Light-activated chemiluminescence detection reagent of APOE4, detection method and application of light-activated chemiluminescence detection reagent

Abstract

The invention relates to a light-activated chemiluminescence detection reagent of APOE4, a detection method and application of the light-activated chemiluminescence detection reagent. The reagent comprises: a luminescent microsphere coated with a first antibody and a first tag molecule; a first pairing molecule capable of specifically recognizing and binding to the at least two first tag molecules; the second antibody and the third antibody are provided with second tag molecules; the photosensitive microsphere is coated with a second pairing molecule capable of specifically recognizing and combining with a second tag molecule; the first antibody and the third antibody can specifically recognize and combine all subtype proteins of APOE, and the second antibody can specifically recognize and combine APOE4 subtype proteins. According to the technical scheme, by increasing the amount of the light-emitting microspheres capable of receiving the active oxygen generated by the light-sensitive microspheres, the utilization rate of the active oxygen is increased, the light-activated chemiluminescence detection signal value is overall increased, the detection performance is improved, the typing detection requirement of APOE is met, and the APOE epsilon 4 genotype in a blood sample is accurately and efficiently detected.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a photoluminescent chemiluminescence detection reagent, detection method and application of APOE4. Background Art

[0002] Apolipoprotein E (APOE) is a secretory protein synthesized by liver cells and brain tissues. It can bind to a variety of receptors on the surface of different cells and play a key role in lipid metabolism, immune regulation and neurology. There are three common subtypes of APOE, APOE2, APOE3, and APOE4. Among them, studies have shown that in addition to being associated with abnormal lipid metabolism in the blood, APOE4 may also promote the deposition of beta-amyloid protein (Aβ) in the brain and promote abnormal phosphorylation of tau protein in the brain. It is considered to be an important associated genetic risk factor for atherosclerosis and Alzheimer's Disease (AD). People carrying the APOE ε4 gene will have a significantly increased risk of developing AD. Therefore, APOE4 testing can indicate the risk of Alzheimer's disease.

[0003] Genotyping based on PCR (Polymerase Chain Reaction) technology is a common method for determining the status of APOE alleles. For example, real-time PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), fluorescent PCR-melting curve method, etc. are used to determine the APOE genotype, thereby judging whether the APOE ε4 allele is carried. It has the advantages of high sensitivity and strong specificity, but it also requires professional laboratory equipment and technicians, and the operation is complicated, time-consuming, and the detection cost is high, which greatly limits its clinical promotion and application. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related technology, the present application provides an APOE4 photochemiluminescence detection reagent, detection method and application thereof, which increases the amount of luminescent microspheres that can receive the reactive oxygen generated by the photosensitive microspheres and generate light signals; and improves the ability of the detection reagent to resist interference from foreign proteins, which can improve the detection signal value and discrimination, and better meet the photochemiluminescence detection needs of APOE.

[0005] In a first aspect, the present application provides a photochemiluminescence detection reagent for APOE4, comprising: Luminescent microspheres coated with a first antibody and a first label molecule; A first pairing molecule capable of specifically recognizing and binding to at least two first tag molecules; The second antibody and the third antibody, both of which carry a second tag molecule; Photosensitive microspheres coated with a second pairing molecule capable of specifically recognizing and binding to the second tag molecule; Among them, the first antibody and the third antibody can specifically recognize and bind to the APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

[0006] In some embodiments, the two sets of specific label systems consisting of the first label molecule and the first pairing molecule, and the second label molecule and the second pairing molecule, respectively, do not react with each other.

[0007] In some embodiments, the specific tag system composed of the first tag molecule and the first pairing molecule is selected from the Tag-Catcher system, and the specific tag system composed of the second tag molecule and the second pairing molecule is selected from the biotin-avidin system.

[0008] In some embodiments, the first pairing molecule is selected from a multimeric protein of Catcher, and the degree of polymerization is not less than 2; preferably, it is an octamer protein of Catcher.

[0009] In some embodiments, the molar amount of the first pairing molecule is less than or equal to the molar amount of the first tag molecule. Preferably, the molar ratio of the first pairing molecule to the first tag molecule is 1:(1-16).

[0010] In some embodiments, the polymerization degree of the first pairing molecule is negatively correlated with its optimal molar weight. The product of the polymerization degree of the first pairing molecule and its optimal molar weight is preferably 1 to 2.5 times the molar weight of the first tag molecule.

[0011] In some embodiments, 1-5 M sodium chloride and / or at least 0.6% Triton X-405 are added to the luminescent microsphere reagent.

[0012] The second aspect of the present application provides a photochemiluminescence detection method for APOE4, which comprises: S1. Mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a second antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere" sandwich immune complex; at the same time, mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a third antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE protein-photosensitive microsphere" sandwich immune complex; S2, adding the first pairing molecule reagent to the two groups of reaction solutions respectively to obtain two groups of mixed solutions; S3, irradiating the two groups of mixed solutions with light of a specific wavelength, and detecting the luminescence values ​​RLU1 and RLU2 to obtain the detection results of APOE4 and APOE; S4. Calculate the ratio of APOE4 to APOE.

[0013] In some embodiments, step S2 adds the first pairing molecule reagent and then incubates for 0 to 15 minutes.

