Light-activated chemiluminescence detection reagent of APOE4, kit and application

Abstract

The invention relates to an APOE4 light-activated chemiluminescence detection reagent, a kit and application. The reagent comprises a first antibody coated on a luminous microsphere, and a series of antibody reagents consisting of a second antibody and a third antibody marked with one of specific binding pairing members, wherein the first antibody and the third antibody can specifically recognize and combine various APOE subtype proteins, and the second antibody can specifically recognize and combine APOE4 subtype proteins; the reagent further comprises no less than 2.5 M of a metal salt, and at least 0.01% (w / v) of a surfactant. According to the technical scheme, the APOE4 subtype protein and the APOE protein can be detected at the same time through cooperation of the three antibody molecules; in addition, the detection reagent is high in impure protein interference resistance, so that the sample discrimination during detection is improved, and efficient typing detection of the APOE epsilon 4 genotype is realized.

Description

Technical Field

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

[0002] Apolipoprotein E (APOE) is the most important lipid transport protein in the body and the apolipoprotein with the greatest impact on lipid metabolism (mainly triglycerides and cholesterol). It is distributed in most tissues of the human body. The mature APOE is a glycoprotein containing 299 amino acids with a molecular weight of approximately 34kDa. There are three common subtypes of APOE, APOE2, APOE3, and APOE4. The genes encoding the three subtypes of APOE are located on the long arm of human chromosome 19. The APOE encoded by the three alleles has different residues at two sites, 112 and 158, resulting in differences in their lipid binding ability and receptor affinity.

[0003] Among them, the ε4 allele of the apolipoprotein E gene (Apolipoprotein ε4, APOE4) is a strong genetic risk factor for Alzheimer's disease (AD). AD is a progressive neurodegenerative disease associated with cognitive decline and is one of the most common types of dementia. Its possible mechanisms are: APOE fragments form toxic products that inhibit the growth of neurons; APOE affects the production and clearance of amyloid β (Aβ), causing abnormal deposition of Aβ and forming plaques. APOE ε4 increases the risk of AD and cognitive dysfunction. Compared with non-APOE ε4 carriers, APOE ε4 carriers in AD show earlier Aβ deposition and clinical symptoms, more severe Aβ plaque load and brain atrophy, and faster disease progression, suggesting that APOE ε4 plays a prominent role in the pathogenesis of AD.

[0004] APOE4 is present in approximately 20% of the global population and in 40-60% of AD patients. The presence of one allele, ε4, in the APOE gene increases the risk of AD by 3-5 times, while ε4 homozygosity increases the risk by 8-12 times. In addition, APOE ε4 carriers develop the disease earlier than APOE ε4 non-carriers, and they progress faster from the prodromal stage of AD (mild cognitive impairment, MCI) to AD. Therefore, the determination of APOE ε4 can provide physicians with meaningful information about their patients' risk of developing brain amyloidosis and AD.

[0005] Genotyping by polymerase chain reaction (PCR) is the most commonly used method to determine the APOE allele status and has high accuracy, but factors such as the need for sample pretreatment, susceptibility to contamination, low automation, low throughput, the need for additional equipment, and high cost limit its application in clinical laboratories. Summary of the invention

[0006] In order to solve or partially solve the problems existing in the related technology, the present application provides an APOE4 photochemiluminescence detection reagent, kit and application. Through the coordination of three antibody molecules, APOE4 subtype proteins and APOE subtype proteins can be simultaneously detected, thereby achieving efficient detection of whether the APOE ε4 genotype exists; and by adjusting the metal salt and surfactant in the reagent, the nonspecific adsorption in the APOE4 detection system can be significantly improved, the sample differentiation can be improved, and the photochemiluminescence detection needs of APOE4 can be met.

[0007] In a first aspect, the present application provides a photo-induced chemiluminescence detection reagent for APOE4, comprising a first antibody coated on a luminescent microsphere, and a series of antibody reagents consisting of a second antibody and a third antibody labeled with one of the specific binding pair members; Among them, the first antibody and the third antibody can specifically recognize and bind to each subtype of APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

[0008] In some embodiments, the mass ratio of the luminescent microspheres to the first antibody is 10:(0.05-0.5).

