A homogeneous immunoassay kit for Alzheimer's disease

Abstract

The present invention discloses a homogeneous immunoassay kit for Alzheimer's disease, and belongs to the field of biological detection technology. The influence of high-abundance proteins and non-specific interfering substances is reduced by dilution, and a sample volume 50-100 times more than the sample volume of other instruments is adopted, and the sensitivity is improved by enriching the antigens in the blood. The method of enrichment can be used to detect with a fast full-automatic biochemical analyzer, and there is no need to purchase expensive special instruments to detect the project. The wash-free reaction of homogeneous immunity is adopted. When the semi-enzyme-labeled double antibody and the antigen to be detected are combined, the semi-enzyme will be complementary and combined to form an active enzyme due to the spatial proximity of the semi-enzyme, and then the multi-enzyme coupling amplification effect is used to enhance the effect of the catalytic substrate and produce a color development effect, so the antigen is quantitatively detected by the change of enzyme activity. The method can improve the sensitivity and specificity of detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a homogeneous immunoassay kit for Alzheimer's disease. Background Art

[0002] Diagnosis of Alzheimer's disease (AD) requires a combination of symptoms, signs, psychological tests, and biomarker measurements. In clinical practice and clinical practice, positron emission tomography (PET) imaging and Ab42, T-Tau, and P-T181-Tau concentrations in cerebrospinal fluid (CSF) are recommended as diagnostic biomarkers for the disease. However, the high cost of PET limits its application in the clinic. CSF is considered the best source of AD biomarkers because direct contact between CSF and the brain allows it to reflect pathophysiological changes occurring in the central nervous system (CNS). Ab42 in CSF is highly correlated with pathological changes in autopsy and PET images of the brain, confirming its effectiveness for AD diagnosis. However, lumbar puncture is an invasive procedure, and repeated collection of CSF is challenging. Compared with CSF, blood measurements are advantageous for AD biomarker screening These findings suggest that peripheral blood may still be a promising source for screening AD biomarkers. In addition, studies have shown that Ab42, T-Tau and P-T181-Tau P-T217-Tau in blood neuronal exosomes can distinguish AD patients from control patients. Tau protein is a phosphoprotein present in normal brain tissue and is a type of microtubule-associated protein. The microtubule system plays an important role in intercellular material transport and signal transmission. Studies have found that in the early stages of Alzheimer's disease, Tau protein in the patient's brain tissue usually undergoes abnormal phosphorylation and accumulation, and the total amount of Tau protein increases significantly. Overphosphorylated Tau protein greatly interferes with the normal operation of the microtubule system, impairs normal axonal transport, causes synaptic loss and neuronal damage, and leads to brain neurodegenerative lesions. Therefore, detecting the content of Tau protein can help to diagnose Alzheimer's disease early and reduce the risk of disease.

[0003] Immunoassays and ultrasensitive platforms for detecting Tau in biological fluids. Several analytical methods have been tested and extensively validated in cerebrospinal fluid and plasma. The current list of the most commonly used assays in the field of tau biomarkers includes (1) standard and second-generation ELISA (INNOTEST, EUROIMLUN, Elecsys), (2) single-molecule enzyme-linked immunosorbent assay (SiMoA), and (3) mass spectrometry, with detection ranges from a few pg / mL to fg / mL depending on the sensitivity of the assay. EUROIMLUN and Elecsys are automated second-generation immunoassays. The initial tests were based on ELISA (enzyme-linked immunosorbent assay), but the results were equivocal at best. In these early attempts, interference from plasma proteins could be significant. ELISA has been gradually improved to achieve higher sensitivity, and other platforms have been developed, including SiMoA (single-molecule array), mass spectrometry. To overcome the complexity of blood and the interference of blood proteins and heterophilic antibodies as well as low concentrations of target biomarkers,

