Light-activated chemiluminescence detection kit for phosphorylated Tau protein p-tau217 as well as use method and application of light-activated chemiluminescence detection kit

Abstract

The invention relates to a light-activated chemiluminescence detection kit for phosphorylated Tau protein p-tau 217 as well as a use method and application of the light-activated chemiluminescence detection kit. The kit comprises a reagent R1, a reagent R2 and a reagent R3, wherein the reagent R1 comprises a luminous microsphere and a first antibody which is coated on the luminous microsphere, is provided with a first tag molecule and can be specifically combined with p-tau 217; the R2 reagent comprises a second antibody with a second tag molecule; the R3 reagent comprises a first pairing molecule capable of being specifically combined with the first tag molecule; the first antibody and the second antibody can be specifically combined with different epitopes of p-tau 217, and one first pairing molecule can be combined with at least two first tag molecules. According to the technical scheme, the capacity of the light-emitting microspheres for receiving active oxygen generated by the light-sensitive microspheres can be improved, the detection sensitivity is improved, and the light-activated chemiluminescence detection requirement of p-tau 217 in plasma is met.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a photoluminescence detection kit for phosphorylated Tau protein p-tau217 and a method for using the kit and its application. Background Art

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is the most common neurodegenerative disease. Neuropathological changes exist 15 to 20 years before the onset of clinical symptoms of AD, such as the appearance of amyloid-β (Aβ) plaques and Tau protein hyperphosphorylation in brain tissue. The accumulation of Aβ and Tau proteins can cause neuronal dysfunction or even death, leading to a significant decline in patients' cognitive functions such as memory, but this process progresses extremely slowly. Therefore, moving the window forward and accurately identifying the disease in its early stages can help early intervention of AD.

[0003] At present, the clinical diagnosis methods for AD include scale assessment, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid (CSF) testing. However, due to the lack of objectivity, strong invasiveness, cumbersome operation, and high price, these methods have greatly limited their clinical promotion and use, especially in primary healthcare institutions. The clinical misdiagnosis rate of AD is as high as 76.8%, making it difficult to screen and intervene in AD at an early stage. Therefore, it is an urgent clinical need to provide an accurate detection of AD blood markers with low cost, low trauma, convenient detection, and standardized results.

[0004] p-tau 217 is a phosphorylated form of Tau protein, which is mainly phosphorylated at the 217th threonine of Tau protein. It has been significantly increased in the preclinical stage of AD patients, which can indicate the occurrence of AD earlier. Studies have shown that the average level of p-tau 217 in the plasma of healthy people is about 0.3 to 0.4 pg / mL, while it reaches 1 to 2 pg / mL in AD patients, and the positive judgment value is below 1pg / mL. Conventional immunoassay technology detects the overall signal after the reaction of the reagent system through optical, electrical and other detection means, and then converts the signal intensity of the standard curve into the detection concentration, but its detection limit is generally around 1 to 10ng / mL, which is difficult to meet such high sensitivity requirements and cannot stably detect p-tau 217 in plasma. Single molecule array (Simoa) technology is a digital detection technology that forms a capture antibody-test antigen-biotinylated detection antibody-avidin-coupled enzyme complex on the surface of magnetic beads. After binding to fluorescent substrates, the magnetic beads fall into arrayed microwells for post-reaction detection. Its detection sensitivity has been exponentially improved compared to ELISA and conventional chemiluminescence detection methods; however, the detection operation process is cumbersome and complicated, and the detection cost is extremely high, which greatly limits its clinical promotion and use. Summary of the invention

[0005] In order to solve or partially solve the problems existing in the related technology, the present application provides a photochemiluminescence detection kit for phosphorylated Tau protein p-tau217 and its use method and application, which can improve the ability of luminescent microspheres to receive reactive oxygen generated by photosensitive microspheres, improve the utilization rate of reactive oxygen, and increase the detection signal value, thereby improving the detection sensitivity and meeting the photochemiluminescence detection needs of phosphorylated Tau protein p-tau 217 in low-value sample plasma.

[0006] The first aspect of the present application provides a photoluminescence detection kit for phosphorylated Tau protein p-tau 217, comprising: R1 reagent, comprising luminescent microspheres coated with a first antibody capable of specifically binding to p-tau 217, wherein the first antibody carries a first label molecule; R2 reagent, which comprises a second antibody with a second tag molecule; R3 reagent, which comprises 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, and one first pairing molecule can bind to at least two first tag molecules.

[0007] In some embodiments, the first tag molecule is linked to the non-specific binding region of the first antibody by co-expression with the first antibody.

[0008] In some embodiments, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag.

[0009] In some embodiments, the first pairing molecule is a polymeric Catcher with a degree of polymerization of not less than 2; preferably an octamer.

[0010] In some embodiments, 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).

