CN114989313B

CN114989313B - P-tau181 epitope peptide and application thereof in Alzheimer disease detection

Info

Publication number: CN114989313B
Application number: CN202210740357.8A
Authority: CN
Inventors: 黄仁杰; 陈佳宇; 汪泽云; 李鑫
Original assignee: Fujian Yitong Biotechnology Co ltd
Current assignee: Fujian Yitong Biotechnology Co ltd
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2024-04-02
Anticipated expiration: 2042-06-28
Also published as: CN114989313A

Abstract

The invention relates to a p-tau181 epitope peptide and application thereof in Alzheimer disease detection. The p-tau181 antigen prepared from 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 human p-tau181 epitope peptide with a protein carrier; the p-tau181 monoclonal antibody or polyclonal antibody is prepared from the Abeta 1-42 antigen of the invention; the p-tau181 monoclonal antibody or polyclonal antibody is used for preparing an in vitro diagnosis kit of the p-tau 181; the human p-tau181 epitope peptide has good antigenicity, and the antigen (immunogen) prepared by the human p-tau181 epitope peptide can produce monoclonal antibodies and polyclonal antibodies with high specificity by immunizing animals, 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 in large scale with high purity.

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, relates to a p-tau181 epitope peptide and a method for preparing the p-tau181 epitope peptide, and the p-tau181 antigen prepared by using the p-tau181 epitope peptide can be used for further preparing corresponding monoclonal antibodies or polyclonal antibodies, in addition, the antibodies can be used for preparing in vitro diagnostic kits of human p-tau181, and the kits can be used for detecting the concentration of p-tau181 protein in serum or plasma samples of Alzheimer disease patients, thereby providing effective scientific basis for diagnosis of Alzheimer disease.

Background

The p-tau181 epitope peptide is known to have the following amino acid sequence: AKTPPAPK-pTPPSSG, amino acid fragment CN114409780A (Chinese patent application No. 202210138280.7) comprising the phosphorylation of threonine 181 (T181) of tau protein. The p-tau181 epitope peptide can be used for detecting phosphorylated tau protein in Alzheimer's patients, and is beneficial to prediction, early discovery and prevention, disease progression and curative effect observation of Alzheimer's disease. In addition, CN111197040a (application No. 2020100721960, a light-weight organism) discloses an epitope peptide, an antigen, an antibody, a use and a kit of chitinase 3-like protein 1 (CHI 3L 1), and it is believed that the CHI3L1 epitope peptide of the present invention exhibits good antigenicity, and an antigen (immunogen) prepared by using the peptide can produce highly specific monoclonal antibodies and polyclonal antibodies, thereby being applicable to in vitro detection of human CHI3L 1. The relevant technical information disclosed in CN103665113a and CN111197040a above is incorporated herein by reference.

At present, china has entered an aging society, and Alzheimer's Disease (AD) is a common disease and frequently occurring disease of the aged, also called Alzheimer's disease, and the incidence rate of the aged over 60 years is more than 5%. AD is a primary degenerative disease of the central nervous system and the progressive mental and cognitive functions of the patient are reduced. Neurodegenerative diseases are nervous system diseases mainly comprising central nervous system damage caused by progressive degeneration and death of neurons, and seriously endanger human health, but have unclear etiology, complex pathogenesis and lack of effective therapeutic measures. In addition to the well-known senile dementia (Alzheimer disease, AD), parkinson's Disease (PD), and the like, diabetic late complications, in which the incidence rate is increasing and the onset age is decreasing, become the leading factor in the group of people, with neurodegenerative diseases. Because of the lack of effective early diagnosis measures, the neurodegenerative diseases bring a series of real problems to treatment and nursing, and simultaneously bring great economic burden and mental stress to society and families. For example, alzheimer's Disease (AD), which is also known as Alzheimer's disease, is expected to reach 2000 thousands in China in 2050. If the government and home spend an average of 1 ten thousand yuan per year for each AD patient, nursing fees, miswork fees for members of the home, etc., then by 2050 China would pay 2000 hundred million yuan per year for the senile dementia patient. Such heavy economic burden would seriously obstruct the economic development of our country and may cause social problems. Therefore, the method has important significance in researching the pathogenesis of the neurodegenerative diseases and delaying and blocking the intervention means of the development of the neurodegenerative diseases.

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

More and more studies have shown that tau protein is expressed in various tissues of humans, such as vascular myocytes, etc., which make it possible to detect the presence of tau protein in plasma, which has been demonstrated to be detectable in plasma, but AD is indistinguishable from the control group. For example, the etiology factor of AD, when it causes the increase of p-tau protein production in brain, also initiates the production of peripheral p-tau protein, or the overflow of p-tau protein in brain caused by the decrease of blood brain barrier function, can make p-tau protein exist in blood plasma, and the increase of p-tau protein is the etiology basis of the pathology of AD, and the detection of p-tau protein is more specific than the detection of tau protein, thus being valuable for clinical diagnosis. There are many documents for detecting the P-tau (181P) protein in the plasma of AD patients, and the result shows that the P-tau (181P) protein exists in the plasma of AD patients, and the plasma concentration of the P-tau (181P) protein of the AD patients in middle and later period is obviously increased, so that the specificity is higher than that of a healthy control group.

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

Phosphorylated Tau181 protein is phosphorylated at threonine position 181 of Tau protein, tau protein (Tau) is a group of six isoforms of highly soluble proteins 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 proteins, which have become highly phosphorylated insoluble aggregates, known as neurofibrillary tangles.

The detection of the human phosphorylated tau-181 protein has the clinical significance that large-scale population screening and early screening of Alzheimer's disease can be performed, and meanwhile, the severity of lesions can be graded, so that the detection method is beneficial to judging prognosis of patients, and has important clinical detection significance. Alzheimer's disease is a degenerative disease of the central nervous system and is mainly manifested by cognitive dysfunction, memory decline and personality, mood abnormalities, and even abnormal behavior. The main cause of the disease is that tau protein in the brain is hyperphosphorylated, so that some amyloid protein in brain tissues is excessively deposited, thereby forming specific senile plaques, and partial neurons are entangled, thereby damaging the neurons and further affecting the functions of the brain neuron system of a patient.

Compared with the detection of the human phosphorylated tau-181 protein, the traditional head MRI detection or extraction of some specific proteins and other detection modes for helping screening and other detection modes have the characteristics of low cost and small damage, and are not suitable for early disease screening and large-scale population screening.

