A kit for detecting phosphorylated Tau protein p-tau217 by luminescence resonance energy transfer, its using method and application

Abstract

The present application relates to a kit for detecting phosphorylated Tau protein p-tau 217 by luminescence immunoassay, and its use method and application. The kit includes R1 reagent, R2 reagent and R3 reagent. The R1 reagent contains luminescent microspheres and a first antibody coated on the luminescent microspheres, which has a first tag molecule and can specifically bind to p-tau 217. The R2 reagent contains a second antibody with a second tag molecule. The R3 reagent contains a first pairing molecule that can specifically bind 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 at least two first tag molecules. The technical solution of the present application can improve the ability of the luminescent microspheres to receive reactive oxygen species generated by the photosensitive microspheres, enhance the detection sensitivity, and meet the needs of luminescence immunoassay for p-tau 217 in plasma.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay, and particularly relates to a photochemiluminescence detection kit for phosphorylated Tau protein p-tau217, its usage method and application. Background Art

[0002] Alzheimer disease (AD), commonly known as senile dementia, is the most common neurodegenerative disease. 15 to 20 years before the clinical symptoms of AD appear, there are neuropathological changes, such as the appearance of β-amyloid protein (Aβ) plaques and over-phosphorylation of Tau protein in brain tissue. The accumulation of Aβ protein and Tau protein will cause neuronal dysfunction and even death, resulting in a significant decline in cognitive functions such as memory in patients. However, this process progresses extremely slowly. Therefore, moving the window forward and accurately identifying the disease in the early stage is helpful for the early intervention of AD.

[0003] Currently, the clinical diagnostic methods for AD include scale assessment, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebro-spinal fluid (CSF) detection, etc. Due to the disadvantages of insufficient objectivity, strong invasiveness, cumbersome operation, high cost, etc., it greatly limits the clinical popularization and use, especially in primary healthcare institutions. The clinical missed diagnosis rate of AD is as high as 76.8%, making it difficult to screen and intervene in AD in the early stage. Therefore, providing a precise detection of AD blood markers with low cost, less trauma, convenient detection, and standardized results has become an urgent need in clinical practice.

[0004] p-tau 217 is a phosphorylated form of the Tau protein, which is mainly phosphorylated at threonine 217 of the Tau protein. It has been significantly elevated in the preclinical stage of AD patients and 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 approximately 0.3 to 0.4 pg / mL, while in AD patients it reaches 1 to 2 pg / mL, and the positive judgment value is below 1 pg / mL. Conventional immunoassay techniques detect the overall signal after the reaction of the reagent system through detection means such as light and electricity, and then convert the signal intensity of the standard curve into the detection concentration. However, its detection limit is generally around 1 to 10 ng / mL, making it difficult to meet such high sensitivity requirements and unable to stably detect p-tau 217 in plasma. Single molecule array (Simoa) technology is a digital detection technology. By forming a capture antibody - antigen to be detected - biotinylated detection antibody - avidin-conjugated enzyme complex on the surface of magnetic beads, after binding the fluorescent substrate, the magnetic beads are dropped into the arrayed microwells for reaction and then detected. Its detection sensitivity has been exponentially improved compared with ELISA and conventional chemiluminescence detection methods; however, the detection operation process is cumbersome and complex, and the detection cost is extremely high, which greatly limits its clinical popularization and use. Summary of the Invention

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

[0006] In the first aspect of the present application, a photochemiluminescence detection kit for phosphorylated Tau protein p-tau 217 is provided, including:

[0007] R1 reagent, which contains luminescent microspheres, and the luminescent microspheres are coated with a first antibody capable of specifically binding to p-tau 217, and the first antibody carries a first tag molecule;

[0008] R2 reagent, which contains a second antibody with a second tag molecule;

[0009] R3 reagent, which contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule;

[0010] The first antibody and the second antibody can specifically bind to different epitopes of p-tau 217, and one first pairing molecule can bind at least two first tag molecules.

[0011] 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.

[0012] In some embodiments, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag.

[0013] In some embodiments, the first pairing molecule is a poly-Catcher with a polymerization degree of not less than 2; preferably an octamer.

[0014] In some embodiments, the molar amount of the first pairing molecule is lower 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 to 16).

[0015] In some embodiments, the kit further comprises:

[0016] R4 reagent, which comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres and capable of specifically binding to the second tag molecule.

[0017] In some embodiments, the two specific tag systems respectively composed 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.

[0018] The second aspect of the present application provides a method for using the kit as described in the first aspect, which includes mixing the R1 reagent, R2 reagent, and R4 reagent with a test sample and incubating, and then adding the R3 reagent to cause at least two luminescent microspheres to aggregate into poly-luminescent microspheres.

[0019] 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 to 15 min.

