CN114539400B

CN114539400B - Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application thereof

Info

Publication number: CN114539400B
Application number: CN202210137460.3A
Authority: CN
Inventors: 王小川; 王喆; 曾宽; 吴刚
Original assignee: Wuhan Tiande Biotechnology Co ltd
Current assignee: Wuhan Tiande Biotechnology Co ltd
Priority date: 2022-02-15
Filing date: 2022-02-15
Publication date: 2024-06-07
Anticipated expiration: 2042-02-15
Also published as: CN114539400A

Abstract

The invention discloses a phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application thereof, wherein the invention uses 217 threonine (T217) phosphorylated antigen sequence immune complex containing Tau protein and antigen sequence immune complex containing Tau protein 412-434 amino acid to carry out animal immunization, so as to prepare phosphorylated Tau pT217 protein monoclonal antibody and Tau (412-434) protein polyclonal antibody, the phosphorylated Tau pT217 protein monoclonal antibody is secreted by hybridoma cell strain Tau-6G12, and has the advantages of strong specificity, high binding force with antigen and the like; then selectively taking a monoclonal antibody of phosphorylated Tau pT217 protein as a capture antibody and a Tau (412-434) protein polyclonal antibody as a detection antibody, so that the sensitivity of the established ELISA kit is higher during detection; in addition, the detection range of the kit is 1.17-800 pg/mL, the detection lower limit is 1.17pg/mL, and the kit has the advantages of high sensitivity and wide detection range.

Description

Phosphorylated Tau pT217 protein monoclonal antibody, ELISA kit and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a phosphorylated Tau pT217 protein monoclonal antibody, an ELISA kit and application thereof.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease characterized clinically by progressive, intellectual decline, and is the leading cause of Alzheimer's disease. The main cause of AD pathogenesis is senile plaques due to the hyperphosphorylation of Tau protein in the brain leading to neuronal tangles (neurofibrillary tangles, NFTs) and the massive deposition of beta amyloid (aβ) in the brain, which in turn leads to abnormal neuronal function.

The Tau protein is a functional protein in brain, contains 2-3 phosphorylation sites in mature normal human brain, and is located on chromosome 17, and because Tau protein mRNA is clipped in different modes, 6 isomorphic isomers containing 352-441 amino acids can be expressed, and the Tau protein is the microtubule-related protein with the highest content, and exists in a great amount in nerve cells in the central system and around the central system, one of the functions of the Tau protein is combined with tubulin to promote the microtubule-related protein to polymerize into microtubules, in addition, the dynamic stability of the microtubule can be enhanced, the dissociation of the microtubule protein can be reduced, the microtubule is induced to be bundled, and the microtubule is not easy to be phosphorylated. Microtubules are the structures necessary to maintain the integrity of the nerve cytoskeleton and transport of nerve cell bodies and axon material. In contrast, tau protein (i.e., PHF-Tau) in AD brain phosphorylates many sites, and PHF-Tau is not bound to microtubules and is highly susceptible to phosphorylation. Abnormal phosphorylation and abnormal glycosylation of tau protein is one of main components of bifilar helix (PHF) in brain Neuron Fiber Tangle (NFT), the effect of tau protein in high phosphorus state connected with microtubules is greatly reduced, and tau protein can be automatically aggregated into Paired Helix (PHF), PHF is aggregated in brain affected neurons of AD patients to form NFT, and becomes one of characteristic pathological changes of AD.

Many studies have shown that in alzheimer's disease and other pathological processes caused by tau protein, hyperphosphorylation of tau protein may occur at the earliest stage, with threonine (T181) phosphorylation at position 181 of tau protein being one of the most common forms of phosphorylation. In literature (Palmqvist S,Janelidze S,Q Uiroz Y,et al.Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders[J].JAMA The Journal of the American Medical Association,2020,324(8).), threonine (T217) phosphorylation at Tau protein is found to be a marker diagnostic for early stages of AD patients, and thus, if phosphorylated Tau pT217 protein can be detected rapidly, alzheimer's disease can be diagnosed early, which is of great importance for the prevention and treatment of alzheimer's disease. At present, few manufacturers develop a kit for detecting phosphorylated Tau pT217 protein on the market, but the kit has the problems of low detection sensitivity, low specificity and the like.

In view of this, there is a need to develop an ELISA kit for detecting human phosphorylated Tau pT217 protein to solve the above problems.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a phosphorylated Tau pT217 protein monoclonal antibody, an ELISA kit and application thereof.

An object of the present invention is to provide a phosphorylated Tau pT217 protein monoclonal antibody.

A phosphorylated Tau pT217 protein monoclonal antibody, wherein the heavy chain amino acid sequence of the phosphorylated Tau pT217 protein monoclonal antibody is shown as SEQ ID NO:3, the light chain amino acid sequence of the phosphorylated Tau pT217 protein monoclonal antibody is shown as SEQ ID NO: 4.