[0004] The Simoa immunoassay is a digital ELISA that determines the analyte concentration digitally without measuring the total analog signal. Simply put, after the immune complex is formed on the magnetic beads, the beads are transferred to thousands of wells, each of which is only large enough to accommodate one bead. By confining the fluorophore produced by a single enzyme to a very small volume, a high concentration of fluorescent product can be obtained. Therefore, even if only a single complex is present on the beads, a readable signal can be detected. The analyzer counts the number of wells containing enzyme-labeled beads and the number of wells containing beads, and the concentration of the target analyte is calculated by the average enzyme label value for each bead number and the standard curve. Although Simoa has a high sensitivity, issues such as antibody combination during the detector capture process should be considered to try to avoid noise signals and maximize analytical performance.

[0005] Mass spectrometry methods This technique uses specific antibodies to separate and enrich P-TAU peptide fragments from plasma by immunoprecipitation (IP), and then uses high-sensitivity mass spectrometry (MS) to identify and relatively quantify individual P-TAU. Although the basic principles of the methods are similar, there are huge differences in their applications. Kaneko and Nakamura et al. used matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF / MS), while Ovod and Keshavan et al. used liquid chromatography-tandem mass spectrometry (LC-MS / MS). Pannee et al. used MALDI-TOF / MS mass spectrometry analysis followed by LC-MS / MS to confirm the identity of the P-Tau peptide. Compared with other mass spectrometry methods, LC-MS / MS generates multiple mass spectra for each fragment peptide, and these mass spectra are compared with a database for protein identification. In MALDI-TOF / MS, only one mass spectrum is generated to compile the masses of all peptides. Therefore, MALDI-TOF / MS provides faster analysis and higher read-in, which is more suitable for large-scale studies. However, MALDI-TOF / MS lacks the real sequence that the control data relies on in protein identification, resulting in low accuracy and reliability of its detection. The above-mentioned new generation platform can perform very high dilution of samples and amplify specific signals, allowing the detection of pg / mL (or lower) concentrations of target proteins. Therefore, a more sophisticated and complex instrument for measuring signal values ​​is required, which results in such instruments being expensive and difficult to use for clinical testing.

[0006] The following are the main problems that interfere with analyte detection in blood tests for markers that are meaningful for predicting Alzheimer's disease, such as P-Tau217 and P-Tau181 (1). The first category includes pre-analytical factors (sample storage, anticoagulants), anti-analyte antibodies, and hormone-binding proteins that can change the measurable concentration of the analyte in the sample; the second group is non-specific antibodies, such as heterophilic antibodies, human anti-animal antibodies (HAAA), and human anti-mouse antibodies (HAMA), which are present in biological fluids and can interfere with antibody binding in the test. In addition, unlike plasma (about 7 grams of protein / 100 ml), there is only about 0.025 grams of protein / 100 ml in CSF - mainly albumin. Given the different compositions of CSF and blood, it is relatively easy to predict the different sensitivities of these two different biological fluids to assay interference, and the difficulty of detecting such markers in CSF is relatively lower than that in blood. Summary of the invention

[0007] In order to solve the above problems existing in the prior art, the present invention aims to provide a homogeneous immunoassay kit for Alzheimer's disease.

[0008] The technical solution adopted by the present invention is: a homogeneous immunoassay kit for Alzheimer's disease, the kit comprising a capture antibody detection solution coupled to a carrier, a specific antibody-B detection solution, a washing solution and an in vitro sample diluent;

[0009] The carrier includes magnetic beads or latex particles.

[0010] The present invention provides a homogeneous immunoassay kit for Alzheimer's disease, which is a new antibody that can be labeled with paramagnetic magnetic beads or latex particles. The labeled antibody is added to the sample to capture the antigen to be tested, thereby enriching the antigen to be tested. The antigen to be tested in the enriched sample improves the sensitivity of detection. The kit increases the sample volume of the reaction (plasma sample volume 500μL-1mL, 10-50 times higher than other methods) and adds a diluent that can remove nonspecific interference in the blood, while reducing the interference of substances such as high-abundance proteins (such as albumin) and rheumatoid factors in the blood on the test object.