[0009] In some embodiments, the series of antibody reagents further include 0.01-0.1 M buffer, 0.01%-5% (w / v) BSA, and 0.01%-5% (w / v) dextran.

[0010] In some embodiments, the buffer is selected from at least one of PBS buffer, Tris-HCl buffer, HEPES buffer, and MES buffer; preferably PBS buffer.

[0011] In some embodiments, the BSA concentration is preferably 0.1% to 3% (w / v); more preferably 0.5% to 1% (w / v).

[0012] In some embodiments, the concentration of the dextran is preferably 0.1% to 3% (w / v); more preferably 0.5% to 2% (w / v).

[0013] In some embodiments, the reagent further comprises no less than 2.5 M of a metal salt and at least 0.01% (w / v) of a surfactant.

[0014] In some embodiments, the metal salt is at least one of sodium chloride and potassium chloride, preferably sodium chloride.

[0015] In some embodiments, the surfactant is selected from at least one of Tween 20, Triton X-100, Triton X-305, and Triton X-405.

[0016] In some embodiments, the surfactant includes Tween 20 and Triton X-405; Preferably, the concentration of Tween 20 is 0.01-1% (w / v); more preferably 0.1-0.6% (w / v); Preferably, the concentration of TritonX-405 is ≥0.01% (w / v); more preferably ≥0.2% (w / v); further preferably 1%~2% (w / v).

[0017] The second aspect of the present application provides a photochemiluminescence detection kit for APOE4, comprising: R1 reagent, which comprises luminescent microspheres coated with a primary antibody, not less than 2.5 M sodium chloride, and at least 1% (w / v) TritonX-405; R2 reagent, which comprises a secondary antibody labeled with biotin; R3 reagent, which comprises a third antibody labeled with biotin; R4 reagent, which includes photosensitive microspheres labeled with streptavidin; Among them, the first antibody and the third antibody can specifically recognize and bind to each subtype of APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

[0018] The third aspect of the present application provides the use of the above-mentioned reagent or kit in detecting APOE4 in a blood sample.

[0019] In some embodiments, the detection method comprises the following steps: S1. Mix the luminescent microspheres containing the first antibody, the second antibody, the photosensitive microspheres and the sample to be tested to obtain a first complex containing "luminescent microspheres-APOE4 subtype protein-photosensitive microspheres"; at the same time, mix the luminescent microspheres containing the first antibody, the third antibody, the photosensitive microspheres and the sample to be tested to obtain a second complex containing "luminescent microspheres-APOE protein-photosensitive microspheres"; S2, irradiating the two complexes with light of a specific wavelength respectively, and detecting the luminescence value RLU1 of the first complex and the luminescence value RLU2 of the second complex to obtain the detection results of APOE4 and APOE; S3. Calculate the ratio of APOE4 to APOE.

[0020] In some embodiments, the detection method further comprises: judging the properties of the sample to be tested according to the ratio of the detection result RLU1 of APOE4 and the detection result RLU2 of APOE; If the ratio of APOE4 to APOE is ≤0.1, the sample to be tested is judged to be a negative sample; If 0.1<APOE4 to APOE ratio≤0.5, the sample to be tested is judged to be an APOE ε4 gene heterozygous positive sample; If the ratio of APOE4 to APOE>0.5, the sample to be tested is judged to be an APOE ε4 gene homozygous positive sample.

[0021] In some embodiments, in step S1, the concentration of the third antibody is ≥ the concentration of the second antibody.

[0022] The technical solution provided by this application may have the following beneficial effects: Through the coordination of three antibody molecules, the signal values ​​of the "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere" and "luminescent microsphere-APOE protein-photosensitive microsphere" complexes can be detected respectively, so as to realize the typing detection of APOE ε4 genotype by calculating the ratio of the signal values ​​of the two complexes, and the sample differentiation degree is high, which can meet the needs of distinguishing APOE4 negative samples, heterozygous samples, and homozygous samples. This APOE ε4 genotyping detection method can not only detect past samples without nucleic acid information, but also be simple and efficient, without the need for complex and expensive detection equipment, easy to operate, high throughput, low cost, and can be widely used in the clinical diagnosis of APOE4, and provide doctors with relevant information about patients' risk of cerebral amyloidosis and AD in a timely manner.