Further, the phosphorylated Tau pT217 protein monoclonal antibody is secreted by a hybridoma cell line Tau-6G12, wherein the hybridoma cell line Tau-6G12 is preserved in China Center for Type Culture Collection (CCTCC); address: university of martial arts in chinese; preservation date: 2021, 12, 30; the preservation number is: cctccc NO: C2021304.

Further, the heavy chain nucleotide sequence of the monoclonal antibody for encoding the phosphorylated Tau pT217 protein is shown in SEQ ID NO:1 is shown in the specification; the light chain nucleotide sequence of the monoclonal antibody for coding the phosphorylated Tau pT217 protein is shown as SEQ ID NO: 2.

Another object of the invention is to provide an ELISA kit.

An ELISA kit comprising the phosphorylated Tau pT217 protein monoclonal antibody, tau (412-434) protein polyclonal antibody, an ELISA plate, a blocking solution, HRP-goat anti-mouse IgG secondary antibody, a chromogenic solution, a stop solution, a coating solution and a washing solution.

Further, the phosphorylated Tau pT217 protein monoclonal antibody is used as a capture antibody, and the Tau (412-434) protein polyclonal antibody is used as a detection antibody.

Further, the dilutions of the phosphorylated Tau pT217 monoclonal antibodies were (1:600) - (1:4800); the dilutions of tau (412-434) protein polyclonal antibodies were (1:600) - (1:9600).

Further, the HRP-goat anti-mouse IgG secondary antibody dilutions were (1:3200) - (1:12800).

The invention also provides a using method of the ELISA kit, which comprises the following steps:

1) Purifying the monoclonal antibody coating: diluting the phosphorylated Tau pT217 protein monoclonal antibody with coating solution, and standing overnight at 4 ℃, wherein 100 mu L of the phosphorylated Tau pT217 protein is used for each well, and standing overnight at 4 ℃;

2) Closing: adding 200 mu L of sealing solution into each hole, and incubating for 0.5-1.5 h at 37 ℃;

3) Adding an antigen: adding 100 mu L of sample into each hole, and incubating for 1-2 h at 37 ℃;

4) Adding purified polyclonal antibody: diluting tau (412-434) protein polyclonal antibody with blocking solution, and incubating for 1-2 h at 37 ℃ with 100 mu L of tau protein polyclonal antibody per well;

5) Adding enzyme-labeled secondary antibodies: diluting the HRP-goat anti-mouse IgG secondary antibody by using a blocking solution, and incubating for 1-2 h at 37 ℃ with 100 mu L of each hole;

6) Developing a substrate: adding 100 mu L of color development liquid into each hole, and developing for 10min;

7) Terminating the reaction: adding 50 mu L/hole of 2mol/L concentrated sulfuric acid into each hole, and measuring an OD _450nm reaction value;

Washing the plate 3-5 times with washing liquid between the steps 1) to 5) for 6-10 min each time.

Further, the blocking solution is selected from one of 5% skim milk powder, 2.5% skim milk powder, 0.5% skim milk powder, 3% BSA, 1% BSA, and 0.5% BSA.

The invention finally provides application of the ELISA kit in preparation of the reagent for detecting Alzheimer's disease.

Compared with the prior art, the invention has the following advantages:

1) The invention uses 217 threonine (T217) phosphorylating antigen sequence immune complex containing Tau protein and antigen sequence immune complex containing Tau protein 412-434 amino acid to make animal immunity, so as to prepare phosphorylating Tau pT217 monoclonal antibody and Tau (412-434) protein polyclonal antibody, the phosphorylating Tau pT217 monoclonal antibody is secreted by hybridoma cell strain Tau-6G12, and has the advantages of strong specificity, high binding force with antigen, etc.; then selectively taking a monoclonal antibody of phosphorylated Tau pT217 protein as a capture antibody and a Tau (412-434) protein polyclonal antibody as a detection antibody, so that the sensitivity of the established ELISA kit is higher during detection;

2) The ELISA kit for detecting the human phosphorylated Tau pT217 protein prepared by the invention can quantitatively detect the content of the phosphorylated Tau pT217 protein in a sample, the detection range of the kit is 1.17-800 pg/mL, the detection lower limit is 1.17pg/mL, and the kit has the advantages of high sensitivity and wide detection range.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a SDS-PAGE electrophoresis of a purified monoclonal antibody of the invention;

FIG. 2 is a standard graph of the detection range of the kit of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and that all other embodiments obtained by persons of ordinary skill in the art without making creative efforts based on the embodiments in the present invention are within the protection scope of the present invention.

Conventional reagents and equipment used in the present invention are commercially available unless otherwise specified.