[0011] The full-length phoA gene of Escherichia coli alkaline phosphatase was used as a template for error-prone PCR, and the product was cloned into the PEK48 vector. The host was transformed to screen a mutant with high enzyme activity. EAP is basically a homodimeric protein, and the complete EAP molecule presents a typical α, β topological structure. The gene fragment was disassembled into the α region active center containing the ASP-SER-Ala triplet region, the β region Arg water molecule and three metal ions and their ligand amino acid region. Hereinafter referred to as α-phoX, β-phoX half enzyme. The two half enzymes prepare the capture antibody α-phoX labeled monoclonal capture antibody, and the specific antibody-B is β-phoX labeled monoclonal specific antibody-B.

[0012] Preferably, the capture antibody detection solution is composed of Α-phoX labeled monoclonal capture antibody, stabilizer and buffer;

[0013] The specific antibody-B detection solution is composed of Β-phoX labeled monoclonal specific antibody-B, a substrate, a stabilizer and a buffer;

[0014] Preferably, the stabilizer includes one or more of disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate manganese, zinc ethylenediaminetetraacetate, polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90;

[0015] Preferably, the buffer comprises one or more of a tris(hydroxymethylaminomethane) buffer, a 3-(N-morpholino)propanesulfonic acid buffer, a 2-(N-morpholino)ethanesulfonic acid buffer and a 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer;

[0016] Preferably, the substrate is prepared from 80-100 mL ol / L AMP buffer having a pH value of 8.0 to 11.0 and 80-100 mL ol / L p-nitrobenzene phosphate.

[0017] Preferably, the A-phoX labeled monoclonal capture antibody and the B-phoX labeled monoclonal specific antibody-B are prepared by splitting the monomeric alkaline phosphatase phoX gene into two half-enzyme genes, A-phoX and B-phoX, respectively, and then cloning and fusion expressing them.

[0018] Preferably, the method for preparing the A-phoX labeled monoclonal capture antibody coupled to a carrier comprises:

[0019] The Α-phoX semi-enzyme gene was selected and cloned into the expression vector Antibody_1-A, and the Α-phoX-labeled monoclonal capture antibody was obtained after fusion expression, and the Α-phoX-labeled monoclonal capture antibody was covalently linked to the magnetic beads to obtain.

[0020] Preferably, the preparation method of the B-phoX labeled monoclonal specific antibody-B comprises:

[0021] The Β-phoX semienzyme gene was selected and cloned into the expression vector Antibody_2-B, and the Β-phoX labeled monoclonal specific antibody-B was obtained after fusion expression.

[0022] Preferably, the in vitro sample diluent is composed of 5-8 mL ol / L buffer, 20-32 mL ol / L sodium chloride solution, and less than 0.1 wt % of a surfactant and a protective agent;

[0023] The pH value of the in vitro sample diluent is 7.0-7.2.

[0024] The above-mentioned in vitro sample diluent is set to solve the shielding effect of high-abundance proteins in the blood matrix on the markers to be tested through dilution and pretreatment, and reduce non-specific antibody interference, so as to ensure the detection amount for subsequent tests.

[0025] Preferably, the protective agent includes one or more of dextran, mannitol, lactitol and sorbitol;

[0026] The surfactant includes one or more of Tween-20, Tween-80, Triton X-100, Brij-35, Thesit and alkyl glycoside.

[0027] Preferably, the washing solution comprises any one of 50 mL pH 6.0 2-morpholineethanesulfonic acid solution, 1 wt% BSA solution, 50 mg / mL EDC solution and 50 mg / mL NHS solution.

[0028] Application of a homogeneous immunoassay kit for Alzheimer's disease in detecting Tau-217 protein in in vitro samples.

