Detection kit for phosphorylated tau protein p18 based on electrochemiluminescence technology and application thereof

Abstract

The application discloses a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology and application thereof, and belongs to the technical field of medical devices. The detection kit comprises a diluent, a capture antibody, a detection antibody and a plate reading solution for detecting human phosphorylated Tau protein p18. The components of the diluent include 6-10 g / L of sodium chloride, 0.1-0.3 g / L of potassium chloride, 2-4 g / L of Tris-base, 5-15 g / L of BSA, 20-40 g / L of mouse negative IgG, and pure water. Compared with the existing diluent (PBS buffer solution), the diluent of the application increases the mouse IgG protein component, reduces the matrix effect of the sample, improves the sensitivity, and avoids false positives caused by the use of the PBS buffer solution. Meanwhile, the electrochemiluminescence technology is used to improve the detection sensitivity of human phosphorylated Tau protein p18.

Description

Technical Field

[0001] This application belongs to the field of medical device technology, specifically relating to a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology and its application. Background Technology

[0002] The microtubule system is a component of the neuronal cytoskeleton and participates in various cellular functions. Microtubules are composed of tubulin and microtubule-associated proteins, with Tau protein being the most abundant microtubule-associated protein. In the normal brain, the cellular function of Tau protein is to bind to tubulin to promote its polymerization and formation of microtubules; to bind to the formed microtubules, maintain microtubule stability, reduce the dissociation of tubulin molecules, and induce microtubule bundle formation. In the brains of Alzheimer's disease (AD) patients, Tau protein is abnormally hyperphosphorylated (pTau), and its binding affinity to tubulin is only 1 / 10 that of normal Tau protein. It also loses its biological function of promoting microtubule assembly and formation and its role in maintaining microtubule stability.

[0003] pTau protein can serve as an important biomarker for the diagnosis of Alzheimer's disease. For example, research from the University of Gothenburg in Sweden has shown that the level of p-Tau181 in plasma can be used to detect whether someone has Alzheimer's disease. Research by Eli Lilly and its affiliates has shown that p-Tau217 has shown promise in the diagnosis of Alzheimer's disease and in the early diagnosis of individuals with known genetic risk factors. p-Tau18, as a newly emerging research site, is gradually attracting clinical attention.

[0004] Currently, the level of human phosphorylated Tau protein is mainly detected by enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich method. This involves adding standards and samples to wells pre-coated with antibodies against human phosphorylated Tau protein, incubating, adding biotin-labeled anti-phosphorylated Tau protein antibodies, which then bind to HRP-labeled streptavidin to form an immune complex. After incubation and washing to remove unbound enzymes, a chromogenic substrate (TMB) is added, producing a blue color that converts to a final yellow color under acidic conditions. Finally, the absorbance (OD) value of the sample in the reaction wells is measured at 450 nm. The concentration of human phosphorylated Tau protein in the sample is directly proportional to the OD value. The concentration of human phosphorylated Tau protein in the sample is calculated by plotting a standard curve.

[0005] However, on the one hand, from the perspective of sensitivity, ELISA has a certain matrix effect, which can only be eliminated by dilution. However, the p18 content is low, and the concentration is even lower after dilution, so it cannot be detected. On the other hand, there is a difficulty in antibody selection because there are very few p-Tau18 antibodies on the market. There are no relevant reports on which antibody to use for sandwich ELISA and what the results are.

[0006] In addition, the diluent used for samples in current tests is mainly phosphate buffered saline (PBS), whose main components are Na2HPO4, KH2PO4, NaCl and KCl, etc. PBS has a certain impact on the detection of phosphorylated proteins and can lead to strong false positives.

[0007] Application content

[0008] 1. The problem to be solved

[0009] This application addresses several issues with ELISA technology, including low sensitivity, lack of readily available commercially available antibody pairs, and false positives caused by using PBS as a diluent. It provides a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology and its application. The kit includes a diluent for detecting human phosphorylated Tau protein p18, comprising sodium chloride, potassium chloride, tris-base, bovine serum albumin, and mouse negative IgG. Using this diluent instead of traditional PBS buffer reduces the false positive rate caused by PBS, while simultaneously improving the detection sensitivity of human phosphorylated Tau protein p18 using electrochemiluminescence technology.

