Methods for antibody identification, polypeptide compositions, kits, and methods of making and using the same

Abstract

The application provides an antibody identification method, a polypeptide composition, a kit and a preparation method and application thereof, and relates to the technical field of biotechnology. The antibody identification method mainly comprises identifying whether an antibody specifically recognizes a 231st phosphorylation site of Tau protein, reacting a to-be-detected antibody with four identification polypeptides respectively, and determining whether the to-be-identified antibody can specifically recognize the 231st phosphorylation site of Tau protein according to a detection result. The four identification polypeptides are based on a basic polypeptide, and the specificity of the to-be-identified antibody to the target phosphorylation site is determined through the phosphorylation difference between the 231st phosphorylation site and potential phosphorylation sites of Tau protein, so that the problem of a complex antibody screening and identification method for recognizing the 231st phosphorylation site of Tau protein in the prior art is solved.

Description

[0001] The present divisional application is based on the Chinese Patent Application No. 2022109111199, filed on July 29, 2022, entitled "Antibody identification method, polypeptide composition, kit and preparation method and application thereof". TECHNICAL FIELD

[0002] The present application relates to the technical field of biotechnology, in particular to an antibody identification method, a polypeptide composition, a kit and a preparation method and application thereof. BACKGROUND

[0003] Neurofibrillary tangles formed by abnormally hyperphosphorylated Tau protein (p-Tau) in nerve cells are one of the pathological features of Alzheimer's disease (AD). Immunological detection of p-Tau is a widely used method for the auxiliary diagnosis of AD, and the specificity of the p-Tau antibody to the phosphorylation site is crucial.

[0004] In the current screening and identification method of antibodies that recognize phosphorylation sites, polypeptides phosphorylated at the site and their corresponding polypeptides that are not phosphorylated at the site are synthesized according to the potential phosphorylation sites, and the two types of polypeptides are reacted with the antibody to be identified, and then the concentration of the polypeptide with cross-reactivity is reduced to obtain the polypeptide with the strongest reactivity, which is determined as the main phosphorylation site recognized by the antibody. Therefore, how to simplify the screening and identification method of antibodies for phosphorylation sites is a problem that needs to be solved at present.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The first object of the present application is to provide a polypeptide composition that can be used to identify whether an antibody can specifically recognize the 231st phosphorylation site of Tau protein, thereby alleviating the technical problem of complex process of screening antibodies that recognize the 231st Tau protein in the prior art.

[0007] The second object of the present application is to provide a method for identifying whether an antibody specifically recognizes the 231st phosphorylation site of Tau protein using the above-mentioned polypeptide composition.

[0008] The third object of the present application is to provide a kit comprising the above-mentioned polypeptide composition, the application of the kit and the preparation method of the kit.

[0009] To solve the above technical problems, the present application adopts the following technical solutions:

[0010] According to one aspect of the present application, the present application provides a polypeptide composition for antibody identification, the identification including identifying whether the antibody specifically recognizes the phosphorylation site at position 231 of Tau protein, the polypeptide composition including four identification polypeptides, the four identification polypeptides being respectively:

[0011] The first identification polypeptide is a polypeptide in which only the phosphorylation site at position 231 of Tau protein in the basic polypeptide is phosphorylated;

[0012] The second identification polypeptide is a polypeptide in which no site in the basic polypeptide is phosphorylated;

[0013] The third identification polypeptide is a polypeptide in which both the phosphorylation site at position 231 of Tau protein and the potential phosphorylation site in the basic polypeptide are phosphorylated;

[0014] The fourth identification polypeptide is a polypeptide in which the phosphorylation site at position 231 of Tau protein is not phosphorylated, while the potential phosphorylation site in the basic polypeptide is phosphorylated;

[0015] The basic polypeptide is a polypeptide starting from the 5th to 7th amino acid residues adjacent to the nitrogen end side of position 231 of Tau protein, ending from the 5th to 7th amino acid residues adjacent to the carbon end side of position 231 of Tau protein, and the amino acid residues therebetween;

[0016] The potential phosphorylation site is: the first threonine residue, serine residue or tyrosine residue adjacent to the nitrogen end side and the carbon end side of position 231 of Tau protein, respectively.

[0017] According to another aspect of the present application, the present application also provides an antibody identification method, wherein the identification includes identifying whether the antibody specifically recognizes the phosphorylation site at position 231 of Tau protein; the antibody identification method includes: reacting the antibody to be identified with the four identification polypeptides respectively, and detecting that the results simultaneously satisfy (I) to (III), then determining that the antibody to be identified is an antibody specifically recognizing the phosphorylation site at position 231 of Tau protein:

[0018] (I) the antibody to be identified reacts positively with the first identification polypeptide and the third identification polypeptide;

[0019] (II) the antibody to be identified reacts negatively with the second identification polypeptide;

[0020] (III) the antibody to be identified reacts negatively or weakly positively with the fourth identification polypeptide;

[0021] The four identification polypeptides are the polypeptide composition described above.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The polypeptide composition for antibody identification provided by the present application can effectively determine whether the antibody to be identified can specifically recognize the 231st phosphorylation site of Tau protein, and the antibody specifically recognizing the target phosphorylation site should have the following characteristics:

[0024] (1) a polypeptide recognizing phosphorylation of the 231st amino acid of Tau protein; (2) a polypeptide not recognizing unphosphorylation of the 231st amino acid of Tau protein, (3) phosphorylation of T (threonine residue) or S (serine residue) or Y (tyrosine residue) adjacent to the 231st amino acid of Tau protein does not affect the sequence recognizing phosphorylation of the 231st amino acid of Tau protein; (4) no cross reaction with adjacent phosphorylated T, S, Y, or weak cross reaction with adjacent phosphorylated T, S, Y. The antibody recognizing the sequence meeting this characteristic should be between the 231st amino acid of Tau protein and the adjacent potential phosphorylated amino acid (T or S or Y).

