Human Tau protein antibody and application

Abstract

The invention discloses a human Tau protein antibody and application thereof. The amino acid sequences of light chains CDR1, CDR2 and CDR3 of the antibody are shown as SEQ ID NO: 1-3 in sequence, and the amino acid sequences of heavy chains CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 4-6 in sequence. The human Tau protein antibody can be specifically combined with phosphorylated and non-phosphorylated human Tau proteins, the affinity is strong, the stability is high, and the EC50 is lower than 3.6 ng / mL. An enzyme-linked immunosorbent assay kit prepared based on the antibody has the characteristic of high sensitivity, the detection sensitivity is less than 19.5 pg / mL, the linear detection range is 0-20000 pg / mL, the specificity is strong, the accuracy is good, the Tau protein content in a sample can be accurately detected at high flux, and the application prospect is excellent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibodies, in particular to a human Tau protein antibody and application. BACKGROUND

[0002] Tau protein is a microtubule-associated protein mainly expressed in neurons, and its core physiological function is to stabilize microtubules and regulate axoplasmic transport. In pathological conditions, the abnormality of Tau protein is the common core of various neurodegenerative diseases. It occurs excessive phosphorylation, misfolding and aggregation to form neurofibrillary tangles, which is not only a hallmark of Alzheimer's disease, but also a major driving factor for primary tauopathy such as progressive supranuclear palsy. This pathological transformation destroys the normal structure and function of neurons, eventually leading to cell death and cognitive decline.

[0003] In neurological diseases, the increase of total Tau protein level is widely regarded as a biomarker reflecting neuronal injury or death, and its application has gone beyond Alzheimer's disease. And the subtypes of different phosphorylation sites have unique pathological significance: phosphorylated Tau181 (p-Tau181) can be elevated in the early stage of AD, which can appear 20 years earlier than clinical symptoms, with a specificity of 89%~98%; phosphorylated Tau217 (p-Tau217) has the strongest correlation with brain amyloid deposition and Tau aggregation, and is one of the most sensitive and specific AD biomarkers; phosphorylated Tau231 (p-Tau231) is involved in the pathological conformational transition of Tau protein, and its level change is related to the speed of disease progression. The coordinated detection of these subtypes provides a multi-dimensional perspective for precise assessment of the disease.

[0004] Based on this, the detection of Tau protein and its phosphorylated subtypes has become a core means for the diagnosis of neurodegenerative diseases. Among them, immunodetection of Tau protein and its phosphorylated subtypes as a high cost-effective detection method is particularly suitable for large-scale screening. In clinical application, combined with cognitive phenotype analysis and multi-marker joint detection, it can significantly improve the early diagnosis accuracy of AD and the reliability of prognosis evaluation, and is expected to become a routine screening tool for primary medical care. However, specific antibodies suitable for immunodetection of Tau protein and its phosphorylated subtypes still need further development and research. SUMMARY

[0005] The purpose of the present application is to provide a human Tau protein antibody with high sensitivity and good specificity; the second purpose is to provide a nucleic acid molecule encoding the human Tau protein antibody, related biological materials and antibody conjugates; the third purpose is to provide applications containing the human Tau protein antibody.

[0006] Technical solution: The human Tau protein antibody or its antigen-binding fragment of the present invention has the following amino acid sequences: light chain CDR1 as shown in SEQ ID NO: 1, light chain CDR2 as shown in SEQ ID NO: 2, light chain CDR3 as shown in SEQ ID NO: 3, heavy chain CDR1 as shown in SEQ ID NO: 4, heavy chain CDR2 as shown in SEQ ID NO: 5, and heavy chain CDR3 as shown in SEQ ID NO: 6.

[0007] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7 or has at least 80% similarity to the sequence shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8 or has at least 80% similarity to the sequence shown in SEQ ID NO: 8.

[0008] Preferably, the antigen-binding fragment includes any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

[0009] Preferably, the antibody further includes a portion or all of the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as a portion or all of the κ chain or λ chain constant region.

[0010] Preferably, the species source of the constant region is selected from any one of cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, and turkeys.

[0011] The nucleic acid molecule described in this invention encodes the human Tau protein antibody or its antigen-binding fragment as described above.

[0012] The biomaterials described in this invention are expression cassettes, vectors, or host cells containing the nucleic acid molecules as described above.