[0014] The third aspect of the present application provides a reagent as described in the first aspect, or the detection method as described in the second aspect, for use in detecting APOE4 in a blood sample.

[0015] The technical solution provided by the present application may include the following beneficial effects: by simultaneously coating the antibody molecule and the label molecule in the luminescent microsphere, and then by adding a tool protein capable of capturing at least two of the label molecules, the free luminescent microspheres in the detection system can be aggregated to the "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere" or "luminescent microsphere-APOE protein-photosensitive microsphere" sandwich immune complex, thereby increasing the amount of luminescent microspheres that can receive the reactive oxygen generated by the photosensitive microspheres, improving the utilization rate of reactive oxygen, and improving the photochemiluminescence detection signal value as a whole, improving the detection performance, and meeting the detection requirements of APOE4. By adjusting the salt ion concentration and the surfactant concentration in the detection reagent, nonspecific adsorption can be reduced, the discrimination of the sample ratio can be improved, and the photochemiluminescence detection requirements of APOE4 can be better met. Automated detection is simple and efficient, does not require complex and expensive detection equipment, is easy to operate, and has high throughput and low cost. It can detect past samples without nucleic acid information and can be widely used in the clinical diagnosis of APOE4.

[0016] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a distribution diagram of the test results of the sample when no sodium chloride and TritonX-405 are added to the reconstituted solution shown in Example 3; Figure 2 This is a distribution diagram of the test results of the sample when 3.5M sodium chloride and 1% (w / v) TritonX-405 are added to the complex solution shown in Example 3; Figure 3 This is a distribution diagram of the test results of the samples shown in Example 6. DETAILED DESCRIPTION

[0018] To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0019] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicitly excluded limits in the specified ranges. Where a specified range includes one or two limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0020] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials or equivalent methods and materials described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0021] I. Terminology The sample to be tested described herein refers to a mixture that may contain an analyte, and the analyte includes but is not limited to proteins, hormones, antibodies or antigens. Typical samples to be tested that can be used in the method disclosed in the present invention include body fluids, such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid and emphysematous effusion, etc. The sample to be tested can be a solution of the sample that may contain the analyte diluted with a diluent or a buffer solution as needed before use. For example, in order to avoid the HOOK effect, the analyte can be diluted with a sample diluent before being tested on the machine and then tested on the detection instrument. At this time, the diluted solution that may contain the analyte is collectively referred to as the sample to be tested.

[0022] Antibodies described herein are used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody can be further conjugated to other moieties, such as specific binding pair members, for example biotin or avidin, etc.

[0023] The antigen described herein refers to a substance that can induce antibody production, which can be divided into complete antigens and incomplete antigens (haptens). The antigen can be a natural antigen extracted from a pathogen or animal tissue or a recombinant antigen with specific antigenic properties prepared by genetic engineering technology. In any desired case, the antigen can be further conjugated with other parts, such as specific binding pair members, such as biotin or avidin.

[0024] As used herein, "binding", "linking" and "coupling" refer to an association between two substances due to interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonds, or interactions including but not limited to salt bridges and water bridges.

[0025] The specific binding described in this article refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the responding reactants from the perspective of three-dimensional structure.

[0026] The specific label system described herein refers to two substances that have the ability to distinguish each other and can be selectively bound through covalent bonds or non-covalent bonds.

[0027] The Tag-Catcher system described herein is a protein covalent connection technology, which includes two parts, Tag and Catcher, composed of multiple amino acid residues, and the covalent connection of proteins can be achieved through the specific reaction between Tag and Catcher. The Tag-Catcher system can be a Spy Tag-Spy Catcher system developed based on the CnaB2 domain in the fibronectin FbaB of Streptococcus pyogenes. Spy Tag is a short peptide containing 13 amino acid residues, and SpyCatcher is a protein containing 138 amino acid residues. During the connection process, the aspartic acid in the Spy Tag can react spontaneously with the lysine on the SpyCatcher under the catalysis of the glutamic acid adjacent to the lysine to form an isopeptide covalent bond. The Catcher protein in the Tag-Catcher system can exist as a monomer or a polymer protein. The Catcher is preferably a multimeric protein, which is a protein composed of two or more polypeptide chains, which may be the same or different, and are interconnected by covalent bonds or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.) to form a multimeric macromolecular structural protein.

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

[0029] The reactive oxygen species mentioned in this article refers to the general term for substances composed of oxygen, containing oxygen and active in the body or natural environment, mainly an excited oxygen molecule, including the one-electron reduction product of oxygen, superoxide anion (O2·-), the two-electron reduction product of oxygen, hydrogen peroxide (H2O2), the three-electron reduction product of oxygen, hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen ( 1 O2) etc.