[0025] The four identification polypeptides designed according to the above rules can effectively determine whether the antibody to be identified can specifically recognize the target phosphorylation site, and the four polypeptides can screen out the antibody specifically recognizing the 231st phosphorylation site of Tau protein through experiments. Due to the presence of the fourth identification polypeptide, the polypeptide composition provided by the present application can also screen out the antibody that may have non-specific reaction with other phosphorylation sites of Tau protein, so as to exclude it. The fourth identification sequence is essential in the method for identifying whether the antibody specifically recognizes the target phosphorylation site.

[0026] The antibody identification method provided by the present application is simple to operate, only four identification polypeptides need to be synthesized, and then the detection results of the four identification polypeptides after reacting with the antibody to be detected are used to determine the specific recognition ability of the antibody to be detected to the 231st phosphorylation site of Tau protein. The antibody identification method is simple and effective. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0028] Generally, the nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present application are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0029] According to one aspect of the present application, the present application provides a polypeptide composition for antibody identification, the identification comprising identifying whether the antibody specifically recognizes the phosphorylation site at position 231 of Tau protein, the polypeptide composition comprising four identification polypeptides, the four identification polypeptides being respectively:

[0030] The first identification polypeptide is a polypeptide in which only the phosphorylation site at position 231 of Tau protein in the basic polypeptide is phosphorylated;

[0031] The second identification polypeptide is a polypeptide in which no site in the basic polypeptide is phosphorylated;

[0032] The third identification polypeptide is a polypeptide in which both the phosphorylation site at position 231 of Tau protein and the potential phosphorylation site in the basic polypeptide are phosphorylated;

[0033] The fourth identification polypeptide is a polypeptide in which the phosphorylation site at position 231 of Tau protein is not phosphorylated, while the potential phosphorylation site in the basic polypeptide is phosphorylated;

[0034] The basic polypeptide is a polypeptide starting from the 5th to 7th amino acid residues adjacent to the nitrogen end side of position 231 of Tau protein, ending from the 5th to 7th amino acid residues adjacent to the carbon end side, and the amino acid residues therebetween; that is, a polypeptide starting from position 224 to 226 and ending from position 236 to 238 of Tau protein. The basic polypeptide in the present application can be understood as having a "arm" of 5 to 7 amino acid residues at the nitrogen end and the carbon end, respectively, with position 231 of Tau protein as the center.

[0035] The potential phosphorylation site is: the first threonine residue, serine residue or tyrosine residue adjacent to position 231 of Tau protein at the nitrogen end side and the carbon end side, respectively.

[0036] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the 7th amino acid residue adjacent to the N-terminal side of position 231 of Tau protein as the start, the 7th amino acid residue adjacent to the C-terminal side of position 231 of Tau protein as the end, and the amino acid residues therebetween; i.e., a polypeptide with the start at position 224 and the end at position 238 of Tau protein.

[0037] In some alternative embodiments, the basic polypeptide is KKVAVVRTPPKSPSS (Seq_14), and the four identification polypeptides consist of:

[0038] The amino acid sequence of the first identification polypeptide is shown in Seq_13: KKVAVVR{pTHR}PPKSPSS.

[0039] The amino acid sequence of the second identification polypeptide is shown in Seq_14: KKVAVVRTPPKSPSS.

[0040] The amino acid sequence of the third identification polypeptide is shown in Seq_15: KKVAVVR{pTHR}PPK{pSER}PSS.

[0041] The amino acid sequence of the fourth identification polypeptide is shown in Seq_16: KKVAVVRTPPK{pSER}PSS.

[0042] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the 6th amino acid residue adjacent to the N-terminal side of position 231 of Tau protein as the start, the 6th amino acid residue adjacent to the C-terminal side of position 231 of Tau protein as the end, and the amino acid residues therebetween; i.e., a polypeptide with the start at position 225 and the end at position 237 of Tau protein.

[0043] In some alternative embodiments, the basic polypeptide is KVAVVRTPPKSPS (Seq_18), and the four identification polypeptides consist of:

[0044] The amino acid sequence of the first identification polypeptide is shown in Seq_17: KVAVVR{pTHR}PPKSPS.

[0045] The amino acid sequence of the second identification polypeptide is shown in Seq_18: KVAVVRTPPKSPS.

[0046] The amino acid sequence of the third identification polypeptide is shown in Seq_19: KVAVVR{pTHR}PPK{pSER}PS.

[0047] The amino acid sequence of the fourth identification polypeptide is shown in Seq_20: KVAVVRTPPK{pSER}PS.