[0020] The third aspect of the present application provides an application of the kit or its use method in detecting p-tau 217 in a blood sample.

[0021] It should be noted that the application in detecting p-tau 217 in a blood sample is for non-disease diagnosis purposes.

[0022] The technical solution provided by this application may include the following beneficial effects: By introducing labeled antibodies into the luminescent microspheres and adding a pairing molecule capable of capturing the label, the specific reaction between the pairing molecule and the label can cause the free luminescent microspheres in the reaction system to aggregate on the immune complex, improving the utilization rate of the reactive oxygen species generated by the photosensitive microspheres, thereby enhancing the detection signal value, overall improving the performance of the photoactivated chemiluminescence detection, and meeting the detection requirements for the sensitivity of the photoactivated chemiluminescence detection of p-tau 217 in low-value sample blood samples. Moreover, the entire detection process does not change the conventional detection sequence, does not interfere with the reagent specificity, is easy to operate, has a low detection cost and a wide application range, and has very important clinical application value.

[0023] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and do not limit this application. Detailed implementation manners

[0024] 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 described specific implementation manners. It should also be understood that the terms used herein are only for describing the specific implementation manners and do not represent any limitation.

[0025] When a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value 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 explicit exclusions in the specified range. When the specified range includes one or both of the limits, the range excluding either or both of the included limits is also included in the present invention.

[0026] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0027] Ⅰ. Terms

[0028] The test sample described in this article refers to a mixture that may contain an analyte, and the analyte includes, but is not limited to, proteins, hormones, antibodies, or antigens. Typical test samples that can be used in the methods disclosed in the present invention include body fluids, such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and pleural effusion. The test sample can be a solution obtained by diluting a sample that may contain an analyte 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 being detected on a detection instrument, and at this time, the diluted solution that may contain the analyte is collectively referred to as the test sample.

[0029] The antibody described in this article is used in the broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins that contain the antigen-binding portion of an antibody and a non-antibody protein. In any case where needed, the antibody can be further conjugated to other moieties, such as specific binding partner members, such as biotin or avidin. The position that can specifically bind to an antigen to produce an immune reaction, namely 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 to it is referred to as the non-specific binding region.

[0030] The antigen described in this article refers to a substance that can induce the generation of antibodies and can be divided into complete antigens and incomplete antigens (hapten). The antigen can be a natural antigen extracted from pathogens or animal tissues or a recombinant antigen with specific antigenic properties prepared by genetic engineering techniques. In any case where needed, the antigen can be further conjugated to other moieties, such as specific binding partner members, such as biotin or avidin.

[0031] "Binding", "linking", and "coupling" as described in this article refer to the association 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.

[0032] Specific binding as described in this article refers to the mutual discrimination and selective binding reaction between two substances. From a three-dimensional structural perspective, it is the conformational correspondence between the corresponding reactants.

[0033] The Tag-Catcher system described in this article is a protein covalent linkage technology, which includes two parts: Tag and Catcher, composed of multiple amino acid residues. Through the specific reaction between Tag and Catcher, the covalent linkage of proteins can be achieved. The Catcher protein in the Tag-Catcher system can exist as a monomer or a multimeric protein. The Catcher used in this application is its multimeric protein. A multimeric protein is a protein composed of two or more polypeptide chains, which can be the same or different, and are interconnected through covalent bonds or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to form a multimeric macromolecular structural protein.

[0034] The biotin-avidin system described in this article is a biological reaction amplification system, which includes two main components: biotin and avidin or streptavidin. Biotin widely exists in animal and plant tissues. It has two ring structures on its molecule, namely an imidazolone ring and a thiophene ring, and the imidazolone ring is the main site for binding to avidin. Activated biotin can be coupled with almost all known biological macromolecules under the mediation of a protein cross-linking agent, including proteins, nucleic acids, polysaccharides, and lipids, etc. Avidin is a protein secreted by streptomyces. The streptavidin molecule is composed of 4 identical peptide chains, and each peptide chain can bind a biotin, with a molecular weight of 65 kD. Each antigen or antibody can be conjugated with multiple biotin molecules at the same time, thus producing a "tentacle effect" with avidin to improve the analysis sensitivity. In any case where needed, any reagent used in the present invention, including antigens, antibodies, receptors, or donors, can be conjugated with any one of the specific binding pair members such as biotin-streptavidin according to actual needs.

[0035] The reactive oxygen species described in this article refers to the general term for substances composed of oxygen, containing oxygen and having active properties in the body or the natural environment. It is mainly an excited oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O 2 ·-), the two-electron reduction product hydrogen peroxide (H 2 O 2 ), the three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and reactive oxygen ( 1 O 2 ) and so on.