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

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

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

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

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

[0035] The inventors of this application have found from long-term experiments and studies that by using photochemiluminescence analysis to detect APOE4, and detecting APOE4 subtype proteins and all APOE subtype proteins in serum or plasma samples at the same time, and making a ratio, it can be determined whether the sample provider carries the APOE ε4 gene, and whether it is homozygous or heterozygous. This automated detection method is simple, efficient, high-throughput, low-cost, and can detect past samples without nucleic acid information. It has important advantages for patient stratification in clinical trials, AD prevention strategies, and clinical assessment of the risk of cerebral amyloidosis. In addition, the performance of the reagent can be effectively improved by increasing the utilization rate of free luminescent microspheres in the detection reagent during photochemiluminescence analysis; by adjusting the salt ion concentration and surfactant concentration in the luminescent microsphere reagent, the detection signal value and discrimination can be further improved to better meet the detection needs of APOE4 in the blood.

[0036] The photoluminescent chemiluminescent detection reagent of APOE4 involved in the present application includes luminescent microspheres, a first pairing molecule, a second antibody and a third antibody, and a photosensitive microsphere. The luminescent microspheres are coated with a first antibody and a first label molecule, and the first pairing molecule can specifically recognize and bind to at least two first label molecules; the second antibody and the third antibody both carry a second label molecule, and the photosensitive microspheres are coated with a second pairing molecule that can specifically recognize and bind to the second label molecule. The first antibody and the third antibody can specifically recognize and bind to all subtypes of APOE proteins, and the second antibody can specifically recognize and bind to APOE4 subtype proteins.

[0037] When the target to be tested, i.e., all subtypes of APOE proteins, are present in the sample to be tested, since the first antibody and the third antibody can recognize the common sequence of all subtypes of APOE proteins, they can bind to all subtypes of APOE proteins to form a sandwich immune complex of "luminescent microspheres-(first antibody)-APOE protein-(third antibody)-photosensitive microspheres"; when the target to be tested, i.e., APOE4 subtype proteins, are present in the sample to be tested, since the second antibody only recognizes the unique sequence of APOE4 subtype proteins, the first antibody and the second antibody can bind to APOE4 subtype proteins to form a sandwich immune complex of "luminescent microspheres-(first antibody)-APOE4 subtype proteins-(second antibody)-photosensitive microspheres". That is, the reagent of the present application can simultaneously detect APOE4 subtype proteins and all subtypes of APOE proteins in blood samples, and the ratio of the two can determine whether the sample to be tested carries the APOE ε4 gene and whether it is homozygous or heterozygous. It can realize automated detection of APOE ε4 genotype, which is simple, efficient, high-throughput, and low-cost. It can detect past samples without nucleic acid information, which has important advantages for patient stratification in clinical trials, AD prevention strategies, and clinical assessment of the risk of cerebral amyloidosis.

[0038] At the same time, in the detection process, the specific pairing reaction of the first label molecule and the first pairing molecule can make the free luminescent microspheres in the detection system aggregate to the luminescent microspheres of the sandwich immune complex, and the aggregation of at least two luminescent microspheres forms multi-luminescent microspheres, so that the free luminescent microspheres are also gathered around the photosensitive microspheres, shortening the distance between more luminescent microspheres and photosensitive microspheres, thereby increasing the amount of luminescent microspheres that can receive the active oxygen generated by the photosensitive microspheres. When the photosensitive microspheres are excited by light, the active oxygen generated can be received by more luminescent microspheres, which improves the utilization rate of the active oxygen, and the intensity of the light signal generated by the luminescent microspheres is increased, so as to significantly improve and enhance the detection performance of APOE4.

[0039] Moreover, in the process of forming the sandwich immune complex, the luminescent microspheres still exist in their original state, without changing the particle size and quality of the luminescent microspheres themselves. They have a good particle migration rate, can ensure the occurrence rate of the antigen-antibody immune reaction, and overall improve the detection performance of APOE4.

[0040] The total number of luminescent microspheres in the polymerized luminescent microspheres described in the present application is equal to or greater than two, which is not limited here.

[0041] In some embodiments of the present application, the first label molecule can be linked to the C-terminus or N-terminus of the first antibody by co-expression, and then the first label molecule can be coated on the luminescent microsphere by coating the first antibody on the luminescent microsphere; the first antibody and the first label molecule can also be coated on the luminescent microsphere separately by step-by-step coating. It should be noted that, by adopting the above-mentioned coating method, the expression of the first antibody and the first label molecule does not affect each other.

[0042] The present application does not impose any limitation on the coating order when the first antibody and the first label molecule are coated step by step on the luminescent microspheres.

[0043] In some embodiments of the present application, the first specific label system composed of the first label molecule and the first pairing molecule does not react with the second specific label system composed of the second label molecule and the second pairing molecule, thereby preventing the specific pairing between the two sets of specific label systems from interfering with the aggregation reaction of the multi-luminescent microspheres and the accuracy and stability of the detection results.

[0044] Specifically, the specific tag system composed of the first tag molecule and the first pairing molecule can be selected from the Tag-Catcher system, and any combination can be selected from the system as the first tag molecule and the first pairing molecule, for example, it can be selected from Spy Tag-Spy Catcher. In the embodiment of the present application, the first pairing molecule can also be called a tool protein.

[0045] Catcher and Tag have excellent specific recognition and binding ability, and the use of Tag tags with smaller molecular weight to coat the luminescent microspheres has no obvious effect on the expression of the first antibody and the properties of the luminescent microspheres.

