Hybridoma cell strain secreting anti-181-site threonine phosphorylated tau monoclonal antibody

Abstract

The invention relates to the technical field of immunodetection, in particular to a hybridoma cell strain secreting an anti-threonine phosphorylated tau protein monoclonal antibody. The hybridoma cell strain for secreting the p-tau181 monoclonal antibody is preserved in the China Center for Type Culture Collection, and the preservation numbers of the hybridoma cell strain and the hybridoma cell strain are respectively CCTCC NO: C2024272 and CCTCC NO: C2024273. The hybridoma cell strain can be used for preparing a monoclonal antibody capable of specifically recognizing p-tau181 and aggregate thereof, and the problem that a targeted p-tau181 antibody is lacked at present is solved. The invention has the advantages that the two monoclonal antibodies can be further applied subsequently to prepare a high-specificity and high-sensitivity p-tau181 detection kit, the passive immunological treatment of specific targeting p-tau181 is developed, the disease progress is slowed down, and the blank of the current immunological treatment of targeting p-tau181 is filled up.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, and in particular to a hybridoma cell line secreting an anti-181-site threonine phosphorylated tau protein monoclonal antibody. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. AD is the most common cause of dementia, accounting for 60% to 80% of dementia cases worldwide. AD has a high prevalence among the elderly, and its incidence increases exponentially with age. With the aggravation of the global aging trend, the prevention and treatment of AD has become the most important public health issue in the world. As of 2024, there will be more than 1 million new cases of Alzheimer's disease in China each year, and the number of AD patients will be close to 9 million. As the population structure enters an aging period, the total number of AD patients in China is expected to exceed 10 million by 2029.

[0003] Currently, there is a lack of effective early diagnosis and treatment methods for AD, which brings great economic burden to patients' families and society. Therefore, early diagnosis of AD is extremely necessary. In addition, a large number of biomarkers are needed clinically to identify early AD patients before clinical symptoms. The cerebrospinal fluid and blood biomarkers tau protein phosphorylated at site 181 (p-tau 181 ), demonstrated high enough diagnostic accuracy to meet this challenge.

[0004] Immunological detection has been increasingly used due to its high sensitivity and rapid speed. 181 The most important factor limiting the quality of immunological test kits is the quality of biologically active raw materials. 181 The tests are all based on specific antibody pairs, and the existing p-tau 181 Most antibodies are imported, which are expensive and have a long purchase cycle. The quality of some domestic antibodies varies greatly, which seriously limits the quality of the detection kits and the accuracy of AD diagnosis. In view of this, it is necessary to develop high-quality specific p-tau 181 Monoclonal antibodies are of great significance for the early diagnosis, detection and targeted treatment of AD. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a hybridoma cell line which secretes anti-threonine phosphorylated tau protein monoclonal antibody.

[0006] The present invention aims to provide a method for secreting p-tau 181The hybridoma cell line of the monoclonal antibody has a deposit number of CCTCC NO: C2024272; the p-tau 181 It is tau protein phosphorylated at threonine 181.

[0007] The present invention also provides a method for secreting p-tau 181 The hybridoma cell line of the monoclonal antibody has a deposit number of CCTCCNO: C2024273; the p-tau 181 It is tau protein phosphorylated at threonine 181.

[0008] The present invention also provides a method for secreting human p-tau 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Application of monoclonal antibodies.

[0009] The present invention also provides a method for secreting p-tau 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Monomer or p-tau 181 Application of oligomer detection reagent; the p-tau 181 The monomer is a single protein formed by human tau protein phosphorylated at site threonine 181; the p-tau 181 Oligomers are proteins of different molecular weights obtained by mutual folding of human tau proteins phosphorylated at threonine 181.

[0010] The present invention also provides a method for secreting human p-tau 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Monomer or p-tau 181 Application in an aggregate detection kit; the p-tau 181 The monomer is a single protein formed by human tau protein phosphorylated at site threonine 181; the p-tau 181 The aggregates are proteins of different molecular weights obtained by mutual folding of human tau proteins phosphorylated at threonine 181.

[0011] The present invention also provides a method for secreting human p-tau 181 Use of hybridoma cell lines of monoclonal antibodies in the preparation of antibody drugs for treating Alzheimer's disease.

[0012] Preferably, the antibody drug for treating Alzheimer's disease is specifically used to inhibit and eliminate the formation of p-tau by tau protein phosphorylated at threonine 181 in humans. 181 Monomers and aggregates.