EXAMPLE 1 immunocomplex preparation

1) The sequence of the antigen comprising threonine at position 217 (T217) phosphorylation of tau protein (amino acid sequence at positions 213 to 227 in tau protein: PSLPT-PPPTREPKKVA as shown in SEQ ID NO. 5) and an antigen sequence comprising amino acids 412 to 434 of tau protein (amino acid sequences 412 to 434 in tau protein: SSTGSIDMVDSPQLATLADEVSA, shown as SEQ ID NO. 6), was performed on an ABI polypeptide synthesizer (431A) in the United states, using the Fmoc (9-fluorenylmethoxycarbonyl) scheme, and the synthesis procedure was performed according to the ABI polypeptide synthesis operating manual. Purifying by high performance liquid chromatography, and performing sequence identification to obtain an antigen sequence containing tau protein at the position 217 of threonine (T217) phosphorylation and an antigen sequence containing tau protein 412-434 amino acids by mass spectrometry.

2) The prepared antigen sequence containing tau protein and the prepared antigen sequence containing tau protein at the position of 217 threonine (T217) phosphorylation and the prepared antigen sequence containing tau protein at the positions of 412-434 amino acids are respectively coupled with KLH through a coupling reagent Sulfo-SMCC to obtain immune complexes.

The above may be achieved by using reagents, instruments, etc. which are common in the art.

EXAMPLE 2 preparation of monoclonal antibodies

2.1 Immunization of animals

Immunization of 4-6 week old BALB/c mice, 5 mice total, with 0.3 mg/dose, with an antigen sequence phosphorylated at position 217 threonine (T217) comprising tau protein (prepared as in example 1) mixed with an adjuvant, emulsified, was performed by subcutaneous injection in the abdomen, the specific procedure being shown in Table 1 below:

TABLE 1 BALB/c mouse immunization procedure

Number of immunizations	Immunization time (d)	Immunization dose	Immunization pathway
				First immunization	1	300 Mug/dose (equivalent complete Freund's adjuvant emulsion)	Intradermal injection
Second immunization	15	300 Mug/dose (equivalent incomplete Freund's adjuvant emulsion)	Subcutaneous injection
				Third immunization	29	300 Mug/dose (equivalent incomplete Freund's adjuvant emulsion)	Subcutaneous injection
Fourth immunization	3D before fusion	200 Μg antigen	Subcutaneous injection

Serum collected before immunization is used as negative serum control, tail vein blood collection is carried out on immunized mice on the tenth day after each immunization, the collected serum is used for detecting antibody titer through indirect ELISA, when the serum titer of the mice reaches more than 10 ⁵, antigen without adjuvant is used for carrying out booster immunization, and then cell fusion can be carried out 3 d.

2.2 Cell fusion

The SP2/0 cells were resuscitated and passaged normally 1 week prior to fusion such that the number of SP2/0 cells in the cell suspension was 1X 10 ⁷. Meanwhile, on the 3 rd day after the booster immunization by using antigen without adjuvant, collecting eyeball blood to kill the mice, collecting serum as a positive control for detection after fusion, soaking the mice in a sterile beaker containing 75% ethanol to sterilize for 5min, taking out the spleen of the mice by using a sterile scalpel after the liquid on the surface of the mice is absorbed by using absorbent paper, placing the spleen on a cell filter screen, lightly grinding the spleen by using a needle core of a sterile syringe, continuously adding a culture medium (incomplete 1640 culture medium) to enable the spleen cells to be in a wet state in the grinding process, collecting filtered cells in a sterile tube by using a disposable sterile Pasteur pipette, repeatedly and lightly blowing the spleen cells to form single cell suspension, centrifuging for 10min under the condition of 1500r/min at room temperature, discarding supernatant, suspending the sediment by using incomplete 1640 culture medium, and keeping the number of the spleen cells in the suspension at 5×10 ⁷ for later use.

The specific steps of cell fusion are as follows:

1) 15mL of the prepared spleen cell suspension is fully and uniformly mixed by a disposable sterile Pasteur pipette, then slowly added into 15mL of prepared SP2/0 cells along the wall, and centrifuged for 8min at 1500r/min at room temperature;

2) Discarding the supernatant, lightly flicking the bottom of the centrifuge tube by hand to loosen and enable the sediment to flow;

3) The PEG 1450 needs to be balanced to the room temperature in a dark place, the centrifuge tube is placed in a water bath kettle at 37 ℃, 1mL of PEG 1450 is added at a constant speed within 1min, and the centrifuge tube is gently rocked while adding;

4) 1mL of incomplete 1640 culture medium preheated at 37 ℃ is added at a constant speed within 1min, 3mL of incomplete 1640 culture medium preheated at 37 ℃ is added at a constant speed within 3min, and finally 10mL of incomplete 1640 culture medium preheated at 37 ℃ is added at a constant speed within 10min, the centrifuge tube is gently rocked while adding, and after the dripping is finished, the centrifuge tube is placed in a water bath kettle at 37 ℃ for 10min;

5) Centrifuging at room temperature for 10min at 800r/min, discarding supernatant, re-suspending cells with 5mL of HAT complete medium containing 10% FBS, and adding HAT complete medium to 50mL;

6) Adding the cell suspension into a 96-well cell culture plate with a row gun, adding 100 mu L/well, then adding 100 mu L/well of HAT for complete culture, and placing into a 37 ℃ cell culture box with 5% CO ₂ for culture;

7) After the fusion, 4d, observing the cell state and half-changing the liquid, namely discarding 100 mu L of in-hole culture medium, and then supplementing 100 mu L of fresh HAT complete culture medium containing 10% FBS;

8) After 8 to 10d of fusion, the cell state was observed under a microscope, and after labeling the wells containing the cell mass, indirect ELISA was performed to determine whether or not the cell culture supernatant contained specific antibodies.