[0013] It should be noted that, in one implementation of the present invention, hybridoma cell technology is specifically used to screen and obtain the human Tau protein antibody of this application; based on this, the coding sequence of the human Tau protein antibody according to the present invention can be artificially synthesized, and then expressed by cloning to obtain the human Tau protein antibody of the present invention. 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.

[0014] The antibody conjugate of the present invention is obtained by conjugating a human Tau protein antibody or its antigen-binding fragment as described above with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, and nanoparticle labeling.

[0015] Preferably, the markers include, but are not limited to, biotin, HRP, alkaline phosphatase, acridine ester, ruthenium tripyridine, luminidine, colloidal gold, fluorescent microspheres, etc.

[0016] The present invention relates to the use of human Tau protein antibodies or antigen-binding fragments thereof, or nucleic acid molecules, or biological materials, or antibody conjugates in the preparation of products for qualitative or quantitative detection of human Tau protein.

[0017] Preferably, the human Tau protein comprises phosphorylated and / or non-phosphorylated human Tau protein.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The human Tau protein antibody provided by the present invention can specifically bind to phosphorylated and non-phosphorylated human Tau protein, with strong affinity and high stability, EC50 < 3.6 ng / mL; 2. The enzyme-linked immunosorbent assay kit prepared based on this antibody has the characteristics of high sensitivity, with a detection sensitivity < 19.5 pg / mL and a linear detection range of 0~20000 pg / mL. It also has strong specificity and good accuracy, and can realize accurate and high-throughput detection of Tau protein content in samples, with excellent application prospects. Attached Figure Description

[0019] Figure 1 The result of titer assay for human Tau protein antibody T-1C1 is shown in the figure.

[0020] Figure 2 Figure showing the results of immunoblotting detection of Tau protein and phosphorylated Tau protein;

[0021] Figure 3 Standard curve for detecting human Tau protein content using an enzyme-linked immunosorbent assay kit based on human Tau protein antibody T-1C1. Detailed Implementation

[0022] The technical solution of the present invention will be further described below.

[0023] Example 1: Preparation of human Tau protein antibody

[0024] 1. Preparation of recombinant human Tau protein

[0025] Based on the publicly available amino acid sequence of human Tau protein (NCBI Reference Sequence: NP_005901.2), a nucleic acid sequence was designed and constructed into the pET-52b(+) vector. The cloning site was XbaI-ScaI, with ampicillin (AMP) resistance and a C-terminal 10×His tag, resulting in pET-52b(+)-Tau.

[0026] Convert pET-52b(+)-Tau to E. coli BL21 strain was then plated onto plates containing ampicillin at a final concentration of 100 μg / mL, and recombinant bacteria were obtained after culturing. E. coli BL21 / pET-52b(+)-Tau; the recombinant bacteria were inoculated into TSB liquid medium containing ampicillin at a final concentration of 100 μg / mL and cultured overnight at 37°C with shaking at 220 rpm until OD. 600 =0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, induce culture at 16℃ for 12 h, centrifuge at 8000×g for 5 min and discard the supernatant, add lysis buffer containing 1% NP40 and sonicate on ice for 40 min; centrifuge at 8000×g for 30 min and centrifuge again, filter the supernatant through a 0.45 μm filter membrane and purify by cobalt ion affinity chromatography to obtain human Tau protein.

[0027] 2. Animal immunization

[0028] The purified recombinant human Tau protein obtained in step 1 was diluted with physiological saline and then mixed with Montanide. TM Gel 01 PR adjuvant was mixed at a volume ratio of 9:1 to prepare an injection solution with a final concentration of 100 μg / mL of recombinant human Tau protein. Then, three Balb / c mice (6-8 weeks old, purchased from Guangdong Provincial Experimental Animal Center) were immunized by intramuscular injection in the calf, with an injection dose of 5 μg / mouse. Booster immunizations were performed in the same manner on day 14 and day 28.

[0029] 3. Preparation of monoclonal cell lines expressing human Tau protein antibodies

[0030] Blood was collected from the tail of mice on day 35. After serum separation, serum titer was detected using an indirect ELISA method. The titer reached a fusion value of 10. 6 Afterwards, the spleen was boosted with 100 μg of human Tau protein; 3 days later, the mice were euthanized by carbon dioxide asphyxiation, disinfected by soaking in 75% alcohol, and the spleen was removed under aseptic conditions. The spleen cells were squeezed and ground to prepare a suspension and counted.

