Light-initiated chemiluminescence detection method, reagent, and use thereof

Abstract

A light-initiated chemiluminescence detection method, a reagent, and a use. The detection method comprises: bringing a sample to be tested into contact with a reaction system comprising a first antibody and a second antibody, so as to obtain an immune reaction product, and measuring the optical signal intensity of the immune reaction product, the first antibody and / or the second antibody directionally coupling a non-specific binding region thereof to a target substance by means of an isopeptide bond. Alternatively, an R3 reagent is added on the basis of a conventional detection method, and at least two label molecules are bound by means of a pairing molecule contained in the R3 reagent, the label molecules being carried by the first antibody in luminescent microspheres, such that the at least two luminescent microspheres are aggregated into a multimeric luminescent microsphere. The detection method can improve the detection performance of light-initiated chemiluminescence, and meet detection requirements of a low-abundance sample such as p-tau217 in a blood sample.

Description

A photo-induced chemiluminescence detection method, reagent and its application

[0001] This application claims priority to Chinese Patent Application No. 2024116293171, filed on November 15, 2024, entitled "A Photoluminescent Detection Kit for Phosphorylated Tau Protein p-tau217 and Its Usage and Application", the entire contents of which are incorporated herein by reference.

[0002] This application claims priority to Chinese Patent Application No. 202411642131X, filed on November 15, 2024, entitled “A Photo-induced Chemiluminescence Detection Reagent, Detection Method and Its Application”, the entire contents of which are incorporated herein by reference.

[0003] This application claims priority to Chinese Patent Application No. 2024116390877, filed on November 15, 2024, entitled "A Photo-induced Chemiluminescence Detection Method and Its Application", the entire contents of which are incorporated herein by reference.

[0004] This application claims priority to Chinese Patent Application No. 2025105363512, filed on April 25, 2025, entitled "A Photo-induced Chemiluminescence Detection Method for p-tau 217 and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0005] This application relates to the field of immunoassay technology, and in particular to a photo-induced chemiluminescence detection method, reagent, and its application. Background Technology

[0006] Alzheimer's disease (AD), commonly known as senile dementia, is the most common neurodegenerative disease. Neuropathological changes occur 15 to 20 years before the onset of clinical symptoms in AD, such as the appearance of β-amyloid plaques and excessive phosphorylation of Tau protein in brain tissue. The accumulation of Aβ and Tau proteins can impair neuronal function and even lead to neuronal death, resulting in a significant decline in cognitive functions such as memory. However, this process progresses extremely slowly. Therefore, early detection and accurate identification in the early stages of the disease are crucial for early intervention in AD.

[0007] Currently, clinical diagnostic methods for Alzheimer's disease (AD) include scale assessments, magnetic resonance imaging (MRI), positron emission tomography (PET), and cerebrospinal fluid analysis. However, these methods suffer from drawbacks such as insufficient objectivity, high invasiveness, cumbersome procedures, and high costs, significantly limiting their clinical application, especially in primary healthcare institutions. The clinical misdiagnosis rate for AD is as high as 76.8%, making early screening and intervention difficult. Therefore, providing a low-cost, minimally invasive, convenient, and standardized blood biomarker for AD is an urgent clinical need.

[0008] p-tau 217 is a phosphorylated form of Tau protein, primarily phosphorylated at threonine 217. It is significantly elevated in the preclinical stage of Alzheimer's disease (AD), providing an earlier indication of AD development. Studies show that the average level of p-tau 217 in the plasma of healthy individuals is approximately 0.3 to 0.4 pg / mL, while in AD patients it reaches 1 to 2 pg / mL, with a positive cutoff value below 1 pg / mL. Conventional immunoassay techniques detect the overall signal after the reagent system reaction using optical or electrical methods, then convert the signal intensity into a detection concentration using a standard curve. However, their detection limits are generally around 1-10 ng / mL, making it difficult to achieve such high sensitivity and reliably detect p-tau 217 in plasma. Single-molecule immunoassay array (SMIA) technology is a digital detection technique that forms an enzyme complex on the surface of magnetic beads, consisting of a capture antibody, a target antigen, a biotinylated detection antibody, and avidin. After binding with a fluorescent substrate, the magnetic beads fall into the arrayed microwells for reaction and detection. Its detection sensitivity is exponentially improved compared to ELISA and conventional chemiluminescence detection methods. However, the detection process is cumbersome and complex, and the detection cost is extremely high, which greatly limits its clinical application. Summary of the Invention

[0009] To address or partially address the problems existing in related technologies, this application provides a photo-induced chemiluminescence detection method, reagent, and its application, which can improve the performance of photo-induced chemiluminescence detection and meet the detection needs of low-abundance proteins such as p-tau 217 in blood samples.

[0010] The photo-induced chemiluminescence detection method of this application involves contacting the sample to be tested with a reaction system containing a first antibody and a second antibody to obtain an immunoreaction product, and detecting the light signal intensity of the immunoreaction product; wherein the first antibody and / or the second antibody are oriented to couple their non-specific binding regions to the target substance via isopeptide bonds.

[0011] In the detection method of this application, the first antibody and / or the second antibody form isopeptide bonds through the Catcher-Tag directional subsystem to directionally couple them to the target substance.

[0012] In the detection method of this application, the Catcher in the Catcher-Tag directional subsystem is a single Catcher.

[0013] In the detection method of this application, the monomer Catcher carries a tagged protein.

[0014] In the detection method of this application, the molecular weight of the monomer Catcher is 12-55 kDa.

[0015] In the detection method of this application, the monomer Catcher contains lysine residues.

[0016] In the detection method of this application, the number of lysine residues in the monomer Catcher is ≥5.

[0017] In the detection method of this application, the monomer Catcher in the Catcher-Tag directing subsystem is coated on the surface of the luminescent microspheres, and the Tag is linked to the non-specific binding region of the first antibody.

[0018] In the detection method of this application, the monomeric Catcher in the Catcher-Tag directional subsystem is marked with one of the specific binding pair members, and the Tag is linked to the non-specific binding region of the second antibody.

[0019] In the detection method of this application, the non-specific binding region of the first antibody is oriented to the luminescent microsphere via a first Catcher-Tag directional subsystem; one of the specific binding pair members is oriented to the non-specific binding region of the second antibody via a second Catcher-Tag directional subsystem.

[0020] In the detection method of this application, the first Catcher-Tag orientation subsystem and the second Catcher-Tag orientation subsystem are different.

[0021] In the detection method of this application, each of the second antibodies is linked to two second Catcher-Tag targeting subsystems, both located in the Fc segment of the second antibody.

[0022] In the detection method of this application, the sample to be tested is a blood sample.

[0023] The photo-induced chemiluminescence detection reagent described in this application includes a reaction reagent containing a first antibody and a second antibody. Both the first antibody and the second antibody are capable of specifically binding to the target molecule to be tested, and the first antibody and / or the second antibody are oriented to couple their non-specific binding regions to the target substance through isopeptide bonds.

[0024] The photo-induced chemiluminescence detection reagent for p-tau 217 described in this application includes a reaction reagent containing a first antibody and a second antibody. Both the first antibody and the second antibody are specifically bound to p-tau 217, and the first antibody and / or the second antibody are oriented to conjugate their non-specific binding regions to the target substance via isopeptide bonds.

[0025] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the first antibody and / or the second antibody form heteropeptide bonds through a Catcher-Tag directional subsystem to directionally couple them to the target substance; preferably, the Catcher in the Catcher-Tag directional subsystem is a monomeric Catcher; more preferably, the monomeric Catcher carries a tag protein.

[0026] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the molecular weight of the monomer Catcher is 12-55 kDa.

[0027] In the photo-induced chemiluminescence detection reagent and the photo-induced chemiluminescence detection reagent of p-tau 217 described in this application, the monomer Catcher contains lysine residues; preferably, the number of lysine residues in the monomer Catcher is ≥5.

[0028] In the photochemiluminescence detection reagent and the p-tau 217 photochemiluminescence detection reagent described in this application, the monomer Catcher in the Catcher-Tag orientation subsystem is coated on the surface of the luminescent microspheres, and the Tag is linked to the non-specific binding region of the first antibody; and / or, in the Catcher-Tag orientation subsystem, the monomer Catcher is coated on the surface of the luminescent microspheres, and the Tag is linked to the non-specific binding region of the first antibody.

[0029] In the photochemiluminescence detection reagent and the p-tau 217 photochemiluminescence detection reagent described in this application, the non-specific binding region of the first antibody is oriented to the luminescent microspheres through a first Cater-Tag orientation subsystem; one of the specific binding pair members is oriented to the non-specific binding region of the second antibody through a second Cater-Tag orientation subsystem; preferably, the first Cater-Tag orientation subsystem and the second Cater-Tag orientation subsystem are different.

[0030] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, each of the second antibodies is linked to two second Catcher-Tag orientation subsystems, both located in the Fc segment of the second antibody.

[0031] The photo-induced chemiluminescence detection kit described in this application includes the aforementioned photo-induced chemiluminescence detection reagent.

[0032] The photochemiluminescence detection kit for p-tau 217 described in this application contains the aforementioned photochemiluminescence detection reagent for p-tau 217.

[0033] The photo-induced chemiluminescence detection method for p-tau 217 described in this application involves mixing and incubating reagents R1, R2, and R4 with the sample to be tested, followed by the addition of reagent R3 to aggregate at least two luminescent microspheres into a polymeric luminescent microsphere. Reagent R1 comprises luminescent microspheres coated with a first antibody capable of specifically binding to the target molecule, the first antibody carrying a first tag molecule. Reagent R2 comprises a second antibody carrying a second tag molecule, the second antibody capable of specifically binding to the target molecule. Reagent R3 comprises a first pairing molecule capable of specifically recognizing and binding to the first tag molecule. Reagent R4 comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres capable of specifically binding to the second tag molecule. One first pairing molecule can bind at least two first tag molecules.

[0034] In the detection method of this application, the first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

[0035] In the detection method of this application, the first tag molecule and the first paired molecule are selected from the Catcher-Tag polymer subsystem.

[0036] In the detection method of this application, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag.

[0037] In the detection method of this application, the first paired molecule is a polycatcher with a degree of polymerization of not less than 2.

[0038] In the detection method of this application, the molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule.

[0039] In the detection method of this application, the molar ratio of the first paired molecule to the first tag molecule is 1:(1-16); preferably 1:(2-8).

[0040] In the detection method of this application, the first paired molecular polymer is negatively correlated with its optimal molar amount.

[0041] In the detection method of this application, the product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

[0042] In the detection method of this application, the molar number of the luminescent microspheres is greater than the molar number of the target molecules to be tested in the sample to be tested.