[0048] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the 5th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein as the start, the 5th amino acid residue adjacent to the C-terminal side of the 231st amino acid residue of the Tau protein as the end, and the amino acid residues therebetween; i.e., a polypeptide with the 226th amino acid residue as the start and the 236th amino acid residue as the end of the Tau protein.

[0049] In some alternative embodiments, the basic polypeptide is VAVVRTPPKSP (Seq_22), and the four identification polypeptides consist of:

[0050] The amino acid sequence of the first identification polypeptide is shown in Seq_21: VAVVR{pTHR}PPKSP;

[0051] The amino acid sequence of the second identification polypeptide is shown in Seq_22: VAVVRTPPKSP;

[0052] The amino acid sequence of the third identification polypeptide is shown in Seq_23: VAVVR{pTHR}PPK{pSER}P;

[0053] The amino acid sequence of the fourth identification polypeptide is shown in Seq_24: VAVVRTPPK{pSER}P.

[0054] In the above polypeptides, {pTHR} refers to a phosphorylated threonine (T) residue; and {pSER} refers to a phosphorylated serine (S) residue.

[0055] According to another aspect of the present application, the present application also provides an antibody identification method for identifying whether an antibody specifically recognizes the 231st phosphorylation site of the Tau protein, the antibody identification method comprising: reacting the antibody to be identified with four identification polypeptides, which are the above polypeptide composition, respectively; and determining that the antibody to be identified is an antibody specifically recognizing the 231st phosphorylation site of the Tau protein if the detection results simultaneously satisfy (I)-(III):

[0056] (I) the antibody to be identified reacts positively with the first identification polypeptide and the third identification polypeptide;

[0057] (II) the antibody to be identified reacts negatively with the second identification polypeptide;

[0058] (III) the antibody to be identified reacts negatively or weakly positively with the fourth identification polypeptide.

[0059] The detection results of the antibodies to be identified after being respectively reacted with the four identification polypeptides are divided into "positive", "negative" and "weak positive" in the present application, and "positive", "negative" and "weak positive" can be set according to the general detection method in the art and the general judgment standard of detection results; the detection results of the antibody and antigen reaction can be divided according to the methods well known in the art and as recorded in various general and more specific textbooks, reference documents, process manuals, product instructions or standard documents, and the present application does not limit this; for example, it can be but is not limited to defining positive, negative and weak positive by OD value in the chemical reflective enzyme-linked immunoassay, or defining by the relative light unit (RLU) signal value of the full-automatic chemiluminescence immunoassay analyzer.

[0060] In some alternative embodiments, the value of the detection result is greater than or equal to the first threshold value for positive, greater than or equal to the second threshold value and less than the first threshold value for weak positive, and less than the second threshold value for negative.

[0061] In some alternative embodiments, the results of the antibody to be tested and the identification sequence are evaluated by the enzyme-linked immunoassay method; the OD value greater than or equal to the first threshold value is set as positive, the OD value greater than or equal to the second threshold value and less than the first threshold value is set as weak positive, and the OD value less than the second threshold value is set as negative.

[0062] In some alternative embodiments, the first threshold value is 1 and the second threshold value is 0.5.

[0063] In some alternative embodiments, when the secondary antibody is labeled with horseradish peroxidase in the enzyme-linked immunoassay method, and the OD value is detected under the condition of wavelength 450 nm, the first threshold value is 1 and the second threshold value is 0.5.

[0064] In addition, the present application is found by experimental screening that when the concentration of the four identification sequences is independently 0.03125-0.0625 μg / mL respectively in the enzyme-linked immunoassay method for detecting the reactivity of the identification polypeptide and the antibody to be tested, a better identification effect is obtained. When the peptide reaction concentration is too high, the antibody to be tested is prone to non-specific reaction with the potential phosphorylation site of the identification polypeptide; when the peptide reaction concentration is too low, the reaction of the antibody and each polypeptide gradually becomes weak positive or negative due to too low content of the polypeptide involved in the reaction. The concentration of the four identification sequences can be but is not limited to 0.03125, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06 or 0.0625 μg / mL respectively, and the detection effect is best when the concentration of the four identification sequences is independently 0.03125 μg / mL.

[0065] In some preferred embodiments, the antibody identification method comprises:

[0066] (a) dilute four identification polypeptides respectively using coating buffer; the coating buffer is sodium carbonate-sodium bicarbonate buffer with pH of 9.6;

[0067] (b) add the four identification polypeptides after dilution respectively to enzyme-labeled plate for incubation, and seal after washing plate;

[0068] (c) dilute the antibody to be detected to 0.5 μg / mL using PBS buffer, and add to the coated enzyme-labeled plate, incubate at 35-39°C for 57-63 min, and wash plate;

[0069] (d) add horseradish peroxidase-labeled secondary antibody, incubate at 37°C for 1-2 h, and wash plate;

[0070] (e) add color developing solution, react at room temperature for 5-10 min, add stop solution to stop, and detect OD450nm; when OD≥1, it is judged as positive; when 0.5≤OD<1, it is judged as weak positive; and when OD<0.5, it is judged as negative.

