contact with the catalyst after mixing, which would not improve the stability, sensitivity, or precision of the detection. After completing step S1, the oxygen-generating agent and catalyst are premixed before being added to the first reaction solution. In some embodiments, the premixing time of the oxygen-generating agent and catalyst is 1–10 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min. Preferably, the premixing time is 3 min. During this premixing time, the oxygen-generating agent and catalyst are in full contact, allowing the oxygen-generating agent to begin producing oxygen under the action of the catalyst, thus increasing the intensity of the detected optical signal. Simultaneously, by controlling the premixing time of the oxygen-generating agent and catalyst, excessive time is avoided to prevent oxygen loss, ensuring timely addition to the first reaction solution and providing sufficient oxygen to the reaction system.

[0074] In some preferred embodiments, the concentration ratio of the oxygen-generating agent to the catalyst premix is ​​(1-10):(1-20), for example, the concentration ratio of the oxygen-generating agent to the catalyst premix can be 1:1, 1:5, 1:10, 1:20, 5:1, 1:2, 5:20, 10:1, etc. Preferably, the premix concentration ratio is 10:1. For example, when the concentration of the oxygen-generating agent is 50 mg / mL, the concentration of the catalyst is 5 mg / mL, and so on. Preferably, the volume ratio of the oxygen-generating agent to the catalyst premix is ​​1:1. The concentration ratio of the oxygen-generating agent to the catalyst premix in this application refers to the ratio of the concentration of the oxygen-generating agent to the concentration of the catalyst before mixing. When the concentration ratio of the oxygen-generating agent to the catalyst is within the above range, the matching of the oxygen-generating agent and the catalyst is higher, the oxygen-generating agent can better generate oxygen under the action of the catalyst, improve the oxygen content in the photo-induced chemiluminescence detection system, and make the detection stability, sensitivity and precision better, while reducing the excess of oxygen-generating agent or catalyst and reducing costs.

[0075] It is understandable that the second reaction solution includes a sandwich immune complex, a catalyst, and an oxygen-generating agent after the reaction. The oxygen-generating agent decomposes under the action of the catalyst to produce ground-state oxygen, thereby increasing the oxygen content in the second reaction solution.

[0076] In some implementations, the second incubation period can be 3 minutes. The incubation temperature in this step can be 37°C to 42°C.

[0077] S3. The second reaction liquid is irradiated with excitation light, and the light signal is detected.

[0078] The second reaction solution is irradiated with excitation light. Under irradiation, for example, with a 680nm laser, the ground-state oxygen in the second reaction solution is converted into reactive oxygen. The luminescent particles react with the reactive oxygen to generate a light signal, which is then detected to obtain the light signal value. In the specific steps described above, to obtain more accurate detection results, in some embodiments, the concentration of the luminescent reagent can be from 10 μg / mL to 100 μg / mL, for example, the concentration of the luminescent reagent can be 10 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL. In some embodiments, the concentration of the labeling reagent can be from 0.5 μg / mL to 5 μg / mL, for example, the concentration of the labeling reagent can be 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, or 5 μg / mL. In some embodiments, the concentration of the donor reagent is 10 μg / mL to 100 μg / mL, for example, the concentration of the donor reagent can be 10 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL. The donor reagent can serve as a universal reagent suitable for photochemiluminescence detection kits that detect different target molecules.

[0079] In some implementations, the incubation temperature is the same for each iteration. This facilitates the binding of the first and second biomolecules to the target molecule, as well as the binding of the donor reagent to the tag in the labeling reagent. It reduces the impact of temperature on detection, improves the stability and accuracy of detection results, and simplifies the detection process while reducing detection costs.

[0080] In some preferred embodiments, the incubation temperature is 37°C to 42°C, for example, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C; more preferably, it is 37°C. When the incubation temperature is within the above range, the activity of each substance in the photo-induced chemiluminescence detection reagent and the oxygen production efficiency of the oxygen-generating agent can be guaranteed, avoiding excessively high temperatures that lead to substance inactivation or excessively low temperatures that lead to low oxygen production and binding efficiency, thereby improving the stability, sensitivity, and precision of the detection.