In addition, the basis for diagnosis of Alzheimer's disease can be increased by simultaneously measuring human phosphorylated tau-181 protein and Abeta 1-42 protein in a biological sample of a subject.

In summary, there remains a need in the art for more efficient means for detection, observation, prediction, etc. of alzheimer's disease, e.g. to improve the manufacturing process of phosphorylated tau protein detection kits. For example, there remains a need in the art for methods of preparing human p-tau181 epitope peptides, such as methods of preparing high purity human p-tau181 epitope peptides, and high purity human p-tau181 epitope peptides prepared by such methods; alternatively, for example, methods for preparing the p-tau181 antigen remain desirable in the art.

Disclosure of Invention

The invention aims to provide a human p-tau181 epitope peptide, which can be used for preparing p-tau181 antigen and corresponding monoclonal antibodies or polyclonal antibodies, 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 aims to provide a method for preparing high-purity human p-tau181 epitope peptide, for example, the present invention aims to provide high-purity human p-tau181 epitope peptide. Alternatively, it is an object of the present invention to provide a p-tau181 antigen, or to provide a method of preparing the p-tau181 antigen. It has been unexpectedly found that high purity human p-tau181 epitope peptides can be obtained by the methods of the present invention, by which p-tau181 antigen can be advantageously prepared. The present invention has been completed based on such findings.

To this end, a first aspect of the invention provides 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

10g of Rink Amide-MBHA resin is placed in a reactor, methylene dichloride is added, the mixture is oscillated and soaked, the mixture is respectively and alternately washed twice with the methylene dichloride, the methanol and the dimethylformamide, the solvent is removed by suction filtration,

adding 20% hexahydropyridine/dimethylformamide solution into the resin, oscillating at room temperature for uncapping reaction, removing nitrogen end Fmoc protecting group, suction-filtering to remove solvent, alternately cleaning the resin with dimethylformamide, methanol and dichloromethane twice, suction-filtering to remove solvent,

adding 30mmol of Fmoc-Gly-OH, HBTU, HOBT into dimethylformamide to dissolve, adding DIEA, stirring uniformly, transferring into a reactor containing the treated resin, performing oscillation reaction at room temperature, removing reaction liquid by suction filtration, alternately cleaning the resin twice with dimethylformamide, methanol and dichloromethane respectively, and removing solvent by suction filtration to obtain Fmoc-Gly-resin;

Step 2: adding 20% of hexahydropyridine/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, filtering to remove a solvent, alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane respectively, filtering to remove the solvent, adding 30mmol of Fmoc-Ser (tBu) -OH, HBTU, HOBT into the dimethylformamide to dissolve, adding DIEA, stirring, transferring into a reactor containing the treated resin, oscillating at room temperature to perform reaction, filtering to remove a reaction solution, alternately cleaning the resin twice by using dimethylformamide, methanol and dichloromethane respectively, and filtering to remove the solvent to obtain Fmoc-Ser (tBu) -Gly-resin;

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

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

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

Step 6: taking the resin obtained in the previous step and 30mmol of Fmoc-Thr (HPO 3 Bzl) -OH charge, and referring to the operation method of the step 2, preparing Fmoc-Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 7: taking the resin obtained in the previous step and 30mmol of Fmoc-Lys (Boc) -OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 8: taking the resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 9: taking the resin obtained in the previous step and 30mmol of Fmoc-Ala-OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 10: taking the resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 11: taking the resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

Step 12: taking the resin obtained in the previous step and 30mmol Fmoc-Thr (Trt) -OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 13: taking the resin obtained in the previous step and 30mmol Fmoc-Lys (Boc) -OH charge, referring to the operation method of step 2, fmoc-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin is prepared;

step 14: taking the resin obtained in the previous step and 30mmol Fmoc-Ala-OH charge, and referring to the operation method of the step 2, obtaining Fmoc-Ala-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin;

step 15: cleavage of peptide chains

Transferring the resin obtained in the previous step to a round-bottom flask, adding precooled cutting fluid (such as 95% trifluoroacetic acid/2% TIS/2% EDT/1% water), stirring at room temperature for reaction, suction filtering to separate filtrate, washing the resin with trifluoroacetic acid, mixing the filtrate and the washing fluid, adding frozen diethyl ether for precipitation, and filtering to obtain a precipitate, namely a pentadecapeptide crude product;

step 16: and (3) sequentially using an ion exchange chromatography system and a high performance liquid chromatography to separate and purify the crude peptide obtained in the previous step to obtain a peptide refined product, namely the human p-tau181 epitope peptide.

The isolation and purification of step 16 of the preparation of the human p-tau181 epitope peptide according to the first aspect of the present invention is performed as follows:

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

(2) The main peak of the peptide was isolated and purified using high performance liquid chromatography as follows:

chromatographic column: c8 10X 100mm of the diameter of the tube,

chromatograph: the liquid phase chromatography is carried out in an industrial manner,

mobile phase: mobile phase a was 0.1% tfa in water, mobile phase B was 70% acetonitrile with 0.1% tfa added, elution gradient 10% B-60% B during 0-50 min,

flow rate: the volume of the solution was 4 ml/min,

detection wavelength: at a wavelength of 219nm,

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

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

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

In the step 1 of preparing the human p-tau181 epitope peptide according to the first aspect of the present invention, 100ml of 20% hexahydropyridine/dimethylformamide solution is added to the treated resin, the uncapping reaction is carried out for 60 minutes by shaking at room temperature, 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, 80ml each, and the solvent is removed by suction filtration.

In the step 1 of the preparation of the human p-tau181 epitope peptide according to the first aspect of the present invention, 30mmol of Fmoc-Gly-OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred uniformly, transferred into a reactor containing the treated resin, reacted for 1 hour at room temperature under shaking, the reaction solution is removed by suction filtration, and the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, respectively, 80ml of each time, and the solvent is removed by suction filtration.

In the step 2 of preparing the human p-tau181 epitope peptide according to the first aspect of the present invention, 100ml of 20% hexahydropyridine/dimethylformamide solution is added, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, 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, 80ml each time, and the solvent is removed by suction filtration.

In step 2 of the preparation of the human p-tau181 epitope peptide according to the first aspect of the present invention, 30mmol of Fmoc-Ser (tBu) -OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred uniformly, transferred into a reactor containing the treated resin, reacted for 1 hour at room temperature under shaking, the reaction solution is removed by suction filtration, and the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, respectively, 80ml each time, and the solvent is removed by suction filtration.