[0071] In some alternative embodiments, the four identification polypeptides have the following compositions: the amino acid sequence of the first identification polypeptide is as shown in Seq_13; the amino acid sequence of the second identification polypeptide is as shown in Seq_14; the amino acid sequence of the third identification polypeptide is as shown in Seq_15; and the amino acid sequence of the fourth identification polypeptide is as shown in Seq_16.

[0072] In some alternative embodiments, the four identification polypeptides have the following compositions: the amino acid sequence of the first identification polypeptide is as shown in Seq_17; the amino acid sequence of the second identification polypeptide is as shown in Seq_18; the amino acid sequence of the third identification polypeptide is as shown in Seq_19; and the amino acid sequence of the fourth identification polypeptide is as shown in Seq_20.

[0073] In some alternative embodiments, the four identification polypeptides have the following compositions: the amino acid sequence of the first identification polypeptide is as shown in Seq_21; the amino acid sequence of the second identification polypeptide is as shown in Seq_22; the amino acid sequence of the third identification polypeptide is as shown in Seq_23; and the amino acid sequence of the fourth identification polypeptide is as shown in Seq_24.

[0074] According to an aspect of the present application, the present application also provides a kit for identifying whether an antibody specifically recognizes the phosphorylation site at position 231 of Tau protein, which comprises the above polypeptide composition.

[0075] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 7th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 7th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 224th amino acid residue and ending from the 238th amino acid residue of the Tau protein.

[0076] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 7th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 7th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 224th amino acid residue and ending from the 238th amino acid residue of the Tau protein.

[0077] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 6th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 6th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 225th amino acid residue and ending from the 237th amino acid residue of the Tau protein.

[0078] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 6th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 6th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 225th amino acid residue and ending from the 237th amino acid residue of the Tau protein.

[0079] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 5th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 5th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 226th amino acid residue and ending from the 236th amino acid residue of the Tau protein.

[0080] In some alternative embodiments, the kit comprises four identification polypeptides whose basic polypeptides consist of the polypeptide starting from the 6th amino acid residue adjacent to the N-terminal side of the 231st amino acid residue of the Tau protein, ending from the 6th amino acid residue adjacent to the C-terminal side, and the amino acid residues therebetween; that is, the polypeptide starting from the 225th amino acid residue and ending from the 237th amino acid residue of the Tau protein.

[0081] The kit for antibody identification provided by the present application can also optionally comprise other detection reagents or consumables acceptable in the art, examples of the detection reagents can be but are not limited to one or more of the following: a buffer, specific examples can be but are not limited to Tris buffer, Bis-Tris buffer, Tris-HCl buffer, HEPES buffer, PIPES buffer, MOPS buffer, Tricine buffer, TEA buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium citrate buffer, sodium acetate-sodium bicarbonate buffer, acetic acid-sodium acetate buffer, glycine-sodium hydroxide buffer, potassium chloride-hydrochloric acid buffer, PBS or PBST; a label, specific examples can be but are not limited to one or more of the following: alkaline phosphatase, horseradish peroxidase, biotin, streptavidin or a fluorescent label; an antibody with the above-mentioned label; an antigen to be detected; a solid carrier, specific examples can be but are not limited to magnetic microparticles, a microwell reaction plate or a nitrocellulose membrane; a calibrator, a negative control or a positive control.

[0082] In some optional embodiments, the concentration of each identified polypeptide in the polypeptide composition is independently 0.03125-0.0625 μg / mL, and the concentration of each of the four identified sequences can be but is not limited to 0.03125, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06 or 0.0625 μg / mL, and the detection effect is best when the concentration of each of the four identified sequences is 0.03125 μg / mL.

[0083] In some optional embodiments, the kit is a kit for detecting the reaction result of the antibody to be detected with the identified polypeptide by an enzyme-linked immunoassay method.

[0084] In some preferred embodiments, the kit for detecting the reaction result of the antibody to be detected with the identified polypeptide by an enzyme-linked immunoassay method further comprises one or more of the following: a sodium carbonate-sodium bicarbonate coating buffer at pH 9.6, a PBST buffer, an enzyme-labeled plate, a blocking solution, a horseradish peroxidase-labeled secondary antibody, preferably a goat anti-mouse IgG, a substrate buffer and a TMB color developing solution.

[0085] According to one aspect of the present application, the present application also provides the use of the above-mentioned kit in antibody identification, and the identification comprises identifying whether the antibody specifically recognizes the 231st phosphorylation site of the Tau protein.

[0086] According to another aspect of the present application, the present application also provides a preparation method of the above-mentioned kit, comprising designing and synthesizing four identified polypeptides according to the following rules:

[0087] The first identified polypeptide is a polypeptide in which only the phosphorylation of the 231st site of Tau protein in the basic polypeptide is identified;

[0088] The second identified polypeptide is a polypeptide in which no phosphorylation of any site in the basic polypeptide is identified;

[0089] The third identified polypeptide is a polypeptide in which both the phosphorylation of the 231st site of Tau protein and the phosphorylation of the potential phosphorylation site in the basic polypeptide are identified;

[0090] The fourth identified polypeptide is a polypeptide in which the phosphorylation of the 231st site of Tau protein is not identified, while the phosphorylation of the potential phosphorylation site in the basic polypeptide is identified;

[0091] The basic polypeptide is a polypeptide in which the 5th-7th amino acid residues adjacent to the nitrogen end of the 231st site of Tau protein are the starting point, the 5th-7th amino acid residues adjacent to the carbon end are the end point, and the amino acid residues therebetween are the polypeptide;

[0092] The potential phosphorylation site is: the 1st threonine residue, serine residue or tyrosine residue adjacent to the nitrogen end and the carbon end of the 231st site of Tau protein, respectively.