[0036] The luminescent microspheres described herein refer to polymer microparticles filled with a luminescent composition, which can react with reactive oxygen species to produce a detectable optical signal. The luminescent microspheres can also be referred to as receptor microspheres or luminescent particles. In some specific embodiments of the present invention, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, and the metastable intermediate can decompose, emitting light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthates, 9-alkylidene-N-alkyl acridans, aryl vinyl ethers, diepoxyethylenes, dimethylthiophenes, aromatic imidazoles or lucigenin. In some other specific embodiments of the present invention, the luminescent composition may further include an europium complex; more preferably, the europium complex is MTTA-EU 3+ .

[0037] The photosensitive microspheres described herein refer to polymer microparticles filled with a photosensitizer, which can generate reactive oxygen species under light excitation, and can also be referred to as donor microspheres or photosensitive particles at this time. A solution containing such photosensitive microspheres can be referred to as a photosensitive solution or a general solution. 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. Examples of some other sensitizers include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or these compounds directly absorbing light will release reactive oxygen species.

[0038] The microparticles described herein can be of any size and any shape, they can be expandable or non-expandable, they can be porous or non-porous, they have any density, but preferably have a density close to that of water, preferably can float in water, and are composed 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, silicone fluids), vesicles (such as synthetic ones such as phospholipids, or natural ones such as cells and cell organs). A non-limiting example of the microparticles suitable for the present invention is carboxyl polystyrene latex microspheres.

[0039] Ⅱ. Specific Embodiments

[0040] The present application will be described in more detail below.

[0041] Luminescence immunoassay 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 the energy level transition, and the number of photons is converted into a relative optical signal through a single photon counter and mathematical fitting. In the presence of the target (such as the antigen / antibody to be detected), the antibody / antigen modified on the surface of the two microspheres reacts with the antigen / antibody to be detected to form a sandwich immune complex, shortening the spatial distance between the two microspheres, and realizing the qualitative or quantitative analysis of the antigen / antibody to be detected through the optical signal generated by the transfer of reactive oxygen species between the two microspheres.

[0042] The inventors of the present application found in their research that in the luminescence immunoassay platform, when the content of the target 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 at this time. However, the vast majority of sandwich immune complexes in the reaction system are formed by bridging one luminescent microsphere and one photosensitive microsphere through one target molecule. When reading the number, a large amount of 1 O 2 generated by the photosensitive microspheres is only received by a small part of the adjacent luminescent microspheres and generates an optical signal, and the un-received 1 O 2 is quenched within 4 μs. Therefore, how to improve the utilization rate of 1 O 2 is the key to improving the detection sensitivity of luminescence immunoassay, especially for the detection ability of low-value samples such as p-tau 217 in plasma. If the method of increasing the particle size of the luminescent microspheres is adopted, more 1 O 2 can be received and a stronger signal can be generated, but at the same time, it will lead to a decrease in its specific surface area and a slowdown in the particle migration rate, directly affecting the efficiency of the immune reaction and reducing the detection sensitivity. The inventors of the present application considered and designed to improve the reagent performance by increasing the utilization rate of free luminescent microspheres in the detection reagent, and provided a new method to improve the utilization rate of 1 O 2 and thus improve the detection sensitivity.

[0043] The chemiluminescence immunoassay kit for phosphorylated Tau protein p-tau 217 involved in the present application includes R1 reagent, R2 reagent and R3 reagent. Among them, the R1 reagent contains luminescent microspheres and a first antibody coated on the luminescent microspheres, which can specifically bind to p-tau 217 and carry a first tag molecule; the R2 reagent contains a second antibody carrying a second tag molecule; the R3 reagent contains a first pairing molecule that can specifically recognize and bind to the first tag molecule; the first antibody and the second antibody can specifically bind to different epitopes of p-tau217, and one first pairing molecule can bind at least two first tag molecules, so that at least two luminescent microspheres bind to form a polymeric luminescent microsphere.

[0044] The first antibody and the second antibody in the R1 reagent and the R2 reagent can undergo an immune reaction with p-tau 217 to form a sandwich immune complex. The first pairing molecule in the R3 reagent can form a specific tag system with the first tag molecule on the luminescent microspheres in the R1 reagent, thereby aggregating free luminescent microspheres (referring to luminescent microspheres where the first antibody does not specifically bind to p-tau 217) onto the main body luminescent microspheres (referring to luminescent microspheres where the first antibody specifically binds to p-tau 217), achieving the effect of increasing the particle size and specific surface area of the effective luminescent microspheres, so that the 1 O 2 utilization rate is increased, and the quenched 1 O 2 is reduced, thereby generating a stronger optical signal. At the same time, the particle size and mass of each luminescent microsphere in the R1 reagent do not change, and it can maintain a good particle migration rate, ensuring the efficiency of the antigen-antibody immune reaction, improving the detection sensitivity as a whole, being beneficial to improving the detection performance of low-value samples, and enabling high-performance detection of the femtogram-level marker p-tau 217 in plasma.