[0081] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0082] The following example uses urea peroxide as the oxygen-generating agent and catalase as the catalyst. The companion reagent (prepared by mixing the oxygen-generating agent and the catalyst) is used to react with the luminescent reagent, the labeling reagent, the donor reagent and the sample to be tested. Finally, the light signal value is detected on the photo-induced chemiluminescence platform.

[0083] Example 1: Preparation of companion reagent

[0084] 1.1 The main experimental materials are shown in Table 1

[0085] Table 1

[0086] peroxyurea Purchased from Sigma catalase Purchased from Sigma

[0087] 1.2 Catalase was diluted with 20 mM HEPES buffer to obtain different concentrations of catalase reagent as catalysts. The concentrations of catalase reagent were 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL. 、 100mg / mL.

[0088] 1.3 Urea peroxide was prepared using 20 mM HEPES buffer to obtain urea peroxide reagents of different concentrations as oxygen generating agents, namely 10 mg / mL, 50 mg / mL and 100 mg / mL.

[0089] 1.4 The catalase reagent and urea peroxide reagent were stored separately, and then mixed at a volume ratio of 1:1 during detection to obtain the companion reagent for use in the following examples. The mixing time was 3 minutes.

[0090] Example 2: Verification of the effect of the companion reagent on the detection signal of glial fibrillary acidic protein (GFAP)

[0091] 2.1 The main experimental materials and equipment are shown in Table 2.

[0092] Table 2

[0093] Glial fibrillary acidic protein (GFAP) Purchased from Oukai Bio GFAP antibody-coated luminescent microspheres (FG-GFAP antibody) Purchased from Huakui Gold Biotin-labeled GFAP antibody (Biotin-GFAP antibody) Purchased from Huakui Gold Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0094] 2.2 The FG-GFAP antibody was diluted with HEPES diluent to obtain an FG-GFAP antibody reagent with a concentration of 25 μg / mL, i.e., the luminescent reagent; the Biotin-GFAP antibody was diluted with HEPES diluent to obtain a Biotin-GFAP antibody reagent with a concentration of 2 μg / mL, i.e., the labeling reagent; the analyte GFAP with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1 to 6 with different concentrations, i.e., CAL1 to CAL6; the concentrations of calibrators 1 to 6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0095] 2.3 The six calibrators prepared in step 2.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding light signal values ​​(see Table 3) and signal enhancement amplitudes (see Table 4). Specifically, 25 μL of calibrator, 25 μL of FG-GFAP antibody reagent, and 25 μL of Biotin-GFAP antibody reagent were mixed sequentially; then, a first incubation was performed at 37°C for 15 min to obtain the first reaction solution; then, 175 μL of donor reagent was added, and a second incubation was performed at 37°C for 10 min to obtain the second reaction solution; then, 50 μL of companion reagent (in which the volume ratio of catalyst to oxygen-generating agent is 1:1) was added, the mixture was shaken evenly, and a third incubation was performed at 37°C for 3 min to obtain the third reaction solution; after photoexcitation reaction in the photo-induced chemiluminescence detector, the light signal value was read. The corresponding signal enhancement amplitude was calculated based on the light signal value to obtain the corresponding signal enhancement amplitude. The calculation formula is: Signal enhancement amplitude (%) = (Light signal value of experimental group - Light signal value of un-partnered reagent) / Light signal value of un-partnered reagent * 100%.

[0096] It should be noted that in the control groups of Tables 3 and 4, "without companion reagent" means that the test is performed according to step 2.3, and the difference from the experimental group is that no companion reagent is added during the reaction process; "HEPES buffer" means that the test is performed according to step 2.3, and the difference from the experimental group is that an equal volume of 20mM HEPES buffer is used instead of companion reagent during the reaction process.

