Polypeptides, polypeptide compositions, kits, and related applications

Abstract

The present application provides a kind of polypeptide, polypeptide composition, kit and related application.The polypeptide is the polypeptide shown in SEQ ID NO:1 to SEQ ID NO:85, can be specifically bound as follows target antibody: human SNRPC / U1C antibody, human PCNA antibody, human CENPB antibody, human NUP210 / gp210 antibody, human FTCD / 58K golgi protein antibody and GAD antibody.The above-mentioned multiple specific binding target antibody polypeptide or polypeptide set can cover the peptide segment including linear epitope and nonlinear epitope, so that the number of polypeptides used can be reasonably selected according to the number of target antibodies to be detected, which can improve the detection sensitivity and specificity of each polypeptide, improve the detection accuracy, and improve the throughput and efficiency of the detected antibody.

Description

Technical Field

[0001] This invention relates to the field of antibody detection, and more specifically, to a polypeptide, a polypeptide composition, a reagent kit, and related applications. Background Technology

[0002] In the immune response, cellular immunity and humoral immunity are two closely related and mutually regulated physiological processes. In clinical laboratory work, the detection of specific antibodies in the humoral immune response is the most widely used. Specific antibody detection is of great clinical significance. It not only assists in clinical diagnosis—for example, the detection of specific antibodies in serum or plasma can serve as an auxiliary reference for the clinical diagnosis of autoimmune diseases, infectious diseases, allergic diseases, and tumors—but also serves as an indicator for observing treatment efficacy and prognosis in some diseases. Furthermore, it has special and important significance in observing the effectiveness of vaccination and in the epidemiological investigation of infectious diseases.

[0003] Currently, there are numerous methods for antibody detection. In addition to traditional precipitation reactions, agglutination tests, and complement fixation tests, labeled immunoassays, such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays, fluorescence immunoassays, and luminescent immunoassays, have become the main immunoassay techniques. Immunoblotting and rapid dot immunobinding assays are also widely used. However, these traditional methods typically can only detect one antibody marker per experiment, and they have low detection sensitivity and require large sample volumes.

[0004] Recently, there have been reports of antibody detection based on peptide microarray methods. However, this method still has some problems in clinical application for the detection of specific antibodies against common diseases, such as:

[0005] 1) Peptide chips designed based on antigen sequence information can detect linear epitopes relatively clearly, and the corresponding peptides can be used for antibody detection. However, this method cannot meet the detection requirements for the ubiquitous nonlinear epitopes. Although there are methods to constrain peptides to simulate discontinuous and conformation-dependent epitopes using one or more disulfide bonds, this only increases the possibility of detecting a small number of nonlinear epitopes.

[0006] 2) Random sequence peptide chips contain a large number of peptides, and the core recognition site of the target antibody conformational epitope may only be a few amino acids. When 4 to 5 amino acid residues in the peptide perfectly match, the antibody can bind. Therefore, the signal peptide after incubation of the random sequence chip with the antibody may contain 1) antigenic epitopes including linear epitopes and / or conformational epitopes; 2) sequences containing core recognition amino acids of linear epitopes and / or conformational epitopes; 3) non-specific binding peptides, etc., but non-specific interference signals cannot be eliminated by sequence alignment and other methods.

[0007] 3) Antibodies in the body are polyclonal antibodies with multiple unique antigenic epitopes. Using peptides to simulate a single epitope for antibody detection can easily lead to missed detections.

[0008] 4) Clinical antibody testing mainly uses serum / plasma samples, which have complex components. The specific peptides obtained by analyzing pure antibodies after incubation on a peptide chip may be affected by interference from other proteins in serum / plasma.

[0009] 5) There are many types of antibodies involved in clinical practice (for example, infectious diseases, allergic diseases, tumors and other diseases require the detection of many types of antibodies; taking autoimmune diseases as an example, one autoimmune disease can produce a variety of autoantibodies, and the same autoantibody can exist in a variety of autoimmune diseases), and the detection frequency is also high.

[0010] Therefore, new specific antibody testing products are still needed to meet the aforementioned clinical testing needs. Summary of the Invention

[0011] The main objective of this invention is to provide a polypeptide, a polypeptide composition, a reagent kit, and related applications, in order to provide an antibody detection product with higher specificity.

[0012] To achieve the above objectives, according to one aspect of the present invention, a polypeptide is provided that is capable of specifically binding to a target antibody, wherein the target antibody is selected from any one of antibodies against human SNRPC / U1C, human PCNA, human CENPB, human NUP210 / gp210, human FTCD / 58K Golgi protein, and GAD antibodies; when the target antibody is an antibody against human SNRPC / U1C, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 10; when the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; or when the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; when the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO: 35; when the target antibody is an antibody against human FTCD / 58K Golgi protein, the polypeptide is selected from SEQ ID NO: 10. Any one or more of SEQ ID NO: 36 to SEQ ID NO: 43; when the target antibody is a GAD antibody, the polypeptide is selected from any one or more of SEQ ID NO: 44 to SEQ ID NO: 85.

[0013] Further, the polypeptide is a modified peptide segment; preferably, the modification is a chemical group modification or an amino acid modification; preferably, the chemical group modification is PEG modification; preferably, the PEG modification is a linear PEG modification, a PEG modification with a monofunctional group, or a PEG modification with a bifunctional group; preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the polypeptide; preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40000; preferably, the amino acid modification is a hydrophilic amino acid modification or a cysteine ​​modification; preferably, the hydrophilic amino acid modification is... One to four hydrophilic amino acids are added to the N-terminus, C-terminus, or NC-terminus of the polypeptide. More preferably, the hydrophilic amino acids are Glu, Lys, Ser, or Gly. More preferably, the one to four hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser. Preferably, cysteine ​​modification is the addition of cysteine ​​at any of the following positions of the polypeptide: N-terminus, C-terminus, NC-terminus, or the middle of the peptide chain. More preferably, adding cysteine ​​to the middle of the peptide chain includes inserting one or more cysteines into the middle of the peptide chain, or one or more cysteines being linked to the middle of the peptide chain in a branched form.

[0014] According to a second aspect of the present invention, a polypeptide product is provided, the polypeptide product comprising the aforementioned polypeptide.

[0015] Furthermore, the polypeptide product also includes a polypeptide stabilizer; preferably, the polypeptide stabilizer includes 150-180 mM NaCl, 100-140 mM polylysine hydrochloride and water; the polypeptide product is a polypeptide chip, and the polypeptides on the polypeptide chip are composed of the aforementioned polypeptides.

[0016] According to a third aspect of the present invention, a polypeptide composition is provided comprising a plurality of the aforementioned polypeptides.

[0017] According to a fourth aspect of the present invention, an antibody detection reagent is provided, the reagent comprising the aforementioned polypeptide.

[0018] According to a fifth aspect of the present invention, an antibody detection kit is provided, the kit comprising the aforementioned polypeptide.

[0019] Furthermore, the kit includes a detection chip, on which peptides are disposed, and the peptides on the detection chip are composed of the aforementioned peptides.

[0020] According to a sixth aspect of the present invention, the use of the aforementioned polypeptide in the preparation of a detection antibody kit is provided.

[0021] According to a seventh aspect of the present invention, an antibody detection method is provided, the method comprising: using a target polypeptide specifically bound by a target antibody to detect a test sample and a negative control of the test sample, and obtaining corresponding detection results for each; if there is a significant difference between the detection results of the test sample and the negative control, it indicates that the test sample contains a target antibody; the target antibody is selected from any one or more of the following: antibodies against human SNRPC / U1C, human PCNA, human CENPB, human NUP210 / gp210, human FTCD / 58K Golgi protein, and GAD antibody, and the target polypeptide is one or more of the aforementioned polypeptides.

[0022] Furthermore, the target peptides include target peptides of multiple target antibodies, and each target antibody has multiple target peptides, which are set on a peptide chip. Using a peptide chip containing target peptides, or a peptide chip composed of target peptides, the test sample and negative control are detected under the condition of blocking the peptide chip, and the corresponding detection results are obtained. If there is a significant difference between the detection results of the target peptides in the test sample and the negative control, it indicates that the test sample contains the target antibody. Preferably, the test sample is a clinical sample diluted with a sample diluent, and the negative control is the sample diluent. More preferably, the clinical sample is a serum sample or a plasma sample. Preferably, the sample diluent is a PBST buffer containing D-mannitol, wherein the D-mannitol content in the PBST is 0.5%–1% by mass / volume. Preferably, the blocking condition for the peptide chip refers to blocking the peptide chip with a blocking solution before detecting the test sample and the negative control of the test sample. Preferably, the blocking solution includes the following components: 130–137 mM sodium chloride, 2.5–2.7 mM potassium chloride, 3.8–4.3 mM disodium hydrogen phosphate, 1.2–1.4 mM potassium dihydrogen phosphate, 0.05%–1% Tween-20 v / v, and 0.05%–0.1% Proclin 950. The blocking solution contains 0.5%–1% D-mannitol w / v and 0.1%–1% casein w / v, with a pH of 7.2–7.6, more preferably 7.38–7.42; more preferably, the blocking solution contains 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium hydrogen phosphate, 1.4 mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin 950 v / v, 1% D-mannitol w / v and 0.1% casein w / v, and the pH of the blocking solution is 7.4.

[0023] Furthermore, the detection method includes: establishing an antibody prediction model, inputting the fluorescence signals corresponding to the test sample and negative control into the antibody prediction model; and outputting the detection results of the test sample. Among them, establishing the antibody prediction model includes: obtaining the signal intensity of the target peptide detected by the peptide chip under closed conditions in the positive and negative serum samples based on multiple positive serum samples known to be positive for the target antibody and multiple negative serum samples known to be negative, and establishing an antibody prediction model based on the relationship between the signal intensity and the positive or negative of the target antibody.

[0024] By applying the technical solution of this invention, the polypeptides provided in this application can specifically bind to corresponding target antibodies. In application, one or more polypeptides targeting a single target antibody can be selected for detection, or multiple polypeptides targeting multiple target antibodies can be used for detection, depending on actual needs. When using multiple polypeptides to detect a single target antibody, since different polypeptides may include both linear and non-linear epitopes that bind to the antibody, the combined use of multiple polypeptides for detection of the target antibody can achieve more sensitive and effective antibody detection. When multiple polypeptides are used to detect multiple target antibodies, not only can the detection sensitivity and specificity of each polypeptide be improved, and the detection accuracy increased, but the throughput and efficiency of the detected antibodies can also be increased. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 A schematic diagram illustrating the principle of antibody detection using a polypeptide chip according to an embodiment of the present invention is shown.

[0027] Figure 2 The following is a heatmap of peptide chip-based detection of single and mixed antibodies provided according to an embodiment of the present invention;

[0028] Figure 3 A statistical graph showing the results of detecting GAD antibodies in clinical serum samples based on a peptide chip according to an embodiment of the present invention, compared with the actual results;

[0029] Figure 4 The diagram shows the ROC curve analysis of GAD antibodies in clinical serum samples based on a peptide chip according to an embodiment of the present invention. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0031] Terminology Explanation:

[0032] Antibodies are glycoproteins found in blood and tissue fluid. They are produced by plasma cells, which differentiate from B cells after they have been stimulated by antigens. Primarily present in body fluids such as serum, they specifically bind to their corresponding antigens and are important effector molecules mediating humoral immunity. B cells activated by antigen stimulation can produce 10 antibodies within one week. 11 A copy of a single-specific antibody.

