Method and kit for detecting AD diagnostic marker based on liquid phase chip

Abstract

The invention provides a method and a kit for detecting an AD diagnostic marker based on a liquid phase chip, and belongs to the technical field of detection.The detection method comprises the steps that calcium ions are added into a sample and a specific microsphere immunoadsorption reaction system or a cross-linking reaction system, the sample comprises a standard substance, a sample to be detected, a control sample or a labeled sample, and the standard substance and the specific microsphere immunoadsorption reaction system or the cross-linking reaction system are sequentially connected; the calcium ions are provided by calcium chloride, calcium glycinate or calcium aspartate, the binding efficiency of two mutually specifically bound proteins in a reaction system is improved through the characteristic that the calcium ions can promote protein binding, and the binding efficiency can be further improved through cooperation with the promoting effect of glycine and asparaginic acid. And the detection lower limit can be greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and specifically relates to a method for detecting AD diagnostic markers based on a liquid phase chip, and also relates to a detection kit matching the detection method. Background Art

[0002] Alzheimer's disease (AD) is a neurodegenerative disease with clinical manifestations of cognitive impairment, decreased living functions, and abnormal mental and behavioral behaviors. The typical pathological characteristics of AD are the appearance of neurofibrillary tangles (NFTs) in neurons, the deposition of senile plaques (SPs) outside cells, and a decrease in the number of neurons.

[0003] There is no clear boundary between the physiology and psychology of early AD patients and normal aging, and it is difficult to diagnose patients in time, so there is still a lack of specific diagnosis and effective treatment. The ideal diagnosis and treatment should be before a large number of synapses and neurons are lost, so the current research focus of the disease has shifted to the diagnosis and treatment of early or very early AD. However, there are no recognized diagnostic criteria except for the confirmation of the disease through the appearance of senile plaques and nerve fiber disorder in the brain after the patient's death. Clinical diagnosis is mainly based on the patient's medical history, brain imaging, and psychological, cognitive and neurological tests. Laboratory-assisted diagnostic methods mainly detect soluble markers contained in body fluids.

[0004] In recent years, many AD research experts and institutions have invested a lot of energy in studying biological detection indicators, and found some biochemical indicators with differential diagnostic significance from the cerebrospinal fluid or blood of AD patients, such as tau protein, β-amyloid protein (Aβ), apolipoprotein E (ApoE), Aβ precursor protein (APP), presenilin (PS-1, PS-2) and aspartate aminotransferase (AST). t-tau (total tau protein), p-tau (phosphorylated tau protein) and Aβ1-42 are three important biological markers for AD auxiliary diagnosis. For example, the invention patent with publication number: CN117471007B discloses a method for detecting plasma β-amyloid protein, which first enriches β-amyloid protein by magnetic beads, then denatures and enzymatically hydrolyzes β-amyloid protein, and then performs LC-MS-MS quantitative analysis, and the analysis results are used for AD auxiliary diagnosis. However, the steps of this method are complicated and cumbersome, the detection efficiency is low, and the detection limit is high, and the role of auxiliary diagnosis still needs to be improved. Summary of the invention

[0005] Based on the above problems in the prior art, the present invention provides a method for detecting AD diagnostic markers based on a liquid phase chip, wherein calcium ions are added to a sample and a specific microsphere immunoadsorption reaction system or a cross-linking reaction system, wherein the sample includes a standard, a sample to be tested, a control sample or a labeled sample.

[0006] Preferably, the specific method of adding calcium ions to the reaction system is to prepare a reaction buffer containing calcium ions, and then place the sample and the specific microspheres in the reaction buffer for immunoadsorption reaction or cross-linking reaction; the reaction buffer comprises calcium chloride and amino acid calcium, calcium chloride: amino acid calcium = 3-4:1, and the working concentration of calcium ions in the reaction buffer is 0.8-2.4 mol / L.

[0007] Preferably, the amino acid calcium is calcium glycinate or calcium aspartate, or a mixture of calcium glycinate and calcium aspartate.

[0008] Preferably, the liquid phase chip comprises:

[0009] Coated microspheres: including microspheres coated with Aβ42 capture antibodies, microspheres coated with Aβ40 capture antibodies, microspheres coated with p-tau181 capture antibodies, microspheres coated with p-tau217 capture antibodies, microspheres coated with t-tau capture antibodies, microspheres coated with GFAP capture antibodies, and microspheres coated with NfL capture antibodies, and the above microspheres are respectively coded with different colors;

[0010] Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin;

[0011] Streptavidin phycoerythrin.