[0023] By adjusting the components included in the APOE4 photochemiluminescence detection reagent, such as the type and concentration of metal salts, the type and concentration of surfactants, etc., the nonspecific adsorption in the APOE4 detection system can be significantly improved, thereby improving the sample differentiation and meeting the photochemiluminescence detection needs of APOE4.

[0024] 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

[0025] 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. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The specific pair members described herein refer to two substances that have the ability to distinguish each other and can selectively bind to each other through covalent bonds or non-covalent bonds.

[0035] The biotin-avidin described in this article is a specific pairing member combination, which includes 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 to 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.

[0036] 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 (O 2 ·-), the two-electron reduction product hydrogen peroxide (H 2 O 2 ), the three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen ( 1 O2 )wait.

[0037] 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+ .

[0038] 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.

[0039] 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.

[0040] II. Specific implementation plan The present application will be described in more detail below.

[0041] 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.

[0042] Alzheimer's disease is a degenerative disease of the central nervous system, characterized by a persistent and comprehensive decline in memory and cognitive function. It is the most common cause of dementia and has become an important public health issue worldwide. The APOE ε4 gene is the most important genetic risk factor for sporadic Alzheimer's disease and is closely related to the pathological changes of its core biological markers, β-amyloid protein (Aβ) and tau protein, making it the most promising genetic biological marker.

[0043] Peripheral blood APOE ε4 gene detection plays an important role in risk prediction and disease assessment of Alzheimer's disease, but there are problems such as insufficient understanding and insufficient attention to APOE ε4 gene detection in clinical practice. In particular, with the development of high-quality clinical drug trials targeting Aβ or the advent of the era of clinical drug treatment, the importance of APOE ε4 gene detection has become increasingly prominent. Currently, the most commonly used APOE ε4 gene detection method, PCR, has problems such as complex sample pre-treatment, low throughput, and high cost.

[0044] The inventors of this application have found through long-term experiments and research that the application of the photochemiluminescence detection and analysis method to APOE4 detection can effectively detect the APOE4 subtype protein in the sample, thereby distinguishing APOE ε4 gene carriers from non-APOE ε4 gene carriers, and has the advantages of high efficiency, simplicity, short time, and high-throughput detection, which is conducive to providing relevant information about the patient's risk of brain amyloidosis and AD as soon as possible. However, when the photochemiluminescence detection and analysis method is applied to the detection of APOE4, since the photochemiluminescence detection and analysis method is a pure homogeneous immunoassay method, the interference of foreign proteins in the sample will increase the background noise in the detection process, thereby affecting the detection sensitivity and specificity, and affecting the accuracy of the detection results. To this end, the inventors of this application have also developed a detection reagent for photochemiluminescence detection of APOE4, which can improve the detection reagent's ability to resist foreign protein interference, reduce nonspecific adsorption in the reaction system, improve detection performance, and meet the APOE ε4 genotype differentiation detection needs.

[0045] The photoluminescent chemiluminescent detection reagent for APOE4 involved in the present application includes a first antibody coated on a luminescent microsphere, and a series of antibody reagents consisting of a second antibody and a third antibody labeled with one of the specific binding pair members; wherein the first antibody and the third antibody can specifically recognize and bind to each subtype of APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

[0046] Through the specificity of the first antibody, the second antibody and the third antibody to different subtypes of APOE proteins, the APOE4 subtype protein and all subtypes of APOE proteins in serum or plasma samples can be detected simultaneously. According to the relationship between the two test values, it is possible to accurately distinguish whether the sample provider is an APOE ε4 gene carrier or a non-APOE ε4 gene carrier. The test has high accuracy and can detect past samples without nucleic acid information. It has the advantages of automated and efficient detection, high-throughput detection, and low cost. It is beneficial for early auxiliary judgment of patients' risk of cerebral amyloidosis and AD, and has important advantages in clinical evaluation.