[0010] 2. Technical Solution

[0011] To solve the above problems, the technical solution adopted in this application is as follows:

[0012] This application provides a diluent for the detection of human phosphorylated Tau protein p18. The diluent comprises: 6-10 g / L sodium chloride, 0.1-0.3 g / L potassium chloride, 2-4 g / L tris-base, 5-15 g / L bovine serum albumin (BSA), 20-40 g / L mouse negative IgG, and purified water. Compared with existing diluents (PBS buffer), this diluent for the detection of human phosphorylated Tau protein p18 increases the IgG protein content, reduces the matrix effect of the sample, improves sensitivity, and avoids false positives that occur when using PBS.

[0013] Furthermore, the components of the above-mentioned diluent include: sodium chloride 6 g / L, potassium chloride 0.1 g / L, Tris-base 2 g / L, BSA 5 g / L, and mouse negative IgG 20 g / L.

[0014] Furthermore, the components of the above-mentioned diluent include: sodium chloride 8 g / L, potassium chloride 0.2 g / L, Tris-base 3 g / L, BSA 10 g / L, and mouse negative IgG 30 g / L.

[0015] Furthermore, the components of the above-mentioned diluent include: sodium chloride 10 g / L, potassium chloride 0.3 g / L, Tris-base 4 g / L, BSA 15 g / L, and mouse negative IgG 40 g / L.

[0016] This application also provides the application of the above-mentioned diluent for the detection of human phosphorylated Tau protein p18 in the preparation of a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology.

[0017] This application also provides a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology. The kit includes: a diluent for the detection of human phosphorylated Tau protein p18 as described above; a capture antibody; a detection antibody; and a reading buffer.

[0018] Furthermore, the aforementioned capture antibody is GTX54658 (genetex).

[0019] Furthermore, the antibody used for detection is F218D (Meso Scale Discovery, MSD).

[0020] Furthermore, the reading solution mentioned above is R92TC-3 (Meso Scale Discovery, MSD).

[0021] This application also provides the application of the above-mentioned detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology in detecting the expression level of human phosphorylated Tau protein p18.

[0022] Furthermore, the expression level of human phosphorylated Tau protein p18 in the above applications includes the expression level of human phosphorylated Tau protein p18 in blood, plasma, brain lysate, and cerebrospinal fluid (CSF).

[0023] Furthermore, the above application includes the following steps:

[0024] S1: Capture antibody coating: Dilute the capture antibody (which needs to be biotinylated) with coating buffer (1×TBS, pH 7.4) and add it to each well of the microplate (MSD, catalog number L45SA). Seal each well with a sealing film to prevent liquid evaporation. Place the microplate flat at room temperature for 1 hour and shake at 800±50 rpm. Wash each well with washing buffer (1×TBS buffered saline containing 0.05% Tween 20). After washing three times, add blocking buffer (1×TBS, 3% BSA) to each well, seal each well with a sealing film, and incubate at room temperature for 1 hour. BSA blocks the exposed sites on the detection plate to prevent non-specific adsorption.

[0025] S2: Sample loading: Add the test sample and / or gradient standards to the corresponding wells. The test sample and / or gradient standards are prepared using any of the diluents described above for the detection of human phosphorylated Tau protein p18. Incubate at 25°C for 1 hour on a shaker at 800±50 rpm. Wash each well with washing buffer (containing 0.05% Tween 20 in 1×TBS buffered saline).

[0026] S3: Antibody incubation. Wash each well with washing buffer (containing 0.05% Tween 20 in 1×TBS buffered saline); dilute the detection antibody with 1×TBS, pH 7.4, and 0.1% BSA and add it to the corresponding well. Incubate at 25°C for 1 hour.

[0027] S4: Reading solution is added to the instrument. Washing solution (containing 0.05% Tween 20 in 1×TBS buffered saline) is added to each microwell for washing; reading solution is added to each microwell for instrument testing.

[0028] Further, in S1 above, the capture antibody (which needs to be biotinylated) is diluted to 2.5 μg / ml with coating buffer, and 50 μl is added to each well of a 96-well microplate; 150 μl of washing buffer is added to each well for washing, for a total of 3 times; 150 μl of blocking buffer is added to each well.

[0029] Furthermore, in S2 above, the gradient standards are 2000 pg / ml, 250 pg / ml, 31.25 pg / ml, and 0 pg / ml; the amount of the test sample and the gradient standards added is 50 μl; 150 μl of washing solution is added to each microwell for washing, for a total of 3 times.

[0030] Further, in S3 above, 150 μl of washing buffer was added to each microwell for washing, for a total of 3 times; the detection antibody was released to 1 μg / ml with 1×TBS, pH 7.4, 0.1% BSA, and the amount added was 50 μl.