[0011] In some embodiments, the kit further comprises: The R4 reagent comprises a photosensitive microsphere and a second pairing molecule coated on the photosensitive microsphere and capable of specifically binding to the second label molecule.

[0012] In some embodiments, the two sets of specific label systems consisting of the first label molecule and the first pairing molecule, and the second label molecule and the second pairing molecule, respectively, do not react with each other.

[0013] The second aspect of the present application provides a method for using the kit as described in the first aspect, which comprises mixing and incubating the R1 reagent, R2 reagent, and R4 reagent with a sample to be tested, and then adding the R3 reagent to aggregate at least two luminescent microspheres into multi-luminescent microspheres.

[0014] In some embodiments, the reaction time after adding the R1 and R2 reagents is ≥45 min; the reaction time after adding the R3 reagent is 0-15 min.

[0015] The third aspect of the present application provides an application of a kit or a method of using the kit for detecting p-tau 217 in a blood sample.

[0016] It is noteworthy that the application of detecting p-tau 217 in a blood sample is for non-disease diagnosis purposes.

[0017] The technical solution provided by the present application may include the following beneficial effects: by introducing labeled antibodies into luminescent microspheres, and by adding paired molecules capable of capturing the labels, the free luminescent microspheres in the reaction system can be aggregated on the immune complexes by utilizing the specific reaction between the paired molecules and the labels, thereby increasing the utilization rate of the reactive oxygen species generated by the photosensitive microspheres, thereby increasing the detection signal value, and improving the photochemiluminescence detection performance as a whole, meeting the sensitivity detection requirements of the photochemiluminescence detection of p-tau 217 in low-value blood samples. Moreover, the entire detection process does not change the conventional detection sequence, does not interfere with the specificity of the reagents, is easy to operate, has low detection costs, and has a wide range of applications, and has very important clinical application value.

[0018] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. DETAILED DESCRIPTION

[0019] To make the present invention easy to understand, the present invention 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 terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0020] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0021] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials or equivalent methods and materials described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0022] I. Terminology The sample to be tested described herein refers to a mixture that may contain an analyte, and the analyte includes but is not limited to proteins, hormones, antibodies or antigens. Typical samples to be tested that can be used in the method disclosed in the present invention include body fluids, such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysematous effusion, etc. The sample to be tested can be a solution of the sample that may contain the analyte diluted with a diluent or a buffer solution as needed before use. For example, in order to avoid the HOOK effect, the analyte can be diluted with a sample diluent before being tested on the machine and then tested on the detection instrument. At this time, the diluted solution that may contain the analyte is collectively referred to as the sample to be tested.

[0023] The antibodies described herein are used in the broadest sense, including antibodies of any isotype, 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 an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated with other parts, such as specific binding pair members, such as biotin or avidin. The position that can specifically bind to an antigen and react with the antigen, i.e., 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 other end opposite thereto is referred to as the non-specific binding region.

[0024] The antigen described herein refers to a substance that can induce antibody production, which can be divided into complete antigens and incomplete antigens (haptens). The antigen can be a natural antigen extracted from a pathogen or animal tissue or a recombinant antigen with specific antigenic properties prepared by genetic engineering technology. In any desired case, the antigen can be further conjugated with other parts, such as specific binding pair members, such as biotin or avidin.

[0025] As used herein, "binding", "linking" and "coupling" refer to an association between two substances due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, or interactions including but not limited to salt bridges and water bridges.

[0026] The specific binding described in this article refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the corresponding reactants from the perspective of three-dimensional structure.

[0027] The Tag-Catcher system described herein is a protein covalent bonding technology, which includes two parts, Tag and Catcher, composed of multiple amino acid residues, and the covalent bonding of proteins can be achieved through the specific reaction between Tag and Catcher. The Catcher protein in the Tag-Catcher system can exist as a monomer or a multimeric protein, and the Catcher used in this application is its multimeric protein. Multimeric proteins are proteins composed of two or more polypeptide chains, which can be the same or different, and are interconnected by covalent bonds or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to form multimeric macromolecular structural proteins.

[0028] The biotin-avidin system described herein is a biological reaction amplification system, which comprises two main components, biotin and avidin or streptavidin. The biotin is widely present in animal and plant tissues, and has two cyclic structures on its molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main site for binding with avidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of a protein cross-linking agent. The avidin is a protein secreted by Streptomyces. The streptavidin molecule consists of 4 identical peptide chains, each of which can bind to one biotin, and its molecular weight is 65kD. Each antigen or antibody can be coupled to multiple biotin molecules at the same time, thereby producing a "tentacle effect" with avidin to improve the analytical sensitivity. In any necessary case, any reagent used in the present invention, including antigens, antibodies, receptors or donors, can be conjugated to any member of the specific binding pair such as biotin-streptavidin according to actual needs.

[0029] The reactive oxygen species mentioned in this article refers to the general term for substances composed of oxygen, containing oxygen and active in the body or natural environment, mainly an excited oxygen molecule, 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 ( 1 O2) etc.

