Light-initiated chemiluminescence assay kit for phosphorylated tau protein p-tau 217, use method thereof, and use thereof

Abstract

A light-initiated chemiluminescence assay kit for phosphorylated Tau protein p-tau 217, a use method thereof and the use thereof. The kit comprises an R1 reagent, an R2 reagent and an R3 reagent. The R1 reagent comprises luminescent microspheres and first antibodies each of which coats a luminescent microsphere, has a first tag molecule and can specifically bind to p-tau 217. The R2 reagent comprises second antibodies carrying second tag molecules. The R3 reagent comprises first pairing molecules that can specifically bind to the first tag molecules. The first antibodies and the second antibodies can specifically bind to different epitopes of p-tau 217, and one first pairing molecule can bind to at least two first tag molecules.

Description

A photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217, its usage and application.

[0001] This application claims priority to Chinese Patent Application No. 2024116293171, filed on November 15, 2024, entitled "A Photochemiluminescence Detection Kit for Phosphorylated Tau Protein p-tau 217 and Its Usage and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of immunoassay technology, and in particular to a photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217, its usage method, and its application. Background Technology

[0003] Alzheimer's disease (AD), commonly known as senile dementia, is the most common neurodegenerative disease. Neuropathological changes occur 15 to 20 years before the onset of clinical symptoms in AD, such as the appearance of β-amyloid (Aβ) plaques and tau protein hyperphosphorylation in brain tissue. The accumulation of Aβ and tau proteins can impair neuronal function and even lead to neuronal death, resulting in a significant decline in cognitive functions such as memory. However, this process progresses extremely slowly. Therefore, early detection and accurate identification in the early stages of the disease are crucial for early intervention in AD.

[0004] Currently, clinical diagnostic methods for Alzheimer's disease (AD) include scale assessments, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid (CSF) testing. However, these methods suffer from drawbacks such as insufficient objectivity, high invasiveness, cumbersome procedures, and high costs, significantly limiting their clinical application, especially in primary healthcare institutions. The clinical misdiagnosis rate for AD is as high as 76.8%, making early screening and intervention difficult. Therefore, providing a low-cost, minimally invasive, convenient, and standardized blood biomarker for AD is an urgent clinical need.

[0005] p-tau 217 is a phosphorylated form of Tau protein, primarily phosphorylated at threonine 217. It is significantly elevated in the preclinical stage of Alzheimer's disease (AD), providing an earlier indication of AD development. Studies show that the average level of p-tau 217 in plasma of healthy individuals is approximately 0.3 to 0.4 pg / mL, while in AD patients it reaches 1 to 2 pg / mL, with a positive cutoff below 1 pg / mL. Conventional immunoassay techniques detect the overall signal after the reagent system reaction using optical or electrical methods, then convert the signal intensity into detectable concentration using a standard curve. However, their detection limits are generally around 1 to 10 ng / mL, making it difficult to achieve such high sensitivity and reliably detect p-tau 217 in plasma. Single molecule array (Simoa) technology is a digital detection technique that forms an enzyme complex on the surface of magnetic beads, consisting of a capture antibody, a target antigen, a biotinylated detection antibody, and an avidin-conjugated complex. After binding with a fluorescent substrate, the magnetic beads fall into the arrayed microwells for reaction and detection. Its detection sensitivity is exponentially improved compared to ELISA and conventional chemiluminescence detection methods. However, the detection process is cumbersome and complex, and the detection cost is extremely high, which greatly limits its clinical application. Summary of the Invention

[0006] To address or partially address the problems existing in related technologies, this application provides a photo-induced chemiluminescence detection kit for phosphorylated Tau protein p-tau 217, along with its usage and applications. This kit enhances the ability of luminescent microspheres to receive reactive oxygen species generated by photosensitive microspheres, improves the utilization rate of reactive oxygen species, and increases the detection signal value, thereby enhancing detection sensitivity and meeting the photo-induced chemiluminescence detection requirements for phosphorylated Tau protein p-tau 217 in low-value plasma samples.

[0007] The photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217 of this application comprises: reagent R1, which contains luminescent microspheres coated with a first antibody capable of specifically binding to p-tau 217, the first antibody carrying a first tag molecule; reagent R2, which contains a second antibody carrying a second tag molecule; and reagent R3, which contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; the first antibody and the second antibody are capable of specifically binding to different epitopes of p-tau 217, and one first pairing molecule can bind to at least two first tag molecules.

[0008] In the kit of this application, the first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

[0009] In the kit of this application, the specific tagging system composed of the first tag molecule and the first paired molecule is selected from the Tag-Catcher system.

[0010] In the kit of this application, in the Tag-Catcher system, a tag with a small molecular weight binds to the first antibody as a first tag molecule.

[0011] In the kit of this application, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag.

[0012] In the kit of this application, the first paired molecule is a polyCatcher with a degree of polymerization of not less than 2.

[0013] In the kit of this application, the molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule.

[0014] In the kit of this application, the molar ratio of the first pairing molecule to the first tag molecule is 1:(1-16).

[0015] In the kit of this application, the degree of polymerization of the first paired molecules is negatively correlated with its optimal molar amount.

