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 181st phosphorylation site of Tau protein, and determining whether the antibody to be identified can specifically recognize the 181st phosphorylation site of Tau protein by reacting the antibody to be detected with four identification polypeptides respectively according to the detection result. The four identification polypeptides are based on a basic polypeptide, and the specificity of the antibody to be identified for the target phosphorylation site is determined by the phosphorylation difference between the 181st phosphorylation site of Tau protein and potential phosphorylation sites, so that the problem of complex screening and identification method of the antibody for recognizing the 181st phosphorylation site of Tau protein in the prior art is solved.

Description

[0001] This divisional application is a divisional application of Chinese patent application No. 2022109111199, filed on July 29, 2022, entitled "Antibody Identification Method, Peptide Composition, Reagent Kit and Preparation Method Thereof and Application". Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to an antibody identification method, a polypeptide composition, a kit, a preparation method thereof, and its application. Background Technology

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

[0004] Current screening and identification methods for antibodies that recognize phosphorylation sites (such as p-Tau181) require synthesizing a peptide that is phosphorylated at a specific site and a corresponding peptide that is not phosphorylated at the same site, based on the inferred potential phosphorylation site. The two types of peptides are then reacted with the antibody to be identified. The concentration of the cross-reactive peptides is then reduced to obtain the peptide with the strongest reactivity, which is then identified as the main phosphorylation site recognized by the antibody.

[0005] For example, in the site identification of a commercial p-Tau181 antibody, 22 site-directed phosphorylation sites and 12 corresponding unphosphorylated peptides were used to react with the antibody, resulting in cross-reactive phosphorylated peptides at sites 181 and 175. After reducing the reaction concentration of these two peptides, the reaction signal of the phosphorylated peptide at site 181 was 18 times that of the phosphorylated peptide at site 175, thus identifying the antibody as a p-Tau181 antibody. Furthermore, to determine if the reaction sequence is within the required range, it is necessary to further truncate the N-terminal and C-terminal amino acids, centered on the phosphorylation site, until the minimum reaction sequence is obtained. This sequence is then used to determine if the antibody recognizes a sequence within the required range. For example, in the site identification of a commercial p-Tau181 antibody, 9 shift-truncation sequences were used to obtain its minimum reaction sequence, thus determining that the antibody recognizes a sequence within the required range. The above-mentioned screening and identification method for antibodies recognizing phosphorylated sites is relatively complex. Therefore, simplifying the screening and identification method for antibodies recognizing phosphorylated sites is a pressing issue that needs to be addressed.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The first objective of this invention is to provide a polypeptide composition that can be used to identify whether an antibody can specifically recognize the 181st phosphorylation site of the Tau protein, thereby alleviating the technical problem of the complex process of screening for antibodies that recognize the 181st phosphorylation site of the Tau protein in the prior art.

[0008] A second objective of this invention is to provide a method for identifying whether an antibody specifically recognizes the 181st phosphorylation site of the Tau protein using the above-described polypeptide composition.

[0009] A third objective of the present invention is to provide a kit comprising the above-described polypeptide composition, the application of the kit, and a method for preparing the kit.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0011] According to one aspect of the present invention, a polypeptide composition for antibody identification is provided, the identification comprising identifying whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein, the polypeptide composition comprising four identification polypeptides, the four identification polypeptides being:

[0012] The first identified polypeptide was a basic polypeptide that only had phosphorylated Tau protein at position 181.

[0013] The second identified polypeptide is a basic polypeptide without any phosphorylation sites.

[0014] The third identified polypeptide is a polypeptide in which both the 181st position and the potential phosphorylation site of the Tau protein in the basic polypeptide are phosphorylated.

[0015] The fourth identified polypeptide is a polypeptide in the basic polypeptide whose Tau protein is unphosphorylated at position 181, while its potential phosphorylation site is phosphorylated.

[0016] The basic polypeptide is a polypeptide that starts with the 5th to 7th amino acid residues adjacent to the N-terminus of the 181st position of the Tau protein, ends with the 5th to 7th amino acid residues adjacent to the C-terminus, and consists of the amino acid residues in between.

[0017] The potential phosphorylation sites are the first threonine, serine, or tyrosine residue adjacent to the 181st position of the Tau protein on the N-terminal and C-terminal sides, respectively.

[0018] According to another aspect of the present invention, the present invention also provides an antibody identification method, wherein the identification includes identifying whether the antibody specifically recognizes the 181st phosphorylation site of the Tau protein; the antibody identification method includes: reacting the antibody to be identified with four identification peptides respectively, and if the detection results simultaneously satisfy (I) to (III), then the antibody to be identified is determined to be an antibody that specifically recognizes the 181st phosphorylation site of the Tau protein.