[0047] Specifically, the series of antibody reagents can be divided into a luminescent microsphere reagent coated with a first antibody, a second antibody reagent, and a third antibody reagent. Each reagent is prepared and stored separately.

[0048] Furthermore, the reagent also includes a metal salt of not less than 2.5M and a surfactant of at least 0.01% (w / v). Metal salts and surfactants can be used as components in reagents such as luminescent microsphere reagents, second antibody reagents, and third antibody reagents to enhance the ability of the APOE4 detection reagent to resist interference from foreign proteins, reduce nonspecific adsorption in the reaction system, thereby improving the detection signal-to-noise ratio, improving the detection signal value and discrimination, and better meeting the APOE4 typing detection requirements. Preferably, metal salts and surfactants are added to the luminescent microsphere reagent.

[0049] In some embodiments of the present application, the mass ratio of the luminescent microspheres to the first antibody is 10:(0.05-0.5). The amount of the first antibody coated on the luminescent microspheres affects the reaction rate between the first antibody and each subtype of APOE protein, thereby affecting the detection performance.

[0050] Furthermore, the luminescent microspheres have modification sites that can be combined with the first antibody in a covalent coupling manner. For example, the luminescent microspheres are modified with amino groups, carboxyl groups, aldehyde groups, etc. to achieve stable coating of the first antibody.

[0051] In some embodiments of the present application, the series of antibody reagents also include 0.01~0.1M (mol / L) buffer, 0.01%~5% (w / v) BSA, 0.01%~5% (w / v) dextran, etc. The solution composed of buffer, BSA, and dextran can be used as a solution to prepare luminescent microsphere reagents, second antibody reagents, and third antibody reagents.

[0052] The buffer may be selected from at least one of PBS buffer, Tris-HCl buffer, HEPES buffer, and MES buffer, preferably PBS buffer. The concentration of PBS buffer may be 0.01M, 0.02M, 0.05M, 0.08M, 0.1M, or any value within the above range, preferably 0.02-0.05M.

[0053] PBS buffer (Phosphate-Buffered Saline) is mainly composed of phosphates (such as sodium dihydrogen phosphate, disodium hydrogen phosphate, etc.) to maintain pH stability, reduce the effect of solution pH on the reaction, and ensure the stability and repeatability of the detection process.

[0054] The BSA is bovine serum albumin, and its concentration can be 0.01% (w / v), 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.03% (w / v), 0.5% (w / v), 0.08% (w / v), 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc., or any value within the above range, preferably 0.1%~3% (w / v); more preferably 0.5%~1% (w / v).

[0055] During the photochemiluminescence detection of APOE4, BSA can act as a stabilizer to maintain the stability and activity of antibodies or other biological molecules. Also, because it contains a large number of amino and carboxyl residues on its surface, it can bind to hydrophilic surfaces, hydrophobic surfaces, and activated covalently bound surfaces, blocking excess active sites, reducing nonspecific adsorption, and improving the accuracy and reliability of detection.

[0056] The glucan is a polysaccharide connected by glycosidic bonds, and there are types such as α-glucan and β-glucan. It can be used as a stabilizer to maintain the stability and activity of antibodies or other biological molecules in the system during the photoluminescence detection of APOE4. Its concentration can be 0.01% (w / v), 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.03% (w / v), 0.5% (w / v), 0.08% (w / v), 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc. or any value within the above range, preferably 0.1%~3% (w / v); more preferably 0.5%~2% (w / v).

[0057] In some embodiments of the present application, the series of antibody reagents include a solution consisting of 0.01~0.1M (mol / L) buffer, 0.01%~5% (w / v) BSA, 0.01%~5% (w / v) dextran, and a metal salt of not less than 2.5M and at least 0.01% (w / v) surfactant. The above solution can be used to prepare luminescent microsphere reagents, secondary antibody reagents, and tertiary antibody reagents; preferably used for luminescent microsphere reagents.

[0058] The metal salt is at least one of sodium chloride and potassium chloride, preferably sodium chloride. Its concentration is not less than 2.5M, for example, it can be 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, 6M, 7M, 8M, etc. or any value within the above range, preferably 2.5-5M.