[0031] Splenic cells were fused with NS1 myeloma cells in logarithmic growth phase at a cell ratio of 5:1. The steps were as follows: Splenic cells and NS1 myeloma cells were mixed and centrifuged at 200×g for 10 min; the cell mixture pellet was placed in a 37°C water bath and 1 mL of 50% PEG1500 preheated to 37°C was added. After stirring and mixing, serum-free DMEM medium preheated to 37°C was added, mixed, and incubated for 2 min; then centrifuged at 200×g for 10 min, the supernatant was discarded, and the cells were cultured in HAT semi-solid selection medium for 8 days. Cell clones were picked and cultured in HT medium for 2 more days.

[0032] Collect cell culture supernatant and detect antibody specificity using an indirect ELISA method. The steps are as follows:

[0033] S1, Take the recombinant human Tau protein obtained in step 1, dilute it with 0.05M carbonate buffer (pH=9.6) to a protein concentration of 2 μg / mL, add 100 μL to each well of the microplate, and coat overnight at 4°C.

[0034] S2, after coating, wash 4 times with PBST solution (washing buffer), add 200 μL of PBST solution (blocking buffer) containing 1 g / 100 mL bovine serum albumin to each well, and block at 37℃ for 2 h;

[0035] S3, after sealing, wash 4 times with washing buffer, then add 100 μL of cell culture supernatant to each well and incubate at 37°C for 1 h;

[0036] S4, after incubation, wash 4 times with washing buffer, then add HRP-labeled goat anti-mouse secondary antibody diluted 1:4000, and incubate at 37℃ for 1 h;

[0037] S5, after incubation, wash 4 times with washing buffer, then add TMB chromogenic solution (purchased from Beijing MecoWander Biotechnology Co., Ltd., catalog number 1001H) for 10 min of color development, then add 50 μL of TMB stop solution (purchased from Beijing MecoWander Biotechnology Co., Ltd., catalog number 1001SA) to stop the color development, and measure the absorbance at 450 nm using an ELISA reader.

[0038] A positive result is defined as an absorbance value greater than 1.5. The positive cells obtained are cloned once using HT semi-solid medium to obtain a monoclonal cell line that can stably secrete antibodies.

[0039] 4. Screening of monoclonal antibodies

[0040] The monoclonal cell lines obtained in step 3 were used to prepare ascites fluid using an in vivo induction method. The ascites antibodies were purified by Protein A affinity chromatography to obtain purified antibodies. The purified antibodies were dialyzed in 0.01 mol / L phosphate buffer at 4°C for 24 h using a dialysis bag with a molecular weight cutoff of 30 kDa. The protein concentration of the purified antibodies was then determined using Nanodrop.

[0041] Referring to the method in step 3, the titer of the purified antibody was detected by indirect ELISA, with the following difference: S3, after blocking, wash four times with washing buffer, then add 100 μL of antibody diluent per well and incubate at 37°C for 1 hour. The antibody diluent was prepared as follows: take the purified monoclonal antibody, dilute it with 0.02M PBS buffer (pH 7.4) to a protein concentration of 10 μg / mL, and perform a 10-fold serial dilution, resulting in a final concentration gradient of 10... 4 10 3 10 2 10, 1, 10 -1 10 -2 ng / mL.

[0042] The titer test results of monoclonal human Tau protein antibody T-1C1 are as follows: Figure 1 As shown, its EC50 is below 3.6 ng / mL.

[0043] Meanwhile, monoclonal antibodies T-1D2, T-1D3, T-2A5, and T-2B3 were also screened and used in subsequent experiments.

[0044] 5. Antibody pairing test

[0045] The optimal combination of coating and detection antibodies is selected using a double-antibody sandwich method.

[0046] S1. Take the purified monoclonal antibodies T-1D2, T-1D3, T-2A5, and T-2B3, and dilute them with carbonate buffer (pH=9.6, 0.05M) to a protein concentration of 2 μg / mL. Add 100 μL to each well of the microplate and coat overnight at 4°C.

