Hybridoma cell line secreting monoclonal antibody against 217 site threonine phosphorylated tau

Abstract

The present application relates to the field of immunodetection technology, and more particularly to a hybridoma cell strain secreting anti-threonine phosphorylated tau protein monoclonal antibody, which is used for secreting p-tau 217 monoclonal antibody, and is preserved in China Center for Type Culture Collection with preservation numbers of CCTCC NO: C2024280 and CCTCC NO: C2024281. The hybridoma cell strain can be used to prepare monoclonal antibody specifically recognizing p-tau 217 and its aggregates, and solves the problem of lack of p-tau 217 targeting antibody at present. The two monoclonal antibodies can be further applied to prepare high-specificity and sensitivity p-tau 217 detection kit, develop specific passive immunological therapy targeting p-tau 217 , slow down the disease progression, and fill the blank of current immunological therapy targeting p-tau 217 .

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunological detection technology, and particularly relates to a hybridoma cell strain secreting a monoclonal antibody against 217-site threonine phosphorylated tau protein. BACKGROUND

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with insidious onset. AD is the most common cause of dementia, accounting for 60% to 80% of dementia cases worldwide. AD has a high prevalence in the elderly, and its incidence increases exponentially with age. As the global aging trend intensifies, preventing and treating AD has become the most important public health issue worldwide. As of 2024, there are more than 1 million new AD patients in China each year, and the number of AD patients is close to 9 million. With the aging of the population structure, 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 methods and treatment methods for AD, which brings a huge economic burden to patients' families and society. Therefore, early diagnosis of AD is extremely needed. In addition, a large number of biomarkers are needed in clinical practice to identify early AD patients before clinical symptoms. The 217-site threonine phosphorylated tau protein (p-tau 217 ) in cerebrospinal fluid and blood has been shown to have high enough diagnostic accuracy to meet this challenge.

[0004] Immunological detection has been increasingly widely used due to its high sensitivity and speed. Human p-tau 217 The most important factor restricting the quality of immunological detection kits is the quality of bioactive raw materials. Current p-tau 217 detection is based on specific antibody pairs, and most of the existing p-tau 217 antibodies on the market are imported, which are expensive and have a long purchase cycle. In addition, the quality of some domestic antibodies is uneven, which seriously limits the quality of detection kits and the accuracy of AD diagnosis. Therefore, it is necessary to develop high-quality specific p-tau 217 monoclonal antibodies, which are of great significance to the early diagnosis and detection of AD and targeted therapy. SUMMARY

[0005] The present application provides a hybridoma cell strain secreting a monoclonal antibody against threonine phosphorylated tau protein to solve the above problems.

[0006] The present application aims to provide a hybridoma cell strain for secreting p-tau 217The hybridoma cell strain of the monoclonal antibody has a preservation number of CCTCC NO: C2024280; the p-tau 217 is tau protein phosphorylated at threonine 217.

[0007] The application also provides a method for secreting p-tau 217 The hybridoma cell strain of the monoclonal antibody has a preservation number of CCTCC NO: C2024281; the p-tau 217 is tau protein phosphorylated at threonine 217.

[0008] The application also provides a method for secreting human p-tau 217 The application also provides a use of the hybridoma cell strain of the monoclonal antibody in the preparation of a p-tau 217 monomer or p-tau

[0009] The application also provides a method for secreting p-tau 217 The application also provides a use of the hybridoma cell strain of the monoclonal antibody in the preparation of a p-tau 217 monomer or p-tau 217 oligomer detection reagent; the p-tau 217 monomer is a single protein formed by tau protein phosphorylated at threonine 217 of human; the p-tau 217 oligomer is a protein with different molecular weights obtained by mutual folding of tau protein phosphorylated at threonine 217 of human.

[0010] The application also provides a method for secreting human p-tau 217 The application also provides a use of the hybridoma cell strain of the monoclonal antibody in the preparation of a p-tau 217 monomer or p-tau 217 aggregate detection reagent; the p-tau 217 monomer is a single protein formed by tau protein phosphorylated at threonine 217 of human; the p-tau 217 aggregate is a protein with different molecular weights obtained by mutual folding of tau protein phosphorylated at threonine 217 of human.

[0011] The application also provides a method for secreting human p-tau 217 The application also provides a use of the hybridoma cell strain of the monoclonal antibody in the preparation of an antibody drug for treating Alzheimer's disease.

[0012] Preferably, the antibody drug for treating Alzheimer's disease is specifically used for inhibiting and eliminating p-tau 217 monomer and aggregate.