[0059] Metal salts such as sodium chloride, potassium chloride and other electrolytes can act as stabilizers to help maintain the stability and uniformity of the solution during the photochemiluminescence detection of APOE4, especially in the configuration of luminescent microspheres and photosensitive microsphere reagents. They can also serve as stabilizers in buffer solutions to maintain appropriate pH values ​​and electrolyte balance, thereby ensuring the accuracy and repeatability of the detection.

[0060] The surfactant is selected from at least one of Tween 20, Triton X-100, Triton X-305, and Triton X-405, preferably Tween 20 and Triton X-405. As non-ionic surfactants, Tween 20 and Triton X-405 can effectively block nonspecific adsorption during the photoluminescence detection of APOE4, improve the signal-to-noise ratio, and improve the sensitivity and specificity of the detection.

[0061] Wherein, the concentration of Tween20 can be ≥0.01% (w / v); for example, 0.01% (w / v), 0.05% (w / v), 0.08% (w / v), 0.1% (w / v), 0.03% (w / v), 0.5% (w / v), 0.08% (w / v), 1% (w / v), 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), etc., or any value within the above range, preferably 0.01~1% (w / v); more preferably 0.1~0.6% (w / v). The concentration of TritonX-405 can be ≥0.01% (w / v), preferably ≥0.2% (w / v); more preferably 0.6%~2% (w / v).

[0062] Furthermore, the luminescent microsphere reagent contains no less than 2.5M sodium chloride and at least 0.6% (w / v) TritonX-405. Adjusting the salt ion concentration and surfactant concentration in the reagent can further improve the detection signal value and discrimination, and better meet the needs of APOE4 typing detection in the blood.

[0063] In some embodiments of the present application, the reagent also includes photosensitive microspheres. The photosensitive microspheres are coated with a substance that can specifically recognize and bind to one of the specific binding pair members labeled on the second antibody and the third antibody. Further, the specific pairing member can be selected from biotin-avidin. The labeling substance on the second antibody and the third antibody is preferably biotin, and the second antibody and the third antibody are labeled with biotin; the substance coated on the photosensitive microspheres is preferably avidin, and further preferably streptavidin, and the second antibody or the third antibody is fixed on the photosensitive microspheres through the specific binding reaction of streptavidin on the photosensitive microspheres and biotin on the second antibody or the third antibody, thereby shortening the distance between the photosensitive microspheres and the luminescent microspheres to meet the needs of photoinduced chemiluminescence detection.

[0064] The photochemiluminescence detection kit for APOE4 involved in the present application comprises: R1 reagent, which comprises luminescent microspheres coated with a primary antibody, not less than 2.5 M sodium chloride, and at least 1% (w / v) TritonX-405; R2 reagent, which comprises a secondary antibody labeled with biotin; R3 reagent, which comprises a third antibody labeled with biotin; R4 reagent, which includes photosensitive microspheres labeled with streptavidin; Among them, the first antibody and the third antibody can specifically recognize and bind to each subtype of APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

[0065] The above detection reagents and kits involved in the present application can be used to detect APOE4 in blood samples, such as serum and plasma samples.

[0066] The photoluminescence detection method of APOE4 involved in the present application specifically comprises the following steps: S1. Mix the luminescent microspheres containing the first antibody, the second antibody, the photosensitive microspheres and the sample to be tested to obtain a first complex containing "luminescent microspheres-APOE4 subtype protein-photosensitive microspheres"; at the same time, mix the luminescent microspheres containing the first antibody, the third antibody, the photosensitive microspheres and the sample to be tested to obtain a second complex containing "luminescent microspheres-APOE protein-photosensitive microspheres"; S2, irradiating the two complexes with light of a specific wavelength respectively, and detecting the luminescence value RLU1 of the first complex and the luminescence value RLU2 of the second complex to obtain the detection results of APOE4 and APOE; S3. Calculate the ratio of APOE4 to APOE.