[0030] The luminescent microspheres described herein refer to polymer particles filled with a luminescent composition, which can react with reactive oxygen to produce detectable light signals. Luminescent microspheres may also be referred to as acceptor microspheres or luminescent particles. In some specific embodiments of the present invention, the luminescent composition undergoes a chemical reaction with reactive oxygen to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of these substances include, but are not limited to, enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridan, aromatic vinyl ethers, diene oxides, dimethylthiophene, aromatic imidazoles or lucigenin. In other specific embodiments of the present invention, the luminescent composition may also include a europium complex; further preferably, the europium complex is MTTA-EU 3+ .

[0031] The photosensitive microspheres described herein refer to polymer particles filled with photosensitizers, which can produce reactive oxygen under light excitation. At this time, they can also be called donor microspheres or photosensitive particles, and the solution containing such photosensitive microspheres can be called photosensitive liquid or universal liquid. The photosensitizer can be a photosensitizer known in the art, such as methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll, and is not limited thereto; the photosensitive microspheres can also be filled with other sensitizers, and non-limiting examples thereof are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water. Some other examples of sensitizers include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or directly absorbing light by these compounds will release reactive oxygen.

[0032] The microparticles described herein can be of any size, any shape, can be expandable or non-expandable, can be porous or non-porous, have any density, but preferably have a density close to that of water, preferably float in water, and are made of transparent, partially transparent or opaque materials. The microparticles can be solids (such as polymers, metals, glasses, organic or inorganic substances such as minerals, salts and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, siliceous fluids), vesicles (such as synthetic such as phospholipids, or natural such as cells and organelles). A non-limiting example of microparticles suitable for use in the present invention is carboxypolystyrene latex microspheres.

[0033] II. Specific implementation plan The present application will be described in more detail below.

[0034] Photochemiluminescence analysis is a new generation of immunoassay technology based on nanoscale polymer particles. Its core principle is the generation and transfer of reactive oxygen species: through the energy transfer of reactive oxygen species between photosensitive microspheres (GG) and luminescent microspheres (FG), high-energy red light is generated during energy level transitions, and the number of photons is converted into relative light signals through single-photon counters and mathematical fitting. In the presence of a target (such as an antigen / antibody to be tested), the antibodies / antigens modified on the surfaces of the two microspheres react with the antigen / antibody to be tested to form a sandwich immune complex, shortening the spatial distance between the two microspheres, and achieving qualitative or quantitative analysis of the antigen / antibody to be tested through the light signal generated by the reactive oxygen species transfer between the two microspheres.

[0035] The inventors of the present application have found in their research that in the photoluminescence analysis platform, when the target content is extremely low, such as p-tau 217 in plasma, the molar amount of the luminescent microspheres is much higher than the molar amount of the target. However, most of the sandwich immune complexes in the reaction system are formed by bridging a luminescent microsphere with a photosensitive microsphere by a target molecule. When reading, the large amount of light generated by the photosensitive microspheres is 1 Only a small part of O2 is received by the adjacent luminescent microspheres and generates light signals, while the unreceived 1 O2 is quenched within 4μs. 1 The utilization rate of O2 is the key to improving the sensitivity of photochemiluminescence detection, especially for low-value samples such as p-tau 217 in plasma. If the particle size of the luminescent microspheres is increased, more 1 O2 and produce a stronger signal, but at the same time it will lead to a decrease in its specific surface area and a slowdown in particle migration, which will directly affect the efficiency of the immune response and reduce the detection sensitivity. The inventors of this application have considered and designed a method to improve the performance of the reagent by increasing the utilization rate of free luminescent microspheres in the detection reagent, providing a method for improving 1 A new way to improve the utilization of O2 and thus enhance detection sensitivity.

[0036] The photoluminescent chemiluminescent detection kit for phosphorylated Tau protein p-tau 217 involved in the present application includes R1 reagent, R2 reagent and R3 reagent. Among them, R1 reagent includes luminescent microspheres and first antibodies with first label molecules coated on luminescent microspheres that can specifically bind to p-tau 217; R2 reagent includes second antibodies with second label molecules; R3 reagent includes first paired molecules that can specifically recognize and bind to first label molecules; the first antibody and the second antibody can specifically bind to different epitopes of p-tau217, and one first paired molecule can bind to at least two first label molecules, so that at least two luminescent microspheres are combined to form multi-luminescent microspheres.

