Antibodies for the detection of phosphorylated tau protein ptau181, immunoassay methods and uses

Abstract

The application discloses an antibody for detecting phosphorylated Tau protein pTau181, an immunodetection method and application. The antibody comprises a T181-1B4 antibody; light chain CDR1, CDR2 and CDR3 of the T181-1B4 antibody are sequentially the sequences shown in SEQ ID NO. 1 to 3, and heavy chain CDR1, CDR2 and CDR3 are sequentially the sequences shown in SEQ ID NO. 4 to 6. The antibody is used for immunodetection of the phosphorylated Tau protein pTau181, and has the advantages of simple operation, high sensitivity and strong specificity, and can realize rapid detection of the phosphorylated Tau protein pTau181, and has important significance for evaluating the Tau protein phosphorylation level and Tau protein pTau181 phosphorylation related detection.

Description

Technical Field

[0001] This application relates to the field of detection technology for phosphorylated Tau protein pTau181, and in particular to an antibody, kit, and application for detecting phosphorylated Tau protein pTau181. Background Technology

[0002] Tau protein is a microtubule-associated protein primarily expressed in neurons. Its core function is to maintain cytoskeleton stability and regulate axoplasmic transport by binding to microtubules. Under physiological conditions, the phosphorylation level of Tau protein is regulated by the dynamic balance of kinases and phosphatases, participating in physiological processes such as fetal development and cell cycle regulation. However, in neurodegenerative diseases such as Alzheimer's disease (AD), Tau protein undergoes abnormal hyperphosphorylation, leading to a decreased ability to bind to microtubules and subsequently accumulating to form neurofibrillary tangles (NFTs).

[0003] Total Tau (t-Tau) refers to the total amount of Tau protein in cells, and elevated levels typically reflect the overall degree of neuronal damage or death. Different phosphorylation sites and their subtypes have unique pathological significance: phosphorylated Tau181 (p-Tau181) can be elevated in the early stages of Alzheimer's disease (AD), up to 20 years before the onset of clinical symptoms, with a specificity of 89%–98%; phosphorylated Tau217 (p-Tau217) has the strongest correlation with amyloid deposition and Tau aggregation in the brain, and is one of the most sensitive and specific biomarkers for AD; phosphorylated Tau231 (p-Tau231) participates in the pathological conformational changes of Tau protein, and its level changes are related to the rate of disease progression. The synergistic detection of these subtypes provides a multidimensional perspective for accurate disease assessment.

[0004] The detection of Tau protein and its phosphorylated isoforms (p-Tau181, p-Tau217, p-Tau231) has become a core method for the diagnosis of neurodegenerative diseases. Immunoassays are cost-effective and suitable for large-scale screening. In clinical applications, combining them with cognitive phenotype analysis and multi-marker detection can significantly improve the accuracy of early diagnosis and the reliability of prognostic assessment in Alzheimer's disease (AD). Plasma Tau testing holds promise as a routine screening tool in primary healthcare, supporting early intervention and precision treatment. Immunoassays rely on specific antibodies; therefore, developing highly sensitive and specific antibodies against phosphorylated Tau protein pTau181 to achieve rapid detection of pTau181 is crucial for assessing Tau protein phosphorylation levels and related detection of pTau181 phosphorylation. Summary of the Invention

[0005] The purpose of this application is to provide a novel antibody, immunoassay method, kit, and application for the detection of phosphorylated Tau protein pTau181.

[0006] The following technical solution is adopted in this application: The first aspect of this application discloses an antibody for detecting phosphorylated Tau protein pTau181, the antibody comprising a T181-1B4 antibody; the T181-1B4 antibody comprises a heavy chain and a light chain, wherein the three complementarity-determining regions CDR1, CDR2 and CDR3 of the light chain variable region are sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and the three complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region are sequentially shown in SEQ ID NO.4 to SEQ ID NO.6.

[0007] It should be noted that highly sensitive and specific antibodies are crucial for the successful development and implementation of immunoassay techniques. This application utilizes monoclonal antibody technology to obtain a specific antibody that recognizes phosphorylated Tau protein pTau181, namely monoclonal antibody T181-1B4. In other words, the antibody in this application is a specific antibody against phosphorylated Tau protein pTau181. Immunoassay using this specific antibody is simple to operate, highly sensitive, and highly specific, enabling rapid detection of phosphorylated Tau protein pTau181.

[0008] In one implementation of this application, the heavy chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.7, and the light chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.8.

[0009] The third aspect of this application discloses a nucleic acid encoding the antibody of this application.