[0016] In the kit of this application, the product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

[0017] In the kit of this application, the mass ratio of the luminescent microspheres to the first antibody is 10:(0.05-5).

[0018] In the kit of this application, the molar ratio of the second antibody to the second tag molecule is 1:(20-50).

[0019] In the kit of this application, the luminescent microspheres are polymeric microparticles filled with a luminescent composition, which can react with reactive oxygen species to generate a detectable light signal.

[0020] In the kit of this application, the luminescent composition includes enol ethers, enamines, 9-alkylxanthan gum, 9-alkyl-N-alkylacridinium, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, luster enhancers, or europium complexes.

[0021] In the kit of this application, the kit further includes: R4 reagent, which comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second tag molecule.

[0022] In the kit of this application, the photosensitive microspheres are polymeric microparticles filled with photosensitizers, which can generate reactive oxygen species under photoexcitation.

[0023] In the kit of this application, the photosensitizer includes methylene blue, rose red, porphyrin, phthalocyanine, chlorophyll, 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, and 9,10-diphenylanthracene-9,10-endoperoxide.

[0024] In the kit of this application, the two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, and the second tag molecule and the second pairing molecule, do not react with each other.

[0025] In the kit of this application, the second tag molecule and the second pairing molecule are selected from the biotin-avidin system.

[0026] The method of using the kit described in this application includes mixing and incubating the R1, R2, and R4 reagents with the sample to be tested, and then adding the R3 reagent to aggregate at least two luminescent microspheres into a polymeric luminescent microsphere.

[0027] In the kit of this application, the reaction time is ≥45 min after adding reagents R1 and R2; the reaction time is 0 to 15 min after adding reagent R3.

[0028] The kit described in this application or its method of use can be used to detect p-tau 217 in blood samples.

[0029] The method for detecting p-tau 217 in blood samples as described in this application includes: mixing the sample to be tested with reagents R1 and R2, which contain a first antibody and a second antibody capable of specifically recognizing p-tau 217; incubating for a period of time; adding reagent R4; incubating for a period of time; adding reagent R3; incubating for a period of time; measuring the intensity of the generated chemiluminescence signal; and determining whether the sample to be tested contains p-tau 217 or the content of p-tau 217 in the sample to be tested based on the intensity of the light signal.

[0030] It should be noted that the detection method described in this application is not for disease diagnosis.

[0031] 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

[0032] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0033] Where numerical ranges are 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 by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0034] 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 invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0035] The sample to be tested as described in this application refers to a mixture that may contain an analyte, including but not limited to proteins, hormones, antibodies, or antigens. Typical samples to be tested that can be used in the methods disclosed in this application include bodily fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion. The sample to be tested can be diluted with a diluent or buffer solution as needed before use. For example, to avoid the hook effect, the analyte can be diluted with a sample diluent before detection on the instrument; in this case, the diluted solution that may contain the analyte is collectively referred to as the sample to be tested.

[0036] The term "antibody" as used in the broadest sense includes any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of the antibody and non-antibody proteins. Where necessary, the antibody may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin. The location capable of specific binding to the antigen in an immune response, known as the complementarity determining region (CDR), is located at the Fab end. Therefore, in this application, the Fab end of the antibody is referred to as the specific binding region, and the opposite end is referred to as the non-specific binding region.

[0037] The antigens described in this application refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). The antigens can be natural antigens extracted from pathogens or animal tissues, or recombinant antigens with specific antigenic properties prepared through genetic engineering technology. Where necessary, the antigens can be further conjugated to other parts, such as specific binding pairing members, for example, biotin or avidin.

[0038] The terms “combination,” “connection,” and “coupling” used in this application refer to the union between two substances caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, or interactions including but not limited to salt bridges and water bridges.

[0039] The specific binding described in this application refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the corresponding reactants.

[0040] The Tag-Catcher system described in this application is a protein covalent linking technology, comprising two parts: a Tag and a Catcher, each consisting of multiple amino acid residues. Covalent linking of proteins is achieved through a specific reaction between the Tag and the Catcher. The Catcher protein in the Tag-Catcher system can exist as a monomer or a multimeric protein; the Catcher used in this application is a multimeric protein. Multimeric proteins are proteins composed of two or more polypeptide chains, which can be identical or different, and are interconnected through covalent or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to form a multimeric macromolecular protein structure.

[0041] The biotin-avidin system described in this application is a bioreaction amplification system comprising two main components: biotin and either avidin or streptavidin. Biotin is widely found in plant and animal tissues and has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary binding site for avidin. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents. Avidin is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" with avidin to enhance analytical sensitivity. Where necessary, any reagent used in this application, including antigens, antibodies, receptors, or donors, can be conjugated to any of the biotin-streptavidin specific binding pairing members as required.

[0042] The reactive oxygen species (ROS) described in this application refer to a general term for oxygen-containing and reactive substances in the body or natural environment. They are primarily excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH),) as well as nitric oxide and reactive oxygen species (ROS). 1 O2), etc.

[0043] The luminescent microspheres described in this application refer to polymeric microparticles filled with a luminescent composition, capable of reacting with reactive oxygen species to generate a detectable light signal. Luminescent microspheres may also be called acceptor microspheres or luminescent microparticles. In some specific embodiments of this application, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridinium, arylate ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of this application, the luminescent composition may further include europium complexes; more preferably, the europium complex is MTTA-EU. 3+ .

