CN114989313A

CN114989313A - P-tau181 epitope peptide and application thereof in Alzheimer disease detection

Info

Publication number: CN114989313A
Application number: CN202210740357.8A
Authority: CN
Inventors: 黄仁杰; 陈佳宇; 汪泽云; 李鑫
Original assignee: Yitong Technology Development Fujian Co ltd
Current assignee: Fujian Yitong Biotechnology Co ltd
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-09-02
Anticipated expiration: 2042-06-28
Also published as: CN114989313B

Abstract

The invention relates to a p-tau181 epitope peptide and application thereof in Alzheimer disease detection. The p-tau181 antigen prepared by the human p-tau181 epitope peptide can be used for preparing antibodies and detection kits; the amino acid sequence of the human p-tau181 epitope peptide is Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly. The p-tau181 antigen can be prepared by coupling the epitope peptide of the human p-tau181 antigen with a protein carrier; the p-tau181 monoclonal antibody or polyclonal antibody is prepared from the A beta 1-42 antigen; the p-tau181 monoclonal antibody or polyclonal antibody is used for preparing a p-tau181 in-vitro diagnostic kit; the human p-tau181 epitope peptide has good antigenicity, and an antigen (immunogen) prepared by the human p-tau181 epitope peptide can be used for immunizing animals to generate a monoclonal antibody and a polyclonal antibody with high specificity, so that the human p-tau181 epitope peptide can be applied to in vitro detection of human p-tau181 protein. The invention can prepare the human p-tau181 epitope peptide with high purity in large batch.

Description

P-tau181 epitope peptide and application thereof in Alzheimer disease detection

Technical Field

The invention belongs to the field of polypeptide chemistry and immunology, and relates to a p-tau181 epitope peptide and a method for preparing the p-tau181 epitope peptide, wherein a p-tau181 antigen prepared by using the p-tau181 epitope peptide can be further used for preparing a corresponding monoclonal antibody or a polyclonal antibody, and in addition, the antibody can be used for preparing a human p-tau181 in-vitro diagnostic kit, and the kit can be used for detecting the concentration of p-tau181 protein in serum or plasma samples of patients suffering from Alzheimer's disease, so that an effective scientific basis is provided for the diagnosis of the Alzheimer's disease.

Background

The p-tau181 epitope peptide is known to have the following amino acid sequence: AKTPPAPK-pTPPSSG, which is 173-186 amino acid fragment CN114409780A phosphorylated by threonine 181 (T181) of tau protein (Chinese patent application No. 202210138280.7). The p-tau181 epitope peptide can be used for detecting phosphorylated tau protein in an Alzheimer patient, and is beneficial to prediction, early discovery and prevention, disease course development and curative effect observation of Alzheimer disease. In addition, CN111197040A (application No. 2020100721960, yi tong bio) discloses an epitope peptide of chitinase 3-like protein 1(CHI3L1), an antigen, an antibody, a use and a kit, and it is believed that the epitope peptide of CHI3L1 of the present invention exhibits good antigenicity, and an antigen (immunogen) prepared by using the epitope peptide can be used for immunizing an animal to produce a highly specific monoclonal antibody and a polyclonal antibody, so that the epitope peptide can be applied to the in vitro detection of human CHI3L 1. The related art information disclosed in the above-mentioned CN103665113A and CN111197040A is incorporated herein by reference.

At present, Alzheimer Disease (AD) is a common and frequently encountered disease in the elderly, also known as senile dementia, with a prevalence of > 5% in elderly over 60 years of age. AD is a primary degenerative disease of the central nervous system with progressive decline in mental and cognitive function in patients. Neurodegenerative diseases are nervous system diseases mainly caused by central nervous system damage due to progressive degeneration and death of neurons, seriously endanger human health, but have unclear etiology, complex pathogenesis and lack of effective treatment measures. In addition to the known senile dementia (AD), Parkinson's Disease (PD), and the like, the late complications of diabetes, in which the incidence of disease is increasing and the age of onset is decreasing, become the first-onset factors as neurodegenerative diseases among people. Due to the lack of effective early diagnosis measures for neurodegenerative diseases, a series of real problems are brought to treatment and nursing, and meanwhile, a heavy economic burden and mental stress are caused to the society and families. Therefore, the method has important significance in researching the pathogenesis of the neurodegeneration and an intervention means for delaying and blocking the development of the neurodegeneration.

As the most common cause of senile dementia, alzheimer's disease is clinically manifested by chronic impairment of intellectual level and chronic loss of memory. With the aging of the global population, the incidence of AD is increasing day by day, so that the timely diagnosis and treatment of AD will be more and more important.

More and more studies have shown that tau is expressed in various tissues of human, such as vascular myocytes, etc., and that it is possible to detect the presence of tau in plasma, which has been confirmed by studies that tau is detectable in plasma, but AD is indistinguishable from the control group. For example, when the causative factor of AD causes the increase of the production of p-tau in the brain, the production of peripheral p-tau is also started, or the function of the blood brain barrier is reduced to cause the overflow of p-tau in the brain, the p-tau exists in the plasma, and the increase of p-tau is the causative basis of the pathology of AD, and the detection of p-tau is more specific than the detection of tau, thus being valuable for clinical diagnosis. A plurality of documents exist for detecting the P-tau (181P) protein in the plasma of AD patients, and the result shows that the P-tau (181P) protein is really present in the plasma of the AD patients, the plasma concentration of the P-tau (181P) protein of the AD patients in the middle and later stages is obviously increased, and the specificity is higher than that of a healthy control group.

P-tau is a pathological protein of protein AD, the detection specificity is high, at present, 21 abnormal phosphate sites are found in AD patients, the phosphorylation of a plurality of phosphate sites is more prone to cause the microtubule binding function loss of tau, a plurality of phosphate sites such as 181, 199, 231, 396 and 404 are researched more, and high-concentration P-tau of the sites is detected in the cerebrospinal fluid of the AD patients, wherein the specificity and the sensitivity of the P-tau (181P) protein are shown to be high.

Phosphorylated Tau181 protein is phosphorylated at threonine 181 of Tau protein, and Tau protein (Tau) is a group of six highly soluble protein isoforms produced by alternative splicing of MAPT genes. They are less common elsewhere, but they have low levels of expression in astrocytes and oligodendrocytes of the central nervous system. Both the pathology of the nervous system and dementia, such as Alzheimer's disease and Parkinson's disease, are associated with the Dow protein, which has become a highly phosphorylated insoluble aggregate known as neurofibrillary tangles.

By detecting the phosphorylated tau-181 protein of the human, the clinical significance of the detection method is that large-scale population screening and early Alzheimer disease screening can be performed, and the severity of pathological changes can be graded, which is helpful for judging the prognosis of patients, so the detection method has important detection significance clinically. Alzheimer's disease is a degenerative disease of the central nervous system, which is manifested mainly by cognitive dysfunction, memory decline and personality, emotional abnormalities, and even abnormal behaviors. The main causes of the disease are that tau protein in the brain is hyperphosphorylated, so that a plurality of amyloid proteins in brain tissues are excessively deposited to form specific senile plaques, and partial neurons are wound to cause neuron damage, and the functions of the cerebral neuron system of a patient are further influenced.

The detection of the human phosphorylated tau-181 protein has the characteristics of low cost and small damage, and compared with the detection of the human phosphorylated tau-181 protein, the traditional detection modes such as skull MRI detection or extraction of some specific proteins to help screening and the like are higher in cost, and are not suitable for early disease screening and large-scale population screening.

In addition, the method can simultaneously measure human phosphorylated tau-181 protein and A beta 1-42 protein in a biological sample of a subject so as to increase the basis for diagnosing the Alzheimer disease.

In conclusion, there remains a need in the art for more efficient means for Alzheimer's disease detection, observation, prediction, etc., such as improving the manufacturing process of phosphorylated tau protein detection kits. For example, methods for producing a human p-tau181 epitope peptide, such as a method for producing a high-purity human p-tau181 epitope peptide, and a high-purity human p-tau181 epitope peptide produced by the method; alternatively, for example, methods for preparing p-tau181 antigen are still contemplated in the art.

Disclosure of Invention

The invention aims to provide a human p-tau181 epitope peptide, which can be used for preparing a p-tau181 antigen and a corresponding monoclonal antibody or polyclonal antibody, and further can be used for preparing an in vitro diagnostic kit for detecting, observing and predicting Alzheimer's disease. For example, the present invention is directed to a method for producing a high-purity human p-tau181 epitope peptide, for example, a high-purity human p-tau181 epitope peptide. Alternatively, the present invention aims to provide a p-tau181 antigen, or a method for producing the p-tau181 antigen. It has been surprisingly found that highly pure human p-tau181 epitope peptide can be obtained by the method of the invention, by which p-tau181 antigen can be advantageously produced. The present invention has been completed based on such findings.

