Kit for joint detection and preparation method thereof

Abstract

The invention discloses a kit for joint detection and a preparation method thereof, and relates to the field of medical detection. The kit for joint detection comprises a magnetic bead coated with a capture antibody, an enzyme-labeled antibody compound, a fluorescein-labeled antibody and a luminous substrate, and the magnetic bead coated with the capture antibody comprises a first antibody specifically bound with Tau217 and a first antibody specifically bound with NfL. The method comprises the following steps: mixing and incubating an object to be detected with captured antibody-coated magnetic beads, an enzyme-labeled antibody compound and a fluorescein-labeled antibody, injecting 630nm exciting light into an instrument, automatically collecting a light signal 1, carrying out a contact reaction on the compound and a luminous substrate, automatically collecting a 470nm light signal 2 emitted by an enzyme catalysis substrate by the instrument, and determining whether the light signal 2 is 470nm or not. The content of two to-be-detected substances, namely Tau217 and NfL, is identified through different optical signals, two items, namely phosphorylated Tau 217 and neural light chain protein, are jointly detected, the detection sensitivity is improved, the time required by the two items of detection is shortened to the time required by one item of detection, and the detection efficiency of the Alzheimer's disease marker is improved.

Description

Technical Field

[0001] This invention relates to the field of medical testing, and more specifically to a reagent kit for combined detection and its preparation method. Background Art

[0002] In the field of neurodegenerative disease research and diagnosis, early identification of Alzheimer's disease (AD), as the most common type of dementia, is crucial for effective intervention. Traditionally, the diagnosis of AD has relied on clinical assessment, changes in biomarkers in cerebrospinal fluid (CSF) analysis, and expensive positron emission tomography (PET) imaging techniques. However, these methods are either invasive or costly, limiting their application in large-scale screening and routine clinical practice.

[0003] Tau protein is a microtubule-associated protein that regulates the stability of microtubules in nerve cells. It contains numerous phosphorylation sites; aberrantly phosphorylated Tau protein inhibits microtubule assembly, leading to its disassembly. Phosphorylation of threonine at site 217 forms Tau217, which has good specificity and sensitivity for the early diagnosis of neurodegenerative diseases. Traditional immunoassay for Tau 217 in cerebrospinal fluid can diagnose early-stage dementia, but cerebrospinal fluid sampling is complex, and it is difficult to sample and test in asymptomatic individuals, limiting its application in early diagnosis. Blood biomarker testing is a more ideal approach, as it can be performed concurrently with routine health checkups.

[0004] Neurofilament light chain protein (NfL) is a subunit of neurofilaments. It is a cylindrical protein located in the cytoplasm of neurons and helps maintain the stability of neuronal structure. NfL has a total protein content of 543 amino acids (aa), containing two glycosylation sites. Aa 2-543 amino acids form the main chain. Aa 2-92 amino acids form the head of the chain, which is the antibody-binding region; aa 93-396 amino acids form the chain body, whose unique mass and high charge density distinguish it from other neurofilament proteins, but it shares very high homology with other neurofilaments; aa 397-543 amino acids form the tail of the chain, influencing its spatial structure. Under normal circumstances, axons release low levels of NfL, which gradually increases with age. Under pathological conditions, axonal damage to the central nervous system caused by inflammation, neurodegeneration, trauma, or vascular injury can cause a sharp increase in NfL release. Released NfL crosses the blood-brain barrier via cerebrospinal fluid into the peripheral blood. NfL is closely related to various neurological diseases. Therefore, the detection of NfL is of great guiding significance for the diagnosis, treatment and prognosis of neurological diseases such as Alzheimer's disease and Parkinson's disease.

[0005] Currently, most diagnostic products for neurodegenerative diseases detect single biomarkers, such as Tau217 and NfL. However, single-marker testing not only has low sensitivity but also takes a long time. Although there are existing technologies that use combined detection methods, such as the combined detection kit and application for Alzheimer's disease disclosed in publication number CN116930510A, this actually involves detecting multiple biomarkers individually and then combining the diagnostic results of each biomarker based on an algorithm model. In essence, it is still a separate detection of each biomarker, and it also suffers from the problems of low sensitivity and long detection time. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing technology has low detection sensitivity and long detection time for Tau217 and NfL. The purpose is to provide a kit for joint detection and its preparation method, which solves the problems of low sensitivity and long detection time.

[0007] This invention is achieved through the following technical solution:

[0008] A kit for combined detection includes magnetic beads coated with capture antibodies, an enzyme-labeled antibody complex, a fluorescently labeled antibody, and a luminescent substrate.