[0019] (I) The antibody to be identified reacts positively with both the first and third identifying polypeptides;

[0020] (II) The antibody to be identified reacts negatively with the second identification polypeptide;

[0021] (III) The antibody to be identified reacts negatively or weakly positively with the fourth identification polypeptide;

[0022] The four identified polypeptides are the aforementioned polypeptide composition.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The polypeptide composition for antibody identification provided by this invention can effectively determine whether the antibody to be identified can specifically recognize the phosphorylation site at position 181 of the Tau protein. An antibody with good specificity in recognizing the above-mentioned target phosphorylation site should possess the following characteristics:

[0025] (1) Recognizes peptides that phosphorylate the 181st amino acid of Tau protein; (2) Does not recognize peptides that do not phosphorylate the 181st amino acid of Tau protein; (3) Phosphorylation of the T (threonine residue), S (serine residue), or Y (tyrosine residue) adjacent to the 181st amino acid of Tau protein does not affect the recognition of the phosphorylated sequence of the 181st amino acid of Tau protein; (4) Does not cross-react with adjacent phosphorylated T, S, or Y, or has a weak cross-reaction with adjacent phosphorylated T, S, or Y. The sequence recognized by an antibody that meets these characteristics should be between the potentially phosphorylated amino acids (T, S, or Y) adjacent to the 181st amino acid of Tau protein.

[0026] The four identification peptides designed according to the above rules can effectively determine whether the antibody to be identified can specifically recognize the target phosphorylation site. The present invention has verified through experiments that the above four peptides can screen out antibodies that specifically recognize the 181st phosphorylation site of Tau protein. Furthermore, due to the presence of the fourth identification peptide, the peptide composition provided by the present invention can also screen out antibodies that may produce non-specific reactions with other phosphorylation sites of Tau protein, thereby excluding them. The fourth identification sequence is essential in the method of identifying whether an antibody specifically recognizes the target phosphorylation site.

[0027] The antibody identification method provided by this invention is simple to operate. It only requires the synthesis of four identification peptides. Then, the detection results after the antibody to be tested reacts with the four identification peptides are used to determine the specific recognition ability of the antibody to be tested for the phosphorylation site at position 181 of Tau protein. This antibody identification method is simple and effective. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0030] According to one aspect of the present invention, a polypeptide composition for antibody identification is provided, the identification comprising identifying whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein, the polypeptide composition comprising four identification polypeptides, the four identification polypeptides being:

[0031] The first identified polypeptide was a basic polypeptide that only had phosphorylated Tau protein at position 181.

[0032] The second identified polypeptide is a basic polypeptide without any phosphorylation sites.

[0033] The third identified polypeptide is a polypeptide in which both the 181st position and the potential phosphorylation site of the Tau protein in the basic polypeptide are phosphorylated.

[0034] The fourth identified polypeptide is a polypeptide in the basic polypeptide whose Tau protein is unphosphorylated at position 181, while its potential phosphorylation site is phosphorylated.

[0035] The basic polypeptide is a polypeptide that begins with the 5th to 7th amino acid residues adjacent to the N-terminus of the Tau protein at position 181, ends with the 5th to 7th amino acid residues adjacent to the C-terminus, and consists of amino acid residues in between; that is, a polypeptide that begins at positions 174 to 176 of the Tau protein and ends at positions 186 to 188. The basic polypeptide in this invention can be understood as having an "arm" of 5 to 7 amino acid residues each at the N-terminus and C-terminus of the Tau protein, centered at position 181.

[0036] The potential phosphorylation sites are the first threonine, serine, or tyrosine residue adjacent to the 181st position of the Tau protein on the N-terminal and C-terminal sides, respectively.

[0037] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the 7th amino acid residue adjacent to the N-terminus of the Tau protein at position 181, the 7th amino acid residue adjacent to the C-terminus, and the amino acid residues in between, i.e., a polypeptide starting at position 174 and ending at position 188 of the Tau protein.

[0038] In some alternative embodiments, the basic polypeptide is KTPPAPKTPPSSGEP(Seq_2), and the four identifying polypeptides are composed of:

[0039] The amino acid sequence of the first identified polypeptide is shown in Seq_1: KTPPAPK{pTHR}PPSSGEP;

[0040] The amino acid sequence of the second identified polypeptide is shown in Seq_2: KTPPAPKTPPSSGEP;

[0041] The amino acid sequence of the third identifying polypeptide is shown in Seq_3: K{pTHR}PPAPK{pTHR}PP{pSER}SGEP;

[0042] The amino acid sequence of the fourth identification polypeptide is shown in Seq_4: K{pTHR}PPAPKTPP{pSER}SGEP.