[0029] The beneficial effects of the present invention are:

[0030] (I) The present invention provides a homogeneous immunoassay kit for Alzheimer's disease, which is a new antibody that can be labeled with paramagnetic magnetic beads or latex particles. The labeled antibody is added to the sample to capture the antigen to be tested, thereby enriching the antigen to be tested. The antigen to be tested in the enriched sample improves the detectable sensitivity. The kit increases the sample volume for the reaction (plasma sample volume 500μL-1mL, 10-50 times higher than other methods) and adds a diluent that can remove nonspecific interference in the blood, while reducing the interference of substances such as high-abundance proteins (such as albumin) and rheumatoid factors in the blood on the test object.

[0031] The impact of high-abundance proteins and non-specific interfering substances is reduced by dilution. At the same time, the sample volume is 50-100 times larger than that of other instruments. The sensitivity is improved by enriching the antigens in the blood.

[0032] The enrichment method can be used with a fast, fully automatic biochemical analyzer, eliminating the need to purchase expensive, dedicated instruments to test the project.

[0033] (ii) Using a homogeneous immune wash-free reaction, the monomeric G6PDH enzyme, alkaline phosphatase, β-D-galactosidase, etc. are split into two semi-monomeric enzymes through genetic recombination. The enzymes are inactive after splitting and are fused and expressed with the antibody pair for immunoassay. When the semi-enzyme-labeled double antibody and the antigen to be tested are combined, the semi-enzymes will complement each other due to their spatial proximity to form an active enzyme. The multi-enzyme coupling amplification effect is then used to enhance the effect of the catalytic substrate and produce a color development effect. Therefore, the antigen quantifies the substance to be tested through changes in enzyme activity. This method can improve the sensitivity and specificity of detection.

[0034] (III) The kit can detect p-Tau217, P-Tau181, or total P-TAU in ex vivo blood samples using reagents of different methodologies, such as ELISA and homogeneous immunochemical analysis, with improved detection sensitivity and specificity compared to existing reagents on the market. DETAILED DESCRIPTION

[0035] The present invention will be further explained below in conjunction with specific embodiments. It will be appreciated by those skilled in the art that the following described embodiments are part of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. All reagents used are conventional products that can be purchased commercially.

[0036] Embodiment 1:

[0037] Process flow:

[0038] 1. Screen suitable Tau protein antibodies, conduct initial affinity screening for purchased and homemade antibodies, find 4-5 groups of antibodies with the best affinity from more than a dozen groups of samples, accurately measure the affinity between antigen and antibody (obtain accurate dissociation constant Kd, binding constant Ka and affinity constant KD), and finally select the antibody with the best affinity.

[0039] 2. Screening of suitable buffers, stabilizers, preservatives and surfactants

[0040] The buffer is one or more of Tris buffer (tris(hydroxymethyl)aminomethane buffer), MOPS buffer (3-(N-morpholino)propanesulfonic acid buffer), MES buffer (2-(N-morpholino)ethanesulfonic acid buffer), and MOPSO buffer (3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer).

[0041] Stabilizer: one or more of EDTA·2Na, EDTA·3Na, EDTA·2NaMn, EDTA·Zn, PVP-K30 (polyvinyl pyrrolidone K30), PVP-K90 (polyvinyl pyrrolidone K90). Stabilizers can be used to maintain the stability of antibodies in the system.

[0042] Protective agent: one or more of dextran, mannitol, lactitol and sorbitol. Used to protect the lactate dehydrogenase-glycocholic acid conjugate in the system to make it more stable and avoid inactivation.

[0043] Surfactant: one or more of Tween-20, Tween-80, Triton X-100, Brij-35, Thesit, and APG (alkyl glycoside).

[0044] Substrate: AMP buffer (pH 8.0-11.0) 80-100mLol / L, p-nitrobenzene phosphate (PNPP) 80-100mLol / L.

[0045] 3. Synthesize the semi-enzyme through genetic engineering, and prepare the capture antibody and specific antibody labeled with the semi-enzyme.

