Phosphorylated 181tau protein fluorescence immunoassay method for improving sensitivity
Through the fluorescence immunoassay method of bead-coated and biotin-labeled antibodies binding to SAPE-PE, the problem of low sensitivity of phosphorylated 181tau protein in the blood is solved, and high sensitivity and low cost detection is achieved, which is suitable for large-scale use.
Patent Information
- Application Number
- CN202411244419.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-05
- Publication Date
- 2024-12-24
AI Technical Summary
In the prior art, the method of detecting phosphorylated 181tau protein in the blood is not sensitive, and the operation is complicated and costly, which limits large-scale use.
The magnetic bead-coated p-tau-181 monoclonal antibody and biotin-labeled tau monoclonal antibody were used, combined with SAPE-PE, and the detection was improved by flow cytometry to achieve fluorescence immunoassay.
High sensitivity detection of p-tau-181 in the blood is achieved, reducing detection cost and operational complexity, and can quantitatively, quickly and simply detect pg/mL-level markers to meet clinical needs.
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Figure CN119178880A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protein fluorescence immunoassay, and in particular to a fluorescence immunoassay method for phosphorylated 181tau protein with improved sensitivity. Background Art
[0002] Alzheimer's disease (AD), commonly known as senile dementia, is a persistent neurological dysfunction that develops in the elderly, progresses slowly, and worsens over time. It is also the main type of brain aging and neurodegeneration. The early stage of this disease is mainly characterized by intellectual impairment, and the clinical manifestations are memory, cognitive impairment, and personality and behavioral changes in the later stage. The typical pathological features are mainly the formation of extracellular senile plaques and intracellular neurofibrillary tangles. Tau protein is a microtubule-binding protein, and its main function is to stabilize the microtubule structure and promote microtubule nucleation, elongation and depolymerization. The activity of tau protein depends on the normal balance of phosphorylation and dephosphorylation. When tau protein is overphosphorylated (imbalanced phosphorylation level), its ability to bind to microtubule protein is reduced, the stability of microtubules is reduced, and intracellular neurofibrils are entangled, resulting in neurodegenerative lesions. Phosphorylated tau-181 protein (hereinafter referred to as p-tau-181) in cerebrospinal fluid has been considered a marker of NFT, which can support the diagnosis of clinical AD and the prediction of mild cognitive impairment.
[0003] Currently, positron emission tomography (PET) and detection of markers in cerebrospinal fluid have become commonly used technical means for diagnosing AD. The content of AD markers in the blood is much lower than that in cerebrospinal fluid, so a more sensitive detection method is needed to detect AD markers in the blood. The detection of markers in the blood is also carried out using the SIMOA single molecule detection platform and the Merck SMCxPRO single molecule immunoassay platform. However, the examination of cerebrospinal fluid is an invasive examination, and the detection costs of other platforms are high and the operation is complicated, which limits the large-scale use of these methods.
[0004] There are not many p-tau-181 test kits on the market. The best-selling one is Anqun Bio, which has enzyme-linked immunosorbent assay and chemiluminescence assay, but the sensitivity is not high, and the blank limit is no more than 5pg / mL. The SIMOA single-molecule detection platform and Merck SMCxPRO single-molecule immunoassay platform have high detection sensitivity, but the detection cost is high and the equipment operation is complicated, which limits the large-scale use of these methods.
[0005] Based on this, the main purpose of the present invention is to provide a fluorescent immunoassay method for rapid detection of p-tau-181 in blood with low detection cost, simple sampling, high sensitivity, quantitative detection and simple operation. Summary of the invention
[0006] The purpose of the present invention is to solve the problems existing in the prior art and to propose a fluorescent immunoassay method for phosphorylated 181tau protein with improved sensitivity.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0009] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0010] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0011] Step 3, preparing SAPE-PE to obtain reagent 3;
[0012] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0013] Preferably, the magnetic beads are coated with p-tau-181 monoclonal antibodies, and the specific process of obtaining reagent 1 is:
[0014] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0015] Preferably, the reactant is used as reagent 1 at a concentration of 40 μg / mL.