[0097] Table 3 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0098]

[0099] Table 4 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0100]

[0101] Table 4 shows that comparing the detection results of the control group without the chaperone reagent and the control group with HEPES buffer, it is clear that adding HEPES buffer has no effect on improving the light signal value. Comparing the experimental group with the chaperone reagent and the control group without the chaperone reagent, the experimental group showed a significant increase in signal strength for all concentrations of calibrators after adding the chaperone reagent, with the highest increase reaching 110%, indicating that adding the chaperone reagent can improve the detected light signal. Comparing the light signal improvement of the chaperone reagents containing different concentrations of catalase in the experimental group, it was found that when the catalase concentration reached 1 mg / mL, the light signal improvement plateaued, and further increasing the catalase concentration had a limited effect on improving the light signal.

[0102] The following steps will involve increasing the concentrations of catalase and urea peroxide compared to the previous year to further investigate the effects of the concentrations of the catalyst and oxygen-generating agent on oxygen production and detection signals.

[0103] The ratio of urea peroxide reagent to catalase reagent concentration in the mixing process was set to 10:1. Various concentrations of urea peroxide reagent were mixed with corresponding concentrations of catalase reagent to obtain the corresponding companion reagents. Detection was performed according to the method in step 2.3, and the corresponding optical signal values ​​were obtained (see Table 5). Subsequently, the signal enhancement amplitude was calculated based on the optical signal values, and the signal enhancement amplitude was obtained (see Table 6).

[0104] Table 5

[0105]

[0106] Table 6

[0107]

[0108]

[0109] Table 5 shows that when the concentration ratio of the catalyst and oxygen-generating agent is fixed, the light signal value increases to a certain extent and then plateaus, meaning that the light signal value does not increase significantly with further increases in the concentration of the catalyst and oxygen-generating agent. Table 6, combined with comparisons of calibrators with different concentrations of catalase and urea peroxide, reveals that when the catalase concentration reaches 5 mg / mL and the urea peroxide concentration reaches 50 mg / mL, the increase in light signal reaches a plateau; further increases in the concentrations of catalase and urea peroxide have a relatively small effect on increasing the light signal.

[0110] In summary, the addition of the companion reagent effectively enhances the optical signal value, which is beneficial for improving the detection capability of GFAP. The preferred concentrations of the companion reagents are: 50 mg / mL for urea peroxide reagent and 5 mg / mL for catalase reagent; the volume ratio of urea peroxide reagent to catalase reagent is 1:1. This design maximizes the enhancement of the optical signal value while controlling the raw material cost of the companion reagent and avoiding waste.

[0111] Example 3: Verification of the effect of the companion reagent on the detection signal of neurofilament light chain (NfL)

[0112] 3.1 The main experimental materials are shown in Table 7.

[0113] Table 7

[0114] Neurofilament light chain (NfL) Purchased from Wuxi Aoyue Dongyuan NfL antibody-coated luminescent microspheres (FG-NfL antibody) Purchased from Wuxi Aoyue Dongyuan Biotin-labeled NfL antibody (Biotin-NfL antibody) Purchased from Wuxi Aoyue Dongyuan Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0115] 3.2 The FG-NfL antibody was diluted with HEPES diluent to obtain an FG-NfL antibody reagent with a concentration of 25 μg / mL, i.e., the luminescent reagent; the Biotin-NfL antibody was diluted with HEPES diluent to obtain a Biotin-NfL antibody reagent with a concentration of 2 μg / mL, i.e., the labeling reagent; the analyte NfL with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1 to 6 with different concentrations, i.e., CAL1 to CAL6; the concentrations of calibrators 1 to 6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0116] 3.3 The six calibrators prepared in step 3.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding optical signal values ​​(see Table 8) and signal enhancement amplitude (see Table 9). Specifically, 25 μL of calibrator, 25 μL of FG-NfL antibody reagent, and 25 μL of Biotin-NfL antibody reagent were mixed sequentially; then, a first incubation was performed at 37°C for 15 min to obtain the first reaction solution; then, 175 μL of donor reagent was added, followed by a second incubation at 37°C for 10 min to obtain the second reaction solution; then, 50 μL of companion reagent was added, shaken thoroughly, and a third incubation was performed at 37°C for 3 min to obtain the third reaction solution; after photoexcitation, the optical signal value was read. The corresponding signal enhancement amplitude was calculated based on the optical signal value. The calculation formula is: Signal enhancement amplitude (%) = (Optical signal value of experimental group - Optical signal value without companion reagent) / Optical signal value without companion reagent * 100%.