[0043] In the detection method of this application, the two sets of specific tag systems, composed of the first tag molecule and the first paired molecule, and the second tag molecule and the second paired molecule, do not react with each other.

[0044] In the detection method of this application, the reaction time after adding reagent R3 is 0 to 15 minutes.

[0045] In the detection method of this application, the sample to be tested is a blood sample.

[0046] The photoluminescence detection reagent described in this application comprises: reagent R1, which contains luminescent microspheres coated with a first antibody capable of specifically binding to a target molecule, the first antibody carrying a first tag molecule; reagent R2, which contains a second antibody carrying a second tag molecule, the second antibody capable of specifically binding to the target molecule; reagent R3, which contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; and reagent R4, which contains photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres capable of specifically binding to the second tag molecule; one first pairing molecule can bind at least two first tag molecules.

[0047] The photo-induced chemiluminescence detection reagent for p-tau 217 described in this application includes the above-mentioned reagents R1, R2, R3, and R4, wherein the first antibody in reagent R1 and the second antibody in reagent R2 can specifically bind to different epitopes of p-tau 217.

[0048] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

[0049] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the first tag molecule and the first pairing molecule are selected from the Catcher-Tag polymer subsystem; preferably, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag; more preferably, the first pairing molecule is a polyCatcher with a degree of polymerization of not less than 2.

[0050] In the photochemiluminescence detection reagent and the p-tau 217 photochemiluminescence detection reagent described in this application, the molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule; preferably, the molar ratio of the first paired molecule to the first tag molecule is 1:(1-16); more preferably, it is 1:(2-8).

[0051] In the photo-induced chemiluminescence detection reagent and the photo-induced chemiluminescence detection reagent of p-tau 217 described in this application, the first paired molecule polymer is negatively correlated with its optimal molar amount; preferably, the product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

[0052] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the molar number of the luminescent microspheres is greater than the molar number of the target molecules to be tested in the sample to be tested.

[0053] In the photo-induced chemiluminescence detection reagent and the p-tau 217 photo-induced chemiluminescence detection reagent described in this application, the two sets of specific tag systems composed of the first tag molecule and the first paired molecule, and the second tag molecule and the second paired molecule, do not react with each other.

[0054] The photo-induced chemiluminescence detection kit described in this application includes the aforementioned photo-induced chemiluminescence detection reagent.

[0055] The photochemiluminescence detection kit for p-tau 217 described in this application contains the aforementioned photochemiluminescence detection reagent for p-tau 217.

[0056] The detection method, detection reagents, and detection kits described in this application are used to detect p-tau 217 in blood samples.

[0057] It should be noted that the method described in this application is not for disease diagnosis purposes.

[0058] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0059] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0060] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered within this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered within this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included within this invention.

[0061] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0062] The sample to be tested as described in this application refers to a mixture that may contain analytes, including but not limited to proteins, hormones, antibodies, or antigens. Typical samples to be tested that can be used in the methods disclosed in this application include bodily fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion. The sample to be tested can be diluted with a diluent or buffer solution as needed before use.

[0063] The term "antibody" as used in the broadest sense includes any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of the antibody and non-antibody proteins. Where necessary, the antibody may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin. The location capable of specific binding to the antigen in an immune response, known as the complementarity determining region (CDR), is located in the Fab segment of the antibody. Therefore, in this application, the Fab segment of the antibody is referred to as the specific binding region, and the opposite segment is referred to as the non-specific binding region.

[0064] The antigens described in this application refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). The antigens can be natural antigens extracted from pathogenic animal tissues or recombinant antigens with specific antigenic properties prepared through genetic engineering technology. Where necessary, the antigens can be further conjugated to other parts, such as specific binding pairing members, for example, biotin or avidin.

[0065] The terms “combination,” “connection,” and “coupling” used in this application refer to the union between two substances caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, or interactions including but not limited to salt bridges and water bridges.

[0066] The specific binding described in this application refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response. In the immune system, it specifically refers to the tight binding between certain molecules and the specific antigens or epitopes they recognize. This binding usually involves covalent or non-covalent bonds, such as hydrogen bonds, van der Waals forces, electrostatic interactions, etc., enabling the two substances to form a stable complex.

[0067] The isopeptide bond described in this application is a special peptide bond formed by the dehydration condensation of amino or carboxyl groups on the side chain of an amino acid.

[0068] The Catcher-Tag system described in this application is a protein covalent linking technology, comprising two parts: a Tag consisting of multiple amino acid residues and a Catcher. Isopeptide bonds can be formed through a specific reaction between the Tag and the Catcher. The Catcher-Tag system includes, but is not limited to, the Spy Catcher-Tag system, the Sdy Catcher-Tag system, the Dog Catcher-Tag system, the Snoop Catcher-Tag system, or other Catcher proteins that can form isopeptide bonds with the corresponding Tag. Furthermore, each Catcher-Tag system can have multiple configurations, and based on their different functions in this application, they are named Catcher-Tag directional subsystems and Catcher-Tag aggregation subsystems.

[0069] The Catcher-Tag orientation subsystem is used to achieve targeted coating or labeling of antibody molecules, preserving the effective affinity terminus (Fab terminus) of the antibody molecule that specifically recognizes the target molecule. This significantly improves the detection signal value and discrimination in photochemiluminescence detection, enabling the detection of femtogram-level biomarkers. The Catcher-Tag orientation subsystem uses a monomer, or a monomeric Catcher with a tagged protein. The tagged protein described in this application refers to a polypeptide or protein molecule fused with a target protein (such as a Catcher) using in vitro DNA recombination technology, facilitating the expression, detection, purification, labeling, and conjugation of the target protein. The tagged protein can help the Catcher increase its occupancy space, further reducing the steric hindrance of the antibody conjugation, increasing the antibody coating amount on the microspheres, and improving detection sensitivity.

[0070] The tags in the Catcher-Tag polymerization subsystem can be selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag. The catcher in the Catcher-Tag polymerization subsystem is a multimer with a degree of polymerization of not less than 2, for example, it can be selected from the octamer protein of the catcher. A multimer is a protein composed of two or more polypeptide chains, which can be the same or different, linked together by covalent bonds or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.). If it is a single tag, the multimer catcher is its corresponding single catcher multimer; if it is multiple tags, the multimer catcher can be multiple catcher multimers corresponding to each tag. The degree of polymerization of the catcher directly affects the catcher's tag capture rate and the number of tag molecules that can be polymerized, thus affecting the aggregation rate and number of luminescent microspheres. Within a certain range, the higher the degree of polymerization of the catcher, the better the effect on improving the light signal intensity.

[0071] The Spy Catcher-Tag system described in this application is developed based on the CnaB2 domain of the fibronectin FbaB from Streptococcus pyogenes. Spy Tag is a short peptide containing 13 amino acid residues, and Spy Catcher is a protein containing 138 amino acid residues. During the linkage process, the aspartic acid in Spy Tag spontaneously reacts with the lysine residue on Spy Catcher, catalyzed by the glutamate adjacent to the lysine residue, to form a heteropeptide covalent bond.

[0072] The Snoop Catcher-Tag system described in this application was developed from the fimbriae protein RrgA of Streptococcus pneumoniae. The D4 domain of RrgA generates a stable isopeptide bond through an E803 catalytic reaction of a lysine (K742) and an asparagine (N854) that are spatially adjacent.

[0073] The biotin-avidin system described in this application is a bioreaction amplification system comprising two main components: biotin and either avidin or streptavidin. Biotin is widely found in plant and animal tissues and has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary site for avidin binding. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents. Avidin is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, creating a "tentacle effect" with avidin to enhance analytical sensitivity. Where necessary, any reagent used in this application, including antigens, antibodies, receptors, or donors, can be conjugated to any member of the biotin-streptavidin specific binding pair, as required.

[0074] The reactive oxygen species (ROS) described in this application refer to a general term for oxygen-containing and reactive substances in the body or natural environment. They are primarily excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH),) as well as nitric oxide and reactive oxygen species (ROS). 1 O2), etc.

[0075] The luminescent microspheres described in this application refer to polymeric microparticles filled with a luminescent composition, capable of reacting with reactive oxygen species to generate a detectable light signal. Luminescent microspheres may also be called acceptor microspheres or luminescent microparticles. In some embodiments of this application, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridine, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other embodiments of this application, the luminescent composition may further include europium complexes; preferably, the europium complex is MTTA-EU. 3+ .

[0076] The photosensitive microspheres described in this application refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microparticles. Solutions containing such photosensitive microspheres can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microspheres can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0077] The microparticles described in this application can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for this application is carboxylated polystyrene latex microspheres.

[0078] This application will now be described in more detail.

[0079] Photocatalytic chemiluminescence analysis is a next-generation immunoassay technique based on nanoscale polymer particles. Its core principle is the generation and transfer of reactive oxygen species (ROS): energy transfer between photosensitive microspheres (GG) and luminescent microspheres (FG) generates high-energy red light during energy level transitions. A single-photon counter and mathematical fitting convert the photon count into a relative light signal. In the presence of the target analyte (e.g., the antigen / antibody to be tested), the antibody / antigen modified on the surfaces of the two microspheres undergoes an antigen-antibody specific binding reaction with the antigen / antibody to be tested, forming a sandwich immune complex. This shortens the spatial distance between the two microspheres, and the light signal generated by the transfer of ROS between the two microspheres enables qualitative or quantitative analysis of the antigen / antibody to be tested.

[0080] In the detection of antigens using a photocatalytic chemiluminescence (PDC) analysis platform, the activity and affinity of antibody molecules modified on the microsphere surface that can react with the target molecule directly affect the detection performance, such as repeatability, sensitivity, and the stability and reliability of the results. The inventors of this application discovered that conventional methods, such as coating and labeling, can bind antibody molecules to the microsphere surface or specifically bind to one of the paired members. However, these binding processes are random, which may result in the regions of the antibody molecule that specifically recognize and bind the target molecule being occupied or not effectively displayed. This prevents effective recognition and binding of the target antigen molecule, affecting the immune reaction process and consequently impacting the sensitivity and accuracy of the PDC detection. This is especially true for low-abundance protein molecules in blood samples, where detection performance is highly susceptible to influences from the conformation and activity of the antibodies in the detection reagents.

[0081] The photo-induced chemiluminescence detection method provided in this application involves contacting the sample to be tested with a reaction system containing a first antibody and a second antibody to obtain an immunoreaction product, and then detecting the light signal intensity of the immunoreaction product; wherein the first antibody and / or the second antibody are oriented to couple their non-specific binding regions to the target substance via isopeptide bonds.