[0010] The fourth aspect of this application discloses a recombinant plasmid containing a nucleic acid encoding a specific antibody that specifically recognizes the phosphorylated Tau protein pTau181 protein.

[0011] The fifth aspect of this application discloses a nucleic acid containing a specific antibody encoding a specific antibody that specifically recognizes the phosphorylated Tau protein pTau181 protein of this application, or a microorganism or cell containing the recombinant plasmid of this application.

[0012] It should be noted that, in one implementation of this application, hybridoma cell technology is specifically used to screen and obtain the specific antibody that specifically recognizes the phosphorylated Tau protein pTau181. It can be understood that, based on this, the coding sequence of the specific antibody can be artificially synthesized according to its sequence, and then expressed through cloning to obtain the specific antibody. The specific nucleic acid sequence can be optimized according to the preferences of the cloning expression microorganism or cell used, and the specific vector sequence and host microorganism or cell can also refer to existing antibody cloning expression technologies, and are not specifically limited here.

[0013] In one implementation of this application, the T181-1B4 antibody is coated on an ELISA plate.

[0014] It should be noted that the key to the immunoassay method of this application lies in the use of the specific antibody of this application. As for the preparation method of the immunoassay, existing technologies can be referenced. In one implementation of this application, a double antibody sandwich method is specifically used. Therefore, the specific antibody T181-1B4 against phosphorylated Tau protein pTau181 protein is used as the detection coating antibody, and another antibody against total Tau protein is used as the detection labeling antibody.

[0015] The sixth aspect of this application discloses the use of the specific antibody of this application, the nucleic acid encoding the specific antibody of this application, the recombinant plasmid of this application, and the microorganism or cell of this application in the preparation of a kit for detecting phosphorylated Tau protein pTau181.

[0016] The seventh aspect of this application discloses a kit for detecting phosphorylated Tau protein pTau181, which contains the specific antibody of this application, a nucleic acid encoding the specific antibody of this application, a recombinant plasmid of this application, a microorganism or cell of this application, or an immunoassay method of this application.

[0017] It should be noted that the kit for detecting phosphorylated Tau protein pTau181 of this application may directly contain the immunoassay method of this application. In use, this immunoassay method is directly employed to detect phosphorylated Tau protein pTau181. The kit may also contain the specific antibody of this application. In use, the specific antibody of this application is used for immunoassay, or the specific antibody of this application is used to prepare the immunoassay method. The kit may also contain the nucleic acid, recombinant plasmid, or cloned microorganisms or cells of this application. In this case, the kit mainly serves as a raw material for preparing the specific antibody of this application to facilitate the detection of phosphorylated Tau protein pTau181.

[0018] The eighth aspect of this application discloses an immunoassay method for detecting phosphorylated Tau protein pTau181, wherein the detection antibody used is the antibody of this application, that is, a specific antibody for detecting phosphorylated Tau protein pTau181. Specifically, the specific antibody of this application, the nucleic acid encoding the specific antibody of this application, the recombinant plasmid of this application, the microorganism or cell of this application, or the kit of this application can be used directly to detect the sample to be tested.

[0019] It should be noted that the key to the detection method in this application lies in the immunodetection of phosphorylated Tau protein pTau181 using the specific antibody described in this application. The beneficial effects of this application are: The antibody for detecting phosphorylated Tau protein pTau181 in this application has high sensitivity and specificity. Immunodetection of phosphorylated Tau protein pTau181 based on the antibody of this application is simple to operate, highly sensitive and specific, and can realize rapid detection of phosphorylated Tau protein pTau181. It is of great significance for assessing the phosphorylation level of Tau protein and related detection of Tau protein pTau181 phosphorylation. Attached Figure Description

[0020] Figure 1 This is the titer test result of the T181-1B4 antibody in the embodiments of this application.

[0021] Figure 2 This is the specific detection result of the T181-1B4 antibody in the embodiments of this application.

[0022] Figure 3 The results of immunoblotting detection of Tau protein and phosphorylated Tau protein in the embodiments of this application are shown.

[0023] Figure 4 This is the standard curve for detecting human phosphorylated Tau protein using a double-antibody sandwich ELISA method in the embodiments of this application. Detailed Implementation

[0024] This application describes the preparation of a monoclonal antibody against phosphorylated Tau protein pTau181 by immunizing animals with a phosphorylated Tau protein pTau181 polypeptide fragment. This monoclonal antibody exhibits high binding affinity and specificity for phosphorylated Tau protein pTau181. This makes the established immunoassay method for phosphorylated Tau protein pTau181 simple to operate, with higher sensitivity and better specificity, enabling rapid detection of phosphorylated Tau protein pTau181.