[0013] Compared with the prior art, the application can achieve the following beneficial effects:

[0014] The two hybridoma cell strains 1A2 and 3B11 secreting the anti-human 217 site threonine phosphorylated tau protein (p-tau 217 ) monoclonal antibodies in the application can be used to prepare the monoclonal antibodies specifically recognizing the human p-tau 217 and its aggregates, and the subsequent application of the anti-human p-tau 217 monoclonal antibodies can be further carried out, filling the blank of the current p-tau 217 antibodies.

[0015] The two hybridoma cell strains in the application can be used for secreting and preparing the anti-human p-tau 217 monoclonal antibodies, and the monoclonal antibodies specifically recognizing the p-tau 217 monomers and aggregates can be further applied to prepare the p-tau 217 detection reagents (such as different forms of p-tau 217 detection reagent kits, etc.), and can also be used for inhibiting the p-tau 217 monomers or the p-tau 217 folding into neurofibrillary tangles, preparing antibody drugs to remove the neurofibrillary tangles, and treating AD; the prepared antibody drugs and related vaccines can inhibit and remove the phosphorylated tau protein from forming neurofibrillary tangles and removing the neurofibrillary tangles.

[0016] Based on the two monoclonal antibodies in the application, the high-specificity and high-sensitivity p-tau 217 detection reagent kit can be prepared to realize the early detection of AD diseases, filling the blank of the current p-tau 217 detection reagent kit. In addition, based on the two monoclonal antibodies developed, the immunological therapy specifically targeting the p-tau 217 can be further developed to slow down the disease progression, filling the blank of the current lack of immunological therapy targeting the p-tau 217 . BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 (A) is the ELISA activity detection results of 2 anti-human p-tau 217 monoclonal antibodies, and (B) is the selectivity results of the monoclonal antibodies 1A2 and 3B11 to different p-tau polypeptides.

[0018] Figure 2 (A) is the titer detection results of the monoclonal antibody 1A2, and (B) is the titer detection results of the monoclonal antibody 3B11.

[0019] Figure 3 (A) 1A2 against p-tau 217 polypeptide; (B) 3B11 against p-tau 217 polypeptide.

[0020] Figure 4 According to the embodiments of the present application, the diagnosis of healthy controls (NC), mild cognitive impairment (MCI) and AD patients based on anti-human p-tau 217 monoclonal antibodies (A) is the ROC curve for distinguishing AD and NC; (B) is the ROC curve for distinguishing MCI and NC; (C) is the ROC curve for distinguishing non-NC and NC. DETAILED DESCRIPTION

[0021] Hereinafter, embodiments of the present application will be described with reference to the accompanying drawings. In the following description, the same modules are denoted by the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, detailed descriptions thereof will not be repeated.

[0022] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed descriptions will be made to the present application in combination with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and do not constitute a limitation on the present application.

[0023] Example 1

[0024] Preparation of anti-human p-tau 217 specific monoclonal antibodies, specifically comprising the following steps:

[0025] 1. Preparation of antigen

[0026] The human tau protein (p-tau 217 ) phosphorylated at position 217 of the human tau protein synthesized by the method known in the prior art is dissolved in a TBS solution to a final concentration of 1 mg / mL.

[0027] 2. Preparation of mouse monoclonal antibodies

[0028] (1) Immunization of mice

[0029] 100 μL of the prepared antigen is mixed with an equal volume of Freund's complete adjuvant and then used for the first immunization; 14 days after the first immunization, 100 μL of the prepared antigen is mixed with an equal volume of Freund's incomplete adjuvant and then used for the second immunization; the third immunization is the same as the second immunization, with an interval of 14 days. After the third immunization, 100 μL of the prepared antigen is used for the tail vein booster immunization.

[0030] (2) Cell fusion screening positive clones

[0031] After 3 days of booster immunization, the mouse spleen cells were collected and fused with SP2 / 0 cells, and the fused cells were cultured with HAT medium. One week later, half of the HT medium was replaced. After 10 days of cell fusion, the first ELISA detection was performed, and the positive wells were subjected to the first subcloning. After 10 days of the first subcloning, the second ELISA detection was performed, and the positive wells were subjected to the second subcloning. After 10 days of the second subcloning, the third ELISA detection was performed. If the ELISA detection results are all positive after 3 times of subcloning, it indicates that it has become a monoclonal, which is then expanded and frozen in liquid nitrogen.

[0032] (3) Preparation and purification of ascites

[0033] Three 6-7 week old female Balb / c mice were injected with 0.5 mL of sterile liquid paraffin into the abdominal cavity, and one week later, 10 8 hybridoma cells were injected into the abdominal cavity of each mouse. One week later, ascites was collected. Ascites was purified using a protein A Sepharose column.