[0045] In the polymeric luminescent microspheres described in the present application, the number of luminescent microspheres is equal to or greater than two, and there is no limitation here.

[0046] In some embodiments of the present application, the R3 reagent may further include luminescent microspheres, that is, the first pairing molecule is also coated on the luminescent microspheres. When the R1 reagent and the R3 reagent 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 the R1 reagent and the R3 reagent to form a polymeric luminescent microsphere.

[0047] In some embodiments of the present application, the specific tag system composed of the first tag molecule and the first pairing molecule can be selected from the Tag-Catcher system, and any combination from the combinations of this system can be selected as the first tag 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 referred to as a tool protein.

[0048] Specifically, the first tag molecule can be selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, Myc-Tag in the Tag-Catcher system, and the first pairing molecule is correspondingly selected from any one of the Catcher proteins. The first tag molecule and the first pairing molecule can be selected from specific tag systems composed of, for example, Spy-Tag and Spy-Catcher, His-Tag and Anti-His (where the Catcher is Anti-His at this time), Snoop-Tag and Snoop-Catcher, etc. Catcher and Tag have excellent specific recognition and binding capabilities, and the relatively small molecular weight Tag label is bound to the first antibody and co-coated on the luminescent microspheres, which has no obvious effect on the expression of the first antibody and the properties of the luminescent microspheres.

[0049] Furthermore, when the first tag molecule is selected from Tag, the first pairing molecule can be selected from the multimeric proteins of Catcher, and the degree of polymerization is not less than 2. For example, it can be selected from the octameric protein of Catcher. The degree of polymerization of Catcher directly affects the rate of Catcher capturing Tag, and thus affects the aggregation rate of the luminescent microspheres. The higher the degree of polymerization of Catcher, the higher the rate of capturing Tag, the higher the aggregation rate of the luminescent microspheres, and the better the effect of enhancing the light signal intensity.

[0050] In some embodiments of the present application, the molar amount of the first pairing molecule is lower 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); preferably 1:(2~8). The usage ratio of the first pairing molecule directly affects the aggregation of the luminescent microspheres. Too low will result in low aggregation efficiency and degree, and the effect of improving the detection performance is not obvious; too high is likely to produce the "hook effect", resulting in difficulty in aggregating the luminescent microspheres, and there is no obvious improvement in the detection performance, and even affects the accuracy of the detection results.

[0051] 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 1~2.5 times the molar amount of the first tag molecule, the effect of enhancing the light signal intensity of the luminescent microspheres is more significant.

[0052] The degree of polymerization and dosage of the first paired molecule can be determined according to the amount of the first labeled molecule. The higher the degree of polymerization of the first paired molecule, the lower the optimal molar amount required, and the faster the reaction rate with the first labeled molecule; the lower the degree of polymerization of the first paired molecule, the higher the optimal molar amount required, and the faster the 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 labeled molecule, the effect of enhancing the light signal intensity of the luminescent microspheres is significant. Excess will easily lead to the HOOK effect and affect the detection results; insufficient amount will affect the aggregation rate and aggregation degree of the polymeric luminescent microspheres, and the performance improvement is not obvious.

[0053] In some embodiments of the present application, the first labeled molecule is connected to the non-specific binding region of the first antibody by co-expression with the first antibody to form the first antibody with the first labeled molecule. The co-expression connection method can be to connect the first labeled molecule to the non-specific binding region of the first antibody through the pClick technology (specific reference: “Synthesis of precision antibody conjugates using proximity-induced chemistry” (Theranostics. 2021 Aug 27;11(18):9107-9117.doi:10.7150 / thno.62444.)), or to insert the sequence of the first labeled molecule into the non-specific binding region of the first antibody to achieve.

[0054] In some embodiments of the present application, the first antibody has 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 has a V-shaped structure, which includes two Fab segments, and the non-specific binding region of the first antibody is the Fab connecting hinge region of the first antibody. When the CDR region of the first antibody or the second antibody is occupied or the Fab end is folded during steps such as coating and labeling, it will cause the Fab end of the antibody to not be effectively displayed, affecting the effective amount or antibody activity of the antibody that can bind to the target molecule p-tau 217 in the reaction system, and further affecting the photochemiluminescence detection performance of p-tau 217. The CDR region, which is the position where specific binding immune reaction can occur with p-tau 217 in the first antibody formed in this way, can be fully and effectively displayed, improving the effective amount and antibody 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.

[0055] 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 between the first antibody and p-tau217 in the sample to be detected, and thus affects the discrimination of the detection results.

[0056] 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, and thus affects the discrimination of the detection results.