To this end, the present invention provides, in a first aspect, a human p-tau181 epitope peptide having the amino acid sequence:

Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys-(p)Thr-Pro-Pro-Ser-Ser-Gly。

in the present invention, the above-mentioned amino acid chain fragment may be referred to as a human p-tau181 epitope peptide, or may be referred to as a p-tau181 epitope peptide, an epitope peptide, a polypeptide, or the like.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by the following method:

step 1: preparation of Fmoc-Gly-resin

Putting 10g of Rink Amide-MBHA resin into a reactor, adding dichloromethane, oscillating and soaking, alternately cleaning twice with dichloromethane, methanol and dimethylformamide respectively, filtering to remove the solvent,

adding 20% piperidine/dimethylformamide solution into the above treated resin, oscillating at room temperature for uncapping reaction, removing nitrogen end Fmoc protecting group, vacuum filtering to remove solvent, cleaning resin with dimethylformamide, methanol and dichloromethane respectively twice, vacuum filtering to remove solvent,

adding 30mmol of Fmoc-Gly-OH, HBTU and HOBT into dimethylformamide for dissolving, adding DIEA, stirring, transferring into a reactor containing the treated resin, oscillating at room temperature for reaction, filtering to remove reaction liquid, respectively washing the resin twice with dimethylformamide, methanol and dichloromethane alternately, and filtering to remove the solvent to obtain Fmoc-Gly-resin;

step 2: adding 20% piperidine/dimethylformamide solution into the resin obtained in the previous step, oscillating at room temperature to perform uncapping reaction, removing a nitrogen end Fmoc protecting group, performing suction filtration to remove the solvent, then respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, performing suction filtration to remove the solvent, adding 30mmol of Fmoc-Ser (tBu) -OH, HBTU and HOBT into dimethylformamide to dissolve, adding DIEA, stirring uniformly, transferring into a reactor containing the treated resin, performing oscillation reaction at room temperature, performing suction filtration to remove reaction liquid, respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, and performing suction filtration to remove the solvent to obtain Fmoc-Ser (tBu) -Gly-resin;

and step 3: taking the resin obtained in the last step and 30mmol of Fmoc-Ser (tBu) -OH for feeding, and preparing Fmoc-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 4, step 4: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing F moc-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 5: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 6: taking the resin obtained in the last step and 30mmol of Fmoc-Thr (HPO3Bzl) -OH, and preparing Fmoc-Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 7: taking the resin obtained in the previous step and 30mmol of Fmoc-Lys (Boc) -OH, preparing Fmoc-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 8: taking the resin obtained in the previous step and 30mmol Fmoc-Pro-OH to prepare F moc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and step 9: taking the resin obtained in the previous step and 30mmol of Fmoc-Ala-OH, and preparing F moc-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 10: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -tBu-Gly-resin by referring to the operation method in the step 2;

step 11: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing F moc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 12: taking the resin obtained in the previous step and 30mmol of Fmoc-Thr (Trt) -OH, and preparing Fmoc-Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 13: taking the resin obtained in the previous step and 30mmol Fmoc-Lys (Boc) -OH, preparing Fmoc-Lys (Boc) -Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method of the step 2;

step 14: taking the resin obtained in the previous step and 30mmol Fmoc-Ala-OH to charge, and preparing F moc-Ala-Lys (Boc) -Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 15: cleavage of peptide chains

Transferring the resin obtained in the last step to a round-bottom flask, adding a precooled cutting fluid (such as 95% trifluoroacetic acid/2% TIS/2% EDT/1% water), stirring and reacting at room temperature, performing suction filtration to obtain a separated filtrate, washing the resin with trifluoroacetic acid, combining the filtrate and a washing solution, adding frozen ether to precipitate, and filtering to obtain a precipitate, namely a crude product of pentadecapeptide;

step 16: and sequentially separating and purifying the crude peptide obtained in the last step by using an ion exchange chromatography system and a high performance liquid chromatography to obtain a refined peptide product, namely the human p-tau181 epitope peptide.

The human p-tau181 epitope peptide according to the first aspect of the present invention, which is prepared by the isolation and purification of step 16, is carried out as follows:

(1) dissolving the crude peptide obtained in the above step in 70% acetonitrile containing 0.1% trifluoroacetic acid, eluting with an ion exchange chromatography system such as Shodex IEC SP-420N, for example, with 70% acetonitrile containing 0.1% trifluoroacetic acid as a solvent, collecting the peptide main peak fraction,

(2) the main peak of the peptide is separated and purified by using the following high performance liquid chromatography:

a chromatographic column: c8, 10X 100mm,

a chromatograph: the industrial preparation of the liquid phase chromatography,

mobile phase: the mobile phase A is 0.1% TFA water solution, the mobile phase B is 70% acetonitrile added with 0.1% TFA, the elution gradient is 10% B-60% B in 0-50 minutes,

flow rate: 4ml of the solution is added in the reaction kettle,

detection wavelength: at the wavelength of 219nm, the molecular weight of the polymer,

(3) and (3) concentrating the collected main peak mobile phase, and freeze-drying by using a freeze dryer to obtain a refined peptide product, namely the human p-tau181 epitope peptide.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by the following steps 1 to 14, wherein the Fmoc-protected amino acids used in each step: HBTU: HOBT: molar ratio of DIEA 1: 1: 1: 4.

according to the human p-tau181 epitope peptide of the first aspect of the present invention, in step 1, when Rink Amide-MBHA resin is treated, 80ml of dichloromethane is added, the resin is shaken and soaked for 60 minutes, and then washed twice with dichloromethane, methanol and dimethylformamide alternately, each time washing with 80ml, and the solvent is removed by suction filtration.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by, in step 1, adding 100ml of 20% piperidine/dimethylformamide solution to a treated resin, shaking at room temperature to perform uncapping reaction for 60 minutes, removing the Fmoc protecting group at the nitrogen terminal, removing the solvent by suction filtration, washing the resin twice with dimethylformamide, methanol and dichloromethane, respectively, each 80ml, and removing the solvent by suction filtration.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by, in step 1, dissolving 30mmol of Fmoc-Gly-OH, HBTU, and HOBT in 100ml of dimethylformamide, adding DIEA, stirring, transferring into a reactor containing the above-mentioned treated resin, oscillating at room temperature for 1 hour, removing the reaction solution by suction filtration, washing the resin twice with dimethylformamide, methanol, and dichloromethane, respectively, and removing the solvent by suction filtration, each 80 ml.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by adding 100ml of 20% piperidine/dimethylformamide solution in step 2, shaking at room temperature to effect decapping for 60 minutes, removing the Fmoc protecting group at the nitrogen terminus, suction-filtering to remove the solvent, then washing the resin twice with dimethylformamide, methanol, and dichloromethane, respectively, in an alternating manner, each 80ml, and suction-filtering to remove the solvent.

The human p-tau181 epitope peptide according to the first aspect of the present invention is prepared by, in step 2, dissolving 30mmol of Fmoc-ser (tbu) -OH, HBTU, and HOBT in 100ml of dimethylformamide, adding DIEA, stirring, transferring into a reactor containing the above-mentioned treated resin, reacting at room temperature with shaking for 1 hour, removing the reaction solution by suction filtration, washing the resin twice with dimethylformamide, methanol, and dichloromethane, respectively, and removing the solvent by suction filtration, each 80 ml.

Further, the second aspect of the present invention provides a method for producing a human p-tau181 epitope peptide having the following amino acid sequence: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly; the method comprises the following steps:

step 1: preparation of Fmoc-Gly-resin

adding 20% piperidine/dimethylformamide solution into the above treated resin, oscillating at room temperature for decapping reaction, removing Fmoc protecting group at nitrogen end, vacuum filtering to remove solvent, cleaning resin with dimethylformamide, methanol and dichloromethane alternately twice, vacuum filtering to remove solvent,

and 2, step: adding 20% piperidine/dimethylformamide solution into the resin obtained in the previous step, oscillating at room temperature to perform uncapping reaction, removing a nitrogen end Fmoc protecting group, performing suction filtration to remove the solvent, then respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, performing suction filtration to remove the solvent, adding 30mmol of Fmoc-Ser (tBu) -OH, HBTU and HOBT into dimethylformamide to dissolve, adding DIEA, stirring uniformly, transferring into a reactor containing the treated resin, performing oscillation reaction at room temperature, performing suction filtration to remove reaction liquid, respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, and performing suction filtration to remove the solvent to obtain Fmoc-Ser (tBu) -Gly-resin;

and step 3: taking the resin obtained in the previous step and 30mmol of Fmoc-Ser (tBu) -OH, and preparing Fmoc-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 6: taking the resin obtained in the previous step and 30mmol of Fmoc-Thr (HPO3Bzl) -OH, and preparing Fmoc-Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and step 8: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 11: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -tBu (tBu) -Gly-resin by referring to the operation method in the step 2;

step 15: cleavage of peptide chains

The method according to the second aspect of the present invention, wherein the separation and purification in step 16 are carried out as follows:

a chromatographic column: c8, 10X 100mm,

a chromatograph: the industrial preparation of the liquid phase chromatography,

mobile phase: mobile phase A is 0.1% TFA water solution, mobile phase B is 70% acetonitrile added with 0.1% TFA, elution gradient is 10% B-60% B in 0-50 min,

flow rate: 4 ml/min of the mixture is added,

The method according to the second aspect of the present invention, wherein the Fmoc-protected amino acid used in each step, in steps 1 to 14, is prepared: HBT U: HOBT: molar ratio of DIEA 1: 1: 1: 4.

the method according to the second aspect of the present invention, wherein in the step 1 of the preparation, when the Rink Amide-MBHA resin is treated, 80ml of dichloromethane is added, shaking and soaking are carried out for 60 minutes, and the resin is alternately washed twice with 80ml of dichloromethane, methanol and dimethylformamide respectively, and is filtered with suction to remove the solvent.