The indirect ELISA detection method comprises the following steps:

1) Antigen coating: the tau protein-containing phosphorylated antigen sequence immunocomplexes at position 217 (T217) prepared in example 1 were diluted with coating solution at 20. Mu.g/mL, 100. Mu.L per well, added to the wells, and chilled overnight at 4 ℃; removing liquid in the plate, spin-drying the water, adding 200 mu L of PBST into each hole, placing the mixture in a micro-oscillator to oscillate for 60s, spin-drying the water, and washing the plate for 3-5 times;

2) Closing: adding 200 mu L of blocking solution/hole into the hole, and blocking overnight at 4 ℃; discarding the liquid in the plate, spin-drying the water, spinning the water by 200 mu L/hole PBST, and washing the plate for 3-5 times;

3) Incubating primary antibody: adding 200 mu L of cell supernatant of the fusion hole and positive, negative and blank controls into an ELISA plate, and allowing the mixture to act for 70min at 37 ℃; discarding the cell supernatant of the fusion hole in the plate, spin-drying water, spin-drying liquid for 3-5 times at 200 mu L/hole PBST;

4) Incubating the secondary antibody: the PBS buffer was used in accordance with 1:12000 dilution HRP-goat anti-mouse IgG, adding 200 mu L diluted secondary antibody into each hole, and allowing the mixture to act at 37 ℃ for 60min; discarding the secondary antibodies in the plate, spin-drying the water for 3-5 times at 200 mu L/hole PBST;

5) Color development: 200 mu L of TMB color developing solution is added into each hole, and the mixture is acted for 15min at 37 ℃ (light-shielding operation);

6) And (3) terminating: the reaction was stopped by adding 50 μl of diluted sulfuric acid to each well, and OD450 _nm was read as soon as possible using a microplate reader.

2.3 Screening of monoclonal antibodies and establishment of monoclonal hybridoma cell lines

Selecting a fusion positive cell clone hole in a 96-hole cell culture plate, when spleen cells are all dead, the fusion positive cell clone grows to 1/3-1/2 of the bottom of the hole, and the cell culture solution is subjected to 3 times of liquid exchange, so that when the antibody secreted by the original spleen cells is completely absent, the hybridoma cell culture solution supernatant is sucked 3-4 days after liquid exchange or when the cell culture solution is yellow, the positive hybridoma cell colony is detected by indirect ELISA, and subcloning is carried out for more than 3 times by a limiting dilution method, and when all clone cell colony clone holes are detected positive, the clone is a monoclonal antibody, and the antibody secreted by the cell line is a monoclonal antibody.

Finally, 5 monoclonal cell lines capable of stably secreting antibodies were obtained, and the OD values of ELISA detection after the stable monoclonal cell lines of the 5 monoclonal cell lines are named as Tau-6G12, tau-2E5, tau-8F3, tau-4C6 and Tau-6B9,5 respectively are shown in Table 2:

ELISA detection values of cell culture supernatants after stabilization of monoclonal cell lines of Table 25

2.4 Purification of monoclonal antibodies and subtype identification

Preparation of ascites: 8-week-old female BALB/c mice are selected, and after 0.5mL of sterile liquid paraffin is injected into the abdominal cavity, 6X 10 ⁶/0.2 mL of hybridoma cells prepared in the step 2.3 in the logarithmic phase are injected into the abdominal cavity of each mouse after 7d, and ascites is collected when the abdomen of each mouse is observed to obviously expand and the pressing fluctuation sense is obvious. Centrifuging the ascites for 10min at 6000r/min, removing liquid paraffin and fat, and sucking the supernatant to obtain the mouse ascites antibody.

Purifying the ascites of the mice by adopting a polypeptide immunoaffinity chromatography column, and the steps are as follows:

1) And (3) filler activation: weighing 1g of agarose 4B, adding 200mL of 1mmol/L ice bath hydrochloric acid for dissolution, removing supernatant, then using 1mmol/L hydrochloric acid (pH is 3.0) to resuspend freeze-dried powder, immediately expanding the filler, then placing the filler on ice for ice bath for 30-40 min, and washing the filler with ultrapure water for 3 times after hydrochloric acid activation for later use;

2) Coupling ligand: firstly, balancing a filler by using coupling buffer with the volume of 5-10 columns, then dissolving amino acid Polypeptide Sequences (PSLPTPPTREPKKVA) at positions 213-227 in tau protein synthesized by a solid phase method in the coupling buffer, and incubating at 4 ℃ overnight;