[0047] S2, after coating, wash 3 times with washing solution, add 200μL of blocking solution to each well and block at 37℃ for 2 h;

[0048] S3, after blocking, wash 4 times with washing buffer, then add 100 μL of recombinant human Tau protein at a concentration of 10 ng / mL or 100 μL of PBS as a negative control to each well, and incubate at 37℃ for 1 h;

[0049] S4, after incubation, wash 3 times with washing buffer, then add HRP-labeled T-1C1 at a concentration of 1:2000 and incubate at 37°C for 1 h;

[0050] S5, after incubation, wash 4 times with washing buffer, then add TMB substrate reaction solution for color development for 10 min, then add TMB stop solution to stop color development, and measure the absorbance (OD) at 450 nm using a microplate reader. 450 .

[0051] HRP-labeled T-1C1 was prepared using an HRP rapid labeling kit (purchased from Luoyang Baitong Experimental Materials Center, catalog number C060220), specifically as follows:

[0052] Monoclonal human Tau protein antibody T-1C1 was dialyzed overnight with 0.01M carbonate buffer at pH 9.6. Then, 1 mg of antibody T-1C1 was added to 1 mg of pre-activated HRP solution, mixed well, and 99 μL of HRP labeling starter solution was added. The reaction was carried out at 4°C in the dark for 24 h. After labeling, 50 μL of reaction stop solution was added, and the reaction was carried out at 4°C in the dark for 2 h. After termination, the solution was washed three times by ultrafiltration with 0.01M phosphate buffer at pH 7.4 and the volume was adjusted to 0.5 mL. Then, an equal volume of labeling preservation solution was added, and the solution was mixed thoroughly to obtain an HRP-labeled T-1C1 solution with a final concentration of 1 mg / mL. The solution was stored at -20°C.

[0053] The absorbance data is processed to calculate the P / N ratio of the reaction. The reaction condition with the highest P / N ratio is used as the criterion for determining the optimal antibody pairing. The formula for calculating the P / N ratio is as follows:

[0054] P / N value = OD of recombinant human Tau protein pores 450 Mean value ÷ OD of negative control well 450 Mean;

[0055] The results are shown in Table 1. The T-2B3 coating paired with the T-1C1-labeled HRP showed the best detection performance.

[0056] Table 1. Results of antibody pairing test

[0057]

[0058] 6. Antibody subtype identification

[0059] Antibody subtypes were identified using a mouse monoclonal antibody Ig class / subclass / subtype identification kit (purchased from 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 2.

[0060] Table 2 Antibody subtype test results

[0061]

[0062] The results in Table 2 indicate that antibody T-1C1 is an IgG1 subtype and the antibody light chain is a Kappa chain.

[0063] Example 2: Monoclonal Antibody Sequencing

[0064] The cell line expressing the human Tau protein antibody T-1C1 was sequenced and identified by General Biotechnology (Anhui) Co., Ltd.

[0065] Based on the sequencing results, the amino acid sequences of the light chain and heavy chain variable regions of antibody T-1C1, and their respective three CDRs, were determined with reference to the Kabat and IMGT numbering methods. Specifically, the amino acid sequences of each CDR of human Tau protein antibody T-1C1 are shown in Table 3, the heavy chain variable region sequence is shown in SEQ ID NO: 7, and the light chain variable region sequence is shown in SEQ ID NO: 8.

[0066] Table 3. Amino acid sequences of three CDRs each in the light and heavy chains of human Tau protein antibody T-1C1.

[0067]

[0068] Example 3: Detection of phosphorylation and non-phosphorylation of human Tau protein 20 μg of recombinant Tau protein was prepared into a 100 μg / mL solution using phosphorylation buffer, and 0.1 μg of phosphokinase GSK-3β protein (purchased from MedChemExpress LLC., catalog number HY-P700591) was added. The mixture was incubated at 30°C for 12 h to obtain phosphorylated Tau protein. The phosphorylation buffer contained 40 mM HEPES, 3 mM MgCl2, 5 mM EGTA, and 10 mM ATP.

[0069] 25 ng of recombinant Tau protein and phosphorylated Tau protein were spotted onto nitrocellulose membranes and dried at 37°C for 4 h. The membranes were then blocked with TBST buffer containing 1% BSA for 1 h. The blocked nitrocellulose membranes were then immersed in TBST buffer and washed three times with shaking for 5 min each time. Then, 1:4000 HRP-labeled T-1C1 antibody solution was added and incubated at room temperature for 1 h. After incubation, the membranes were washed three times with TBST buffer for 5 min each time. After washing, the membranes were developed using the SuperPico ECL Master Mix kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., E432-01).