The method according to the second aspect of the present invention, wherein the preparation step 1 is carried out by adding 100ml of 20% piperidine/dimethylformamide solution to the treated resin, subjecting the mixture to uncapping reaction for 60 minutes with shaking at room temperature, removing the Fmoc protecting group at the nitrogen terminal, removing the solvent by suction filtration, washing the resin twice with dimethylformamide, methanol and dichloromethane, respectively, in an amount of 80ml each time alternately, and removing the solvent by suction filtration.

The method according to the second aspect of the present invention, wherein in the preparation step 1, 30mmol of Fmoc-Gly-OH, HBTU, HOB T was added to 100ml of dimethylformamide to dissolve, DIEA was added, stirred, transferred to a reactor containing the above treated resin, reacted for 1 hour with shaking at room temperature, the reaction solution was removed by suction filtration, and then the resin was washed twice with dimethylformamide, methanol, and dichloromethane, respectively, each 80ml, and the solvent was removed by suction filtration.

The method according to the second aspect of the present invention, wherein the preparation step 2 is carried out by adding 100ml of 20% piperidine/dimethylformamide solution, conducting the decapping reaction for 60 minutes at room temperature with shaking, removing the nitrogen-terminal Fmoc protecting group, removing the solvent by suction filtration, washing the resin twice with dimethylformamide, methanol and dichloromethane, respectively, in an alternating manner, each 80ml, and removing the solvent by suction filtration.

The method according to the second aspect of the present invention, wherein in the preparation step 2, 30mmol of Fmoc-ser (tbu) -OH, HBTU, H OBT was added to 100ml of dimethylformamide to dissolve, DIEA was added, stirred, transferred into a reactor containing the above treated resin, reacted at room temperature with shaking for 1 hour, the reaction solution was removed by suction filtration, and then the resin was washed twice with dimethylformamide, methanol, and dichloromethane, respectively, each 80ml, and the solvent was removed by suction filtration.

Further, the third aspect of the present invention provides a p-tau181 antigen prepared by coupling a human p-tau181 epitope peptide with a carrier protein KLH using a double nitrogenated benzidine dichloride method; the amino acid sequence of the human p-tau181 epitope peptide is as follows: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly.

The p-tau181 antigen according to the third aspect of the invention, which is prepared by using the following method: dissolving the human p-tau181 epitope peptide with PBS buffer solution, dissolving KLH with borate buffer solution, mixing the two solutions, cooling to 0 ℃, adding 110 mu L of double-nitriding benzidine dichloride, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the p-tau181 epitope peptide.

The p-tau181 antigen according to the third aspect of the present invention, wherein the formulation of the PBS buffer is: 0.2mol/L of Na2HPO 481 ml and 0.2mol/L of NaH2PO 419 ml.

The p-tau181 antigen according to the third aspect of the invention, wherein the borate buffer is formulated as: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are added and mixed to obtain the product.

The p-tau181 antigen according to the third aspect of the invention, which is prepared by using the following method: dissolving 10.0mg of human p-tau181 epitope peptide in 1ml of 0.1M PBS buffer (pH7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); and then mixing the two, cooling to 0 ℃, adding 110 mu L of BDBCl2, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the product.

The p-tau181 antigen according to the third aspect of the invention, wherein the borate buffer is replaced with an equal volume of carbonate buffer formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate and 0.12g of sodium nitrite were weighed, dissolved in water and made up to 1000ml with water to give a carbonate buffer solution of 50mM, pH 9.6.

The p-tau181 antigen according to the third aspect of the present invention, wherein said human p-tau181 epitope peptide is prepared by:

step 1: preparation of Fmoc-Gly-resin

Putting 10g of Rink Amide-MBHA resin into a reactor, adding dichloromethane, oscillating and soaking, respectively and alternately cleaning twice by dichloromethane, methanol and dimethylformamide, filtering to remove the solvent,

step 2: adding 20% piperidine/dimethylformamide solution into the resin obtained in the last step, oscillating at room temperature for uncapping reaction, removing a nitrogen end Fmoc protecting group, performing suction filtration to remove the solvent, then respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, performing suction filtration to remove the solvent, adding 30mmol of Fmoc-Ser (tBu) -OH, HBTU and HOBT into dimethylformamide for dissolving, adding DIEA, stirring uniformly, transferring into a reactor containing the treated resin, performing oscillation reaction at room temperature, performing suction filtration to remove reaction liquid, respectively and alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane, and performing suction filtration to remove the solvent to obtain Fmoc-Ser (tBu) -Gl-resin;

and 5: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 6: taking the resin obtained in the last step and 30mmol of Fmoc-Thr (HPO3Bzl) -OH, and preparing Fmoc-Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 7: taking the resin obtained in the previous step and 30mmol of Fmoc-Lys (Boc) -OH, and preparing Fmoc-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and step 8: taking the resin obtained in the previous step and 30mmol Fmoc-Pro-OH to prepare F moc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 10: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing F moc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 12: taking the resin obtained in the last step and 30mmol of Fmoc-Thr (Trt) -OH for feeding, and preparing Fmoc-Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Ser (Ser tBu) -Gly-resin by referring to the operation method of the step 2;

step 15: cleavage of peptide chains

The isolation and purification in step 16 of preparing the human p-tau181 epitope peptide according to the p-tau181 antigen of the third aspect of the present invention is carried out as follows:

a chromatographic column: c8, 10X 100mm,

a chromatograph: the industrial preparation of the liquid phase chromatography,

flow rate: 4 ml/min of the mixture is added,

In steps 1 to 14 of preparing the human p-tau181 epitope peptide according to the p-tau181 antigen of the third aspect of the present invention, the Fmoc-protected amino acids used in each step: HBTU: HOBT: molar ratio of DIEA 1: 1: 1: 4.

according to the third aspect of the invention, in the step 1 of preparing the human p-tau181 epitope peptide, when the Rink Amide-MBHA resin is treated, 80ml of dichloromethane is added, the resin is shaken and soaked for 60 minutes, dichloromethane, methanol and dimethylformamide are respectively and alternately washed twice, 80ml of each time, and the solvent is removed by suction filtration.

According to the third aspect of the present invention, in the step 1 of preparing the human p-tau181 epitope peptide, a 20% piperidine/dimethylformamide solution (100ml) was added to the treated resin, the mixture was shaken at room temperature to conduct a decapping reaction for 60 minutes, the nitrogen-terminal Fmoc protecting group was removed, the solvent was removed by suction filtration, and then the resin was washed twice with dimethylformamide, methanol and dichloromethane, respectively, each by 80ml, and the solvent was removed by suction filtration.

In step 1 of preparing the human p-tau181 epitope peptide according to the p-tau181 antigen of the third aspect of the present invention, 30mmol of Fmoc-Gly-OH, HBTU, and HOBT was dissolved in 100ml of dimethylformamide, DIEA was added, the mixture was stirred, the mixture was transferred to a reactor containing the above-mentioned treated resin, the reaction mixture was stirred at room temperature for 1 hour, the reaction mixture was removed by suction filtration, and the resin was washed twice with 80ml each time of dimethylformamide, methanol, and dichloromethane, and the solvent was removed by suction filtration.

According to the p-tau181 antigen of the third aspect of the present invention, in step 2 of preparing the human p-tau181 epitope peptide, 100ml of a 20% piperidine/dimethylformamide solution is added, a decapping reaction is performed at room temperature by shaking for 60 minutes, the Fmoc protecting group at the nitrogen terminal is removed, after removing the solvent by suction filtration, the resin is washed twice with dimethylformamide, methanol and dichloromethane alternately, 80ml each time, and the solvent is removed by suction filtration.

In step 2 of preparing the human p-tau181 epitope peptide according to the p-tau181 antigen of the third aspect of the present invention, 30mmol of Fmoc-Ser (tBu) -OH, HBTU, and HOBT was dissolved in 100ml of dimethylformamide, DIEA was added, the mixture was stirred, the mixture was transferred to a reactor containing the above-mentioned treated resin, the reaction was stirred at room temperature for 1 hour, the reaction solution was removed by suction filtration, and the resin was washed twice with 80ml of dimethylformamide, methanol, and dichloromethane, respectively, and the solvent was removed by suction filtration.