3) Closing: washing away unbound ligand using 5-8 column volumes of coupling buffer; adding a sealing buffer solution with the volume of 4-6 times of the column volume, rotating at room temperature, incubating for 2-3 hours, and cleaning with ultrapure water for 3 times after sealing is finished for standby;

4) Purifying monoclonal antibodies: and 3-5 times of the column volume of PBS buffer solution is used for balancing and filling the 4B filler coupled with the polypeptide prepared in the step 3), the supernatant of the mouse ascites filtered by a 0.45 mu m filter is loaded into the filler column, then the PBS buffer solution is used for eluting nonspecific binding, when the OD _280nm value of the eluate is smaller than 0.001, tris-HCl buffer solution with the pH value of 7.2-7.4 is used for eluting the specific monoclonal antibody, the eluate is collected, the collected eluate is dialyzed for 3-5 times by the PBS buffer solution, each time for 2-4 hours, and the dialyzed liquid is the purified monoclonal antibody.

The purified monoclonal antibodies were detected by SDS-PAGE and the results are shown in FIG. 1.

Subtype identification of monoclonal antibodies: analysis was performed using a mouse monoclonal antibody immunoglobulin typing kit from Serotec company, and the purified monoclonal antibody was suitably diluted and then detected, and the operation was strictly performed according to the kit instructions, which revealed that Tau-6G12 was of the IgG1 class and light chain was of the kappa chain.

Because the hybridoma cell strain Tau-6G12 can secrete specific monoclonal antibodies, the titer of the monoclonal antibodies is also high, and therefore, the hybridoma cell strain Tau-6G12 is preserved in China Center for Type Culture Collection (CCTCC); address: university of martial arts in chinese; preservation date: 2021, 12, 30; the preservation number is: cctccc NO: C2021304.

EXAMPLE 3 identification of monoclonal antibodies

3.1 Determination of the sequence of monoclonal antibody secreted by hybridoma cell line Tau-6G12

Extracting total RNA of hybridoma cell strain Tau-6G12, using general primer to make reverse transcription into cDNA, amplifying light chain and heavy chain of antibody, separating light chain and heavy chain, cloning them into standard cloning vector and expressing them, single colony PCR identifying light chain and heavy chain, selecting 5-10 single colonies with correct light chain and heavy chain length and sequencing them, in which the 5 sequencing results are almost identical, then the real sequence of antibody can be regarded. (sequencing Process and antibody mass expression of the Committee health organism (Wuhan) technology Co., ltd.)

The heavy chain coding gene sequence of the monoclonal antibody secreted by the hybridoma cell strain Tau-6G12 is 1380bp long, and the sequence is shown as SEQ ID NO. 1; the monoclonal antibody light chain coding gene sequence secreted by the hybridoma cell strain Tau-6G12 is 711bp long, and the sequence is shown as SEQ ID NO. 2; deducing that the heavy chain encoded by the gene sequence consists of 460 amino acids according to the obtained gene sequence, wherein the sequence is shown as SEQ ID NO. 3; the light chain consists of 237 amino acids and has a sequence shown in SEQ ID NO. 4.

EXAMPLE 4 preparation of polyclonal antibodies

The BALB/c mice of 4-6 weeks old were immunized with an antigen immune complex (prepared in example 1) comprising amino acids 412-434 of tau protein as immunogen, emulsified with equal amount of complete Freund's adjuvant at the time of primary immunization, and with equal amount of incomplete Freund's adjuvant at the time of the last 3 times, the neck and back subcutaneous multipoint immunized mice were given an immunization dose of 500. Mu.L/mouse, and the second immunization was carried out on day 14, the third immunization was carried out on day 28, and the fourth immunization was carried out on day 42. Collecting blood from the neck vein on day 45, collecting blood at 4deg.C overnight, centrifuging the blood stored overnight at 5000r/min for 10min, collecting serum, and purifying polyclonal antibody in a part of serum by octanoic acid-ammonium sulfate salting-out method, which comprises the following steps:

1) Centrifuging 5mL of serum at 10000r/min for 10min at low temperature, and removing impurities;

2) Adding 10mL of 0.06M acetic acid solution with pH of 5.0 into the pretreated serum, and adjusting the pH to 4.8 by using 1M HCl; dropwise adding octanoic acid at room temperature under stirring at a ratio of 11 mu L of octanoic acid per mL of diluted serum, adding completely within 30min, standing at 4 ℃ for 2h, centrifuging at 13000r/min for 30min, discarding precipitate, sieving supernatant with 125 mu M nylon, adding 1/10 volume of 0.01M PBS buffer solution, and adjusting pH to 7.4 with 1M NaOH;

3) Slowly adding an equal volume of saturated SAS into serum supernatant with stirring, wherein the final concentration is 1:1 (V/V), and the mixture acts for 3 hours at 4 ℃ to fully precipitate protein;

4) Centrifuging at 13000r/min for 10min, discarding supernatant, re-suspending the precipitate with 12mL of 0.01M PBS buffer, slowly adding 8mL of saturated SAS under stirring, and standing at 4deg.C for 1 hr;