[0046] Further, when the first tag molecule is selected from Tag, the first pairing molecule can be selected from the multimeric protein of Catcher, and the degree of polymerization is not less than 2. For example, it can be selected from the dimer protein, pentamer protein, octamer protein, etc. of Catcher, preferably octamer protein. The degree of polymerization of Catcher directly affects the rate at which Catcher captures Tag, and then affects the aggregation rate of luminescent microspheres. The higher the degree of polymerization of Catcher, the higher the rate of capturing Tag, the higher the aggregation rate of luminescent microspheres, and the better the effect of improving the intensity of the light signal.

[0047] In some embodiments of the present application, the molar amount of the first pairing molecule is less than or equal to the molar amount of the first label molecule. Preferably, the molar ratio of the first pairing molecule to the first label molecule is 1:(1~16); preferably 1:(2~8). The use ratio of the first pairing molecule directly affects the aggregation of the luminescent microspheres. If it is too low, the aggregation efficiency and aggregation degree will be low, and the effect of improving the detection performance will not be obvious; if it is too high, it will easily produce a "hook effect", which will make it difficult for the luminescent microspheres to aggregate, and there will be no obvious improvement in the detection performance, and even affect the accuracy of the detection results.

[0048] When the first tag molecule and the first pairing molecule are selected from the Tag-Catcher system, the higher the polymerization degree of the first pairing molecule Catcher, the lower the required molar amount of the first pairing molecule, that is, the polymerization degree of the first pairing molecule is negatively correlated with its optimal molar amount. Preferably, the product of the polymerization degree of the first pairing molecule and its optimal molar amount is 1 to 2.5 times the molar amount of the first tag molecule.

[0049] The degree of polymerization and dosage of the first pairing molecule can be determined according to the amount of the first label molecule, and the higher the degree of polymerization of the first pairing molecule, the lower the required optimal molar amount, and the faster the reaction rate with the first label molecule; the lower the degree of polymerization of the first pairing molecule, the higher the required optimal molar amount, and the faster the aggregation rate. When the product of the degree of polymerization of the first pairing molecule and its molar amount is 1 to 2.5 times the molar amount of the first label molecule, the light signal intensity of the luminescent microspheres is significantly improved. Excessive amounts are prone to cause HOOK effects, affecting the detection results; insufficient amounts affect the aggregation rate and degree of aggregation of the multi-polymerized luminescent microspheres, and the performance improvement is not obvious.

[0050] In some embodiments of the present application, the second label molecule and the second pairing molecule can be selected from the biotin-avidin system, for example, one of them is selected from biotin and the other is selected from avidin or streptavidin; preferably, streptavidin is coated on the photosensitive microsphere, the second antibody is bound to biotin, and the second antibody and the third antibody are labeled with biotin.

[0051] In some embodiments of the present application, the reagent further includes a buffer, a stabilizer, etc. The buffer and the stabilizer can be used as a storage and dilution solution for the luminescent microspheres, the second antibody and the third antibody, the photosensitive microspheres, and the first paired molecule. Among them, the buffer can be selected from at least one of PBS buffer, Tris-hydrochloric acid buffer, HEPES buffer, and MES buffer. The stabilizer can be selected from at least one of bovine serum albumin (BSA), dextran, sorbitol, glycerol, glycine, alanine, Tween 20, Tween 80, etc.

[0052] In order to further improve the detection signal value and discrimination, and better meet the detection needs of APOE4 in the blood, salt ions and / or surfactants are also added to the luminescent microsphere reagent. Specifically, the salt ion can be sodium chloride, and the surfactant can be TritonX-405. Preferably, 1~5M sodium chloride and / or at least 0.6% TritonX-405 are added to the luminescent microsphere reagent. By adjusting the type and concentration of salt ions and surfactants, the ability of the luminescent microsphere reagent to resist interference from foreign proteins can be adjusted, thereby reducing nonspecific adsorption in the reaction system during the detection process, improving the signal-to-noise ratio, and improving detection sensitivity and specificity.

[0053] The reagent involved in the present application, wherein the first pairing molecule is added after the sandwich immune complex is formed. Specifically, the present application also provides a photoluminescence detection method for APOE4, that is, a method for using the above reagent, which includes: S1. Mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a second antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere" sandwich immune complex; at the same time, mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a third antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE protein-photosensitive microsphere" sandwich immune complex; S2, adding the first pairing molecule reagent to the two groups of reaction solutions respectively, so that the free luminescent microspheres are aggregated to the luminescent microspheres of the sandwich immune complex to form multi-luminescent microspheres, and obtaining two groups of mixed solutions; S3, irradiating the two mixed solutions with light of a specific wavelength in separate cups to stimulate the photosensitive microspheres to produce reactive oxygen species, and detecting the luminescence values ​​RLU1 and RLU2 of the luminescent microspheres to obtain the detection results of APOE4 and APOE; S4. Calculate the ratio of APOE4 to APOE.