[0082] The detection method can be applied to detect p-tau 217 markers in body fluid samples, wherein the sample to be tested is a blood or cerebrospinal fluid sample, and the blood sample can be whole blood, plasma or serum.

[0083] The detection method of this application achieves targeted coating or labeling of antibody molecules through isopeptide bonds, which can increase the effective antibody molecular weight that can interact with the target molecule without interfering with the specificity of the reagent, and allows the effective conformation of the antibody molecule to be displayed, retaining the activity of the antibody molecule and improving the photo-induced chemiluminescence detection performance to meet the detection requirements of femtogram-level markers such as p-tau217 in the sample.

[0084] In some embodiments of this application, the non-specific binding regions of any one or two antibody molecules in the detection reagent are directionally coupled to the target substance via isopeptide bonds. That is, a first antibody is directionally coated onto luminescent microspheres, and one of the specific binding pairs is non-directionally labeled onto a second antibody; or, a first antibody is non-directionally coated onto luminescent microspheres, and one of the specific binding pairs is directionally labeled onto a second antibody; or, a first antibody is directionally coated onto luminescent microspheres, and one of the specific binding pairs is directionally labeled onto a second antibody.

[0085] This application utilizes isopeptide bonds in the antibody coating or labeling process to effectively expose the antibody's specific binding region, preserving the effective affinity ends that can specifically bind to target molecules such as p-tau217. This ensures the antigen-antibody immune reaction after mixing with the test sample, guarantees the efficient transfer of reactive oxygen species between the luminescent and photosensitive microspheres in the reaction system, and ensures that the light signal intensity emitted by the luminescent microspheres meets the requirements of photochemiluminescence clinical detection. Consequently, it ensures that photochemiluminescence detection has good sensitivity, consistent and reproducible results, and high accuracy.

[0086] In some embodiments of this application, isopeptide bonds are formed via a Catcher-Tag directional subsystem. In some preferred embodiments of this application, the Catcher-Tag directional subsystem may be selected from the Spy Catcher-Tag system, Sdy Catcher-Tag system, Dog Catcher-Tag system, or Snoop Catcher-Tag system, etc. In some embodiments of this application, the monomeric Catcher in the Catcher-Tag directional subsystem carries a tagged protein.

[0087] In some embodiments of this application, the tag protein can be attached to the N-terminus and / or C-terminus of the monomeric Catcher protein. When a tag protein is introduced into both the N-terminus and C-terminus of the monomeric Catcher protein, the introduced tag proteins may be the same or different.

[0088] In some embodiments of this application, the tag protein can bind to the monomer Catcher via a linker peptide at one end. Preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; more preferably, n is a natural number from 1 to 5.

[0089] For example, genetic engineering techniques can be used to insert the sequence of the tag protein into the beginning or end of the sequence of the monomeric Catcher protein to construct a fusion expression vector. Then, through methods such as induced expression and purification, the monomeric Catcher protein fused with the tag protein can be obtained, enabling it to be expressed simultaneously.

[0090] In some embodiments of this application, the tag protein may be selected from at least one of commonly used protein tags, such as solubilization tags, molecular chaperones, enzyme tags, and purification tags. Specifically, the tag protein may be selected from at least one of Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, and SUMO, or it may be a polypeptide sequence containing a specific amino acid constructed through gene editing.

[0091] To further increase the amount of effective antibody in the detection reagent, the monomeric catcher can be selected from molecules with a molecular weight greater than 12 kDa; preferably 12–55 kDa; more preferably 30–40 kDa; and even more preferably 37–39 kDa. If the monomeric catcher carries a tag protein, the above molecular weight refers to the total molecular weight of the monomeric catcher and the tag protein.

[0092] To further increase the amount of effective antibody in the detection reagent and improve the antigen-antibody immune reaction rate in photochemiluminescence detection, the monomeric catcher needs to have as many lysine molecules as possible. For example, the tag protein can be selected from molecules containing lysine residues. The number of lysine residues in the monomeric catcher can be ≥5. If the monomeric catcher carries a tag protein, the above-mentioned number of lysine residues refers to the total number of lysine residues in the monomeric catcher and the tag protein.

[0093] The conventional process for coating antibodies onto luminescent microspheres typically includes activation, coupling, and blocking. Activation usually involves using an activator to activate active groups on the microspheres, such as carboxyl groups, enabling the activated microspheres to covalently couple with the antibody through active sites. Then, a blocking agent, such as bovine serum albumin, is added to block unreacted sites on the microspheres. However, in this conventional coating process, the coupling of antibodies to the surface of the luminescent microspheres is random, resulting in some antibody Fab fragments not being effectively displayed. Therefore, these fragments cannot bind to target molecules such as p-tau 217 in the sample, affecting the effective amount of antibody on the surface of the luminescent microspheres that can bind to the target molecule, thus impacting detection performance.

[0094] This application aims to increase the effective antibody content on the surface of luminescent microspheres by employing a Catcher-Tag directional coating system for the directional coating of antibody molecules. The process includes: first, coating the surface of the luminescent microspheres with the monomer Catcher from the Catcher-Tag directional coating system; second, linking the Tag to the non-specific binding region (Fc segment) of the first antibody; and third, connecting the Fc end of the first antibody to the luminescent microspheres via a spontaneously formed isopeptide bond between the Catcher and the Tag, while leaving the Fab end of the antibody unaffected.

[0095] The tag can be linked to the non-specific binding region of the first antibody in two ways: First, by linking the coding sequences of two or more genes together using gene recombination technology to form a fusion gene. After expression, this fusion gene produces a fusion protein containing different functional domains encoded by multiple genes. Second, by using technologies such as pClick (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18):9107-9117. doi:10.7150 / thno.62444.)) to link two or more peptides, proteins, etc., with different or identical gene sequences together to form a fusion protein.

[0096] The targeted coating process for antibody molecules may specifically include:

[0097] (1) Insert the Tag sequence into the antibody sequence and express it in cells to obtain Tag-antibody;

[0098] (2) Mix the luminescent microspheres with the monomer Catcher to couple the monomer Catcher to the surface of the luminescent microspheres, thereby sealing and reducing them;

[0099] (3) The luminescent microspheres coupled with the monomer Catcher were mixed with the first antibody coupled with the Tag, washed and reconstituted to obtain luminescent microspheres oriented to be coated with the first antibody.

[0100] In step (2) above, the luminescent microsphere coupling monomer Catcher can be carried out at (37±2.5)℃; a mixture of glycine and NaBH4 can be used in the blocking and reduction steps; the blocking and reduction are carried out at low temperature, for example at (4±0.5)℃.

[0101] In step (3) above, the coating of the luminescent microspheres with the first antibody is performed under low temperature conditions, such as (4±0.5)℃; the reconstitution solution can be a mixture of PBS, BSA, Tween 20, and dextran. The luminescent microspheres treated with the reconstitution solution have better stability, which helps to ensure detection sensitivity and accuracy of detection results.

[0102] The conventional labeling process for secondary antibodies typically involves the specific binding of one of the pairing members, such as biotin, to a random label (conjugation) onto the lysine residues of the secondary antibody. However, the conventional labeling process is random, which may result in the labeling molecule occupying the CDR region of the secondary antibody. This can impair the activity of the secondary antibody in recognizing and binding the target molecule, thus affecting the detection sensitivity.

[0103] This application aims to improve the activity of antibodies labeled with one of their specific binding pairing members by employing a Catcher-Tag directing subsystem for the directional labeling of antibody molecules. This involves labeling one of the specific binding pairing members, such as biotin, onto a monomeric Catcher in the Catcher-Tag directing subsystem, directionally linking the Tag end to the non-specific binding region of the second antibody, i.e., the Fc region of the second antibody, and then directionally labeling the specific binding pairing member onto the second antibody through an isopeptide bond formed between the monomeric Catcher and the Tag.

[0104] The way in which the tag is attached to the non-specific binding region of the second antibody is the same as the way the tag is attached to the non-specific binding region of the first antibody.

[0105] In some embodiments of this application, the monomeric Catcher of the Catcher-Tag directional subsystem used in the directional labeling of the second antibody may be selected from molecules with or without tagged proteins; preferably, molecules with tagged proteins.

[0106] The molecular weight of the monomeric catcher influences its motility in liquids to some extent. Motility is related to capture ability. In the catcher-tag orientation subsystem, the ability of the monomeric catcher to capture the tag affects the amount of effectively oriented secondary antibody (Bio-Ab2). The amount of effectively oriented antibody significantly affects detection sensitivity. Furthermore, the molecular weight of the monomeric catcher also affects the diffusion rate of the secondary antibody (Bio-Ab2) and the number of Bio-Catchers that can be coupled to the Fc fragment of the secondary antibody, thus affecting detection sensitivity. The molecular weight of the monomeric catcher can be greater than 12 kDa; preferably 12–55 kDa; more preferably 30–40 kDa; and even more preferably 37–39 kDa. If the monomeric catcher carries a tag protein, the above molecular weight refers to the total molecular weight of the monomeric catcher and the tag protein.

[0107] The number of lysine residues on the monomeric catcher directly affects the number of biotin tags, and thus the reaction equilibrium and rate between Bio-Ab2 and the photosensitive microspheres. The number of lysine residues can be ≥5; preferably greater than 16–30; more preferably 23–28. If the monomeric catcher carries a tag protein, the above-mentioned number of lysine residues refers to the total number of lysine residues in the monomeric catcher and the tag protein.

[0108] The targeted labeling step of antibody molecules may include:

[0109] (a) The Tag sequence is inserted into the antibody sequence and expressed in cells to obtain Tag-antibody;

[0110] (b) Mix one of the specific binding pair members with the monomer Catcher to conjugate one of the specific binding pair members with the monomer Catcher.

[0111] (c) Mix one of the specific binding pair members of the monomer Catcher with a second antibody conjugated with a Tag, purify, and obtain a second antibody that specifically binds to one of the pair members and is directionally labeled.

[0112] Steps (b) and (c) above can be performed under low temperature conditions, such as (4±0.5)℃.

[0113] The method of this application can achieve the purpose of specifically binding one of the paired members to the Fc end of the second antibody, avoiding the CDR region of the second antibody being occupied by the labeled molecule, maximizing the preservation of the activity of the second antibody, increasing the effective amount of antibody in the detection reagent that can recognize and bind to the target molecule, thereby rapidly and effectively identifying the target molecule, improving detection sensitivity and the consistency and repeatability of detection results, and reducing the impact of batch-to-batch differences of detection reagents on detection results.

[0114] To further improve detection sensitivity, isopeptide bonds can be used to directionally couple the target substance to both the coating end of the first antibody and the labeling end of the second antibody.