Further, the fourth aspect of the present invention provides a method for producing p-tau181 antigen by coupling human p-tau181 epitope peptide to carrier protein KLH using the double nitriding benzidine dichloride method; the amino acid sequence of the human p-tau181 epitope peptide is as follows: al a-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly.

The method according to the fourth aspect of the invention, comprising the steps of: dissolving the human p-tau181 epitope peptide with PBS buffer solution, dissolving KLH with borate buffer solution, mixing the two solutions, cooling to 0 ℃, adding 110 mu L of double-nitriding benzidine dichloride, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the p-tau181 epitope peptide.

The method according to the fourth aspect of the present invention, wherein the formulation of the PBS buffer is: 0.2mol/L of Na2HPO 481 ml and 0.2mol/L of NaH2PO 419 ml.

The method according to the fourth aspect of the present invention, wherein the formulation of the borate buffer is: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are mixed to obtain the borax.

The method according to the fourth aspect of the present invention is prepared by using the following method: dissolving 10.0M g human p-tau181 epitope peptide in 1ml of 0.1M PBS buffer solution (pH7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); and then mixing the two, cooling to 0 ℃, adding 110 mu L of BDBCl2, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the product.

The method according to the fourth aspect of the invention, wherein the borate buffer is replaced with an equal volume of carbonate buffer formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate and 0.12g of sodium nitrite were weighed, dissolved in water and made up to 1000ml with water to give a carbonate buffer solution of 50mM, pH 9.6.

The method according to the fourth aspect of the present invention, wherein the human p-tau181 epitope peptide is prepared by:

step 1: preparation of Fmoc-Gly-resin

adding 30mmol of Fmoc-Gly-OH, HBTU and HOBT into dimethylformamide for dissolving, adding DIEA, stirring, transferring into a reactor containing the treated resin, oscillating at room temperature for reaction, filtering to remove reaction liquid, respectively washing the resin twice with dimethylformamide, methanol and dichloromethane alternately, filtering to remove the solvent, and preparing Fmoc-Gly-resin;

and 4, step 4: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH to be fed, and preparing F moc-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 13: taking the resin obtained in the previous step and 30mmol of Fmoc-Lys (Boc) -OH, and preparing Fmoc-Lys (Boc) -Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method of the step 2;

step 15: cleavage of peptide chains

According to the method of the fourth aspect of the present invention, the isolation and purification of step 16 for preparing the human p-tau181 epitope peptide is carried out as follows:

(1) dissolving the crude peptide obtained in the above step in 70% acetonitrile containing 0.1% trifluoroacetic acid, eluting with an ion exchange chromatography system such as Shodex IEC SP-420N, for example, with 70% acetonitrile containing 0.1% trifluoroacetic acid as a solvent, collecting the peptide main peak fractions,

a chromatographic column: c8, 10X 100mm,

chromatograph: the industrial preparation of the liquid phase chromatography,

flow rate: 4 ml/min of the mixture is added,

According to the method of the fourth aspect of the present invention, in steps 1 to 14 of preparing the human p-tau181 epitope peptide, the amino acid protected by F moc used in each step: HBTU: HOBT: molar ratio of DIEA 1: 1: 1: 4.

according to the method of the fourth aspect of the invention, in the step 1 of preparing the human p-tau181 epitope peptide, when the Rink Amide-MB HA resin is treated, 80ml of dichloromethane is added, the resin is shaken and soaked for 60 minutes, and the resin is alternately washed twice with 80ml of dichloromethane, methanol and dimethylformamide, and is filtered to remove the solvent by suction.

According to the method of the fourth aspect of the present invention, in step 1 of preparing the human p-tau181 epitope peptide, 100ml of 20% piperidine/dimethylformamide solution is added to the treated resin, the mixture is shaken at room temperature to carry out uncapping reaction for 60 minutes, the nitrogen-terminal Fmoc protecting group is removed, after the solvent is removed by suction filtration, the resin is washed twice with 80ml each time of dimethylformamide, methanol and dichloromethane alternately, and the solvent is removed by suction filtration.

According to the method of the fourth aspect of the invention, in step 1 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Gly-OH, HBTU and HOBT are added into 100ml of dimethylformamide to be dissolved, DIEA is added, the mixture is stirred uniformly and is transferred into a reactor containing the treated resin, the reaction is carried out for 1 hour under the oscillation at room temperature, the reaction solution is removed by suction filtration, and then the resin is washed twice with 80ml of dimethylformamide, methanol and dichloromethane alternately and respectively, and the solvent is removed by suction filtration.

According to the method of the fourth aspect of the invention, in the step 2 of preparing the human p-tau181 epitope peptide, 100ml of 20% piperidine/dimethylformamide solution is added, the mixture is shaken at room temperature to carry out uncapping reaction for 60 minutes, the nitrogen-terminal Fmoc protecting group is removed, after the solvent is removed by suction filtration, the resin is washed twice with dimethylformamide, methanol and dichloromethane alternately, 80ml of each time, and the solvent is removed by suction filtration.

According to the method of the fourth aspect of the invention, in the step 2 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Ser (tBu) -OH, HBTU and HOBT are added into 100ml of dimethylformamide to be dissolved, DIEA is added, the mixture is stirred evenly and transferred into a reactor containing the treated resin, the reaction is shaken at room temperature for 1 hour, the reaction solution is removed by suction filtration, and the resin is washed twice by dimethylformamide, methanol and dichloromethane alternately, and 80ml of the resin is removed by suction filtration each time.

The invention also provides a p-tau181 in vitro diagnostic kit, which is prepared by preparing p-tau181 antigen by using the human p-tau181 epitope peptide of the first aspect of the invention, preparing human p-tau181 monoclonal antibody or human p-tau181 polyclonal antibody by using the antigen, and then constructing the human p-tau181 in vitro diagnostic kit by using the antigen and/or antibody by the principles of fluorescence chromatography, ELISA, chemiluminescence, or colloidal gold immunoassay.

Further, the invention provides application of the p-tau181 epitope peptide or the p-tau181 antigen in the preparation of a kit for detecting p-tau181 protein in an Alzheimer patient.

Any reagent or means required for detection may be further included in the various kits of the present invention, including but not limited to pre-coated plates, washing solutions, color-developing solutions, stop solutions, etc., depending on the detection principle.

In the above-described steps of the preparation method of the present invention, although the specific steps described therein are distinguished in some detail or in language description from the steps described in the preparation examples of the detailed embodiments below, those skilled in the art can fully summarize the above-described method steps in light of the detailed disclosure throughout the present disclosure.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

The p-tau181 epitope peptide of the invention has the following advantages and positive effects:

1. the human p-tau181 epitope peptide has good antigenicity, and an antigen (immunogen) prepared by the peptide can be used for immunizing animals to produce monoclonal antibodies and polyclonal antibodies with high specificity.

2. The p-tau181 monoclonal and polyclonal antibodies prepared by the present invention are capable of binding with high specificity to p-tau181 in blood samples.

3. The human p-tau181 in-vitro diagnostic kit can predict, early discover and prevent AD and monitor the course of disease, thereby providing a basis for early clinical diagnosis of AD and making an important contribution to timely treatment of patients.

Drawings

FIG. 1 is a schematic synthesis of the p-tau181 epitope peptide.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention generally and/or specifically describes the materials used in the tests, as well as the test methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. The following examples further illustrate the invention without limiting it.

The invention is further illustrated or explained below by means of examples: unless otherwise stated, all solutions described below are aqueous solutions; when referring to percentages, the percentages of the mixtures formulated as liquid/liquid are volume/volume percentages, the percentages of the mixtures formulated as solid/liquid are mass/volume percentages, and the percentages of the mixtures formulated as solid/solid are mass/mass percentages.

CN111197040A (application No. 20201)00721960, yi tong bio) case some of the following typical raw materials used in the case Materials may also be used in the present invention if necessary:

HMP resin (HMP resin, P-hydroxymethylphenoxymethyl polyethylene resin available from Sigma-Aldrich),

Fmoc-AA (9-fluorenylmethoxycarbonyl protected amino acids, available as required for polypeptide synthesis, from Merck),

NMP (Nitrogen methyl pyrrolidone, from Sigma-Aldrich Co.),

DCM (dichloromethane, available from original chemical Co.),

MeOH (methanol, available from Central plains chemical Co., Ltd.),

piperidine (Piperidine, from Sigma-Aldrich),

DMAP (dimethylaminopyridine, from Sigma-Aldrich),

HOBT (hydroxybenzotriazole, available from Sigma-Aldrich),

DCC (dicyclohexylcarbodiimide, available from Sigma-Aldrich),

TFA (trifluoroacetic acid, from Sigma-Aldrich),

EDT (1, 2-ethanedithiol, from Sigma-Aldrich),

thioanisole, available from Guangzhou Weber chemical Co., Ltd,

crystalline phenol, available from national pharmaceutical group chemical reagents ltd,

acetonitrile, purchased from national pharmaceutical group chemical reagents, ltd.

The raw materials not mentioned are also readily commercially available.