[0043] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the sixth amino acid residue adjacent to the N-terminus of the Tau protein at position 181, the sixth amino acid residue adjacent to the C-terminus, and the amino acid residues in between; that is, a polypeptide starting at position 175 and ending at position 187 of the Tau protein.

[0044] In some alternative embodiments, the basic polypeptide is TPPAPKTPPSSGE(Seq_6), and the four identifying polypeptides are composed of:

[0045] The amino acid sequence of the first identified polypeptide is shown in Seq_5: TPPAPK{pTHR}PPSSGE;

[0046] The amino acid sequence of the second identification polypeptide is shown in Seq_6: TPPAPKTPPSSGE;

[0047] The amino acid sequence of the third identifying polypeptide is shown in Seq_7: {pTHR}PPAPK{pTHR}PP{pSER}SGE;

[0048] The amino acid sequence of the fourth identified polypeptide is shown in Seq_8: {pTHR}PPAPKTPP{pSER}SGE.

[0049] In some alternative embodiments, the basic polypeptide is a polypeptide consisting of the fifth amino acid residue adjacent to the N-terminus of the Tau protein at position 181, the fifth amino acid residue adjacent to the C-terminus, and the amino acid residues in between; that is, a polypeptide starting at position 176 and ending at position 186 of the Tau protein.

[0050] In some optional embodiments, the basic polypeptide is PPAPKTPPSSG(Seq_10). The four identifying polypeptides are composed of:

[0051] The amino acid sequence of the first identified polypeptide is shown in Seq_9: PPAPK{pTHR}PPSSG;

[0052] The amino acid sequence of the second identification polypeptide is shown in Seq_10: PPAPKTPPSSG;

[0053] The amino acid sequence of the third identification polypeptide is shown in Seq_11: PPAPK{pTHR}PP{pSER}SG;

[0054] The amino acid sequence of the fourth identification polypeptide is shown in Seq_12: PPAPKTPP{pSER}SG.

[0055] In the above polypeptides, {pTHR} refers to phosphorylated threonine (T) residues; {pSER} refers to phosphorylated serine (S) residues.

[0056] According to another aspect of the present invention, the present invention also provides an antibody identification method for identifying whether an antibody specifically recognizes the 181st phosphorylation site of Tau protein. The antibody identification method includes: reacting the antibody to be identified with four identification peptides, wherein the four identification peptides are the aforementioned peptide composition; if the detection results simultaneously satisfy (I) to (III), then the antibody to be identified is determined to be an antibody that specifically recognizes the 181st phosphorylation site of Tau protein.

[0057] (I) The antibody to be identified reacts positively with both the first and third identifying polypeptides;

[0058] (II) The antibody to be identified reacts negatively with the second identification polypeptide;

[0059] (III) The antibody to be identified reacts negatively or weakly positively with the fourth identification polypeptide.

[0060] This invention classifies the detection results of the antibody to be identified reacting with four identification peptides into "positive", "negative", and "weakly positive". The terms "positive", "negative", and "weakly positive" can be set according to general detection methods and general judgment criteria in the art. The detection results after the antibody and antigen reaction can be classified according to methods well known in the art and described in various general and more specific textbooks, references, process manuals, product instructions or standard documents. This invention does not limit this. For example, it can be, but is not limited to, defining positive, negative and weakly positive by OD value in chemiluminescent enzyme-linked immunosorbent assay, or by relative photon number (RLU) signal value of a fully automated chemiluminescence immunoassay analyzer.

[0061] In some optional implementations, a test result value greater than or equal to a first threshold is considered positive, a value greater than or equal to a second threshold but less than the first threshold is considered weakly positive, and a value less than the second threshold is considered negative.

[0062] In some optional implementations, an enzyme-linked immunosorbent assay (ELISA) is used to evaluate the results of the antibody to be tested and the identified sequence; an OD value greater than or equal to a first threshold is defined as positive, an OD value greater than or equal to a second threshold and less than the first threshold is defined as weakly positive, and an OD value less than the second threshold is defined as negative.

[0063] In some alternative implementations, the first threshold is 1 and the second threshold is 0.5.

[0064] In some optional embodiments, when the secondary antibody is labeled with horseradish peroxidase in the enzyme-linked immunosorbent assay (ELISA) and the OD value is detected at a wavelength of 450 nm, the first threshold is 1 and the second threshold is 0.5.