[0115] In some embodiments of this application, the non-specific binding region of the first antibody can be oriented to the luminescent microspheres via a first Cater-Tag directional subsystem; one of the specific binding pair members can be oriented to the non-specific binding region of the second antibody via a second Cater-Tag directional subsystem.

[0116] At this point, the first and second Catcher-Tag orientation subsystems can be the same or different; preferably, they are different. Using different types of Catcher-Tag orientation subsystems for the coated end of the first antibody and the labeled end of the second antibody can reduce cross-interference between the two systems in the detection reagent, avoid generating additional background signals that affect the accuracy of the detection results, and further improve detection performance.

[0117] In some embodiments of this application, each second antibody may be linked to two second Catcher-Tag targeting subsystems, with the linking sites all located in the Fc region of the second antibody. This minimizes the amount of free antibody in the detection reagent, improves antibody utilization, fully leverages the ability of the first and second antibodies in the detection reagent to specifically recognize target molecules, and enhances photochemiluminescence detection performance to achieve the effect of detecting extremely low-value samples.

[0118] In some embodiments of this application, a specific binding pair refers to a pair of substances capable of specifically binding to each other, such as an enzyme-substrate pair, an antigen-antibody pair, or a ligand-receptor pair. A specific example of a specific binding pair suitable for this application is the biotin-avidin system. Therefore, one of the specific binding pairs described in this application, capable of being linked to the non-specific binding region of a second antibody via an isopeptide bond, can be biotin or an activated biotin derivative; preferably a biotin derivative; more preferably Biotin-PEG-NHs.

[0119] The photo-induced chemiluminescence detection reagent provided in this application includes reagent 1 and reagent 2. Reagent 1 includes luminescent microspheres and a first antibody coated on its surface that can specifically recognize the target molecule to be tested. Reagent 2 includes a second antibody that can specifically recognize the target molecule to be tested and one of the specific binding pair members labeled therein. The first antibody and / or the second antibody are oriented to couple their non-specific binding regions to the target substance through isopeptide bonds.

[0120] The photo-induced chemiluminescence detection reagent described in this application is suitable for the detection of the low-abundance protein p-tau 217. The photo-induced chemiluminescence detection reagent for p-tau 217 provided in this application includes reagent 1 and reagent 2. Reagent 1 includes luminescent microspheres and a first antibody coated on its surface that specifically recognizes p-tau 217. Reagent 2 includes a second antibody that specifically recognizes p-tau 217 and one of its specific binding pair members labeled therein. The first antibody and / or the second antibody directionally couple their non-specific binding regions to the target substance via isopeptide bonds.

[0121] Specifically, the non-specific binding region of the first antibody in reagent 1 is directionally coated onto the luminescent microspheres using a first Catcher-Tag directional system, and / or, the non-specific binding region of the second antibody in reagent 2 is directionally labeled with one of the specific binding pair members using a second Catcher-Tag directional system. The first and second Catcher-Tag directional systems are preferably different Catcher-Tag systems. The directional coating of the first antibody in reagent 1 and the directional labeling of the second antibody in reagent 2 are performed in the same manner as described above.

[0122] In some embodiments of this application, the detection reagent further includes a universal solution comprising photosensitive microspheres and a substance coated therein corresponding to one of the specific binding pairing members. In embodiments of this application, one of the specific binding pairing members labeled on the second antibody may be selected from biotin or a derivative thereof, and one of the specific binding pairing members coated on the photosensitive microspheres may be avidin, which is capable of specifically binding to biotin or a derivative thereof.

[0123] The photo-induced chemiluminescence detection kit provided in this application includes reagent 1, reagent 2 and a universal solution, wherein reagent 1, reagent 2 and the universal solution are as described above.

[0124] The photocatalytic chemiluminescence detection kit described in this application is suitable for the detection of the low-abundance protein p-tau 217. The p-tau 217 photocatalytic chemiluminescence detection kit provided in this application includes reagent 1, reagent 2, and a universal solution, wherein the first antibody in reagent 1 and the second antibody in reagent 2 can specifically recognize and bind to p-tau 217.

[0125] The detection methods, reagents, and kits described above can all be used to detect p-tau 217 in blood samples.

[0126] The technical solution provided in this application improves the performance of photo-induced chemiluminescence detection by introducing directional coupling, enabling the sensitivity of photo-induced chemiluminescence detection of target molecules to reach the femtogram level, which can meet the detection requirements of low-abundance proteins such as p-tau 217 in blood samples.

[0127] The researchers of this invention have creatively discovered that this method can increase the amount of effective antibodies in photochemiluminescence detection reagents that can specifically recognize the target molecules, and can also maximize the preservation of the activity of the capture antibody (i.e., the primary antibody) and the detection antibody (i.e., the secondary antibody) in the detection reagent, thereby significantly improving the performance of photochemiluminescence detection. Furthermore, the detection cost is low, the detection procedure is simple, and it can rapidly achieve large-scale, high-throughput sample detection, which has significant clinical application value.

[0128] The inventors of this application also discovered in their research that in a photocatalytic chemiluminescence analysis platform, when the target analyte concentration is extremely low, such as p-tau 217 in plasma, the molar amount of the luminescent microspheres is much higher than that of the target analyte. However, in most cases, the sandwich immune complexes in the reaction system are formed by bridging a luminescent microsphere with a photosensitive microsphere through a target analyte molecule. During reading, the photosensitive microspheres generate a large amount of... 1 Only a small portion of the O2 was received by the adjacent luminescent microspheres and generated light signals; the rest was not received. 1 O2 is quenched within 4 μs. Therefore, how to improve... 1 The utilization rate of O2 is crucial for improving the sensitivity of photochemiluminescence detection, especially for low-value samples such as plasma containing p-tau 217. Increasing the particle size of the luminescent microspheres can allow them to receive more O2. 1O2 generates a stronger signal, but at the same time, it reduces the specific surface area and slows down particle migration rate, directly affecting the efficiency of the immune response and reducing detection sensitivity. The inventors of this application considered and designed a method to improve reagent performance by increasing the utilization rate of free luminescent microspheres in the detection reagent, providing an improved... 1 A new way to improve detection sensitivity by increasing the utilization rate of O2.

[0129] The photo-induced chemiluminescence detection method provided in this application involves mixing and incubating reagents R1, R2, and R4 with the sample to be tested, and then adding reagent R3 to aggregate at least two luminescent microspheres into a polymeric luminescent microsphere.

[0130] The reagent R1 comprises luminescent microspheres and a first antibody coated with a first tag molecule; the reagent R2 comprises a second antibody coated with a second tag molecule; the reagent R3 comprises a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; the reagent R4 comprises photosensitive microspheres and a second pairing molecule coated with the photosensitive microspheres capable of specifically binding to the second tag molecule; the first antibody and the second antibody are capable of specifically binding to different epitopes of the target molecule to be tested, and one first pairing molecule can bind to at least two first tag molecules, so that at least two luminescent microspheres combine to form a polymeric luminescent microsphere.

[0131] In this application, the first and second antibodies in reagents R1 and R2 can react with the target molecule to form a sandwich immune complex. The first pairing molecule in reagent R3 can form a specific tagging system with the first tag molecule on the luminescent microspheres in reagent R1. This aggregates free luminescent microspheres (those where the first antibody has not specifically bound to the target molecule) onto the main luminescent microspheres (those where the first antibody has specifically bound to the target molecule), increasing the effective particle size and specific surface area of ​​the luminescent microspheres, thus enhancing the photosensitive microsphere production. 1 Increased O2 utilization and quenching 1 The reduction in O2 leads to a stronger light signal. Simultaneously, the particle size and mass of each luminescent microsphere in reagent R1 remain unchanged, maintaining a good particle migration rate and ensuring the efficiency of the antigen-antibody immune reaction. This improves overall detection sensitivity and enhances the detection performance of low-value samples, enabling high-performance detection of femtogram-level biomarkers such as p-tau 217 in plasma.

[0132] The number of luminescent microspheres in the polymeric luminescent microspheres described in this application is equal to or greater than two, and no limitation is made herein.

[0133] In some embodiments of this application, reagent R3 may further include luminescent microspheres, i.e., the first pairing molecule is also coated on the luminescent microspheres. When reagents R1 and R3 are mixed, the specific recognition and binding ability between the first tag molecule and the first pairing molecule can promote the aggregation of the luminescent microspheres of reagents R1 and R3 to form polymeric luminescent microspheres.

[0134] In some embodiments of this application, the specific tagging system composed of the first tag molecule and the first pairing molecule can be selected from the Catcher-Tag polymer subsystem, and any combination from the system can be selected as the first tag molecule in reagent R1 and the first pairing molecule in reagent R3. In this application, the first pairing molecule can also be referred to as a tool protein.

[0135] In some embodiments of this application, in the Catcher-Tag polymer subsystem, the first tag molecule may be selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag, and the first pairing molecule is selected from any Catcher multimer protein. Preferably, the first tag molecule and the first pairing molecule may be selected from specific tag systems such as Spy-Tag and Spy-Catcher, His-Tag and Anti-His, Snoop-Tag and Snoop-Catcher, etc.

[0136] In some embodiments of this application, the multimeric protein of the Catcher has a degree of polymerization of not less than 2, for example, it can be selected from the octamer protein of the Catcher. The degree of polymerization of the Catcher directly affects the rate at which the Catcher captures tags, and thus affects the aggregation rate of the luminescent microspheres. The higher the degree of polymerization of the Catcher, the higher the tag capture rate, the higher the aggregation rate of the luminescent microspheres, and the better the effect on improving the light signal intensity.

[0137] In some embodiments of this application, the molar amount of the first pairing molecule is less than or equal to the molar amount of the first tag molecule. Preferably, the molar ratio of the first pairing molecule to the first tag molecule is 1:(1-16); more preferably, it is 1:(2-8). The proportion of the first pairing molecule directly affects the aggregation of the luminescent microspheres. If it is too low, the aggregation efficiency and degree will be low, and the effect on improving the detection performance will not be obvious; if it is too high, it is easy to produce a "hook effect", which makes it difficult for the luminescent microspheres to aggregate, resulting in no significant improvement in detection performance, and even affecting the accuracy of the detection results.

[0138] When the first tag molecule and the first pairing molecule are selected from the Catcher-Tag polymer subsystem, the higher the degree of polymerization of the first pairing molecule polymer Catcher, the lower the required molar amount of the first pairing molecule; that is, the degree of polymerization of the first pairing molecule is negatively correlated with its optimal molar amount. Preferably, when the product of the degree of polymerization of the first pairing molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, the effect on improving the light signal intensity of the luminescent microspheres is more significant.