[0044] The photosensitive microspheres described in this application refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microparticles. Solutions containing such photosensitive microspheres can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microspheres can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0045] The microparticles described in this application can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for this application is carboxylated polystyrene latex microspheres.

[0046] This application will now be described in more detail.

[0047] Photocatalytic chemiluminescence analysis is a next-generation immunoassay technique based on nanoscale polymer particles. Its core principle is the generation and transfer of reactive oxygen species (ROS): energy transfer between photosensitive microspheres (GG) and luminescent microspheres (FG) generates high-energy red light during energy level transitions. A single-photon counter and mathematical fitting convert the photon count into a relative light signal. In the presence of the target analyte (e.g., the antigen / antibody to be tested), the antibody / antigen modified on the surfaces of the two microspheres undergoes an antigen-antibody specific binding reaction with the antigen / antibody to be tested, forming a sandwich immune complex. This shortens the spatial distance between the two microspheres, and the light signal generated by the transfer of ROS between the two microspheres enables qualitative or quantitative analysis of the antigen / antibody to be tested.

[0048] The inventors of this application discovered in their research that in a photochemiluminescence analysis platform, when the target analyte concentration is extremely low, such as p-tau 217 in plasma, the molar amount of the luminescent microspheres is much higher than that of the target analyte. However, in most sandwich immune complexes in the reaction system, a luminescent microsphere and a photosensitive microsphere are formed by bridging a target analyte molecule with the other microsphere. During reading, the photosensitive microsphere generates a large amount of... 1 Only a small portion of the O2 was received by the adjacent luminescent microspheres and generated light signals; the rest was not received. 1 O2 is quenched within 4 μs. Therefore, how to improve... 1 The utilization rate of O2 is crucial for improving the sensitivity of photochemiluminescence detection, especially for low-value samples such as plasma containing p-tau 217. Increasing the particle size of the luminescent microspheres can allow them to receive more O2. 1 O2 generates a stronger signal, but at the same time, it reduces the specific surface area and slows down particle migration rate, directly affecting the efficiency of the immune response and reducing detection sensitivity. The inventors of this application considered and designed a method to improve reagent performance by increasing the utilization rate of free luminescent microspheres in the detection reagent, providing an improved... 1 A new way to improve detection sensitivity by increasing the utilization rate of O2.

[0049] The photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217 provided in this application includes reagents R1, R2, and R3. Reagent R1 comprises luminescent microspheres and a first antibody coated on the luminescent microspheres, carrying a first tag molecule capable of specifically binding to p-tau 217; reagent R2 comprises a second antibody carrying a second tag molecule; and reagent R3 comprises a first pairing molecule capable of specifically recognizing and binding to the first tag molecule. The first and second antibodies can specifically bind to different epitopes of p-tau 217, and one first pairing molecule can bind to at least two first tag molecules, thereby causing at least two luminescent microspheres to bind and form a polymeric luminescent microsphere.

[0050] In this application, the first and second antibodies in reagents R1 and R2 can react with p-tau 217 to form a sandwich immune complex. The first pairing molecule in reagent R3 can form a specific tagging system with the first tag molecule on the luminescent microspheres in reagent R1. This aggregates free luminescent microspheres (those whose first antibody has not specifically bound to p-tau 217) onto the main luminescent microspheres (those whose first antibody has specifically bound to p-tau 217), increasing the effective particle size and specific surface area of ​​the luminescent microspheres, thus enhancing the production of photosensitive microspheres. 1 Increased O2 utilization and quenching 1 The reduction in O2 leads to a stronger light signal. Simultaneously, the particle size and mass of each luminescent microsphere in reagent R1 remain unchanged, maintaining a good particle migration rate and ensuring the efficiency of the antigen-antibody immune reaction. This improves overall detection sensitivity and enhances the detection performance of low-value samples, enabling high-performance detection of the femtogram-level biomarker p-tau 217 in plasma.

[0051] The number of luminescent microspheres in the polymeric luminescent microspheres described in this application is equal to or greater than two, and no limitation is made herein.

[0052] In some embodiments of this application, reagent R3 may further include luminescent microspheres, i.e., the first pairing molecule is also coated on the luminescent microspheres. When reagents R1 and R3 are mixed, the specific recognition and binding ability between the first tag molecule and the first pairing molecule can promote the aggregation of the luminescent microspheres of reagents R1 and R3 to form polymeric luminescent microspheres.

[0053] In some embodiments of this application, the specific tagging system consisting of the first tag molecule and the first pairing molecule can be selected from the Tag-Catcher system, and any combination from the system can be selected as the first tag molecule in reagent R1 and the first pairing molecule in reagent R3. In this application, the first pairing molecule can also be referred to as a tool protein.

[0054] In some embodiments of this application, the first tag molecule may be selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag in the Tag-Catcher system, and the first pairing molecule may be selected from any Cater protein. The first tag molecule and the first pairing molecule may be selected from specific tag systems such as Spy-Tag and Spy-Catcher, His-Tag and Anti-His (where the Cater is Anti-His), Snoop-Tag and Snoop-Catcher, etc. Catcher and Tag have excellent specific recognition and binding capabilities, and the use of a small molecular weight Tag tag to bind to the first antibody and co-coat the luminescent microspheres has no significant impact on the expression of the first antibody or the properties of the luminescent microspheres.