[0033] An epitope, also called an antigenic determinant, is an antigenic portion that is specifically recognized by antigen receptors TCR and BCR. There are two types of epitopes: linear epitopes (epitaxes that are continuous in sequence) and conformational epitopes (epitaxes that are spatially adjacent but not continuous in sequence).

[0034] Polypeptide: In this application, polypeptide or peptide segment has the same meaning and can be used interchangeably, both referring to amino acid sequence fragments.

[0035] Peptide chip: A chip based on a substrate material, which includes features with pre-designed quantity, location and sequence. Each feature is a cluster of peptides with the same sequence. The peptide sequences between features are often different. These features form a high-density peptide array.

[0036] Peptide chip technology is a detection technology based on peptide chips. It utilizes the contact between a variety of peptides on a peptide chip and the sample, then employs image acquisition technology to capture various characteristic signals from the peptide chip (specifically, fluorescence images carrying these signals). The signal intensity of each feature in the chip is then output, representing the peptide chip detection result data. Based on the sample detection signal output from the peptide chip detection result data, analysis of analytes in samples bound to peptides on the peptide chip, and other sample analysis can be achieved.

[0037] ROC curve: A curve reflecting the relationship between sensitivity and specificity. The horizontal axis (X-axis) represents 1 - specificity, also known as the false positive rate; the closer the X-axis is to zero, the higher the accuracy. The vertical axis (Y-axis) represents sensitivity, also known as the true positive rate; a larger Y-axis value indicates better sensitivity. Based on the curve's position, the entire graph is divided into two parts. The area under the curve is called the AUC (Area Under Curve), used to represent predictive accuracy; a higher AUC value indicates higher predictive accuracy. The closer the curve is to the upper left corner (smaller X, larger Y), the higher the predictive accuracy.

[0038] Peptide chip antibody detection technology is a cutting-edge technology in the biochip industry. It primarily relies on peptides to specifically capture antibodies in bodily fluid samples, enabling high-throughput antibody detection. Compared to traditional antibody detection methods, chip detection technology can detect multiple antibody indicators in a single experiment, requires less sample volume, offers faster analysis and detection speeds, is safer and more environmentally friendly, and allows for advanced automation and integration. Furthermore, compared to protein chip antibody detection technology, peptide chip manufacturing processes are more stable, resulting in more reliable and accurate data, and offering a more competitive price.

[0039] For example, the polypeptide chip (HealtTell V13) used in this application contains over 130,000 synthetic polypeptides with known sequences. Each polypeptide contains 5-13 amino acids. The amino acid combination of the polypeptides is an unbiased random combination, measuring the overall level of free antibodies in body fluids according to the immunological principles of antigen-antibody competitive binding and amino acid-amino acid interactions. A schematic diagram of the principle of polypeptide chip antibody detection is shown below. Figure 1 As shown.

[0040] As mentioned in the background section, it is difficult to design all conformational epitopes recognized by antibodies based on sequence information. Peptide chips (HealthTell's V13 chip) contain 130,000 synthetic peptides with known sequences. Their amino acid combinations cover 99.9% of 4-mer amino acid combinations (4-mer refers to a set of peptide sequences consisting of 4 amino acids, with adjacent peptide sequences differing by one amino acid, such as MGAS, GAST, ASTC, etc. Similarly, 5-mer refers to a peptide sequence consisting of 5 amino acids, with adjacent peptide sequences differing by one amino acid) and 48.3% of 5-mer amino acid combinations, making them more suitable for antibody binding analysis of linear and conformational epitopes.

[0041] However, random peptide chips contain a large number of peptides and have high detection sensitivity. When the chip is incubated with an antibody and then a fluorescently labeled secondary antibody is added, the signal may include the following: 1) antigenic epitopes including linear epitopes and / or conformational epitopes; 2) sequences containing core recognition amino acids of linear epitopes and / or conformational epitopes; 3) non-specific binding peptides; 4) background signals of the secondary antibody itself, etc. For non-linear epitopes, the target antigen sequence alignment method is not suitable for determining whether an antibody corresponds to an antigenic epitope peptide among numerous binding signal peptides. In addition, for polyclonal antibodies, antigenic epitopes are not unique. Detecting as many target antigenic epitopes as possible is crucial for the sensitivity of detecting antibodies corresponding to target antigens in subsequent complex samples. Furthermore, from the perspective of subsequent clinical antibody detection applications, complex samples such as serum / plasma are often used, increasing interference from other proteins and antibodies in the sample, resulting in complex signals. Traditional specific peptides based on direct detection of a single antibody may not necessarily indicate the presence or absence of antibodies in clinical samples. Therefore, only specific peptides with high specificity and unaffected by interference from other proteins in complex samples can be used for the development of subsequent diagnostic kits, etc.

[0042] Therefore, this application improves upon existing peptide chip antibody detection methods based on the detection principle of random peptide chips. The screening method in this application systematically eliminates interfering signals, particularly simulating the interference of complex signals in clinical serum samples on antibody detection, thereby identifying a set of antibody-specific anti-interference peptides for stable detection in complex samples. Furthermore, in the screening process for antibody-specific binding peptides with strong anti-interference capabilities, a standardized screening procedure was developed, providing guidance for the screening and identification of such antibody-specific binding peptides.

[0043] In order to develop detection products for one or more antibodies suitable for clinical auxiliary diagnosis of diseases, the peptide collection of this application is screened by using antibody samples of different concentrations and different solution backgrounds under closed or unclosed conditions of peptide chip to screen peptide libraries with high sensitivity and specificity for specific binding to target antibodies. Validated using serum samples from clinically known antibody-negative or antibody-positive cohorts, the peptides or sets thereof screened in this application (including both linear antigenic epitopes and conformational epitopes, where linear epitopes can be identified by comparison with the protein sequence of the corresponding antigen, while conformational epitopes are independent of the antigen sequence but can simulate the state of antigen-antibody binding, and even have stronger binding ability than the antigen) exhibit higher sensitivity than existing antibody detection methods (such as mainstream IVD antibody detection kits, i.e., ELISA methods) not only when detecting samples containing a single antibody, but also when detecting samples containing multiple antibodies (the limit of detection of the peptide chip detection method in this application reaches 0.05 ng / mL, while the limit of detection of the reference ELISA is 1.56 ng / mL). This demonstrates that these peptides can serve as antigenic epitope peptides for detecting the corresponding antibodies, and can be used to detect the presence or absence of the corresponding antibodies in the serum sample to be tested. Based on this, the applicant has proposed the technical solution of this application.

[0044] To more clearly illustrate the solution of this application, the types of samples involved in this application are described herein:

[0045] Positive samples include: sample diluents containing single or mixed antibodies (such as PBST containing single or mixed antibodies) and serum samples containing single or mixed antibodies. Serum samples containing single or mixed antibodies further include: simulated clinical positive serum samples prepared by combining single or mixed antibodies with negative serum, and real clinical positive serum samples containing single or mixed antibodies from known population cohorts.

[0046] Negative samples include: sample diluents that do not contain a single antibody or a mixture of antibodies (such as PBST samples that do not contain a single antibody or a mixture of antibodies) and serum samples that do not contain a single antibody or a mixture of antibodies. Serum samples that do not contain a single antibody or a mixture of antibodies further include: serum samples from healthy individuals.

[0047] In one typical embodiment of this application, a polypeptide is provided, which can specifically bind to a target antibody. The target antibody is selected from any one of the following: antibodies against human SNRPC / U1C, human PCNA, human CENPB, human NUP210 / gp210, human FTCD / 58K Golgi protein, and GAD antibodies. When the target antibody is an antibody against human SNRPC / U1C, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 10; when the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; or when the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; when the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO: 35; when the target antibody is an antibody against human FTCD / 58K Golgi protein, the polypeptide is selected from SEQ ID NO: 36 to SEQ ID NO: 10. Any one or more of SEQ ID NO: 43; when the target antibody is a GAD antibody, the polypeptide is selected from any one or more of SEQ ID NO: 44 to SEQ ID NO: 85.

[0048] The peptides provided in this application can specifically bind to their corresponding target antibodies. In application, one or more peptides targeting a single target antibody, or multiple peptides targeting multiple target antibodies, can be selected for detection, depending on actual needs. When using multiple peptides to detect a single target antibody, since different peptides may include both linear and non-linear epitopes that bind to the antibody, the combined use of multiple peptides for detection can achieve more sensitive and effective antibody detection. When using multiple peptides to detect multiple target antibodies, not only can the detection sensitivity and specificity of each peptide be improved, thus increasing detection accuracy, but the throughput and efficiency of the detected antibodies can also be increased.

[0049] To further enhance the affinity of certain peptides, in some preferred embodiments, one or more peptide segments of the aforementioned peptides are modified peptides. Specific modifications can be chemical group modifications or amino acid modifications. Preferably, the chemical group modification is PEG modification. More preferably, the PEG modification is linear PEG modification, PEG modification with a monofunctional group, or PEG modification with a difunctional group (the functional group can be -NHS, -OH, -Mal, -NH2, or -COOH; the monofunctional group is selected from any one of these, and the difunctional group is selected from any two of these). Preferably, the PEG modification site is selected from any one or more of the N-terminus, C-terminus, Lys side chain, and Cys side chain of the polypeptide. Preferably, the PEG modification is a PEG modification with a molecular weight of 500 to 40,000. Preferably, the amino acid modification is hydrophilic amino acid modification or cysteine ​​modification. Preferably, the amino acid modification is hydrophilic amino acid modification or cysteine ​​modification. Preferably, the hydrophilic amino acid modification involves adding 1-4 hydrophilic amino acids at the N-terminus, C-terminus, or both N and C-termini. Preferably, the hydrophilic amino acid is Glu, Lys, Ser, or Gly. Preferably, the 1-4 hydrophilic amino acids are selected from any one of the following: Glu-Glu, Lys-Lys, or Ser-Gly-Ser.

[0050] The aforementioned PEG modification or hydrophilic amino acid modification can increase the hydrophilicity of peptides. PEG modification has advantages such as prolonged half-life, reduced toxicity, and enhanced physical, chemical, and biological stability.

[0051] In some embodiments, to better achieve targeted coupling of the peptide, any one or more peptide segments in the aforementioned peptide can be cysteine-modified peptide segments. Specifically, this includes, but is not limited to, adding cysteine ​​at the N-terminus, C-terminus, or NC-terminus of the peptide segment, or adding cysteine ​​in the middle of the peptide chain. When adding cysteine ​​in the middle of the peptide chain, one or more cysteines can be inserted into the middle of the peptide chain (i.e., inserted between two amino acid residues), or one or more cysteines can be linked to the middle of the peptide chain in a branched form (i.e., as a side chain of a certain amino acid in the middle of the peptide chain).

[0052] In a second typical embodiment of this application, a polypeptide product is provided, comprising any of the aforementioned polypeptides. The polypeptides, as detection products for detecting corresponding target antibodies, can be in any product form capable of utilizing these polypeptides, such as an ELISA kit or a polypeptide chip. When it is an ELISA kit, one or more of the aforementioned polypeptides are detected by encapsulating them in a solid-phase support (such as microspheres). When the polypeptide product is a polypeptide chip, the polypeptide chip is composed of the aforementioned multiple polypeptides.

[0053] The aforementioned polypeptide product is used as a testing reagent. To further improve the stability of its active ingredient, the polypeptide, the product also includes a polypeptide stabilizer.