[0139] The degree of polymerization and amount of the first paired molecule can be determined based on the amount of the first tag molecule. A higher degree of polymerization of the first paired molecule results in a lower required optimal molar amount and a faster reaction rate with the first tag molecule; conversely, a lower degree of polymerization of the first paired molecule results in a higher required optimal molar amount and a faster aggregation rate. When the product of the degree of polymerization of the first paired molecule and its molar amount is 1 to 2.5 times the molar amount of the first tag molecule, it significantly enhances the light signal intensity of the luminescent microspheres. Excessive amounts can easily lead to the hook effect, affecting the detection results; insufficient amounts affect the aggregation rate and degree of aggregation of the polymeric luminescent microspheres, resulting in minimal performance improvement.

[0140] In some embodiments of this application, a first tag molecule is co-expressed with a first antibody and linked to the non-specific binding region of the first antibody to form a first antibody with the first tag molecule. The co-expression linking can be achieved by linking the first tag molecule to the non-specific binding region of the first antibody using pClick technology (see reference "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18):9107-9117. doi:10.7150 / thno.62444.)), or by inserting the sequence of the first tag molecule into the non-specific binding region of the first antibody.

[0141] In some embodiments of this application, the first antibody has a Y-shaped structure, including two Fab segments and one Fc segment, with the non-specific binding region of the first antibody being its Fc terminus. In some embodiments of this application, the first antibody has a V-shaped structure, including two Fab segments, with the non-specific binding region of the first antibody being its Fab-linked hinge region. When the CDR region of the first or second antibody is occupied or the Fab terminus is folded during coating, labeling, or other steps, the Fab terminus of the antibody may not be effectively displayed, affecting the effective amount of antibody or antibody activity that can bind to the target molecule such as p-tau 217 in the reaction system, thereby affecting the photochemiluminescence detection performance. The first antibody formed by linking the first tag molecule to the non-specific binding region of the first antibody allows the CDR region, the site where a specific binding immune reaction with the target molecule can occur, to be fully and effectively displayed, thereby increasing the effective amount of antibody and antibody activity that can bind to the target molecule in the reaction system, and improving the sensitivity of photochemiluminescence detection.

[0142] Catcher and Tag exhibit excellent specific recognition and binding capabilities. Furthermore, the use of a small molecular weight Tag tag to bind to the primary antibody and co-coating it onto the luminescent microspheres has no significant impact on the expression of the primary antibody or the properties of the luminescent microspheres.

[0143] In some embodiments of this application, the mass ratio of luminescent microspheres to the first antibody is 10:(0.05-5); preferably 10:(0.1-1); more preferably 10:0.5. The amount of the first antibody coated on the luminescent microspheres affects the reaction efficiency between the first antibody and the target molecule in the sample to be tested, thereby affecting the discrimination of the detection results.

[0144] In some embodiments of this application, the molar ratio of the second antibody to the second tag molecule is 1:(20-50); preferably 1:(25-35); more preferably 1:30. The amount of the second tag molecule on the second antibody affects its reaction efficiency with the photosensitive microspheres, thereby affecting the discrimination of the detection results.

[0145] This application utilizes the specific binding ability of a second pairing molecule and a second tag molecule on photosensitive microspheres to enable the second antibody to bind to the photosensitive microspheres, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres, and enabling the photosensitive microspheres to produce... 1 O2 can be effectively received by the luminescent microspheres and generate light signals, meeting the requirements of photo-induced chemiluminescence detection.

[0146] In some embodiments of this application, the two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, the second tag molecule and the second pairing molecule, do not react with each other, thereby avoiding the occurrence of specific pairing interference reactions and improving the accuracy and stability of the detection results.

[0147] The second tag molecule and the second pairing molecule in reagents R2 and R4 can be selected from the biotin-avidin system, for example, one of which is selected from biotin and the other is selected from avidin or streptavidin; preferably, avidin is coated on the photosensitive microspheres, and the second antibody is bound to streptavidin, thereby labeling the second antibody with biotin.

[0148] In some embodiments of this application, 15 μL of reagent R1, 15 μL of reagent R2, and 175 μL of reagent R4 can be mixed with 100 μL of the sample to be tested and incubated before adding 15 μL of reagent R3 to initiate the reaction. That is, in the photochemiluminescence detection process, by controlling the volume ratio of reagent R1, reagent R2, and the sample to be tested to 3:3:20, the volume ratio of reagent R2 to reagent R4 to 3:35, and the volume ratio of reagent R1 to reagent R3 to 1:1, the reagent volume can be reduced, the reagent concentration increased, and the sample proportion increased. This shifts the reaction equilibrium towards the binding direction, which is beneficial for increasing the amount of the formed luminescent microsphere-target molecule-photosensitive microsphere sandwich immune complex, thereby improving detection performance.

[0149] Furthermore, reagents R1 and R2 can be mixed with the sample to be tested, incubated for a period of time, then reagent R4 can be added, and after incubation for a period of time, reagent R3 can be added.

[0150] In some embodiments of this application, the method of use includes:

[0151] S1. Mix reagents R1 and R2 with the sample to be tested and incubate at 37°C to obtain the first reactant;

[0152] S2, add reagent R4, incubate at 37°C to obtain the second reactant;

[0153] S3, add reagent R3, incubate at 37°C to obtain the third reactant;

[0154] S4. Irradiate the third reactant with light of a specific wavelength and detect the luminescence value; and determine whether the sample contains the target molecule or the content of the target molecule in the sample based on the intensity of the light signal.

[0155] This application first combines luminescent microspheres and their coated first antibody with a first tag molecule and a second antibody with a second tag molecule with the target molecule in the sample to form a sandwich immune complex of "first antibody-target molecule-second antibody". Then, the target luminescent microsphere is immobilized on the photosensitive microsphere by the specific binding of the second tag molecule on the second antibody with the second pairing molecule coated on the photosensitive microsphere to obtain the "luminescent microsphere-target molecule-photosensitive microsphere" complex. Finally, the free luminescent microsphere is bound to the target luminescent microsphere by the specific recognition and binding ability of the first pairing molecule and the first tag molecule on the first antibody to form a polymeric luminescent microsphere.

[0156] After the conventional reaction is completed, the free luminescent microspheres aggregate around the composite luminescent microspheres, causing the photosensitive microspheres to produce... 1 O2 can be received by multiple luminescent microspheres in the polymer luminescent microspheres and simultaneously generate light signals, effectively improving... 1 This approach improves O2 utilization, enhances signal intensity, and thus increases detection sensitivity, meeting the requirements for photochemiluminescence detection of low-value samples. Furthermore, the specific tagging system used in this scheme, namely the first tag molecule and the first paired molecule, employs the Catcher-Tag polymer subsystem. This system boasts high specificity and affinity, significantly improving reagent performance without interfering with the original immunoreaction. It is highly applicable and has a wide range of applications, representing a platform-type improvement. Particularly when used for detecting p-tau 217 in extremely low-value samples such as plasma, it significantly enhances detection sensitivity, meeting the needs of detecting extremely low-value samples. Simultaneously, the detection method is simple, easy to operate, and low in cost.

[0157] In some embodiments of this application, the reaction time after adding reagent R3 is 0–15 min, that is, the aggregation time of the luminescent microspheres is 0–15 min. As the aggregation time increases, the aggregation reaction of the luminescent microspheres gradually increases and tends to equilibrium. Therefore, the aggregation time is preferably 3–9 min, more preferably 5–7 min.

[0158] In some embodiments of this application, when using each detection reagent, the concentration of luminescent microspheres in reagent R1 is 75–100 μg / mL, preferably 80–90 μg / mL, and more preferably 83.3 μg / mL. The concentrations of the luminescent microspheres and the first antibody affect the efficiency of their immunoreaction with the target molecule, thereby affecting the discrimination of the detection results. The concentration of the second antibody in reagent R2 is 1–10 μg / mL, preferably 2–5 μg / mL, and more preferably 3.3 μg / mL. The concentration of the second antibody affects its immunoreaction efficiency with the target molecule and its ability to resist biotin interference, thereby affecting the discrimination of the detection results.

[0159] The photoluminescence detection reagent provided in this application includes the aforementioned R1, R2, and R3 reagents. The R1 reagent comprises luminescent microspheres and a first antibody coated with a first-tagged molecule; the R2 reagent comprises a second antibody coated with a second-tagged molecule; the R3 reagent comprises a first pairing molecule capable of specifically recognizing and binding to the first-tagged molecule; and the R4 reagent comprises photosensitive microspheres and a second pairing molecule coated with the photosensitive microspheres capable of specifically binding to the second-tagged molecule. The first and second antibodies can specifically bind to different epitopes of the target molecule, and one first pairing molecule can bind to at least two first-tagged molecules, so that at least two luminescent microspheres combine to form a polymeric luminescent microsphere.

[0160] The photo-induced chemiluminescence detection kit described in this application includes the aforementioned reagents R1, R2, R3, and R4.

[0161] The photoluminescence detection reagent provided in this application embodiment is suitable for the detection of p-tau 217. The photoluminescence detection reagent for p-tau 217 described in this application embodiment includes reagents R1, R2, R3, and R4. The first antibody in reagent R1 and the second antibody in reagent R2 are capture and detection antibodies that specifically bind to different epitopes of p-tau 217, respectively. Reagent R3 is a reagent containing a polycatcher from the aforementioned Catcher-Tag polymer subsystem. Reagent R4 is stored separately as a universal liquid reagent.

[0162] This application first combines luminescent microspheres with a first antibody and a second antibody coated thereon that specifically recognize and bind to p-tau 217, and then binds them to the target analyte p-tau 217 in the test sample to form a sandwich immune complex of "first antibody-(p-tau 217)-second antibody". Then, the target luminescent microspheres are immobilized onto the photosensitive microspheres by the specific binding of a second tag molecule on the second antibody to a second pairing molecule coated thereon, resulting in a "luminescent microsphere-(p-tau 217)-photosensitive microsphere" complex. Finally, the free luminescent microspheres are bound to the target luminescent microspheres by the specific recognition and binding ability of the first pairing molecule and the first tag molecule, forming polymeric luminescent microspheres.

[0163] The photochemiluminescence detection kit for p-tau 217 described in this application includes the aforementioned reagents R1, R2, R3, and R4.

[0164] In some embodiments of this application, the kit further includes buffers, stabilizers, etc. The buffer may be selected from at least one of PBS buffer, Tris-hydrochloric acid buffer, HEPES buffer, and MES buffer. The stabilizer may be selected from at least one of bovine serum albumin, dextran, sorbitol, glycerol, glycine, alanine, Tween 20, and Tween 80.

[0165] The detection methods, reagents, and kits described above can all be used to detect p-tau 217 in blood samples.