[0037] The first antibody and the second antibody in the R1 reagent and the R2 reagent can react with p-tau 217 to form a sandwich immune complex, and the first pairing molecule in the R3 reagent can form a specific label system with the first label molecule on the luminescent microsphere in the R1 reagent, thereby aggregating the free luminescent microspheres (referring to the luminescent microspheres that the first antibody does not specifically bind to p-tau 217) to the main luminescent microspheres (referring to the luminescent microspheres that the first antibody specifically binds to p-tau 217), thereby increasing the effective luminescent microsphere particle size and specific surface area, so that the photosensitive microspheres produce 1 The utilization rate of O2 is improved, and the quenching 1 O2 is reduced, thereby generating a stronger light signal. At the same time, the particle size and mass of each luminescent microsphere in the R1 reagent do not change, which can maintain a good particle migration rate, ensure the efficiency of the antigen-antibody immune response, and improve the overall detection sensitivity, which is conducive to improving the detection performance of low-value samples, and can achieve high-performance detection of femtogram-level markers p-tau 217 in plasma.

[0038] The number of luminescent microspheres in the polymerized luminescent microspheres described in the present application is equal to or greater than two, which is not limited here.

[0039] In some embodiments of the present application, the R3 reagent may further include luminescent microspheres, that is, the first pairing molecule is also coated in the luminescent microspheres. When the R1 reagent and the R3 reagent are mixed, the specific recognition and binding ability between the first label molecule and the first pairing molecule can cause the luminescent microspheres of the R1 reagent and the R3 reagent to aggregate to form multi-luminescent microspheres.

[0040] In some embodiments of the present application, the specific label system composed of the first label molecule and the first pairing molecule can be selected from the Tag-Catcher system, and any combination can be selected from the combination of the system as the first label molecule in the R1 reagent and the first pairing molecule in the R3 reagent. In the embodiments of the present application, the first pairing molecule can also be called a tool protein.

[0041] Specifically, the first tag molecule can 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 is selected from any one of the Catcher proteins. The first tag molecule and the first pairing molecule can be selected from a specific tag system composed of, for example, Spy-Tag and Spy-Catcher, His-Tag and Anti-His (in this case, the Catcher is Anti-His), Snoop-Tag and Snoop-Catcher, etc. Catcher and Tag have excellent specific recognition and binding capabilities, and a Tag tag with a smaller molecular weight is used to bind to the first antibody and is co-coated on the luminescent microspheres, which has no significant effect on the expression of the first antibody and the properties of the luminescent microspheres.

[0042] Furthermore, when the first tag molecule is selected from Tag, the first pairing molecule can be selected from a multimeric protein of Catcher, and the degree of polymerization is not less than 2, for example, it can be selected from an 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 rate of capturing Tag, the higher the aggregation rate of luminescent microspheres, and the better the effect of improving the intensity of the light signal.

[0043] In some embodiments of the present application, the molar amount of the first pairing molecule is less than or equal to the molar amount of the first label molecule. Preferably, the molar ratio of the first pairing molecule to the first label molecule is 1:(1~16); preferably 1:(2~8). The use ratio of the first pairing molecule directly affects the aggregation of the luminescent microspheres. If it is too low, the aggregation efficiency and aggregation degree will be low, and the effect of 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, and there is no obvious improvement in the detection performance, and even affects the accuracy of the detection results.

[0044] When the first tag molecule and the first pairing molecule are selected from the Tag-Catcher system, the higher the polymerization degree of the first pairing molecule Catcher, the lower the molar amount of the required first pairing molecule, that is, the polymerization degree of the first pairing molecule is negatively correlated with its optimal molar amount. Preferably, when the polymerization degree of the first pairing molecule multiplied by its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, the effect of improving the light signal intensity of the luminescent microsphere is more significant.

[0045] The degree of polymerization and dosage of the first pairing molecule can be determined according to the amount of the first label molecule, and the higher the degree of polymerization of the first pairing molecule, the lower the required optimal molar amount, and the faster the reaction rate with the first label molecule; the lower the degree of polymerization of the first pairing molecule, the higher the required optimal molar amount, and the faster the aggregation rate. 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 label molecule, the light signal intensity of the luminescent microspheres is significantly improved. Excessive amounts are prone to cause HOOK effects, affecting the detection results; insufficient amounts affect the aggregation rate and degree of aggregation of the multi-polymerized luminescent microspheres, and the performance improvement is not obvious.

[0046] In some embodiments of the present application, the first tag molecule is connected to the non-specific binding region of the first antibody by co-expression with the first antibody to form a first antibody with the first tag molecule. The co-expression connection method can be achieved by connecting the first tag molecule to the non-specific binding region of the first antibody through pClick technology (specific reference "Synthesis of precisionantibody conjugates using proximity-induced chemistry" (Theranostics.2021Aug27;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.