[0012] Preferably, the method for detecting AD diagnostic markers based on a liquid phase chip comprises the following detailed steps:

[0013] Step S1, taking equal proportions of the coated microspheres and adding them to a reaction buffer, shaking to make the coated microspheres uniformly suspended in the reaction buffer, to obtain a mixed system a;

[0014] Step S2, adding the sample to be tested into the mixed system a, incubating in the dark with shaking, to obtain the mixed system b;

[0015] Step S3, adding the biotin-labeled detection antibody to the mixed system b, incubating in the dark with shaking to obtain a mixed system c, wherein the final concentration of each detection antibody is 1-3 μg / ml;

[0016] Step S4, adding streptavidin phycoerythrin to the mixed system c, incubating in the dark with shaking, to obtain a mixed system d;

[0017] Step S5, using a liquid phase chip analyzer to detect and analyze the mixed system d.

[0018] Preferably, the final concentration of each coated microsphere in the mixed system a in step S1 is 145-165 / μl; the amount of reaction buffer in step S1 is 1 ml of reaction buffer for every 1 ml of sample to be tested.

[0019] Preferably, the incubation temperature in steps S2, S3 and S4 is 25°C, and the shaking is performed on a microplate shaker at a speed of 600 rpm.

[0020] Preferably, the step S1 further includes ultrasonic treatment after shaking, and the ultrasonic treatment time is 30 seconds.

[0021] The present invention also provides a kit for detecting AD diagnostic markers based on a liquid phase chip, which includes an immunobinding or cross-linking reaction buffer, wherein the immunobinding or cross-linking reaction buffer contains calcium chloride and amino acid calcium, calcium chloride: amino acid calcium = 3-4 mol: 1 mol, and the working concentration of calcium ions in the reaction buffer is 0.8-2.4 mol / L; the amino acid calcium is calcium glycinate or calcium aspartate, or a mixture of calcium glycinate and calcium aspartate.

[0022] Preferably, the kit for detecting AD diagnostic markers based on a liquid phase chip further comprises:

[0023] Capture antibodies are capture antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL, respectively, and the capture antibodies are monoclonal antibodies that can specifically bind to the corresponding proteins;

[0024] Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin, wherein the detection antibodies are monoclonal antibodies or polyclonal antibodies that can specifically bind to the corresponding proteins respectively;

[0025] Standards, which are Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP, and NfL;

[0026] As well as streptavidin phycoerythrin, uncoated original microspheres, quality control solution and matrix solution.

[0027] The beneficial effects of the present invention are as follows: calcium ions are introduced into the immune reaction or cross-linking reaction system, and the binding efficiency of two proteins specifically binding to each other in the reaction system is improved by the property of calcium ions that can promote protein binding. The promoting effect of glycine and aspartic acid can further improve the binding efficiency, which can greatly reduce the detection limit, thereby further reducing the sampling amount, reducing damage or performing more tests with the same sampling amount. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is described below in conjunction with specific embodiments.

[0029] Example 1: A method for detecting AD diagnostic markers based on liquid phase chip.

[0030] A method for detecting AD diagnostic markers based on a liquid phase chip, in the process of preparing a reaction buffer, calcium chloride, calcium glycinate and calcium aspartate are added to a 50mM MES buffer (pH5.0) in a ratio (calcium chloride: calcium glycinate: calcium aspartate = 3:0.5:0.5), so as to prepare a reaction buffer containing calcium ions, wherein the working concentration of calcium ions in the reaction buffer is 1.2 mol / L, and then a sample and a specific microsphere are placed in the reaction buffer for an immunoadsorption reaction or a cross-linking reaction, wherein the sample includes a standard, a sample to be tested, a control sample or a labeled sample;

[0031] The liquid phase chip comprises:

[0032] Coated microspheres: including microspheres coated with Aβ42 capture antibodies, microspheres coated with Aβ40 capture antibodies, microspheres coated with p-tau181 capture antibodies, microspheres coated with p-tau217 capture antibodies, microspheres coated with t-tau capture antibodies, microspheres coated with GFAP capture antibodies, and microspheres coated with NfL capture antibodies, and the above microspheres are respectively coded with different colors;

[0033] Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin;

[0034] Streptavidin-phycoerythrin;

[0035] The immunoadsorption reaction or cross-linking reaction includes the following detailed steps:

[0036] Step S1, measure the same volume of reaction buffer as the sample to be tested, that is, use 1 ml of reaction buffer for every 1 ml of sample to be tested, and then take each coated microsphere in equal proportion according to the detection requirements and add them to the reaction buffer. If Aβ42 and Aβ40 need to be detected, take microspheres coated with Aβ42 capture antibody and microspheres coated with Aβ40 capture antibody in equal proportion, without taking other microspheres; first take out the corresponding microsphere suspension from the kit and mix it with a vortex mixer for 30 seconds, then ultrasonically treat it for 30 seconds, then take an appropriate amount of the two microspheres in equal proportion and mix them, and then add them to the reaction buffer, and then use the vortex mixer to mix the mixed system again for 30 seconds and ultrasonically treat it for 30 seconds to make the coated microspheres uniformly suspended in the reaction buffer to obtain a mixed system a, and the final concentration of each coated microsphere in the mixed system a is 150 / μl; the coated microspheres should be mixed before use, and should be used immediately after mixing, otherwise the microspheres will precipitate again if left for too long;

[0037] Step S2, take a 96-well plate and determine the positions of the standard, quality control, sample to be tested and blank wells on the 96-well plate, first add 25 μl of reaction buffer to each well, then add 25 μl of the corresponding sample to be tested (the sample to be tested refers to the detection object in each parallel detection experiment, including the aforementioned standard, quality control and sample to be tested, and the blank well is to add 25 μl of reaction buffer), seal all the sample addition holes with sealing film, and wrap the 96-well plate with tin foil to avoid light, place it on a microplate oscillator at 25°C and shake at 600 rpm, incubate for 60 minutes, and obtain a mixed system b;

[0038] Step S3, similarly, according to the detection requirements, biotin-labeled detection antibodies are taken in equal proportions and mixed. In this embodiment, only the detection antibodies of Aβ42 and Aβ40 labeled with biotin are taken as described above, and then added to the mixed system b. The final concentration of each detection antibody is 2 μg / ml. All the sample addition holes are sealed with sealing film, and the 96-well plate is wrapped with tin foil to avoid light. The plate is placed on a microplate oscillator at 25° C. and shaken at 600 rpm, and then incubated for 60 min to obtain a mixed system c.

[0039] Step S4, add 25ul streptavidin phycoerythrin to each well of the mixed system c, seal all the sample addition holes with sealing film, and wrap the 96-well plate with tin foil to avoid light, place it on a microplate shaker at 25°C and shake at 600 rpm, and incubate for 30 minutes to obtain a mixed system d;

[0040] Step S5, using a Luminex series liquid phase chip analyzer to analyze the mixed system d, automatically draw a standard curve, and calculate the measurement value of the sample to be tested.

[0041] Example 2: A kit for detecting AD diagnostic markers based on a liquid phase chip.

[0042] A kit for detecting AD diagnostic markers based on a liquid phase chip, comprising:

[0043] (1) an immunobinding or cross-linking reaction buffer, wherein the immunobinding or cross-linking reaction buffer is a 50 mM MES buffer and contains calcium chloride and amino acid calcium, wherein calcium chloride:amino acid calcium=3 mol:1 mol, and the working concentration of calcium ions in the reaction buffer is 1.2 mol / L; the amino acid calcium is a mixture of calcium glycinate and calcium aspartate, and the mixing ratio is 1:1;

[0044] (2) capture antibodies, which are capture antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL, respectively, and the capture antibodies are monoclonal antibodies that can specifically bind to the corresponding proteins;

[0045] (3) Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin, wherein the detection antibodies are monoclonal antibodies or polyclonal antibodies that can specifically bind to the corresponding proteins respectively;

[0046] (4) standards, namely Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP, and NfL;

[0047] and (5) streptavidin phycoerythrin, (6) uncoated original microspheres, (7) quality control solution, (8) matrix solution, (9) sealing film and (10) 96-well plate.

[0048] In order to use the kit more flexibly and conveniently and to ensure the accuracy of the detection, the coated microspheres and detection antibodies of the kit provided in this embodiment need to be prepared by themselves. The coated microspheres can be prepared according to the following steps:

[0049] Select microspheres with different numbers for different required markers, and suspend the microspheres with a vortex oscillator or ultrasound for about 30 seconds; take 50 μL of microspheres in a 1.5 ml centrifuge tube and centrifuge at 15000 rpm for 10 minutes; carefully remove the supernatant, resuspend the microspheres in 100 μL of double distilled water, vortex for about 30 seconds, and ultrasonicate for 1 minute; centrifuge at 15000 rpm for 10 minutes; discard the supernatant, add 80 μL of phosphate buffer (pH 6.2), vortex for about 30 seconds, and ultrasonicate for 1 minute; add 10 μL of 50 mg / mLSulfo-NHS, mix gently with a vortex oscillator, and ultrasonicate for 1 minute; add 10 μL of 50 mg / mL EDC, mix gently with a vortex oscillator, and then activate the microspheres by shaking at room temperature for about 20 minutes.