[0025] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Balb / c mice: Guangdong Provincial Experimental Animal Center, 6-8 weeks old.

[0027] Example 1: Preparation of monoclonal antibody against phosphorylated Tau protein pTau181 I. Animal Immunization and Cell Fusion Screening The phosphorylated Tau protein pTau181 peptide fragment was conjugated with OVA protein, diluted with physiological saline, and then mixed with MontanideGel 01 PR adjuvant at a ratio of 9:1 to prepare an injection solution with a protein concentration of 400 μg / mL. Three Balb / c mice were then immunized intramuscularly in the calf, with an injection dose of 20 μg / mouse. A booster immunization was performed on day 14 using the same method. Blood was collected from the tail of the mice on day 21, and serum titers were detected using an indirect ELISA method. The titers met the fusion requirement, i.e., reaching 10-1. 6 Three days before cell fusion, spleen booster immunization was performed using 100 μg of pTau181 conjugated with OVA protein.

[0028] Three days after spleen booster immunization, mice were euthanized according to biosafety procedures, specifically carbon dioxide asphyxiation. After alcohol disinfection, spleen cells were fused with logarithmically growing NS1 myeloma cells at a 5:1 ratio in 50% PEG1500. The fused cells were cultured in HAT semi-solid selection medium for 8-10 days. Cell clones were then picked and cultured in HT medium for 2 days. Antibody specificity in the cell culture supernatant was detected using an indirect ELISA method. Positive cells were cloned again in HT semi-solid medium to obtain a stable antibody-secreting monoclonal cell line. After expansion culture, the cells were cryopreserved in a cell cryopreservation medium containing 10% DMSO at a cell density of 10-1. 6 Ascites fluid was prepared using an in vivo induction method. The ascites antibody was purified by Protein A affinity chromatography, dialyzed in 0.01 mol / L phosphate buffer, and the protein concentration was determined using Nanodrop. Multiple monoclonal cell lines were obtained. After antibody titer identification, a cell line with optimal specificity was screened from the monoclonal cell lines and named hybridoma cell line T181-1B4.

[0029] In the above methods, the indirect ELISA method for detecting serum titer and identifying antibody titer is the same as in "II. Titer Detection". The phosphorylated Tau protein pTau181 polypeptide fragment APK(p)TPPSSGEPP, the phosphorylated Tau protein pTau181 polypeptide conjugated with OVA protein, and the phosphorylated Tau protein pTau181 polypeptide conjugated with BSA protein were all synthesized and prepared by Jier Biochemical (Shanghai) Co., Ltd.

[0030] II. Potency Testing (Indirect ELISA Method) 1. Antigen coating: Take phosphorylated Tau protein pTau181 peptide conjugated with BSA protein, dilute with pH 9.6, 0.05M carbonate buffer to a protein concentration of 2 μg / mL, add 100 μL / well to the microplate, and coat overnight at 4℃.

[0031] 2. After completing step 1, take the microplate, wash it three times with PBST solution, then add blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Blocking buffer: PBST solution containing 1 g / 100 mL BSA.

[0032] 3. After completing step 2, take the ELISA plate, wash it three times with PBST solution, then add antibody dilution buffer (100 μL / well) and incubate at 37°C for 1 hour. Antibody dilution: Take monoclonal antibody T181-1B4 and dilute it with 0.02M PBS buffer (pH 7.4) to a protein concentration of 10 μg / mL, then perform 10-fold serial dilutions.

[0033] 4. After completing step 3, take the ELISA plate, wash it three times with PBST solution, then add HRP-labeled secondary antibody working solution and incubate at 37°C for 1 hour. HRP-labeled secondary antibody working solution: 1:4000 dilution of HRP-labeled goat anti-mouse secondary antibody.

[0034] 5. After completing step 4, take the microplate, wash it three times with PBST solution, then add 100 μL of TMB substrate reaction solution (purchased from Beijing MecoWander Biotechnology Co., Ltd., catalog number 1001H) and develop the color for 10 minutes. Then add 50 μL of stop solution (purchased from Beijing MecoWander Biotechnology Co., Ltd., catalog number 1001SA) to stop the color development. Finally, measure the absorbance at 450 nm using a microplate reader. The results are shown in the figure. Figure 1 . Figure 1 The horizontal axis represents the exponent of 10, -2 is equivalent to 10. -2 ng / mL, that is, 0.01ng / mL, and so on.