[0046] 4. The preparation method of the reagent comprises the following steps:

[0047] a) A-phoX labeled monoclonal capture antibody-A was covalently attached to carboxyl-terminated magnetic beads with a diameter of 10 μm using a standard coupling method based on 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0048] b) Take 200 microliters of magnetic beads containing monoclonal capture antibodies labeled with Α-phoX, add them to the diluted sample and incubate for 30 minutes, then place them on a magnetic base for 1 minute, remove the supernatant, and add 100 microliters of sample diluent to mix. Load the above-treated capture antigen sample into the fully automatic biochemical analyzer, aspirate 35 microliters of sample, then add 100 microliters of R1 component Β-phoX labeled monoclonal specific antibody-B, incubate for 5 minutes, and then add R2 alkaline phosphatase substrate, detect the efficiency of enzyme catalysis of substrate by rate method, and quantify the content of P-Tau217 in the sample.

[0049] The principle of homogeneous immunoassay for Alzheimer's disease is a sandwich-based reaction. Through genetic recombination, monomeric G6PDH enzyme, alkaline phosphatase, β-D-galactosidase, etc. are split into two semi-monomer enzymes. After being split, the enzymes are inactive and fused with the antibody pair for immunoassay. When the semi-enzyme-labeled double antibody and the antigen to be detected are combined, the semi-enzyme will be complementary due to the spatial proximity of the semi-enzyme to form an active enzyme. Then, the multi-enzyme coupling amplification effect is used to enhance the effect of the catalytic substrate and produce a color development effect. Therefore, the antigen quantifies the substance to be detected through the change of enzyme activity. This method can improve the sensitivity and specificity of the detection. At the same time, in order to improve the detection sensitivity and accuracy of the method, the sample volume of the reaction is increased (plasma sample volume 500μL-1mL, 10-50 times higher than other methods) and the antigen to be detected in the sample to be enriched with the capture antibody is added to improve the detectable sensitivity, and a diluent that can remove nonspecific interference in the blood is added. At the same time, the interference of substances such as high-abundance proteins (such as albumin) and rheumatoid factors in the blood on the substance to be detected can be reduced.

[0050] 5. Select standard substances, prepare manufacturer calibration products, set instrument detection parameters, and generate calibration curves

[0051] 6. Reagent performance verification and stability testing

[0052] 7. Preparation of recombinant capture monoclonal antibodies

[0053] Each Tau deletion mutant was verified by DNA sequencing. All Tau deletion mutants and Tau peptides were cloned into the expression plasmid pET-17b (Novagen) based on the longest human Tau and expressed in bacteria.

[0054] The production of Tau protein involves the following steps:

[0055] a) Expression of Tau in bacteria;

[0056] b) purifying Tau by ion exchange chromatography;

[0057] c) purifying Tau by gel filtration;

[0058] d) Concentrating and storing the isolated Tau.

[0059] a) Bacterial expression of human full-length Tau (2N4R) and recombinant Tau deletion mutants: Human Tau (above) expression plasmid 84 was introduced into Escherichia coli (E. coli) production strain BL21 (DE3). Bacterial cells containing appropriate expression plasmids were cultured and induced. A single colony of BL21 (DE3) bacteria transformed with pET-17b plasmid driving the expression of Tau protein or its fragments was cultured at 37°C in 500 mL of Luria broth medium with 100 μg / ml ampicillin at 300 rpm and induced by adding isopropyl-β-D-1-thiogalactoside (IPTG) to a final concentration of 0.4 mL. After further incubation at 37°C for 3 hours, the bacteria were collected by centrifugation at 3,000 x g for 15 minutes at 4°C.