[0047] In some embodiments of the present application, the first antibody is in a Y-shaped structure, which includes two Fab segments and one Fc segment, and the non-specific binding region of the first antibody is the Fc end of the first antibody. In some embodiments of the present application, the first antibody is in a V-shaped structure, which includes two Fab segments, and the non-specific binding region of the first antibody is the Fab connection hinge region of the first antibody. When the CDR region is occupied or the Fab end is folded in the steps of coating and labeling of the first antibody or the second antibody, the Fab end of the antibody will fail to be effectively displayed, affecting the effective amount of antibody or antibody activity that can bind to the target molecule p-tau 217 to be detected in the reaction system, thereby affecting the photochemical detection performance of p-tau 217. The position of the first antibody formed in this way that can specifically bind to p-tau 217, that is, the CDR region, can be fully and effectively displayed, thereby increasing the effective amount of antibody and antibody activity that can bind to the target molecule p-tau 217 to be detected in the reaction system, and improving the detection sensitivity of p-tau 217.

[0048] In some embodiments of the present application, the mass ratio of the 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 of the first antibody and p-tau217 in the sample to be tested, thereby affecting the discrimination of the test results.

[0049] In some embodiments of the present application, the molar ratio of the second antibody to the second label molecule is 1: (20-50); preferably 1: (25-35); more preferably 1: 30. The amount of the second label molecule on the second antibody affects its reaction efficiency with the photosensitive microspheres, thereby affecting the discrimination of the detection results.

[0050] The kit involved in the present application also includes R4 reagent, which includes photosensitive microspheres and second pairing molecules coated on the photosensitive microspheres that can specifically bind to the second label molecule. The second antibody can be bound to the photosensitive microspheres through the specific binding ability of the second pairing molecules on the photosensitive microspheres and the second label molecules, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres, so that the photosensitive microspheres produce 1 O2 can be effectively received by the luminescent microspheres and generate light signals, meeting the needs of photo-induced chemiluminescence detection.

[0051] Furthermore, the two groups of specific label systems composed of the first label molecule and the first pairing molecule, and the second label molecule and the second pairing molecule respectively do not react with each other, thereby avoiding the occurrence of specific pairing interference reactions generated by the two and the accuracy and stability of the detection results.

[0052] The second label molecule and the second pairing molecule in the R2 reagent and the R4 reagent can be selected from the biotin-avidin system, for example, one of them is selected from biotin and the other is selected from avidin or streptavidin; preferably, avidin is coated on the photosensitive microsphere, the second antibody is bound to streptavidin, and the second antibody is labeled with biotin.

[0053] The kit involved in the present application can be used in the following method: after mixing and incubating the R1 reagent, the R2 reagent, the R4 reagent and the sample to be tested, the R3 reagent is added to react with the first label molecule of the R1 reagent to aggregate at least two luminescent microspheres into a multi-luminescent microsphere.

[0054] Furthermore, 15 μL of R1 reagent, 15 μL of R2 reagent, 175 μL of R4 reagent and 100 μL of the sample to be tested can be mixed and incubated, and then 15 μL of R3 reagent can be added for reaction. That is, in the photoluminescence detection process of p-tau217, by controlling the volume ratio of R1 reagent, R2 reagent and the sample to be tested to 3:3:20, the volume ratio of R2 reagent and R4 reagent to 3:35, and the volume ratio of R1 reagent to R3 reagent to 1:1, the reagent volume can be reduced, the reagent concentration can be increased, and the sample proportion can be increased, so that the balance of the reaction moves toward the binding direction, which is conducive to increasing the amount of the formed luminescent microsphere-(p-tau217)-photosensitive microsphere sandwich immune complex, thereby improving the detection performance.

[0055] Furthermore, the R1 reagent, the R2 reagent and the sample to be tested may be mixed first, and then the R4 reagent may be added after incubation for a period of time, and then the R3 reagent may be added after incubation for a period of time.

[0056] Specifically, the method of use includes: S1, mixing the R1 reagent, the R3 reagent and the sample to be tested and incubating at 37° C. to obtain a first reactant; S2, adding R4 reagent, incubating at 37°C to obtain the second reactant; S3, adding R2 reagent, incubating at 37°C to obtain the third reactant; S4. Irradiate the third reactant with light of a specific wavelength and detect the luminescence value.

[0057] The present application first combines the luminescent microspheres and the first antibody coated thereon that can specifically recognize and bind to p-tau 217, and the second antibody that can specifically recognize and bind to p-tau 217 with the target p-tau 217 in the sample to be tested to form a "first antibody-(p-tau 217)-second antibody" sandwich immune complex, and then fixes the target luminescent microspheres to the photosensitive microspheres through the specific binding of the second label molecule carried by the second antibody and the second pairing molecule coated with the photosensitive microspheres to obtain a "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex, and finally, through the specific recognition and binding ability of the first pairing molecule and the first label molecule, the free luminescent microspheres are bound to the target luminescent microspheres to form multi-luminescent microspheres.