[0093] In some alternative embodiments, the following polypeptide compositions are synthesized respectively to prepare a kit for identifying whether an antibody specifically recognizes the phosphorylation site of the 231st site of Tau protein:

[0094] The polypeptides with the amino acid sequences shown in Seq_13-16, respectively; or, the polypeptides with the amino acid sequences shown in Seq_17-20, respectively; or, the polypeptides with the amino acid sequences shown in Seq_21-24, respectively.

[0095] The technical solutions and beneficial effects of the present application are further illustrated below in combination with preferred embodiments.

[0096] The optimal working concentration of the polypeptide composition is screened by using a polypeptide composition for identifying whether an antibody specifically recognizes the phosphorylation site of the 181st site of Tau protein, which is consistent with the design idea of the polypeptide composition of the present application, as shown in Embodiment 1-Embodiment 4.

[0097] Embodiment 1

[0098] The present embodiment provides a polypeptide composition for identifying p-Tau181 antibody, which includes four identified polypeptides, and the arm length of the identified polypeptide is 7 amino acids.

[0099] The first identified polypeptide: p-Tau181 polypeptide-1, the amino acid sequence is Seq_1: KTPPAPK{pTHR}PPSSGEP;

[0100] Second identification polypeptide: p-Tau181 polypeptide-2, with an amino acid sequence of Seq_2: KTPPAPKTPPSSGEP;

[0101] Third identification polypeptide: p-Tau181 polypeptide-3, with an amino acid sequence of Seq_3: K{pTHR}PPAPK{pTHR}PP{pSER}SGEP;

[0102] Fourth identification polypeptide: p-Tau181 polypeptide-4, with an amino acid sequence of Seq_4: K{pTHR}PPAPKTPP{pSER}SGEP.

[0103] Example 2

[0104] The present example provides a polypeptide composition for identifying p-Tau181 antibodies, comprising four identification polypeptides, with an arm length of 6 amino acids for the identification polypeptides:

[0105] First identification polypeptide: p-Tau181 polypeptide-5, with an amino acid sequence of Seq_5: TPPAPK{pTHR}PPSSGE;

[0106] Second identification polypeptide: p-Tau181 polypeptide-6, with an amino acid sequence of Seq_6: TPPAPKTPPSSGE;

[0107] Third identification polypeptide: p-Tau181 polypeptide-7, with an amino acid sequence of Seq_7: {pTHR}PPAPK{pTHR}PP{pSER}SGE;

[0108] Fourth identification polypeptide: p-Tau181 polypeptide-8, with an amino acid sequence of Seq_8: {pTHR}PPAPKTPP{pSER}SGE.

[0109] Example 3

[0110] The present example provides a polypeptide composition for identifying p-Tau181 antibodies, comprising four identification polypeptides, with an arm length of 5 amino acids for the identification polypeptides:

[0111] First identification polypeptide: p-Tau181 polypeptide-9, with an amino acid sequence of Seq_9: PPAPK{pTHR}PPSSG;

[0112] Second identification polypeptide: p-Tau181 polypeptide-10, with an amino acid sequence of Seq_10: PPAPKTPPSSG;

[0113] Third identification polypeptide: p-Tau181 polypeptide-11, with an amino acid sequence of Seq_11: PPAPK{pTHR}PP{pSER}SG;

[0114] Fourth identified polypeptide: p-Tau181 polypeptide-12, amino acid sequence is Seq_12: PPAPKTPP{pSER}SG.

[0115] Example 4

[0116] Verification of site specificity of antibody AT270:

[0117] p-Tau181 antibody (Phospho-Tau (Thr181) Monoclonal Antibody (AT270), manufacturer: Fujirebio, product number: 90007) has been identified in the literature as having the property of specifically recognizing the 181 -position phosphorylated threonine of Tau protein. When the polypeptides of the 181 -position amino acid phosphorylation and the 175-position amino acid phosphorylation are at a higher concentration, the antibody has a positive reaction with both; as the concentration of the polypeptides decreases, the antibody's reaction signal to the polypeptide of the 175-position amino acid phosphorylation decreases faster than that of the polypeptide of the 181 -position amino acid phosphorylation, and eventually at a low concentration, the reaction signal to the polypeptide of the 181 -position amino acid phosphorylation is 18 times that of the polypeptide of the 175-position amino acid phosphorylation.

[0118] The 12 identified sequence polypeptides in Examples 1-3 were diluted to 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.01563 μg / mL, 0.00781 μg / mL, 0.00391 μg / mL, 0.00195 μg / mL, respectively, using coating buffer (pH 9.6, Na2CO3 1.59 mg / mL, NaHCO3 2.93 mg / mL).

[0119] The diluted identified sequence polypeptides were added to a 96-well enzyme-labeled plate, 100 μL per well, and incubated at 37°C for 2 h. The plate was washed 3 times with PBST buffer, 60 s each time, and then dried. Blocking solution 200 μL / well was added, and incubated at 37°C for 2 h, then washed and dried.