Some of the following typical instruments used in CN111197040A (application No. 2020100721960, Yitong Bio) If necessary, can also be used in the present invention:

an automated polypeptide synthesizer, model 431A, available from ABI,

a rotary evaporator, model R-201, available from Shanghai Zhongshun company,

high performance liquid chromatography, Waters600, available from Waters corporation,

a freeze dryer, model VFD-2000, available from Beijing Bo Yi kang, Inc.

Instruments and devices not mentioned in the present invention are also readily available commercially.

Example 1: preparation of p-tau181 epitope peptide

The p-tau181 epitope peptide can be synthesized by a solid phase method. The main idea of solid phase peptide synthesis is: the carboxyl group of the carboxyl terminal amino acid of the peptide chain to be synthesized is firstly connected with an insoluble high molecular compound (resin) in a covalent bond mode, then the amino acid combined on the solid phase carrier is taken as an amino component, and the peptide chain is lengthened by removing the amino protecting group and reacting with excessive activated carboxyl component. Such steps may be repeated several times until the desired length of the peptide chain is reached. A schematic synthesis is shown in FIG. 1. The target polypeptide is obtained after deprotection in step (5) of the synthetic process shown in FIG. 1.

The Rink Amide-MBHA resin and Fmoc-AA-OH used in the synthesis of the p-tau181 epitope peptide of the present invention are commercially available from Gill Biochemical company, HBTU, HOBT, DIEA, etc., reagents are commercially available from Alantin reagent company, and other reagents are also readily available, for example, CN111197040A (application No. 2020100721960, Yitong organism) described in example 1.

This example 1 uses a classical method to prepare a p-tau181 epitope peptide of the invention of the following sequence:

Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys-(p)Thr-Pro-Pro-Ser-Ser-Gly，

or as: AKTPPAPK-pTPPSSG in which (p) Thr is phosphorylated threonine.

In each of the following preparative procedures, an amino acid (Fmoc-AA-OH) was used: peptide coupling agent HBTU: amide bond formation accelerator HOBT: the molar ratio of organic base DIEA is 1: 1: 1: 4;

the amount of the solvent to be used is determined empirically and by the particular operation, and is, for example, 50 to 150ml for washing in 10g of the resin, uncapping reaction, and coupling reaction, and is particularly determined by using as little solvent as possible each time washing is carried out. Some typical commercially available protected amino acids used in the assay are as follows: Fmoc-Ala-OH [ CAS No.35661-39-3],

Fmoc-Gly-OH[CAS No.29022-11-5]、

Fmoc-Lys(Boc)-OH[CAS No.71989-26-9]、

Fmoc-Pro-OH[CAS No.71989-31-6]、

Fmoc-Ser(tBu)-OH[CAS No.71989-33-8]、

Fmoc-Thr(HPO3Bzl)-OH[CAS No.175291-56-2]、

Fmoc-Thr(Trt)-OH[CAS No.133180-01-5]。

Step 1: preparation of Fmoc-Gly-resin

In the step, 10g of Rink Amide-MBHA resin (0.79mmol/g) and 30mmol (8.91g) of Fmoc-Gly-OH are taken for feeding.

The Rink Amide-MBHA resin is placed in a reactor, 80ml of dichloromethane is added, the reactor is shaken and soaked for 60 minutes, dichloromethane, methanol and dimethylformamide are used for alternately washing twice, 80ml of dichloromethane, methanol and dimethylformamide are used for washing each time, and the solvent is removed by suction filtration.

To the resin obtained in the previous step, 20% piperidine/dimethylformamide (100ml) was added, and the mixture was subjected to decapping reaction for 60 minutes with shaking at room temperature to remove the nitrogen-terminal Fmoc protecting group. After removing the solvent by suction filtration, the resin was washed twice with 80ml of dimethylformamide, methanol and dichloromethane, respectively, and the solvent was removed by suction filtration. The ninhydrin should be blue, and if not, the procedure should be repeated. (the ninhydrin test is also called KT test, and a trace amount of 1-2 mg of resin is used for the test, as follows). Fmoc-Gly-OH, HBTU and HOBT were dissolved in dimethylformamide (100ml) in a predetermined ratio, DIEA was added thereto, and the mixture was stirred, transferred to a reactor containing the above-mentioned treated resin, and reacted at room temperature for 1 hour with shaking. Filtering to remove reaction solution, washing the resin twice with dimethylformamide, methanol and dichloromethane respectively, each time washing with 80ml, filtering to remove solvent; the KT should be yellow, and if not, the reaction time is prolonged. The coupling ratio of the title resin obtained in step 1, i.e. Fmoc-Gly-resin, was determined to be 0.91. The resin was used in its entirety in the subsequent reaction step (the same applies below).

Step 2: preparation of Fmoc-Ser (tBu) -Gly-resin

Referring to the operation method of the step 1, taking the resin obtained in the previous step and 30mmol of Fmoc-Ser (tBu) -OH for feeding;

to the resin obtained in the previous step, 20% piperidine/dimethylformamide (100ml) was added, and the mixture was subjected to decapping reaction for 60 minutes with shaking at room temperature to remove the nitrogen-terminal Fmoc protecting group. After removal of the solvent by suction filtration, the resin was washed twice with 80ml of dimethylformamide, methanol and dichloromethane, respectively, and the solvent was removed by suction filtration. The ninhydrin should be blue, and if not, the procedure should be repeated. The Fmoc-protected amino acid, HBTU and HOBT were dissolved in dimethylformamide (100ml), and DIE A was added thereto, stirred, transferred to a reactor containing the above-mentioned treated resin, and reacted at room temperature for 1 hour with shaking. Removing reaction liquid by suction filtration, respectively washing the resin twice by using dimethylformamide, methanol and dichloromethane alternately, wherein each time is 80ml, and removing the solvent by suction filtration; the KT should be yellow, and if not, the reaction time is prolonged. The title resin obtained in this step was found to have a coupling ratio of 0.87.

And step 3: preparation of Fmoc-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Ser (tBu) -OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was found to have a coupling ratio of 0.89.

And 4, step 4: preparation of Fmoc-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Pro-OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was examined to have a coupling ratio of 0.90.

And 5: preparation of Fmoc-Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Pro-OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was examined to have a coupling ratio of 0.91.

Step 6: preparation of Fmoc-Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step was charged with 30mmol of Fmoc-Thr (HPO3Bzl) -OH, and the procedure of step 2 was followed to obtain the title resin, which was found to have a coupling ratio of 0.87.

And 7: preparation of Fmoc-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step was charged with Fmoc-Lys (Boc) -OH (30 mmol) and the procedure of step 2 was followed to obtain the title resin, which was found to have a coupling ratio of 0.90.

And 8: preparation of Fmoc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Pro-OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was tested to have a coupling ratio of 0.88.

And step 9: preparation of Fmoc-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Ala-OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was found to have a coupling ratio of 0.91.

Step 10: preparation of Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

Step 11: preparation of Fmoc-Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step was charged with 30mmol of Fmoc-Pro-OH, and the procedure of step 2 was followed to obtain the title resin, which was examined to have a coupling ratio of 0.89.

Step 12: preparation of Fmoc-Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Thr (Trt) -OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was found to have a coupling ratio of 0.89.

Step 13: preparation of Fmoc-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Lys (Boc) -OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was found to have a coupling ratio of 0.88.

Step 14: preparation of Fmoc-Ala-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Se r (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol of Fmoc-Ala-OH were charged, and the procedure of step 2 was referenced to obtain the title resin, which was found to have a coupling ratio of 0.90.

And (3) after the peptide grafting reaction in the step (14) is finished, filtering the resin, putting the resin into a vacuum drier for drying overnight, and weighing to obtain the protected tetradecapeptide resin.

Step 15: cleavage of peptide chains

Transferring the resin obtained in the previous step to a 500ml round-bottom flask, adding 100ml of precooled cutting fluid (95% trifluoroacetic acid/2% TIS/2% EDT/1% water), stirring and reacting for 2 hours at room temperature, performing suction filtration to separate filtrate, washing the resin for 2 times with trifluoroacetic acid, wherein each time is 25ml, and combining the filtrate and washing liquid; adding 1300ml of frozen ether to precipitate for 4 hours, and filtering to obtain a precipitate, namely a crude product of the tetradecapeptide.

Step 16: separating and purifying

(1) Dissolving the crude peptide product obtained in the previous step in 70% acetonitrile (containing 0.1% trifluoroacetic acid), eluting with 70% acetonitrile (containing 0.1% trifluoroacetic acid) as a solvent by using an ion exchange chromatography system Shodex IEC SP-420N (Beijing operone), and collecting a main peak fraction of the peptide;

a chromatographic column: c8, 10X 100mm, Waters corporation, USA;

chromatograph: YMC industrial preparative liquid chromatography;

mobile phase: the mobile phase A is 0.1% TFA (trifluoroacetic acid) aqueous solution, the mobile phase B is 70% acetonitrile added with 0.1% TFA (trifluoroacetic acid), and the elution gradient is 10% B-60% B in 0-50 minutes;

flow rate: 4 ml/min;

detection wavelength: 219 nm;

(3) and (3) concentrating the collected main peak mobile phase, and freeze-drying by using a freeze dryer to obtain a refined peptide product, namely the p-tau181 epitope peptide.