[0009] The capture antibody-coated magnetic beads include a first antibody that specifically binds to Tau217 and a first antibody that specifically binds to NfL. Specifically, the capture antibody-coated magnetic beads include magnetic beads coated with Tau217 antibody and magnetic beads coated with NfL antibody.

[0010] The first antibody that specifically binds to Tau217 can specifically recognize the Tau217 fragment of the tau protein, facilitating the capture of the target antigen in the analyte and enabling convenient enrichment of these antigen-antibody complexes from the mixture via magnetic separation.

[0011] The first antibody that specifically binds to NfL can specifically recognize NfL, facilitating the capture of target antigens in the analyte, and these antigen-antibody complexes can be conveniently enriched from the mixture by magnetic separation.

[0012] The enzyme-labeled antibody complex is a complex containing alkaline phosphatase (ALP), which is linked to a Tau217 monoclonal antibody 2 via a cross-linking agent. The ALP-labeled antibody 2 can specifically bind to other sites of the antigen in the preceding complex, forming an immune complex with an "antibody-antigen-antibody" sandwich structure. ALP catalyzes the emission of light from the APS-5 substrate, and this signal can be monitored. The magnitude of the light intensity indirectly reflects the concentration of the analyte.

[0013] The fluorescein-labeled antibody is a fluorescently labeled antibody obtained by conjugating an NfL monoclonal secondary antibody with APC (allophycocyanin) fluorescein. During detection, this labeled antibody directly binds to NfL in the analyte, forming a fluorescent signal that can be detected by the optical receiving module of a flow cytometer or other equipment. The fluorescence properties of APC allow it to be excited and detected at a specific wavelength, thereby quantifying the NfL content.

[0014] Preferably, the magnetic beads coated with the above-mentioned capture antibody comprise the following raw materials: carboxyl magnetic beads, Tau217 monoclonal primary antibody, NfL monoclonal primary antibody, 1% bovine serum albumin (BSA), 0.1M Tris (tris(hydroxymethyl)aminomethane), PC300 (preservative ProClin300), tween20 (polyoxyethylene sorbitan monolaurate), and PC950 (preservative ProClin950).

[0015] Preferably, the Tau217 antibody-coated magnetic beads are prepared by the following method:

[0016] 1. Activation of carboxyl magnetic beads: Carboxyl magnetic beads (concentration 1.0 mg / ml) were activated at room temperature for 30 min using 20 mg / ml carbodiimide (EDC), with a mass ratio of EDC to magnetic beads of 1:5.

[0017] 2. Carboxyl magnetic beads coated with protein A: At room temperature, the activated carboxyl magnetic beads and protein A were mixed, and 20 μg of protein A was added per milligram of magnetic beads. The reaction was carried out for 1 hour.

[0018] 3. Protein A magnetic beads linked to Tau217 monoclonal antibody: Add 20 μg of Tau217 monoclonal antibody to each milligram of protein A magnetic beads and react at room temperature for 1 hour;

[0019] 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads. Add 50 μl of 20% BSA per milligram of magnetic beads, mix well at room temperature, and incubate for 1 hour.

[0020] 5. Clean and adjust the volume of the magnetic beads.

[0021] NfL antibody-coated magnetic beads:

[0022] 1. Activation of carboxyl magnetic beads: Carboxyl magnetic beads (concentration 1.0 mg / ml) were activated at room temperature for 30 min using 20 mg / ml carbodiimide (EDC), with a mass ratio of EDC to magnetic beads of 1:5.

[0023] 2. Carboxyl magnetic beads coated with protein A: At room temperature, the activated carboxyl magnetic beads and protein A were mixed, and 20 μg of protein A was added per milligram of magnetic beads. The reaction was carried out for 1 hour.

[0024] 3. Protein A magnetic beads are linked to NfL monoclonal primary antibody: 20 μg NfL monoclonal primary antibody is added to each milligram of protein A magnetic beads, and the reaction is carried out at room temperature for 1 hour;

[0025] 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads. Add 50 μl of 20% BSA per milligram of magnetic beads, mix well at room temperature, and incubate for 1 hour.

[0026] 5. Clean and adjust the volume of the magnetic beads.

[0027] Preferably, the enzyme-labeled antibody complex comprises the following raw materials: Tau217 monoclonal secondary antibody, alkaline phosphatase, 1% bovine serum albumin, 0.1M Tris, PC300, Tween20, PC950, 0.1M MgCl2 buffer, and 0.01M ZnCl2 buffer.