[0054] In a preferred embodiment of this application, the peptide stabilizer comprises 150–180 mM NaCl, 100–140 mM polylysine hydrochloride, and water. More preferably, the peptide stabilizer comprises 153–158 mM NaCl, 110–130 mM polylysine hydrochloride, and water; even more preferably, the peptide stabilizer comprises 154 mM NaCl, 126.4 mM polylysine hydrochloride, and water. Specifically, in the peptide stabilizer, the concentration of NaCl can be 150mM, 151mM, 152mM, 153mM, 154mM, 155mM, 156mM, 157mM, 158mM, 159mM, 160mM, 161mM, 162mM, 163mM, 164mM, 165mM, 166mM, 167mM, 168mM, 169mM, 170mM, 171mM, 172mM, 173mM, 174mM, 175mM, 176mM, 177mM, 178mM, 179mM, or 18... 0 mM; the concentration of polylysine hydrochloride can be 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, 120 mM, 121 mM, 122 mM, 123 mM, 124 mM, 125 mM, 126 mM, 127 mM, 128 mM, 129 mM, 130 mM, 131 mM, 132 mM, 133 mM, 134 mM, 135 mM, 136 mM, 137 mM, 138 mM, 139 mM or 140 mM.

[0055] In a preferred embodiment of this application, the concentration of the polypeptide in the above-mentioned polypeptide product is 3-10 μM, preferably 5-8 μM, and more preferably 5 μM. More specifically, it can be 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM.

[0056] In a preferred embodiment of this application, as described above, the polypeptide product is a polypeptide chip, and the polypeptides on the polypeptide chip are composed of the aforementioned polypeptides. All of the polypeptides capable of specifically binding to the target antibody can be disposed on the polypeptide chip, thereby enabling the simultaneous detection of multiple samples or multiple antibodies. This improves detection throughput and efficiency, and also facilitates the accurate determination of the target antibody status of the sample under test by comprehensively considering the detection results of multiple target antibodies.

[0057] Therefore, in a preferred embodiment, the use of any of the above-mentioned peptide products in the detection of the corresponding target antibody is provided.

[0058] In a third typical embodiment of this application, a polypeptide composition is provided, comprising multiple of the aforementioned polypeptides. These polypeptides can be disposed on a chip for detection in the form of a composition, or they can be encapsulated in a solid-phase support for detection using a method similar to an ELISA kit.

[0059] In a preferred embodiment, the use of any of the above-described polypeptide compositions in the detection of the corresponding target antibody is also provided.

[0060] In a fourth typical embodiment of this application, a reagent for detecting a target antibody is provided, the reagent comprising any one or more of the above-described polypeptides.

[0061] In a fifth exemplary embodiment of this application, a kit for detecting a target antibody is provided, the kit comprising any one or more of the aforementioned peptides. Preferably, the kit comprises a detection chip on which the aforementioned peptides are disposed.

[0062] The application of any one or more of the above-mentioned peptides in the preparation of kits for detecting target antibodies.

[0063] In a preferred embodiment, the use of any of the above reagents or kits in detecting the corresponding target antibody is also provided.

[0064] The above applications can be prepared into various types of detection kits according to specific needs. The specific form of the kit is not limited; for example, it can be an ELISA kit, an immunofluorescence kit, or an immunogold kit. From the perspective of convenient detection and easy interpretation of test results, the peptides in the kit are preferably pre-coated peptides. Preferably, the pre-coated peptides are coated on a solid support; the specific pre-coated solid support is designed reasonably according to needs. More preferably, the solid support includes an ELISA plate (mostly made of polystyrene), a membrane carrier, or microspheres; even more preferably, the membrane carrier includes a nitrocellulose membrane (the most widely used), a glass cellulose membrane, or a nylon membrane; even more preferably, the membrane carrier is also coated with a positive control, and the peptide-carrier protein conjugate and the positive control are sequentially arranged on the nitrocellulose membrane according to the detection order.

[0065] Depending on the specific detection method of the kit, the specific reagents in the kit will also vary accordingly, but they can all be combined according to the known preparation method of the kit. Preferably, the above kit also includes at least one of the following: (1) enzyme-labeled secondary antibody, more preferably HRP-labeled secondary antibody (corresponding to ELISA detection kit); (2) colloidal gold conjugate pad, the colloidal gold conjugate pad is coated with a specific conjugate of colloidal gold-labeled peptide and positive control (corresponding to immunogold detection kit); (3) label pad, the label pad is coated with fluorescently labeled microspheres, the microspheres are loaded with a specific conjugate of positive control (corresponding to immunofluorescence detection kit).

[0066] The aforementioned immunochromatographic and immunofluorescence assay kits offer greater convenience, requiring only the establishment of a C-line for the positive control and a T-line for the test sample. The positive control pre-coated at the C-line can be any specific conjugate carrying a detection marker that binds to the sample during serum chromatography; there are no specific limitations on the specific peptide or antibody used. Preferably, the positive control is selected from mouse immunoglobulin, human immunoglobulin, goat immunoglobulin, or rabbit immunoglobulin, and correspondingly, the specific conjugate is selected from anti-mouse immunoglobulin, anti-human immunoglobulin, anti-goat immunoglobulin, or anti-rabbit immunoglobulin.

[0067] The aforementioned anti-mouse immunoglobulins, depending on the target animal, can be sheep anti-mouse immunoglobulins, rabbit anti-mouse immunoglobulins, or anti-mouse immunoglobulins from other immunizable animals. Similarly, anti-human, anti-sheep, or anti-rabbit immunoglobulins can also be derived from different species depending on the immunized animal. These immunoglobulins can be any one of IgM, IgG, IgA, IgD, or IgE. These anti-immunoglobulin antibodies can be monoclonal or polyclonal antibodies.

[0068] The specifications of the microplates used in the above kits vary depending on the number of samples to be tested, and can be reasonably selected from 12 to 384-well microplates.

[0069] In a sixth typical embodiment of this application, an antibody detection method is provided. The detection method includes: using a target polypeptide specifically bound by a target antibody to detect a test sample and a negative control of the test sample, and obtaining corresponding detection results for each; if there is a significant difference between the detection results of the test sample and the negative control, it indicates that the test sample contains the target antibody; wherein, the target antibody is selected from any one or more of the following: antibody against human SNRPC / U1C, antibody against human PCNA, antibody against human CENPB, antibody against human NUP210 / gp210, antibody against human FTCD / 58K Golgi protein, and GAD antibody, and the target polypeptide is one or more of the above-mentioned polypeptides of this application.

[0070] By using the aforementioned peptides that can specifically bind to the corresponding target antibodies to detect the test samples, multiple peptides can be used to detect multiple samples at once, and / or multiple target antibodies at once. By combining the detection results of multiple peptides, it is possible to efficiently, rapidly, with high throughput, high sensitivity, and high accuracy detect whether the test samples contain the corresponding target antibodies.

[0071] The specific operation of the above detection method varies depending on whether the peptide is set in an ELLISA-like form or in a peptide chip form. In a preferred embodiment of this application, the target peptide includes target peptides of multiple target antibodies, and there are multiple target peptides for each target antibody. The multiple target peptides are set on a peptide chip (that is, the target peptides for detection are set on the chip to form a peptide array chip for detection). Using a peptide chip containing target peptides, or a peptide chip composed of target peptides (that is, the peptide chip may consist only of multiple or all of the peptides selected in this application from SEQ ID NO:1 to SEQ ID NO:85, or it may also include other peptide fragments, which may be the target antibodies mentioned in this application or other antibodies of interest), under the condition of blocking the peptide chip, the test sample and the negative control are detected to obtain their respective corresponding detection results; if there is a significant difference between the detection results of the target peptide in the test sample and the negative control, it indicates that the test sample contains the target antibody.

[0072] By utilizing a sealed peptide chip to detect the presence or absence of target antibodies in the test sample, false positives caused by non-specific binding of the chip itself to the test sample can be reduced or eliminated. Depending on the number of peptides on the peptide chip and the types of antibodies they bind to, this method can detect one or more test samples, or one or more antibodies. Because these peptides have high binding specificity to their respective antibodies, the detection accuracy is also relatively high. Therefore, it is more suitable for clinical samples with complex antibody environments. Preferably, the test sample is a clinical sample diluted with a sample diluent, and the negative control is the sample diluent; more preferably, the clinical sample is a serum sample or a plasma sample.

[0073] The sample diluent can be any buffer system that can stabilize the sample to be tested and the antibodies contained therein. In this application, when loading samples for peptide chip detection, it is preferable to use clinical serum or clinical plasma samples diluted with the sample diluent, as the diluted samples are more conducive to detection on the peptide chip.

[0074] In a preferred embodiment of this application, the sample diluent is a PBST buffer containing D-mannitol, wherein the D-mannitol content in the PBST is 5%–1% by mass / volume. Using this sample diluent not only provides a stable ionic environment and pH buffering capacity, maintaining the activity of the antibody protein, but also provides a stable solution environment under sample incubation conditions. This avoids interference with experimental results without affecting the normal binding of the peptide-antibody, providing accurate, stable, and effective data for subsequent fluorescence imaging. Preferably, the sample dilution formulation is as follows: 130–137 mM sodium chloride, 2.5–2.7 mM potassium chloride, 3.8–4.3 mM disodium hydrogen phosphate, 1.2–1.4 mM potassium dihydrogen phosphate, 0.05%–1% Tween-20 v / v, 0.05%–0.1% Proclin 950 v / v, 0.5%–1% D-mannitol w / v, with a pH of 7.4; more preferably, the sample diluent formulation is: 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium hydrogen phosphate, 1.4 mM potassium dihydrogen phosphate, 0.05% Tween 20 (v / v), 0.1% Proclin 950 (v / v), and 1% D-mannitol (w / v), with a pH of 7.4.

[0075] When the peptide chip contains not only some or all of the peptides shown in SEQ ID NO:1 to SEQ ID NO:85, but also other peptide fragments, the detection method of this application can use the chip's built-in analysis software to analyze the detection results, or write an analysis program to perform relevant analysis. However, it is only necessary to analyze whether there is a significant difference between the target peptide of interest in the test sample and the control sample. This helps to reduce the amount of data and improve the analysis speed and efficiency.

[0076] In the preferred embodiments described above, "blocking conditions" refers to blocking the peptide chip with a blocking solution before using the blocked peptide chip for antibody detection of the sample. Blocking the peptide chip with a blocking solution before loading the sample helps reduce the signal intensity of non-specifically binding peptides. The blocking solution can also be a commonly used blocking solution. To more effectively reduce the background signal of the high-density peptide array, in the preferred embodiments of this application, the composition of the blocking solution differs from that of commonly used blocking reagents.

[0077] The active ingredients in typical blocking reagents include BSA, animal serum, and Fab fragment single-chain secondary antibodies. BSA is a relatively simple component and may contain bovine IgG, thus exhibiting strong cross-reactivity with anti-bovine, goat, sheep, and horse secondary antibodies, leading to background noise. Some animal blocking sera may contain sodium azide, making them unsuitable for HRP-labeled detection systems. Fab fragment single-chain secondary antibodies are complex to prepare, expensive, and not suitable for large-scale use. In short, existing blocking solutions have potential problems such as causing non-specific binding and background signals, high cost, and unstable blocking effects.