By HPLC purity determination, the total yield of 17 steps from the initial feeding of 10g of resin in step 1 to the final product peptide refined product was calculated to be 26.4%, and the purity of the peptide refined product was 98.2%.

[ HPLC purity assay ]:

the purity of the p-tau181 epitope peptide is measured by HPLC, and the main measurement conditions are as follows:

a chromatographic column: c18, 4.6X 150mm, Waters,

chromatograph: agilent 1260 type high performance liquid chromatograph, Agilent,

mobile phase: mobile phase a with 0.1% TFA in water, mobile phase B with acetonitrile containing 0.1% TFA, elution gradient 0-60% B over 30min,

flow rate: 1 ml/min of the mixture is added,

detection wavelength: 219 nm.

[ Mass Spectrometry of polypeptide molecular weight ]:

the method uses mass spectrum to determine the molecular weight of the polypeptide obtained by the invention, and the main determination conditions are as follows:

(1) reagent raw materials: TFA (trifluoroacetic acid, from Sigma-Aldrich), HCCA (α -cyano-4-hydroxycinnamic acid, from Sigma-Aldrich), acetonitrile (from Chemicals, Inc., national drug group);

(2) the instrument comprises the following steps: MALDI-TOF-MS (model: REFLEX III, Bruk er, Germany) of matrix-assisted laser desorption ionization time-of-flight mass spectrometer;

(3) matrix liquid: dissolving alpha-CCA in 50% ACN solution containing 0.1% TFA to prepare saturated solution, centrifuging, and taking supernatant;

(4) the detection conditions of the instrument are as follows: a reflection detection mode; the length of the flight tube is 3 m; nitrogen laser: the wavelength is 337nm, and the acceleration voltage is 20 KV; reflected voltage 23 KV;

(5) the method comprises the following operation steps: mu.L of the purified polypeptide sample (dissolved in acetonitrile to a concentration of 50 mg/ml) was mixed with 10. mu.L of the matrix solution, and 1. mu.L of the mixture was spotted onto the sample target and sent to the ion source for detection.

As a result: the molecular weight of the p-tau181 epitope peptide 1431.47 obtained in this example is consistent with its theoretical molecular weight of 1431.54, which proves that the synthesized polypeptide is the target product.

[ polypeptide sequence assay ]:

the method uses a polypeptide amino acid sequence analyzer to determine the amino acid sequence of the polypeptide obtained by the invention, and the main determination conditions/operations are as follows:

(1) the principle is as follows: the basic principle of polypeptide amino acid sequence analysis is Edman degradation, which is a cyclic chemical reaction process comprising three main chemical steps:

coupling: the phenylisothiocyanate reacts with the N-terminal residue of the protein and polypeptide to form a Phenylaminothiocarbonyl (PTC) derivative, i.e., a PTC-peptide;

cyclizing and cracking: (iii) PTC-peptide cyclization cleavage;

and (3) transformation: converting thiazolinone phenylamino (ATZ) to benzethionam amino acid (PTH-amino acid), and repeating the above reaction process with the peptide remaining in solution reduced by one amino acid residue, wherein the whole sequencing process is automatically performed by a sequencer;

(2) the instrument comprises the following steps: model 491 protein/polypeptide N-terminal amino acid sequence analyzer manufactured by ABI of America;

(3) reagent raw materials: phenyl isothiocyanate PITC (Sigma-Aldrich), n-heptane (national group chemical limited), trimethylamine TMA aqueous solution (national group chemical limited), trifluoroacetic acid (TFA, Sigma-Aldrich), ethyl acetate (national group chemical limited), chlorobutane (Sigma-Aldrich), acetonitrile (national group chemical limited);

(4) and (3) determination: according to the instrument instruction.

As a result, it was identified that the sequence of the p-tau181 epitope peptide obtained in example 1 was:

Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys-(p)Thr-Pro-Pro-Ser-Ser-Gly。

wherein threonine 181 is phosphorylated. The above results are consistent with the target synthetic peptide fragments prepared in the examples.

Example 21: preparation of antigen of p-tau181 epitope peptide

This example prepared p-tau181 epitope peptide obtained in example 11 by linking to a carrier protein to prepare p-tau181 antigen as follows:

preparing p-tau181 antigen by connecting p-tau181 epitope peptide with KLH (keyhole limpet hemocyanin) carrier protein by using a BDB (Bis-diazotizedbenzidine dichloride) method;

dissolving 10.0mg of p-tau181 epitope peptide in 1ml of 0.1M PBS buffer (pH7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); and then mixing the two, cooling to 0 ℃, taking 110 mu L of BDBCl2, reacting for 1.5h at room temperature, dialyzing overnight (12-15 h), subpackaging, and storing at-20 ℃ (if necessary, freeze drying is carried out), thereby preparing the p-tau181 epitope peptide-KLH coupled protein, namely the p-tau181 antigen.

In this example, the formulation of PBS buffer is: 0.2mol/L of Na2HPO 481 ml and 0.2mol/L of NaH2PO 419 ml;

the formula of the borate buffer solution is as follows: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are mixed to obtain the borax.

[ Bradford method ]: diluting the conjugated antigen to different concentrations (according to the pre-test, the absorbance of A595 is in the absorbance range falling in the standard curve series of concentrations); 0.1ml of Bradford staining solution with 5ml of the sample and the KLH standard protein solution are taken and reacted for 5-30 min at room temperature, and the A595 value is measured; the CHI3L1 epitope peptide is a small peptide, which is connected to KLH to become a part of protein to increase the protein concentration; the excess of A595 was the concentration of CHI3L1 epitope peptide from which the conjugate protein binding ratio (peptide/KLH value) was calculated. In the above process, the Bradford stain: dissolving 100mg of Coomassie brilliant blue G-250 in 50ml of 96% ethanol solution, adding 100ml of phosphoric acid, and adding water to 200ml of constant volume; KLH standard curve: an appropriate amount of KLH was precisely weighed and dissolved in PBS (10mM, pH7.4), and diluted with PBS to prepare a series of solutions having final concentrations of 50, 100, 200, 300, 400, 500, and 600g/L, respectively. The [ Bradford method ] described herein can be used to determine the binding ratio of a conjugated protein. Given that the relative molecular weight of KLH is not readily determinable, the binding ratio of the two above-described conjugated proteins is determined and calculated using the Bradford method of the present invention; the Bradford method (Coomassie brilliant blue method) is a classical protein quantitative method in the field, the method is established by Bradford in 1976, the reagent preparation is simple, the operation is simple, convenient and quick, the reaction is very sensitive, the sensitivity is 4 times higher than that of the Lowry method, the microgram-grade protein content can be measured, the measured protein concentration range is 0-1000 mu g/mL, the minimum measured protein content is 2.5 mu g/mL, and the method is a common rapid measurement method for the trace protein; the principle of the Bradford method is that Coomassie brilliant blue G-250 has two forms of red and blue with different colors, can be prepared into a light red solution under certain concentration of ethanol and acidic conditions, and forms a blue compound after being combined with protein, the compound has a maximum absorption value at 595nm, and the color depth of the compound is in direct proportion to the concentration of the protein.

The binding ratio of the p-tau181 epitope peptide-KLH conjugate protein obtained in this example was measured to be 4.84 using the Bradford method.

Example 21 a: preparation of p-tau181 antigen

Referring to example 21, except that the borate buffer used was changed to an equal volume of carbonate buffer, p-tau181 epitope peptide-KLH conjugated protein was prepared as p-tau181 antigen; the carbonate buffer used was formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate and 0.12g of sodium nitrite are weighed, dissolved in water and made up to 1000ml with water to obtain carbonate buffer solution with the concentration of 50m M and the pH value of 9.6. The binding ratio of the p-tau181 epitope peptide-KLH conjugate protein obtained in this example was determined to be 9.11 using the Bradford method.

Example 21 b: preparation of p-tau181 antigen

Referring to example 21, except that the borate buffer used was changed to an equal volume of carbonate buffer, p-tau181 epitope peptide-KLH conjugated protein was prepared as p-tau181 antigen; the carbonate buffer used was formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3 and 0.25g of ammonium pyruvate were weighed out, dissolved in water and made up to 1000ml with water to give 50mM, pH9.6 carbonate buffer. The binding ratio of the p-tau181 epitope peptide-KLH conjugate protein obtained in this example was measured to be 4.64 using the Bradford method.

Example 21 c: preparation of p-tau181 antigen

Referring to example 21, except that the borate buffer used was changed to an equal volume of carbonate buffer, p-tau181 epitope peptide-KLH conjugated protein was prepared as p-tau181 antigen; the carbonate buffer used was formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3 and 0.12g of sodium nitrite were weighed, dissolved in water and made up to 1000ml with water to give 50mM carbonate buffer, pH 9.6. The binding ratio of the p-tau181 epitope peptide-KLH conjugate protein obtained in this example was measured to be 4.89 using the Bradford method.