[0013] Compared with the prior art, the present invention can achieve the following beneficial effects: The two secreted anti-human 181-threonine phosphorylated tau protein (p-tau 181 ) monoclonal antibody hybridoma cell lines HNU (181) mab-1E9 and HNU (181) mab-1H8, which can be used to prepare antibodies that specifically recognize human p-tau 181 and its aggregates, and further studies have been conducted on these anti-human p-tau 181 Subsequent application of monoclonal antibodies fills the gap of current simultaneous targeting of p-tau 181 Antibody blank.

[0014] The two hybridoma cell lines of the present invention can be used to secrete and prepare anti-human p-tau 181 Monoclonal antibodies were obtained that specifically recognized p-tau 181 Monoclonal antibodies to monomers and aggregates can be further used to prepare p-tau 181 Detection reagents (such as different forms of p-tau 181 detection kits, etc.), can also be used to inhibit p-tau 181 Monomer or p-tau 181 Folding into neurofibrillary tangles, preparing antibody drugs to clear neurofibrillary tangles, and treating AD; the prepared antibody drugs and related vaccines can inhibit and clear phosphorylated tau protein to form neurofibrillary tangles and clear neurofibrillary tangles.

[0015] The two monoclonal antibodies of the present invention can prepare high specificity and sensitivity p-tau 181 The detection kit can realize the early detection of AD disease and fill the gap of current p-tau 181 In addition, the two monoclonal antibodies developed can be further developed to specifically target p-tau 181 Immunological treatment to slow down the disease progression and fill the current lack of targeting p-tau 181 gaps in immunological treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 According to the embodiment of the present invention, (A) is 2 strains of anti-human p-tau 181 ELISA activity test results of monoclonal antibodies; (B) is the selectivity results of monoclonal antibodies 1E9 and 1H8 for different p-tau peptides.

[0017] Figure 2(A) is the titer test result of monoclonal antibody 1E9; (B) is the titer test result of monoclonal antibody 1H8 provided according to an embodiment of the present invention.

[0018] Figure 3 According to the embodiment of the present invention, (A) is 1E9 for p-tau 181 Peptide affinity results; (B) 1H8 to p-tau 181 Affinity results for peptides.

[0019] Figure 4 According to an embodiment of the present invention, the anti-human p-tau 181 Diagnosis of monoclonal antibodies in healthy controls (NC), mild cognitive impairment (MCI) and AD patients (A) is the ROC curve for distinguishing AD from NC; (B) is the ROC curve for distinguishing MCI from NC; (C) is the ROC curve for distinguishing non-NC from NC. DETAILED DESCRIPTION

[0020] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are represented by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, the detailed description thereof will not be repeated.

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0022] Example 1 Anti-human p-tau 181 The preparation of specific monoclonal antibodies specifically includes the following steps: 1. Preparation of Antigen The human tau protein (p-tau) phosphorylated at serine 181 was synthesized by a method known in the prior art. 181 ), dissolved in TBS solution to a final concentration of 1 mg / mL.

[0023] 2. Preparation of mouse monoclonal antibodies (1) Immunization of mice The first immunization was performed by mixing 100 μL of the prepared antigen with an equal volume of Freund's complete adjuvant; the second immunization was performed 14 days after the first immunization, and 100 μL of the prepared antigen was mixed with an equal volume of Freund's incomplete adjuvant for immunization; the third immunization was the same as the second one, with an interval of 14 days. After the third immunization, 100 μL of the prepared antigen was taken for tail vein booster immunization.

[0024] (2) Screening of positive clones by cell fusion Three days after booster immunization, spleen cells of mice were collected and fused with SP2 / 0 cells, and the fused cells were cultured with HAT medium. Half of the HT medium was replaced after one week. The first ELISA test was performed 10 days after cell fusion, and the positive wells were taken for the first subcloning. The second ELISA test was performed 10 days after the first subcloning, and the positive wells were taken for the second subcloning. The third ELISA test was performed 10 days after the second subcloning. If the ELISA test results were positive after three subcloning, it means that it has become a monoclonal clone, and it is frozen in liquid nitrogen after expanded culture.

[0025] (3) Preparation and purification of ascites Three 6-7 week old female Balb / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. One week later, each mouse was intraperitoneally injected with 10 8 hybridoma cells; one week later, the ascites was collected; and the ascites was purified using a protein A Sepharose column.

[0026] Results: After cell fusion, two strains of anti-human p-tau were successfully screened 181 The monoclonal antibody cell lines were named 1E9 and 1H8. They were deposited in the China Center for Type Culture Collection (CCTCC), where the deposit number of 1E9 is CCTCC No: C2024272, the classification name is Hybridoma cell line 1E9, and the deposit time is August 23, 2024; the deposit number of 1H8 is CCTCC No: C2024273, the classification name is Hybridoma cell line 1H8, and the deposit time is August 23, 2024; the deposit units are all China Center for Type Culture Collection; the deposit address is Wuhan University Collection Center, Luojiashan, Wuchang, Wuhan, Hubei Province.