[0055] In some embodiments of this application, when the first tag molecule is selected from Tag, the first pairing molecule can be selected from the multimeric protein of Catcher, and the degree of polymerization is not less than 2, for example, it can be selected from the octamer protein of Catcher. The degree of polymerization of Catcher directly affects the rate at which Catcher captures Tag, and thus affects the aggregation rate of luminescent microspheres. The higher the degree of polymerization of Catcher, the higher the Tag capture rate, the higher the aggregation rate of luminescent microspheres, and the better the effect on improving the light signal intensity.

[0056] In some embodiments of this application, the molar amount of the first pairing molecule is less than or equal to the molar amount of the first tag molecule. Preferably, the molar ratio of the first pairing molecule to the first tag molecule is 1:(1-16); more preferably, it is 1:(2-8). The proportion of the first pairing molecule directly affects the aggregation of the luminescent microspheres. If it is too low, the aggregation efficiency and degree will be low, and the effect on improving the detection performance will not be obvious; if it is too high, it is easy to produce a "hook effect", which makes it difficult for the luminescent microspheres to aggregate, resulting in no significant improvement in detection performance, and even affecting the accuracy of the detection results.

[0057] When the first tag molecule and the first pairing molecule are selected from the Tag-Catcher system, the higher the degree of polymerization of the first pairing molecule (Catcher), the lower the required molar amount of the first pairing molecule; that is, the degree of polymerization of the first pairing molecule is negatively correlated with its optimal molar amount. Preferably, when the product of the degree of polymerization of the first pairing molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, the effect on improving the light signal intensity of the luminescent microspheres is more significant.

[0058] The degree of polymerization and amount of the first paired molecule can be determined based on the amount of the first tag molecule. A higher degree of polymerization of the first paired molecule results in a lower required optimal molar amount and a faster reaction rate with the first tag molecule; conversely, a lower degree of polymerization of the first paired molecule results in a higher required optimal molar amount and a faster aggregation rate. When the product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, it significantly enhances the light signal intensity of the luminescent microspheres. Excessive amounts can easily lead to the hook effect, affecting the detection results; insufficient amounts affect the aggregation rate and degree of aggregation of the polymeric luminescent microspheres, resulting in minimal performance improvement.

[0059] In some embodiments of this application, a first tag molecule is co-expressed with a first antibody and linked to the non-specific binding region of the first antibody to form a first antibody with the first tag molecule. The co-expression linking can be achieved by linking the first tag molecule to the non-specific binding region of the first antibody using pClick technology (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18):9107-9117. doi:10.7150 / thno.62444.)), or by inserting the sequence of the first tag molecule into the non-specific binding region of the first antibody.

[0060] In some embodiments of this application, the first antibody has a Y-shaped structure, comprising two Fab segments and one Fc segment, with the non-specific binding region of the first antibody being its Fc terminus. In other embodiments of this application, the first antibody has a V-shaped structure, comprising two Fab segments, with the non-specific binding region of the first antibody being its Fab-linked hinge region. When the CDR region of the first or second antibody is occupied or the Fab terminus is folded during coating, labeling, or other steps, the Fab terminus of the antibody may not be effectively displayed, affecting the effective amount or activity of the antibody that can bind to the target molecule p-tau 217 in the reaction system, thereby affecting the photochemical detection performance of p-tau 217. The CDR region, the site in the first antibody formed in this manner that can specifically bind to p-tau 217 in the immunoreaction, can be fully and effectively displayed, increasing the effective amount and activity of the antibody that can bind to the target molecule p-tau 217 in the reaction system, and improving the detection sensitivity of p-tau 217.

[0061] In some embodiments of this application, the mass ratio of luminescent microspheres to the first antibody is 10:(0.05-5); preferably 10:(0.1-1); more preferably 10:0.5. The amount of the first antibody coated on the luminescent microspheres affects the reaction efficiency between the first antibody and p-tau 217 in the sample to be tested, thereby affecting the discrimination of the detection results.

[0062] In some embodiments of this application, the molar ratio of the second antibody to the second tag molecule is 1:(20-50); preferably 1:(25-35); more preferably 1:30. The amount of the second tag molecule on the second antibody affects its reaction efficiency with the photosensitive microspheres, thereby affecting the discrimination of the detection results.

[0063] The kit provided in this application also includes R4 reagent, which comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second tag molecule. The specific binding ability of the second pairing molecule on the photosensitive microspheres to the second tag molecule enables the second antibody to bind to the photosensitive microspheres, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres, allowing the photosensitive microspheres to produce… 1 O2 can be effectively received by the luminescent microspheres and generate light signals, meeting the requirements of photo-induced chemiluminescence detection.

[0064] In some embodiments of this application, the two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, the second tag molecule and the second pairing molecule, do not react with each other, thereby avoiding the occurrence of specific pairing interference reactions and improving the accuracy and stability of the detection results.

[0065] The second tag molecule and the second pairing molecule in reagents R2 and R4 can be selected from the biotin-avidin system, for example, one of which is selected from biotin and the other is selected from avidin or streptavidin; preferably, avidin is coated on the photosensitive microspheres, and the second antibody is bound to streptavidin, thereby labeling the second antibody with biotin.