Based on the results of example 21 above, and example 21a, example 21b, and example 21c, it was unexpectedly found that the binding ratio of peptide/KLH coupled protein could be significantly increased by using 50mM, pH9.6 carbonate buffer containing ammonium pyruvate and sodium nitrite.

Example 22: preparation of monoclonal antibody and polyclonal antibody

This example used the p-tau181 antigen obtained in example 21 to immunize animals, and thus used the p-tau181 antigen to prepare specific monoclonal and polyclonal antibodies.

1. Immunization of animals human p-tau181 monoclonal antibody was prepared:

1.1. after the p-tau181 antigen (immunogen) prepared in example 21 was mixed well with Freund's complete adjuvant (purchased from Shanghai-derived Biotech Co., Ltd.), Balb/c mice were immunized with 60. mu.g of antigen per mouse and injected subcutaneously at multiple sites; after 4 weeks, serum titers were measured, and mice with good immunoreactivity were selected for boosting: taking antigen and Freund incomplete adjuvant with the same volume, mixing the antigen with 35 μ g/per cell, injecting subcutaneously at multiple points for 6 times, continuously boosting twice before fusing, taking spleen cells and Sp2/0 myeloma cells, mediating with 50% PEG (M W4000) (purchased from Central chemical industry Co.) according to a conventional method, fusing, and selectively culturing with HAT conditioned medium (purchased from Sigma-Aldrich Co.); after the fusion is placed into a CO2 incubator and cultured for 12-13 days at 37 ℃, larger cell clones appear in the pores; screening with indirect ELISA starting 13 days; the primary-screened positive wells were subjected to 4 times of cloning culture (even if the selected cells were proliferated by dividing a large amount) by the limiting dilution method, and then the cells were expanded, frozen and prepared into ascites. Balb/c mice were purchased from Fujian medical university (SYXK (Min) 2020-.

1.2. Balb/c mice were treated with 0.5 ml/mouse of pristane (purchased from Sigma-Aldrich Co.), and one week later were intraperitoneally inoculated with 2X 10 hybridoma cells ⁶ Ascites was collected 10 days later.

1.3. And (3) measuring the antibody titer: the titer of the human p-tau181 monoclonal antibody prepared by using the p-tau181 antigen is measured by an indirect ELISA method, and the result shows that the titer of the monoclonal antibody reaches 1: 32000.

2. immunization of animals human p-tau181 polyclonal antibody was prepared:

2.1. selecting a New Zealand white rabbit with the age of three months and the weight of about 2kg as an immune animal; in the basic immunization, 2mg of p-tau181 antigen prepared in example 21 above was mixed with Freund's complete adjuvant-emulsified well and injected subcutaneously at multiple sites on the back of rabbits; the booster was administered every 4 weeks, and the antigen was thoroughly emulsified with Freund's incomplete adjuvant and injected subcutaneously at 100. mu.g/dorsum. Carotid bleeding was performed 10 days after the last booster immunization and serum was isolated. New Zealand white rabbits were purchased from Fujian university of medicine (SYXK (Min) 2020-.

2.2. And (3) measuring the antibody titer:

the titer of the human p-tau181 polyclonal antibody prepared using the human p-tau181 antigen was measured by indirect ELISA, which showed that the antibody titer reached 1: 29200.

2.3. blood sampling and serum separation: blood was taken from the carotid artery by intubation and serum was isolated.

3. Separating and purifying the antibody:

the ascites fluid or serum obtained above was precipitated with ammonium sulfate and affinity-purified with Protein G (purchased from Sigma-Aldrich).

4. Antibody freezing storage:

and (5) subpackaging the antibody, freeze-drying and storing at low temperature.

Example 22 a: preparation of monoclonal antibody and polyclonal antibody

This example used p-tau181 antigen obtained in example 21a to immunize animals, and thus used p-tau181 antigen to prepare specific monoclonal and polyclonal antibodies

1. Immunization of animals human p-tau181 monoclonal antibody was prepared:

1.1. after p-tau181 antigen (immunogen) prepared in example 21a above was mixed well with Freund's complete adjuvant (purchased from Shanghai-derived Biotech Co., Ltd.), Balb/c mice were immunized with 35. mu.g of antigen per mouse, and injected subcutaneously at multiple sites; after 4 weeks, serum titers were measured, and mice with good immunoreactivity were selected for boosting: fully mixing the antigen with an equal volume of Freund incomplete adjuvant, carrying out subcutaneous multi-point injection with the antigen dose of 20 mu G/cell, carrying out boosting immunization for 6 times, continuously boosting immunization twice before fusion, then carrying out fusion on splenocytes and Sp2/0 myeloma cells by using 50% PE G (MW4000) (purchased from the original chemical company) mediated by a conventional method, and selectively culturing the splenocytes and the Sp2/0 myeloma cells by using HAT conditioned medium (purchased from Sigma-Aldrich company); after the fusion is placed into a CO2 incubator and cultured for 12-13 days at 37 ℃, larger cell clones appear in the pores; screening with indirect ELISA starting 13 days; the primary-screened positive wells were subjected to 4 times of cloning culture (even if the selected cells were proliferated by dividing a large amount) by the limiting dilution method, and then the cells were expanded, frozen and prepared into ascites. Balb/c mice were purchased from Fujian medical university (SYXK (Min) 2020-.

1.2. Balb/c mice were treated with 0.5 ml/mouse of pristane (purchased from Sigma-Aldrich Co.), and one week later were intraperitoneally inoculated with hybridoma cells2× 10 ⁶ Ascites were collected 10 days later.

1.3. And (3) measuring the antibody titer: the titer of the human p-tau181 monoclonal antibody prepared by using the p-tau181 antigen is measured by an indirect ELISA method, and the result shows that the titer of the monoclonal antibody reaches 1: 29800.

2. immunization of animals human p-tau181 polyclonal antibody was prepared:

2.1. selecting a New Zealand white rabbit with the age of three months and the weight of about 2kg as an immune animal; in the basic immunization, 2mg of p-tau181 antigen prepared in example 21a above was mixed with Freund's complete adjuvant-emulsified well and injected subcutaneously at multiple sites on the back of rabbits; the booster was administered every 4 weeks, and the antigen was thoroughly emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites at the back at a dose of 60. mu.g/patient. Carotid bleeding was performed 10 days after the last booster immunization and serum was isolated. New Zealand white rabbits were purchased from Fujian university of medicine (SYXK (Min) 2020-.

2.2. And (3) measuring the antibody titer: the titer of human p-tau181 polyclonal antibody prepared by using human p-tau181 antigen was measured by indirect ELISA method, and the result showed that the antibody titer reached 1: 31600.

2.3. blood sampling and serum separation: blood was taken from carotid artery by cannulation and serum was isolated.

3. Separating and purifying the antibody:

4. Freezing and storing the antibody:and (5) subpackaging the antibody, freeze-drying and storing at low temperature.

Example 3: specific identification of human p-tau181 monoclonal antibody

ELISA is used for detection, p-tau181, Abeta 1-40 and Abeta 1-42 proteins are used as detection antigens to coat an ELISA plate, the p-tau181 monoclonal antibody prepared in the ELISA detection example 22 reacts with the specificity of the monoclonal antibody, the serum of a normal BALB/c mouse is used as a negative control, and PBS liquid is used as a blank control; as a result: the P-tau181 monoclonal antibody reacted positively with P-tau181 (P/N >2.1) and negatively with A β 1-40 and A β 1-42 proteins (P/N <2.1), indicating the specificity of the P-tau181 monoclonal antibody.

Example 3 a:referring to the method of example 3, ELISA was used to detect the specific reaction of the p-tau181 monoclonal antibody prepared in example 22a, and normal BALB/c mouse serum was used as a negative control, and PBS solution was used as a blank control; as a result: the P-tau181 monoclonal antibody reacted positively with P-tau181 (P/N)>2.1) and the reactions with the A beta 1-40 and A beta 1-42 proteins are negative (P/N)<2.1), indicating that the p-tau181 monoclonal antibody has specificity.

Example 4: specific identification of human p-tau181 polyclonal antibodies

The p-tau181 polyclonal antibody prepared in example 22 was identified by the same method as that for identifying the monoclonal antibody specificity described above; the results show that: the P-tau181 polyclonal antibody reacts positively with P-tau181 (P/N >2.1) and negatively with A beta 1-40 and A beta 1-42 proteins (P/N <2.1), indicating that the P-tau181 polyclonal antibody is specific.

Example 4 a:the polyclonal antibody against p-tau181 prepared in example 22a was identified by the method described in example 4; the results show that: the polyclonal antibody to P-tau181 reacted positively with P-tau181 (P/N)>2.1) and the reaction with the A.beta.1-40 and A.beta.1-42 proteins is negative (P/N)<2.1), indicating that the p-tau181 polyclonal antibody has specificity.