[0031] Furthermore, in S4 above, 150 μl of washing solution is added to each microwell for washing, for a total of 3 times; the amount of reading solution added is 150 μl.

[0032] 3. Beneficial effects

[0033] Compared with the prior art, the advantages of this application are as follows:

[0034] This application provides a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology and its application. The kit includes a diluent for the detection of human phosphorylated Tau protein p18, which includes sodium chloride, potassium chloride, tris-base, bovine serum albumin, and mouse negative IgG. Diluting the sample with this diluent increases the IgG protein content, reduces the matrix effect of the sample, improves sensitivity, and avoids false positives that occur when using PBS. At the same time, the electrochemiluminescence technology is used to improve the detection sensitivity of human phosphorylated Tau protein p18. Attached Figure Description

[0035] Figure 1 It is a standard curve of the standard sample tested with different dilutions. Detailed Implementation

[0036] The present application will be further described below with reference to specific embodiments.

[0037] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0039] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0040] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0041] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0042] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0043] Example 1

[0044] This embodiment provides a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology. The kit includes: a diluent for detecting human phosphorylated Tau protein p18; a capture antibody; a detection antibody; and a reading buffer.

[0045] in:

[0046] The capture antibody was GTX54658 (genetex);

[0047] The detection antibody was F218D (Meso Scale Discovery, MSD);

[0048] The reading solution was R92TC-3 (Meso Scale Discovery, MSD);

[0049] The diluent consists of: sodium chloride 6–10 g / L, potassium chloride 0.1–0.3 g / L, Tris-base 2–4 g / L, BSA 5–15 g / L, mouse negative IgG 20–40 g / L, and pure water.

[0050] In the examples, the diluent consisted of: 6 g / L sodium chloride, 0.1 g / L potassium chloride, 2 g / L Tris-base, 5 g / L BSA, and 20 g / L mouse negative IgG.

[0051] In the examples, the diluent consisted of: 8 g / L sodium chloride, 0.2 g / L potassium chloride, 3 g / L Tris-base, 10 g / L BSA, and 30 g / L mouse negative IgG.

[0052] In the examples, the diluent consisted of: 10 g / L sodium chloride, 0.3 g / L potassium chloride, 4 g / L Tris-base, 15 g / L BSA, and 40 g / L mouse negative IgG.

[0053] Example 2

[0054] This embodiment provides a standard curve for the application of the detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology from Example 1. Specifically, it includes the following steps:

[0055] S1: Dilute the capture antibody (which needs to be biotinylated) with coating buffer (1×TBS, pH 7.4) and add it to each well of the ELISA plate (MSD, catalog number L45SA). Seal each well with a sealing film to prevent liquid evaporation. Place the ELISA plate flat at room temperature for 1 hour for coating and shake at 800±50 rpm. Wash each well with washing buffer (1×TBS buffered saline containing 0.05% Tween 20). After washing three times, add blocking buffer (1×TBS, 3% BSA) to each well, seal each well with a sealing film, and incubate at room temperature for 1 hour. BSA blocks the exposed sites on the detection plate to prevent non-specific adsorption.

[0056] S2: Add the test sample and / or gradient standards to the corresponding wells. The test sample and / or gradient standards are prepared using a dilution solution for the detection of human phosphorylated Tau protein p18 as described in Example 1. Incubate at 25°C for 1 h on a shaker at 800±50 rpm. Wash each well with washing buffer (containing 0.05% Tween 20 in 1×TBS buffered saline).

[0057] S3: Antibody incubation. Wash each well with washing buffer (containing 0.05% Tween 20 in 1×TBS buffered saline); dilute the detection antibody with 1×TBS, pH 7.4, and 0.1% BSA and add it to the corresponding well. Incubate at 25°C for 1 hour.

[0058] S4: Add reading solution to the well and wash with washing solution; add reading solution to each well and then test.

[0059] The concentrations of capture antibodies, detection antibodies, and standards are shown in Table 1.

[0060] Table 1. Concentrations of capture antibodies, detection antibodies, and standards.

[0061]

[0062]

[0063] Table 2 Standard curves for different ratios of capture antibody and detection antibody

[0064] 2000pg / ml 46266 73168 61688 250pg / ml 7315 14106 9754 125pg / ml 1065 2975 1419 31.25 pg / ml 852 965 1136 0 210 230 260 Signal-to-noise ratio 220 318 237

[0065] The results are shown in Table 2. From the perspective of signal-to-noise ratio, the ratio of capture antibody to detection antibody in standard curve 2 is optimal (highest signal-to-noise ratio), and subsequent experiments will be conducted at this concentration.