[0066] In some embodiments of this application, the kit further includes buffers, stabilizers, etc. The buffer may be selected from at least one of PBS buffer, Tris-hydrochloric acid buffer, HEPES buffer, and MES buffer. The stabilizer may be selected from at least one of bovine serum albumin, dextran, sorbitol, glycerol, glycine, alanine, Tween 20, and Tween 80.

[0067] The kit provided in this application can be used as follows: after mixing and incubating reagents R1, R2, and R4 with the sample to be tested, reagent R3 is added and reacts with the first tag molecule of reagent R1 to cause at least two luminescent microspheres to aggregate into multiple luminescent microspheres.

[0068] In some embodiments of this application, 15 μL of reagent R1, 15 μL of reagent R2, and 175 μL of reagent R4 can be mixed with 100 μL of the sample to be tested and incubated before adding 15 μL of reagent R3 to initiate the reaction. That is, in the photochemiluminescence detection process of p-tau 217, by controlling the volume ratio of reagent R1, reagent R2, and the sample to be tested to 3:3:20, the volume ratio of reagent R2 to reagent R4 to 3:35, and the volume ratio of reagent R1 to reagent R3 to 1:1, the reagent volume can be reduced, the reagent concentration increased, and the sample proportion increased. This shifts the reaction equilibrium towards the binding direction, which is beneficial for increasing the amount of the formed luminescent microsphere-(p-tau 217)-photosensitive microsphere sandwich immune complex, thereby improving detection performance.

[0069] In some embodiments of this application, reagents R1 and R2 can be mixed with the sample to be tested, incubated for a period of time, and then reagent R4 can be added. After incubating for a period of time, reagent R3 can be added.

[0070] In some embodiments of this application, the method of use includes:

[0071] S1. Mix reagents R1 and R3 with the sample to be tested and incubate at 37°C to obtain the first reactant;

[0072] S2, add reagent R4, incubate at 37°C to obtain the second reactant;

[0073] S3. Add reagent R2 and incubate at 37°C to obtain the third reactant;

[0074] S4. Irradiate the third reactant with light of a specific wavelength and detect the luminescence value.

[0075] This application first combines luminescent microspheres with a first antibody and a second antibody coated thereon that specifically recognize and bind to p-tau 217, and then binds the target analyte p-tau 217 in the test sample to form a sandwich immune complex of "first antibody-(p-tau 217)-second antibody". Then, the target luminescent microspheres are immobilized onto the photosensitive microspheres by the specific binding of a second tag molecule on the second antibody to a second pairing molecule coated thereon, resulting in a "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex. Finally, the free luminescent microspheres are bound to the target luminescent microspheres by the specific recognition and binding ability of the first pairing molecule and the first tag molecule, forming polymeric luminescent microspheres.

[0076] After the conventional reaction is completed, the free luminescent microspheres aggregate around the composite luminescent microspheres, causing the photosensitive microspheres to produce... 1O2 can be received by multiple luminescent microspheres in the polymer luminescent microspheres and simultaneously generate light signals, effectively improving... 1 This approach improves O2 utilization, enhances signal intensity, and thus increases the detection sensitivity of the reagent kit, meeting the requirements for photochemiluminescence detection of low-value samples. Furthermore, the specific tagging system used in this scheme, namely the first tag molecule and the first paired molecule, employs a Tag-Catcher system. This system boasts high specificity and affinity, significantly improving reagent performance without interfering with the original immunoreaction. It is highly applicable and has a wide range of applications, representing a platform-type improvement. In particular, when used for the detection of p-tau 217 in extremely low-value plasma samples, it significantly enhances detection sensitivity, meeting the detection needs of extremely low-value samples. Simultaneously, the detection method is simple, easy to operate, and low in cost.

[0077] In some embodiments of this application, the reaction time after adding reagents R1 and R2 is ≥45 min, preferably ≥60 min; more preferably 60–75 min. The reaction time of the first antibody, the second antibody, and the target analyte p-tau 217 affects the degree of reaction. Extending the reaction time ensures the formation of the "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex, improving the discrimination of the detection results. The reaction time after adding reagent R3 is 0–15 min, i.e., the aggregation time of the luminescent microspheres is 0–15 min. As the aggregation time increases, the aggregation reaction of the luminescent microspheres gradually increases and tends to equilibrium. Therefore, the aggregation time is preferably 3–9 min, more preferably 5–7 min.

[0078] In some embodiments of this application, when using each detection reagent, the concentration of luminescent microspheres in reagent R1 is 75–100 μg / mL, preferably 80–90 μg / mL, and more preferably 83.3 μg / mL. The concentrations of the luminescent microspheres and the first antibody affect the efficiency of their immunoreaction with p-tau 217, thereby affecting the discrimination of the detection results. The concentration of the second antibody in reagent R2 is 1–10 μg / mL, preferably 2–5 μg / mL, and more preferably 3.3 μg / mL. The concentration of the second antibody affects its immunoreaction efficiency with p-tau 217 and its ability to resist biotin interference, thereby affecting the discrimination of the detection results.

[0079] The test kit described above can be used to detect p-tau 217 in blood samples.