[0067] When the target substance to be tested, i.e., APOE4 subtype protein, is present in the sample to be tested, since the second antibody only recognizes the unique sequence of APOE4 subtype protein, and the first antibody can recognize the common sequence of all subtypes of APOE protein, the first antibody and the second antibody can bind to the APOE4 subtype protein to form a sandwich immune complex of "luminescent microsphere-(first antibody)-APOE4 subtype protein-(second antibody)-photosensitive microsphere", i.e., the first complex. When the target substance to be tested, i.e., all subtypes of APOE protein, is present in the sample to be tested, since the first antibody and the third antibody can both recognize the common sequence of all subtypes of APOE protein, they can bind to all subtypes of APOE protein to form a sandwich immune complex of "luminescent microsphere-(first antibody)-APOE protein-(third antibody)-photosensitive microsphere", i.e., the second complex. Therefore, the detection reagent or kit of the present application can simultaneously detect the APOE4 subtype protein and all APOE subtype proteins in the blood sample, and then determine whether the sample to be tested carries the APOE ε4 gene through the ratio of the two, and judge whether it is homozygous or heterozygous. This automated detection method can realize the automated detection of the APOE ε4 genotype, which 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 evaluation of the risk of cerebral amyloidosis.

[0068] In order to further improve the detection signal value and discrimination and better meet the detection needs of APOE4 in the blood, metal salts such as sodium chloride at a concentration of not less than 2.5 M and surfactants such as Triton X-405 at a concentration of not less than 0.6% (w / v) are added to the luminescent microsphere reagent.

[0069] The step S1 further comprises: S11, using a reagent containing 0.01~0.1M (mol / L) buffer, 0.01%~5% (w / v) BSA, 0.01%~5% (w / v) dextran, a metal salt of not less than 2.5M, and at least 0.6% (w / v) surfactant to redissolve the luminescent microspheres containing the first antibody, and obtain the R1 reagent; using a reagent containing 0.01~0.1M (mol / L) buffer, 0.01%~5% (w / v) BSA, and 0.01%~5% (w / v) dextran to redissolve the second antibody and the third antibody, respectively, to obtain the R2 reagent and the R3 reagent; S12, mixing the R1 reagent, the R2 reagent and the photosensitive microsphere reagent with the sample to be tested to obtain a first complex comprising "luminescent microsphere-APOE4 subtype protein-photosensitive microsphere"; S13. Mix the R1 reagent, R3 reagent and photosensitive microsphere reagent with the sample to be tested to obtain a second complex containing "luminescent microsphere-APOE protein-photosensitive microsphere".

[0070] The detection method involved in the present application utilizes three antibody molecules, namely, the first antibody, the second antibody and the third antibody, to respectively capture the APOE4 subtype protein and all APOE subtype proteins in the sample to be tested, and through the ratio of the two, efficient typing detection of the APOEε4 genotype can be achieved.

[0071] Specifically, when the above-mentioned reagent, kit or detection method of the present application detects APOE4 in a blood sample, the concentration of the third antibody used in step S1 is ≥ the concentration of the second antibody.

[0072] In some embodiments of the present application, the detection method further comprises: S4, judging the properties of the sample to be tested according to the ratio of the detection result of APOE4 to the detection result of APOE. If the ratio of APOE4 to APOE is ≤0.1, the sample to be tested is judged to be a negative sample (non-APOE ε4 gene carrier); If 0.1<APOE4 to APOE ratio≤0.5, the sample to be tested is judged to be an APOE ε4 gene heterozygous positive sample (i.e., an APOE ε4 gene carrier); If the ratio of APOE4 to APOE is greater than 0.5, the sample to be tested is judged to be an APOE ε4 gene homozygous positive sample (APOE ε4 gene carrier).

[0073] By forming two sandwich immune complexes, "luminescent microspheres-(first antibody)-APOE4 subtype protein-(second antibody)-photosensitive microspheres" and "luminescent microspheres-(first antibody)-APOE protein-(third antibody)-photosensitive microspheres", the APOE4 subtype protein and all APOE subtype protein contents in blood samples can be detected simultaneously. The APOE4 / APOE 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 judgment and detection of the APOE ε4 genotype. It is simple and efficient, does not require complex and expensive detection equipment, is easy to operate, has low requirements for complex equipment and detection personnel, and can achieve high throughput, low cost, and can detect past samples without nucleic acid information. It can be widely used in the clinical diagnosis of APOE4 and has important advantages for patient stratification in clinical trials, AD prevention strategies, and clinical assessment of the risk of cerebral amyloidosis.