[0027] Example 2 Identification of the biological characteristics of monoclonal antibodies, including: 1. Identification of monoclonal antibody activity ① Antigen coating: dilute the antigen to 10 μg / mL with TBS, then add 100 μL of antigen to each well and coat overnight at 4°C; ② Blocking: After coating, discard the liquid in the wells, wash with TBS-T three times, add 200 μL of blocking solution to each well, and block at 37 ℃ for 2 h; ③ Add primary antibody: After blocking, discard the liquid in the wells, wash three times with TBS-T, add 100 μL of monoclonal antibody diluted with skim milk (1:50000) to each well, and react at 37°C for 2 h; ④ Add enzyme-labeled secondary antibody: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each well, and react at 37 °C for 1 h; ⑤ Color development: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL TMB to each well, and react at 37°C for 15 min; ⑥Termination: Add 50 μL 2 M H2SO4 to each well and measure the absorbance at OD450 nm.

[0028] Results: The prepared monoclonal antibody ascites was tested for ELISA activity; the ELISA results showed that ( Figure 1 A in the figure), both monoclonal antibodies can bind to p-tau 181 reaction.

[0029] 2. Monoclonal antibody specificity identification ① Coating antigen: p-tau 181 , p-tau 217 , p-tau 231 , p-tau 396,404 Dilute to 10 μg / mL with TBS, then add 100 μL of antigen to each well and coat overnight at 4°C; ② Blocking: After coating, discard the liquid in the wells, wash with TBS-T three times, add 200 μL of blocking solution to each well, and block at 37°C for 2 h; ③ Add primary antibody: After blocking, discard the liquid in the wells, wash three times with TBS-T, add 100 μL of monoclonal antibody diluted with skim milk (1:50000) to each well, and react at 37°C for 2 h; ④ Add enzyme-labeled secondary antibody: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each well, and react at 37°C for 1 h; ⑤ Color development: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL TMB to each well, and react at 37°C for 15 min; Results: The prepared monoclonal antibody only specifically binds to p-tau 181 Reaction, but not reaction with phosphorylated tau protein at other sites; ELISA specificity test results are as follows Figure 1 As shown in B.

[0030] (2) Hybridoma cell ascites titer detection ① Antigen coating: dilute the antigen to 10 μg / mL with TBS, then add 100 μL of antigen to each well and coat overnight at 4°C; ② Blocking: After coating, discard the liquid in the wells, wash with TBS-T three times, add 200 μL of blocking solution to each well, and block at 37 ℃ for 2 h; ③ Add primary antibody: After blocking, discard the liquid in the wells, wash with TBS-T three times, add 100 μL of different dilutions of monoclonal antibody (from 1:6400 to 1:12107200) to each well, and react at 37°C for 2 h; ④ Add enzyme-labeled secondary antibody: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each well, and react at 37 °C for 1 h; ⑤ Color development: After the reaction is completed, discard the liquid in the wells, wash with TBS-T three times, add 100 μL TMB to each well, and react at 37°C for 15 min; ⑥Termination: Add 50 μL 2 M H2SO4 to each well and measure the absorbance at OD450 nm.

[0031] Results: ELISA test results showed that the titer of 1E9 hybridoma cell ascites was as high as 1:6553600. Figure 2 (A); the titer of 1H8 hybridoma cell ascites is as high as 1:3276800. Figure 2 (B) in.

[0032] (3) Monoclonal antibody affinity detection ① Coating antigen: p-tau 181 Dilute the solution to 2.5 μg / mL and add 100 μL / well of the ELISA plate to coat overnight at 4°C.

[0033] ② Blocking: After coating, discard the liquid in the wells, wash three times with TBS-T, add 200 μL of blocking solution to each well, and block at 37°C for 2 h.

[0034] ③ Add antiserum: After blocking, discard the liquid in the wells, wash three times with TBS-T, add 100 μL of antibody (10-20000 ng) diluted in blocking solution to each well, and incubate the 96-well plate at 37°C for 2 hours.

[0035] ④ Add enzyme-labeled secondary antibody: After 2 hours, discard the liquid in the wells, wash with TBS-T three times, and add 100 μL HRP-labeled goat anti-mouse IgG secondary antibody (1:5000) to each well. Place the 96-well plate at 37°C for 1 hour.

[0036] ⑤ Color development: After 1 hour, discard the liquid in the wells, wash with TBS-T three times, add 100 μL of soluble TMB substrate color development solution to each well, and react at 37°C for 15 minutes.