[0080] The photo-induced chemiluminescence detection method for phosphorylated Tau protein p-tau 217 includes: mixing the sample to be tested with reagents R1 and R2, which contain a first antibody and a second antibody that specifically recognize p-tau 217; incubating for a period of time; adding reagent R4; incubating for a period of time; adding reagent R3; incubating for a period of time; and then performing photo-induced chemiluminescence detection. The intensity of the generated chemiluminescence signal is measured, and the presence or content of p-tau 217 in the sample is determined based on the intensity of the light signal.

[0081] The technical solution provided in this application introduces tagged antibodies into luminescent microspheres and adds pairing molecules capable of capturing the tag. The specific reaction between the pairing molecules and the tag allows the free luminescent microspheres in the reaction system to aggregate on the immune complex, improving the utilization rate of reactive oxygen species generated by the photosensitive microspheres, thereby enhancing the detection signal value and improving the overall performance of photochemiluminescence detection. This meets the sensitivity requirements for photochemiluminescence detection of p-tau 217 in low-value blood samples. Furthermore, the entire detection process does not alter the conventional detection sequence, does not interfere with reagent specificity, is easy to operate, has low detection cost, and a wide range of applications, making it of significant clinical value.

[0082] To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0083] Next, taking p-tau 217 as the target molecule, antibody molecules specifically recognizing p-tau 217 were modified with different tags and coated onto luminescent microparticles in the same proportion. These microparticles were then used in a double-antibody sandwich assay with biotin-labeled antibodies that specifically recognize different sites on p-tau 217 to detect the target molecule p-tau 217 in the sample. After the conventional detection sequence (sample + reagent R1 + reagent R3, followed by reagent R4), luminescent microparticles containing polymerized free molecules corresponding to the tags were added. By comparing the detection discrimination between different experimental groups, the improvement effect of the multi-polymer luminescent microparticle mode on the photochemiluminescence detection performance of p-tau 217 was determined.

[0084] Example 1: Preparation of antibody-coated luminescent microspheres, biotin-labeled antibodies, and Spy Catcher

[0085] 1. The main experimental materials and equipment are shown in Table 1.

[0086] Table 1

[0087] 2. Experimental Procedure

[0088] 2.1 Preparation of luminescent microspheres using the Spy Tag system as an example

[0089] 2.1.1 Antibody (p-tau 217) Ab1 linked to Tag

[0090] The Spy Tag (sequence AHIVMVDAYKPTK) was linked to the antibody (p-tau 217)Ab1 using pClick technology (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18):9107-9117. doi:10.7150 / thno.62444.)) to obtain the Fc-linked Tag antibody. The protein concentration of the collected fraction was determined by the BCA method, and the fraction containing protein was then subjected to electrophoresis. The fractions that met the molecular weight of the Tag-antibody were collected and dialyzed into the buffer required for the subsequent coupling reaction to obtain the antibody (p-tau 217)Ab1-Spy Tag co-expressed by the tag molecule Spy Tag and (p-tau 217)Ab1.

[0091] 2.1.2 Luminescent microparticles coated with antibody (p-tau 217) Ab1

[0092] ① Take 10 mg of aldehyde-based luminescent microparticles into a centrifuge tube, wash once with 0.02 M PBS buffer, and then adjust the volume to 20 mg / mL with 0.02 M PBS;

[0093] ② Take (p-tau 217)Ab1-Spy Tag, dialyze it with 0.02M PBS buffer, and then determine its concentration using the BCA method;

[0094] ③ The treated luminescent microparticles FG and (p-tau 217)Ab1-Spy Tag were thoroughly mixed at a mass ratio of 10:0.5 and then reacted at 37℃ for 16 hours.

[0095] ④ Add 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH4 (8 mg / mL, 0.05 M CB) to the reaction system, mix thoroughly, and react at 4 °C for 2 h to obtain the reaction solution;

[0096] ⑤ The reaction solution was washed twice with PBST buffer, then washed once with reconstitution solution (0.02M PBS + 0.5% BSA + 0.5% Tween 20 + 1% dextran), and then reconstituted by sonication to obtain luminescent microparticles coated with (p-tau 217)Ab1-Spy Tag with a final concentration of 83.3 μg / mL, i.e., reagent R1.

[0097] 2.2 Biotin-labeled antibody (p-tau 217) Ab2

[0098] ① Dialyze (p-tau 217)Ab2 with 0.1M NaHCO3 solution to remove impurities, and then determine the concentration using the BCA method;

[0099] ② Add 30 molar amounts of Biotin-PEG12-NHs to the dialyzed (p-tau 217)Ab2 solution, mix well, and react at 4℃ for 12 hours to obtain Bio-(p-tau 217)Ab2 solution;

[0100] ③ Dialyze with 0.02M PBS buffer to remove free biotin, then determine the concentration using BCA.

[0101] ④ Prepare a 3.3 μg / mL biotin-labeled antibody Bio-(p-tau 217)Ab2, i.e., reagent R2, using a reconstitution solution.