[0078] In a preferred embodiment of this application, the blocking solution comprises the following components: 130–137 mM sodium chloride, 2.5–2.7 mM potassium chloride, 3.8–4.3 mM disodium hydrogen phosphate, 1.2–1.4 mM potassium dihydrogen phosphate, 0.05%–1% Tween-20 v / v, 0.05%–0.1% Proclin 950 v / v, 0.5%–1% D-mannitol w / v, and 0.1%–1% casein w / v; the pH is 7.2–7.6, preferably 7.38–7.42. In a more preferred embodiment of this application, the blocking solution is 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium hydrogen phosphate, 1.4 mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin 950 v / v, 1% D-mannitol w / v, 0.1% casein w / v, and the pH is adjusted to 7.4 with 1N hydrochloric acid or sodium hydroxide.

[0079] The blocking solution provided in this application offers a stable solution environment under secondary antibody incubation conditions. It reduces non-specific adsorption of the secondary antibody, particularly to solid-phase components outside the peptide array on the peptide chip, without affecting the normal binding of the peptide-antibody pair. This avoids interference with experimental results and facilitates the provision of accurate, stable, and reliable data for subsequent fluorescence imaging. Furthermore, the blocking solution is low-cost and stable, and it enhances specificity and sensitivity in immunological detection, especially when using peptide array chips or in other scientific research.

[0080] The species source of the secondary antibody used in the detection method of this application can be any of the following: rabbit, goat, sheep, rat, mouse, guinea pig, chicken or donkey.

[0081] In the detection method of this application, since the more peptides used, the more data is generated, the less accurate and more labor-intensive it is to use conventional ELISA kits to detect whether fragment antibodies are positive or negative. Therefore, this application preferably uses artificial intelligence to process such "big data". For example, an antibody prediction model can be established using peptide chip detection results data corresponding to clinically known positive or negative samples, and then the antibody prediction model can be used to detect whether the target antibody is positive or negative in the sample to be tested. The specific modeling method can be based on existing data modeling methods, such as ridge regression. In a preferred embodiment, the above detection method includes: establishing an antibody prediction model; inputting the fluorescence signals corresponding to the sample to be tested and the negative control into the antibody prediction model; and outputting the detection results of the sample to be tested. The establishment of the antibody prediction model includes: obtaining the signal intensity of the target peptide detected by the peptide chip under closed conditions in the positive and negative serum samples based on multiple positive serum samples known to be positive for the target antibody and multiple negative serum samples known to be negative, and establishing an antibody prediction model based on the relationship between the signal intensity and the positive or negative of the target antibody.

[0082] The following section uses commercially available antibodies to further explain the peptide screening method of this application (peptide microarray detection with progressively increasing interference conditions), which mainly includes the following steps:

[0083] 1) Under non-blocking conditions, the antibody is diluted with sample diluent (preferably multiple concentrations, or two concentrations, such as 10ng / mL-1μg / mL) and then tested on the instrument;

[0084] 2) Under closed conditions, the sample is diluted with a sample diluent (preferably multiple concentrations, or two concentrations, such as 10 ng / mL-1 μg / mL) and then tested on the instrument.

[0085] 3) Under closed conditions, the antibody is mixed with the negative serum background and then diluted (preferably multiple concentrations, or two concentrations, such as 10ng / mL-1μg / mL) for instrument detection;

[0086] Then, by eliminating blocking fluid and serum background interference signals step by step, the peptides specifically bound to the antibody samples were analyzed.

[0087] For example: Experimental design with progressively increasing interference conditions

[0088] Table 1:

[0089]

[0090] It should be noted that: the blocking condition refers to incubating the sample (i.e. the specific commercial antibody in this application) with the peptide chip in a blocking solution before adding it to the peptide chip; after incubation, the sample is added to the peptide chip for further incubation and detection.

[0091] Final antibody concentration for assay: The concentration of antibody in the reaction system when performing peptide chip detection;

[0092] The detection background refers to the solution system in which the antibody is placed when performing peptide chip detection.

[0093] The testing process is as follows:

[0094] 1. Sample Preparation: Antibody samples were dissolved in sample diluent and negative serum respectively to obtain high and low antibody concentrations, denoted as [low antibody + sample diluent], [high antibody + sample diluent], [low antibody + serum], and [high antibody + serum], respectively. Separately, [antibody-free + sample diluent] and [antibody-free + serum] samples were prepared as negative controls.

[0095] 2. Sample dilution: Dilute the above sample 1000 times with sample diluent and shake well.

[0096] 3. Sample incubation: Under non-blocking conditions, add 1000-fold diluted samples of [low antibody + sample diluent], [high antibody + sample diluent], and negative control samples of [antibody-free + sample diluent], along with blank (washing buffer), incubate at constant temperature, and wash the plate. Under blocking conditions, add samples of [low antibody + sample diluent], [high antibody + sample diluent], [low antibody + serum], [high antibody + serum], and negative control samples of [antibody-free + sample diluent] and [antibody-free + serum] in sequence, incubate with blank (washing buffer) at constant temperature, and wash the plate.

[0097] 4. Secondary Antibody Incubation: Add secondary antibodies according to the species of the antibody (e.g., if the antibody is mouse-derived, choose anti-mouse secondary antibody; if the antibody is rabbit-derived, choose anti-rabbit secondary antibody; if the antibody is human-derived, choose anti-human secondary antibody), incubate at a constant temperature, and wash the plate. The negative control secondary antibody should be selected according to the species of the antibody detected on the same detection chip. If there is more than one species of antibody on the same detection chip, add a corresponding control.

[0098] 5. Fluorescence Imaging: The chip is transferred to an imaging device, fluorescence signals are scanned, and high-resolution images are generated.

[0099] 6. Image processing: Convert the images obtained from fluorescence imaging into fluorescence intensity values ​​to obtain the corresponding numerical matrix.

[0100] 7. Preliminary Data Processing and Quality Control: Perform logarithmic transformation and standardization on the sample values ​​to output a standardized matrix. Also perform single-sample quality control and system stability quality control.

[0101] Data analysis process:

[0102] 1. Using antibody-peptide chip detection experiments with progressively increasing interference conditions, we aim to identify antibody-specific peptide sets that are unaffected by blocking solutions and other serum proteins. The hierarchical peptide analysis approach is as follows:

[0103] 1) For a single detection antibody, first screen out the high-affinity peptides (denoted as Lv1) that the peptide chip platform can stably capture;

[0104] 2) For multiple detection antibodies, exclude peptides that have cross-identification, thereby obtaining the specific peptide for detecting a single antibody (denoted as Lv2).

[0105] 3) The number of specific peptides detected under the blocking conditions is lower than that detected under the non-blocking conditions, thus eliminating the interference of the blocking solution on the screened peptides and obtaining a specific binding peptide resistant to the interference of the blocking solution (denoted as Lv3).

[0106] 4) Detection was performed in both blocked and serum environments to obtain a specific binding polypeptide (denoted as Lv4) that resists dual interference (blocking solution and serum environment);

[0107] 5) When the screened peptides are applied, the peptides that specifically bind to multiple antibodies are combined (denoted as Lv5) to detect whether the target antibody is present in the serum at one time, and whether the target antibody is one or more.

[0108] 2. The specific steps are as follows:

[0109] (1) By detecting the same antibody at different concentrations under non-blocking conditions, the signal intensity of the peptide signal in the sample is obtained. Peptides that are significantly higher than the signal intensity threshold of the negative control sample with no antibody + sample dilution are selected as the initial seed peptide library. False positive signals introduced by production and experimental processes are excluded, and the set of high-signal peptides stably captured by each antibody platform - Lv1 peptide set - is obtained. The Lv1 peptide sets of each antibody that have been detected are compared with each other to exclude peptides shared by antibodies against different immunogens or homologous immunogens. These peptides include systematic interference signals caused by factors such as antibody preparation and purification methods. The set of specific peptides for each antibody - Lv2 peptide set - is output, which can indicate the presence or absence of a specific antibody under non-blocking conditions.

[0110] (2) Analyze the peptide signals detected under two blocking conditions (high and low concentration) of the same antibody. Screen for peptide sets that are significantly higher than the signal intensity threshold of the negative control sample (no antibody + sample dilution). Eliminate false positive signals introduced in the production experiment to obtain a set of high-signal peptides stably captured by the platform under blocking conditions for each antibody. Then compare the various antibodies to exclude peptides shared by antibodies against different immunogens or homologous immunogens, and preliminarily obtain a set of antibody-specific peptides under blocking conditions. Eliminate false positive signals caused by the interaction between blocking solution components and antibodies. Take the intersection of antibody-specific peptides under blocking and non-blocking conditions -Lv3, which can indicate the presence or absence of a specific antibody under blocking conditions.

[0111] (3) Analyze the peptide signals detected under high and low concentration blocking conditions when the same antibody is mixed into the serum background. Screen for peptide sets that are significantly higher than the signal intensity threshold of the negative control sample (no antibody + serum). Exclude false positive signals introduced by the production experiment as indicated. Obtain the set of high-signal peptides stably captured by the platform under blocking conditions and in the presence of serum background. Then compare the various antibodies to exclude peptides shared by antibodies against different immunogens or homologous immunogens. Preliminarily obtain the set of antibody-specific peptides under blocking conditions and in the serum background. To eliminate false positive signals caused by the interaction between the blocking solution components and serum background components and the antibody, take the intersection of the above-mentioned specific peptide set and the Lv3 peptide set - Lv4, which can indicate the presence or absence of a specific antibody in the serum background under blocking conditions.

[0112] The beneficial effects of the present invention are further illustrated below with reference to specific embodiments. It should be noted that the sample diluent used in the following embodiments has the following composition: PBST containing 1% D-mannitol, where PBST is PBS containing 0.05%-1% Tween 20. Specifically, the PBS 1×PBS (pH 7.9, Teknova) has the following formulation: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin 950 v / v, and 1% D-mannitol w / v.

[0113] Blocking solution: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin 950 v / v, 1% D-mannitol w / v and 0.1% casein w / v, pH of the blocking solution is 7.4.

[0114] Example 1: Highly reliable and specific antibody-binding peptides for the recognition of antibody components in mixed antibodies

[0115] Eight antibodies were included, and their highly reliable specific binding peptides were analyzed using a step-by-step filtering method to remove interference. Four of these antibodies were selected for analysis using two-antibody, three-antibody, and four-antibody mixed assays, respectively. This step-by-step approach, increasing interference conditions, employed peptide chip technology for detection, including direct detection under non-blocking conditions, detection under blocked conditions, and detection of antibodies mixed with negative serum samples. The final concentrations for individual antibody assays were set to 50 ng / ml and 500 ng / ml, while the final concentration for mixed antibody assays was set to 50 ng / ml for all assays.

[0116] A antibody alone detection

[0117] I. Sample Preparation

[0118] 1. Antibody sample information confirmation: including pretreatment operation requirements, concentration, source species, etc. See the table below for details.

[0119] 2. Information on the detection antibodies involved in this application is shown in the table below:

[0120] Table 2:

[0121]

[0122] Note:

[0123] PCNA antibodies: Proliferating Cell Nuclear Antigen (PCNA) was first discovered and named by Miyachi et al. in 1978 in the serum of SLE (Systemic Lupus Erythematosus) patients. It is named for its presence only in normal proliferating cells and tumor cells. PCNA is a 36 kDa protein synthesized and present in the cell nucleus, serving as a cofactor for DNA polymerase δ. PCNA antibodies have long been used clinically for the specific detection of SLE, but the positive rate is very low, with an IIF (intracellular infusion) method positive rate of only 2%–5%. Recent studies have shown that it can be detected in various autoimmune diseases, with SLE remaining the primary detectable disease. Patients with SLE who are positive for anti-PCNA are more likely to experience skin rashes, Raynaud's phenomenon, neuropsychiatric lupus, and kidney involvement, which are associated with disease activity.