[0034] Results: After cell fusion, two strains of anti-human p-tau 217 monoclonal cell lines were successfully screened, named Hybridoma cell line 1A2 and Hybridoma cell line 3B11. They were preserved in the China Center for Type Culture Collection (CCTCC). The preservation number of 1A2 strain is CCTCC No: C2024280, the classification name is Hybridoma cell line 1A2, the preservation time is August 23, 2024, and the preservation unit is China Center for Type Culture Collection, Wuhan University Preservation Center, Luojia Mountain, Wuchang, Wuhan, Hubei Province, China.

[0035] Example 2

[0036] Identification of the biological characteristics of the monoclonal antibody, specifically including:

[0037] 1. Identification of the activity of the monoclonal antibody

[0038] ① Coating antigen: dilute the antigen with TBS to 10 μg / mL, then add 100 μL of the antigen per well to coat at 4°C overnight;

[0039] ②Blocking: After coating, discard the liquid in the hole, wash 3 times with TBS-T, then add 200 μL blocking solution to each hole, block at 37 °C for 2 h;

[0040] ③Add primary antibody: After blocking, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of single antibody diluted with skim milk (1:50000) to each hole, react at 37 °C for 2 h;

[0041] ④Add enzyme-labeled secondary antibody: After the reaction is completed, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each hole, react at 37 °C for 1 h;

[0042] ⑤Color development: After the reaction is completed, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of TMB to each hole, react at 37 °C for 15 min;

[0043] ⑥Termination: Add 50 μL of 2 M H2SO4 to each hole to terminate, and measure the OD450 nm absorbance.

[0044] Results: The prepared monoclonal antibody ascites was subjected to ELISA activity detection; the ELISA results showed (A in FIG. 1) Figure 1 , and the ascites of the two monoclonal antibodies could react with p-tau 217 .

[0045] 2. Specific identification of monoclonal antibody

[0046] ①Coat antigen: Dilute p-tau 181 , p-tau 217 , p-tau 231 , and p-tau 396,404 to 10 μg / mL with TBS, then add 100 μL of the antigen to each hole to coat at 4 °C overnight;

[0047] ②Blocking: After coating, discard the liquid in the hole, wash 3 times with TBS-T, then add 200 μL blocking solution to each hole, block at 37 °C for 2 h;

[0048] ③Add primary antibody: After blocking, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of single antibody diluted with skim milk (1:50000) to each hole, react at 37 °C for 2 h;

[0049] ④Add enzyme-labeled secondary antibody: After the reaction is completed, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each hole, react at 37 °C for 1 h;

[0050] Color development: after the reaction, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL TMB to each hole, react at 37°C for 15 min;

[0051] Results: The prepared monoclonal antibody ascites is only specific to p-tau 217 reacts with other site phosphorylated tau protein; ELISA specific detection results are shown in Figure 1 B.

[0052] (2) Ascites titer detection of hybridoma cell strain

[0053] ① Coating antigen: dilute the antigen to 10 μg / mL with TBS, then add 100 μL of the antigen to each hole for coating at 4°C overnight;

[0054] ② Blocking: after coating, discard the liquid in the hole, wash 3 times with TBS-T, then add 200 μL of blocking solution to each hole, block at 37°C for 2 h;

[0055] ③ Add primary antibody: after blocking, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of ascites of different dilutions to each hole, react at 37°C for 2 h;

[0056] ④ Add enzyme-labeled secondary antibody: after the reaction, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of goat anti-mouse enzyme-labeled secondary antibody (1:8000) diluted with blocking solution to each hole, react at 37°C for 1 h;

[0057] ⑤ Color development: after the reaction, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL TMB to each hole, react at 37°C for 15 min;

[0058] ⑥ Termination: add 50 μL 2 M H2SO4 to each hole to terminate, and measure OD450 nm absorbance.

[0059] Results: ELISA detection results show that the ascites titer of 1A2 hybridoma cell strain is as high as 1:3276800, as shown in Figure 2 (A); the ascites titer of 3B11 hybridoma cell strain is as high as 1:12288000, as shown in Figure 2 (B).

[0060] (3) Monoclonal antibody affinity detection

[0061] ① Coating antigen: dilute p-tau 217 to 2.5 μg / mL with coating solution, add 100 μL / hole to the enzyme-labeled plate for coating at 4°C overnight.

[0062] ②Blocking: After coating, discard the liquid in the hole, wash 3 times with TBS-T, then add 200 μL blocking solution to each hole, block at 37°C for 2h.