[0102] 2.3 Preparation and purification of Spy Catcher

[0103] ① Construction of Catcher plasmid expression vector: The expression vector of Spy Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0104] ②Catcher expression induced by *E. coli*: The *E. coli* expression vector induces the expression of Spy Catcher protein. Culture conditions: ① Self-induction medium (10g peptone, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x 5052, pH 7.4), with 100nM ampicillin added; ② Induction conditions: overnight culture at 30℃ and 200rpm in a shaker.

[0105] ③Catcher purification: The cultured bacterial solution was centrifuged at 8000 rpm, and the bacterial pellet was collected. The bacterial cells were resuspended in 100 mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20 mM ID, pH=7.4). The resuspended bacterial solution was centrifuged at 18000 rpm for 40 minutes, and the supernatant was collected and subjected to nucleic acid disruption, filtration, affinity column purification, elution, collection of protein, and reconstitution solution was added and sonicated to obtain Spy Catcher.

[0106] Example 2: Effects of degree of polymerization and usage ratio on the photochemiluminescence detection performance of p-tau 217

[0107] 1. Experimental Procedure

[0108] 1.1 Dimers, pentamers, and octamers of Spy Catcher were prepared according to the method described in Example 1, and then diluted with a reconstitution solution to reagent R3 with a Tag molar ratio of 1:16, 1:8, 1:4, 1:2, 1:1, and 2:1, respectively. These were then mixed with a universal photosensitive microparticle solution, reagent R4. The solutions were then applied to the test sample... The detection was performed on a 500 chemiluminescence detection system.

[0109] 1.2 Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of universal solution (R4), and incubate at 37 °C for 15 min; add 15 μL of R3 reagent, and add 15 μL of reconstitution solution to the control group, and incubate at 37 °C for 15 min respectively; after photoexcitation reaction, read the light signal value of each group. The experimental data are shown below.

[0110] 2. Experimental Results

[0111] 2.1 Effects of the Spy Catcher dimer reaction:

[0112] Table 2 Signal Values

[0113] Table 3 Discrimination

[0114] 2.2 Spy Catcher pentamer reaction effect:

[0115] Table 4 Signal Values

[0116] Table 5 Discrimination

[0117] 2.3 Spy Catcher Octamer Reaction Effect:

[0118] Table 6 Signal Values

[0119] Table 7 Discrimination

[0120] 3. Results Analysis

[0121] ① As shown in Table 2-7, within the current gradient range, the polymer particle size (degree of polymerization) does not affect the particle size at which the luminescent microparticles receive singlet oxygen. The higher the degree of polymerization of the first paired molecule corresponding to the first tag molecule, the better the improvement effect. When the first paired molecule is a Spy Catcher octamer, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more.

[0122] ② As shown in Table 2-7, when the molar ratio of Spy Tag in Spy Catcher and R1 reagent is 1:(16~1), both the positive signal and the discrimination are improved; among them, the molar ratio of 1:4 (Spy Catcher octamer: Spy Tag) has the best effect, at which the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more.

[0123] ③ The degree of polymerization and the proportion of the first paired molecule depend on the molecular weight of the first tag. The molar amount of the first paired molecule should be lower than or equal to the molar amount of the first tag molecule, and the degree of polymerization of the first paired molecule is negatively correlated with the optimal molar amount. As shown in Table 2-7, when the product of the degree of polymerization and the molar amount of the first paired molecule is 1 to 2.5 times the molar amount of the first tag molecule, the positive signal and discrimination are significantly improved.

[0124] ④ As shown in Table 2-7, using Spy Tag as a tag and Spy Catcher as a pairing molecule significantly improves the positive signal and discrimination for detecting p-tau 217 recombinant antigen and actual samples.

[0125] Therefore, using a specific tagging system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres can improve the positive signal and discrimination, and significantly enhance the sensitivity of photo-induced chemiluminescence detection, thus meeting the detection requirements of p-tau 217 in low-value samples such as blood samples.

[0126] Example 3: Effect of aggregation time on the photochemiluminescence detection performance of p-tau 217

[0127] 1. Experimental Procedure

[0128] 1.1 Dilute the Spy Catcher octamer with a redissolved solution to a final concentration of reagent R3 (1:4 Tag molar ratio with reagent R1). Prepare the general-purpose photosensitive microparticle solution R4. Mix with the sample to be tested... The detection was performed on a 500 chemiluminescence detection system.

[0129] 1.2 Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of universal solution, and incubate at 37 °C for 15 min; add 15 μL of R3 reagent, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 3, 6, 9, 12, and 15 min, respectively; after photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0130] 2. Experimental Results

[0131] Table 8 Signal Values

[0132] Table 9 Discrimination Index

[0133] 3. Results Analysis

[0134] As shown in Tables 8-9, with the addition of reagent R3 (containing the first pairing molecule) and the extension of reaction time, the positive signal and discrimination gradually increased and tended to reach equilibrium; when the reaction reached about 6 minutes, the aggregation reaction of free luminescent particles reached equilibrium.

[0135] Therefore, in the process of using a specific tagging system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres, from the perspective of balancing reaction time and performance, the reaction time after adding reagent R3 can be 0 to 15 min, with the preferred reaction time being 3 to 9 min and the optimal reaction time being 5 to 7 min.