[0028] Preferably, the above-mentioned enzyme-labeled antibody complex is prepared by the following method:

[0029] 1. ALP activation: Add 30 μL of 50 mL MSulfo-SMCC to a 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight;

[0030] 2. Activated ALP-labeled Tau217 monoclonal secondary antibody;

[0031] (1) Add 2.4 μL of 2IT (2-iminothiacyclopentane, 50 mM) to 0.2 mg of Tau217 monoclonal secondary antibody, mix and react at pH 8.5 and room temperature for 30 min, and dialyze overnight;

[0032] (2) 0.2 mg of 2IT-conjugated Tau217 monoclonal secondary antibody was added to 48 μL of activated ALP and reacted at pH 7.4 and room temperature for 2 hours.

[0033] 3. Dilute and bring the enzyme working solution to a fixed volume.

[0034] Preferably, the fluorescein-labeled antibody comprises the following raw materials: NfL monoclonal secondary antibody, APC fluorescein, sodium dihydrogen phosphate, disodium hydrogen phosphate, 1% bovine serum albumin, and PC300.

[0035] Preferably, the APC-labeled antibody is prepared by the following method:

[0036] 1. APC preparation: Weigh an appropriate amount of APC, dialyze overnight with PBS, recover and calculate the concentration;

[0037] 2. Activation of APC: Prepare a 10 mg / ml Sulfo-SMCC solution with DMSO (dimethyl sulfoxide), add 6 μl of Sulfo-SMCC per mg of APC, rotate in an aluminum foil-wrapped reaction tube at room temperature for 60 minutes, dialyze overnight with PBS (phosphate buffered saline) to obtain SMCC-APC;

[0038] 3. NfL monoclonal secondary antibody reduction: NfL monoclonal secondary antibody was dialyzed with PBS, recovered and adjusted to a final antibody concentration of 4 mg / ml. 1 MDTT (dithiothreitol) was prepared with pure water. 20 μl of DTT solution was added to 1 ml of antibody, mixed well and allowed to stand at room temperature for 30 minutes.

[0039] 4. Activated fluorescein conjugates reduced NfL monoclonal secondary antibody: 1 mg NfL monoclonal secondary antibody is added to 1.5 mg SMCC-APC and rotated for 60 minutes in a foil-wrapped reaction tube at room temperature;

[0040] 5. Termination: Dissolve the prepared 10 mg / ml NEM solution in DMSO, add 34 μg of NEM (N-ethylmaleimide) solution to 1 mg of reduced NfL monoclonal secondary antibody, and rotate at room temperature for 20 minutes.

[0041] 6. Purification: Separate the above substances by chromatography column and bring the volume up to a final volume using fluorescent antibody working solution.

[0042] As one possible design, the enzyme-labeled antibody complex described above is a secondary antibody that specifically binds to Tau217; the fluorescein-labeled antibody is a secondary antibody that specifically binds to NfL.

[0043] As one possible design, both the enzyme-labeled antibody complex and the fluorescein-labeled antibody described above are monoclonal antibodies.

[0044] As one possible design, both the first antibody that specifically binds to Tau217 and the first antibody that specifically binds to NfL are monoclonal antibodies.

[0045] As one possible design, the aforementioned luminescent substrate is the APS-5 substrate.

[0046] Preferably, the above kit also includes a cleaning solution containing 50 mM Tris, 1.8% NaCl, 0.1% ProClin 950, and 0.05% Tween 20.

[0047] A method for preparing a reagent kit for combined detection includes the following steps:

[0048] Preparation of magnetic beads coated with capture antibodies;

[0049] Preparation of enzyme-labeled antibody complexes;

[0050] Prepare fluorescently labeled antibodies.

[0051] As one possible design, the preparation of the capture antibody-coated magnetic beads described above includes the following steps:

[0052] Activation of magnetic beads;

[0053] The activated magnetic beads were linked to a first antibody that specifically binds to Tau217 and a first antibody that specifically binds to NfL, respectively.

[0054] Magnetic beads specifically bound to a first antibody that binds to Tau217 and a first antibody that binds to NfL were blocked.

[0055] By activating the magnetic beads, it is easier to covalently link the primary antibody to the magnetic beads. After the activated magnetic beads bind to the Tau217 primary antibody and the NfL primary antibody respectively, they are blocked, which can block the remaining active groups on the surface of the magnetic beads that are not occupied by the antibody, reduce non-specific binding, and improve the specificity of detection.