[0054] Through the formation of two sandwich immune complexes, "luminescent microspheres-(first antibody)-APOE protein-(third antibody)-photosensitive microspheres" and "luminescent microspheres-(first antibody)-APOE4 subtype protein-(second antibody)-photosensitive microspheres", the APOE4 subtype protein and all APOE subtype protein contents in blood samples can be detected simultaneously. The APOE4 / APOE subtype protein ratio can be calculated to determine whether the sample carries the APOE ε4 gene and whether it is homozygous or heterozygous. This method can realize the automated detection of the APOE ε4 genotype. It is simple and efficient, does not require complex and expensive detection equipment, is easy to operate, and has high throughput and low cost. It can detect past samples without nucleic acid information and can be widely used in the clinical diagnosis of APOE4. It has important advantages for patient stratification in clinical trials, AD prevention strategies, and clinical assessment of the risk of cerebral amyloidosis.

[0055] Moreover, the free luminescent microspheres after the conventional reaction are concentrated around the luminescent microspheres of the complex, so that the reactive oxygen generated by the photosensitive microspheres can be received by multiple luminescent microspheres in the polymerized luminescent microspheres (including the main luminescent microspheres and at least one free luminescent microsphere bound to the main luminescent microspheres) and generate light signals at the same time, effectively improving the utilization rate of reactive oxygen, increasing the signal intensity, and significantly improving the photochemiluminescence detection performance of APOE4.

[0056] In some embodiments of the present application, the incubation time after adding the first pairing molecule reagent in step S2 is 0 to 15 minutes, that is, the aggregation time of the luminescent microspheres is 0 to 15 minutes. As the reaction time increases, the aggregation reaction of the luminescent microspheres gradually increases and tends to equilibrium, so the reaction time is preferably 3 to 9 minutes, and more preferably 5 to 7 minutes.

[0057] The detection reagent or detection method described above can be used to detect the APOE4 subtype protein content in a blood sample, and then determine whether the sample carries the APOE ε4 gene and whether it is homozygous or heterozygous.

[0058] APOE4 and APOE in serum or plasma samples are detected by photochemiluminescence technology, and the APOE4 / APOE ratio is calculated to determine whether the sample carries the APOE ε4 gene. If the APOE4 to APOE ratio is ≤0.1, the sample provider is determined to be negative (non-APOE ε4 gene carrier); if 0.1 < APOE4 to APOE ratio ≤0.5, the sample provider is determined to be an APOE ε4 gene heterozygote (APOE ε4 gene carrier); if the APOE4 to APOE ratio is >0.5, the sample provider is determined to be an APOE ε4 gene homozygote (APOE ε4 gene carrier).

[0059] III. Specific Examples In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0060] Next, taking the target molecule to be detected as APOE protein as an example, the antibody molecules with specific recognition of APOE4 subtype protein are coated on the luminescent microspheres, and different labels are modified on the surface of the luminescent microspheres. The antibody molecules that can specifically recognize all subtype protein sequences of APOE and can specifically recognize the single subtype protein sequence of APOE4 are labeled with biotin, and the double antibody sandwich method is used to detect the target APOE4 subtype protein or APOE protein in the sample. After the conventional detection sequence is completed, the pairing molecules corresponding to the label are added to aggregate the free luminescent microspheres. By comparing the detection discrimination between different experimental groups, the improvement effect of the multi-luminescent microsphere mode on the photoluminescence detection performance of APOE4 subtype protein is judged.

[0061] The main experimental materials and equipment are shown in Table 1: Table 1

[0062] APOE Ab1 and APOE Ab3 are APOE protein antibodies, and APOE Ab2 is an APOE4 subtype protein antibody.

[0063] Example 1: Reagent preparation 1.1 Preparation of luminescent microparticles coated with antibody APOE Ab1: ① Take 10 mg of luminescent microparticles in a centrifuge tube, wash twice with 0.05 M CB buffer and redissolve; ② Take APOE Ab1 and dialyze it with 0.05M CB buffer, then determine the concentration using the BCA method; ③ After the treated luminescent particles FG and APOE Ab1 are fully mixed at a mass ratio of 10: (0.05-0.5), react at 37°C for 24 hours; ④ Add 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH4 (20 mg / mL, 0.05 M CB) to the reaction system, mix thoroughly, and react at 4°C for 2 h to obtain a reaction solution; ⑤ The reaction solution was washed twice with PBST buffer, and then washed once with reconstitution solution (prepared according to Table 2), and then 1 mL of reconstitution solution (prepared according to Table 2) was added for ultrasonic reconstitution to obtain luminescent microparticles coated with APOE Ab1.

[0064] Table 2 Basic reconstitution solution

[0065] 1.2 Preparation of biotin-labeled antibodies APOE Ab2 and APOE Ab3 ① Dialyze APOE Ab2 or antibody APOE Ab3 with 0.05M NaHCO3 solution, remove impurities and determine the concentration using the BCA method; ② Add 30 times the molar amount of Biotin-LC-LC-NHs to the dialyzed APOE Ab2 or antibody APOE Ab3 solution, mix well, and react at 4°C for 24 hours to obtain Bio-APOE Ab2 solution and antibody Bio-APOE Ab3 solution; ③ Dialyze with 0.02M PBS buffer to remove free Biotin and then determine the concentration with BCA to obtain biotin-labeled antibodies Bio-APOE Ab2 and Bio-APOE Ab3.