[0124] CENPB antibodies: Centromere proteins consist of three components: centromere protein A (CENPA), centromere protein B (CENPB), and centromere protein C (CENPC); among them, CENPB, with a relative molecular mass of 80,000, is the main target antigen of anti-centromere antibodies. Anti-centromere proteins have long been considered to have high specificity for systemic sclerosis (SSc), especially the CREST subtype, whose main clinical manifestations include calcification, Raynaud's phenomenon, esophageal motility dysfunction, finger sclerosis, and telangiectasia. The positive rate of anti-centromere antibodies in patients with CREST syndrome is 40%–90%.

[0125] gp210 antibody: Anti-gp210 antibody is a type of anti-nuclear envelope protein antibody that exhibits a nuclear membrane-type fluorescent staining pattern. Its target antigen is a 210kD transmembrane glycoprotein located on the nuclear pore complex. This antibody is present in the serum of some patients with primary biliary cirrhosis (PBC). Antimitochondrial antibody (AMA) is a specific antibody for diagnosing PBC, with a sensitivity of up to 90%. However, 10% of PBC patients are AMA antibody negative, indicating that AMA has certain limitations in the diagnosis of PBC. Anti-gp210 antibody is present in 20%–47% of AMA-negative PBC patients. For patients with clinical, biochemical, and histological presentations suggestive of PBC but negative for AMA, or for patients with AMA positive but atypical clinical symptoms or overlapping syndromes (such as overlap with Sjögren's syndrome), anti-gp210 antibody detection is of significant value. In addition, the incidence of cirrhosis is significantly higher in gp210-positive patients than in negative patients. Therefore, a positive result for this antibody suggests a poor prognosis and can also serve as a prognostic indicator for PBC patients.

[0126] LC-1 antibody: In 1988, Martini et al. first confirmed the presence of anti-LC-1 antibody in the serum of 6 adult AIH patients using immunofluorescence (IIF) and ID methods. Its target antigen is present in the cytosol of hepatocytes, and is detected by Western blotting (IB) as a 58kD–62kD hepatocyte cytoplasmic polypeptide protein. Anti-hepatocyte cytoplasmic antigen type 1 antibody (anti-LC-1 antibody), also known as anti-hepatocyte cytoplasmic antigen type 1 antibody, is a specific and sensitive marker antibody for type II autoimmune hepatitis (AIH-II), with a positive rate of 56%–72% in AIH-II, and is closely related to the diagnosis of autoimmune hepatitis.

[0127] HIB antibody: Hib is invasive Haemophilus influenzae type b, a common commensal bacterium in the nasopharynx of children. It is an estimated bacterium that causes serious illness in about 3 million people and causes an estimated 386,000 deaths annually, mainly through meningitis and pneumonia.

[0128] SARS-CoV-E: The SARS virus causes "atypical pneumonia". Its E protein is an envelope protein. Antibody testing for this protein can help diagnose whether a sample has been infected with the SARS virus.

[0129] GAD Antibody: Anti-glutamate decarboxylase antibodies are suitable for the diagnosis of late-onset autoimmune diabetes mellitus in adults. Glutamate decarboxylase (GAD) is the rate-limiting enzyme that converts glutamate into the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). GAD is present in pancreatic β cells, which also synthesize and secrete GABA. Glutamate decarboxylase antibody (GADA) is an immunomarker in the early stages of type 1 diabetes and also serves as an indicator for monitoring the efficacy of treatment in type 1 diabetes patients. The positive rate of GADA is significantly higher in patients with type 1 diabetes and Graves' disease than in type 1 diabetes patients without Graves' disease. GADA levels are significantly elevated in patients with Graves' disease. The presence of GADA in non-diabetic patients does not always predict the occurrence of type 1 diabetes.

[0130] Pf: Antibody against Plasmodium falciparum ferroredoxin-NADP+ reductase (pfFNR). Plasmodium falciparum is one of the four types of Plasmodium parasites that parasitize humans and is the pathogen that causes falciparum malaria. Plasmodium falciparum uses humans and female Anopheles mosquitoes as hosts. In humans, it initiates schizogony and gametogenesis. In mosquitoes, it completes gametogenesis and spore proliferation. Both parasites develop in hepatocytes and erythrocytes within the human body.

[0131] 3. Sample pretreatment: Some of the powdered antibodies were dissolved and then combined with some antibodies for which concentration information was not available for analysis. All antibodies to be tested were uniformly diluted to 100 μg / ml using sample diluent.

[0132] 4. Preparation of total IgG for quality control (total IgG sample obtained from the same serum sample by ammonium persulfate precipitation method; in other examples, unless otherwise specified, the total IgG for quality control is the same).

[0133] II. Detection of pure antibodies under non-closed conditions

[0134] Each antibody was diluted with sample diluent to 50 ng / ml and 500 ng / ml respectively for peptide chip detection;

[0135] The secondary antibody for antibody detection should be selected according to the species of origin information (for example, if the antibody is rabbit-derived, then the secondary antibody is an anti-rabbit secondary antibody).

[0136] Use antibodies from the same species as much as possible on the same chip, and add sample diluent to each test chip as a control with an antibody concentration of 0 (i.e., a negative control without antibody);

[0137] The secondary antibody for control is selected according to the species of the antibody detected on the same detection chip. If the antibody species on the same detection chip are from more than one source, a control is added accordingly (i.e., if the antibody on the same detection chip is from both rabbit and mouse sources, then rabbit and mouse control secondary antibodies are set on that chip).

[0138] Detection operation

[0139] 1. Sample dilution

[0140] a) Dilution of total IgG in quality control samples

[0141] Dilute the IgG using a 5ml centrifuge tube. Add 16μL of IgG to 1584μL of sample diluent to form a 100-fold diluted IgG, and vortex to mix.

[0142] b) Antibody dilution

[0143] The antibodies were diluted to 500 ng / ml and 50 ng / ml using sample diluent, respectively.

[0144] 1) Take 2 μL of antibody with a concentration of 100 μg / ml and add it to 398 μL of sample dilution buffer to form a diluted antibody with a concentration of 500 ng / ml (gradient 1), and vortex to mix.

[0145] 2) Add 20 μL of gradient 1 to 180 μL of sample dilution buffer to form a diluted antibody (gradient 2) with a concentration of 50 ng / ml, and vortex to mix.

[0146] 2. Layout and proofing

[0147] According to the sample arrangement design, dispense 120 μL each of the 500 ng / ml diluted antibody, 50 ng / ml diluted antibody, 100-fold diluted IgG and sample diluent into the corresponding wells of the PCR plate. Then, use a multi-channel pipette to transfer 90 μL into the corresponding wells of the assayCassette and seal the plate.

[0148] 3. First incubation

[0149] 1) Turn on the thermostat mixer in advance and stabilize the temperature at 37℃.

[0150] 2) Incubate the assay cassette on a constant temperature mixer for 60 minutes, shaking the cassette for 15 seconds every 6 minutes.

[0151] 4. First wash of the board

[0152] After stopping the oscillation and tearing off the film, place the plate into the plate washer. Ensure that the waste liquid bottle of the plate washer has sufficient capacity, and use the automatic plate washer to wash the plate. 405LS Microplate Magnetic Washing Machine.

[0153] 5. Add secondary antibody

[0154] Preparation of secondary antibody dilution buffer: Under light-protected conditions (lamp off), use a 15ml centrifuge tube to dilute the rabbit secondary antibody 1500 times (9μL of secondary antibody added to 13500μL of secondary antibody dilution buffer). Then, according to the well position information in the layout table, use a multi-channel pipette to add 40μL / well to the corresponding well of the corresponding Cassette, and seal the membrane.

[0155] It should be noted that in other embodiments, depending on the species origin of the antibody, such as mouse or human, the corresponding secondary antibody and its corresponding dilution conditions are selected. For example, human secondary antibody (i.e., antibody of human antibody) can be diluted 1500 times (4 μL of secondary antibody added to 6 ml of secondary antibody diluent), and mouse secondary antibody (i.e. antibody of mouse antibody) can be diluted 3000 times (2 μL of secondary antibody added to 6 ml of secondary antibody diluent).

[0156] 6. Second incubation

[0157] Seal the cassette and incubate it on a thermostat mixer for 60 minutes, shaking the cassette for 15 seconds every 6 minutes.

[0158] 7. Second wash

[0159] After stopping the oscillation and tearing off the film, place the plate into the plate washer. Ensure the waste liquid bottle of the plate washer has sufficient capacity, and use the automatic plate washer to wash the plate. 405LS Microplate Magnetic Washing Machine.

[0160] 8. Chip Removal

[0161] 1) Prepare the operating area in the biosafety cabinet, including a microchip centrifuge, a cotton absorbent pad, a 90% isopropyl alcohol spray bottle, a basin containing MilliQ chips with chip slots and handles, a hydration box containing MilliQ chips, and a clean chip storage box. Carefully remove each chip from the assay cassette, one at a time, and place it on the chip slot.

[0162] 2) Dry the chips and pack them into boxes.

[0163] 9. Assemble the chip in the imaging cassette.

[0164] 10. Fluorescence Imaging

[0165] The Imaging System uses a robotic arm to transfer the imaging cassette to the barcode scanning location. After both the imaging cassette and the chip barcode are scanned, the cassette is transferred to Image Xpress for imaging. The system guides the operator through Venus software, requiring the operator to set the exposure time and the number of imaging cassettes. Once imaging begins, the operator can continue adding imaging cassettes to be imaged.

[0166] The imaging process uses a Molecular Devices Image Xpress 4 imager to generate high-resolution TIFF images for each array. Gridding Software extracts feature intensity data from the TIFF image files.

[0167] Once the imaging cassette is assembled, it will be transferred to the cassette hotel. The automated imaging process will then commence.

[0168] The specific data preprocessing process is as follows:

[0169] 1) Extract the fluorescence intensity values ​​of the features and output one GPR5 data file and one corner images file. The GPR5 file contains all the information of a sample and the fluorescence intensity information of all features.

[0170] 2) Extract feature fluorescence intensity information from the GPR5 data files of all samples to generate a raw fluorescence intensity (FG, foreground) data matrix. Then, perform a logarithmic transformation on the data of each sample to obtain an LFG (log-transferred foreground) data matrix. Finally, subtract the median of the LFG data matrix for each sample to obtain an NLFG (normalized and log-transferred foreground) data matrix. This step also generates a sample chip information file, which includes information such as the sample array location and the chip number used.

[0171] 3) Quality control

[0172] The samples and system passed quality control using Health Tell's built-in quality control methods.

[0173] III. Detection of Pure Antibody under Closed Conditions

[0174] Under closed conditions, the antibody was diluted to a final concentration of 50 ng / ml and 500 ng / ml with sample diluent and then detected by peptide chip.

[0175] For antibody testing, the secondary antibody should be selected according to the information on the species of origin.

[0176] Antibodies from the same species should be used as much as possible on the same chip, and one well of pure sample diluent should be added to each detection chip as a control with an antibody concentration of 0.