[0037] ⑥Termination: Add 50 μL 2M H2SO4 to each well and measure the absorbance at OD450nm.

[0038] Results: The affinity test results of the two monoclonal antibodies showed that the Kd value of the 1E9 antibody was 0.079nM. Figure 3 In (A), the Kd value of 1H8 antibody is 0.272 nM. Figure 3 (B) in.

[0039] Example 3 Based on anti-human p-tau 181 Performance of monoclonal antibodies in distinguishing healthy controls (NC), mild cognitive impairment (MCI) and AD patients Plasma samples from AD (n=38), mild cognitive impairment (MCI, n=30), and healthy elderly controls (NC, n=24) were collected to evaluate the anti-human p-tau 181 The detection method established by monoclonal antibodies (RPA-CRISPR) can distinguish the performance of NC, MCI and AD. The specific steps are as follows: 1) Take 50 μL of 5 mg / mL anti-tau modified Fe3O4 magnetic beads, magnetically separate and discard the supernatant. Then add 100 μL of AD, MCI and NC plasma samples in turn and incubate at room temperature for 30 min.

[0040] 2) Magnetic separation, discard the supernatant, wash 3 times with TBS-T, and add the corresponding anti-human p-tau 181 The monoclonal antibody-ssDNA conjugate was incubated at room temperature for 30 minutes, and then washed three times with TBS-T, and the bound antibody-ssDNA conjugate was washed off with elution buffer.

[0041] 3) RPA amplification: The RPA amplification system was prepared including forward primer, reverse primer, 2×Reaction buffer, 10×Basic E-mix, DNTP, 20×Core Reaction Mix, MgOAc and antibody-ssDNA. After mixing, the mixture was incubated at 37°C for 30 min.

[0042] 4) CRISPR detection: The CRISPR detection system was prepared including: the product of step 3), crRNA with different universal structural sequences, CRISPR Cas12a, 10×NEB buffer, RNA inhibitor and fluorescent reporter probe. After mixing, incubate at 37°C for 60 minutes on a fluorescent quantitative PCR instrument, and record the fluorescent signal every 30 seconds.

[0043] 5) Record the fluorescence signals of the samples and evaluate the diagnostic effects on NC, MCI, and AD based on the receiver operating characteristic (ROC) curve.

[0044] Results: Based on anti-human p-tau 181 The detection method established by monoclonal antibody can accurately distinguish AD and NC with an area under the curve (AUC) value of 0.91. Figure 4 In (A), the AUC value for distinguishing between MCI and NC is 0.788. Figure 4 In (B), the AUC value for distinguishing non-NC from NC is 0.856. Figure 4 (C) It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps described in the disclosure of the present invention can be performed in parallel, sequentially or in different orders, as long as the desired results of the technical solution disclosed in the present invention can be achieved, and this document does not limit this.

[0045] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for secreting p-tau 181 A hybridoma cell line of a monoclonal antibody, characterized in that: The deposit number is CCTCCNO: C2024272; the p-tau 181 It is tau protein phosphorylated at threonine 181.

2. A method for secreting p-tau 181 A hybridoma cell line of a monoclonal antibody, characterized in that: The deposit number is CCTCCNO: C2024273; the p-tau 181 It is tau protein phosphorylated at threonine 181.

3. The method for secreting human p-tau according to claim 1 or 2 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Application of monoclonal antibodies.

4. The method for secreting p-tau according to claim 1 or 2. 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Monomer or p-tau 181 Application in oligomer detection reagent, characterized in that: p-tau 181 The monomer is a single protein formed by human tau protein phosphorylated at site threonine 181; the p-tau 181 Oligomers are proteins of different molecular weights obtained by mutual folding of human tau proteins phosphorylated at threonine 181.

5. The method for secreting human p-tau according to claim 1 or 2 181 Hybridoma cell lines producing monoclonal antibodies for the preparation of p-tau 181 Monomer or p-tau 181 Application in an aggregate detection kit, characterized in that: p-tau 181 The monomer is a single protein formed by human tau protein phosphorylated at site threonine 181; the p-tau 181 The aggregates are proteins of different molecular weights obtained by mutual folding of human tau proteins phosphorylated at threonine 181.

6. The method for secreting human p-tau according to claim 1 or 2 181 Use of hybridoma cell lines of monoclonal antibodies in the preparation of antibody drugs for treating Alzheimer's disease.

7. The use according to claim 6, characterized in that: The antibody drug for treating Alzheimer's disease is specifically used to inhibit and eliminate the formation of p-tau by tau protein phosphorylated at site threonine 181 in humans. 181 Monomers and aggregates.

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