[0057] The kit involved in the present application further includes R4 reagent, which contains photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second label molecule. Through the specific binding ability between the second pairing molecule on the photosensitive microspheres and the second label molecule, the second antibody can be bound to the photosensitive microspheres, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres, so that the 1 O 2 generated by the photosensitive microspheres can be effectively received by the luminescent microspheres and generate an optical signal, meeting the requirements of photochemiluminescence detection.

[0058] Furthermore, the two specific label systems respectively composed of the first label molecule and the first pairing molecule, and the second label molecule and the second pairing molecule do not react with each other, avoiding the occurrence of specific pairing interference reactions between the two and the accuracy and stability of the detection results.

[0059] 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 microspheres, and the second antibody binds to streptavidin, and the second antibody is labeled with biotin.

[0060] The using method of the kit involved in the present application can be: after mixing and incubating the R1 reagent, R2 reagent, R4 reagent with the sample to be detected, then adding the R3 reagent to react with the first label molecule of the R1 reagent to aggregate at least two luminescent microspheres into polymeric luminescent microspheres.

[0061] Further, 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 first, and then 15 μL of R3 reagent is added for reaction. That is, in the photochemiluminescence detection process of p-tau217, by controlling the volume ratio of R1 reagent, R2 reagent and the sample to be tested to be 3:3:20, the volume ratio of R2 reagent and R4 reagent to be 3:35, and the volume ratio of R1 reagent to R3 reagent to be 1:1, the reagent volume can be reduced and the reagent concentration can be increased, thereby increasing the sample proportion, shifting the reaction equilibrium towards the binding direction, being conducive to increasing the amount of the formed luminescent microsphere-(p-tau217)-photosensitive microsphere sandwich immune complex, and further improving the detection performance.

[0062] Furthermore, the R1 reagent, R2 reagent and the sample to be tested can be mixed first, the R4 reagent is added after incubation for a period of time, and the R3 reagent is added after incubation for a period of time.

[0063] Specifically, the usage method includes:

[0064] S1. Mix the R1 reagent, R3 reagent and the sample to be tested and incubate at 37 °C to obtain a first reaction product;

[0065] S2. Add the R4 reagent and incubate at 37 °C to obtain a second reaction product;

[0066] S3. Add the R2 reagent and incubate at 37 °C to obtain a third reaction product;

[0067] S4. Irradiate the third reaction product with light of a specific wavelength and detect the luminescence value.

[0068] In this application, first, the luminescent microsphere and the first antibody coated thereon that can specifically recognize and bind p-tau 217 and the second antibody that can specifically recognize and bind p-tau 217 are combined 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. Then, the target luminescent microsphere is fixed to the photosensitive microsphere through the specific binding of the second tag molecule carried by the second antibody and the second paired molecule coated on the photosensitive microsphere to obtain a "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex. Finally, through the specific recognition and binding ability of the first paired molecule and the first tag molecule, the free luminescent microspheres are bound to the target luminescent microspheres to form poly-luminescent microspheres.

[0069] After the conventional reaction, the free luminescent microspheres aggregate around the complex luminescent microspheres, enabling the 1 O 2 to be jointly received by multiple luminescent microspheres in the poly-luminescent microspheres and simultaneously generate optical signals, effectively improving1 O 2 Utilization rate, improve the signal intensity, and then enhance the detection sensitivity of the kit to meet the requirements of photochemiluminescence detection for low-value samples. Moreover, the specific tag system used in this solution, namely the first tag molecule and the first pairing molecule, adopts the Tag-Catcher system, which has strong specificity and high affinity, can significantly improve the reagent performance without interfering with the original immune reaction, has strong applicability and a wide range of applications, and belongs to the improvement of the platform type. Especially when it is used for the detection of p-tau 217 in plasma of extremely low-value samples, it can significantly improve the detection sensitivity, meet the detection requirements of extremely low-value samples, and at the same time, the detection method is simple, easy to operate, and the detection cost is low.

[0070] In some embodiments of the present application, after adding the R1 reagent and the R2 reagent, the reaction time ≥ 45 min, preferably ≥ 60 min; preferably 60 - 75 min. The reaction time of the first antibody, the second antibody and the target analyte p-tau217 affects the reaction degree. 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. After adding the R3 reagent, the reaction time is 0 - 15 min, that is, the aggregation time of the luminescent microspheres is 0 - 15 min. As the aggregation time prolongs, the aggregation reaction of the luminescent microspheres gradually increases and tends to be balanced. Therefore, the aggregation time is preferably 3 - 9 min, more preferably 5 - 7 min.