Example 5: an exemplary application:

in this example, the human p-tau181 polyclonal antibody obtained in example 22a was used as the binding antibody in the kit; p-tau181 monoclonal antibody prepared in example 22a was used as a coating antibody; then, with reference to the CN103665113A under various conditions and operating conditions of example 5, the concentration of p-tau181 protein was measured and calculated from 36 plasma of healthy group (age 71-79), 32 plasma of early-stage AD patients (age 74-82, 19 ≦ simple intellectual state checklist (MMSE) value ≦ 27, clinical dementia scale (CDR) value ≦ 0.8-1.3), and 35 plasma of late-stage AD patients (age 73-80, MMSE < 19, CDR ≧ 2) (all of the patients diagnosed/treated and volunteers provided in a hospital), and as a result: healthy group plasma p-tau181 protein concentration 9.2 ± 2.7ng/L (n ═ 36), early AD patient plasma p-tau181 protein concentration 13.3 ± 4.5ng/L (n ═ 32), late AD patient plasma p-tau181 protein concentration 21.1 ± 4.1ng/L (n ═ 35); these results indicate that the epitope peptide of human p-tau181 prepared by the present invention, the antigen prepared therefrom and the antibody further prepared therefrom are effective in constructing p-tau181 in vitro diagnostic kits.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A p-tau181 antigen prepared by coupling a human p-tau181 epitope peptide with a carrier protein KLH using a double nitrogenated benzidine dichloride method; the amino acid sequence of the human p-tau181 epitope peptide is as follows: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Ly s- (p) Thr-Pro-Pro-Ser-Ser-Gly.

2. The p-tau181 antigen according to claim 1, which is prepared by using the following method: dissolving the human p-tau181 epitope peptide with PBS buffer solution, dissolving KLH with borate buffer solution, mixing the two solutions, cooling to 0 ℃, adding 110 mu L of double-nitriding benzidine dichloride, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the p-tau181 epitope peptide.

3. The p-tau181 antigen of claim 1, wherein:

the formula of the PBS buffer solution is as follows: 0.2mol/L of Na2HPO 481 ml and 0.2mol/L of NaH2PO 419 ml;

the formula of the borate buffer solution comprises: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are mixed to obtain the borax;

it is prepared by using the following method: dissolving 10.0mg of human p-tau181 epitope peptide in 1ml of 0.1M PBS buffer (pH7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); then mixing the two, cooling to 0 ℃, adding 110 mu L of BDBCl2, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the product;

the borate buffer was replaced with an equal volume of carbonate buffer, formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate and 0.12g of sodium nitrite were weighed, dissolved in water and made up to 1000ml with water to give a carbonate buffer solution of 50mM, pH 9.6.

4. The p-tau181 antigen of claim 1, wherein said human p-tau181 epitope peptide is prepared by:

step 1: preparation of Fmoc-Gly-resin

and 4, step 4: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 5: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 8: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and step 9: taking the resin obtained in the previous step and 30mmol of Fmoc-Ala-OH, and preparing Fmoc-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 10: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 11: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method of the step 2;

step 14: taking the resin obtained in the previous step and 30mmol of Fmoc-Ala-OH, preparing Fmoc-Ala-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 15: cleavage of peptide chains

5. The p-tau181 antigen according to claim 1, the isolation and purification of step 16 for the preparation of said human p-tau181 epitope peptide is carried out as follows:

(1) dissolving the crude peptide obtained in the above step in 70% acetonitrile containing 0.1% trifluoroacetic acid, eluting with an ion exchange chromatography system such as ShodexIEC SP-420N, for example, with 70% acetonitrile containing 0.1% trifluoroacetic acid as a solvent, collecting the peptide main peak fraction,

and (3) chromatographic column: c8, 10X 100mm,

chromatograph: the industrial preparation of liquid-phase chromatography is carried out,

flow rate: 4ml of the solution is added in the reaction kettle,

6. The p-tau181 antigen of claim 1, wherein:

in the steps 1 to 14 for preparing the human p-tau181 epitope peptide, the Fmoc protected amino acid used in each step: HBTU: HO BT: molar ratio of DIEA 1: 1: 1: 4;

in the step 1 of preparing the human p-tau181 epitope peptide, 80ml of dichloromethane is added when Rink Amide-MBHA resin is processed, the mixture is shaken and soaked for 60 minutes, dichloromethane, methanol and dimethylformamide are respectively and alternately washed twice, each time, 80ml of dichloromethane is used, and the solvent is removed by suction filtration;

in the step 1 of preparing the human p-tau181 epitope peptide, 100ml of 20% piperidine/dimethylformamide solution is added into the treated resin, the mixture is oscillated at room temperature to carry out uncapping reaction for 60 minutes, the nitrogen end Fmoc protecting group is removed, after the solvent is removed by suction filtration, the resin is alternately washed twice by dimethylformamide, methanol and dichloromethane, each time, the volume of the resin is 80ml, and the solvent is removed by suction filtration;

in the step 1 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Gly-OH, HBTU and HOBT are added into 100ml of dimethylformamide to be dissolved, DIEA is added, the mixture is stirred uniformly and is moved into a reactor containing the treated resin, the oscillation reaction is carried out for 1 hour at room temperature, the reaction liquid is removed by suction filtration, the resin is washed twice by dimethylformamide, methanol and dichloromethane respectively, each time is 80ml, and the solvent is removed by suction filtration;

in the step 2 of preparing the human p-tau181 epitope peptide, 100ml of 20% piperidine/dimethylformamide solution is added, the mixture is oscillated at room temperature to carry out uncapping reaction for 60 minutes, the nitrogen end Fmoc protecting group is removed, after the solvent is removed by suction filtration, the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, each time, the volume of the resin is 80ml, and the solvent is removed by suction filtration;

in the step 2 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Ser (tBu) -OH, HBTU and HOBT are added into 100ml of dimethylformamide to be dissolved, DIEA is added, the mixture is stirred evenly and is moved into a reactor containing the treated resin, the oscillation reaction is carried out for 1 hour at room temperature, the reaction solution is removed by suction filtration, the resin is washed twice by dimethylformamide, methanol and dichloromethane alternately, each time is 80ml, and the solvent is removed by suction filtration.

7. A method for producing the p-tau181 antigen of any of claims 1 to 6, which comprises coupling a human p-tau181 epitope peptide to a carrier protein KLH by using a double nitriding benzidine dichloride method; the amino acid sequence of the human p-tau181 epitope peptide is as follows: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly; or, it comprises the following steps: dissolving human p-tau181 epitope peptide with PBS buffer solution, dissolving KLH with borate buffer solution, mixing the two solutions, cooling to 0 ℃, adding 110 mu L of double-nitriding benzidine dichloride, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the p-tau181 epitope peptide; or the formula of the PBS buffer solution is as follows: 0.2mol/L of Na2HPO 481 ml and 0.2mol/L of NaH2PO 419 ml; or the formula of the borate buffer solution is as follows: 80ml of 0.05mol/L borax and 20ml of 0.2mol/L boric acid are added and mixed to obtain the product; alternatively, it is prepared by using the following method: dissolving 10.0mg of human p-tau181 epitope peptide in 1ml of 0.1M PBS buffer (pH7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); then mixing the two, cooling to 0 ℃, adding 110 mu L of BDBCl2, reacting for 1.5h at room temperature, and dialyzing for 12-15 h to obtain the product; alternatively, the borate buffer is replaced with an equal volume of carbonate buffer, formulated as follows: 2.94g of NaHCO3, 1.58g of Na2CO3, 0.25g of ammonium pyruvate and 0.12g of sodium nitrite were weighed, dissolved in water and made up to 1000ml with water to give a carbonate buffer solution of 50mM, pH 9.6.

8. A human p-tau181 epitope peptide having the amino acid sequence: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly; for example, it is prepared by the following method:

step 1: preparation of Fmoc-Gly-resin

and 5: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH to be fed, and preparing Fmoc-Pro-Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

and 8: taking the resin obtained in the previous step and 30mmol of Fmoc-Pro-OH to prepare Fmoc-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 11: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -tBu-Gly-resin by referring to the operation method in the step 2;

step 14: taking the resin obtained in the previous step and 30mmol of Fmoc-Ala-OH to charge, and preparing Fmoc-Ala-Lys (Boc) -Thr (Trt) -Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 15: cleavage of peptide chains

9. A method for preparing a human p-tau181 epitope peptide having the amino acid sequence: Ala-Lys-Thr-Pro-Pro-Ala-Pro-Lys- (p) Thr-Pro-Pro-Ser-Ser-Gly; the method comprises the following steps:

step 1: preparation of Fmoc-Gly-resin

step 10: taking the resin obtained in the last step and 30mmol of Fmoc-Pro-OH, and preparing Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO3Bzl) -Pro-Ser (tBu) -Gly-resin by referring to the operation method in the step 2;

step 15: cleavage of peptide chains

10. Use of the p-tau181 epitope peptide of claim 8 or the p-tau181 antigen of any of claims 1 to 6 in the preparation of a kit for detecting p-tau181 protein in an alzheimer's patient.