Further, a second aspect of the present invention provides a method of 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 15: cleavage of peptide chains

The method according to the second aspect of the invention, wherein the isolation and purification of step 16 is performed as follows:

chromatographic column: c8 10X 100mm of the diameter of the tube,

flow rate: the volume of the solution was 4 ml/min,

detection wavelength: at a wavelength of 219nm,

The method according to the second aspect of the present invention, wherein in the steps 1 to 14 of the preparation, fmoc protected amino acids used in each step: HBTU: HOBT: DIEA molar ratio = 1:1:1:4.

According to the method of the second aspect of the present invention, in the preparation of step 1, 80ml of dichloromethane is added when the Rink Amide-MBHA resin is processed, and the mixture is shaken and soaked for 60 minutes, and the mixture is alternately washed twice with dichloromethane, methanol and dimethylformamide, 80ml each, and filtered with suction to remove the solvent.

According to the method of the second aspect of the present invention, in the preparation step 1, 100ml of 20% hexahydropyridine/dimethylformamide solution is added to the treated resin, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, 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, 80ml each, and the solvent is removed by suction filtration.

According to the method of the second aspect of the present invention, in the preparation step 1, 30mmol of Fmoc-Gly-OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred and transferred into a reactor containing the treated resin, the reaction solution is removed by suction filtration after 1 hour of shaking reaction at room temperature, and the resin is alternately washed with dimethylformamide, methanol and dichloromethane twice each of 80ml, and the solvent is removed by suction filtration.

According to the method of the second aspect of the invention, in the preparation step 2, 100ml of 20% hexahydropyridine/dimethylformamide solution is added, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, 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 respectively, 80ml of each time, and the solvent is removed by suction filtration.

According to the method of the second aspect of the present invention, in the preparation step 2, 30mmol of Fmoc-Ser (tBu) -OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred and transferred into a reactor containing the treated resin, the reaction solution is removed by suction filtration at room temperature for 1 hour, and the resin is alternately washed with dimethylformamide, methanol and dichloromethane twice, 80ml each, and the solvent is removed by suction filtration.

Further, a 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 present 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, then mixing the two, cooling to 0 ℃, adding 110 mu L of double nitridation benzidine dichloride, reacting for 1.5 hours at room temperature, and dialyzing for 12-15 hours to obtain the antigen peptide.

The p-tau181 antigen according to the third aspect of the present invention, wherein the PBS buffer is formulated as follows: 0.2mol/L Na2HPO4 (81 ml) and 0.2mol/L NaH2PO4 (19 ml) were mixed.

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

The p-tau181 antigen according to the third aspect of the present invention, which is prepared by using the following method: 10.0mg of human p-tau181 epitope peptide is taken and dissolved in 1ml of 0.1M PBS buffer (pH 7.4); 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 1.5 hours at room temperature, and dialyzing for 12-15 hours to obtain the product.

The p-tau181 antigen according to the third aspect of the present invention, wherein the borate buffer is replaced with an equal volume of carbonate buffer formulated in the following manner: 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 the volume is fixed to 1000ml by water, thus obtaining a carbonate buffer with the concentration of 50mM and the pH value of 9.6.

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

Step 1: preparation of Fmoc-Gly-resin

step 15: cleavage of peptide chains

According to the p-tau181 antigen of the third aspect of the present invention, the isolation and purification of step 16 of preparing the human p-tau181 epitope peptide is performed as follows:

chromatographic column: c8 10X 100mm of the diameter of the tube,

Flow rate: the volume of the solution was 4 ml/min,

detection wavelength: at a wavelength of 219nm,

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

according to the p-tau181 antigen of the third aspect of the present invention, in the step 1 of preparing the human p-tau181 epitope peptide, 80ml of dichloromethane is added when the RinkAmide-MBHA resin is processed, the mixture is shaken and soaked for 60 minutes, and the mixture is alternately washed twice with dichloromethane, methanol and dimethylformamide, respectively, and 80ml of each solution is filtered by suction to remove the solvent.

In the step 1 of preparing the human p-tau181 epitope peptide according to the third aspect of the present invention, 100ml of 20% hexahydropyridine/dimethylformamide solution is added to the treated resin, the uncapping reaction is carried out for 60 minutes by shaking at room temperature, the Fmoc protecting group at the nitrogen end is removed, after the solvent is removed by suction filtration, the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, respectively, 80ml each time, and the solvent is removed by suction filtration.

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

According to the p-tau181 antigen of the third aspect of the present invention, in the step 2 of preparing the human p-tau181 antigen epitope peptide, 100ml of 20% hexahydropyridine/dimethylformamide solution is added, the uncapping reaction is carried out for 60 minutes by shaking at room temperature, 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 respectively, 80ml of each time, and the solvent is removed by suction filtration.

In step 2 of preparing the human p-tau181 epitope peptide according to the third aspect of the present invention, 30mmol of Fmoc-Ser (tBu) -OH, HBTU, HOBT was added to 100ml of dimethylformamide to dissolve, DIEA was added, stirred and transferred into a reactor containing the above-mentioned treated resin, and the reaction solution was removed by suction filtration, and the resin was alternately washed twice with dimethylformamide, methanol and dichloromethane, 80ml each time, and the solvent was removed by suction filtration.

Further, a fourth aspect of the present invention provides a method for preparing p-tau181 antigen by coupling human p-tau181 epitope peptide to carrier protein KLH using the double nitridation 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 method according to the fourth aspect of the invention comprises the steps of: dissolving the human p-tau181 epitope peptide with PBS buffer solution, dissolving KLH with borate buffer solution, then mixing the two, cooling to 0 ℃, adding 110 mu L of double nitridation benzidine dichloride, reacting for 1.5 hours at room temperature, and dialyzing for 12-15 hours to obtain the antigen peptide.

The method according to the fourth aspect of the present invention, wherein the PBS buffer is formulated as follows: 0.2mol/L Na2HPO4 (81 ml) and 0.2mol/L NaH2PO4 (19 ml) were mixed.

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

The method according to the fourth aspect of the invention is prepared by using the following method: 10.0mg of human p-tau181 epitope peptide is taken and dissolved in 1ml of 0.1M PBS buffer (pH 7.4); 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 1.5 hours at room temperature, and dialyzing for 12-15 hours 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, the carbonate buffer being formulated in the following manner: 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 the volume is fixed to 1000ml by water, thus obtaining a carbonate buffer with the concentration of 50mM and the pH value of 9.6.