[0074] 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.

[0075] Next, taking the target molecule to be detected as APOE protein as an example, the antibody molecules with specific recognition of all subtypes of APOE protein sequences were coated onto luminescent microspheres, and the antibody molecules that can specifically recognize all subtypes of APOE protein sequences and can specifically recognize a single subtype of APOE4 protein sequence were labeled with biotin, and the double antibody sandwich method was used to detect the target APOE4 protein or APOE protein in the sample. The conventional detection sequence (sample + R1 reagent + R2 or R3 reagent reaction followed by addition of universal reagent (R4 reagent)) was used for detection, and the detection discrimination between different experimental groups was compared to determine the effects of the photochemiluminescence detection analysis method and the method of adjusting the salt ion concentration and surfactant concentration in the diluent on the typing detection performance of APOEε4.

[0076] Example 1: Preparation of luminescent microspheres coated with antibodies and biotin-labeled antibodies The main experimental materials and equipment are shown in Table 1: Table 1

[0077] APOE Ab1 and APOE Ab3 are APOE protein antibodies, and APOE Ab2 is an APOE4 subtype protein antibody.

[0078] 2. Experimental Procedure 2.1 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 in a ratio of 10: (0.05-0.5), react at 37°C for 24 hours; ④ Add 80 μL glycine (75 mg / mL, 0.05 M CB) and 10 μL NaBH to the reaction system. 4 (20 mg / mL, 0.05 M CB), 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 was added for ultrasonic reconstitution to obtain luminescent microparticles coated with APOE Ab1.

[0079] Table 2 Basic reconstitution solution

[0080] 2.2 Biotin-labeled Antibody APOE Ab2 and Antibody APOE Ab3 ① Use 0.05M NaHCO 3 The solution was dialyzed against APOE Ab2 or antibody APOE Ab3 to remove impurities and the concentration was determined 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.

[0081] Example 2: APOE4 typing test 1. Experimental Procedure 1.1 Preparation of 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).

[0082] 1.2 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.

[0083] 1.3 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.

[0084] 1.4 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).

[0085] Example 3: Effects of sodium chloride and Triton X-405 on APOE ε4 typing test results 1. Experimental Procedure 1.1 Based on Example 1, reconstituted solutions with different sodium chloride concentrations were prepared as shown in Table 3: Table 3 Reconstitution solution with sodium chloride added

[0086] 1.2 Based on Example 1, different Triton surfactant solutions were prepared as shown in Table 4: Table 4 Reconstitution solution with Triton surfactant added

[0087] 1.3 Based on Example 1, TritonX-405 reconstitution solutions with different concentrations were prepared as shown in Table 5: Table 5 Reconstitution solution with TritonX-405 added

[0088] 1.4 Based on Example 1, a complex solution containing sodium chloride and TritonX-405 was prepared as shown in Table 6: Table 6 Adjustment of sodium chloride and TritonX-405 concentrations in the reconstitution solution

[0089] 1.5 APOE4 typing test was performed as shown in Example 2. The R1 reagent in the experimental group used the reconstitution solution shown in Tables 3 to 6 above; the control group, as well as the R2 reagent and the R3 reagent in the experimental group all used the reconstitution solution shown in Table 2.

[0090] 2. Experimental results Table 7 Test results of control group

[0091] Table 8 Test results of adjusting sodium chloride concentration

[0092] Table 9 Adjustment of Triton surfactant test results

[0093] Table 10 Test results of adjusting TritonX-405 concentration

[0094] Table 11 Test results of adjusting sodium chloride and TritonX-405 content

[0095] 3. Results Analysis 3.1 Experimental data analysis ① Combined with Table 7 and Figure 1 As shown, when sodium chloride and TritonX-405 were not added to the luminescent microsphere reagent of the detection reagent, 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 to the luminescent microsphere reagent 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 surfactants such as TritonX-100, TritonX-305, and TritonX-405 to the luminescent microsphere reagent, 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 to the luminescent microsphere reagent increases, the ratio of negative samples decreases and the ratio of homozygous samples increases, but there are still samples that cannot be distinguished; ⑤ Combined with Table 11 and Figure 2 As shown, when sodium chloride and TritonX-405 were added to the luminescent microsphere reagent at the same time, and 3.5M sodium chloride + 1% (w / v) 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.