[0177] The secondary antibody should be selected according to the species of the detection antibody on the same detection chip. If there is more than one species of antibody on the same detection chip, a control should be added accordingly.

[0178] Detection operation

[0179] 1. Sample dilution:

[0180] a) Dilution of total IgG in quality control samples

[0181] Dilute the IgG using a 1.5 ml centrifuge tube. Add 16 μL of IgG to 784 μL of sample diluent to form a 50-fold diluted IgG. Shake well.

[0182] b) Antibody dilution

[0183] The antibodies were diluted to 1000 ng / ml and 100 ng / ml using the sample dilution buffer, respectively.

[0184] 1) Take 2 μL of antibody with a concentration of 100 μg / ml and add it to 18 μL of sample dilution buffer to form a diluted antibody with a concentration of 10 μg / ml (gradient 1). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0185] 2) Add 15 μL of gradient 1 antibody to 135 μL of sample dilution buffer to form a diluted antibody concentration of 1000 ng / ml (gradient 2). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0186] 3) Add 12 μL of gradient 2 to 108 μL of sample dilution buffer to form a diluted antibody concentration of 100 ng / ml (gradient 3). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0187] c) Preparation of sealing solution

[0188] Add 2 ml of 1% casein to 18 ml of sample diluent, mix well and set aside.

[0189] 2. Layout and proofing

[0190] 1) Chip encapsulation

[0191] Dispense 45 μL of the prepared blocking solution into the wells of the Cassette using a multi-pipette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate at 37°C for 1 hour.

[0192] 2) Sample loading for sealed Cassette (chip)

[0193] According to the sample arrangement design, 45 μL of diluted antibody (1000 ng / ml) and diluted antibody (100 ng / ml) were transferred into the corresponding wells of the Cassette using a multi-channel pipette.

[0194] 3. First incubation

[0195] Seal the Cassette film and place it on the incubation module of the automated instrument for 1 hour (see the first incubation under non-sealed conditions for specific procedures).

[0196] 4. First wash of the board

[0197] Tear off the film from the Cassette and place it in an automatic plate washer for washing (see the first plate wash under non-enclosed conditions for specific operation).

[0198] 5. Add secondary antibody

[0199] Under light-protected conditions (lights off), using 15ml centrifuge tubes, dilute the human secondary antibody 1500-fold (4μL of secondary antibody added to 6ml of secondary antibody diluent), and the mouse secondary antibody 3000-fold (2μL of secondary antibody added to 6ml of secondary antibody diluent). Using 50ml centrifuge tubes, dilute the rabbit secondary antibody 1500-fold (9μL of secondary antibody added to 13500μL of secondary antibody diluent). Then, according to the well position information in the layout table, use a multipipe to add 40μL to the corresponding well of the corresponding Cassette.

[0200] 6. Second incubation

[0201] Seal the cassette and incubate it on a thermostatic mixer for 60 minutes, shaking the cassette for 15 seconds every 6 minutes.

[0202] 7. Second wash

[0203] After stopping the oscillation and tearing off the film, place the plate into the plate washer. Ensure the waste liquid bottle of the plate washer has sufficient capacity, and use the automatic plate washer to wash the plate. 405LS Microplate Magnetic Washing Machine.

[0204] 8. Chip Removal

[0205] 1) Prepare the operating area in the biosafety cabinet, including a microchip centrifuge, a cotton absorbent pad, a 90% isopropyl alcohol spray bottle, a basin containing MilliQ chips with chip slots and handles, a hydration box containing MilliQ chips, and a clean chip storage box. Carefully remove each chip from the assay cassette, one at a time, and place it on the chip slot.

[0206] 2) Dry the chips and pack them into boxes.

[0207] 9. Assemble the chip in the imaging cassette.

[0208] 10. Fluorescence Imaging

[0209] The Imaging System uses a robotic arm to transfer the imaging cassette to the barcode scanning location. After both the imaging cassette and the chip barcode are scanned, the cassette is transferred to Image Xpress for imaging. The system guides the operator through Venus software, requiring the operator to set the exposure time and the number of imaging cassettes. Once imaging begins, the operator can continue adding imaging cassettes to be imaged.

[0210] The imaging process uses a Molecular Devices Image Xpress 4 imager to generate high-resolution TIFF images for each array. Gridding Software extracts feature intensity data from the TIFF image files.

[0211] Once the imaging cassette is assembled, it will be transferred to the cassette hotel. The automated imaging process will then commence.

[0212] IV. Detection of antibody contamination with negative serum under closed conditions

[0213] Under closed conditions, antibodies were added to negative serum at concentrations of 50 μg / ml and 500 μg / ml, respectively. The serum was then diluted 1000 times for peptide chip detection. (In practice, the serum can be diluted first, and then the antibody can be added according to the final concentration for the reaction. The final concentration for the reaction is the concentration of the antibody in the system during the peptide chip detection reaction. In specific operations, considering the consumption of serum and sample, the serum and antibody sample can be diluted separately to achieve the requirement of 1000-fold dilution of background serum and antibody concentrations of 50 ng / ml and 500 ng / ml in the reaction system before adding the antibody.)

[0214] For antibody testing, the secondary antibody should be selected according to the information on the species of origin.

[0215] Antibodies from the same species should be arranged on the same chip as much as possible, and a control with a 1:1000 dilution of negative serum should be added to each chip as an antibody concentration of 0.

[0216] The secondary antibody should be selected according to the species of the antibody detected by the same detection chip. If the antibody species on the same detection chip are from more than one source, a control should be added accordingly.

[0217] Detection operation

[0218] 1. Sample dilution:

[0219] a) Dilution of total IgG in quality control samples

[0220] Dilute the IgG using a 1.5 ml centrifuge tube. Add 16 μL of IgG to 784 μL of sample diluent to form a 50-fold diluted IgG. Shake well.

[0221] b) Serum dilution

[0222] Using a 50ml centrifuge tube, take 60μL of serum sample and add it to 29940μL of sample dilution buffer to form a 500-fold diluted serum. Shake to mix.

[0223] c) Antibodies are mixed into serum for dilution

[0224] The antibodies were diluted to 1000 ng / ml and 100 ng / ml using a 500-fold dilution of serum, respectively. The dilution steps are as follows:

[0225] 1) Take 2 μL of antibody with a concentration of 100 μg / ml and add it to 18 μL of sample dilution buffer to form a diluted antibody with a concentration of 10 μg / ml (gradient 1). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0226] 2) Take 10 μL of gradient 1 antibody and add it to 90 μL of 500 times serum to form a diluted antibody concentration of 1000 ng / ml (gradient 2). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0227] 3) Take 10 μL of gradient 2 and add it to 90 μL of 500 times serum to form a diluted antibody concentration of 100 ng / ml (gradient 3). Seal the membrane, vortex to mix, and centrifuge (4000 rpm for 1 min).

[0228] d) Preparation of sealing solution

[0229] Add 2 ml of 1% Casein to 18 ml of sample diluent, mix well and set aside.

[0230] 2. Layout and proofing

[0231] 1) Chip encapsulation

[0232] Dispense 45 μL of the prepared blocking solution into the wells of the Cassette using a multi-pipette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate at 37°C for 1 hour.

[0233] 2) Sample loading for sealed Cassette (chip)

[0234] According to the sample arrangement design, 45 μL of diluted antibody (1000 ng / ml and 100 ng / ml) diluted with 500 times serum were transferred to the corresponding wells in the Cassette using a multi-channel pipette.

[0235] 3. First incubation

[0236] Seal the Cassette film and place it on the incubation module of the automated instrument for 1 hour (see the first incubation under non-sealed conditions for specific procedures).

[0237] 4. First wash of the board

[0238] Tear off the film from the Cassette and place it in an automatic plate washer for washing (see the first plate wash under non-enclosed conditions for specific operation).

[0239] 5. Add secondary antibody

[0240] Under light-protected conditions (lights off), using 15ml centrifuge tubes, dilute the human secondary antibody 1500-fold (4μL of secondary antibody added to 6ml of secondary antibody diluent), and the mouse secondary antibody 3000-fold (2μL of secondary antibody added to 6ml of secondary antibody diluent). Using 50ml centrifuge tubes, dilute the rabbit secondary antibody 1500-fold (9μL of secondary antibody added to 13500μL of secondary antibody diluent). Then, according to the well position information in the layout table, use a multipipe to add 40μL to the corresponding well of the corresponding Cassette.

[0241] 3. Second incubation

[0242] Seal the cassette and incubate it on a thermostatic mixer for 60 minutes, shaking the cassette for 15 seconds every 6 minutes.

[0243] 4. Second wash

[0244] After stopping the oscillation and tearing off the film, place the plate into the plate washer. Ensure the waste liquid bottle of the plate washer has sufficient capacity, and use the automatic plate washer to wash the plate. 405LS Microplate Magnetic Washing Machine.

[0245] 5. Imaging Scan (For detailed instructions, please refer to the sections on chip disassembly under non-enclosed conditions, chip assembly in the imaging cassette, and fluorescence imaging).

[0246] V. Data Analysis

[0247] The analysis was performed following a step-by-step approach to eliminate interference, yielding the specific binding peptides for each of the eight antibodies. The statistical results and sequences of some of the antibody-specific binding peptides are shown in the following two tables:

[0248] Table 3:

[0249] Antibody code Number of Lv4 peptide segments RNPC 55 PCNA 30 CENPB 65 gp210 57 LC-1 31 HIB 201 SARS-CoV-E 80 Pf 508 GAD 339

[0250] Table 4:

[0251]

[0252]

[0253]

[0254] B. Take a portion of the antibody for mixed antibody detection.

[0255] Hybrid antibody detection design:

[0256] Table 5:

[0257]

[0258]

[0259] In addition, add one well of total IgG quality control material to each test chip; the same procedure applies to both closed and unclosed chips.

[0260] a. In the absence of sealing fluid

[0261] Each antibody component of the mixed antibody was diluted with the same sample diluent to prepare a mixed antibody sample for peptide microarray detection. The final concentration of each antibody component in the sample was 50 ng / ml.

[0262] Select the appropriate anti-rabbit secondary antibody according to the species information of each antibody source. Add pure sample diluent to each detection chip to create a control with an antibody concentration of 0.

[0263] b. In the presence of a sealing liquid

[0264] Each antibody component of the mixed antibody was diluted with the same sample diluent to prepare a mixed antibody sample for peptide microarray detection. The final concentration of each antibody component in the sample was 50 ng / ml.

[0265] Select the appropriate anti-rabbit secondary antibody according to the species information of each antibody source. Add pure sample diluent to each detection chip to create a control with an antibody concentration of 0.

[0266] c. In the presence of a sealing liquid

[0267] Each antibody component of the mixed antibody was added to the same negative serum sample at a concentration of 50 μg / ml. The serum was then diluted 1000 times to prepare a mixed antibody sample with a serum background for use in peptide chip detection.

[0268] Select the appropriate anti-rabbit secondary antibody according to the species information of each antibody source. Add one well of negative serum diluted 1:1000 to each detection chip as a control with an antibody concentration of 0.

[0269] C. Antibody-specific binding peptides are used for antibody component analysis in mixed antibodies.