[0056] Preferably, the magnetic beads are carboxyl magnetic beads.

[0057] Preferably, the above-mentioned magnetic bead activation is carried out by coupling agents DCC (N,N'-dicyclohexylcarboimide) and NHS (N-hydroxysuccinimide).

[0058] Preferably, the magnetic beads are activated and then coupled with protein A.

[0059] By activating magnetic beads with DCC and NHS and then coupling them with protein A, protein A is chemically immobilized onto the magnetic beads, increasing the chain length of the linker and reducing steric hindrance. Protein A can purify the target linker protein, improving labeling sensitivity. The magnetic beads can also wash away other impurities in the raw materials, reducing the need for dialysis. Furthermore, protein A can link the Fc terminus of the monoclonal antibody, causing the Fab terminus of the monoclonal antibody to face outwards in a specific direction, thus enabling more efficient binding to the analyte antigen in the sample.

[0060] Preferably, the above-mentioned closure is carried out with 20% BSA.

[0061] As one possible design, the preparation of the enzyme-labeled antibody complex includes the following steps:

[0062] Alkaline phosphatase is coupled with cross-linking agent 1;

[0063] The second antibody that specifically binds to Tau217 is coupled with cross-linking agent 2 to obtain the labeled second antibody;

[0064] The conjugated alkaline phosphatase is conjugated with a conjugated labeled second antibody to obtain an enzyme-labeled antibody complex.

[0065] Alkaline phosphatase is bound by cross-linking agent 1, and a stable covalent bond is formed by the reaction of the NHS ester group with the amino group on ALP, allowing ALP to carry the end of Sulfo-SMCC. Then, Tau217 secondary antibody is bound by cross-linking agent 2, and its thiol group reacts with the lysine residue of the antibody to introduce a thiol group that readily reacts with Sulfo-SMCC. The reaction of the two results in an enzyme-labeled antibody complex (ALP-SMCC-2-IT-antibody complex).

[0066] Preferably, the crosslinking agent 1 is Sulfo-SMCC (succinimide-4-[N-maleimidemethyl]cyclohexane-1-carboxylate), and the crosslinking agent 2 is 2-IT (2-iminothione).

[0067] As one possible design, zinc chloride and magnesium chloride were also added to the enzyme-labeled antibody complex.

[0068] Adding zinc chloride and magnesium chloride can increase ALP activity and enhance the structural stability of the enzyme.

[0069] As one possible design, the preparation of the fluorescently labeled antibody includes the following steps:

[0070] The fluorescein was activated with an activation solution to obtain an activated fluorescein solution;

[0071] The second antibody that specifically binds to NfL is activated with a reducing agent to obtain a reduced second antibody;

[0072] The reduced second antibody is mixed with the activated fluorescein and reacted to obtain a fluorescein-labeled antibody.

[0073] Preferably, the activating solution is Sulfo-SMCC and the reducing agent is DTT.

[0074] Preferably, the above-mentioned activated second antibody and fluorescein are mixed and reacted at room temperature for 60 minutes. After the reaction, a stop agent is added to terminate the reaction at room temperature, and finally the mixture is separated by a chromatography column.

[0075] The NfL secondary antibody was reduced to enable it to conjugate with fluorescein; then it was covalently bound to fluorescein. After the reaction, a terminator was added to reduce nonspecific fluorescence, and the unbound fluorescein and other impurities were removed by chromatography to ensure the purity of the labeled antibody.

[0076] This invention involves coating magnetic beads with Tau217 primary antibody and NfL primary antibody, respectively. The analyte is then mixed with the coated magnetic beads, enzyme-labeled antibody complex, and enzyme-labeled antibody complex and incubated. After washing, a mixture of magnetic beads (Tau217 monoclonal primary antibody) - analyte - enzyme (Tau217 monoclonal secondary antibody) and magnetic beads (NfL monoclonal primary antibody) - APC fluorescein (NfL monoclonal secondary antibody) is formed. The mixture is then excited with light at a wavelength of 630 nm. The light signal 1 (approximately 660 nm) emitted by the fluorescein and the light signal 2 (approximately 470 nm) emitted by the enzyme-catalyzed substrate are received. The content of NfL and Tau217 are calculated based on the light signal 1 and light signal 2, respectively.