[0058] After the conventional reaction, the free luminescent microspheres gather around the composite luminescent microspheres, making the photosensitive microspheres 1 O2 can be received by multiple luminescent microspheres in the multi-luminescent microspheres and generate light signals at the same time, effectively improving 1O2 utilization rate, improve signal strength, and then improve the detection sensitivity of the kit to meet the photochemiluminescence detection requirements of low-value samples. Moreover, the specific label system used in this scheme, that is, the first label molecule and the first pairing molecule adopt the Tag-Catcher system, which has strong specificity and high affinity, and can significantly improve the performance of the reagent without interfering with the original immune response. It has strong applicability and a wide range of applications, and is a platform-type improvement. Especially when it is used for the detection of p-tau 217 in extremely low-value sample plasma, it can significantly improve the detection sensitivity and meet the detection needs of extremely low-value samples. At the same time, the detection method is simple, easy to operate, and the detection cost is low.

[0059] In some embodiments of the present application, the reaction time after adding the R1 reagent and the R2 reagent is ≥45 min, preferably ≥60 min; preferably 60 to 75 min. The reaction time of the first antibody, the second antibody and the target p-tau217 to be detected affects the degree of reaction. Prolonging the reaction time can ensure the formation of the "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex and improve the discrimination of the detection results. The reaction time after adding the R3 reagent is 0 to 15 min, that is, the aggregation time of the luminescent microspheres is 0 to 15 min. As the aggregation time increases, the aggregation reaction of the luminescent microspheres gradually increases and tends to equilibrium, so the aggregation time is preferably 3 to 9 min, more preferably 5 to 7 min.

[0060] In some embodiments of the present application, the kit further comprises a buffer, a stabilizer, etc. The buffer may be selected from at least one of PBS buffer, Tris-HCl 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.

[0061] In some embodiments of the present application, when each detection reagent is used, the concentration of the luminescent microspheres in the R1 reagent 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 immune reaction with p-tau217, thereby affecting the discrimination of the detection results. The concentration of the second antibody in the R2 reagent 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 immune reaction efficiency with p-tau217 and its anti-biotin interference ability, thereby affecting the discrimination of the detection results.

[0062] The detection kit as described above can be used to detect p-tau 217 in a blood sample.

[0063] The detection method specifically includes: mixing the sample to be tested with R1 reagent and R3 reagent containing a first antibody and a second antibody that can specifically recognize p-tau 217, adding R4 reagent after incubation for a period of time, adding R2 reagent after incubation for a period of time, performing photoluminescence detection after incubation 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 determining the content of p-tau 217 in the sample to be tested based on the intensity of the light signal.

[0064] III. Specific Examples In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0065] Next, taking p-tau 217 as the target molecule to be detected, the antibody molecules with specific recognition of p-tau 217 are modified with different labels and coated on luminescent microparticles in the same ratio, and the target p-tau 217 in the sample is detected by double antibody sandwich method with antibody molecules labeled with biotin that can specifically recognize different sites of p-tau 217. After the conventional detection sequence (sample + R1 reagent + R3 reagent reaction and then add R4 reagent), the luminescent microparticles containing the paired molecules corresponding to the label are added. By comparing the detection discrimination between different experimental groups, the improvement effect of the multi-luminescent microparticle mode on the photoluminescence detection performance of p-tau 217 is judged.

[0066] Example 1: Preparation of luminescent microspheres coated with antibodies, biotin-labeled antibodies and Spy Catcher 1. The main experimental materials and equipment are shown in Table 1.

[0067] Table 1

[0068] 2. Experimental Procedure 2.1 Preparation of luminescent microspheres using the Spy Tag system as an example 2.1.1 Antibody (p-tau217) Ab1 linked to Tag The Spy Tag (sequence AHIVMVDAYKPTK) was connected to the antibody (p-tau217) Ab1 by pClick technology (for details, refer to "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021Aug27;11(18):9107-9117.doi:10.7150 / thno.62444.)) to obtain the antibody with the Fc segment connected to the Tag. The protein concentration of the collected part was determined by the BCA method, and then the protein part was taken for electrophoresis detection. The part that met the molecular weight of the Tag-antibody was selected and dialyzed into the buffer required for the subsequent coupling reaction to obtain the antibody (p-tau217) Abl-SpyTag co-expressed with the tag molecule Spy Tag and (p-tau217) Ab1.

[0069] 2.1.2 Luminescent microparticles coated with antibody (p-tau 217) Ab1 ① Take 10 mg of aldehyde-based luminescent microparticles in a centrifuge tube, wash once with 0.02 M PBS buffer, and then dilute to 20 mg / mL with 0.02 M PBS; ② Take (p-tau 217) Ab1-Spy Tag and dialyze it with 0.02M PBS buffer, then determine the concentration using the BCA method; ③ The treated luminescent particles FG and (p-tau 217) Ab1-Spy Tag were fully mixed at a mass ratio of 10:0.5 and reacted at 37°C for 16 hours; 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 a reaction solution; ⑤ The reaction solution was washed twice with PBST buffer, and then washed once with reconstitution solution (0.02M PBS + 0.5% BSA + 0.5% Tween20 + 1% dextran), and then ultrasonically reconstituted with reconstitution solution to obtain luminescent microparticles coated with (p-tau217) Ab1-Spy Tag with a final concentration of 83.3μg / mL, namely reagent R1.