[0120] The p-Tau181 antibody (AT270) was diluted to 0.5 μg / mL using PBS buffer, and added to the coated luminescent plate, 100 μL per well, and incubated at 37±2°C for 60±3 min, then washed and dried.

[0121] HRP-labeled goat anti-mouse IgG was added, 100 μL per well, and incubated at 37°C for 1-2 h, then washed and dried.

[0122] Substrate buffer was diluted with substrate solution TMB at 1:1, mixed well, then 100 μL was added to each well, and reacted at room temperature for 5-10 min. 50 μL of termination solution was added to each well, mixed well, and OD450nm value of each well was read by using an enzyme label meter. OD≥1 was judged as positive; 0.5≤OD<1 was judged as weak positive; and OD<0.5 was judged as negative. The results are shown in Tables 1-3.

[0123] Table 1 Reactivity of polypeptide composition of Example 1 with AT270 antibody and p-Tau181 antibody, respectively.

[0124]

[0125]

[0126] Table 2 Reactivity of polypeptide composition of Example 2 with AT270 antibody and p-Tau181 antibody, respectively.

[0127]

[0128]

[0129] Table 3 Reactivity of polypeptide composition of Example 3 with AT270 antibody and p-Tau181 antibody, respectively.

[0130]

[0131]

[0132] From the detection results of antibody AT270 and 4 identified polypeptides (Example 1, arm length 7), when the polypeptide coating concentration was 0.125-2 μg / mL, the polypeptide reaction concentration was too high, and antibody AT270 had certain non-specificity to the phosphorylated groups of some sequences (such as reported in the literature that it had cross reaction with the phosphorylated polypeptide of amino acid 175), so it reacted positively with p-Tau181 polypeptide-1, p-Tau181 polypeptide-3, and p-Tau181 polypeptide-4, and reacted negatively with p-Tau181 polypeptide-2.

[0133] When the coating concentration of the polypeptide decreased, the reaction signal of the antibody to p-Tau181 polypeptide-4 decreased faster, and at a concentration of 0.03125 ug / mL, p-Tau181 polypeptide-4 was 1 / 3 of the signal of p-Tau181 polypeptide-1 and p-Tau181 polypeptide-3. When the coating concentration was less than 0.03125 ug / mL, the content of the polypeptide involved in the reaction was too low, and the reaction of the antibody with each polypeptide gradually turned to weak positive (had weak cross reaction) or negative (had no cross reaction).

[0134] The above results show that the antibody is not specific to the phosphate group, but mainly targets the phosphorylated threonine at position 181 of Tau protein, which is consistent with the literature reports, and the method is effective.

[0135] The reaction of the antibody with the four identified polypeptides of Arm Length 5 (Example 3) and Arm Length 6 (Example 2) and the like of Arm Length 7.

[0136] Example 5

[0137] This example provides a polypeptide composition for identifying a p-Tau231 antibody, comprising four identified polypeptides, the identified polypeptide arm length being 7 amino acids:

[0138] The first identified polypeptide: p-Tau231 polypeptide-1, the amino acid sequence is Seq_13: KKVAVVR{pTHR}PPKSPSS;

[0139] The second identified polypeptide: p-Tau231 polypeptide-2, the amino acid sequence is Seq_14: KKVAVVRTPPKSPSS;

[0140] The third identified polypeptide: p-Tau231 polypeptide-3, the amino acid sequence is Seq_15: KKVAVVR{pTHR}PPK{pSER}PSS;

[0141] The fourth identified polypeptide: p-Tau231 polypeptide-4, the amino acid sequence is Seq_16: KKVAVVRTPPK{pSER}PSS.

[0142] Example 6

[0143] This example provides a polypeptide composition for identifying a p-Tau231 antibody, comprising four identified polypeptides, the identified polypeptide arm length being 6 amino acids:

[0144] The first identified polypeptide: p-Tau231 polypeptide-5, the amino acid sequence is Seq_17: KVAVVR{pTHR}PPKSPS;

[0145] The second identified polypeptide: p-Tau231 polypeptide-6, the amino acid sequence is Seq_18: KVAVVRTPPKSPS;

[0146] The third identified polypeptide: p-Tau231 polypeptide-7, the amino acid sequence is Seq_19: KVAVVR{pTHR}PPK{pSER}PS;

[0147] The fourth identified polypeptide: p-Tau231 polypeptide-8, the amino acid sequence is Seq_20: KVAVVRTPPK{pSER}PS.

[0148] Example 7

[0149] This embodiment provides a polypeptide composition for identifying p-Tau231 antibodies, comprising four identification polypeptides, each with a 5-amino acid arm:

[0150] First identified polypeptide: p-Tau231 polypeptide-9, amino acid sequence is Seq_21:VAVVR{pTHR}PPKSP;

[0151] Second identified polypeptide: p-Tau231 polypeptide-10, amino acid sequence is Seq_22:VAVVRTPPKSP;

[0152] The third identified polypeptide is p-Tau231 polypeptide-11, with the amino acid sequence Seq_23: VAVVR{pTHR}PPK{pSER}P;

[0153] The fourth identified polypeptide is p-Tau231 polypeptide-12, with the amino acid sequence Seq_24: VAVVRTPPK{pSER}P.