[0050] The activated microspheres were centrifuged at a speed greater than 15000rpm / min for 10min; the supernatant was discarded, the microspheres were resuspended in 250μL 50mM MES (pH5.0) solution, vortexed for about 30s, sonicated for 1min, and centrifuged at a speed greater than 15000rpm / min for 10min. The above steps were repeated once, the supernatant was discarded, the microspheres were resuspended in 100μL 50mM MES (pH5.0) solution, vortexed for about 30s, sonicated for 1min, 1μg of the corresponding capture antibody was added to the suspended microspheres, the total volume was filled to 500μL with 50mM MES (pH5.0) solution, mixed with a vortex oscillator, and oscillated for 2hr (speed 900rpm) at 25°C in the dark to complete the coupling. The antibody-coupled microspheres were centrifuged at a speed greater than 12000g for 10min, the supernatant was discarded, and the microspheres were resuspended in 500μL In PBS-TBN solution, vortex for about 30 seconds, ultrasonicate for 1 minute, and oscillate for 30 minutes at room temperature in the dark. The microspheres were centrifuged at a speed greater than 12,000 g for 10 minutes, and the supernatant was discarded.

[0051] The microspheres were resuspended in 1 mL of PBS-TBN solution, vortexed for about 30 seconds, and ultrasonically treated for 1 minute; the microspheres were centrifuged at a speed greater than 12,000 g for 5 minutes; this step was repeated once; the supernatant was discarded, and the microspheres were resuspended in 600 μL of PBS-TBN solution to obtain coated microspheres. The coated microspheres were counted by a luminex instrument. After counting, the microspheres can be stored at 2-8°C away from light. Each antibody-coupled microsphere was stored separately and mixed in equal proportions according to the test items when used.

[0052] The detection antibody can be prepared according to the following steps:

[0053] The volume of the antibody solution when diluted to 1 mg / ml was calculated based on the protein concentration and regarded as the target volume (V); NHS-Biotin reaction solution was prepared (dissolved in DMSO to a concentration of 10 mg / ml); the detection antibody and NHS-Biotin (10 mg / ml) reaction solution were added to the reaction tube at a molar ratio of 1:100; 1 / 10 of the target volume of NaHCO3 (pH 8.9) solution was added; PBS (pH 7.4) was added to make up to the target volume; aluminum foil was wrapped, the reaction tube was placed on an oscillator, and incubated in a 25°C thermostat for 4 hours in the dark at a speed of 900 rpm; the reaction solution was transferred to a dialysis box and dialyzed in PBS buffer overnight to remove unreacted NHS-Biotin, thus preparing the required detection antibody and determining the protein concentration.

[0054] Example 3: Testing of the detection method and the kit.

[0055] The test kit provided in Example 2 was used to carry out the test experiment of Example 1, and compared with other control experiments, as follows:

[0056] Three sets of parallel experiments were performed according to the method of Example 1. Three repeated experiments with completely identical experimental conditions were set up in each set of experiments. The differences between the three sets of parallel experiments are as follows:

[0057] Group 1: 50 mM MES buffer was used as the reaction buffer;

[0058] Group 2: 50 mM MES buffer was used as the basic reaction buffer and calcium chloride was added;

[0059] Group 3: 50 mM MES buffer was used as the basic reaction buffer and calcium glycinate and calcium aspartate were added;

[0060] Group 4: 50 mM MES buffer was used as the basic reaction buffer and calcium chloride, calcium glycinate and calcium aspartate were added;

[0061] In the second, third and fourth groups, the working concentration of calcium ions in the reaction buffer was 1.2 mol / L.

[0062] A series of mixed standards with concentration gradients (mixed standards are prepared by mixing marker standards with the same concentration) were used as the test objects. The concentration gradient range was 2-14pg / ml. The results are as follows:

[0063]

[0064] It can be seen from the above experimental results that the addition of calcium chloride and amino acid calcium to the reaction buffer can reduce the detection limit, and the error between the detected concentration data and the corresponding preset preparation concentration does not exceed 0.05pg / ml. In the subsequent supplementary experiment, the detection limit of the fourth group was measured, and the results showed that the detection limit can reach 4.8pg / ml, but the repeatability is not ideal. In actual use, in order to ensure the accuracy of the detection, the detection limit is defined as 5.5pg / ml, which can have ideal repeatability and accuracy.