[0070] 2.2 Biotin-labeled antibody (p-tau 217) Ab2 ① Dialyze (p-tau 217) Ab2 with 0.1M NaHCO3 solution, remove impurities and determine the concentration using the BCA method; ② Add 30 times the molar amount of Biotin-PEG12-NHs to the dialyzed (p-tau 217) Ab2 solution, mix well, and react at 4°C for 12 hours to obtain Bio-(p-tau 217) Ab2 solution; ③ Dialyze with 0.02M PBS buffer to remove free Biotin and then determine the concentration with BCA.

[0071] ④ Use the complex solution to prepare 3.3 μg / mL of biotin-labeled antibody Bio-(p-tau 217) Ab2, namely reagent R2.

[0072] 2.3 Preparation and purification of Spy Catcher ① Construction of Catcher plasmid expression vector: The expression vector of Spy Catcher (E. coli vector-pet series expression plasmid) was constructed according to the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0073] ②Catcher E. coli induced expression: E. coli expression vector was induced to express Spy Catcher protein. Its culture conditions: ① Auto-induction medium (10g peptone per liter, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x5052, PH=7.4), with an added concentration of 100nM ampicillin; ② Induction conditions: 30℃, 200rpm shaker overnight culture.

[0074] ③Catcher purification: The cultured bacterial solution was centrifuged at 8000rpm, the bacterial precipitate was collected, and the bacteria were resuspended in 100mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20mM ID, PH=7.4). The resuspended bacterial solution was centrifuged at 18000rpm for 40 minutes, the supernatant was collected and nucleic acid was fragmented, filtered, purified by affinity column, eluted, the protein was collected and added to the resolubilization solution for ultrasonic resolubilization to obtain Spy Catcher.

[0075] Example 2: Effects of polymerization degree and usage ratio on the photochemiluminescence detection performance of p-tau 217 1. Experimental Procedure 1.1 Prepare the Spy Catcher dimer, pentamer and octamer according to the method of Example 1, and then dilute them with the reconstituted solution to the R3 reagent with a molar ratio of 1:16, 1:8, 1:4, 1:2, 1:1 and 2:1 to the Tag in the R1 reagent. Use the photosensitive particle universal liquid reagent R4. ® Detection was performed on a 500 chemiluminescence detection system.

[0076] 1.2 Mix 100 μL of the sample to be tested, 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 reconstitution solution to the control group, and incubate at 37°C for 15 min respectively; after light excitation reaction, read the light signal value of each test group respectively. The experimental data are shown below.

[0077] 2. Experimental results 2.1 Spy Catcher dimer reaction effect: Table 2 Signal values

[0078] Table 3 Discrimination

[0079] 2.2 Spy Catcher pentamer reaction effect: Table 4 Signal values

[0080] Table 5 Discrimination

[0081] 2.3 Spy Catcher octamer reaction effect: Table 6 Signal values

[0082] Table 7 Discrimination

[0083] 3. Results Analysis ① As shown in Table 2-7, within the current gradient range, the polymer particle size (polymerization degree) does not affect the particle size of the luminescent particle receiving singlet oxygen. The higher the polymerization degree of the first paired molecule corresponding to the first label molecule, the better the improvement effect. When the first paired molecule is the Spy Catcher octamer, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved to a greater extent.

[0084] ② As shown in Table 2-7, when the molar ratio of Spy Catcher and Spy Tag in R1 reagent is 1:(16~1), the positive signal and discrimination are improved. Among them, the best effect is achieved when the molar ratio is 1:4 (Spy Catcher octamer: Spy Tag). At this time, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more.

[0085] ③ The polymerization degree and usage ratio of the first pairing molecule depend on the molecular weight of the first tag. The molar weight of the first pairing molecule should be lower than or equal to the molar weight of the first tag molecule, and the polymerization degree of the first pairing molecule is negatively correlated with the optimal molar weight. As shown in Table 2-7, when the product of the polymerization degree and molar weight of the first pairing molecule is 1 to 2.5 times the molar weight of the first tag molecule, the positive signal and discrimination are significantly improved.

[0086] ④ As shown in the results of Table 2-7, using Spy Tag as a label and Spy Catcher as a pairing molecule, the positive signal and discrimination are significantly improved for detecting p-tau 217 recombinant antigen and actual samples.

[0087] Therefore, using a specific label system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form multi-luminescent microspheres can enhance the positive signal and discrimination, and significantly improve the sensitivity of photochemiluminescence detection, thus meeting the detection needs of p-tau 217 in low-value samples such as blood samples.