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

step 1: preparation of Fmoc-Gly-resin

step 15: cleavage of peptide chains

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

chromatographic column: c8 10X 100mm of the diameter of the tube,

flow rate: the volume of the solution was 4 ml/min,

detection wavelength: at a wavelength of 219nm,

According to the method of the fourth aspect of the present invention, in step 1 to step 14 of preparing the human p-tau181 epitope peptide, fmoc-protected amino acids used in each step: HBTU: HOBT: DIEA molar ratio = 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, 80ml of dichloromethane is added when the Rink Amide-MBHA resin is processed, the mixture is shaken and soaked for 60 minutes, and the mixture is alternately washed twice with dichloromethane, methanol and dimethylformamide respectively, 80ml each time, and the solvent is removed by suction filtration.

According to the method of the fourth aspect of the invention, in the step 1 of preparing the human p-tau181 epitope peptide, 100ml of 20% hexahydropyridine/dimethylformamide solution is added to the treated resin, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, the Fmoc protecting group at the nitrogen end is removed, after the solvent is removed by suction filtration, the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane respectively, 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 1 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Gly-OH, HBTU, HOBT is added into 100ml of dimethylformamide to be dissolved, DIEA is added, stirred uniformly, transferred into a reactor containing the treated resin, subjected to oscillation reaction at room temperature for 1 hour, the reaction solution is removed by suction filtration, and the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane respectively, 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, 100ml of 20% hexahydropyridine/dimethylformamide solution is added, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, 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 respectively, 80ml of each time, and the solvent is removed by suction filtration.

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

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

Furthermore, the invention provides the use of the p-tau181 epitope peptide or the p-tau181 antigen of the invention in the preparation of a kit for detecting p-tau181 protein in Alzheimer's patients.

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

Among the steps of the above-described preparation method of the present invention, although the specific steps described therein are distinguished in some details or language description from the steps described in the preparation examples of the following detailed description, the above-described method steps can be fully summarized by one skilled in the art based on the detailed disclosure of the present invention as a whole.

Any of the embodiments of any of the aspects of the invention may be combined with other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.

All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.

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 the antigen (immunogen) prepared by the human p-tau181 epitope peptide can produce monoclonal antibodies and polyclonal antibodies with high specificity by immunizing animals.

2. The p-tau181 monoclonal antibodies and polyclonal antibodies prepared by the invention are capable of highly specific binding 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 disease course, thus providing basis for early clinical diagnosis of AD and making important contribution for patients to timely treatment.

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. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.

The following further explains or illustrates the contents of the present invention by means of examples: unless otherwise indicated, the solutions described below are aqueous solutions; when referring to percentages, the percentages of the mixture formulated with liquid/liquid are all volume/volume percentages, the percentages of the mixture formulated with solid/liquid are all mass/volume percentages, and the percentages of the mixture formulated with solid/solid are all mass/mass percentages.

Some of the following typical raw materials used by CN111197040A (application No. 2020100721960, yi-Tong biological) are The materials can also be used in the present invention if necessary:

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

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

NMP (azamethylpyrrolidone, available from Sigma-Aldrich),

DCM (dichloromethane, available from Zhongyuan chemical company),

MeOH (methanol, available from chinese chemical company),

piperidine (Piperidine, available from Sigma-Aldrich),

DMAP (dimethylaminopyridine, available 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, available from Sigma-Aldrich),

thioanisole, available from Weber chemical Co., guangzhou,

crystalline phenol, purchased from national pharmaceutical group chemical reagent company,

acetonitrile, available from national pharmaceutical group chemical company, inc.

The raw materials not mentioned are also readily available from the market.

CN111197040a (application No. 2020100721960, yitong biological) uses some of the following typical instruments If necessary, can also be used in the present invention:

an automatic polypeptide synthesizer, model 431A, available from ABI company,

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

high performance liquid chromatographs, waters600, available from Waters company,

freeze dryer, model VFD-2000, available from Beijing Bo Yikang.

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

Example 1: preparation of p-tau181 epitope peptide

The p-tau181 epitope peptide can be synthesized by a solid phase method. The main ideas of solid phase peptide synthesis are: the carboxyl of the carboxyl terminal amino acid of the peptide chain to be synthesized is connected with an insoluble high molecular compound (resin) in a covalent bond form, and then the amino acid combined on the solid phase carrier is taken as an amino component, and the amino protecting group is removed and reacts with excessive activated carboxyl component to grow the peptide chain. Such a step may be repeated a number of times to finally reach the desired length of the synthesized peptide chain. An exemplary synthesis is shown in FIG. 1. The target polypeptide is obtained after deprotection in step (5) of the synthesis 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 were purchased from Gill Biochemical company, agkis Ding Shiji, HBTU, HOBT, DIEA, and other reagents were readily available commercially, such as described in example 1 of CN111197040A (application No. 2020100721960, yitong organism), unless otherwise indicated.

This example 1 uses classical methods to prepare p-tau181 epitope peptides of the following sequences:

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

or is marked as: AKTPPAPK-pTPPSSG wherein (p) Thr is phosphorylated threonine.

In each of the following preparation steps, amino acid (Fmoc-AA-OH) was used: peptide coupling agent HBTU: amide bond formation promoter HOBT: the molar ratio of the organic base DIEA is 1:1:1:4, a step of;

the amount of solvent used is empirically and specifically controlled, for example, 50 to 150ml of solvent is used in the washing, uncapping and coupling reactions of 10g of resin, and particularly, as little solvent as possible is used at each washing. 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 this step, 10g and 30mmol (8.91 g) Fmoc-Gly-OH of Rink Amide-MBHA resin (0.79 mmol/g) were taken for dosing.

The Rink Amide-MBHA resin was placed in a reactor, 80ml of dichloromethane was added, shaken and soaked for 60 minutes, and the solvent was removed by suction filtration with two alternating washes of dichloromethane, methanol, dimethylformamide, each 80 ml.