[0016] Preferably, the specific process of obtaining reagent 2 by biotin-labeling tau monoclonal antibody is as follows: biotin and antibody are mixed in a molar ratio of 20:1, reacted at room temperature for 40 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0017] Preferably, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0018] Preferably, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0019] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0020] Preferably, the SAPE-PE is used as reagent 3 at a concentration of 1 μg / mL.
[0021] Preferably, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL p-tau-181 antigen + 50 μL reagent 1 + 50 μL reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL PBST cleaning solution, and then add 50 μL reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL PBST cleaning solution; add 60 μL PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0022] Preferably, the flow cytometer parameters are set as follows: medium speed, stop condition 10 μL, threshold FSC>50000, FSC gate critical value 200, center area r1, PE gate critical value 900, center area r2, and r2 / r1 ratio (i.e., positive percentage) analysis.
[0023] Preferably, the magnetic beads are carboxyl magnetic beads with a particle size of 3 um, and the p-tau-181 antibody coated with the magnetic beads is a monoclonal antibody that recognizes the p-tau-181 protein.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) The reaction system of the present invention uses the brightest fluorescent substance, phycoerythrin (PE), as the detection substrate, and adopts SAPE and BIO-PE cyclic reaction to make a single antibody carry more fluorescent molecules for identification, thereby improving the sensitivity of detection.
[0026] (2) The final reaction product of the present invention is detected by flow cytometry. Under the irradiation of the laser light device of the flow cytometer, a single magnetic bead is identified and SAPE-PE on the surface of the magnetic bead is excited in sequence, which is similar to the single molecule technology, thereby improving the sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1This is a schematic diagram of the process of a fluorescent immunoassay method for phosphorylated 181tau protein with improved sensitivity proposed by the present invention. DETAILED DESCRIPTION
[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.
[0029] Example 1
[0030] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0031] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0032] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0033] Step 3, preparing SAPE-PE to obtain reagent 3;
[0034] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0035] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0036] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0037] In this example, the reactant is used as reagent 1 at a concentration of 40 μg / mL.
[0038] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 40 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0039] In this embodiment, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0040] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0041] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0042] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0043] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0044] Example 2
[0045] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0046] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0047] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0048] Step 3, preparing SAPE-PE to obtain reagent 3;
[0049] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0050] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0051] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0052] In this example, the concentration of the reactant used as reagent 1 is 35 μg / mL.
[0053] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 30 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0054] In this embodiment, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0055] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0056] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4.3:1, and reacted at 37°C for 1.2 h to obtain SAPE-PE, which was used as reagent 3.
[0057] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0058] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL p-tau-181 antigen + 50 μL reagent 1 + 50 μL reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, use 205 μL PBST cleaning solution to wash three times, and then add 52 μL reagent 3; magnetically absorb, discard the supernatant, and use 205 μL PBST cleaning solution to wash three times; add 62 μL PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0059] Example 3
[0060] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0061] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0062] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0063] Step 3, preparing SAPE-PE to obtain reagent 3;
[0064] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0065] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0066] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 25 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of antibody to magnetic beads of 1:50, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0067] In this example, the concentration of the reactant used as reagent 1 is 39 μg / mL.
[0068] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 45 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0069] In this embodiment, the marker is used as reagent 2 at a concentration of 1.1 μg / mL.
[0070] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0071] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 3.95:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0072] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0073] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0074] Example 4
[0075] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0076] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0077] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0078] Step 3, preparing SAPE-PE to obtain reagent 3;
[0079] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0080] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0081] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0082] In this example, the reactant is used as reagent 1 at a concentration of 50 μg / mL.
[0083] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 40 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0084] In this embodiment, the marker is used as reagent 2 at a concentration of 2 μg / mL.
[0085] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0086] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0087] In this example, the concentration of SAPE-PE used as reagent 3 is 2 μg / mL.
[0088] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0089] Example 5
[0090] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0091] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0092] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0093] Step 3, preparing SAPE-PE to obtain reagent 3;
[0094] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0095] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0096] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 33 minutes. After being collected and washed twice, antibodies were added at a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with post-group washing solution to obtain a reactant, which was used as reagent 1.