[0166] The technical solution provided in this application introduces tagged antibodies into luminescent microspheres and adds pairing molecules capable of capturing the tag. The specific reaction between the pairing molecules and the tag allows the free luminescent microspheres in the reaction system to aggregate on the immune complex, improving the utilization rate of reactive oxygen species generated by the photosensitive microspheres, thereby enhancing the detection signal value and improving the overall performance of photochemiluminescence detection. This meets the sensitivity requirements for photochemiluminescence detection of p-tau 217 in low-value samples such as blood samples. Furthermore, the entire detection process does not change the conventional detection sequence, does not interfere with reagent specificity, is easy to operate, has low detection cost, and a wide range of applications, making it of significant clinical value.

[0167] To make this application easier to understand, the following will use p-tau 217 as the target molecule to be tested as an example, and combine it with embodiments to further describe this application in detail. These embodiments are for illustrative purposes only and are not limited to the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained by commercial means or conventional methods.

[0168] 1. The main experimental materials and equipment are shown in Table 1:

[0169] Table 1

[0170] Example 1: Preparation of luminescent microspheres with directionally coated antibodies and directionally labeled biotin

[0171] 1. Preparation and purification of the catcher

[0172] (1) Construction of Catcher plasmid expression vector: The expression vector of Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1) or Snoop Catcher (GenBank: KU500646.1).

[0173] (2) Catcher expression induced by E. coli: The E. coli expression vector was used to induce the expression of Catcher. The culture conditions were as follows: self-induction medium (10 g peptone, 5 g yeast extract, 1 x NPS, 1 mM MgCl2, 1 x 5052, pH = 7.4), with 100 nM ampicillin added; induction conditions: overnight culture at 30°C and 200 rpm in a shaker.

[0174] (3) Catcher purification: After overnight reaction, centrifuge at 8000 rpm and collect the bacterial pellet; after culture, centrifuge the bacterial solution at 8000 rpm for 2 minutes, discard the supernatant, and retain the bacterial cells; resuspend the bacterial cells in 100 mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20 mM ID, pH = 7.4), and after homogenization in a high-pressure homogenizer, centrifuge at 18000 rpm for 40 minutes to separate the supernatant and inclusion bodies; collect the supernatant, sonicate for 1 minute (sonication conditions: 2s sonication, 3s interval, 60% power) to disrupt the nucleic acid, and filter with a 0.22 μm needle filter after sonication; obtain Catcher protein using conventional Ni-NTA affinity chromatography gravity column purification method, purification buffer: loading supernatant (soluble protein) of solution A and equilibration buffer (50 mM PBS, 150 mM NaCl, 25 mM imidazole, pH = 7.4). 7.4) + B solution supernatant (soluble protein) elution buffer (50mM PBS, 150mM NaCl, 500mM imidazole, pH 7.4), the target protein is obtained by adjusting the imidazole concentration; the elution conditions are: 5%, 15%, 50% and 100% of solution B for fractional elution.

[0175] (4) The protein concentration of the collected portion was determined by the BCA method. Then, the portion containing protein was subjected to reduction and non-reduction electrophoresis. A collection tube that matches the molecular weight of the Catcher protein was selected and dialyzed into the subsequently labeled buffer.

[0176] Prepare the Catchers in Table 2 according to the steps described above, where Spy Catcher and Snoop Catcher are untagged protein Catchers, and Spy D\G\K\L\M Catcher and Snoop D\G\K\L\M Catcher are tagged protein Catchers.

[0177] Table 2

[0178] 2. Antibody Fc fragment linked to Tag

[0179] The Spy Tag (sequence AHIVMVDAYKPTK) or Snoop Tag (sequence KLGDIEFIKVNK) was linked to the antibody (p-tau 217 Ab1 or p-tau 217 Ab2) using pClick technology to obtain the corresponding Fc fragment-linked Tag antibody. The protein concentration of the collected fraction was determined using the BCA method, and the protein-containing fraction was then subjected to electrophoresis to obtain the Tag-antibody molecule, which was dialyzed into the buffer required for subsequent conjugation reactions.

[0180] 3. Luminescent microparticles coated with antibodies

[0181] 3.1 The luminescent microparticles were directionally coated with antibodies using the Catcher-Tag system.

[0182] 10 mg of luminescent microparticles were placed in a centrifuge tube, washed twice with 0.05 M CB buffer, and reconstituted. Catcher was dialyzed with 0.05 M CB buffer and its concentration was determined by the BCA method. The treated luminescent microparticles FG and Catcher were thoroughly mixed at a mass ratio of 10:0.8 and reacted at 37℃ for 24 hours. 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH4 (20 mg / mL, 0.05 M CB) were added to the reaction system, and the mixture was thoroughly mixed and reacted at 4℃ for 2 hours to obtain the reaction solution. The reaction solution was washed with 0.02 M PBST buffer and PBS buffer, and then reconstituted by sonication with 0.1 mL of 0.02 M PBS buffer to obtain the FG-Catcher solution. p-tau 217 Ab1-Tag was dialyzed with 0.02 M PBS buffer and its concentration was determined by the BCA method. The treated FG-Catcher and p-tau... After thoroughly mixing 217 Ab1-Tag at a mass ratio of 10:0.5, the mixture was reacted at 4℃ for 24 h. After washing twice with PBST buffer, the mixture was washed once with reconstitution solution (0.02M PBS + 0.5% BSA + 0.5% Tween 20 + 1% dextran), and then 1 mL of reconstitution solution was added and sonicated to reconstitute the mixture, thus obtaining antibody-coated luminescent microparticles FG-Ab1.

[0183] 3.2 Luminescent microparticles directly coat antibody molecules

[0184] Take 10 mg of luminescent microparticles into a centrifuge tube, wash once with 0.02 M PBS buffer, and then adjust the volume to 20 mg / mL with 0.02 M PBS. Dialyze (p-tau 217)Ab1 or (p-tau 217)Ab1-Spy Tag with 0.02 M PBS buffer and determine the concentration using the BCA method. Mix the treated luminescent microparticles FG with (p-tau 217)Ab1 or (p-tau 217)Ab1-Spy Tag at a mass ratio of 10:0.5 and react at 37 °C for 16 hours. 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. After thorough mixing, the reaction solution was reacted at 4°C for 2 hours to obtain the reaction solution. The reaction solution was washed twice with PBST buffer, washed once with reconstitution solution, and then reconstituted by sonication with reconstitution solution to obtain luminescent microparticles coated with (p-tau 217)Ab1 or (p-tau 217)Ab1-Spy Tag.

[0185] 4. Biotin-labeled antibodies

[0186] 4.1 Biotin-directed labeled antibody

[0187] Catcher was dialyzed with 0.05M NaHCO3 solution to remove impurities, and the concentration was determined by BCA method. 10 molar amounts of Biotin-PEG12-NHs were added to the dialyzed Catcher solution, mixed, and reacted at 4℃ for 24 hours to obtain Bio-Catcher solution. The solution was dialyzed with 0.02M PBS buffer to remove free Biotin-PEG12-NHs, and the concentration was determined by BCA. p-tau 217 Ab2-Tag was dialyzed with 0.02M PBS buffer, and the concentration was determined by BCA method. The treated Bio-Catcher solution and p-tau 217 Ab2-Tag solution were mixed at a molar ratio of 4:1 and reacted at 4℃ for 24 hours. The solution was purified using a Protein A column to remove free Bio-Catcher, yielding the directionally labeled biotinylate antibody Bio-Ab2.

[0188] 4.2 Biotin-labeled antibodies

[0189] The process of directly labeling antibodies with biotin is the same as the process of directional labeling antibodies using Catcher-Tag described above, the only difference being that the Catcher-Tag system is not used, and will not be described in detail here.

[0190] Example 2: Performance testing of directional coupling of the light-emitting end using a Catcher-Tag system

[0191] 1. Experimental Procedure

[0192] The luminescent microparticles obtained by directionally coating antibody Ab1 onto luminescent microparticles using 12 different types of Catcher-Tag systems, and luminescent microparticles directly coated using conventional conjugation methods (without using the Catcher-Tag system), were diluted with a reconstitution solution to a luminescent microparticle concentration of 83.3 μg / mL to obtain Reagent 1. Biotin-labeled Bio-Ab2 was diluted with a reconstitution solution to 3.3 μg / mL to obtain Reagent 2, which was then used to detect the sample on a photo-induced chemiluminescence detection system.

[0193] Mix 50 μL of the test sample, 15 μL of reagent 1, and 15 μL of reagent 2 thoroughly and incubate at 37 °C for 60 min. Add 175 μL of universal solution (containing photosensitive microparticles) and incubate at 37 °C for 15 min. After photoexcitation, read the light signal values ​​for each test group. The experimental data are shown in Tables 3 and 4 below.

[0194] 2. Experimental Results

[0195] Table 3

[0196] Table 4

[0197] Note: ① Conventional coupling; ② Spy-Catcher; ③ Spy-DCatcher; ④ Spy-GCatcher; ⑤ Spy-KCatcher; ⑥ Spy-LCatcher; ⑦ Spy-MCatcher; ⑧ Snoop-Catcher; ⑨ Snoop-DCatcher; ⑩ Snoop-GCatcher; Snoop-KCatcher; Snoop-LCatcher; Snoop-MCatcher.

[0198] 3. Experimental Data Analysis

[0199] As shown in Tables 3 and 4, after the luminescent end (luminescent microparticles) was coated with antibodies using the Catcher-Tag system for directional conjugation, the positive signal and discrimination were significantly improved, enabling the detection of the picogram or even femtogram level marker p-tau 217. The improvement was further amplified when the Catcher was tagged with proteins (such as Spy D\G\K\L\M Catcher and Snoop D\G\K\L\M Catcher), with a significant increase in both positive signal and discrimination. Specifically, when the Catcher molecular weight was between 12 and 55 kDa, both the positive signal and discrimination were improved compared to conventional conjugation methods, with a more significant improvement observed in the 30-40 kDa range. The optimal Catcher molecular weight for detection was around 38 kDa.

[0200] As shown in Tables 3 and 4, the detection of recombinant antigens and actual samples was significantly improved after the luminescent end (luminescent microparticles) was directionally coated with antibodies using the Catcher-Tag system.

[0201] Therefore, by directionally conjugating antibodies to the luminescent end, the effective antibody amount on the luminescent microparticle FG-Ab1 is increased, the positive signal and discrimination are significantly improved, and the detection capability reaches the femtogram level, which is suitable for the clinical detection of p-tau 217.