5) Centrifuging at 13000r/min for 10min, discarding supernatant, re-suspending the precipitate with 13.3mL of 0.01M PBS buffer, slowly adding 6.7mL of saturated SAS under stirring, and standing at 4deg.C for 1 hr;

6) Centrifuging at 13000r/min for 10min at low temperature, discarding supernatant, suspending the precipitate with appropriate amount of 0.01M PBS buffer solution, placing into dialysis bag, placing into 50 times volume of 0.01M PBS buffer solution, stirring on magnetic stirrer for 6h, changing dialysate for 3 times, and removing ammonium sulfate completely;

7) And recovering the dialysis product and centrifuging to obtain the purified polyclonal antibody.

The titers of polyclonal antibodies in the other fraction of the collected serum were determined using an indirect ELISA and were considered positive when the absorbance/negative absorbance of the sample was > 2.1, the results are shown in Table 3:

TABLE 3 detection of titers of polyclonal antibodies by indirect ELISA

Dilution factor	1:2000	1:4000	1:8000	1:16000	1:32000	1:64000	1:128000	1:256000
									Polyclonal antibodies	2.756	2.425	2.132	1.712	1.356	0.914	0.652	0.456
Negative control	0.235	0.189	0.098	0.071	0.056	0.041	0.038	0.025

From the results in the table, the obtained serum titers can reach 1:256000, which shows that the antigen immune complex containing tau protein 412 th to 434 th amino acids is used as an immunogen to prepare the high-titer specific polyclonal antibody.

EXAMPLE 5 establishment of ELISA detection method for human phosphorylated Tau pT217 protein

5.1 Basic reaction scheme of antibody Sandwich ELISA

The ELISA basic reaction steps are as follows:

1) Purifying the monoclonal antibody coating: overnight at 4 ℃, 100 μl per well;

2) Closing: incubation at 37℃for 1h, 200. Mu.L per well;

3) Adding an antigen: incubation at 37℃for 1h, 100. Mu.L per well;

4) Adding purified polyclonal antibody: incubation at 37℃for 1h, 100. Mu.L per well;

5) Adding enzyme-labeled secondary antibodies: incubation at 37℃for 1h, 100. Mu.L per well;

6) Developing a substrate: 100 mu L of each hole, and developing for 10min;

7) Terminating the reaction: the OD _450nm reaction value was determined by adding 50. Mu.L/well of 2mol/L concentrated sulfuric acid to each well.

Washing the plate 3-5 times between the steps 1) to 5), processing the data tested each time for 8 minutes, calculating the P/N value of the reaction, taking the reaction condition with the maximum P/N value as the judgment basis of the ELISA optimal reaction condition, wherein the calculation formula of the P/N value is as follows:

P/N value = positive control well OD _450nm mean/negative control well OD _450nm mean

5.2 Screening of optimal working concentration of monoclonal and polyclonal antibodies

Diluting the purified polyclonal antibody from 1:600 to 1:19200, diluting the purified monoclonal antibody from 1:300 to 1:9600, and detecting other reaction conditions in the same way as in step 5.1 by using a checkerboard titration method, and reading an OD _450nm value by using an enzyme-labeling instrument to determine the optimal combined dilution of the two, wherein the result is shown in Table 4:

TABLE 4 ELISA matrix titration results for different concentrations of polyclonal antibody and monoclonal antibody under working conditions

As can be seen from the data in the table, the multiple dilution ratio is 1:2400, and the P/N value of the reaction is higher than that of the other dilutions when the single dilution ratio is 1:1200, so that the multiple dilution ratio is 1:2400, and the single dilution ratio is 1:1200, which is the optimal reaction dilution.

5.3 Screening of blocking fluid and blocking time

The reaction was performed using different dilutions of BSA and skim milk as blocking agents, and the other reaction conditions were the same as in step 5.1, and the experimental results are shown in Table 5 below:

TABLE 5 optimization results of the blocking solution

	5% Skim milk	2.5% Skim milk	0.5% Skim milk	3％BSA	1％BSA	0.5％BSA
							P value	1.412	1.312	1.245	1.219	1.168	1.201
N value	0.113	0.151	0.165	0.169	0.171	0.185
							P/N value	12.49	8.68	7.54	7.21	6.83	6.49

As can be seen from the data in the table, 5% skim milk is the best blocking, and therefore 5% skim milk is selected as the blocking solution.

0.5, 1 And 1.5h were chosen as the blocking time for the reaction, incubated at 37℃and other reaction conditions were identical to those in step 5.1, the experimental results are shown in Table 6 below:

TABLE 6 optimization results of closing time

As can be seen from the data in the table, the blocking for 1h works best, so 1h is chosen as the blocking reaction time.