To the resin obtained in the previous step, 20% hexahydropyridine/dimethylformamide solution (100 ml) was added, and the uncapping reaction was carried out at room temperature with shaking for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After removal of the solvent by suction filtration, the resin was washed twice alternately with dimethylformamide, methanol and dichloromethane, each 80ml, and the solvent was removed by suction filtration. The ninhydrin test should be blue, and if not, the procedure should be repeated. (the ninhydrin test is also referred to as KT test, and a trace amount of 1 to 2mg of the resin is used for the test, which will be described later). Fmoc-Gly-OH, HBTU, HOBT in the prescribed ratio was dissolved in dimethylformamide (100 ml), DIEA was added thereto, and the mixture was stirred uniformly and transferred into a reactor containing the above-mentioned treated resin, and reacted at room temperature for 1 hour with shaking. Removing the reaction liquid by suction filtration, alternately cleaning the resin twice with dimethylformamide, methanol and dichloromethane respectively, and removing the solvent by suction filtration, wherein 80ml of each resin is used for each time; KT detection should appear yellow, and if not, the reaction time is prolonged. The title resin obtained in step 1, fmoc-Gly-resin, was found to have a coupling ratio of 0.91. The resin was used in its entirety for 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 Fmoc-Ser (tBu) -OH for feeding;

to the resin obtained in the previous step, 20% hexahydropyridine/dimethylformamide solution (100 ml) was added, and the uncapping reaction was carried out at room temperature with shaking for 60 minutes to remove the Fmoc protecting group at the nitrogen end. After removal of the solvent by suction filtration, the resin was washed twice alternately with dimethylformamide, methanol and dichloromethane, each 80ml, and the solvent was removed by suction filtration. The ninhydrin test should be blue, and if not, the procedure should be repeated. Fmoc-protected amino acid, HBTU and HOBT in the prescribed proportions were added to dimethylformamide (100 ml) to dissolve, DIEA was added thereto, and the mixture was stirred and transferred into a reactor containing the above-mentioned treated resin, and reacted at room temperature with shaking for 1 hour. Removing the reaction liquid by suction filtration, alternately cleaning the resin twice with dimethylformamide, methanol and dichloromethane respectively, and removing the solvent by suction filtration, wherein 80ml of each resin is used for each time; KT detection should appear yellow, and if not, the reaction time is prolonged. The title resin obtained in this step had a coupling ratio of 0.87.

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

The resin obtained in the previous step and 30mmol of Fmoc-Ser (tBu) -OH charge were taken, and the procedure of step 2 was referred to, to obtain the title resin, whose coupling ratio was examined to be 0.89.

Step 4: preparation of Fmoc-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.90.

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

The resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.91.

Step 6: preparation of Fmoc-Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Thr (HPO 3 Bzl) -OH charge were taken, and the procedure of step 2 was referred to, to give the title resin, which was examined for coupling ratio of 0.87.

Step 7: preparation of Fmoc-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Lys (Boc) -OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.90.

Step 8: preparation of Fmoc-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.88.

Step 9: preparation of Fmoc-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Ala-OH charge were taken, and the procedure of step 2 was referred to give the title resin, which was examined for coupling ratio of 0.91.

Step 10: preparation of Fmoc-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

Step 11: preparation of Fmoc-Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Pro-OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.89.

Step 12: preparation of Fmoc-Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Thr (Trt) -OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.89.

Step 13: preparation of Fmoc-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Lys (Boc) -OH charge were taken, and the procedure of step 2 was referred to obtain the title resin, which was examined for coupling ratio of 0.88.

Step 14: preparation of Fmoc-Ala-Lys (Boc) -Thr (Trt) -Pro-Pro-Ala-Pro-Lys (Boc) -Thr (HPO 3 Bzl) -Pro-Pro-Ser (tBu) -Ser (tBu) -Gly-resin

The resin obtained in the previous step and 30mmol Fmoc-Ala-OH charge were taken, and the procedure of step 2 was referred to give the title resin, which was examined for coupling ratio of 0.90.

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

Step 15: cleavage of peptide chains

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

Step 16: separation and purification

(1) Dissolving the crude peptide obtained in the last step in 70% acetonitrile (containing 0.1% trifluoroacetic acid), eluting with ion exchange chromatography system Shodex IEC SP-420N (Beijing spectral pun) with 70% acetonitrile (containing 0.1% trifluoroacetic acid), and collecting main peak fraction of peptide;

chromatographic column: c8 10X 100mm, waters, USA;

chromatograph: liquid chromatography for YMC industrial preparation;

mobile phase: mobile phase a was 0.1% tfa (trifluoroacetic acid) in water and mobile phase B was 70% acetonitrile with 0.1% tfa (trifluoroacetic acid) added, with an elution gradient of 10% B-60% B during 0-50 minutes;

flow rate: 4 ml/min;

detection wavelength: 219nm;

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

The total yield was calculated to be 26.4% for 17 steps of starting from 10g of resin from step 1 and feeding to the final product peptide concentrate, which was 98.2% pure, as determined by [ HPLC purity assay ].

[ HPLC purity assay ]:

the purity of the p-tau181 epitope peptide is determined by using HPLC, and the main determination conditions are as follows:

Chromatographic column: c18, 4.6X105 mm, waters company,

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

mobile phase: aqueous 0.1% TFA as mobile phase A, acetonitrile containing 0.1% TFA as mobile phase B, elution gradient 0-60% B over 30 minutes,

flow rate: 1 ml/min of the total volume of the solution,

detection wavelength: 219nm.

Mass spectrometry of polypeptide molecular weight:

the method uses mass spectrometry 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, available from Sigma-Aldrich), HCCA (α -cyano-4-hydroxycinnamic acid, available from Sigma-Aldrich), acetonitrile (available from national pharmaceutical systems chemical company, inc);

(2) Instrument: matrix assisted laser desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS (model: REFLEX III, bruker Corp., germany);

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

(4) Instrument detection conditions: a reflection detection mode; the length of the flying tube is 3m; nitrogen laser: wavelength 337nm, acceleration voltage 20KV; the reflected voltage is 23KV;

(5) The operation steps are as follows: 10. Mu.L of purified polypeptide sample (50 mg/ml concentration dissolved in acetonitrile) was mixed with 10. Mu.L of matrix solution, 1. Mu.L of the mixture was spotted on the sample target, and the mixture was fed into an ion source for detection.

Results: the molecular weight 1431.47 of the p-tau181 epitope peptide obtained in the embodiment is consistent with the theoretical molecular weight 1431.54, and the synthetic polypeptide is proved to be a target product.