[0065] Furthermore, through experimental screening, this invention has found that when using enzyme-linked immunosorbent assay (ELISA) to detect the reactivity of the identified peptides and the test antibodies, the concentrations of the four identification sequences, each independently ranging from 0.03125 to 0.0625 μg / mL, exhibit good identification results. When the peptide reaction concentration is too high, the test antibody is prone to non-specific reactions with the potential phosphorylation sites of the identified peptides; when the peptide reaction concentration is too low, due to the low content of the peptides involved in the reaction, the reactions between the antibody and each peptide gradually turn weakly positive or negative. The concentrations of the four identification sequences can be, for example, but 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 optimal when the concentrations of the four identification sequences are each independently 0.03125 μg / mL.

[0066] In some preferred embodiments, the antibody identification method includes:

[0067] (a) Dilute the four identification peptides separately with a coating buffer; the coating buffer is a sodium carbonate-sodium bicarbonate buffer with a pH of 9.6;

[0068] (b) Add the four diluted identification peptides to the ELISA plate for incubation, wash the plate, and then block it.

[0069] (c) Dilute the antibody to be tested to 0.5 μg / mL with PBS buffer, add it to the coated microplate, incubate at 35-39°C for 57-63 min, and then wash the plate.

[0070] (d) Add horseradish peroxidase-labeled secondary antibody, incubate at 37°C for 1-2 hours, and wash the plate;

[0071] (e) Add the colorimetric reagent and react at room temperature for 5-10 min. Add the stop solution to terminate the reaction and detect OD at 450 nm. When OD ≥ 1, it is interpreted as positive. When 0.5 ≤ OD < 1, it is interpreted as weakly positive. When OD < 0.5, it is interpreted as negative.

[0072] In some optional embodiments, the four identification peptides are composed of: the amino acid sequence of the first identification peptide is shown in Seq_1; the amino acid sequence of the second identification peptide is shown in Seq_2; the amino acid sequence of the third identification peptide is shown in Seq_3; and the amino acid sequence of the fourth identification peptide is shown in Seq_4.

[0073] In some optional embodiments, the four identification peptides are composed of: the amino acid sequence of the first identification peptide is shown in Seq_5; the amino acid sequence of the second identification peptide is shown in Seq_6; the amino acid sequence of the third identification peptide is shown in Seq_7; and the amino acid sequence of the fourth identification peptide is shown in Seq_8.

[0074] In some optional embodiments, the four identification peptides are composed of: the amino acid sequence of the first identification peptide is shown in Seq_9; the amino acid sequence of the second identification peptide is shown in Seq_10; the amino acid sequence of the third identification peptide is shown in Seq_11; and the amino acid sequence of the fourth identification peptide is shown in Seq_12.

[0075] According to one aspect of the invention, the invention also provides a kit for identifying whether an antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein, the kit comprising the above-described polypeptide composition.

[0076] In some optional embodiments, the kit contains four basic identification peptides, each consisting of a peptide that starts at the 7th amino acid residue adjacent to the N-terminus of the Tau protein at position 181, ends at the 7th amino acid residue adjacent to the C-terminus, and consists of amino acid residues in between; that is, a peptide that starts at position 174 and ends at position 188 of the Tau protein.

[0077] In some alternative embodiments, the kit contains the amino acid sequence of a first identification peptide as shown in Seq_1; the amino acid sequence of a second identification peptide as shown in Seq_2; the amino acid sequence of a third identification peptide as shown in Seq_3; and the amino acid sequence of a fourth identification peptide as shown in Seq_4.

[0078] In some alternative embodiments, the kit contains four basic identification peptides, each consisting of a peptide that starts at the 6th amino acid residue adjacent to the N-terminus of the Tau protein at position 181, ends at the 6th amino acid residue adjacent to the C-terminus, and consists of amino acid residues in between; that is, a peptide that starts at position 175 and ends at position 187 of the Tau protein.

[0079] In some alternative embodiments, the kit contains the amino acid sequence of a first identification peptide as shown in Seq_5; the amino acid sequence of a second identification peptide as shown in Seq_6; the amino acid sequence of a third identification peptide as shown in Seq_7; and the amino acid sequence of a fourth identification peptide as shown in Seq_8.

[0080] In some optional embodiments, the kit contains four basic peptides for identifying peptides, each consisting of a peptide that starts at the 5th amino acid residue adjacent to the N-terminus of the Tau protein at position 181, ends at the 5th amino acid residue adjacent to the C-terminus, and consists of amino acid residues in between; that is, a peptide that starts at position 176 and ends at position 186 of the Tau protein.

[0081] In some alternative embodiments, the kit contains the amino acid sequence of a first identification peptide as shown in Seq_9; the amino acid sequence of a second identification peptide as shown in Seq_10; the amino acid sequence of a third identification peptide as shown in Seq_11; and the amino acid sequence of a fourth identification peptide as shown in Seq_12.