[0270] See the heatmap for mixed antibody detection. Figure 2 .exist Figure 2 In the figure, a heatmap of all Lv4 signal peptides of mixed antibodies 1-4 and control antibodies 5-8 is created. Each row represents one peptide, and each column represents one antibody sample. The first eight columns represent individual antibodies 1-8, respectively; the last three columns represent mixed antibody samples, namely mixed samples of antibodies 1 and 2, mixed samples of antibodies 1, 2, and 3, and mixed samples of antibodies 1, 2, 3, and 4, respectively. The brightness of each band represents the relative signal intensity of a single peptide in a single sample. As shown in the figure, antibodies 1 to 8 each have their own positive peptides. Signal peptides from one antibody only show high signal levels in that antibody, and the mixed samples simultaneously contain the specific peptide signals of each antibody component. Moreover, the detection signal level at the same concentration is comparable to the detection signal level of the antibody alone, and no abnormal signals are generated due to antibody mixing.

[0271] The above results indicate that antibodies and antibody combinations obtained by gradually eliminating interference (including removing cross-peptide fragments between multiple antibodies under different conditions, and taking the intersection of peptide sets detected by the same antibody under different conditions) have the advantage of high specificity.

[0272] Example 2: Highly reliable and specific antibody-binding peptides for identifying the presence or absence of specific antibodies in clinical samples.

[0273] A. Antibody-peptide microarray detection and analysis:

[0274] Screening of GAD antibody-specific binding peptides: This was performed in conjunction with peptide microarray detection of other monoclonal antibodies in Example 1, and the main procedures are as follows:

[0275] (1) Peptide chip detection

[0276] a. In the absence of sealing fluid

[0277] The antibodies were diluted with sample diluent before being detected using a peptide chip. The final antibody concentrations for detection were 50 ng / ml and 500 ng / ml.

[0278] Select the appropriate anti-rabbit secondary antibody according to the antibody's species of origin information. Add pure sample diluent to the detection chip to create a control with an antibody concentration of 0.

[0279] b. In the presence of a sealing liquid

[0280] The antibodies were diluted with sample diluent before being detected using a peptide microarray. The final antibody concentrations for detection were 50 ng / ml and 500 ng / ml.

[0281] Select the appropriate anti-rabbit secondary antibody according to the antibody's species of origin information. Add pure sample diluent to the detection chip to create a control with an antibody concentration of 0.

[0282] c. In the presence of a sealing liquid

[0283] The antibody to be tested was added to a negative serum sample at a concentration of 50 μg / ml. The serum was then diluted 1000 times to prepare an antibody sample with a serum background for use in peptide chip detection.

[0284] Select the appropriate anti-rabbit secondary antibody according to the antibody source species information. Add one well to the detection chip with a 1:1000 diluted negative serum as a control with an antibody concentration of 0.

[0285] d. Scanning imaging and data analysis (for specific operations, see the sections on chip disassembly under non-enclosed conditions, chip assembly in an imaging cassette, and fluorescence imaging).

[0286] After scanning and imaging, data processing is performed, and analysis is carried out according to the step-by-step analysis approach and specific steps to obtain the target antibody-specific binding peptide (used to detect the clinical serum samples of the confirmed cases below, in order to verify the detection effect of the peptide obtained here).

[0287] B. Clinical peptide microarray detection:

[0288] Sample set: Serum samples from clinically diagnosed type 1 diabetes patients and healthy controls (see table below), the presence of GAD antibody and IA2A antibody were determined by radioimmunoassay.

[0289] Table 6:

[0290]

[0291] Peptide microarray detection: Clinical serum sample detection under closed conditions

[0292] Under closed conditions, each serum sample was diluted with sample diluent and then subjected to peptide chip detection. The overall sample dilution factor was 1000 times during the detection.

[0293] Add one well of pure sample diluent to each detection chip as a control;

[0294] Detection operation

[0295] 1. Sample dilution:

[0296] a) Dilution of total IgG in quality control samples

[0297] Dilute the IgG using a 1.5 ml centrifuge tube. Add 16 μL of IgG to 784 μL of sample diluent to form a 50-fold diluted IgG. Shake well.

[0298] b) Sample dilution

[0299] Dilute the serum sample 500 times using sample diluent.

[0300] 1) Take 2 μL of serum sample and add it to 48 μL of sample diluent to form a dilution sample with a dilution factor of 25 (gradient 1). Seal the sample, shake to mix, and centrifuge (4000 rpm for 1 min).

[0301] 2) Take 5 μL of the sample dilution buffer of gradient 1 and add it to 95 μL of the sample dilution buffer to form a diluted sample with a dilution of 500 (gradient 2). Seal the sample, shake to mix, and centrifuge (4000 rpm for 1 min).

[0302] c) Preparation of sealing solution

[0303] Add 2 ml of 1% Casein to 18 ml of sample diluent, mix well and set aside.

[0304] 2. Layout and proofing

[0305] 2) Chip encapsulation

[0306] Dispense 45 μL of the prepared blocking solution into the wells of the Cassette using a multi-pipette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate at 37°C for 1 hour.

[0307] 3) Sample loading for sealed Cassette (chip)

[0308] According to the sample arrangement design, 45 μL of the 500-fold diluted sample was transferred to the corresponding well in the Cassette using a multi-channel pipette.

[0309] 3. First incubation

[0310] Seal the Cassette film and place it on the incubation module of the automated instrument for 1 hour (see the first incubation under non-sealed conditions for specific procedures).

[0311] 4. First wash of the board

[0312] Tear off the film from the Cassette and place it in an automatic plate washer for washing (see the first plate wash under non-enclosed conditions for specific operation).

[0313] 5. Add secondary antibody

[0314] Under light-protected conditions (lights off), use a 15ml centrifuge tube to dilute the human secondary antibody 1500 times (4μL of secondary antibody added to 6ml of secondary antibody dilution buffer), and use a multi-channel pipette to add 40μL to the corresponding well of the corresponding Cassette.

[0315] 6. Second incubation

[0316] Seal the cassette and incubate it on a thermostatic mixer for 60 minutes, shaking the cassette for 15 seconds every 6 minutes.

[0317] 7. Second wash

[0318] After stopping the oscillation and tearing off the film, place the plate into the plate washer. Ensure the waste liquid bottle of the plate washer has sufficient capacity, and use the automatic plate washer to wash the plate. 405LS Microplate Magnetic Washing Machine.

[0319] 8. Imaging Scan (For detailed instructions, please refer to the sections on chip disassembly under non-enclosed conditions, chip assembly in the imaging cassette, and fluorescence imaging).

[0320] 9. Data Processing

[0321] C. A set of GAD-specific binding peptides is used to identify whether GAD antibodies are positive or not in clinical samples:

[0322] Using the specific binding peptide of the GAD antibody (rabbit polyclonal antibody) screened in step A of this embodiment, the serum samples from patients in the diabetes cohort mentioned in this embodiment who tested positive for GAD antibodies alone were trained with the serum samples from healthy individuals (the test results here refer to the results after the aforementioned data processing flow from FG to LFG, and then processed to NLFG), and a ridge regression model was used for modeling (parameter alpha = 1000). The obtained model was tested with the serum samples from patients who tested positive for multiple antibodies, including those who were positive for GAD (and not only GAD), and with the serum samples from another group of healthy individuals.

[0323] In this embodiment, the selected peptide sets from Lv1 to Lv4 were modeled according to the above approach. The prediction results of the model built using the Lv1 peptide set are as follows. Figure 3 and Figure 4 As shown. The prediction results after modeling the peptide sets from Lv2 to Lv4 are similar to those of Lv1 (not shown). When using the Lv4 peptide set for modeling and prediction, the processing speed is faster due to the relatively smaller number of peptides. Furthermore, for the Lv4 peptide set, when not using model prediction to detect the presence of the target antibody, and instead using a detection method similar to conventional ELISA, the smaller number of peptides in the Lv4 peptide set, coupled with its higher specificity, allows for direct comparison with negative samples to determine the test result.

[0324] It should be noted that the test results of serum samples (16 cases) from a cohort of individuals who were positive for GAD antibodies alone, and the test results of serum samples (24 cases) from healthy individuals were used as the training set (a total of 40 cases). Then, the test results of the remaining samples were used as the test set (including the test results of 13 healthy individuals and 24 serum samples from diabetic patients who were positive for GAD and at least one of IA2A and ZnT8 tests, a total of 37 cases) to verify the predictive accuracy of the constructed model (in the case of samples that were positive for more than one GAD antibody alone).

[0325] Figure 3 The results shown are statistical (accuracy 35 / 37 = 95%). Figure 4 This demonstrates the performance of ROC analysis in determining the specific binding peptide set of GAD antibodies. A comprehensive analysis of all GAD antibody-specific binding peptide sets showed an accuracy of 97% in distinguishing between GAD antibody-positive and GAD antibody-negative (non-diabetic) individuals in the validation set (i.e., the test set), with an AUC of 0.98. These results indicate that the specific binding peptides screened by antibody peptide microarray detection have practical significance in detecting the presence or absence of specific antibodies in clinical cohorts.

[0326] As can be seen from the above embodiments, this application leverages the unique advantages of high-density random peptide chip technology platforms (such as HealthTell's V13 chip containing 130,000 peptides) in antigen epitope analysis—it is compatible with the binding of linear and nonlinear epitope antibodies. By employing experimental methods and analytical approaches that progressively eliminate potential background interference, it obtains highly sensitive and specific peptide sets and combinations, with corresponding peptides that can be used for the detection of their respective antibodies. During detection, the antibody specifically binds to the peptide and then to the antibody in the blood. After incubation with a fluorescently labeled secondary antibody, the fluorescence value is detected in an ELISA reader, providing a comprehensive and unbiased reflection of the antibody profile in the blood.