[0066] 1.3 Preparation of detection reagents: The luminescent microparticles coated with APOE Ab1 were diluted to 50 μg / mL with the reconstitution solution (prepared according to Table 2) to obtain the R1 reagent. The Bio-APOE4 Ab2 was diluted to 1.0 μg / mL with the reconstitution solution (prepared according to Table 2) to obtain the R2 reagent. The Bio-APOE Ab3 was diluted to 1.2 μg / mL with the reconstitution solution (prepared according to Table 2) to obtain the R3 reagent. The reagent containing the SA photosensitive microspheres is the universal solution (or R4 reagent).

[0067] Example 2: APOE4 typing test 1.1 APOE4 detection: Mix 20 μL of the sample to be tested, 25 μL of R1 reagent and 25 μL of R2 reagent, and incubate at 37°C for 17 min; add 175 μL of universal solution and incubate at 37°C for 15 min; after the light excitation reaction, read the light signal value of each group of tests to obtain the APOE4 test results.

[0068] 1.2 APOE testing: Mix 20 μL of the same test sample, 25 μL of R1 reagent and 25 μL of R3 reagent, and incubate at 37°C for 17 min; add 175 μL of universal solution and incubate at 37°C for 15 min; after the light excitation reaction, read the light signal value of each group of tests to obtain the APOE test results.

[0069] 1.3 The APOE4 test results and APOE test results of the same sample are compared to obtain the APOE4 / APOE ratio. If the ratio is ≤0.1, the sample provider is determined to be negative (non-APOE ε4 gene carrier); if 0.1<ratio≤0.5, the sample provider is determined to be APOE ε4 gene heterozygote (APOE ε4 gene carrier); if the ratio is >0.5, the sample provider is determined to be APOE ε4 gene homozygote (APOE ε4 gene carrier).

[0070] Example 3: Effects of sodium chloride and Triton X-405 on APOE ε4 typing detection 1.1 Based on Example 1, reconstitution solutions with different sodium chloride concentrations were prepared as shown in Table 3, and the luminescent microparticles coated with APOE Ab1 were diluted to 50 μg / mL with the reconstitution solution in Table 3 to obtain R1 reagent.

[0071] Table 3 Sodium chloride for adjusting the reconstitution solution

[0072] 1.2 Based on Example 1, different Triton surfactant reconstitution solutions were prepared as shown in Table 4, and the luminescent microparticles coated with APOE Ab1 were diluted to 50 μg / mL with the reconstitution solution in Table 4 to obtain R1 reagent.

[0073] Table 4 Triton surfactant for adjusting the reconstitution solution

[0074] 1.3 Based on Example 1, TritonX-405 reconstitution solutions with different concentrations were prepared as shown in Table 5, and the luminescent microparticles coated with APOE Ab1 were diluted to 50 μg / mL with the reconstitution solution in Table 5 to obtain R1 reagent.

[0075] Table 5 TritonX-405 for adjusting the reconstitution solution

[0076] 1.4 Based on Example 1, a reconstitution solution containing sodium chloride and TritonX-405 was prepared as shown in Table 6, and the luminescent microparticles coated with APOE Ab1 were diluted to 50 μg / mL with the reconstitution solution in Table 6 to obtain R1 reagent.

[0077] Table 6 Sodium chloride and TritonX-405 for adjusting the reconstitution solution

[0078] 1.5 Perform APOE4 typing test as shown in Example 2 to obtain the test results of APOE 4 and APOE.

[0079] 2. Experimental results Table 7 Test results of control group

[0080] Table 8 Test results of adjusting sodium chloride concentration

[0081] Table 9 Adjustment of Triton surfactant test results

[0082] Table 10 Adjusted TritonX-405 test results

[0083] Table 11 Adjusted sodium chloride and TritonX-405 test results

[0084] 3. Results Analysis 3.1 Experimental data analysis ① Combined with Table 7 and Figure 1 As shown in the figure, when sodium chloride and TritonX-405 were not added to the luminescent microsphere complex solution, negative samples were distinguished from heterozygous samples and homozygous samples to a certain extent, but the values ​​were higher than the theoretical value, which may be caused by nonspecific adsorption of foreign proteins, and there was no distinction between heterozygous and homozygous; ② As shown in Table 8, as the amount of sodium chloride added increases, the ratio of negative samples decreases, the ratio of heterozygous samples decreases, and the ratio of homozygous samples increases, but there are still samples that cannot be distinguished; ③ As shown in Table 9, after adding TritonX-100, TritonX-305, and TritonX-405, the ratio of negative samples decreased, the ratio of heterozygous samples decreased, and the ratio of homozygous samples increased. When TritonX-405 was added, the effect was better, but there were still samples that could not be distinguished; ④ As shown in Table 10, as the amount of TritonX-405 added increased, the ratio of negative samples decreased and the ratio of homozygous samples increased, but there were still samples that could not be distinguished; ⑤ Combined with Table 11 and Figure 2 As shown, sodium chloride and TritonX-405 were added to the luminescent microsphere complex solution at the same time. When 3.5M sodium chloride + 1% TritonX-405 was added, the ratio of negative samples decreased and approached 0; the degree of distinction between heterozygous samples and homozygous samples was optimal.