[ polypeptide sequencing method ]:

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) Principle of: 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: phenylisothiocyanates react with the N-terminal residues of proteins and polypeptides to form Phenylsulfamoyl (PTC) derivatives, i.e., PTC-peptides;

cyclized cleavage: PTC-peptide cyclized cleavage;

conversion: the thiazolyl-ketotifen (ATZ) is converted into benzisothiourea amino acid (PTH-amino acid), the peptide which is left in solution and reduced by one amino acid residue is repeatedly subjected to the reaction process, and the whole sequencing process is automatically performed by a sequencer;

(2) Instrument: an AbI company 491 U.S.A.protein/polypeptide N-terminal amino acid sequence analyzer;

(3) Reagent raw materials: phenyl isothiocyanate PITC (Sigma-Aldrich), n-heptane (national pharmaceutical systems chemical company, inc.), trimethylamine TMA aqueous solution (national pharmaceutical systems chemical company, TFA, sigma-Aldrich), trifluoroacetic acid (TFA, sigma-Aldrich), ethyl acetate (national pharmaceutical systems chemical company, inc.), chlorobutane (Sigma-Aldrich), acetonitrile (national pharmaceutical systems chemical company, inc.);

(4) And (3) measuring: according to the instruction of the instrument.

As a result, the p-tau181 epitope peptide obtained in example 1 was identified as having the sequence:

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

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

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

This example illustrates the preparation of p-tau181 antigen by linking the p-tau181 epitope peptide obtained in example 11 to a carrier protein as follows:

the p-tau181 antigen epitope peptide is connected with carrier protein KLH (keyhole limpet hemocyanin, keyhole limpet haemocyanin) by BDB (Bis-diazotizedbenzidine dichloride, double-nitrided benzidine dichloride) method to prepare the p-tau181 antigen;

10.0mg of p-tau181 epitope peptide is taken and dissolved in 1ml of 0.1M PBS buffer (pH 7.4); 10mg of KLH was dissolved in 20ml of 0.2M borate buffer (pH 9.0); then mixing the two, cooling to 0 ℃, taking 110 mu L of BDBCl2, reacting for 1.5 hours at room temperature, sub-packaging after dialysis overnight (12-15 hours), and preserving at-20 ℃ (freeze drying can be carried out if necessary), thereby obtaining the p-tau181 epitope peptide-KLH coupling protein, namely the p-tau181 antigen.

In this example, the formulation of the PBS buffer is: 0.2mol/L Na2HPO4 81ml and 0.2mol/L NaH2PO4 19 ml;

The borate buffer solution comprises the following components: 80ml of borax 0.05mol/L and 20ml of boric acid 0.2 mol/L.

[ Bradford method ]: diluting the coupled antigen to different concentrations (according to the pre-test such that the absorbance of a595 falls within the absorbance range of the standard curve series concentration); taking 0.1ml of Bradford dye solution added with 5ml of Bradford dye solution into each sample and KLH standard protein solution, reacting for 5-30min at room temperature, and measuring A595 value; the CHI3L1 epitope peptide is a small peptide, and the concentration of the protein is increased when the small peptide is connected to KLH to form a part of the protein; the excess of A595 was the concentration of CHI3L1 epitope peptide, from which the conjugated protein binding ratio (peptide/KLH value) was calculated. In the above method, bradford dye liquor: 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 (10 mM, pH 7.4) and diluted with PBS to give a series of solutions with final concentrations of 50, 100, 200, 300, 400, 500, 600g/L, respectively. The [ Bradford method ] of the present invention can be used to determine the binding ratio of coupled proteins. Knowing that the relative molecular weight of KLH is not easily determinable, the present invention uses the Bradford method to determine and calculate the binding ratio of the two coupled proteins; the Bradford method (Coomassie Brilliant blue method) is a classical protein quantification method in the field, the method is established in 1976, the reagent preparation is simple, the operation is simple and quick, the reaction is very sensitive, the sensitivity is 4 times higher than that of the Lowry method, the content of microgram-level protein can be measured, the concentration range of the measured protein is 0-1000 mug/mL, the minimum measurable protein is 2.5 mug/mL, and the method is a commonly used rapid trace protein measurement method; the principle of the Bradford method is that coomassie brilliant blue G-250 has two different colors of red and blue, and can be prepared into a light red solution under the conditions of ethanol with a certain concentration and acidity, and the light red solution is combined with protein to form a blue compound, 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 p-tau181 epitope peptide-KLH conjugate protein binding ratio obtained in this example was measured using the method [ Bradford ] = 4.84.

Example 21a: preparation of p-tau181 antigen

Referring to example 21, the p-tau181 epitope peptide-KLH conjugated protein was prepared by changing the borate buffer used to an equal volume of carbonate buffer, and was the 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 the volume is fixed to 1000ml by water, thus obtaining a carbonate buffer with the concentration of 50mM and the pH value of 9.6. The binding ratio of the conjugated protein of the p-tau181 epitope peptide-KLH obtained in this example was measured using the method [ Bradford ] = 9.11.

Example 21b: preparation of p-tau181 antigen

Referring to example 21, the p-tau181 epitope peptide-KLH conjugated protein was prepared by changing the borate buffer used to an equal volume of carbonate buffer, and was the 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 are weighed, dissolved in water and the volume is fixed to 1000ml by water to obtain a carbonate buffer with the concentration of 50mM and the pH value of 9.6. The binding ratio of the conjugated protein of the p-tau181 epitope peptide-KLH obtained in this example was measured using the method [ Bradford ] = 4.64.

Example 21c: preparation of p-tau181 antigen

Referring to example 21, the p-tau181 epitope peptide-KLH conjugated protein was prepared by changing the borate buffer used to an equal volume of carbonate buffer, and was the 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 are weighed, dissolved in water and fixed to volume to 1000ml with water to obtain a carbonate buffer with a concentration of 50mM and pH 9.6. The binding ratio of the conjugated protein of the p-tau181 epitope peptide-KLH obtained in this example was measured using the method [ Bradford ] = 4.89.

From the results of example 21 and examples 21a, 21b, and 21c described above, it was unexpectedly found that the use of a 50mM, pH9.6 carbonate buffer comprising ammonium pyruvate and sodium nitrite significantly increased the conjugated protein binding ratio of peptide/KLH.

Example 22: preparation of monoclonal and polyclonal antibodies

This example uses the p-tau181 antigen obtained in example 21 to immunize animals, thereby producing specific monoclonal and polyclonal antibodies using the p-tau181 antigen.