[0077] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0078] This invention involves incubating the analyte with magnetic beads coated with capture antibodies, an enzyme-labeled antibody complex, and a fluorescein-labeled antibody. First, the analyte is injected at the wavelength required to excite fluorescence and the light signal 1 is collected. Then, after contact with the luminescent substrate, a photomultiplier tube is used to receive the light signal 2 emitted by the enzyme-catalyzed substrate at approximately 470 nm. By collecting different light signals, the levels of two analytes, Tau217 and NfL, are identified. This invention combines the detection of phosphorylated Tau 217 (Tau217) and neurolactic light chain protein (NfL), improving detection sensitivity while reducing the time required for two separate detections to that of a single detection, thus increasing the detection efficiency of Alzheimer's disease biomarkers.

[0079] Specifically, this invention coats carboxyl magnetic beads with protein A. The binding of protein A and monoclonal antibodies has the following three advantages: 1. It increases the chain length of the linker and reduces steric hindrance; 2. Protein A can purify the target linker protein, improve labeling sensitivity, and wash away other impurities in the raw material through the adsorption of magnetic beads, reducing dialysis steps; 3. Protein A is linked to the Fc end of the monoclonal antibody, which can make the Fab end of the monoclonal antibody face outward in a specific direction, thus binding to the antigen to be tested in the sample more efficiently.

[0080] For Alzheimer's disease projects, existing technologies use chemiluminescence reagents for Alzheimer's disease testing as separate tests, and testing all tests simultaneously takes a long time. This invention, which combines two tests, can save half the time. DETAILED DESCRIPTION

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The illustrative embodiments and descriptions of this invention are for explanation only and are not intended to limit the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0082] Current Alzheimer's disease detection products typically detect only a single biomarker, resulting in low sensitivity. Improving sensitivity usually involves detecting multiple biomarkers and analyzing the combined results, but this leads to time-consuming testing, complex processing, and low efficiency.

[0083] Based on this, the inventors combined and simultaneously detected Tau 217 and NfL. By considering the different wavelengths of the light signals emitted by the substrate, the detection and content calculation of the two markers can be carried out simultaneously, thereby improving detection sensitivity and shortening the retrieval time.

[0084] The magnetic beads used in this embodiment and comparative example were purchased from JSR's MS300 carboxylated magnetic beads.

[0085] Example 1

[0086] A kit for combined detection, comprising:

[0087] Capture antibody-coated magnetic beads: Tau217 antibody-coated carboxyl magnetic beads, where the Tau217 antibody is a Tau217 monoclonal antibody specific to the Tau217 antigen in the sample; NfL antibody-coated carboxyl magnetic beads, where the NfL antibody is an NfL monoclonal antibody specific to the NfL antigen; the ratio of Tau217 antibody-coated carboxyl magnetic beads to NfL antibody-coated carboxyl magnetic beads is 1:1.

[0088] Enzyme-labeled antibody complex: a Tau217 monoclonal antibody solution conjugated with alkaline phosphatase that is specific to the Tau217 antigen in the sample;

[0089] Fluorescein-labeled antibody: NfL monoclonal second antibody linked with fluorescein; enzyme-labeled antibody complex: fluorescein-labeled antibody = 1:2;

[0090] Cleaning solution: contains 50 mM Tris, 1.8% NaCl, 0.1% ProClin 950, and 0.05% Tween 20;

[0091] Luminescent substrate: APS-5, sourced from HanHai New Enzyme.

[0092] In the samples to be tested, the concentrations of Tau217 were 0 ng / mL, 0.6 ng / mL, 1.5 ng / mL, 3 ng / mL, 10 ng / mL, and 20 ng / mL, respectively; and the concentrations of NfL were 0 ng / mL, 15 ng / mL, 50 ng / mL, 250 ng / mL, 1000 ng / mL, and 2000 ng / mL, respectively.

[0093] The magnetic beads coated with capture antibodies were prepared by the following method:

[0094] A. Carboxyl magnetic beads coated with Tau217 antibody:

[0095] 1. Activation of carboxyl magnetic beads: Carboxyl magnetic beads (concentration 1.0 mg / ml) were activated at room temperature for 30 min using 20 mg / ml carbodiimide (EDC), with a mass ratio of EDC to magnetic beads of 1:5.

[0096] 2. Carboxyl magnetic beads coated with protein A: At room temperature, the activated carboxyl magnetic beads and protein A were mixed, and 20 μg of protein A was added per milligram of magnetic beads. The reaction was carried out for 1 hour.

[0097] 3. Protein A magnetic beads linked to Tau217 monoclonal antibody: Add 20 μg of Tau217 monoclonal antibody to each milligram of protein A magnetic beads and react at room temperature for 1 hour;

[0098] 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads. Add 50 μl of 20% BSA per milligram of magnetic beads, mix well at room temperature, and incubate for 1 hour.