[0082] The antibody identification kit provided by this invention may optionally include other detection reagents or consumables acceptable in the art. Examples of detection reagents include, but are not limited to, buffer solutions, specifically, but not limited to, one or more of 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, citrate-sodium citrate buffer, sodium bicarbonate-sodium acetate buffer, acetic acid-sodium acetate buffer, glycine-sodium hydroxide buffer, potassium chloride-hydrochloric acid buffer, PBS, or PBST; labels, specifically, but not limited to, one or more of alkaline phosphatase, horseradish peroxidase, biotin, streptavidin, or fluorescent labels; antibodies carrying the above labels; detection antigens; solid-phase carriers, specifically, but not limited to, magnetic microparticles, microplates, or nitrocellulose membranes; calibrators, negative controls, or positive controls.

[0083] In some optional embodiments, the concentration of each identifying peptide in the peptide composition is independently 0.03125 to 0.0625 μg / mL, and the concentrations of the four identifying sequences are independently, for example, but not limited to, 0.03125, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, or 0.0625 μg / mL. The detection effect is optimal when the concentrations of the four identifying sequences are independently 0.03125 μg / mL.

[0084] In some alternative embodiments, the kit is a kit for detecting the reaction results of the antibody to be tested and the identified peptide using an enzyme-linked immunosorbent assay (ELISA).

[0085] In some preferred embodiments, the kit for detecting the reaction results of the antibody and the identified peptide using an enzyme-linked immunosorbent assay (ELISA) further comprises a sodium carbonate-sodium bicarbonate coating buffer at pH 9.6, PBST buffer, an ELISA plate, a blocking solution, and a horseradish peroxidase-labeled secondary antibody, preferably one or more of goat anti-mouse IgG, substrate buffer, and TMB chromogenic solution.

[0086] According to one aspect of the invention, the invention also provides the application of the above-described kit in antibody identification, wherein the identification includes identifying whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein.

[0087] According to another aspect of the present invention, the present invention also provides a method for preparing the above-mentioned kit, comprising designing and synthesizing four identification peptides according to the following rules:

[0088] The first identified polypeptide was a basic polypeptide that only had phosphorylated Tau protein at position 181.

[0089] The second identified polypeptide is a basic polypeptide without any phosphorylation sites.

[0090] The third identified polypeptide is a polypeptide in which both the 181st position and the potential phosphorylation site of the Tau protein in the basic polypeptide are phosphorylated.

[0091] The fourth identified polypeptide is a polypeptide in the basic polypeptide whose Tau protein is unphosphorylated at position 181, while its potential phosphorylation site is phosphorylated.

[0092] The basic polypeptide is a polypeptide that starts with the 5th to 7th amino acid residues adjacent to the N-terminus of the 181st position of the Tau protein, ends with the 5th to 7th amino acid residues adjacent to the C-terminus, and consists of the amino acid residues in between.

[0093] The potential phosphorylation sites are the first threonine, serine, or tyrosine residue adjacent to the 181st position of the Tau protein on the N-terminal and C-terminal sides, respectively.

[0094] In some alternative embodiments, the following polypeptide compositions are synthesized to prepare a kit for identifying whether an antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein:

[0095] The polypeptides with amino acid sequences as shown in Seq_1 to 4; or, the polypeptides with amino acid sequences as shown in Seq_5 to 8; or, the polypeptides with amino acid sequences as shown in Seq_9 to 12.

[0096] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0097] Example 1

[0098] This embodiment provides a polypeptide composition for identifying p-Tau181 antibodies, comprising four identification polypeptides, each with a 7-amino acid arm:

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

[0100] Second identified polypeptide: p-Tau181 polypeptide-2, amino acid sequence Seq_2: KTPPAPKTPPSSGEP;

[0101] The third identified polypeptide is p-Tau181 polypeptide-3, with the amino acid sequence Seq_3: K{pTHR}PPAPK{pTHR}PP{pSER}SGEP;

[0102] The fourth identified polypeptide was p-Tau181 polypeptide-4, with the amino acid sequence Seq_4: K{pTHR}PPAPKTPP{pSER}SGEP.

[0103] Example 2

[0104] This embodiment provides a polypeptide composition for identifying p-Tau181 antibodies, comprising four identification polypeptides, each with a 6-amino acid arm:

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

[0106] The second identified polypeptide is p-Tau181 polypeptide-6, with the amino acid sequence Seq_6: TPPAPKTPPSSGE;

[0107] The third identified polypeptide: p-Tau181 polypeptide-7, with the amino acid sequence Seq_7: {pTHR}PPAPK{pTHR}PP{pSER}SGE;

[0108] The fourth identified polypeptide was p-Tau181 polypeptide-8, with the amino acid sequence Seq_8: {pTHR}PPAPKTPP{pSER}SGE.