[0097] In this example, the reactant is used as reagent 1 at a concentration of 50 μg / mL.
[0098] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 50 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0099] In this embodiment, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0100] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0101] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 5:1, and reacted at 38°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0102] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0103] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0104] Example 6
[0105] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0106] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0107] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0108] Step 3, preparing SAPE-PE to obtain reagent 3;
[0109] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0110] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0111] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0112] In this example, the reactant is used as reagent 1 at a concentration of 30 μg / mL.
[0113] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 40 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0114] In this embodiment, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0115] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0116] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.15 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0117] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0118] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0119] Example 7
[0120] A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity comprises the following steps:
[0121] Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1;
[0122] Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2;
[0123] Step 3, preparing SAPE-PE to obtain reagent 3;
[0124] Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0125] In this embodiment, the magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1 in the following specific process:
[0126] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of antibody to magnetic beads of 1:40, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
[0127] In this example, the reactant is used as reagent 1 at a concentration of 30 μg / mL.
[0128] In this embodiment, the specific process of biotin-labeling tau monoclonal antibody to obtain reagent 2 is: biotin and antibody are mixed in a 20:1 molar ratio, reacted at room temperature for 40 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
[0129] In this embodiment, the marker is used as reagent 2 at a concentration of 1 μg / mL.
[0130] In this embodiment, the specific process of preparing SAPE-PE to obtain reagent 3 is:
[0131] First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
[0132] In this example, the concentration of SAPE-PE used as reagent 3 is 1 μg / mL.
[0133] In this embodiment, the specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0134] The present invention adopts a double antibody sandwich fluorescent immunoassay to quantitatively detect p-tau-181 in human serum, wherein a monoclonal antibody of p-tau-181 is used to coat magnetic microparticles to prepare reagent 1, and another monoclonal antibody of tau is labeled with biotin to prepare reagent 2, the sample is incubated with reagent 1 and reagent 2 to form a triple complex (antibody-coated magnetic beads-antigen-biotin antibody), and then an external magnetic field is applied for precipitation, the supernatant is removed, the triple complex is washed 3 times with a washing solution, and then a SAPE-PE working solution is added and mixed, thereby SAPE-PE is bound to magnetic beads to form a quadruplex (antibody-coated magnetic beads-antigen-biotin antibody-SAPE-PE), and then a magnetic field is applied for precipitation, the supernatant is removed, the quadruplex is washed three times with a washing solution, a transfer solution is added to resuspend the quadruplex, and the sample is loaded on a flow cytometer. Under laser irradiation, a single magnetic bead and SAPE-PE on the surface of the magnetic bead are sequentially identified and an optical signal is obtained. The concentration of the measured p-tau-181 can be calculated by data processing. Compared with the prior art, the present invention:
[0135] (1) The reaction system of the present invention uses the brightest fluorescent substance, phycoerythrin (PE), as the detection substrate, and adopts SAPE and BIO-PE cyclic reaction to make a single antibody carry more fluorescent molecules for identification, thereby improving the sensitivity of detection.
[0136] (2) The final reaction product of the present invention is detected by flow cytometry. Under the irradiation of the laser light device of the flow cytometer, a single magnetic bead is identified and SAPE-PE on the surface of the magnetic bead is excited in sequence, which is similar to the single molecule technology, thereby improving the sensitivity of the detection.
[0137] The specific method flow of the present invention is as follows:
[0138] Reagent preparation:
[0139] (1) Magnetic beads coated with p-tau-181 monoclonal antibody:
[0140] Magnetic beads: JSR MS300 / Carboxy l;
[0141] Antibodies: thermo, tau181 monoclonal antibody AT270;
[0142] Coating process:
[0143] The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and the beads were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added at a mass ratio of 1:50 between the antibodies and the magnetic beads, and the beads were reacted on a rotator for 2 hours. Blocking solution was then added, and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain the reactant.
[0144] Use concentration: 40 μg / mL, as reagent 1.