[0085] 3.2 Experimental Conclusion When high-concentration salt ions and high-concentration surfactants are added to the complex solution of the luminescent microsphere reagent, it has a strong ability to resist interference from foreign proteins, can significantly reduce nonspecific adsorption during detection, reduce the negative sample ratio, and make the heterozygous samples and homozygous samples clearly distinguishable, meeting the needs of distinguishing negative samples from heterozygous samples and homozygous samples of APOE ε4.

[0086] Example 4: Preparation of luminescent microspheres coated with label molecules and antibody molecules Antibody APOE Ab1 and label molecule Spy-Tag are coated on the luminescent microparticles: ① Take 10 mg of luminescent microparticles in a centrifuge tube, wash twice with 0.05 M CB buffer and redissolve; ② Take APOE Ab1 and dialyze it with 0.05M CB buffer, then determine the concentration using the BCA method; ③ After the treated luminescent particles FG and APOE Ab1 are fully mixed at a mass ratio of 10: (0.05-0.5), react at 37°C for 24 hours; ④ Take Spy-Tag and dialyze it with 0.05M CB buffer, then determine the concentration using the BCA method; ⑤ After the treated luminescent particles FG-Ab1 and Spy-Tag are fully mixed, react at 37°C for 24 hours; ⑥ Add 80 μL of glycine (75 mg / mL, 0.05 M CB) and 10 μL of NaBH4 (20 mg / mL, 0.05 M CB) to the reaction system, mix thoroughly, and react at 4°C for 2 h to obtain a reaction solution; ⑦ The reaction solution was washed twice with PBST buffer, and then washed once with reconstitution solution (prepared according to Table 2). Then, 1 mL of reconstitution solution (prepared according to Table 2) was added and ultrasonically reconstituted to obtain luminescent microparticles coated with APOE Ab1 and Spy Tag.

[0087] Example 5: Effects of tool protein polymerization degree and usage ratio on APOE4 detection performance 1.1 Dilute the luminescent microparticles coated with APOE Ab1 and Spy Tag with a reconstitution solution (prepared according to Table 2) to 50 μg / mL to obtain R1 reagent. Dilute Bio-APOE4 Ab2 with a reconstitution solution (prepared according to Table 2) to 1.0 μg / mL to obtain R2 reagent. R4 reagent containing photosensitive microspheres is a universal solution. Dilute Spy Catcher dimer, pentamer and octamer with a reconstitution solution (prepared according to Table 2) to R5 reagents with a molar ratio of 1:16, 1:8, 1:4, 1:2, 1:1 and 2:1 to the Tag in R1 reagent. ® Detection was performed on a 500 chemiluminescence detection system.

[0088] 1.2 Mix 20μL of the sample to be tested, 25μL of R1 reagent and 25μL of R2 reagent, and incubate at 37℃ for 17min; add 175μL of universal solution and incubate at 37℃ for 15min; add 15μL of R5 reagent to the experimental group and 15μL of complex solution (prepared according to Table 2) to the control group, and incubate at 37℃ for 15min respectively; after light excitation reaction, read the light signal value of each group test respectively. The experimental data are shown below.

[0089] 2. Experimental results 2.1 Spy Catcher dimer reaction effect: Table 12 Signal values

[0090] 2.2 Spy Catcher pentamer reaction effect: Table 13 Signal values

[0091] 2.3 Spy Catcher octamer reaction effect: Table 14 Signal values

[0092] 3. Results Analysis ① Within the current gradient range, the polymer particle size (polymerization degree) does not affect the particle size of the luminescent particle receiving singlet oxygen. The higher the polymerization degree of the first paired molecule (tool protein) corresponding to the first label molecule, the better the improvement effect. When the tool protein is the Spy Catcher octamer, the positive signal is significantly improved, and the detection performance of low-value samples is improved to a greater extent. ② When the molar ratio of Spy Catcher to Spy Tag in R1 reagent is 1:(1-16), the positive signal is improved. The best effect is achieved when the molar ratio is 1:4 (Spy Catcher octamer: Spy Tag). At this time, the positive signal is significantly improved, and the detection performance of low-value samples is improved even more.

[0093] ③ Using Spy Tag as a label and Spy Catcher as a pairing molecule (tool protein), the positive signal for detecting APOE4 samples is significantly improved.

[0094] ④ The polymerization degree and concentration of the first pairing molecule depend on the molecular weight of the first tag. The molar weight of the first pairing molecule should be lower than or equal to the molar weight of the first tag molecule, and the polymerization degree of the first pairing molecule is negatively correlated with the optimal molar weight. The results show that when the polymerization degree of the first pairing molecule multiplied by its molar weight is 1 to 2.5 times the molar weight of the first tag molecule, the positive signal is significantly improved. Excessive amounts will lead to the Hook effect and affect aggregation; insufficient amounts will lead to low aggregation efficiency and no obvious performance improvement.

[0095] Therefore, the positive signal can be enhanced by using a specific tag system such as Spy Catcher-Tag to aggregate free luminescent microspheres into complexes to form multimeric luminescent microspheres.