[0099] 5. Clean and adjust the volume of the magnetic beads.

[0100] B. Carboxyl magnetic beads coated with NfL antibody:

[0101] 1. Activation of carboxyl magnetic beads: Carboxyl magnetic beads (concentration 1.0 mg / ml) were activated at room temperature for 30 min using 20 mg / ml carbodiimide (EDC), with a mass ratio of EDC to magnetic beads of 1:5.

[0102] 2. Carboxyl magnetic beads coated with protein A: At room temperature, the activated carboxyl magnetic beads and protein A were mixed, and 20 μg of protein A was added per milligram of magnetic beads. The reaction was carried out for 1 hour.

[0103] 3. Protein A magnetic beads are linked to NfL monoclonal primary antibody: 20 μg NfL monoclonal primary antibody is added to each milligram of protein A magnetic beads, and the reaction is carried out at room temperature for 1 hour;

[0104] 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads. Add 50 μl of 20% BSA per milligram of magnetic beads, mix well at room temperature, and incubate for 1 hour.

[0105] 5. Clean and adjust the volume of the magnetic beads.

[0106] The enzyme-labeled antibody complex was prepared by the following method:

[0107] 1. ALP activation: Add 30 μL of 50 mM Sulfo-SMCC dropwise to a 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight;

[0108] 2. Activated ALP-tagged Tau217 monoclonal secondary antibody;

[0109] (1) Add 2.4 μL of 2IT (2-iminothiacyclopentane, 50 mM) to 0.2 mg of Tau217 monoclonal secondary antibody, mix and react at pH 8.5 and room temperature for 30 min, and dialyze overnight;

[0110] (2) 0.2 mg of 2IT-conjugated Tau217 monoclonal secondary antibody was added to 48 μL of activated ALP and reacted at pH 7.4 and room temperature for 2 hours.

[0111] 3. Dilute and bring the enzyme working solution to a fixed volume.

[0112] APC-labeled antibodies were prepared by the following method:

[0113] 1. APC preparation: Weigh an appropriate amount of APC, dialyze overnight with PBS, recover and calculate the concentration;

[0114] 2. Activation of APC: Prepare a 10 mg / ml Sulfo-SMCC solution with DMSO (dimethyl sulfoxide), add 6 μl of Sulfo-SMCC per mg of APC, rotate in an aluminum foil-wrapped reaction tube at room temperature for 60 minutes, dialyze overnight with PBS (phosphate buffered saline) to obtain SMCC-APC;

[0115] 3. NfL monoclonal secondary antibody reduction: NfL monoclonal secondary antibody was dialyzed with PBS, recovered and adjusted to a final antibody concentration of 4 mg / ml. 1M DTT (dithiothreitol) was prepared with pure water. 20 μl of DTT solution was added to 1 ml of antibody, mixed well and allowed to stand at room temperature for 30 minutes.

[0116] 4. Activated fluorescein conjugates reduced NfL monoclonal secondary antibody: 1 mg NfL monoclonal secondary antibody is added to 1.5 mg SMCC-APC and rotated for 60 minutes in a foil-wrapped reaction tube at room temperature;

[0117] 5. Termination: Dissolve the prepared 10 mg / ml NEM solution in DMSO, add 34 μg of NEM (N-ethylmaleimide) solution to 1 mg of reduced NfL monoclonal secondary antibody, and rotate at room temperature for 20 minutes.

[0118] 6. Purification: Separate the above substances by chromatography column and bring the volume up to a final volume using fluorescent antibody working solution.

[0119] Comparative Example 1

[0120] This comparative example is basically the same as Example 1, except that: the magnetic beads are carboxyl magnetic beads that have not been treated with protein A; and the label is a conventional alkaline phosphatase-labeled antibody.

[0121] A kit for detecting Tau217 includes:

[0122] Capture antibody-coated magnetic beads: Carboxyl magnetic beads coated with Tau217 antibody, which is a Tau217 monoclonal antibody that is specific to the Tau217 antigen in the sample;

[0123] Enzyme-labeled antibody complex: a Tau217 monoclonal antibody solution conjugated with alkaline phosphatase that is specific to the Tau217 antigen in the sample;

[0124] Cleaning solution: contains 50 mM tris, 1.8% NaCl, 0.1% ProClin 950, and 0.05% Tween 20.