[0145] (2) Biotinylated tau monoclonal antibody:
[0146] Biotin: Thermo company su l fo-NHS-LC-LC-biotin;
[0147] Antibodies: thermo, tau181 monoclonal antibody HT7;
[0148] Marking process:
[0149] Biotin and antibody were mixed in a molar ratio of 20:1, reacted at room temperature for 40 min, and the reactants were purified by a desalting column to obtain the labeled substance.
[0150] Use concentration: 1 μg / mL, as reagent 2.
[0151] (3) Preparation of SAPE-PE:
[0152] PE: Langya Biotechnology;
[0153] SAPE: Longya Biotech;
[0154] Preparation process: First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4:1, and reacted at 37°C for 1 hour to obtain SAPE-PE.
[0155] Use concentration: 1 μg / mL, as reagent 3.
[0156] Reaction process:
[0157] Preparation of instrument consumables: 96-well ELISA plate, 96-well plate magnetic stand, 37°C incubator, Agilent flow cytometer.
[0158] Experimental steps: Take a 96-well ELISA plate, add 50μL p-tau-181 antigen + 50μL reagent 1 + 50μL reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200μL PBST cleaning solution, and then add 50μL reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200μL PBST cleaning solution; add 60μL PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
[0159] Flow cytometer parameter settings: medium speed, stop condition 10 μL, threshold FSC>50000, FSC gate critical value 200, center area r1, PE gate critical value 900, center area r2. Analyze the ratio of r2 / r1 (i.e., positive percentage).
[0160] Data Analysis:
[0161] Preparation of standard curve:
[0162] The p-tau-181 antigen standard was diluted to 50pg / mL, 25pg / mL, 10pg / mL, 5pg / mL, 2.5pg / mL, 1pg / mL, and 0.5pg / mL with phosphate buffer containing 1% BSA, and used as sample detection to analyze the ratio of r2 / r1 (i.e., the positive percentage). As the concentration increased, the positive percentage gradually increased until it gradually saturated. The specific data are as follows:
[0163]
[0164] Blank Limit Assessment:
[0165] According to the test results of the antigen standard, the concentration was used as the X-axis, the positive percentage * coefficient 1000000 was used as the Y-axis, and the cubic spline calculation method was used for fitting to make a standard curve.
[0166] Use blank PBS buffer as the sample test, repeat the measurement 20 times, and obtain the corresponding percentage coefficients. Substitute the mean of the percentage coefficient plus twice the standard deviation into the standard curve, and the calculated concentration value is used as the blank limit of the kit.
[0167]
[0168]
[0169] Through calculation, the blank limit of this test is 0.0352pg / mL. The sensitivity can reach the fg / mL level, far exceeding the pg / mL sensitivity performance of the test kits on the market.
[0170] Detection range assessment:
[0171] Without diluting the sample, the detection range can be set at (0.1-50pg / mL). If the sample is diluted 4 times, the detection range can be set at (0.4-200pg / mL). The upper limit of detection can meet clinical requirements, and the lower limit of detection is superior to the current test kits on the market. The overall detection range has obvious advantages.
[0172] Experimental conclusion
[0173] The present invention proposes a method for detecting phosphorylated 181tau protein by fluorescence immunoassay with improved sensitivity, the minimum detection limit of which can reach the level of fg / mL, and the detection range after dilution is (0.4-200pg / mL), which can meet clinical needs. Compared with the SIMOA single molecule detection platform and the Merck SMCxPRO single molecule immunoassay platform, the experimental operation is simple, the detection cost is low, and this detection method can be used on a large scale.
[0174] It should be noted here that:
[0175] In this embodiment, the minimum detection limit of the p-tau-181 antibody-coated magnetic beads, the biotin-labeled p-tau-181 antibody, and SAPE-PE can reach the level of fg / mL, and the detection range is (0.4-200pg / mL);
[0176] In this embodiment, the magnetic beads are MS300 / Carboxy l from JSR Company, carboxyl magnetic beads, with a particle size of 3 um.