[0109] Example 3

[0110] This embodiment provides a polypeptide composition for identifying p-Tau181 antibodies, comprising four identification polypeptides, each with a 5-amino acid arm:

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

[0112] The second identified polypeptide is p-Tau181 polypeptide-10, with the amino acid sequence Seq_10: PPAPKTPPSSG;

[0113] The third identified polypeptide is p-Tau181 polypeptide-11, with the amino acid sequence Seq_11: PPAPK{pTHR}PP{pSER}SG;

[0114] The fourth identified polypeptide was p-Tau181 polypeptide-12, with the amino acid sequence Seq_12: PPAPKTPP{pSER}SG.

[0115] Example 4

[0116] Verify the site specificity of antibody AT270:

[0117] p-Tau181 antibody (Phospho-Tau(Thr181) Monoclonal Antibody (AT270), manufacturer: Fujirebio, catalog number: 90007) has been identified in the literature as having the property of specifically recognizing phosphorylated threonine at position 181 of the Tau protein. At higher concentrations of peptides phosphorylated at both amino acids 181 and 175 of the Tau protein, this antibody shows a positive reaction to both. As the peptide concentration decreases, the reaction signal of the antibody to the peptide phosphorylated at amino acid 175 decreases faster than that to the peptide phosphorylated at amino acid 181, and ultimately at a low concentration, the reaction signal with the peptide phosphorylated at amino acid 181 is 18 times that with the peptide phosphorylated at amino acid 175.

[0118] The 12 identified sequence peptides from Examples 1 to 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, and 0.00195 μg / mL using coating buffer (pH 9.6, Na2CO3 1.59 mg / mL, NaHCO3 2.93 mg / mL).

[0119] Add 100 μL of the diluted identification sequence peptide to each well of a 96-well microplate and incubate at 37°C for 2 h. Wash the plate three times with PBST buffer for 60 s each time, and blot dry. Add 200 μL of blocking buffer per well, incubate at 37°C for 2 h, wash, and blot dry.

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

[0121] 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.

[0122] 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 1–3.

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

[0124]

[0125]

[0126] Table 2 shows the reactivity of the polypeptide composition from Example 2 with AT270 antibody and p-Tau181 antibody, respectively.

[0127]

[0128]

[0129] Table 3 shows the reactivity of the polypeptide composition in Example 3 with AT270 antibody and p-Tau181 antibody, respectively.

[0130]

[0131]

[0132] Based on the detection results of antibody AT270 and four identification peptides (Example 1, arm length 7), when the peptide coating concentration is 0.125-2 μg / mL, the reaction concentration of the peptide is too high, and antibody AT270 has a certain degree of non-specificity to certain phosphorylation groups (such as cross-reactivity with peptides phosphorylated at amino acid position 175 as reported in the literature). Therefore, the reaction with p-Tau181 peptide-1, p-Tau181 peptide-3, and p-Tau181 peptide-4 is positive, while the reaction with p-Tau181 peptide-2 is negative.

[0133] As the coating concentration of the peptides decreased, the antibody's response signal to p-Tau181 peptide-4 decreased even more rapidly. At a concentration of 0.03125 μg / mL, the signal of p-Tau181 peptide-4 was one-third that of p-Tau181 peptide-1 and p-Tau181 peptide-3. When the coating concentration fell below 0.03125 μg / mL, due to the extremely low concentration of participating peptides, the antibody's reaction with each peptide gradually shifted to weakly positive (weak cross-reactivity) or negative (no cross-reactivity).

[0134] The above results indicate that although this antibody has some nonspecificity to phosphate groups, it mainly targets phosphorylated threonine at position 181 of the Tau protein, which is consistent with the literature reports, and this method is effective.

[0135] The reaction of the antibody with the four identification peptides of arm length 5 (Example 3) and arm length 6 (Example 2) was similar to that of arm length 7.

[0136] Example 5

[0137] Site specificity verification of commercially available p-Tau181 antibodies

[0138] The four p-Tau181 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.

[0139] p-Tau181 antibody-1 to p-Tau181 antibody-12 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.

[0140] 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.

[0141] Dilute the substrate buffer and the substrate solution TMB 1:1, mix thoroughly, and add 100 μL to each well. Incubate at room temperature for 5-10 min. Add 50 μL of stop solution to each well, mix well, and read the OD450 nm value of each well using a microplate reader. OD ≥ 1 indicates a positive result; 0.5 ≤ OD < 1 indicates a weak positive result; and OD < 0.5 indicates a negative result.