[0065] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting AD diagnostic markers based on a liquid phase chip, characterized in that: Calcium ions are added to a sample and a specific microsphere immunoadsorption reaction system or a cross-linking reaction system, wherein the sample includes a standard, a sample to be tested, a control sample or a labeled sample.

2. A method for detecting AD diagnostic markers based on a liquid phase chip according to claim 1, characterized in that: The specific method of adding calcium ions to the reaction system is to prepare a reaction buffer containing calcium ions, and then place the sample and the specific microspheres in the reaction buffer for immunoadsorption reaction or cross-linking reaction; the reaction buffer comprises calcium chloride and amino acid calcium, the ratio of calcium chloride to amino acid calcium is 3-4:1, and the working concentration of calcium ions in the reaction buffer is 0.8-2.4 mol / L.

3. A method for detecting AD diagnostic markers based on a liquid phase chip according to claim 2, characterized in that: The amino acid calcium is calcium glycinate or calcium aspartate, or a mixture of calcium glycinate and calcium aspartate.

4. A method for detecting AD diagnostic markers based on a liquid phase chip according to any one of claims 1 to 3, characterized in that: The liquid phase chip comprises: Coated microspheres: including microspheres coated with Aβ42 capture antibodies, microspheres coated with Aβ40 capture antibodies, microspheres coated with p-tau181 capture antibodies, microspheres coated with p-tau217 capture antibodies, microspheres coated with t-tau capture antibodies, microspheres coated with GFAP capture antibodies, and microspheres coated with NfL capture antibodies, and the above microspheres are respectively coded with different colors; Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin; Streptavidin phycoerythrin.

5. The method for detecting AD diagnostic markers based on a liquid phase chip according to claim 4, characterized in that: It includes the following detailed steps: Step S1, taking equal proportions of the coated microspheres and adding them to a reaction buffer, shaking to make the coated microspheres uniformly suspended in the reaction buffer, to obtain a mixed system a; Step S2, adding the sample to be tested into the mixed system a, incubating in the dark with shaking, to obtain the mixed system b; Step S3, adding the biotin-labeled detection antibody to the mixed system b, incubating in the dark with shaking to obtain a mixed system c, wherein the final concentration of each detection antibody is 1-3 μg / ml; Step S4, adding streptavidin phycoerythrin to the mixed system c, incubating in the dark with shaking, to obtain a mixed system d; Step S5, using a liquid phase chip analyzer to detect and analyze the mixed system d.

6. The method for detecting AD diagnostic markers based on a liquid phase chip according to claim 5, characterized in that: In the step S1, the final concentration of each coated microsphere in the mixed system a is 145-165 / μl; the amount of the reaction buffer in the step S1 is 1 ml of the reaction buffer for every 1 ml of the sample to be tested.

7. The method for detecting AD diagnostic markers based on a liquid phase chip according to claim 5, characterized in that: The incubation temperature in steps S2, S3 and S4 is 25°C, and the shaking is performed on a microplate shaker at 600 rpm.

8. The method for detecting AD diagnostic markers based on a liquid phase chip according to claim 5, characterized in that: The step S1 further includes ultrasonic treatment after shaking, and the ultrasonic treatment time is 30 seconds.

9. A kit for detecting AD diagnostic markers based on a liquid phase chip, characterized in that: The invention comprises an immunobinding or cross-linking reaction buffer, wherein the immunobinding or cross-linking reaction buffer comprises calcium chloride and amino acid calcium, wherein the ratio of calcium chloride:amino acid calcium is 3-4 mol:1 mol, and the working concentration of calcium ions in the reaction buffer is 0.8-2.4 mol / L; the amino acid calcium is calcium glycinate or calcium aspartate, or a mixture of calcium glycinate and calcium aspartate.

10. A kit for detecting AD diagnostic markers based on a liquid phase chip according to claim 9, characterized in that: It also includes: Capture antibodies are capture antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL, respectively, and the capture antibodies are monoclonal antibodies that can specifically bind to the corresponding proteins; Biotin-labeled detection antibodies: including detection antibodies for Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP and NfL respectively labeled with biotin, wherein the detection antibodies are monoclonal antibodies or polyclonal antibodies that can specifically bind to the corresponding proteins respectively; Standards, which are Aβ42, Aβ40, p-tau181, p-tau217, t-tau, GFAP, and NfL; As well as streptavidin phycoerythrin, uncoated original microspheres, quality control solution and matrix solution.

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