1. Immunization of animals human p-tau181 monoclonal antibodies were prepared:

1.1. after thoroughly mixing the p-tau181 antigen (immunogen) prepared in example 21 above with Freund's complete adjuvant (purchased from Shanghai Source Polymer Co.), balb/c mice were immunized, 60. Mu.g antigen/mouse, and injected subcutaneously at multiple points; serum titers were measured after 4 weeks, and mice with good immunoreactivity were selected for re-immunization: taking antigen and fully mixing with equal volume of Freund's incomplete adjuvant, performing subcutaneous multipoint injection with the antigen dosage of 35 mug/dose, performing boosting for 6 times, performing continuous boosting twice before fusion, then taking spleen cells and Sp2/0 myeloma cells to perform fusion with 50% PEG (MW 4000) (purchased from the original chemical company) mediated by a conventional method, and selecting and culturing by using HAT conditioned medium (purchased from Sigma-Aldrich company); placing the fused cells into a CO2 incubator to culture for 12-13 days at 37 ℃, and allowing larger cell clones to appear in the holes; screening with an indirect ELISA was started 13 days; the primary screening positive wells were subjected to 4 cloning cultures (even if the screened cells were propagated by mass division) by limiting dilution, followed by cell expansion, cryopreservation and ascites preparation. Balb/c mice were purchased from university of Fujian medical science (SYXK (Min) 2020-0005).

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

1.3. Determination of antibody titers: the potency of human p-tau181 monoclonal antibodies prepared using the p-tau181 antigen was determined by indirect ELISA, and showed that the potency of the monoclonal antibodies reached 1:32000.

2. immunization of animals human p-tau181 polyclonal antibodies were prepared:

2.1. selecting New Zealand white rabbits with the age of three months and the weight of about 2kg as immunized animals; in basic immunization, 2mg of the p-tau181 antigen prepared in example 21 above was mixed with Freund's complete adjuvant-fully emulsified and then injected subcutaneously in multiple spots on the back of rabbits; once every 4 weeks, the antigen was injected subcutaneously at 100 μg/back multiple spot after sufficient emulsification with incomplete freund's adjuvant. Carotid artery bleeding was performed on day 10 after last booster immunization, and serum was isolated. New Zealand white rabbits were purchased from the university of Fujian medical science (SYXK (Min) 2020-0004).

2.2. Determination of antibody titers:

the potency of human p-tau181 polyclonal antibodies prepared using human p-tau181 antigen was determined by indirect ELISA, and the results showed that the antibody potency reached 1:29200.

2.3. blood collection and serum separation: carotid cannulation was used to remove blood and isolate serum.

3. And (3) separating and purifying the antibody:

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

4. Antibody cryopreservation:

the antibody is frozen and dried after being packaged and stored at low temperature.

Implementation of the embodimentsExample 22a: preparation of monoclonal and polyclonal antibodies

This example uses the p-tau181 antigen obtained in example 21a to immunize animals, thereby using the p-tau181 antigen to generate specific monoclonal and polyclonal antibodies

1. Immunization of animals human p-tau181 monoclonal antibodies were prepared:

1.1. after thoroughly mixing the p-tau181 antigen (immunogen) prepared in example 21a above with Freund's complete adjuvant (purchased from Shanghai Source Polymer Co.), balb/c mice were immunized with 35. Mu.g antigen/mouse, and injected subcutaneously at multiple points; serum titers were measured after 4 weeks, and mice with good immunoreactivity were selected for re-immunization: taking antigen and fully mixing with equal volume of Freund's incomplete adjuvant, performing subcutaneous multipoint injection with the antigen dosage of 20 mug/dose, performing boosting for 6 times, performing continuous boosting twice before fusion, then taking spleen cells and Sp2/0 myeloma cells to perform fusion with 50% PEG (MW 4000) (purchased from the original chemical company) mediated by a conventional method, and selecting and culturing by using HAT conditioned medium (purchased from Sigma-Aldrich company); placing the fused cells into a CO2 incubator to culture for 12-13 days at 37 ℃, and allowing larger cell clones to appear in the holes; screening with an indirect ELISA was started 13 days; the primary screening positive wells were subjected to 4 cloning cultures (even if the screened cells were propagated by mass division) by limiting dilution, followed by cell expansion, cryopreservation and ascites preparation. Balb/c mice were purchased from university of Fujian medical science (SYXK (Min) 2020-0005).

1.3. Determination of antibody titers: the potency of human p-tau181 monoclonal antibodies prepared using the p-tau181 antigen was determined by indirect ELISA, and showed that the potency of the monoclonal antibodies reached 1:29800.

2. immunization of animals human p-tau181 polyclonal antibodies were prepared:

2.1. selecting New Zealand white rabbits with the age of three months and the weight of about 2kg as immunized animals; in basic immunization, 2mg of the p-tau181 antigen prepared in example 21a above was mixed with Freund's complete adjuvant-fully emulsified and then injected subcutaneously in multiple spots on the back of rabbits; once every 4 weeks, the antigen was injected subcutaneously at 60 μg/back multiple spot after sufficient emulsification with incomplete freund's adjuvant. Carotid artery bleeding was performed on day 10 after last booster immunization, and serum was isolated. New Zealand white rabbits were purchased from the university of Fujian medical science (SYXK (Min) 2020-0004).

2.2. Determination of antibody titers: the potency of human p-tau181 polyclonal antibodies prepared using human p-tau181 antigen was determined by indirect ELISA, and the results showed that the antibody potency reached 1:31600.

3. And (3) separating and purifying the antibody:

4. Antibody cryopreservation:the antibody is frozen and dried after being packaged and stored at low temperature.

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

ELISA is used for detection, p-tau181, abeta 1-40 and Abeta 1-42 proteins are respectively used as detection antigens to coat ELISA plates, the p-tau181 monoclonal antibody prepared in ELISA detection example 22 specifically reacts with the ELISA plates, normal BALB/c mouse serum is used as negative control, and PBS solution is used as blank control; results: the P-tau181 monoclonal antibody reacted positively with P-tau181 (P/N > 2.1) and negatively with the aβ1-40 and aβ1-42 proteins (P/N < 2.1), indicating that this P-tau181 monoclonal antibody has specificity.

Example 3a: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, with normal BALB/c mouse serum as negative control, and PBS as blank control; results: p-tau181 monoclonal antibodies react positively with P-tau181 (P/N)>2.1 While the reaction with the Abeta 1-40 and Abeta 1-42 proteins is negative (P/N)<2.1 Indicating that this 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 using the same method as described above for identifying monoclonal antibody specificity; the results show that: the P-tau181 polyclonal antibody reacted positively with P-tau181 (P/N > 2.1) and negatively with the Aβ1-40 and Aβ1-42 proteins (P/N < 2.1), indicating that the P-tau181 polyclonal antibody has specificity.