[0060] b) Includes basic and neutral Tau mutants:

[0061] Cation exchange chromatography purification of Thr217Ala, Ser231Ala, Tau221-441, Tau99-441, Tau188-44, Tau31-441, Tau151-391, Tau2N4RΔ(134-168), Tau2N4RΔ(49-243) was performed essentially as described above. After expression, the bacterial pellet was resuspended in 10 ml lysis buffer (50 mL 1,4-piperazinediethanesulfonic acid (PIPES) pH 6.9, 50 mL sodium chloride (NaCl), 1 mL ethylenediaminetetraacetic acid (EDTA), 5 mL dithiothreitol (DTT), 0.1 mL phenylmethylsulfonyl fluoride (PMSF), 5% (v / v) glycerol), quickly frozen in liquid nitrogen and stored at -80°C until used for purification of Tau protein.

[0062] For Tau protein purification, frozen bacterial suspensions were quickly thawed and placed on ice. Bacterial cell walls were disrupted by sonication on ice and by the following conditions: lysate was clarified by centrifugation (21,000 x g, 15 min at 4°C) and the supernatant was filtered through a 0.45 μm membrane filter. Large-scale purification of recombinant Tau protein was performed at 6°C.

[0063] Fractions containing Tau protein were pooled and purified by a second cation exchange chromatography step. Acidic Tau protein was purified by anion exchange chromatography.

[0064] c) In the final gel filtration step of the purification (same for all Tau proteins), the

[0065] The resulting combined Tau protein fractions were injected onto a gel filtration column at 3 ml / min in PIPES or histidine lysis buffer supplemented with 100 mL NaCl for alkaline / neutral or acidic Tau proteins, respectively. The eluted Tau proteins were combined. Protein quantification was performed on the purified samples. The Tau proteins were divided into working aliquots, snap-frozen in liquid nitrogen, and stored at -70°C.

[0066] Step 2: Obtaining a secondary antibody-specific monoclonal antibody cell line: Referring to the amino acid sequence of p-Tau217, select a site with strong antigenicity to artificially synthesize a polypeptide sequence of about 40 amino acids, and use a standard monoclonal antibody preparation method to prepare a monoclonal antibody cell line with high specificity and affinity. Perform pairing experiments and affinity determination experiments on the monoclonal antibodies corresponding to the obtained cell lines, and determine the capture antibody and detection antibody based on the experimental results;

[0067] Step 2: Preparation of monoclonal antibodies: Prepare and purify the p-Tau monoclonal antibodies for detection using the standard ascites production process, and store them at -20°C for later use after aliquoting;

[0068] Step 3: Split the monomeric alkaline phosphatase phoX gene into two half-enzyme genes, Α-phoX and Β-phoX;

[0069] Biochemical analyzer parameter setting example: P-TAU-217 homogeneous enzyme immunoassay;

[0070] Obtain standard curve: Set the reaction parameters of Roche 702 fully automatic biochemical analyzer (see Table 1).

[0071]

[0072]

[0073] 5 samples of different concentrations were collected from the clinic, and the test was repeated 21 times to find the mean value and calculate the coefficient of variation. The coefficient of variation was less than 10%.

[0074] serum Mean (pg / ml) Coefficient of variation Sample 1 36.7 6.8％ Sample 2 67.8 3.7％ Sample 3 129.6 3.3％ Sample 4 266.8 2.8％ Sample 5 466.8 2.9％

[0075] Preparation of P-TAU217 Standard

[0076] Three custom peptides produced by New England Peptide were used to calibrate the assay (calibration peptides), which were reconstituted to 5 mg / ml in 0.1% phosphoric acid / water, aliquoted into 20 μl and frozen. When ready for use, the calibration peptide aliquots were thawed and diluted 1:1000 (e.g., 1.5 μl was diluted into 1498.5 μl), and the dilutions were diluted 1:100 to a final concentration of 500 pg / ml of the peptide. Five concentration point lines were made starting from 30 pg / ml: 60 pg / ml, 120 pg / ml, 240 pg / ml, 500 pg / ml.