[0066] Example 3

[0067] This embodiment provides the detection of human phosphorylated Tau protein p18 with different sample dilutions. The specific steps are as described in Embodiment 2. The concentration of the capture antibody is 0.5 μg / ml and the concentration of the detection antibody is 1 μg / ml. The signal values ​​of different dilutions and corresponding standards are shown in Table 3.

[0068] The components of the following diluent include:

[0069] Diluent 1: PBS buffer, specifically including: Absin 10x PBS, abs961;

[0070] Diluent 2: TBS buffer, specifically comprising: sodium chloride 6 g / L, potassium chloride 0.1 g / L, Tris-base 2 g / L, BSA 1 g / L;

[0071] Diluent 3: Sodium chloride 8 g / L, potassium chloride 0.2 g / L, Tris-base 3 g / L, BSA 10 g / L, mouse negative IgG 10 g / L;

[0072] Diluent 4: Sodium chloride 8 g / L, potassium chloride 0.2 g / L, Tris-base 3 g / L, BSA 10 g / L, mouse negative IgG 30 g / L.

[0073] Table 3. Signal values ​​of different diluents and corresponding standard samples

[0074]

[0075]

[0076] Result Analysis: Based on the above signal values, we can obtain... Figure 1 The standard curve shows that the dilution 4 in this invention has higher sensitivity and signal-to-noise ratio, making it easier to detect trace amounts of p-TAU18 protein expression and exhibiting stronger resistance to mechanism interference.

Claims

1. A diluent for detecting human phosphorylated Tau protein p18, characterized in that, The diluent comprises: sodium chloride 6-10 g / L, potassium chloride 0.1-0.3 g / L, Tris-base 2-4 g / L, BSA 5-15 g / L, mouse negative IgG 20-40 g / L, and pure water.

2. The diluent for detecting human phosphorylated Tau protein p18 according to claim 1, characterized in that, The diluent comprises: 6 g / L sodium chloride, 0.1 g / L potassium chloride, 2 g / L Tris-base, 5 g / L BSA, and 20 g / L mouse negative IgG.

3. The diluent for detecting human phosphorylated Tau protein p18 according to claim 1, characterized in that, The diluent comprises: 8 g / L sodium chloride, 0.2 g / L potassium chloride, 3 g / L Tris-base, 10 g / L BSA, and 30 g / L mouse negative IgG.

4. The diluent for detecting human phosphorylated Tau protein p18 according to claim 1, characterized in that, The diluent comprises: 10 g / L sodium chloride, 0.3 g / L potassium chloride, 4 g / L Tris-base, 15 g / L BSA, and 40 g / L mouse negative IgG.

5. The use of the diluent for detecting human phosphorylated Tau protein p18 as described in any one of claims 1-4 in the preparation of a detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology.

6. A detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology, characterized in that, The kit comprises: a diluent for the detection of human phosphorylated Tau protein p18 as described in any one of claims 1-4; a capture antibody; a detection antibody; and a reading buffer; wherein the capture antibody is GTX54658 and the detection antibody is F218D.

7. The application of the detection kit for human phosphorylated Tau protein p18 based on electrochemiluminescence technology as described in claim 6 in the detection of human phosphorylated Tau protein p18 expression level for non-diagnostic purposes.

8. The application according to claim 7, characterized in that, The expression level of human phosphorylated Tau protein p18 includes the expression level of human phosphorylated Tau protein p18 in blood, plasma, brain lysate, and cerebrospinal fluid.

9. The application according to claim 8, characterized in that, Includes the following steps: S1: Capture antibody coating: Dilute the capture antibody with coating buffer and add it to each well of the microplate. Seal each well with sealing film to prevent liquid evaporation. Place the microplate flat at room temperature for 1 hour and shake at 800±50 rpm. Add washing buffer to each well for washing. Add blocking buffer to each well, seal each well with sealing film, and incubate at room temperature for 1 hour. S2: Sample loading: Add the test sample and / or gradient standards to the corresponding wells. The test sample and / or gradient standards are prepared using the dilution solution for the detection of human phosphorylated Tau protein p18 as described in any one of claims 1-4; incubate at 25°C for 1 h on a shaker at 800±50 rpm; add washing solution to each well for washing. S3: Incubate the detection antibody, add washing buffer to each well for washing; dilute the detection antibody and add it to the corresponding well, and react at 25°C for 1 h. S4: Reading solution is added to the plate, and washing solution is added to each microwell for washing; Add reading solution to each well and perform instrument testing.

Images