Example 4a:the p-tau181 polyclonal antibody prepared in example 22a was identified by the method of example 4; the results show that: p-tau181 polyclonal antibodies reacted positively with P-tau181 (P/N)>2.1 While the reaction with the Abeta 1-40 and Abeta 1-42 proteins is negative (P/N)<2.1 Indicating that the p-tau181 polyclonal antibody has specificity.

Example 5: exemplary application:

in this example, the human p-tau181 polyclonal antibody obtained in example 22a was used as the binding antibody in the present kit; the p-tau181 mab prepared in example 22a was used as a coating antibody; next, referring to various conditions and operation modes of example 5 of CN103665113a, the concentration of p-tau181 protein was detected and calculated for plasma from 36 healthy groups (age 71 to 79 years), plasma from 32 early patients with AD (age 74 to 82 years, 19.ltoreq.simple mental state checkup (MMSE) value.ltoreq.27 minutes, clinical dementia level scale (CDR) value=0.8 to 1.3 minutes), plasma from 35 patients with AD late patients (age 73 to 80 years, MMSE <19 minutes, cdr.gtoreq.2 minutes), which are all samples of definitive/therapeutic patients and volunteers provided by a certain hospital, and the results were: healthy group plasma p-tau181 protein concentration=9.2±2.7ng/L (n=36), early stage AD patient plasma p-tau181 protein concentration=13.3±4.5ng/L (n=32), late stage AD patient plasma p-tau181 protein concentration=21.1±4.1ng/L (n=35); these results indicate that the human p-tau181 epitope peptide prepared by the present invention, the antigen prepared therefrom and the further prepared antibody are effective in constructing a p-tau181 in vitro diagnostic kit.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.

Claims

1. A method of making a p-tau181 antigen comprising the steps of: 10.0mg of human p-tau181 epitope peptide is taken and dissolved in 1ml of 0.1M PBS buffer of pH 7.4; 10mg of KLH was dissolved in 20ml of carbonate buffer; then mixing the two, cooling to 0 ℃, adding 110 mu L of BDBCl ₂ Reacting for 1.5 hours at room temperature, and dialyzing for 12-15 hours to obtain the product; wherein:

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 formula of the PBS buffer solution is as follows: na of 0.2mol/L ₂ HPO ₄ 81ml of NaH added with 0.2mol/L ₂ PO ₄ 19ml of the mixture;

the carbonate buffer is formulated as follows: weigh 2.94g NaHCO ₃ 1.58g of Na ₂ CO ₃ 0.25g of ammonium pyruvate and 0.12g of sodium nitrite are dissolved in water, and the volume is fixed to 1000ml by water, so that a carbonate buffer with a concentration of 50mM and a pH of 9.6 is obtained.

2. The method according to claim 1, wherein the human p-tau181 epitope peptide is prepared by:

Step 1: preparation of Fmoc-Gly-resin

step 15: cleavage of peptide chains

Transferring the resin obtained in the previous step to a round bottom flask, adding precooled cutting fluid, stirring at room temperature for reaction, filtering to separate filtrate, washing the resin with trifluoroacetic acid, mixing the filtrate and the washing fluid, adding frozen diethyl ether for precipitation, and filtering to obtain a precipitate, namely a pentadecapeptide crude product;

3. The method of claim 2, wherein the pre-chilled cutting fluid is a mixture of 95% trifluoroacetic acid, 2% tis, 2% edt, 1% water.

4. The method according to claim 2, wherein the separation and purification of step 16 is performed as follows:

(1) Dissolving the crude peptide obtained in the last step in 70% acetonitrile containing 0.1% trifluoroacetic acid, eluting with ion exchange chromatography system Shodex IEC SP-420N with 70% acetonitrile containing 0.1% trifluoroacetic acid as solvent, collecting main peak fraction of peptide,

chromatographic column: c8 10X 100mm of the diameter of the tube,

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

Flow rate: the volume of the solution was 4 ml/min,

detection wavelength: at a wavelength of 219nm,

5. The method according to claim 2, wherein in step 1 to step 14 of preparing the human p-tau181 epitope peptide, the Fmoc protecting amino acid used in each step: HBTU: HOBT: DIEA molar ratio = 1:1:1:4.

6. the method according to claim 2, wherein in step 1 of preparing the human p-tau181 epitope peptide, 80ml of methylene chloride is added, and the mixture is shaken and soaked for 60 minutes, and washed with methylene chloride, methanol, dimethylformamide alternately twice, 80ml each, and suction-filtered to remove the solvent.

7. The method according to claim 2, wherein in step 1 of preparing the human p-tau181 epitope peptide, 100ml of 20% hexahydropyridine/dimethylformamide solution is added to the treated resin, the uncapping reaction is carried out for 60 minutes with shaking at room temperature, the nitrogen-terminal Fmoc protecting group is removed, the solvent is removed by suction filtration, the resin is alternately washed with dimethylformamide, methanol and dichloromethane twice each of 80ml, and the solvent is removed by suction filtration.

8. The method according to claim 2, wherein in step 1 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Gly-OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred and transferred into a reactor containing the treated resin, the reaction solution is removed by suction filtration for 1 hour under shaking at room temperature, and the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, respectively, 80ml each time, and the solvent is removed by suction filtration.

9. The method according to claim 2, wherein in step 2 of preparing the human p-tau181 epitope peptide, 100ml of 20% hexahydropyridine/dimethylformamide solution is added, the uncapping reaction is carried out for 60 minutes at room temperature by shaking, the nitrogen-terminal Fmoc protecting group is removed, after the solvent is removed by suction filtration, the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, 80ml each, and the solvent is removed by suction filtration.

10. The method according to claim 2, wherein in step 2 of preparing the human p-tau181 epitope peptide, 30mmol of Fmoc-Ser (tBu) -OH, HBTU, HOBT is added to 100ml of dimethylformamide to dissolve, DIEA is added, stirred, transferred into a reactor containing the above treated resin, reacted for 1 hour at room temperature with shaking, the reaction solution is removed by suction filtration, and the resin is alternately washed twice with dimethylformamide, methanol and dichloromethane, respectively, 80ml each time, and the solvent is removed by suction filtration.