[0096] 3.2 Experimental Conclusion The detection reagent of APOE4, especially when high concentration of salt ions and high concentration of surfactants are added to the luminescent microsphere reagent, can help improve the ability of the detection reagent of APOE4 to resist interference from foreign proteins, significantly improve nonspecific adsorption during detection, reduce the ratio of negative samples, and make the distinction between heterozygous samples and homozygous samples clear, thus meeting the needs of distinguishing and detecting negative samples, heterozygous samples and homozygous samples of APOE4.

[0097] 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: A series of antibody reagents comprising a first antibody coated on a luminescent microsphere, and a second antibody and a third antibody labeled with one of the specific binding pair members; Among them, the first antibody and the third antibody can specifically recognize and bind to each subtype of 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 mass ratio of the luminescent microspheres to the first antibody is 10:(0.05-0.5).

3. The reagent according to claim 1, characterized in that The series of antibody reagents also include 0.01-0.1M buffer, 0.01%-5% (w / v) BSA, and 0.01%-5% (w / v) dextran.

4. The reagent according to any one of claims 1 to 3, characterized in that The reagent further comprises no less than 2.5 M of a metal salt and at least 0.01% (w / v) of a surfactant.

5. The reagent according to claim 4, characterized in that The metal salt is at least one of sodium chloride and potassium chloride; The surfactant is selected from at least one of Tween 20, Triton X-100, Triton X-305 and Triton X-405.

6. The reagent according to claim 5, characterized in that The surfactant includes Tween 20 and Triton X-405; the concentration of the Tween 20 is 0.01-1% (w / v), and the concentration of the Triton X-405 is ≥ 0.01% (w / v).

7. A photochemiluminescence detection kit for APOE4, characterized in that: include: R1 reagent, which comprises luminescent microspheres coated with a primary antibody, not less than 2.5 M sodium chloride, and at least 0.6% (w / v) TritonX-405; R2 reagent, which comprises a secondary antibody labeled with biotin; R3 reagent, which comprises a third antibody labeled with biotin; R4 reagent, which includes photosensitive microspheres labeled with streptavidin; Among them, the first antibody and the third antibody can specifically recognize and bind to each subtype of APOE protein, and the second antibody can specifically recognize and bind to the APOE4 subtype protein.

8. Use of the reagent according to any one of claims 1 to 6, or the kit according to claim 7, in detecting APOE4 in a blood sample.

9. The use according to claim 8, characterized in that: The detection method comprises the following steps: S1. Mix the luminescent microspheres containing the first antibody, the second antibody, the photosensitive microspheres and the sample to be tested to obtain a first complex containing "luminescent microspheres-APOE4 subtype protein-photosensitive microspheres"; at the same time, mix the luminescent microspheres containing the first antibody, the third antibody, the photosensitive microspheres and the sample to be tested to obtain a second complex containing "luminescent microspheres-APOE protein-photosensitive microspheres"; S2, irradiating the two complexes with light of a specific wavelength respectively, and detecting the luminescence value RLU1 of the first complex and the luminescence value RLU2 of the second complex to obtain the detection results of APOE4 and APOE; S3. Calculate the ratio of APOE4 to APOE.

10. The use according to claim 9, characterized in that: The detection method further includes judging the properties of the sample to be tested based on the ratio of the detection result of APOE4 to the detection result of APOE: If the ratio of APOE4 to APOE is ≤0.1, the sample to be tested is judged to be a negative sample; If 0.1<APOE4 to APOE ratio≤0.5, the sample to be tested is judged to be an APOE ε4 gene heterozygous positive sample; If the ratio of APOE4 to APOE>0.5, the sample to be tested is judged to be an APOE ε4 gene homozygous positive sample.

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