[0071] In some embodiments of the present application, the kit further includes a buffer solution, a stabilizer, etc. Among them, the buffer solution can be selected from at least one of PBS buffer solution, Tris-hydrochloric acid buffer solution, HEPES buffer solution, and MES buffer solution. The stabilizer can be selected from at least one of bovine serum albumin, dextran, sorbitol, glycerol, glycine, alanine, Tween 20, and Tween 80.

[0072] 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, more preferably 83.3 μg / mL. The concentration of the luminescent microspheres and the first antibody affects their immune reaction efficiency with p-tau217, and then affects 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, more preferably 3.3 μg / mL. The concentration of the second antibody affects its immune reaction efficiency with p-tau217 and the ability to resist biotin interference, and then affects the discrimination of the detection results.

[0073] The detection kit as described above can be applied to detect p-tau 217 in blood samples.

[0074] The detection method specifically includes: mixing a 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 photochemiluminescence detection after incubation for a period of time, measuring the intensity of the chemiluminescence signal generated, 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 according to the intensity of the optical signal.

[0075] Ⅲ. Specific Embodiments

[0076] To make the present invention easier to understand, the following will further illustrate the present application in detail with reference to embodiments. These embodiments are only illustrative and are not limited to the application scope of the present application. The raw materials or components used in the present application can be obtained through commercial channels or conventional methods without special instructions.

[0077] Next, taking the target molecule to be tested as p-tau 217 as an example, antibody molecules that can specifically recognize p-tau 217 are modified with different tags and coated onto luminescent microparticles in the same proportion, and antibody molecules labeled with biotin that can specifically recognize different sites of p-tau 217 are used to detect the target p-tau 217 in the sample by the sandwich immunoassay method. After the reaction in the conventional detection sequence (sample + R1 reagent + R3 reagent, and then adding R4 reagent) is completed, a paired molecule corresponding to the tag that can polymerize free luminescent microparticles is added. By comparing the detection discrimination between different experimental groups, the improvement effect of the multi-polymerized luminescent microparticle mode on the photochemiluminescence detection performance of p-tau 217 is judged.

[0078] Example 1: Preparation of Luminescent Microspheres Coated with Antibodies, Biotin-Labeled Antibodies, and Spy Catcher

[0079] 1. The main experimental raw materials and equipment are shown in Table 1.

[0080] Table 1

[0081]

[0082] 2. Experimental Procedures

[0083] 2.1 Preparation of Luminescent Microspheres Using the Spy Tag System as an Example

[0084] 2.1.1 Linking Tag to Antibody (p-tau217) Ab1

[0085] The Spy Tag (sequence AHIVMVDAYKPTK) was conjugated to the antibody (p-tau217) Ab1 via the pClick technology (specific reference: "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 antibody with the Tag conjugated to the Fc segment. The protein concentration of the collected portion was determined by the BCA method, and then the portion containing protein was subjected to electrophoresis detection. The collected portion that met the molecular weight size of the Tag-antibody was selected and dialyzed into the buffer required for subsequent coupling reactions to obtain the antibody (p-tau217) Abl-SpyTag in which the tag molecule Spy Tag and (p-tau217) Ab1 were co-expressed.

[0086] 2.1.2 Coating the antibody (p-tau 217) Ab1 on luminescent particles

[0087] ① Take 10 mg of aldehyde-functionalized luminescent particles in a centrifuge tube, wash them once with 0.02 M PBS buffer, and then make up the volume to 20 mg / mL with 0.02 M PBS;

[0088] ② Take (p-tau 217) Ab1-Spy Tag, dialyze it with 0.02 M PBS buffer, and then determine the concentration by the BCA method;

[0089] ③ After thoroughly mixing the treated luminescent particles FG and (p-tau 217) Ab1-Spy Tag at a mass ratio of 10:0.5, react them at 37 °C for 16 hours;

[0090] ④ Add 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH 4 (8 mg / mL, 0.05 M CB) to the reaction system, mix well, and then react at 4 °C for 2 h to obtain the reaction solution;

[0091] ⑤ Wash the reaction solution twice with PBST buffer, then add the reconstitution solution (0.02 M PBS + 0.5% BSA + 0.5% Tween20 + 1% dextran) and wash it once, and then add the reconstitution solution and ultrasonically re-dissolve it to obtain luminescent particles coated with (p-tau217) Ab1-Spy Tag with a final concentration of 83.3 μg / mL, namely reagent R1.

[0092] 2.2 Biotin-labeling the antibody (p-tau 217) Ab2

[0093] ① Dialyze (p-tau 217) Ab2 with 0.1M NaHCO 3 solution to remove impurities, and then determine the concentration by BCA method;

[0094] ② Add 30-fold 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;

[0095] ③ Dialyze with 0.02M PBS buffer to remove free Biotin, and then determine the concentration by BCA.