[0035] Figure 1 The results showed that the EC50 of the monoclonal antibody T181-1B4 was less than 1.7 ng / mL.

[0036] III. Antibody Specific Detection Indirect ELISA was used for detection. Phosphorylated Tau protein pTau181 peptide, non-phosphorylated Tau181 peptide, phosphorylated Tau protein pTau217 peptide, non-phosphorylated recombinant human Tau protein, and phosphorylated recombinant human Tau protein were diluted to a protein concentration of 2 μg / mL with 0.05 M carbonate buffer (pH 9.6). 100 μL / well was added to an ELISA plate, and the plate was incubated overnight at 4°C. The plate was washed three times with PBST. Blocking buffer containing 1 g / 100 mL BSA was added, and the plate was incubated at 37°C for 1 h. The plate was washed three times with PBST. 100 μL of 0.1 μg / mL T181-1B4 was added, and the plate was incubated at 37°C for 1 h. The plate was washed three times with PBST. 100 μL of... HRP-labeled goat anti-mouse secondary antibody diluted 1:4000 was incubated at 37°C for 1 hour, followed by washing three times with PBST. TMB substrate solution was then added for color development for 10 minutes, and stop solution was added to terminate the color development. The absorbance at 450 nm was measured using a microplate reader. Results are shown below. Figure 2 T181-1B4 only recognizes phosphorylated Tau protein pTau181 peptide and phosphorylated recombinant human Tau protein, and does not recognize non-phosphorylated Tau181 peptide, phosphorylated Tau protein pTau217 peptide, and non-phosphorylated full-length Tau protein. This indicates that the anti-human phosphorylated Tau181 monoclonal antibody T181-1B4 of this application specifically recognizes the phosphorylation epitope at threonine (T) at position 181 in human Tau protein.

[0037] IV. Antibody Subtype Identification Antibody subtypes were identified using a commercially available antibody Ig class / subclass / subtype identification kit (Luoyang Baitong Experimental Materials Center, catalog number C060101-L). Specific methods were described in the kit's instruction manual. The results are shown in Table 1.

[0038] Table 1. Antibody subtype test results The results in Table 1 indicate that antibody T181-1B4 is an IgG1 subtype and its light chain is a Kappa chain.

[0039] Example 2: Monoclonal Antibody Sequencing The amino acid sequences of the antibody T181-1B4, consisting of three CDRs each in the light and heavy chains, were obtained by sequencing and identification by General Biotech (Anhui) Co., Ltd.

[0040] Based on the sequencing results, the amino acid sequences of the light chain and heavy chain variable regions of antibody T181-1B4, and their respective three CDRs, were determined with reference to the Kabat and IMGT numbering methods. Specifically, the heavy chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.7, and the light chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.8; the amino acid sequences of each CDR of the antibody are shown in Table 2.

[0041] SEQ ID NO.7: EVQLEESGPGLVAPSQSLSITCTVSGFSLTDYGVSWIRQSPGKGLEWLGIIWKTGSTYYNSGLKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAKHRGDWGQGTLVTVSA SEQ ID NO.8: QIVLTQSPAILSASPGDKVTLTCRASSSISYMHWYQQKPGSSPKPWISATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAAIYYCQQWSSNPYTFGGGTKLEIK Table 2. Amino acid sequences of three CDRs each from the antibody light chain and heavy chain. Example 3: Detection of phosphorylation of human Tau protein 20 μg of recombinant Tau protein was prepared to a concentration of 100 μg / mL using phosphorylation buffer (40 mM HEPES, 3 mM MgCl2, 5 mM EGTA, 10 mM ATP). 0.1 μg of phosphokinase GSK-3β protein was added, and the mixture was incubated at 30°C for 12 h to obtain phosphorylated Tau protein. 25 ng of Tau protein and phosphorylated Tau protein were spotted onto a nitrocellulose membrane, and the membrane was detected by Western blotting using antibody T181-1B4 and anti-total Tau antibody T-1C1. The results are as follows: Figure 3 As shown, antibody T181-1B4 only recognizes phosphorylated Tau protein.