[0154] Example 8

[0155] Site specificity verification of commercially available p-Tau231 antibodies:

[0156] The four p-Tau231 peptides were serially diluted to 0.03125 μg / mL using CB buffer. 100 μL of the diluted peptides was added to each well of a 96-well microplate and incubated at 37°C for 2 h. The plates were washed three times with PBST buffer for 60 s each time, and then blotted dry. 200 μL of blocking buffer was added to each well, and the plates were incubated at 37°C for 2 h. After washing, the plates were blotted dry.

[0157] p-Tau231 antibody-1 and p-Tau231 antibody-2 were diluted to 0.5 g / mL using PBS buffer and added to the coated luminescent plates accordingly, 100 μL per well. The plates were incubated at 37±2℃ for 60±3 min, washed, and then dried.

[0158] Add 100 μL of HRP-labeled goat anti-mouse IgG to each well, incubate at 37°C for 1–2 h, wash the plate and pat dry.

[0159] The substrate buffer and substrate solution TMB were diluted 1:1, thoroughly mixed, and 100 μL was added to each well. The mixture was incubated at room temperature for 5–10 min. 50 μL of stop solution was added to each well, mixed, and the OD450 nm value of each well was read using a microplate reader. An OD ≥ 1 was interpreted as positive; 0.5 ≤ OD < 1 was interpreted as weakly positive; and OD < 0.5 was interpreted as negative. The results are shown in Tables 4–6.

[0160] Table 4. Reactivity of two commercially available p-Tau231 antibodies and that of Example 7

[0161]

[0162]

[0163] Table 5. Reactivity of two commercially available p-Tau231 antibodies and that of Example 8

[0164]

[0165] Table 6. Reactivity of two commercially available p-Tau231 antibodies and that of Example 9

[0166]

[0167] The reactions of p-Tau231 antibody-2 and p-Tau231 peptide-1, p-Tau231 peptide-5, p-Tau231 peptide-9, p-Tau231 peptide-3, p-Tau231 peptide-7, and p-Tau231 peptide-11 were all positive, while the reactions of p-Tau231 peptide-2, p-Tau231 peptide-6, p-Tau231 peptide-10, p-Tau231 peptide-4, p-Tau231 peptide-8, and p-Tau231 peptide-12 were all negative, meeting the screening requirements. These are specific antibodies against the threonine phosphorylation at position 231 of the Tau protein. p-Tau231 antibody-1 did not meet the screening requirements.

[0168] Example 1: Specificity of the reaction between p-Tau antibody and antigen

[0169] Three p-Tau181 antigens, one p-Tau217 antigen, one p-Tau231 antigen, and four Tau antigens were purchased from the market. These four antigens have very similar sequences and structures, differing only in whether amino acids at positions 181, 217, and 231 are phosphorylated. Using p-Tau231 antibody-2 that meets the screening requirements, combined with total Tau antibody, a double-antibody sandwich chemiluminescent immunoassay was employed to react with the aforementioned antigens.

[0170] Cross-reactivity evaluation of p-Tau antibody with antigen and its analogues

[0171] (1) Preparation of p-Tau antibody-conjugated toluenesulfonyl (Tysol) magnetic beads and working solution

[0172] p-Tau231 antibody-2 and Tysol magnetic beads (JSR, MS160 Tosylactivated 10%) were conjugated according to the manufacturer's instructions to obtain a stock solution of p-Tau antibody-magnetic bead conjugate at a concentration of 10 mg / mL.

[0173] Prepare a 0.05M Tris buffer solution with a pH of 7.4, containing 5 mg / mL NaCl2, 9 mg / mL Proclin 300, and 0.5 mg / mL Proclin 300. Then, use this buffer solution to dilute the stock solution of the p-Tau antibody magnetic bead conjugate to 0.2 mg / mL.

[0174] (2) Preparation of alkaline phosphatase (AP) labeled total Tau antibody and working solution

[0175] Zhuhai Lihe Medical Diagnostic Products Co., Ltd. was commissioned to label the total Tau antibody with AP enzyme, and a stock solution of 0.5 mg / mL total Tau antibody AP enzyme-labeled product was obtained.

[0176] Prepare a 0.1M MES buffer with a pH of 6.5, containing 1 mM MgCl2, 0.1 mM ZnCl2, 1 mg / mL BSA, and 0.5 mg / mL Proclin 300. Then, use this buffer to dilute the stock solution of total Tau antibody AP enzyme-conjugated ester to 1 μg / mL.

[0177] (3) Antigen preparation

[0178] A phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 M was prepared, containing 9 mg / mL NaCl2, 10 mg / mL BSA, 0.5 mg / mL Tween 20, and 0.5 mg / mL Proclin 300. Then, three p-Tau181 antigens, one p-Tau217 antigen, one p-Tau231 antigen, and four Tau antigens were diluted with this buffer to concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL, and 1 pg / mL, respectively.

[0179] (4) On-machine test

[0180] We used the Shinei2900 fully automated chemiluminescence immunoassay analyzer manufactured by Shenzhen Yingkai Technology Co., Ltd.