5.4 Screening of the working concentration and blocking time of the second enzyme-labeled antibody

Coating ELISA plates with monoclonal antibodies with optimal dilution concentration of 1:1200, blocking the ELISA plates with 5% skim milk for 1h at 4 ℃ overnight, adding antigen with certain dilution, continuously adding polyclonal antibodies with optimal dilution concentration of 1:2400, washing, diluting HRP-goat anti-mouse IgG secondary antibodies to different reaction concentrations (1:800, 1:1600, 1:3200, 1:6400 and 1:12800), adding the secondary antibodies into a reaction system, incubating the secondary antibodies for 1h at 37 ℃, developing the secondary antibodies for 10min at 37 ℃ in a dark place, stopping the reaction for 10min with 2mol/L H ₂SO₄, and reading OD _450nm values with an ELISA reader to determine the optimal working concentration of the ELISA antibodies, wherein the results are shown in Table 7:

TABLE 7 optimization results of the working concentration of the second enzyme-labeled antibody

	1:800	1:1600	1:3200	1:6400	1:12800
						P value	1.278	1.301	1.432	1.789	1.612
N value	0.081	0.099	0.081	0.051	0.068
						P/N value	15.7	13.1	17.7	35.1	23.7

As can be seen from the data in the table, the optimal dilution of the second enzyme-labeled antibody was 1:6400, and therefore, a dilution of 1:6400 was selected as the optimal working concentration at which the second enzyme-labeled antibody works.

Coating an ELISA plate with monoclonal antibody with optimal dilution concentration of 1:1200, blocking with 5% skim milk for 1h at 4 ℃ overnight, adding antigen with certain dilution, continuously adding polyclonal antibody with optimal dilution concentration of 1:2400, washing, diluting HRP-goat anti-mouse IgG secondary antibody to 1:6400, adding into a reaction system, incubating (1, 1.5 and 2 h) at 37 ℃, developing in dark at 37 ℃ for 10min, stopping reaction with 2mol/L H ₂SO₄ for 10min, reading OD _450nm value with an ELISA instrument to determine the optimal working concentration of the ELISA antibody, and the result is shown in Table 8:

table 8 optimization results of the incubation time of the second enzyme-labeled antibody

	1h	1.5h	2h
				P value	1.682	1.987	1.875
N value	0.063	0.041	0.049
				P/N value	26.70	48.46	39.26

From the data in the table, the time for incubation of the second enzyme-labeled antibody for 1.5 hours was most effective, and therefore, 1.5 hours was selected as the incubation time for the second enzyme-labeled antibody.

5.5 Establishment of a Standard Curve for a kit

Total tau protein phosphorylated at position 217 (T217) comprising tau protein was diluted to 800, 700, 600, 300, 75, 18.75, 4.6875, 1.171875pg/mL, 3 replicates each, and the standard curve results obtained were measured as described in step 5.1, see FIG. 2.

As can be seen from the graph, when the total tau protein concentration is between 1.17 and 800pg/mL, the linear relation between the natural logarithm of the total tau protein concentration and the OD value is better, the linear regression equation y=0.1449x+0.0289, and R ² =0.9989, wherein x is the natural logarithm of the total tau protein concentration, the detection range of the kit is 1.17 to 800pg/mL, and the detection lower limit is 1.17pg/mL.

Example 5 repeatability experiments

3 Different samples (positive and negative) were selected, each sample was subjected to 4 batch experiments in 4 different ELISA plates, and the results obtained were subjected to statistical analysis, and the specific results are shown in Table 9 below:

Table 9 results of repeatability experiments

As can be seen from the results in the table, the variation coefficient of 3 different samples is less than 10% when the batch detection is carried out, which indicates that the repeatability of the kit is better.

Through the experiment, the ELISA kit for detecting the human phosphorylated Tau pT217 protein is obtained, and the ELISA kit comprises the following components:

Phosphorylated Tau pT217 protein monoclonal antibody (example 2, optimal dilution 1:1200), tau (412-434) protein polyclonal antibody (example 4, optimal dilution 1:2400), ELISA plate, blocking solution (5% skim milk powder), HRP-goat anti-mouse IgG secondary antibody (optimal dilution 1:6400), chromogenic solution (tetramethylbenzidine), stop solution (2 mol/L H ₂SO₄), coating solution (20 mmol/L pH8.5, tris-HCl) and washing solution (PBST solution).

The detection range of the kit is 1.17-800 pg/mL, and the detection lower limit is 1.17pg/mL.

The ELISA kit is used as follows:

1) Purifying the monoclonal antibody coating: the phosphorylated Tau pT217 monoclonal antibody was diluted to 1:1200 with coating solution overnight at 4℃and 100. Mu.L per well overnight at 4℃in a refrigerator;

2) Closing: 200 mu L of 5% skim milk powder is added to each well, and incubated for 1h at 37 ℃;

4) Adding purified polyclonal antibody: diluting tau (412-434) protein polyclonal antibody to 1:2400 with 5% skim milk powder, incubating for 1-2 h at 37 ℃ and 100 μl per well;

5) Adding enzyme-labeled secondary antibodies: HRP-goat anti-mouse IgG secondary antibody was diluted to 1:6400 with 5% skim milk powder, incubated at 37 ℃ for 1.5h, 100 μl per well;

Washing the plate 3-5 times with washing liquid for 10min each time between the steps 1) to 5).