[0125] The concentrations of Tau217 in the samples tested were 0 ng / mL, 0.6 ng / mL, 1.5 ng / mL, 3 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0126] The magnetic beads coated with capture antibodies were prepared by the following method:

[0127] Tau217 antibody-coated carboxyl magnetic beads:

[0128] 1. Activation of carboxyl magnetic beads: Carboxyl magnetic beads (concentration 1.0 mg / ml) were activated at room temperature for 30 min using 20 mg / ml carbodiimide (EDC), with a mass ratio of EDC to magnetic beads of 1:5.

[0129] 2. Coating Tau217 monoclonal antibody with carboxyl magnetic beads: Add 20 μg of Tau217 monoclonal antibody to 1 magnetic bead and react at room temperature for 1 hour;

[0130] 3. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads. Add 50 μl of 20% BSA to 1 magnetic bead, mix well at room temperature, and incubate for 1 hour.

[0131] 4. Clean and stabilize the magnetic beads.

[0132] The enzyme-labeled antibody complex was prepared by the following method:

[0133] 1. ALP activation: Add 30 μL of 50 mM Sulfo-SMCC dropwise to a 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight;

[0134] 2. Activated ALP-tagged Tau217 monoclonal secondary antibody;

[0135] (1) Add 2.4 μL of 2IT (2-iminothiacyclopentane, 50 mM) to 0.2 mg of Tau217 monoclonal secondary antibody, mix and react at pH 8.5 and room temperature for 30 min, and dialyze overnight;

[0136] (2) 0.2 mg of 2IT-conjugated Tau217 monoclonal secondary antibody was added to 48 μL of activated ALP and reacted at pH 7.4 and room temperature for 2 hours.

[0137] Comparative Example 2

[0138] This comparative example is basically the same as comparative example 1, except that: the magnetic beads are carboxyl magnetic beads coated with NfL antibody that have not been treated with protein A; and the label is a conventional alkaline phosphatase-labeled antibody.

[0139] Example 1 and Comparative Examples 1-2 were used to test Alzheimer's disease. Blood samples from Alzheimer's patients were obtained, processed, and tested using a reagent kit. The data were recorded.

[0140] Detection Procedure: The instrument is operated on a fully automated chemiluminescence immunoassay analyzer. 50 μL of the plasma sample to be tested is added to the reaction vessel using the sampling needle. After cleaning the needle, 30 μL of a mixture of magnetic beads coated with Tau217 monoclonal primary antibody and NfL monoclonal primary antibody is aspirated. Then, 100 μL of a mixture of enzyme working solution labeled with Tau217 monoclonal secondary antibody and fluorescein working solution labeled with NfL monoclonal secondary antibody is added to the reaction vessel. The reaction is incubated at 37°C for 20 min. After adsorption, the waste liquid is aspirated and discarded. 300 μL of washing buffer is added, and the waste liquid is discarded after washing (repeated 3 times). At this point, 200 μL of washing buffer is added to the reaction vessel, and the instrument is switched to the excitation position. Excitation light at a wavelength of 630 nm is introduced, and the first reading light signal is automatically acquired. Then, after adsorption, the waste liquid is aspirated and discarded. APS-5 is added to the reaction vessel. After incubating with 200 μL for 2 min, the instrument rotates the reaction vessel to the PMT reading position. The instrument automatically collects the light signal 2 emitted by the enzyme-catalyzed substrate. The two emission values ​​are substituted into the two standard curves of the kit, and the instrument automatically fits and calculates the concentration of the analyte.

[0141] The data analysis results are as follows:

[0142] I. Experimental data for Tau217:

[0143] Control reagents: The magnetic beads are carboxyl magnetic beads coated with antibodies using the current process; the label is alkaline phosphatase-labeled antibody with a wavelength of 470 nm.

[0144] Reagents for the experimental group: The magnetic beads were carboxyl magnetic beads coated with the antibody of the present invention; the label was alkaline phosphatase-labeled antibody with a wavelength of 470 nm.

[0145] Table 1

[0146]

[0147] Table 2

[0148]

[0149]

[0150] Reference range: <1.8 pg / mL

[0151] II. NfL Experimental Data:

[0152] Control reagents: The magnetic beads are carboxyl magnetic beads coated with antibodies using the current process; the label is alkaline phosphatase-labeled antibody with a wavelength of 470 nm.

[0153] Reagents for the experimental group: The magnetic beads are carboxyl magnetic beads coated with the antibody of the present invention; the label is a fluorescently labeled antibody with a wavelength of 630 nm.