[0063] ③Add antisera: After blocking, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of antibody diluted with blocking solution to each hole, and incubate the 96-well plate at 37°C for 2h.

[0064] ④Add enzyme-labeled secondary antibody: After 2h, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000) to each hole. Incubate the 96-well plate at 37°C for 1h.

[0065] ⑤Color development: After 1h, discard the liquid in the hole, wash 3 times with TBS-T, then add 100 μL of soluble TMB substrate color developing solution to each hole, and react at 37°C for 15min.

[0066] ⑥Termination: Add 50 μL of 2M H2SO4 to each hole to terminate, and measure the OD450nm absorbance.

[0067] Results: The affinity detection results of the two monoclonal antibodies showed that the Kd value of 1A2 antibody was 0.022nM as shown in (A) of Figure 3 , and the Kd value of 3B11 antibody was 0.015nM as shown in B of Figure 3 .

[0068] Example 3

[0069] Based on anti-human p-tau 217 monoclonal antibody in differentiating healthy controls (NC), mild cognitive impairment (MCI) and AD patients

[0070] Plasma samples of AD (n=56), mild cognitive impairment (MCI, n=52) and healthy elderly controls (NC, n=47) were collected to evaluate the detection method (RPA-CRISPR) based on anti-human p-tau 217 monoclonal antibody in differentiating NC, MCI and AD. The specific steps are as follows:

[0071] 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.

[0072] 2) Magnetic separation, discard the supernatant, wash 3 times with TBS-T, and add the corresponding anti-human p-tau 217The monoclonal antibody-ssDNA conjugate was incubated at room temperature for 30 minutes. Then, after washing with TBS-T for 3 times, the bound antibody-ssDNA conjugate was eluted with the elution solution.

[0073] 3) RPA amplification: The RPA amplification system was configured to include forward primer, reverse primer, 2x Reaction buffer, 10x Basic E-mix, DNTP, 20x Core Reaction Mix, MgOAc and antibody-ssDNA, respectively, and then mixed and incubated at 37°C for 30 min.

[0074] 4) CRISPR detection: The CRISPR detection system was configured to include the product of step 3), different universal structure sequences crRNA, CRISPR Cas12a, 10x NEB buffer, RNA inhibitor and fluorescent reporter probe, respectively, and then mixed and incubated at 37°C for 60 min on a fluorescence quantitative PCR instrument, and the fluorescence signal was recorded every 30 seconds.

[0075] 5) The fluorescence signal of the sample was recorded, and the diagnostic effect on NC, MCI and AD was evaluated based on the receiver operating characteristic curve (ROC).

[0076] Results: Based on the anti-human p-tau 217 The detection method established by the monoclonal antibody can accurately distinguish AD and NC, and the area under the curve (AUC) value is 0.9012, as shown in (A) of Figure 4 ; The AUC value for distinguishing MCI and NC is 0.7390, as shown in (B) of Figure 4 ; The AUC value for distinguishing non-NC and NC is 0.8231, as shown in (C) of Figure 4 .

[0077] It should be understood that the various forms of the flow shown above can be reordered, added or deleted steps. For example, the steps described in the present disclosure can be performed in parallel, sequentially or in different order, as long as the desired results of the technical solutions of the present disclosure can be achieved, which is not limited herein.

[0078] The above specific embodiments do not constitute a limitation on the scope of protection of the present application. Those skilled in the art should understand that various modifications, combinations, sub-combinations and substitutions can be made according to design requirements and other factors. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for secreting p-tau 217 Hybridoma cell lines of monoclonal antibodies, characterized by: The accession number is CCTCCNO: C2024280; the p-tau 217 It is a tau protein phosphorylated with threonine at position 217.

2. A method for secreting p-tau 217 Hybridoma cell lines of monoclonal antibodies, characterized by: The accession number is CCTCCNO: C2024281; the p-tau 217 It is a tau protein phosphorylated with threonine at position 217.

3. The method for secreting human p-tau according to claim 1 or 2 217 Hybridoma cell lines for monoclonal antibodies in the preparation of p-tau 217 Applications of monoclonal antibodies.

4. The method for secreting p-tau according to claim 1 or 2 217 Hybridoma cell lines for monoclonal antibodies in the preparation of p-tau 217 Monomer or p-tau 217 Application in oligomer detection reagents; characterized by: The p-tau 217 A single protein formed by phosphorylation of human tau protein at threonine position 217; the p-tau 217 Oligomers are proteins of different molecular weights obtained by folding together tau protein phosphorylated at threonine at position 217.

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