[0202] Example 3: Performance testing of directional coupling at the tag end using a Catcher-Tag system

[0203] 1. Experimental Procedure

[0204] Biotinylated antibodies obtained by biotin-directed labeling of antibody Ab2 using 12 different types of Catcher-Tag systems were diluted with reconstitution solution to a Bio-Ab2 concentration of 4.6 μg / mL. Biotinylated antibodies directly labeled using conventional conjugation methods (without using the Catcher-Tag system) were diluted with reconstitution solution to 3.3 μg / mL to prepare reagent 2 (the amount of Ab2 in each reagent 2 is approximately the same). Luminescent microparticles directly coated with antibody Ab1 using conventional processes were diluted with reconstitution solution to 83.3 μg / mL to obtain reagent 1, which was then used to detect the sample in a photo-induced chemiluminescence detection system.

[0205] Mix 50 μL of the test sample, 15 μL of reagent 1, and 15 μL of reagent 2 thoroughly and incubate at 37 °C for 60 min. Add 175 μL of universal solution (containing photosensitive microparticles) and incubate at 37 °C for 15 min. After photoexcitation, read the light signal values ​​for each test group. The experimental data are shown in Tables 5 and 6 below.

[0206] 2. Experimental Results

[0207] Table 5

[0208] Table 6

[0209] Note: ① Conventional coupling; ② Spy-Catcher; ③ Spy-DCatcher; ④ Spy-GCatcher; ⑤ Spy-KCatcher; ⑥ Spy-LCatcher; ⑦ Spy-MCatcher; ⑧ Snoop-Catcher; ⑨ Snoop-DCatcher; ⑩ Snoop-GCatcher; Snoop-KCatcher; Snoop-LCatcher; Snoop-MCatcher.

[0210] 3. Experimental Data Analysis

[0211] As shown in Tables 5 and 6, antibody labeling using the Cater-Tag system for targeted conjugation at the biotin end significantly improved the positive signal and discrimination, enabling the detection of p-tau 217 markers at the picogram or even femtogram level. The improvement was further amplified when the Cater was labeled with a tagged protein, resulting in a significant increase in both positive signal and discrimination. Specifically, when the molecular weight of the tagged Cater was between 12 and 55 kDa, both the positive signal and discrimination were improved compared to conventional conjugation. However, at larger molecular weights (e.g., ≥53 kDa), Ab2 could only conjugate one Cater, leading to a decrease in the amount of biotin on the immunoreaction product and a decline in detection capability. The number of lysine residues directly affected the biotin labeling amount; the greatest improvement in detection performance was observed when the number of lysine residues was greater than 5, especially ≥23, after targeted labeling of the antibody at the label end.

[0212] After using the Catcher-Tag system to directionally label antibodies at the labeling end, the detection of recombinant antigens and actual samples was significantly improved.

[0213] Example 4: Labeled end protein reduction electrophoresis detection

[0214] Electrophoresis experiments were performed on the directionally labeled Bio-Ab2 prepared in Example 1 above, and the following results were obtained (restored electrophoretic heavy chain band distribution):

[0215] Antibody protein molecules labeled with the above-mentioned ②Spy-Catcher or ⑧Snoop-Catcher contain two heavy chain bands: 50kDa (heavy chain) and 63kDa (heavy chain + Bio-Catcher).

[0216] Using the aforementioned tagged protein Catchers, such as DCatcher, GCatcher, KCatcher, and LCatcher, the corresponding targeted labeling antibody protein molecules all have only one heavy chain band: approximately 90 kDa (heavy chain + BioCatcher).

[0217] Using the aforementioned tagged protein Catcher—MCatcher, the corresponding targeted labeling antibody protein molecule has two heavy chain bands: 50kDa (heavy chain) and 90kDa (heavy chain + Bio Cacther).

[0218] The results showed that among the catchers containing tagged proteins, DCatcher, GCatcher, KCatcher, and LCatcher had better performance. The molecular weight of the catcher had a consistent effect on improving detection performance as in the label-terminal directional labeling antibody experiment of Example 3.

[0219] Therefore, it can be inferred that the labeling end reduces the loss of Ab2 activity by directionally conjugating antibodies, thereby increasing the amount of effective Ab2 antibodies, significantly improving the positive signal and discrimination, and achieving femtogram-level detection capability, which is suitable for clinical detection of low-value samples such as p-tau 217.

[0220] Example 5: Both the light-emitting end and the tag end are directionally coupled using a Catcher-Tag system.

[0221] 1. Experimental Procedure

[0222] The luminescent microparticles FG-Ab1, which were directionally coated with Catcher as described in Example 2, were diluted to 83.3 μg / mL with a reconstitution solution (Reagent 1). The biotinylated antibody Bio-Ab2, which was directionally labeled as described in Example 3, was diluted to 4.6 μg / mL with a reconstitution solution (Reagent 2). Both were then detected on a photo-induced chemiluminescence detection system with the sample to be tested.

[0223] Mix 50 μL of sample buffer, 15 μL of reagent 1, and 15 μL of reagent 2, and incubate at 37 °C for 60 min. Add 175 μL of universal solution (containing photosensitive microparticles) and incubate at 37 °C for 15 min. After photoexcitation, read the light signal value for each test group. The experimental data are shown in Table 7 below.

[0224] 2. Experimental Results

[0225] Table 7. Background signals of the experimental group

[0226] 3. Experimental Data Analysis

[0227] As shown in Table 7, compared with using two different types of Catcher-Tag systems, the background signal of the light-emitting end and the tag end using the same type of Catcher-Tag system is significantly improved and non-specific adsorption is serious.

[0228] Example 6: The light-emitting end and the tag end are directionally coupled using different Catcher-Tag systems.

[0229] 1. Experimental Procedure

[0230] The two pairs of combinations that showed the most significant signal value enhancement in Experiments 2 and 3 were selected for the experiment, and the experimental results are shown in Tables 8 and 9.

[0231] 2. Experimental Results

[0232] Table 8

[0233] Table 9

[0234] 3. Experimental Data Analysis

[0235] As shown in Tables 8 and 9, when both the luminescent end and the labeling end are directionally conjugated with antibodies using the Catcher-Tag system, the positive signal value and discrimination are significantly improved; and the detection of recombinant antigens and actual samples are also significantly improved.

[0236] As shown in Tables 8 and 9, selecting two different Cater-Tag systems for the luminescent and labeled ends yields better results in improving positive signal values ​​and discrimination, and can increase the detection sensitivity of p-tau 217 by 1 to 2 orders of magnitude (Dafiq level) compared to before optimization, meeting the detection requirements of p-tau 217 in low-value samples such as plasma.

[0237] Therefore, using two different Catcher-Tag systems alternately at the light-emitting end and the tagging end can reduce cross-reaction and reduce the impact of background signals on the detection results.

[0238] Example 7: The Influence of Catcher Polymerization Degree and Usage Ratio on Photochemiluminescence Detection Performance

[0239] 1. Experimental Procedure

[0240] 1.1 The luminescent microparticles FG-Ab1 directly coated with Tag-antibody molecules as described in Example 1, 3.2 above were diluted with a reconstitution solution to 83.3 μg / mL to obtain reagent R1; the biotinylated antibody Bio-Ab2 directly labeled as described in Example 1, 4.2 above was diluted with a reconstitution solution to 4.6 μg / mL to obtain reagent R2.

[0241] Dimers, pentamers, and octamers of Spy Catcher were prepared according to the method described in Example 1, and then diluted with a reconstituted solution to reagent R3 at a Tag molar ratio of 1:16, 1:8, 1:4, 1:2, 1:1, and 2:1, respectively. These were then mixed with a universal photosensitive microparticle solution, reagent R4. The solutions were then detected using a photo-induced chemiluminescence detection system with the sample to be tested.

[0242] 1.2 Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of universal solution, and incubate at 37 °C for 15 min; add 15 μL of R3 reagent, and add 15 μL of reconstitution solution to the control group, and incubate at 37 °C for 15 min respectively; after photoexcitation reaction, read the light signal value of each group. The experimental data are shown below.

[0243] 2. Experimental Results

[0244] 2.1 Spy Catcher dimer reaction effect:

[0245] Table 10 Signal Values

[0246] Table 11 Discrimination Index

[0247] 2.2 Spy Catcher pentamer reaction effect:

[0248] Table 12 Signal Values

[0249] Table 13 Discrimination Index

[0250] 2.3 Spy Catcher Octamer Reaction Effect:

[0251] Table 14 Signal Values

[0252] Table 15 Discrimination Index

[0253] 3. Results Analysis

[0254] ① As shown in Table 10-15, within the current gradient range, the polymer particle size (degree of polymerization) does not affect the particle size at which the luminescent microparticles receive singlet oxygen. The higher the degree of polymerization of the first paired molecule corresponding to the first tag molecule, the better the improvement effect. When the first paired molecule is a Spy Catcher octamer, the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more.

[0255] ②As shown in Table 10-15, when the molar ratio of Spy Tag in Spy Catcher and R1 reagent is 1:(16~1), both the positive signal and the discrimination are improved; among them, the molar ratio of 1:4 (Spy Catcher octamer: Spy Tag) has the best effect, at which the positive signal and discrimination are significantly improved, and the detection performance of low-value samples is improved even more.

[0256] ③ The degree of polymerization and the proportion of the first paired molecule depend on the molecular weight of the first tag. The molar amount of the first paired molecule should be lower than or equal to the molar amount of the first tag molecule, and the degree of polymerization of the first paired molecule is negatively correlated with the optimal molar amount. As shown in Table 2-7, when the product of the degree of polymerization and the molar amount of the first paired molecule is 1 to 2.5 times the molar amount of the first tag molecule, the positive signal and discrimination are significantly improved.

[0257] ④ As shown in Table 10-15, using Spy Tag as a tag and Spy Catcher as a pairing molecule significantly improves the positive signal and discrimination for detecting p-tau 217 recombinant antigen and actual samples.

[0258] Therefore, using a specific tagging system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres can improve the positive signal and discrimination, and significantly enhance the sensitivity of photo-induced chemiluminescence detection, thus meeting the detection requirements of p-tau 217 in low-value samples such as blood samples.

[0259] Example 8: Effect of aggregation time on the photochemiluminescence detection performance of p-tau 217

[0260] 1. Experimental Procedure

[0261] 1.1 The Spy Catcher octamer described in Example 7 was diluted with a complex solution to form reagent R3, with a Tag molar ratio of 1:4 to that in reagent R1. This was then mixed with the universal photosensitive microparticle solution reagent R4. The solution was then used to detect the sample on a photo-induced chemiluminescence detection system.