[0154] Table 3

[0155]

[0156] Table 4

[0157]

[0158]

[0159] Reference range: <17 pg / mL

[0160] Conclusion: 1. In both Example 1 and Comparative Example 1 of the tau217 project, the alkaline phosphatase label is in liquid phase. However, the solid phase of Example 1 is magnetic beads coated with a special process, which effectively improves the reagent sensitivity and the test results of the sample are more consistent with the clinical positive and negative judgment.

[0161] 2. Example 1 of the NfL project uses a reagent with a special solid-phase labeling process and a fluorescein-labeled liquid phase. Comparative Example 2 uses reagents with current solid-phase antibody coating and alkaline phosphatase labeling processes. Example 1, being a fluorescent label, has a lower signal, but its sensitivity and linearity meet the requirements. Clinical sample testing is consistent with clinical diagnosis and can be used.

[0162] 3. The reagent in Example 1 takes 32 minutes to produce results for two items from one sample. Comparative Examples 1 and 2 are single-item tests, with each item taking approximately 30 minutes to test.

[0163] This invention involves incubating the analyte with magnetic beads coated with capture antibodies, an enzyme-labeled antibody complex, and a fluorescein-labeled antibody. After washing, the mixture is first incubated at a wavelength that excites fluorescence and the light signal 1 is collected. Then, after contacting the luminescent substrate, a photomultiplier tube is used to receive the light signal 2 emitted by the enzyme-catalyzed substrate at approximately 470 nm. By collecting different light signals, the levels of two analytes, Tau217 and NfL, are identified. The combined detection of phosphorylated Tau 217 (Tau217) and neurolactic light chain protein (NfL) improves detection sensitivity and reduces the time required for two detections to that required for one detection, thereby increasing the detection efficiency of Alzheimer's disease biomarkers.

[0164] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for combined detection, characterized in that: It includes capture antibody-coated magnetic beads, enzyme-labeled antibody complexes, fluorescein-labeled antibodies, and luminescent substrates. The capture antibody-coated magnetic beads include a first antibody that specifically binds to Tau217 and a first antibody that specifically binds to NfL.

2. A kit for combined detection according to claim 1, characterized in that: The enzyme-labeled antibody complex is a second antibody that specifically binds to Tau217; the fluorescein-labeled antibody is a second antibody that specifically binds to NfL.

3. A kit for combined detection according to claim 1 or 2, characterized in that: The enzyme-labeled antibody complex and the fluorescein-labeled antibody are both monoclonal antibodies.

4. A kit for combined detection according to claim 1, characterized in that: The first antibody that specifically binds to Tau217 and the first antibody that specifically binds to NfL are both monoclonal antibodies.

5. A kit for combined detection according to claim 1, characterized in that: The luminescent substrate is an APS-5 substrate.

6. A method for preparing a kit for combined detection according to any one of claims 1 to 5, characterized in that: The steps include: preparing capture antibody-coated magnetic beads; preparing enzyme-labeled antibody complexes; Prepare fluorescein-labeled antibodies.

7. The method for preparing a kit for combined detection according to claim 6, characterized in that: The preparation of magnetic beads coated with capture antibodies comprises the following steps: Activate magnetic beads; The activated magnetic beads are respectively connected to a first antibody that specifically binds to Tau217 and a first antibody that specifically binds to NfL; The magnetic beads to which the primary antibody specifically binding to Tau217 and the primary antibody specifically binding to NfL were respectively linked were blocked.

8. The method for preparing a kit for combined detection according to claim 6, characterized in that: The preparation of the enzyme-labeled antibody complex comprises the following steps: Alkaline phosphatase was coupled to cross-linker 1; coupling a second antibody that specifically binds to Tau217 with a cross-linking agent 2 to obtain a labeled second antibody; The coupled alkaline phosphatase is coupled to the coupled labeled second antibody to obtain an enzyme-labeled antibody complex.

9. The method for preparing a kit for combined detection according to claim 8, characterized in that: Zinc chloride and magnesium chloride are also added to the enzyme-labeled antibody complex.

10. The method for preparing a kit for combined detection according to claim 6, characterized in that: The preparation of the fluorescein-labeled antibody comprises the following steps: Activating the fluorescein with an activation solution to obtain an activated fluorescein solution; activating the second antibody that specifically binds to NfL with a reducing agent to obtain a reduced second antibody; The reduced secondary antibody is mixed with the activated fluorescein to react and obtain the fluorescein-labeled antibody.

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