[0096] ④ Prepare biotin-labeled antibody Bio-(p-tau 217) Ab2 at 3.3 μg / mL with the reconstitution solution, namely reagent R2.

[0097] 2.3 Preparation and purification of Spy Catcher

[0098] ① Construction of Catcher plasmid expression vector: Construct the expression vector of Spy Catcher (E. coli vector - pet series expression plasmid) according to the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0099] ② Induced expression of Catcher in E. coli: The E. coli expression vector induces the expression of Spy Catcher protein. The culture conditions are as follows: ① Auto-induction medium (10 g peptone, 5 g yeast extract, 1x NPS, 1 mM MgCl2, 1x5052, PH = 7.4 per liter), add 100 nM ampicillin; ② Induction conditions: Incubate overnight at 30°C in a shaker at 200 rpm.

[0100] ③ Purification of Catcher: Centrifuge the cultured bacterial solution at 8000 rpm to collect the bacterial cell precipitate, resuspend the cell precipitate with 100 mL of purification loading buffer solution A (50 mM PBS, 150 mM NaCl, 20 mM ID, PH = 7.4), centrifuge the resuspended bacterial solution at 18000 rpm for 40 minutes, collect the supernatant and perform nucleic acid fragmentation, filtration, affinity column purification, elution, collect the protein and add the reconstitution solution for ultrasonic reconstitution to obtain Spy Catcher.

[0101] Example 2: Influence of polymerization degree and usage ratio on the performance of p-tau 217 photochemiluminescence detection

[0102] 1. Experimental procedure

[0103] 1.1 Prepare the dimers, pentamers, and octamers of Spy Catcher according to the method of Example 1, and then dilute them with the complex solution to R3 reagents with a molar ratio of Tag in the R1 reagent of 1:16, 1:8, 1:4, 1:2, 1:1, and 2:1. Combine with the photosensitive particle universal liquid reagent R4. Detect on the LiCA ® 500 chemiluminescence detection system.

[0104] 1.2 Mix 100 μL of the test sample, 15 μL of the R1 reagent, and 15 μL of the R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of the universal liquid and incubate at 37 °C for 15 min; add 15 μL of the R3 reagent, add 15 μL of the complex solution to the control group, and incubate at 37 °C for 15 min respectively; after the photoexcitation reaction, read the light signal values of each group of tests. The experimental data are shown below.

[0105] 2. Experimental Results

[0106] 2.1 Reaction effect of Spy Catcher dimer:

[0107] Table 2 Signal values

[0108]

[0109] Table 3 Discrimination

[0110]

[0111] 2.2 Reaction effect of Spy Catcher pentamer:

[0112] Table 4 Signal values

[0113]

[0114] Table 5 Discrimination

[0115]

[0116] 2.3 Reaction effect of Spy Catcher octamer:

[0117] Table 6 Signal values

[0118]

[0119] Table 7 Discrimination

[0120]

[0121] 3. Result Analysis

[0122] 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 of the luminescent particles receiving singlet oxygen. The higher the degree of polymerization of the first pairing molecule corresponding to the first tag molecule, the better the improvement effect. When the first pairing molecule is 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.

[0123] ② As shown in Table 2-7, when the molar ratio of Spy Catcher to Spy Tag in the R1 reagent is 1:(16~1), both the positive signal and discrimination are improved; among them, when the molar ratio is 1:4 (Spy Catcher octamer: Spy Tag), the effect is the best. At this time, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved to a greater extent.

[0124] ③ The degree of polymerization and usage ratio of the first pairing molecule depend on the molecular weight of the first tag. The molar amount of the first pairing molecule should be lower than or equal to the molar amount of the first tag molecule, and the degree of polymerization of the first pairing molecule is negatively correlated with the optimal molar amount. As shown in the results of Table 2-7, when the product of the degree of polymerization and molar amount of the first pairing molecule is 1~2.5 times the molar amount of the first tag molecule, both the positive signal and discrimination are significantly improved.

[0125] ④ As shown in the results of Table 2-7, using Spy Tag as the tag and Spy Catcher as the pairing molecule, both the positive signal and discrimination are significantly improved for the detection of p-tau 217 recombinant antigen and actual samples.

[0126] Therefore, adopting a specific tag system such as Spy Catcher-Tag to aggregate free luminescent microspheres into complex to form multi-polymer luminescent microspheres can improve the positive signal and discrimination, and significantly improve the detection sensitivity of photochemiluminescence, meeting the detection requirements of low-value samples such as p-tau 217 in blood samples.

[0127] Example 3: Influence of aggregation time on the detection performance of p-tau 217 by photochemiluminescence

[0128] 1. Experimental procedure

[0129] 1.1 Dilute Spy Catcher octamer with a reconstitution solution to an R3 reagent with a molar ratio of 1:4 to Tag in the R1 reagent. Combine it with the photosensitive particle universal reagent R4. Detect with the test sample on the LiCA ® 500 chemiluminescence detection system.