[0096] Example 6: Preparation and performance testing of APOE4 typing detection reagents 1. Reagent Preparation 1.1 Take the luminescent microspheres of Example 4 (coated with the antibody molecule APOE Ab1 and the label molecule Spy-Tag), and match them with the biotin-labeled antibodies Bio-APOE Ab2 and Bio-APOE Ab3 of Example 1.

[0097] 1.2 Dilute the luminescent microparticles coated with APOE Ab1 and Spy Tag with the reconstitution solution (prepared according to Table 6) to 50μg / mL to obtain R1 reagent. Dilute Bio-APOE4 Ab2 with the reconstitution solution (prepared according to Table 2) to 1.0μg / mL to obtain R2 reagent. Dilute Bio-APOE Ab3 with the reconstitution solution (prepared according to Table 2) to 1.2μg / mL to obtain R3 reagent. R4 reagent containing photosensitive microspheres is a universal solution. Dilute Spy Catcher octamer with the reconstitution solution (prepared according to Table 2) to R5 reagent with a molar ratio of 1:4 to Tag in R1 reagent.

[0098] 2. Incubate the sample under test in LiCA ® Detection was performed on a 500 chemiluminescence detection system.

[0099] 2.1 Mix 20 μL of the sample to be tested, 25 μL of R1 reagent and 25 μL of R2 reagent, and incubate at 37°C for 17 min; add 175 μL of universal solution and incubate at 37°C for 15 min; add 15 μL of R5 reagent and incubate at 37°C for 15 min; after light excitation reaction, read the light signal value of each group of tests respectively to obtain the APOE4 test results.

[0100] 2.2 Mix 20 μL of the same sample to be tested, 25 μL of R1 reagent and 25 μL of R3 reagent, and incubate at 37°C for 17 min; add 175 μL of universal solution and incubate at 37°C for 15 min; add 15 μL of R5 reagent and incubate at 37°C for 15 min; after light excitation reaction, read the light signal value of each group of tests respectively to obtain the APOE test results.

[0101] 2.3 The APOE4 test results and APOE test results of the same sample are compared to obtain the APOE4 / APOE ratio. If the ratio is ≤0.1, the sample provider is determined to be negative (non-APOE ε4 gene carrier); if 0.1 < ratio ≤0.5, the sample provider is determined to be an APOE ε4 gene heterozygote (APOE ε4 gene carrier); if the ratio is >0.5, the sample provider is determined to be an APOE ε4 gene homozygote (APOE ε4 gene carrier).

[0102] 3. Experimental results Table 15 Signal values

[0103] 4. Results Analysis As shown in Table 15 and Figure 3It shows that when 3.5M sodium chloride + 1% TritonX-405 is added to the luminescent microsphere complex solution and the molar ratio of Spy Tag in SpyCatcher and R1 reagent is 1:4, the sample signal is high, and the ratio of negative samples is close to 0, and the degree of distinction between heterozygous samples and homozygous samples is obvious.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A photoluminescent chemiluminescent detection reagent for APOE4, characterized in that: include: Luminescent microspheres coated with a first antibody and a first label molecule; A first pairing molecule capable of specifically recognizing and binding to at least two first tag molecules; The second antibody and the third antibody, both of which carry a second tag molecule; Photosensitive microspheres coated with a second pairing molecule capable of specifically recognizing and binding to the second tag molecule; Among them, the first antibody and the third antibody can specifically recognize and bind to the APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

2. The reagent according to claim 1, characterized in that The two groups of specific label systems respectively composed of the first label molecule and the first matching molecule, and the second label molecule and the second matching molecule do not react with each other.

3. The reagent according to claim 1 or 2, characterized in that The specific label system composed of the first label molecule and the first pairing molecule is selected from the Tag-Catcher system, and the specific label system composed of the second label molecule and the second pairing molecule is selected from the biotin-avidin system.

4. The reagent according to claim 3, characterized in that The first pairing molecule is selected from the multimeric protein of Catcher.

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

6. The reagent according to claim 5, characterized in that The molar ratio of the first pairing molecule to the first tag molecule is 1:(1-16).

7. The reagent according to claim 5, characterized in that The product of the polymerization degree of the first pairing molecule and its optimal molar weight is 1 to 2.5 times the molar weight of the first tag molecule.

8. The reagent according to claim 1, characterized in that The luminescent microsphere reagent is added with 1-5 M sodium chloride and at least 0.6% TritonX-405.

9. A photochemiluminescence detection method for APOE4, comprising: S1. Mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a second antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere" sandwich immune complex; at the same time, mix a luminescent microsphere reagent containing a first antibody and a first label molecule, a third antibody reagent containing a second label molecule, and a photosensitive microsphere reagent containing a second pairing molecule with the sample to be tested to obtain a reaction solution containing a "luminescent microsphere-APOE protein-photosensitive microsphere" sandwich immune complex; S2, adding the first pairing molecule reagent to the two groups of reaction solutions respectively to obtain two groups of mixed solutions; S3, irradiating the two groups of mixed solutions with light of a specific wavelength, and detecting the luminescence values ​​RLU1 and RLU2 to obtain the detection results of APOE4 and APOE; S4. Calculate the ratio of APOE4 to APOE.

10. Use of the reagent according to any one of claims 1 to 8, or the detection method according to claim 9, in detecting APOE4 in a blood sample.

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