[0088] Example 3: Effect of aggregation time on the photoluminescence detection performance of p-tau 217 1. Experimental Procedure 1.1 Dilute the Spy Catcher octamer with the reconstituted solution to a 1:4 ratio of the molar number of Tag in the R1 reagent to the R3 reagent. Use the photosensitive particle universal liquid reagent R4. ® Detection was performed on a 500 chemiluminescence detection system.

[0089] 1.2 Mix 100 μL of the sample to be tested, 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 reconstitution solution to the control group, and incubate at 37°C for 3, 6, 9, 12 and 15 min respectively; after light excitation reaction, read the light signal value of each test group respectively. The experimental data are shown below.

[0090] 2. Experimental results Table 8 Signal values

[0091] Table 9 Discrimination

[0092] 3. Results Analysis As shown in the results of Tables 8-9, with the extension of the reaction time after the addition of R3 reagent (including the first pairing molecule), the positive signal and discrimination gradually increased and tended to equilibrium; when the reaction reached about 6 minutes, the aggregation reaction of the free luminescent particles reached equilibrium.

[0093] Therefore, in the reaction process of using a specific label system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form multi-luminescent microspheres, from the perspective of the balance between reaction time and performance, the reaction time after adding the R3 reagent can be 0~15min, the better reaction time is 3~9min, and the optimal reaction time is 5~7min.

[0094] Example 4: Verification of the applicability of different labeling systems for forming “polymerized luminescent microspheres” 1. Experimental Procedure 1.1 According to the method of coating luminescent microspheres with antibodies in Example 1 above, luminescent microspheres coated with (p-tau 217) Ab1 with different tag molecules (Spy-Tag, His-Tag, Snoop-Tag) were prepared, and diluted to 83.3 μg / mL with a complexing solution to obtain R1 reagent.

[0095] 1.2 Bio-(p-tau 217) Ab2 was diluted to 3.3 μg / mL with the complex solution to obtain R2 reagent.

[0096] 1.3 The first paired molecule (octamer Spy-Catcher, octamer Anti-His, octamer Snoop-Catcher corresponding to the tag molecule) is diluted with the complex solution to R3 reagents with molar ratios of 1:16, 1:4 and 1:1 to the tag molecule in R1 reagent.

[0097] 1.4 With the sample to be tested in LiCA ® Detection on 800 chemiluminescence detection system: Mix 100μL of the sample to be tested, 15μL of R1 reagent and 15μL of R2 reagent, and incubate at 37℃ for 60min; add 175μL of photosensitive particle universal solution and incubate at 37℃ for 15min; add 15μL of R3 reagent, and add 15μL of reconstitution solution to the control group, and incubate at 37℃ for 15min respectively; after light excitation reaction, read the light signal value of each test group respectively. The experimental data is shown below.

[0098] 2. Experimental results Table 10 Signal values

[0099] Table 11 Discrimination

[0100] 3. Results Analysis As shown in the results of Tables 10-11, the use of different specific label systems such as Spy-Catcher / Spy-Tag, His-Tag / Anti His-Ab, Snoop-Tag / Catcher, etc. can improve the positive signal value and low discrimination sensitivity of the detection to varying degrees.

[0101] Therefore, the specific label system described in the present application has a good effect on the aggregation of free luminescent microspheres to form polymerized luminescent microspheres to improve the detection performance. The sensitivity of photoinduced chemiluminescence detection is effectively improved, which can meet the detection needs of p-tau 217 in low-value samples such as blood samples.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A photoluminescence detection kit for phosphorylated Tau protein p-tau217, It is characterized in that include: R1 reagent, comprising luminescent microspheres coated with a first antibody that can specifically bind to p-tau 217, wherein the first antibody carries a first label molecule; R2 reagent, which comprises a second antibody with a second tag molecule; R3 reagent, which comprises 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; and one first pairing molecule can bind to at least two first tag molecules.

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

3. The kit according to claim 1, It is 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.

4. The kit according to claim 1, It is characterized in that The first pairing molecule is a polymerized Catcher with a degree of polymerization of no less than 2.

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

6. The kit according to any one of claims 1 to 5, It is characterized in that Also includes: The R4 reagent comprises a photosensitive microsphere and a second pairing molecule coated on the photosensitive microsphere and capable of specifically binding to the second label molecule.

7. The kit according to claim 6, It is characterized in that The two groups of specific label systems respectively composed of the first label molecule and the first matching molecule, and the second label molecule and the second matching molecule do not react with each other.

8. A method for using the kit according to any one of claims 1 to 7, It is characterized in that After the R1 reagent, R2 reagent, and R4 reagent are mixed and incubated with the sample to be tested, the R3 reagent is added to aggregate at least two luminescent microspheres into multiple luminescent microspheres.

9. A method for using the kit according to claim 8, It is characterized in that The reaction time after adding R1 and R2 reagents is ≥45min; the reaction time after adding R3 reagent is 0~15min.

10. Use of the kit according to any one of claims 1 to 7 for detecting p-tau 217 in a blood sample.

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