[0136] Example 4: Applicability verification of different labeling systems for forming "multi-luminescent microspheres"

[0137] 1. Experimental Procedure

[0138] 1.1 Following the method of coating antibody onto luminescent microspheres in Example 1 above, luminescent microspheres coated with (p-tau 217)Ab1 carrying different tag molecules (Spy-Tag, His-Tag, Snoop-Tag) were prepared and diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1.

[0139] 1.2 Bio-(p-tau 217)Ab2 was diluted to 3.3 μg / mL with a reconstitution solution to obtain reagent R2.

[0140] 1.3 The first paired molecules (octamers Spy-Catcher, Anti-His, and Snoop-Catcher corresponding to the tag molecules) are diluted with a reconstituted solution to reagent R3 in molar ratios of 1:16, 1:4, and 1:1 with the tag molecules in reagent R1.

[0141] 1.4 with the sample to be tested Detection was performed on a 500 chemiluminescence detection system:

[0142] Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min. Add 175 μL of universal photosensitive microparticle solution and incubate at 37 °C for 15 min. Add 15 μL of R3 reagent, and add 15 μL of reconstitution solution to the control group, and incubate at 37 °C for 15 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0143] 2. Experimental Results

[0144] Table 10 Signal Values

[0145] Table 11 Discrimination Index

[0146] 3. Results Analysis

[0147] As shown in Table 10-11, different specific tagging systems such as Spy-Catcher / Spy-Tag, His-Tag / Anti His-Ab, and Snoop-Tag / Catcher can all improve the positive signal value and low-discrimination resolution to varying degrees.

[0148] Therefore, the specific labeling system described in this application has a good effect on the aggregation of free luminescent microspheres into polymeric luminescent microspheres to improve detection performance, and the photo-induced chemiluminescence detection sensitivity is effectively improved, which can meet the detection requirements of p-tau 217 in low-value samples such as blood samples.

[0149] The embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Any modifications, equivalent substitutions, improvements, etc., made without departing from the scope and spirit of the described embodiments should be included within the scope of protection of this invention. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to the technology in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

Claims

1. A photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217, characterized in that, include: R1 reagent comprises luminescent microspheres coated with a first antibody capable of specifically binding to p-tau 217, the first antibody carrying a first tag molecule; R2 reagent contains a second antibody with a second tag molecule; R3 reagent contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; The first antibody and the second antibody can specifically bind to different epitopes of p-tau 217; one first pairing molecule can bind to at least two first tag molecules.

2. The reagent kit according to claim 1, characterized in that, The first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

3. The reagent kit according to claim 1, characterized in that, The specific tagging system consisting of the first tag molecule and the first paired molecule is selected from the Tag-Catcher system.

4. The reagent kit according to claim 3, characterized in that, In the Tag-Catcher system, a tag with a small molecular weight binds to the first antibody as a first tag molecule.

5. The reagent kit according to claim 3, characterized in that, The first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag.

6. The reagent kit according to claim 3, characterized in that, The first paired molecule is a polycatcher with a degree of polymerization of not less than 2.

7. The kit according to claim 1, characterized in that, The molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule.

8. The reagent kit according to claim 7, characterized in that, The molar ratio of the first paired molecule to the first tag molecule is 1:(1-16).

9. The reagent kit according to claim 1, characterized in that, The degree of polymerization of the first paired molecules is negatively correlated with its optimal molar amount.

10. The reagent kit according to claim 9, characterized in that, The product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

11. The reagent kit according to claim 1, characterized in that, The mass ratio of the luminescent microspheres to the first antibody is 10:(0.05-5).

12. The kit according to claim 1, characterized in that, The molar ratio of the second antibody to the second tag molecule is 1:(20-50).

13. The reagent kit according to claim 1, characterized in that, The luminescent microspheres are high molecular particles filled with a luminescent composition, which can react with reactive oxygen species to generate detectable light signals.

14. The kit according to claim 13, characterized in that, The luminescent composition includes enol ethers, enamines, 9-alkylxanthan gum, 9-alkyl-N-alkylacridinium, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, gloss enhancers, or europium complexes.

15. The kit according to any one of claims 1 to 14, characterized in that, Also includes: The R4 reagent comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second tag molecule.

16. The reagent kit according to claim 15, characterized in that, The photosensitive microspheres are high molecular particles filled with photosensitizers, which can generate reactive oxygen species under photoexcitation.

17. The kit according to claim 16, characterized in that, The photosensitizers include methylene blue, rose red, porphyrin, phthalocyanine, chlorophyll, 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, and 9,10-diphenylanthracene-9,10-endoperoxide.

18. The reagent kit according to claim 15, characterized in that, The two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, the second tag molecule and the second pairing molecule, do not react with each other.

19. The reagent kit according to claim 15, characterized in that, The second tag molecule and the second pairing molecule are selected from the biotin-avidin system.

20. The method of using the kit as described in any one of claims 1 to 19, characterized in that, After mixing and incubating the R1, R2, and R4 reagents with the sample to be tested, the R3 reagent is then added to cause at least two luminescent microspheres to aggregate into a polymeric luminescent microsphere.

21. The method of using the reagent kit according to claim 20, characterized in that, The reaction time after adding reagents R1 and R2 is ≥45 min; the reaction time after adding reagent R3 is 0–15 min.

22. The use of the kit according to any one of claims 1 to 19 in the detection of p-tau217 in a blood sample.

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