[0070] The results are as follows Figure 2 As shown, both unphosphorylated and phosphorylated Tau proteins can be detected by human Tau protein antibody T-1C1.

[0071] Example 4: Preparation and application of immunoassay kit

[0072] 1. Preparation of Immunoassay Kit

[0073] S1. Coating: Dilute the T-2B3 antibody with PBS to prepare a 2 μg / mL coating antibody solution. Add 100 μL to each well of a 96-well microplate and coat overnight at 2-8°C.

[0074] S2. Washing: Take out the 96-well microplate coated overnight and place it on the plate washer rack. Set the washing solution volume to 300 μL / well and wash 3 times. After washing, gently pat dry on clean absorbent paper.

[0075] S3. Blocking: Add 240 μL of blocking solution to each well of the ELISA plate. After the sample is added, attach the sealing film and incubate at 37°C for 1 h.

[0076] S4. Washing the plate: Wash the blocked ELISA plate according to step S2. After washing 3 times, gently pat it dry on clean absorbent paper to obtain the immunoassay kit.

[0077] 2. Immunological testing

[0078] S1, Sample addition: Dilute the recombinant Tau protein to prepare samples with concentrations of 20000, 5000, 1250, 312.5, 78.1, 19.5, 4.9, and 0 pg / mL. Add 100 μL / well of the sample to the wells of the aforementioned immunoassay kit and incubate at 37°C for 1 h.

[0079] S2, Washing: After incubation, place the plate on the plate washer rack, set the washing liquid volume to 300 μL / well, wash 3 times, and gently pat dry on clean absorbent paper after washing.

[0080] S3, Add enzyme-labeled antibody: Add 100 μL of HRP-labeled T-1C1 antibody solution diluted 1:2000 to each well and let stand at 37℃ for 1 h.

[0081] S4, 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.

[0082] S5, color development: Add 100 μL of TMB substrate reaction solution to each well and let it stand at room temperature for 10 min.

[0083] S6, Termination: After the color development is complete, add 50 μL of TMB stop solution to each well to terminate the color development reaction.

[0084] S7, Measure absorbance: Within 5 minutes after the color development is terminated, use an ELISA reader to measure the absorbance at 450 nm.

[0085] The preparation method of HRP-labeled T-1C1 is the same as in Example 1.

[0086] The test results are shown in Table 4.

[0087] Table 4 Results of human Tau protein detection using the immunoassay kit

[0088]

[0089] Based on the measured absorbance values ​​shown in Table 4, a four-parameter standard curve was plotted, as follows: Figure 3 As shown, the standard curve R 2 =0.9995, linear detection range 0-20000 pg / mL, detection sensitivity less than 19.5 pg / mL.

Claims

1. A human Tau protein antibody or its antigen-binding fragment, characterized in that, The amino acid sequence of the light chain CDR1 of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 1, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO:

6.

2. The human Tau protein antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region amino acid sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7 or has at least 80% similarity to the sequence shown in SEQ ID NO: 7, and the light chain variable region amino acid sequence is as shown in SEQ ID NO: 8 or has at least 80% similarity to the sequence shown in SEQ ID NO:

8.

3. The human Tau protein antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragments include any one or more of F(ab')2, Fab', Fab, Fv, ScFv, and dsFv.

4. The human Tau protein antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody also includes partial or complete sequences of the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as partial or complete sequences of the constant region of the κ chain or λ chain.

5. The human Tau protein antibody or its antigen-binding fragment according to claim 4, characterized in that, The species source of the constant region is selected from any one of the following: cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, and turkeys.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the human Tau protein antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. A biomaterial, characterized in that, The biomaterial is an expression cassette, vector, or host cell containing the nucleic acid molecule described in claim 6.

8. An antibody conjugate, characterized in that, The antibody conjugate is obtained by conjugating the human Tau protein antibody or its antigen-binding fragment as described in any one of claims 1-5 with a label, wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, and nanoparticle labeling.

9. The use of the human Tau protein antibody or antigen-binding fragment thereof according to any one of claims 1-5, or the nucleic acid molecule according to claim 6, or the biological material according to claim 7, or the antibody conjugate according to claim 8 in the preparation of products for qualitative or quantitative detection of human Tau protein.

10. The application according to claim 9, characterized in that, The human Tau protein includes phosphorylated and / or non-phosphorylated human Tau protein.

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