[0117] It should be noted that in the control groups of Tables 8 and 9, "without companion reagent" means that the test is performed according to step 3.3, and the difference from the experimental group is that no companion reagent is added during the reaction process.

[0118] Table 8

[0119]

[0120] Table 9

[0121]

[0122] As shown in Tables 8 and 9, when comparing the detection light signal values ​​of the experimental group with the added chaperone reagent and the control group without the chaperone reagent, the signal enhancement of each concentration of calibrator in the experimental group with the added chaperone reagent was greater, with the highest reaching 103%. This indicates that adding the chaperone reagent can improve the detection light signal, which means that adding the chaperone reagent can improve the detection capability of NfL.

[0123] Example 4: Verification of the effect of the companion reagent on the detection signal of ubiquitin carboxyl-terminal hydrolase (UCH-L1)

[0124] 4.1 The main experimental materials are shown in Table 10.

[0125] Table 10

[0126] Ubiquitin C-terminal hydrolase (UCH-L1) Purchased from Huamei Bio UCH-L1 antibody-coated luminescent microspheres (FG-UCH-L1 antibody) Purchased from Baixinyi Biotin-labeled UCH-L1 antibody (Biotin-UCH-L1 antibody) Purchased from Wuxi Aoyue Dongyuan Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0127] 4.2 The FG-UCH-L1 antibody was diluted with HEPES diluent to obtain an FG-UCH-L1 antibody reagent with a concentration of 25 μg / mL, i.e., a luminescent reagent; the Biotin-UCH-L1 antibody was diluted with HEPES diluent to obtain a Biotin-UCH-L1 antibody reagent with a concentration of 2 μg / mL, i.e., a labeling reagent; the analyte UCH-L1 with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1 to 6 with different concentrations, i.e., CAL1 to CAL6; the concentrations of calibrators 1 to 6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0128] 4.3 The six calibrators prepared in step 4.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding optical signal values ​​(see Table 11) and signal enhancement amplitudes (see Table 12). Specifically, 25 μL of calibrator, 25 μL of FG-UCH-L1 antibody reagent, and 25 μL of Biotin-UCH-L1 antibody reagent were mixed sequentially; then incubated at 37°C for 15 min; followed by the addition of 175 μL of donor reagent and incubation at 37°C for 10 min to obtain the first reaction solution; then, 50 μL of companion reagent was added, shaken thoroughly, and incubated a second time at 37°C for 3 min to obtain the second reaction solution; after photoexcitation in the photo-induced chemiluminescence detector, the optical signal value was read. The corresponding signal enhancement amplitude was calculated based on the optical signal value to obtain the corresponding signal enhancement amplitude. The calculation formula is: Signal enhancement amplitude (%) = (Light signal value of experimental group - Light signal value without mate reagent) / Light signal value without mate reagent * 100%, which gives the signal enhancement amplitude.

[0129] It should be noted that in the control groups of Tables 11 and 12, "without companion reagent" means that the test is performed according to step 4.3, and the difference from the experimental group is that no companion reagent is added during the reaction process.

[0130] Table 11

[0131]

[0132] Table 12

[0133]

[0134] As shown in Tables 11 and 12, the experimental group with added chaperone reagent and the control group without chaperone reagent showed a greater signal enhancement for each concentration of calibrator in the experimental group with added chaperone reagent. In particular, the signal enhancement was as high as 101% for the detection of calibrators with concentrations as low as 50 pg / mL. This indicates that adding chaperone reagent can improve the detection light signal, which also means that adding chaperone reagent can improve the detection capability for low concentrations of UCH-L1.

[0135] Example 5: Verification of the effect of the companion reagent on the detection signal of the laminin (LN) assay kit (photochemical photoimmunoassay).

[0136] 5.1 The main experimental materials are shown in Table 13.

[0137] Table 13

[0138] Laminin (LN) Assay Kit (Photochemical Immunoassay) Purchased from Komei Diagnostics (batch 2301)

[0139] 5.2 Follow the instructions for the laminin (LN) assay kit (photochemical immunoassay).