Example 6 clinical application of antibody Sandwich ELISA kit

Samples of cases at the university of science and technology affiliated with the medical college of ataxia were selected, 30 cases of cases with early Alzheimer's disease, 15 cases of normal persons were examined using the ELISA kit of the invention, and the results are shown in Table 10:

table 10 ELISA clinical application test results of the kit

The number of people	Results	The number of people	Results
				Case 1	-	Normal 1	-
Case 2	+	Normal 2	-
				Case 3	+	Normal 3	-
Case 4	+	Normal 4	-
				Case 5	+	Normal 5	-
Case 6	+	Normal 6	+
				Case 7	+	Normal 7	-
Case 8	+	Normal 8	-
				Case 9	+	Normal 9	-
Case 10	+	Normal 10	-
				Case 11	+	Normal 11	-
Case 12	+	Normal 12	-
				Case 13	+	Normal 13	-
Case 14	+	Normal 14	-
				Case 15	+	Normal 15	-
Case 16	+
				Case 17	+
Case 18	+
				Case 19	+
Case 20	+
				Case 21	-
Case 22	+
				Case 23	+
Case 24	+
				Case 25	+
Case 26	+
				Case 27	+
Case 28	+
				Case 29	+
Case 30	+

In the table, + represents positive, -represents negative

As shown by the results in the table, the accuracy of the ELISA kit in the case of early Alzheimer's disease detection reaches more than 93%, so the ELISA kit has good application prospect in early Alzheimer's disease diagnosis.

The above examples are only specific embodiments of the present invention for illustrating the technical solution of the present invention, but not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the foregoing examples, it will be understood by those skilled in the art that the present invention is not limited thereto: any person skilled in the art may modify or easily conceive of the technical solution described in the foregoing embodiments, or perform equivalent substitution of some of the technical features, while remaining within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. The phosphorylated Tau pT217 protein monoclonal antibody is characterized in that the heavy chain amino acid sequence of the phosphorylated Tau pT217 protein monoclonal antibody is shown as SEQ ID NO:3, the light chain amino acid sequence of the phosphorylated Tau pT217 protein monoclonal antibody is shown as SEQ ID NO: 4.

2. The phosphorylated Tau pT217 protein monoclonal antibody of claim 1, wherein the phosphorylated Tau pT217 protein monoclonal antibody is secreted by hybridoma cell line Tau-6G12 which has been deposited by the chinese collection of classical cultures (cctccc); address: university of martial arts in chinese; preservation date: 2021, 12, 30; the preservation number is: cctccc NO: C2021304.

3. The phosphorylated Tau pT217 protein monoclonal antibody of claim 1, wherein the heavy chain nucleotide sequence encoding the phosphorylated Tau pT217 protein monoclonal antibody is set forth in SEQ ID NO:1 is shown in the specification; the light chain nucleotide sequence of the monoclonal antibody for coding the phosphorylated Tau pT217 protein is shown as SEQ ID NO: 2.

4. An ELISA kit, which is characterized by comprising the phosphorylated Tau pT217 monoclonal antibody of any one of claims 1-3, and polyclonal antibody, an ELISA plate, a blocking solution, HRP-goat anti-mouse IgG secondary antibody, a chromogenic solution, a stop solution, a coating solution and a washing solution;

The preparation method of the polyclonal antibody comprises the steps of coupling an antigen sequence containing tau protein 412-434 amino acids with KLH through a coupling reagent Sulfo-SMCC to obtain an immune complex, and obtaining the polyclonal antibody by taking the immune complex as an immunogen; the amino acids at positions 412-434 of the tau protein are shown as SEQ ID NO. 6.

5. The ELISA kit of claim 4, wherein the phosphorylated Tau pT217 protein monoclonal antibody is used as a capture antibody and the polyclonal antibody is used as a detection antibody.

6. The ELISA kit of claim 4, wherein the dilution of the phosphorylated Tau pT217 monoclonal antibody is (1:600) - (1:4800); the polyclonal antibody dilutions were (1:600) - (1:9600).

7. The ELISA kit of claim 4, wherein the HRP-goat anti-mouse IgG secondary antibody dilutions are (1:3200) - (1:12800).

8. The method of using the ELISA kit of any one of claims 4 to 7 for non-diagnostic purposes, comprising the steps of:

4) Adding purified polyclonal antibody: diluting the polyclonal antibody by using a blocking solution, and incubating for 1-2 h at 37 ℃ with 100 mu L of each hole;

9. The method of claim 8, wherein the blocking solution is selected from the group consisting of 5% skim milk powder, 2.5% skim milk powder, 0.5% skim milk powder, 3% bsa, 1% bsa, and 0.5% bsa.

10. Use of an ELISA kit according to any of claims 4 to 7 for the preparation of a reagent to detect alzheimer's disease.