[0262] 1.2 Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min; add 175 μL of universal solution, and incubate at 37 °C for 15 min; add 15 μL of R3 reagent, and add 15 μL of reconstituted solution to the control group, and incubate at 37 °C for 3, 6, 9, 12, and 15 min, respectively; after photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0263] 2. Experimental Results

[0264] Table 16 Signal Values

[0265] Table 17 Discrimination Index

[0266] 3. Results Analysis

[0267] As shown in Table 16-17, with the addition of reagent R3 (containing the first pairing molecule) and the extension of reaction time, the positive signal and discrimination gradually increased and tended to reach equilibrium; when the reaction reached about 6 minutes, the aggregation reaction of free luminescent particles reached equilibrium.

[0268] Therefore, in the process of using a specific tagging system such as Spy Catcher-Tag to aggregate free luminescent microspheres into a complex to form polymeric luminescent microspheres, from the perspective of balancing reaction time and performance, the reaction time after adding reagent R3 can be 0 to 15 min, with the preferred reaction time being 3 to 9 min and the optimal reaction time being 5 to 7 min.

[0269] Example 9: Applicability verification of different labeling systems for forming "multi-luminescent microspheres"

[0270] 1. Experimental Procedure

[0271] 1.1 Following the method described in Example 1, 3.2 above for coating Tag-antibody molecules with luminescent microspheres, luminescent microspheres coated with (p-tau 217)Ab1 carrying different tag molecules (Spy-Tag, His-Tag, Snoop-Tag) were prepared and diluted to 83.3 μg / mL with a reconstitution solution to obtain reagent R1.

[0272] 1.2 Bio-(p-tau 217)Ab2 was diluted to 3.3 μg / mL with a reconstitution solution to obtain reagent R2.

[0273] 1.3 The first paired molecules (octamers Spy-Catcher, Anti-His, and Snoop-Catcher corresponding to the tag molecules) are diluted with a reconstituted solution to reagent R3 in molar ratios of 1:16, 1:4, and 1:1 with the tag molecules in reagent R1.

[0274] 1.4 The sample to be tested is detected using a photo-induced chemiluminescence detection system:

[0275] Mix 100 μL of the test sample, 15 μL of R1 reagent, and 15 μL of R2 reagent, and incubate at 37 °C for 60 min. Add 175 μL of universal photosensitive microparticle solution and incubate at 37 °C for 15 min. Add 15 μL of R3 reagent, and add 15 μL of reconstitution solution to the control group, and incubate at 37 °C for 15 min respectively. After photoexcitation reaction, read the light signal value of each test group. The experimental data are shown below.

[0276] 2. Experimental Results

[0277] Table 18 Signal Values

[0278] Table 19 Discrimination Index

[0279] 3. Results Analysis

[0280] As shown in Tables 18-19, different specific tagging systems such as Spy-Catcher / Spy-Tag, His-Tag / Anti His-Ab, and Snoop-Tag / Catcher can all improve the positive signal value and low-discrimination resolution to varying degrees.

[0281] Therefore, the specific labeling system described in this application has a good effect on the aggregation of free luminescent microspheres into polymeric luminescent microspheres to improve detection performance, and the photo-induced chemiluminescence detection sensitivity is effectively improved, which can meet the detection requirements of p-tau 217 in low-value samples such as blood samples.

[0282] The embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Any modifications, equivalent substitutions, improvements, etc., made without departing from the scope and spirit of the described embodiments should be included within the scope of protection of this invention. The terminology used herein is chosen to best explain the principles, practical applications, or improvements to the technology in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method of photochemiluminescence detection, characterized in that The sample to be tested is brought into contact with a reaction system containing a first antibody and a second antibody to obtain an immune reaction product, and the light signal intensity of the immune reaction product is detected. The first antibody and / or the second antibody are coupled to the target substance via isopeptide bonds, targeting their non-specific binding regions.

2. The detection method according to claim 1, characterized in that, The first antibody and / or the second antibody form isopeptide bonds through the Catcher-Tag directional subsystem to directionally couple them to the target substance.

3. The detection method according to claim 2, characterized in that, In the Catcher-Tag targeting subsystem, the Catcher is a monomeric Catcher; preferably, the monomeric Catcher carries a tag protein.

4. The detection method according to claim 3, characterized in that, The molecular weight of the monomer Catcher is 12–55 kDa; Preferably, the monomer Catcher contains lysine residues; Preferably, the number of lysine residues in the monomer Catcher is ≥5.

5. The detection method according to claim 3, characterized in that, In the Catcher-Tag directing subsystem, the monomer Catcher is coated on the surface of the luminescent microspheres, and the Tag is linked to the non-specific binding region of the first antibody. And / or, in the Catcher-Tag directional subsystem, the monomeric Catcher is labeled with one of the specific binding pair members, and the Tag is linked to the non-specific binding region of the second antibody.

6. The detection method according to any one of claims 2 to 5, characterized in that, The non-specific binding region of the first antibody is directionally connected to the luminescent microspheres via a first Catcher-Tag directional subsystem. One of the specific binding pair members is directionally linked to the non-specific binding region of the second antibody via a second Catcher-Tag directional subsystem; Preferably, the first Catcher-Tag orientation subsystem and the second Catcher-Tag orientation subsystem are different.

7. The assay method according to any one of claims 2 to 6, characterized in that, Each of the second antibodies is linked to two second Catcher-Tag targeting subsystems, both located in the Fc region of the second antibody.

8. A method of photochemiluminescent detection, characterized in that After mixing and incubating reagents R1, R2, and R4 with the sample to be tested, reagent R3 is added to aggregate at least two luminescent microspheres into multiple luminescent microspheres. The R1 reagent comprises luminescent microspheres coated with a first antibody capable of specifically binding to the target molecule to be tested, the first antibody carrying a first tag molecule. The R2 reagent contains a second antibody with a second tag molecule, which can specifically bind to the target molecule to be tested. The R3 reagent contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; The R4 reagent comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second tag molecule; One of the first pairing molecules can bind to at least two first tag molecules.

9. The detection method according to claim 8, characterized in that, The first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

10. The detection method of claim 8, wherein, The first tag molecule and the first pairing molecule are selected from the Catcher-Tag polymer subsystem; preferably, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag; more preferably, the first pairing molecule is a polyCatcher with a degree of polymerization of not less than 2.

11. The detection method of claim 8, wherein, The molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule; preferably, the molar ratio of the first paired molecule to the first tag molecule is 1:(1-16); more preferably, it is 1:(2-8).

12. The detection method of claim 8, wherein, The first paired polymer molecules are negatively correlated with their optimal molar amount; preferably, the product of the degree of polymerization of the first paired molecules and their molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

13. The method of claim 8, wherein, The number of moles of the luminescent microspheres is greater than the number of moles of the target molecules in the sample to be tested.

14. The method of claim 8, wherein, The two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, the second tag molecule and the second pairing molecule, do not react with each other.

15. The method of claim 8, wherein, The reaction time after adding reagent R3 is 0–15 min.

16. A photochemiluminescent detection reagent, characterized by The reagent includes a reaction reagent containing a first antibody and a second antibody, both of which are capable of specifically binding to the target molecule to be tested, and the first antibody and / or the second antibody are coupled to the target substance via isopeptide bonds through which their non-specific binding regions are directed.

17. The agent of claim 16, wherein The first antibody and / or the second antibody form isopeptide bonds through the Catcher-Tag directional subsystem to directionally couple them to the target substance.

18. The agent of claim 17, wherein In the Catcher-Tag targeting subsystem, the Catcher is a monomeric Catcher; preferably, the monomeric Catcher carries a tag protein.

19. The agent of claim 18, wherein The molecular weight of the monomer Catcher is 12–55 kDa; Preferably, the monomer Catcher contains lysine residues; Preferably, the number of lysine residues in the monomer Catcher is ≥5.

20. The agent of claim 18, wherein In the Catcher-Tag directing subsystem, the monomer Catcher is coated on the surface of the luminescent microspheres, and the Tag is linked to the non-specific binding region of the first antibody. And / or, in the Catcher-Tag directional subsystem, the monomeric Catcher is labeled with one of the specific binding pair members, and the Tag is linked to the non-specific binding region of the second antibody.

21. The agent of any one of claims 17 to 20, wherein, The non-specific binding region of the first antibody is directionally connected to the luminescent microspheres via a first Catcher-Tag directional subsystem. One of the specific binding pair members is directionally linked to the non-specific binding region of the second antibody via a second Catcher-Tag directional subsystem; Preferably, the first Catcher-Tag orientation subsystem and the second Catcher-Tag orientation subsystem are different.

22. The agent of any one of claims 17 to 21, wherein Each of the second antibodies is linked to two second Catcher-Tag targeting subsystems, both located in the Fc region of the second antibody.

23. A photochemiluminescent detection reagent, characterized by include: R1 reagent comprises luminescent microspheres coated with a first antibody capable of specifically binding to the target molecule to be tested, the first antibody carrying a first tag molecule; R2 reagent contains a second antibody with a second tag molecule that can specifically bind to the target molecule to be tested. R3 reagent contains a first pairing molecule capable of specifically recognizing and binding to the first tag molecule; The R4 reagent comprises photosensitive microspheres and a second pairing molecule coated on the photosensitive microspheres that can specifically bind to the second tag molecule; One of the first pairing molecules can bind to at least two first tag molecules.

24. The agent of claim 23, wherein The first tag molecule is co-expressed with the first antibody and linked to the non-specific binding region of the first antibody.

25. The agent of claim 23, wherein The first tag molecule and the first pairing molecule are selected from the Catcher-Tag polymer subsystem; preferably, the first tag molecule is selected from one or more of Spy-Tag, His-Tag, HA-Tag, Snoop-Tag, Flag-Tag, and Myc-Tag; more preferably, the first pairing molecule is a polyCatcher with a degree of polymerization of not less than 2.

26. The agent of claim 23, wherein The molar amount of the first paired molecule is less than or equal to the molar amount of the first tag molecule; preferably, the molar ratio of the first paired molecule to the first tag molecule is 1:(1-16); more preferably, it is 1:(2-8).

27. The agent of claim 23, wherein The first paired polymer molecules are negatively correlated with their optimal molar amount; preferably, the product of the degree of polymerization of the first paired molecules and their molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

28. The agent of claim 23, wherein The number of moles of the luminescent microspheres is greater than the number of moles of the target molecules in the sample to be tested.

29. The agent of claim 23, wherein The two sets of specific tag systems, consisting of the first tag molecule and the first pairing molecule, the second tag molecule and the second pairing molecule, do not react with each other.

30. The application of the detection method according to any one of claims 1 to 15 or the photo-induced chemiluminescence detection reagent according to any one of claims 16 to 29 in the detection of p-tau 217 in blood samples.

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