[0140] 5.3 The concentrations of urea peroxide reagent and catalase reagent in the companion reagent were controlled at 50 mg / mL and 5 mg / mL, respectively, and then mixed at a volume ratio of 1:1 during detection. 50 μL of the mixed companion reagent was added to the laminin (LN) assay kit, shaken to mix, and the optical signal value was obtained. The corresponding signal enhancement amplitude was then calculated based on the optical signal value, as shown in Table 14.

[0141] Table 14

[0142]

[0143] As shown in Table 14, based on the comparison between the experimental group with the chaperone reagent and the control group without the chaperone reagent, the signal enhancement of the calibrators of each concentration in the experimental group was relatively large after the addition of the chaperone reagent, with the highest reaching 104%, and the signal enhancement of low concentration CAL2 reached 97%. This indicates that the addition of the chaperone reagent can effectively enhance the light signal value of low concentration target molecules during detection.

[0144] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A reagent for detecting neurodegenerative markers, characterized in that, This includes oxygen-generating agents, catalysts, and immunoassay reagents that can specifically bind to the neurodegenerative markers to be tested in the sample. The oxygen-generating agent reacts under the action of the catalyst to generate oxygen, and the generated oxygen is used to increase the oxygen content of the reaction system; the immunoassay reagent can generate a light signal using reactive oxygen species in the reaction solution, and the target molecule to be tested is selected from neurodegenerative markers.

2. The reagent according to claim 1, characterized in that, The oxygen-generating agent includes metal-peroxide salts and / or hydrogen peroxide complexes; Preferably, the oxygen-generating agent includes at least one of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxide.

3. The reagent according to claim 1, characterized in that, The catalyst includes at least one of enzyme compounds, halides, and metal oxides; Preferably, the catalyst comprises at least one selected from catalase, iodide, manganese dioxide, iron(III), silver and dichromate.

4. The reagent according to any one of claims 1 to 3, characterized in that: The concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL, and / or The concentration of the catalyst is 0.5 mg / mL to 100 mg / mL.

5. The reagent according to any one of claims 1 to 4, characterized in that: The sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma; and / or, The target molecules to be tested are selected from neurofilament light chains, glial fibrous acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

6. The reagent according to any one of claims 1 to 5, characterized in that, The immunoassay reagent is a photo-induced chemiluminescence assay reagent, which includes: A luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles are capable of reacting with reactive oxygen species to generate a detectable light signal; The labeling reagent includes a tag-labeled second biomolecule; wherein both the first biomolecule and the second biomolecule are capable of specifically binding to the target molecule in the test sample. Preferably, it also includes a donor reagent, which can convert ground-state oxygen into reactive oxygen under laser irradiation of a certain wavelength; More preferably, the donor reagent comprises photosensitive microparticles filled with a photosensitive substance.

7. A method for detecting neurodegenerative markers according to any one of claims 1 to 6, characterized in that, The method includes: After the sample to be tested and the immunoassay reagent are mixed and reacted, an oxygen-generating agent and a catalyst are added, and the amount of emitted photons is measured to obtain the light signal value.

8. The detection method according to claim 7, characterized in that, The specific steps include: S1. Mix the sample to be tested with the immunoassay reagent, and incubate for the first time to obtain the first reaction solution; S2. The oxygen-generating agent and catalyst are added to the first reaction solution, and the second reaction solution is obtained after a second incubation. S3. The second reaction solution is irradiated with excitation light, and the light signal is detected.

9. The method according to claim 8, characterized in that, The oxygen-generating agent and catalyst are premixed and then added to the first reaction solution; Preferably, the concentration ratio of the oxygen-generating agent and the catalyst premix is ​​(1-10):(1-20); more preferably, the concentration ratio of the premix is ​​10:

1. Preferably, the volume ratio of the oxygen-generating agent to the catalyst premix is ​​1:1; Preferably, the premixing time of the oxygen-generating agent and the catalyst is 1 to 10 minutes; more preferably, the premixing time is 3 minutes.

10. The method according to claim 9, characterized in that: The incubation temperature is the same for each incubation; preferably, the incubation temperature is 37-42°C; more preferably, it is 37°C.

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