[0077] The present invention is not limited to the above optional implementation modes. Anyone can obtain other various forms of products under the enlightenment of the present invention, which belong to the protection scope of the present invention. The above specific implementation modes should not be understood as limiting the protection scope of the present invention. Ordinary technicians in this field should understand that without departing from the scope of the present invention, the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features therein can be replaced by equivalents. At the same time, these modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A homogeneous immunoassay kit for Alzheimer's disease, characterized in that: The kit comprises a capture antibody detection solution coupled to a carrier, a specific antibody-B detection solution, a washing solution and an in vitro sample diluent; The in vitro sample diluent is composed of 5-8 mLol / L buffer, 20-32 mLol / L sodium chloride solution, less than 0.1 wt% of a surfactant and a protective agent; the pH value of the in vitro sample diluent is 7.0-7.2; The capture antibody is an A-phoX labeled monoclonal capture antibody, and the specific antibody-B is a B-phoX labeled monoclonal specific antibody-B; The carrier includes magnetic beads; The kit also includes standards, and the preparation steps of the standards are as follows: three standards produced by New England Peptide are used to calibrate the assay, the standards are reconstituted to 5 mg / ml in 0.1% phosphoric acid / water, aliquoted into 20 μl and frozen; when ready for use, the standard aliquot is thawed and diluted 1:1000, and the diluent is diluted 1:100 so that the final concentration of the standard is 500 pg / ml; starting from 30 pg / ml, five concentration point lines of 60 pg / ml, 120 pg / ml, 240 pg / ml, and 500 pg / ml are made; The Α-phoX labeled monoclonal capture antibody and the Β-phoX labeled monoclonal specific antibody-B are prepared by splitting the monomeric alkaline phosphatase phoX gene into two half-enzyme genes, Α-phoX and Β-phoX, respectively, and cloning and fusion expression; The method for preparing the carrier-coupled Α-phoX labeled monoclonal capture antibody comprises: The Α-phoX semi-enzyme gene was selected and cloned into the expression vector Antibody_1-A, and the Α-phoX-labeled monoclonal capture antibody was obtained after fusion expression, and the Α-phoX-labeled monoclonal capture antibody was covalently linked to the magnetic beads to obtain; The preparation method of the B-phoX labeled monoclonal specific antibody-B comprises: The Β-phoX semienzyme gene was selected and cloned into the expression vector Antibody_2-B, and the Β-phoX labeled monoclonal specific antibody-B was obtained after fusion expression; The kit detects Tau-217 protein.

2. The homogeneous immunoassay kit for Alzheimer's disease according to claim 1, characterized in that: The capture antibody detection solution coupled to the carrier is composed of Α-phoX labeled monoclonal capture antibody, stabilizer and buffer; The specific antibody-B detection solution is composed of Β-phoX labeled monoclonal specific antibody-B, a substrate, a stabilizer and a buffer; The stabilizer includes one or more of disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate manganese, zinc ethylenediaminetetraacetate, polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90; The buffer comprises one or more of a tris(hydroxymethylaminomethane) buffer, a 3-(N-morpholino)propanesulfonic acid buffer, a 2-(N-morpholino)ethanesulfonic acid buffer and a 3-(N-morpholino)-2-hydroxypropanesulfonic acid buffer; The substrate is prepared from 80-100 mol / L AMP buffer with a pH value of 8.0-11.0 and 80-100 mol / L p-nitrobenzene phosphate.

3. The homogeneous immunoassay kit for Alzheimer's disease according to claim 2, characterized in that: The protective agent includes one or more of dextran, mannitol, lactitol and sorbitol; The surfactant includes one or more of Tween-20, Tween-80, Triton X-100, Brij-35, Thesit and alkyl glycoside.

4. The homogeneous immunoassay kit for Alzheimer's disease according to claim 1, characterized in that: The washing solution includes any one of 50 mL of pH 6.0 2-morpholineethanesulfonic acid solution, 1 wt% BSA solution, 50 mg / mL EDC solution and 50 mg / mL NHS solution.

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