[0327] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Guangdong Carbon Cloud Crystal Core Intelligent Technology Co., Ltd. <120> Peptides, Peptide Compositions, Kits and Related Applications <130> PN153167SZTY <140> 202110378999 .3 <141> 2021-04-08 <160> 85 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Specific binding of antibodies to human SNRPC / U1C <400> 1 Tyr His Ser Lys Ala Asn Asp Ala Leu Asp 1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthesized peptide specifically binds to the human SNRPC / U1C antibody. <400> 2 Val Ser Lys Trp Arg Asp His Leu Ser 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthesized peptide specifically binds to the human SNRPC / U1C antibody. <400> 3 Gln Gly Glu Asp Asn Val Asn Leu Ser Asp 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthesized peptide specifically binds to the human SNRPC / U1C antibody. <400> 4 His Ser Lys Lys His Asp Tyr Gln Ser Gly 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 5 Ala Ala Lys Arg Asp Asp Ala Leu Asp 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 6 Ala Ser Lys Glu His Asp Leu Asp 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 7 Leu Ser Lys Leu Asn Asp His Leu Glu 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(7) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 8 Arg Ser Lys Lys Asp Asp Gly 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 9 His Asp Ala Leu Gly Glu Asp Asn Leu Gly 1 5 10 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic peptide specifically binds to the human SNRPC / U1C antibody. <400> 10 Arg Ser Lys Trp His Asp Leu Asp 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic sequence specifically binds to antibodies against human PCNA. <400> 11 Glu Gln Pro Leu Gln Lys Val Gly 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to antibodies against human PCNA. <400> 12 Asp Gln Gly Lys Gln Phe Glu Asp 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies against human PCNA. <400> 13 Asp Gln Pro Tyr Gln Arg Ser His Gly 1 5 <210> 14 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to antibodies against human PCNA. <400> 14 Asn Gln Pro Gln Gln His Val Tyr Val Glu Gly 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies against human PCNA. <400> 15 Asp Gln Pro Lys Gln Arg His Val Phe 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 16 Pro Asn Phe Asn Asn Arg His Ala Gly 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 17 Asn Tyr Asn Asn Leu Ala Phe Ser Gly 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 18 His Asn His Asn Asn His Phe Asp 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 19 Arg Asn Ala Asn Asn His Leu Phe Gly 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 20 Asn Phe Asn Asn Glu His Glu Gly 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> twenty one Asn His Asn Asn Pro Asn Phe Glu Gly 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(7) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> twenty two Tyr Gly Ser Arg Asn Leu Gly 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> twenty three Asn His Asn Asn Gly Pro Tyr Arg Gly 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> twenty four Asn Tyr Asn Asn Ala Gln Ser Gly 1 5 <210> 25 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 25 Arg Pro Trp Asn Ser Asn Asn Gln His Gly 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(7) <223> The synthetic polypeptide specifically binds to the antibody against human CENPB. <400> 26 Arg Asn Phe Asn Asn Asp Gly 1 5 <210> 27 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 27 Phe Asp Val Pro Pro Asn Gln Lys Leu Ser 1 5 10 <210> 28 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 28 Gln His Leu Asn Pro Asn Leu Tyr Glu Gly 1 5 10 <210> 29 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 29 His Val Phe Gly Ala Ser Asp His Tyr Gln Gly 1 5 10 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 30 Tyr Leu Asp Gly Gly Arg Arg Val Asp 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 31 Asn Asp Asn Gln Asn Pro Asn Leu Ser 1 5 <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 32 Asn Phe Leu Asn Pro Arg Leu Gly 1 5 <210> 33 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 33 Lys His Ala Trp Asn Ala Asn Pro Arg Leu Asp 1 5 10 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(7) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 34 Phe Gly Val Gly Asp Gly Gly 1 5 <210> 35 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> The synthetic polypeptide specifically binds to the antibody against human gp210. <400> 35 Asn Gln Ser Gly Pro Glu Tyr Lys Pro His Gly 1 5 10 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 36 Arg Phe Val Ser Leu Ser Asp Ala Asp 1 5 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 37 Pro Ala Asn Leu Lys Asp Ala Asp Ala Gly 1 5 10 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 38 Lys Phe Phe Ser Gln Lys Glu Val Asp 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 39 Gln Trp Val Ser Phe Ser Gln Lys Gly 1 5 <210> 40 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 40 Ala Gln Leu Asp Ala Asp Ala Lys Asp Tyr Leu Glu 1 5 10 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 41 Arg Val Asn Leu Arg Asp Ala Asp Gly 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 42 Pro Pro Glu Arg Gly Pro Trp Asp 1 5 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to antibodies that target human 58K Golgi protein. <400> 43 Arg Leu Phe Asp Ala Asp Gly Ala Pro Lys Asp 1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 44 Tyr Gly Glu Tyr Asn Lys Glu Leu Phe Gly 1 5 10 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 45 Tyr His Trp Pro Asn Val His Val Ser 1 5 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 46 Phe Phe His Leu Pro Asn Asp Gly 1 5 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 47 Tyr Ser Asn Glu Leu Gly Tyr Asn Gln Phe Glu 1 5 10 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 48 Ala Leu Phe Gly Phe Pro Asn Asp Pro Lys Val Ser 1 5 10 <210> 49 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 49 Tyr Leu Asn Glu Arg Phe Glu Ala Gln Val Ser 1 5 10 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 50 Arg Lys Phe Pro Asn Glu Leu Phe Asp 1 5 <210> 51 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 51 Asn His Ala Pro Asn Gln Pro Trp Lys His Gly 1 5 10 <210> 52 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 52 Trp His Pro His Tyr Pro Asn Arg Ser Asp 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 53 Trp Asn Pro Asn Val His Phe Pro Asn Ser Glu 1 5 10 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 54 Lys Trp Leu Lys Tyr Ala Asn Glu Asp 1 5 <210> 55 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 55 Trp Lys Tyr Trp Lys Leu Glu Tyr Pro Asn Asp 1 5 10 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 56 Ser Tyr Gln Asn Glu Tyr Asn Leu Asp 1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 57 Tyr Arg Asn Glu Val Asn His Val Glu 1 5 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 58 Gly His Tyr Ala Asn Glu Asn His Gly 1 5 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 59 Tyr Lys Glu Phe Asn His Gly Val Asp Gly 1 5 10 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 60 Tyr Gln Asn Glu Phe Gly Leu Asp Gly 1 5 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 61 Tyr Ala Asn Glu Lys His Glu Phe His Ser Asp 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 62 Phe Arg Ser Gly Tyr Ala Asn Glu Arg Val Leu 1 5 10 <210> 63 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 63 Asn Val His Lys Pro Asn His Asp Gly 1 5 <210> 64 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 64 Trp His Lys Gly Ala His Val Pro Asn Glu Gly 1 5 10 <210> 65 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 65 Tyr Pro Asn Asp Tyr Arg Val Pro Leu Ser Gly 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 66 Pro Trp Lys His Arg Phe His Phe Pro Asn His Val 1 5 10 <210> 67 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 67 Tyr Gln Asn Glu Pro Tyr Arg Pro His Phe Gly 1 5 10 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 68 Ala Val His Gln Pro Asn Val Phe Ser 1 5 <210> 69 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 69 Ala Tyr Pro Asn Glu Phe Glu Gly 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 70 Phe Asn Gln Pro Asn Gln Leu Leu Gly 1 5 <210> 71 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 71 Val His Glu Pro Asn Glu Asp Ala Asn Arg Phe 1 5 10 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 72 Phe Gly Phe His Gly Pro Asn Asp 1 5 <210> 73 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 73 Asn Pro Tyr Lys Arg Val Tyr Pro Asn Glu Asp 1 5 10 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 74 Asp Tyr Pro Asn Glu Ala Lys His Asp 1 5 <210> 75 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 75 Ser Tyr Pro Asn Glu Asp Pro Lys Arg Asp 1 5 10 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 76 Tyr Pro Asn Glu His Gln Lys Leu Asp 1 5 <210> 77 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(11) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 77 Tyr Pro Asn Glu Val Tyr Trp Gln Lys Arg Ser 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 78 His Tyr His Glu Pro Asn Ser Val Phe Gly 1 5 10 <210> 79 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 79 His Ala His Phe Pro Asn Ala Trp Arg Ser 1 5 10 <210> 80 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 80 Val His Val Pro Asn Gln Gln Arg Val Leu 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 81 Asn Tyr His Gln Pro Asn Lys Arg Leu Glu 1 5 10 <210> 82 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(8) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 82 Ala Phe His Lys Pro Asn Ser Gly 1 5 <210> 83 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(12) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 83 Gln Gln His Ser Tyr His Leu Pro Asn Arg Leu Gly 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 84 Ala Asn Ser Ala His Tyr Pro Asn Leu Gly 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Synthetic peptides specifically bind to human GAD antibodies. <400> 85 Phe His Glu Pro Asn Gln Leu Lys Arg Asp 1 5 10

Claims

1. A polypeptide composition, characterized in that, The polypeptide composition contains one or more polypeptides, which can specifically bind to one or more target antibodies, wherein the one or more target antibodies are selected from any one of the following: human SNRPC / U1C antibody, human SNRPC / U1C antibody + human CENPB antibody, human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody, or human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody + human NUP210 / gp210 antibody; When the target antibody is a human SNRPC / U1C antibody, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NOs: 9-10; When the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; When the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; When the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO:

35.

2. A polypeptide product, characterized in that, The polypeptide product contains one or more polypeptides, which can specifically bind to one or more target antibodies. The one or more target antibodies are selected from any one of the following: human SNRPC / U1C antibody, human SNRPC / U1C antibody + human CENPB antibody, human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody, or human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody + human NUP210 / gp210 antibody. When the target antibody is a human SNRPC / U1C antibody, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NOs: 9-10; When the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; When the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; When the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO:

35.

3. The polypeptide product according to claim 2, characterized in that, The polypeptide products also include polypeptide stabilizers.

4. The polypeptide product according to claim 3, characterized in that, The polypeptide stabilizer comprises 150-180 mM NaCl, 100-140 mM polylysine hydrochloride, and water; The polypeptide product is a polypeptide chip, and the polypeptide on the polypeptide chip is composed of the multiple polypeptides.

5. An antibody detection reagent, characterized in that, The reagent contains one or more polypeptides, which can specifically bind to one or more target antibodies. The one or more target antibodies are selected from any one of the following: human SNRPC / U1C antibody, human SNRPC / U1C antibody + human CENPB antibody, human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody, or human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody + human NUP210 / gp210 antibody. When the target antibody is a human SNRPC / U1C antibody, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NOs: 9-10; When the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; When the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; When the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO:

35.

6. An antibody detection kit, characterized in that, The kit contains multiple peptides, each of which can specifically bind to one or more target antibodies. The one or more target antibodies are selected from any of the following: human SNRPC / U1C antibody, human SNRPC / U1C antibody + human CENPB antibody, human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody, or human SNRPC / U1C antibody + human CENPB antibody + human PCNA antibody + human NUP210 / gp210 antibody. When the target antibody is a human SNRPC / U1C antibody, the polypeptide is selected from any one or more of SEQ ID NO: 1 to SEQ ID NO: 7 and SEQ ID NO: 9-10; When the target antibody is an antibody against human PCNA, the polypeptide is selected from any one or more of SEQ ID NO: 11 to SEQ ID NO: 15; When the target antibody is an antibody against human CENPB, the polypeptide is selected from any one or more of SEQ ID NO: 16 to SEQ ID NO: 26; When the target antibody is an antibody against human NUP210 / gp210, the polypeptide is selected from any one or more of SEQ ID NO: 27 to SEQ ID NO:

35.

7. The reagent kit according to claim 6, characterized in that, The kit includes a detection chip, on which multiple peptides are disposed, and the peptides on the detection chip are composed of the multiple peptides.

8. The use of the polypeptide composition of claim 1 in the preparation of an antibody detection kit; wherein, The antibody is selected from any one or more of the following: human SNRPC / U1C antibody, human CENPB antibody, human PCNA antibody, or human NUP210 / gp210 antibody.

9. The use of the polypeptide composition of claim 1 in the preparation of antibody products for detection; wherein, The antibody is selected from any one or more of the following: human SNRPC / U1C antibody, human CENPB antibody, human PCNA antibody, or human NUP210 / gp210 antibody.

10. The application according to claim 9, characterized in that, The detection antibody product also includes a sample diluent, which is a PBST buffer containing D-mannitol, wherein the D-mannitol content in the PBST buffer is 0.5% to 1% by mass and volume.

11. The application according to claim 10, characterized in that, The detection antibody product also includes a blocking solution comprising the following components: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM disodium hydrogen phosphate, 1.2-1.4 mM potassium dihydrogen phosphate, 0.05%-1% Tween-20 v / v, 0.05%-0.1% Proclin950 v / v, 0.5%-1% D-mannitol w / v, and 0.1%-1% casein w / v. The pH of the blocking solution is 7.2-7.

6.

12. The application according to claim 11, characterized in that, The pH of the sealing solution is 7.38~7.

42.

13. The application according to claim 12, characterized in that, The blocking solution consists of 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium hydrogen phosphate, 1.4 mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin950 v / v, 1% D-mannitol w / v, and 0.1% casein w / v, and the pH of the blocking solution is 7.4.

Images