[0130] 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 the universal solution and incubate at 37 °C for 15 min; add 15 μL of R3 reagent, and add 15 μL of the reconstitution 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 values of each group of tests. The experimental data are shown below.

[0131] 2. Experimental results

[0132] Table 8 Signal values

[0133]

[0134] Table 9 Discrimination

[0135]

[0136] 3. Result analysis

[0137] As shown in the results of Tables 8 - 9, with the extension of the reaction time after adding R3 reagent (containing the first paired molecule), the positive signal and discrimination gradually increase and tend to balance; at about 6 min of the reaction, the aggregation reaction of free luminescent particles reaches equilibrium.

[0138] Therefore, in the reaction process of aggregating free luminescent microspheres into composite multi - luminescent microspheres using a specific tag system such as Spy Catcher - Tag, considering the balance between reaction time and performance, the reaction time can be 0 - 15 min after adding R3 reagent, the preferred reaction time is 3 - 9 min, and the optimal reaction time is 5 - 7 min.

[0139] Example 4: Verification of the applicability of different tag systems for forming "multi - luminescent microspheres"

[0140] 1. Experimental procedure

[0141] 1.1 Prepare luminescent microspheres coated with (p - tau 217) Ab1 with different tag molecules (Spy - Tag, His - Tag, Snoop - Tag) according to the method of coating antibodies on luminescent microspheres in Example 1 above, and dilute to 83.3 μg / mL with the reconstitution solution to obtain R1 reagent.

[0142] 1.2 Dilute Bio - (p - tau 217) Ab2 to 3.3 μg / mL with the reconstitution solution to obtain R2 reagent.

[0143] 1.3 The first pairing molecules (octamer Spy-Catcher, octamer Anti-His, octamer Snoop-Catcher corresponding to the tag molecules) are diluted with the reconstitution solution to R3 reagents with a molar ratio of 1:16, 1:4, and 1:1 to the tag molecules in the R1 reagent.

[0144] 1.4 Detection is performed with the LiCA ® 800 chemiluminescence detection system:

[0145] Mix 100 μL of the test sample, 15 μL of the R1 reagent, and 15 μL of the R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of the photosensitive particle universal solution, and incubate at 37 °C for 15 min; add 15 μL of the R3 reagent, and add 15 μL of the reconstitution solution to the control group, and incubate at 37 °C for 15 min respectively; after the photoexcitation reaction, read the light signal values of each group of tests. The experimental data are shown below.

[0146] 2. Experimental results

[0147] Table 10 Signal values

[0148]

[0149] Table 11 Discrimination

[0150]

[0151] 3. Result analysis

[0152] As shown in the results of Tables 10 - 11, different specific tag systems such as Spy-Catcher / Spy-Tag, His-Tag / Anti His-Ab, and Snoop-Tag / Catcher can all improve the positive signal value and the discrimination in the low region of the detection to varying degrees.

[0153] Therefore, the use of the specific tag system described in this application has a good effect on the aggregation of free luminescent microspheres to form multi-polymer luminescent microspheres to improve the detection performance, effectively improving the sensitivity of the photoinduced chemiluminescence detection and meeting the detection requirements of low-value samples such as p-tau 217 in blood samples.

[0154] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A photoluminescence detection kit for phosphorylated Tau protein p-tau217, characterized in that: include: R1 reagent, comprising luminescent microspheres, wherein the luminescent microspheres are coated with a first antibody that can specifically bind to p-tau 217, wherein the first antibody carries a first label molecule; the luminescent microspheres can react with reactive oxygen to generate detectable light signals; R2 reagent, which comprises a second antibody with a second tag molecule; the first antibody and the second antibody can specifically bind to different epitopes of p-tau217; R3 reagent, which comprises a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; R4 reagent, which comprises a photosensitive microsphere and a second pairing molecule coated on the photosensitive microsphere and capable of specifically binding to the second label molecule; The specific label system composed of the first label molecule and the first pairing molecule is selected from the Tag-Catcher system, the first pairing molecule is selected from the multimeric protein of Catcher, and one first pairing molecule can bind to at least two first label molecules; the two groups of specific label systems composed of the first label molecule and the first pairing molecule, the second label molecule and the second pairing molecule respectively do not react with each other.

2. The kit according to claim 1, 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, 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, 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.

5. The method for using the kit according to any one of claims 1 to 4, 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.

6. The method for using the kit according to claim 5, characterized in that: The reaction time after adding R1 and R2 reagents is ≥45min; the reaction time after adding R3 reagent is 0~15min.

7. Use of the kit according to any one of claims 1 to 4 in detecting p-tau217 in a blood sample.

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