[0142] Table 4. Reactivity of 12 commercially available p-Tau181 antibodies and that of Example 1

[0143]

[0144]

[0145] Table 5. Reactivity of 12 commercially available p-Tau181 antibodies and that in Example 2

[0146]

[0147]

[0148] Table 6. Reactivity of 12 commercially available p-Tau181 antibodies and that in Example 3

[0149]

[0150]

[0151] The reactions of p-Tau181 antibody-12 and p-Tau181 peptide-1, p-Tau181 peptide-5, p-Tau181 peptide-9, p-Tau181 peptide-3, p-Tau181 peptide-7, and p-Tau181 peptide-11 were all positive, while the reactions of p-Tau181 peptide-2, p-Tau181 peptide-6, p-Tau181 peptide-10, p-Tau181 peptide-4, p-Tau181 peptide-8, and p-Tau181 peptide-12 were all negative, meeting the screening requirements and indicating that they are specific antibodies against the phosphorylation of threonine at position 181 of the Tau protein. The remaining antibodies did not meet the screening requirements.

[0152] Among them, the reactions of p-Tau181 antibody-7 and p-Tau181 peptide-2, p-Tau181 peptide-6, and p-Tau181 peptide-10 were negative, while the reactions of p-Tau181 peptide-1, p-Tau181 peptide-5, p-Tau181 peptide-9, p-Tau181 peptide-3, p-Tau181 peptide-7, p-Tau181 peptide-11, p-Tau181 peptide-4, p-Tau181 peptide-8, and p-Tau181 peptide-12 were all positive, indicating that they may produce non-specific reactions with phosphate groups at non-target sites.

[0153] The identification peptides designed from the fourth identification sequence—p-Tau181 peptide-4, p-Tau181 peptide-8, and p-Tau181 peptide-12—are crucial. Without this fourth identification sequence, since the identification results of the first three sequences of p-Tau181 antibody-7 and p-Tau181 antibody-12 are consistent, it would be impossible to detect that p-Tau181 antibody-7 may react nonspecifically with phosphate groups at non-target sites, thus preventing its exclusion.

[0154] Example 1: Specificity of the reaction between p-Tau antibody and antigen.

[0155] 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-Tau181 antibody-12 (meeting screening requirements) and p-Tau antibody-7 (which recognizes p-Tau181 but has poor specificity), total Tau antibody was added to each antigen, and a double-antibody sandwich chemiluminescent immunoassay was used to react with the antigens.

[0156] Cross-reactivity evaluation of p-Tau antibody with antigen and its analogues

[0157] (1) Preparation of p-Tau antibody-conjugated toluenesulfonyl (Tysol) magnetic beads and working solution

[0158] p-Tau181 antibody-12 and p-Tau181 antibody-7 were conjugated with Tysol magnetic beads (JSR, MS160 Tosylactivated 10%) 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.

[0159] 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.

[0160] (2) Preparation of alkaline phosphatase (AP) labeled total Tau antibody and working solution

[0161] 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.

[0162] Prepare a 0.1M MES buffer with a pH of 6.5, containing 1mM MgCl2, 0.1mM ZnCl2, 1mg / mL BSA, and 0.5mg / mL Proclin 300. Then, use this buffer to dilute the stock solution of total Tau antibody AP enzyme-conjugated ester to 1μg / mL.

[0163] (3) Antigen preparation

[0164] 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.

[0165] (4) On-machine test

[0166] We used the Shine i2900 fully automated chemiluminescence immunoassay analyzer manufactured by Shenzhen Yingkai Technology Co., Ltd.

[0167] 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.

[0168] The results are shown in Table 7:

[0169] Table 7 Reactivity of p-Tau antibody with antigens and analogues

[0170]

[0171]

[0172] When p-Tau181 antibody-12 reacted with three p-Tau181 antigens, signals changed with the concentration gradient in all cases, with the highest RLU values ​​exceeding 100,000. For some p-Tau181 antigens at concentrations below 1 ng / mL, the concentration was too low, falling below the affinity level for the antibody-antigen reaction, resulting in RLU values ​​less than 1000 and no gradient signal. However, this still indicates that p-Tau181 antibody-12 can recognize the three p-Tau181 antigens. When p-Tau181 antibody-12 reacted with four Tau antigens, one p-Tau217 antigen, and one p-Tau231 antigen, the measured RLU values ​​for all antigen concentrations showed no gradient and were less than 1000, indicating that p-Tau181 antibody-12 does not recognize Tau antigen, p-Tau217 antigen, or p-Tau231 antigen. In conclusion, p-Tau181 antibody-12 specifically recognizes the p-Tau181 antigen.