[0042] Example 4: Preparation of Immunoassay Kit I. Preparation of HRP-labeled antibodies HRP-labeled antibodies were prepared using the EZ-Link™ Activated Peroxidase Labeling Kit (Thermo, catalog number 31489). Specifically, anti-total Tau antibody T-1C1 was dialyzed overnight with carbonate buffer (pH 9.4). Then, 1 mg of antibody T-1C1 was added to 1 mg of activated HRP, mixed well, and reacted at 4°C in the dark for 12 hours. 10 µL of sodium cyanoborohydride was added, and the reaction was carried out at room temperature for 15 minutes. 20 µL of stop buffer was added, and the reaction was carried out at room temperature for 15 minutes. After labeling, the antibody was dialyzed at 4°C in the dark for 24 hours, and then an equal volume of glycerol was added before storage at -20°C.

[0043] II. Preparation of coated ELISA plates 1. Coating: Dilute the T181-1B4 antibody with PBS to prepare a 2 μg / ml coating antibody solution. Add 100 μl to each well of a 96-well microplate and incubate overnight at 2-8°C.

[0044] 2. Washing: Take out the 96-well microplate coated overnight and place it on the plate washer rack. Set the washing buffer volume to 300 μl / well and wash 3 times. After washing, gently pat dry on clean absorbent paper.

[0045] 3. Blocking: Add 240 μl of blocking solution to each well of the microplate. After adding the samples, attach the sealing film and incubate the 96-well microplate at 37°C for 1 hour.

[0046] 4. Washing the plate: Wash the blocked microplate as in step 2, wash 3 times, and gently pat dry on clean absorbent paper after washing.

[0047] III. Immunological Testing 1. Sample addition: Dilute phosphorylated recombinant human Tau protein to prepare a series of concentration samples. Add 100 μl of each sample to a 96-well microplate and incubate at 37°C for 1 hour.

[0048] 2. Washing the plate: After incubation, set the washing buffer volume to 300 μl / well and wash 3 times. After washing, gently pat dry on clean absorbent paper.

[0049] 3. Add enzyme-labeled antibody: Add 100 μl of T-1C1-labeled HRP (1:2000 dilution) to each well of a 96-well microplate and incubate at 37°C for 1 hour.

[0050] 4. Washing: After incubation, set the washing solution volume to 300 μl / well, wash 3 times, and gently pat dry on clean absorbent paper after washing.

[0051] 5. Color development: Add 100 μl of TMB color development solution to each well of a 96-well microplate and let stand at room temperature for 10 minutes.

[0052] 6. Termination: After the color development is complete, add 50 μl of the stop solution to each well of the 96-well microplate.

[0053] 7. Absorbance Measurement: After the termination procedure, use a microplate reader to measure the absorbance at a wavelength of 450 nm. The detection should be completed within 5 minutes of termination. The results are shown in Table 4.

[0054] Table 4 Results of phosphorylated human Tau protein detected by double-antibody sandwich ELISA Plot a four-parameter standard curve based on the measured absorbance values, such as... Figure 4 As shown, the standard curve R 2 =0.9983, linear detection range 0-10000 pg / mL, detection sensitivity less than 9.8 pg / mL.

[0055] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. An antibody for the detection of phosphorylated Tau protein pTau181, characterized in that: The antibody includes the T181-1B4 antibody; The T181-1B4 antibody comprises a heavy chain and a light chain. The three complementary determinant regions (CDR1, CDR2, and CDR3) of the light chain are sequentially shown in SEQ ID NO.1 to SEQ ID NO.3, and the three complementary determinant regions (CDR1, CDR2, and CDR3) of the heavy chain are sequentially shown in SEQ ID NO.4 to SEQ ID NO.

6.

2. The antibody of claim 1, wherein: The heavy chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.7, and the light chain variable region of T181-1B4 is the sequence shown in SEQ ID NO.

8.

3. The nucleic acid encoding the antibody of claim 1 or 2.

4. A recombinant plasmid containing the nucleic acid of claim 3.

5. Microorganisms or cells containing the nucleic acid of claim 3 or the recombinant plasmid of claim 4.

6. The use of the antibody of claim 1 or 2, the nucleic acid of claim 3, the recombinant plasmid of claim 4, or the microorganism or cell of claim 5 in the preparation of a kit for detecting phosphorylated Tau protein pTau181.

7. A kit for the detection of phosphorylated Tau protein pTau181, characterized in that: It contains the antibody as described in claim 1 or 2, the nucleic acid as described in claim 3, the recombinant plasmid as described in claim 4, or the microorganism or cell as described in claim 5.

8. An immunoassay method for detecting phosphorylated Tau protein pTau181, characterized in that: The sample to be tested is made using the antibody as described in claim 1 or 2, the nucleic acid as described in claim 3, the recombinant plasmid as described in claim 4, the microorganism or cell as described in claim 5, or the kit as described in claim 7.

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