[0181] The instrument aspirates 50 μL of sample, adds 50 μL of p-Tau antibody magnetic bead conjugate working solution and 50 μL of total Tau antibody AP enzyme-conjugated working solution; incubates at 37℃ for 5 min; after incubation, magnetic separation is performed to remove the supernatant, and the sample is washed three times; substrate solution is added, and signal values ​​are read. A gradient of relative photon number (RLU) signals is obtained for serially diluted antigens, and a maximum RLU ≥ 100,000 indicates that the antibody can recognize the antigen.

[0182] The results are shown in Table 7:

[0183] Table 7 Reactivity of p-Tau antibody with antigens and analogues

[0184]

[0185]

[0186] When p-Tau231 antibody-2 reacted with one p-Tau231 antigen, a signal change with the concentration gradient was observed, and the highest RLU was greater than 100,000, indicating that p-Tau231 antibody-2 can recognize the p-Tau231 antigen. When p-Tau231 antibody-2 reacted with four Tau antigens, three p-Tau181 antigens, and one p-Tau217 antigen, the RLU measured for all antigen concentrations showed no gradient and was less than 2,000, indicating that p-Tau231 antibody-2 does not recognize Tau antigen, p-Tau181 antigen, or p-Tau217 antigen. In conclusion, p-Tau231 antibody-2 specifically recognizes the p-Tau231 antigen.

[0187] The above results demonstrate that, through reactivity evaluation with four peptides, the phosphorylation site-specific antibodies screened in Example 8 only recognize antigens phosphorylated at the target site, and show no cross-reactivity with antigens that are very similar in sequence and structure.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide composition for antibody identification, characterized in that, The identification comprises identifying whether the antibody specifically recognizes the phosphorylation site at position 231 of the Tau protein, the polypeptide composition comprises four identification polypeptides, and the four identification polypeptides are respectively: The amino acid sequence of the first identification polypeptide is shown as Seq_13; the amino acid sequence of the second identification polypeptide is shown as Seq_14; the amino acid sequence of the third identification polypeptide is shown as Seq_15; and the amino acid sequence of the fourth identification polypeptide is shown as Seq_16. Alternatively, the amino acid sequence of the first identification polypeptide is shown as Seq_17; the amino acid sequence of the second identification polypeptide is shown as Seq_18; the amino acid sequence of the third identification polypeptide is shown as Seq_19; and the amino acid sequence of the fourth identification polypeptide is shown as Seq_20. Alternatively, the amino acid sequence of the first identification polypeptide is shown as Seq_21; the amino acid sequence of the second identification polypeptide is shown as Seq_22; the amino acid sequence of the third identification polypeptide is shown as Seq_23; and the amino acid sequence of the fourth identification polypeptide is shown as Seq_24.

2. A method for antibody identification, characterized in that The identification comprises identifying whether the antibody specifically recognizes the phosphorylation site at position 231 of the Tau protein; The antibody identification method comprises: reacting the antibody to be identified with the four identification polypeptides respectively, and determining that the antibody to be identified is an antibody specifically recognizing the phosphorylation site at position 231 of the Tau protein when the detection results simultaneously satisfy (I)-(III): (I) the antibody to be identified reacts positively with the first identification polypeptide and the third identification polypeptide; (II) the antibody to be identified reacts negatively with the second identification polypeptide; (III) the antibody to be identified reacts negatively or weakly positively with the fourth identification polypeptide; The four identification polypeptides are the polypeptide composition of claim 1.

3. The method of claim 2, wherein, The value of the detection result greater than or equal to the first threshold value is positive, greater than or equal to the second threshold value and less than the first threshold value is weakly positive, and less than the second threshold value is negative; The first threshold value is 1, and the second threshold value is 0.5; The working concentrations of the four identification polypeptides are independently 0.03125-0.0625 μg / mL.

4. The method of claim 3, wherein, The working concentrations of the four identification polypeptides are independently 0.03125 μg / mL.

5. The method of claim 2, wherein, The method comprises: (a) diluting the four identification polypeptides with a coating buffer respectively; the coating buffer is a sodium carbonate-sodium bicarbonate buffer with a pH of 9.6; (b) adding the diluted four identification polypeptides to an enzyme-labeled plate for incubation, and then washing the plate and blocking; (c) diluting the antibody to be detected to 0.5 μg / mL with a PBS buffer, and adding it to the coated enzyme-labeled plate for incubation at 35-39°C for 57-63 min, and then washing the plate; (d) adding a horseradish peroxidase-labeled secondary antibody, incubating at 37°C for 1-2 h, and then washing the plate; (e) adding a color developing solution for reaction at room temperature for 5-10 min, adding a stop solution to terminate the reaction, detecting OD450nm, and determining that the result is positive when OD≥1, weakly positive when 0.5≤OD<1, and negative when OD<0.

5.

6. A kit for identifying whether an antibody specifically recognizes the phosphorylation site at position 231 of the Tau protein, characterized in that, The polypeptide composition of claim 1.

7. Use of a kit according to claim 6 in the identification of an antibody, said identification comprising the identification of whether the antibody specifically recognizes the phosphorylation site at position 231 of the Tau protein.

8. A process for the preparation of a kit as claimed in claim 6, characterized in that, comprising synthesizing four identification polypeptides.

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