[0173] When p-Tau181 antibody-7 reacted with three p-Tau181 antigens and one p-Tau231 antigen, signals changed with the concentration gradient, and the highest RLU was greater than 100,000, indicating that p-Tau181 antibody-7 can recognize not only p-Tau181 antigen but also p-Tau231 antigen. When p-Tau181 antibody-7 reacted with four Tau antigens and one p-Tau217 antigen, the measured RLU for all antigen concentrations showed no gradient and was less than 3,000, indicating that p-Tau181 antibody-7 does not recognize Tau antigen or p-Tau217 antigen. Therefore, although p-Tau181 antibody-7 does not cross-react with Tau antigen or p-Tau217 antigen and can recognize p-Tau181 antigen, it does exhibit significant cross-reactivity with p-Tau231 antigen. Consistent with the site-specific verification conclusions in Example 5, p-Tau181 antibody-7 may exhibit non-specific reactions with phosphate groups at non-target sites. Therefore, the fourth identification sequence is essential in the method for determining whether an antibody specifically recognizes the target phosphorylation site.

[0174] The above results demonstrate that, based on the reactivity evaluation with four peptides, the phosphorylation site-specific antibody screened in Example 5 only recognizes the antigen phosphorylated at position 181 of the Tau protein, and shows no cross-reactivity with antigens that are very similar in sequence and structure.

[0175] 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 is as follows: to identify whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein. The polypeptide composition consists of four identification polypeptides, which are as follows: The amino acid sequence of the first identified peptide is shown in Seq_1; the amino acid sequence of the second identified peptide is shown in Seq_2; the amino acid sequence of the third identified peptide is shown in Seq_3; and the amino acid sequence of the fourth identified peptide is shown in Seq_4. Alternatively, the amino acid sequence of the first identified polypeptide is shown in Seq_5; the amino acid sequence of the second identified polypeptide is shown in Seq_6; the amino acid sequence of the third identified polypeptide is shown in Seq_7; and the amino acid sequence of the fourth identified polypeptide is shown in Seq_8. Alternatively, the amino acid sequence of the first identified polypeptide is shown in Seq_9; the amino acid sequence of the second identified polypeptide is shown in Seq_10; the amino acid sequence of the third identified polypeptide is shown in Seq_11; and the amino acid sequence of the fourth identified polypeptide is shown in Seq_12.

2. An antibody identification method, characterized in that, The identification is as follows: to identify whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein; The antibody identification method includes: reacting the antibody to be identified with the four identification peptides described in claim 1, and if the detection results simultaneously satisfy (I) to (III), then the antibody to be identified is determined to be an antibody that specifically recognizes the phosphorylation site at position 181 of the Tau protein. (I) The antibody to be identified reacts positively with both the first and third identifying polypeptides; (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.

3. The method according to claim 2, characterized in that, A test result value greater than or equal to the first threshold is considered positive; a value greater than or equal to the second threshold and less than the first threshold is considered weakly positive; and a value less than the second threshold is considered negative. The first threshold is 1, and the second threshold is 0.

5.

4. The method according to claim 2, characterized in that, The working concentrations of the four identified peptides were independently 0.03125–0.0625 μg / mL.

5. The method according to claim 4, characterized in that, The working concentrations of the four identified peptides were each 0.03125 μg / mL.

6. The method according to claim 2, characterized in that, The method includes: (a) Dilute the four identification peptides separately with a coating buffer; the coating buffer is a sodium carbonate-sodium bicarbonate buffer with a pH of 9.6; (b) Add the four diluted identification peptides to the ELISA plate for incubation, wash the plate, and then block it; (c) Dilute the antibody to be tested to 0.5 μg / mL with PBS buffer, add it to the coated microplate, incubate at 35~39℃ for 57~63 min, and then wash the plate. (d) Add horseradish peroxidase-labeled secondary antibody, incubate at 37°C for 1-2 hours, and wash the plate; (e) Add the colorimetric reagent and react at room temperature for 5-10 min. Add the stop solution to terminate the reaction and detect OD at 450 nm. When OD ≥ 1, it is interpreted as positive; when 0.5 ≤ OD < 1, it is interpreted as weakly positive; when OD < 0.5, it is interpreted as negative.

7. A kit for identifying whether an antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein, characterized in that, The polypeptide composition comprising claim 1.

8. The application of the kit according to claim 7 in antibody identification, wherein the identification is: identifying whether the antibody specifically recognizes the phosphorylation site at position 181 of the Tau protein.

9. The method for preparing the reagent kit according to claim 7, characterized in that, This includes designing and synthesizing the polypeptide composition as described in claim 1.

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