[0177] In this embodiment, the p-tau-181 antibody coated with the magnetic beads is a monoclonal antibody that recognizes the p-tau-181 protein (thermo, p-tau-181 monoclonal antibody AT270).
[0178] In this example, the biotin-labeled p-tau-181 antibody is a monoclonal antibody that recognizes the p-tau-181 protein (thermo, p-tau-181 monoclonal antibody HT7).
[0179] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A fluorescent immunoassay method for phosphorylated 181tau protein with improved sensitivity, characterized in that: The following steps are involved: Step 1, magnetic beads are coated with p-tau-181 monoclonal antibodies to obtain reagent 1; Step 2, biotin-labeling tau monoclonal antibody to obtain reagent 2; Step 3, preparing SAPE-PE to obtain reagent 3; Step 4, take a 96-well ELISA plate, add Lp-tau-181 antigen and reagent 1 and reagent 2 to each well, and incubate in an incubator; magnetically absorb, discard the supernatant, wash three times with PBST cleaning solution, and then add reagent 3; magnetically absorb, discard the supernatant, and wash with PBST cleaning solution; add PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
2. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 1, characterized in that: The specific process of coating the magnetic beads with p-tau-181 monoclonal antibody to obtain reagent 1 is as follows: The magnetic beads were collected with 0.1M MES (pH5.0) and washed twice, EDC was added, and they were activated on a rotator for 30 minutes. After being collected and washed twice, antibodies were added according to a mass ratio of 1:50 between antibodies and magnetic beads, and the reaction was carried out on a rotator for 2 hours. Then, blocking solution was added and the beads were blocked on a rotator for 30 minutes. The beads were washed with a post-group washing solution to obtain a reactant, which was used as reagent 1.
3. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 2, characterized in that: The reactant is used as reagent 1 at a concentration of 40 to 50 μg / mL.
4. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 1, characterized in that: The specific process of obtaining reagent 2 by biotin-labeling tau monoclonal antibody is as follows: biotin and antibody are mixed in a molar ratio of 20:1, reacted at room temperature for 40 to 50 minutes, the reactant is purified by a desalting column to obtain a marker, and the marker is used as reagent 2.
5. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 4, characterized in that: The marker is used as reagent 2 at a concentration of 1 to 2 μg / mL.
6. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 1, characterized in that: The specific process of preparing SAPE-PE to obtain reagent 3 is as follows: First, biotin and antibody were mixed at a molar ratio of 20:1 to prepare BIO-PE, and then SAPE and the BIO-PE prepared above were diluted to 0.1 mg / mL, mixed at a volume ratio of 4 to 5:1, and reacted at 37°C for 1 hour to obtain SAPE-PE, which was used as reagent 3.
7. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 6, characterized in that: The SAPE-PE is used as reagent 3 at a concentration of 1 to 2 μg / mL.
8. A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity according to claim 1, characterized in that: The specific process of step 4 is: take a 96-well ELISA plate, add 50 μL of p-tau-181 antigen + 50 μL of reagent 1 + 50 μL of reagent 2 to each well, and incubate in a 37°C incubator for 30 minutes; magnetically absorb, discard the supernatant, wash three times with 200 μL of PBST cleaning solution, and then add 50 μL of reagent 3; magnetically absorb, discard the supernatant, and wash three times with 200 μL of PBST cleaning solution; add 60 μL of PBS to each well to resuspend the magnetic beads, and load the sample on the flow cytometer for detection.
9. A method for fluorescent immunoassay of phosphorylated 181 tau protein with improved sensitivity according to claim 8, characterized in that: The flow cytometer parameters were set as follows: medium speed, stop condition 10 μL, threshold FSC>50000, FSC gate critical value 200, center area r1, PE gate critical value 900, center area r2, and r2 / r1 ratio (i.e., positive percentage) analysis.
10. A fluorescent immunoassay method for phosphorylated 181 tau protein with improved sensitivity according to claim 2, characterized in that: The magnetic beads are carboxyl magnetic beads with a particle size of 3um, and the p-tau-181 antibodies coated with the magnetic beads are monoclonal antibodies that recognize the p-tau-181 protein.