Method for screening polypeptides specifically bound by antibodies based on polypeptide chips and application of the polypeptides screened

Abstract

The present invention provides a method for screening antibody-specifically binding polypeptides based on a polypeptide chip and the application of the polypeptides screened therefrom. The screening method comprises using a polypeptide chip to perform a first screening on a sample diluent containing an antibody and a sample diluent not containing an antibody under closed and non-closed conditions, respectively, to obtain a first seed peptide library under closed conditions and a first seed peptide library under non-closed conditions; taking the intersection of the polypeptides in the first seed peptide library under the two conditions to obtain a second seed peptide library; using a polypeptide chip to perform a second screening on a serum sample containing an antibody and a serum sample not containing an antibody under closed conditions to obtain a third seed peptide library; taking the intersection of the third seed peptide library and the second seed peptide library to obtain a fourth seed peptide library; the antibody is one or more single antibodies, or a multi-mixed antibody. The method excludes interfering polypeptides under different conditions, and the obtained polypeptide set has higher specificity, which is suitable for the detection of antibodies in complex clinical samples.

Description

Technical Field

[0001] The present invention relates to the field of polypeptide chips, and in particular to a method for screening polypeptides specifically bound by antibodies based on polypeptide chips and applications of the screened polypeptides. Background Art

[0002] Biochip technology originated in the 1980s and is also known as "microfluidics technology" and "chip laboratory". Biochip technology can conduct more complex biological, chemical, physical and other experiments on a chip the size of a stamp. Biochip technology provides technical support for medical instruments with low production costs, small samples, short time and simple operation. Depending on the molecules fixed on the chip, biochips can be divided into oligonucleotide chips, protein chips, small molecule chips and peptide chips. Protein chips can be used for protein-level interaction research, enzyme substrate analysis and small molecule protein target analysis, but because the expression and purification of large amounts of proteins, directional fixation on the surface of the support to form a high-throughput chip and maintaining protein activity are all technically challenging and cost-controlled in terms of transformation and application, peptide chips have become a favorable tool for high-throughput protein research due to their advantages such as easy synthesis and good stability.

[0003] There are many technical implementation methods for peptide chips, depending on the selection of solid support, surface chemical treatment and fixation methods, and array peptide synthesis methods. According to different implementation technologies, it can be divided into several technical routes such as Merrifield synthesis, SPOT, photolithography synthesis, SAMDI, and particle-based synthesis. The current commercially available peptide chip machine technology comparison is as follows. Among them, the technology based on photolithography in situ synthesis has a small physical size of a single feature point, which can achieve high-density chip preparation and better match the complexity of biomolecular interactions.

[0004] Peptide chips can be used for antigen epitope analysis, protein and peptide ligand binding analysis, and enzyme activity substrate analysis. Among them, the analysis of antibody-bound mapped epitopes (i.e., peptides that specifically bind to antibodies on peptide chips, which may be linear epitopes or conformational epitopes) is one of the most basic applications of peptide chips. Based on the PEPperPRINT peptide chip, Loeffler and his colleagues used the PEPperPRINT peptide chip to perform antibody epitope analysis on the Lyme disease infectious characteristic protein VlsE. Overlapping peptides with a length of 15 amino acids were designed along the VlsE sequence, and a total of 335 peptide segments formed a peptide array. The array was used to detect and analyze the sera of 17 patients infected with Lyme disease and 7 uninfected patients to identify the peptides that the antibodies can recognize. The results showed that the sera of infected patients were divided into two major categories. The first group of antibodies mainly bound to the N-terminal or C-terminal region of the VlsE antigen. The second group of antibodies mainly recognized the variable region.

[0005] The peptide chip designed based on the antigen sequence contains multiple partially overlapping peptide segments to cover the antigen sequence as a whole, which is suitable for linear epitope detection. B cells and antibodies derived from them mainly recognize antigenic determinants located on the surface of antigen molecules, which are adjacent in spatial structure but connected or disconnected in sequence, that is, in addition to linear epitopes, antibody recognition epitopes also include conformational epitopes / discontinuous epitopes.

[0006] Stafford et al. used the Merrifield synthesis method to prepare random peptide chips and designed peptides based on antigen sequences based on the previous use of random peptide chips to screen specific peptides for coccidiosis patients. The two peptide chips were used to detect coccidiosis patients and control samples respectively. One chip contains 96 random 20-mer peptides (based on the screening of 10,000 random peptide chips in previous work), and the second contains 83 20-mer overlapping peptides designed based on known coccidiosis antigen epitope sequences, which are used to detect coccidiosis antibody binding in patients' serum. Both peptide chips have been processed with coccidiosis-infected and non-infected serum samples for binding signal detection, and both have been tested with known coccidiosis IgG and IgM antibodies to compare the sensitivity and specificity of the two peptide chips in detecting coccidiosis-related antibodies. The results show that random peptide arrays can diagnose infections at different stages more accurately than peptide arrays designed based on epitope sequences.

[0007] As mentioned above, the peptides of nonlinear epitopes that bind to antibodies in the random peptide array are not related to the antigen sequence, but the antibody binding may be as strong as or even stronger than the original antigen. The above experiment also indirectly confirmed that the random peptide array can be used to find peptide sequences that are more representative of the real epitope binding situation, which can be used for antibody detection in clinical samples and judgment of antibody-positive related diseases.

[0008] However, the current antibody detection and epitope analysis based on peptide chip method mainly have the following problems:

[0009] 1. Peptide chips designed based on sequence information can detect linear epitopes more clearly, and accordingly, the peptides corresponding to the epitopes can be used for antibody detection. However, this method cannot meet the detection needs of ubiquitous nonlinear epitopes. Although there are currently methods that constrain peptides to simulate discontinuous and conformation-dependent epitopes through one or more disulfide bonds, it still only increases the possibility of detecting a small number of nonlinear epitopes.

[0010] 2. There are many peptides in the random sequence peptide chip, and the core recognition site of the target antibody conformational epitope may be only a few amino acids. When 4 to 5 amino acid residues in the peptide are perfectly matched, the antibody can be bound. Therefore, the signal peptide after the random sequence chip is incubated with the antibody may contain 1) antigen 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 it is impossible to exclude non-specific interference signals through methods such as sequence alignment.

[0011] 3. Clinical antibody detection is mainly based on serum / plasma samples, which have complex components. The specific peptides obtained by incubating pure antibodies on peptide chips may be interfered by other proteins in serum / plasma and may not be used for signal detection of target antibodies in serum. In addition, antibodies in the body are polyclonal antibodies with non-unique antigenic epitopes, and using peptides to simulate single epitopes for antibody detection is prone to missed detection.

[0012] Therefore, the method of detecting antigen epitopes based on peptide chips still needs to be improved. Summary of the invention

[0013] The main purpose of the present invention is to provide a method for screening polypeptides that specifically bind to antibodies based on a polypeptide chip and the application of the polypeptides screened, so as to improve the sensitivity of the screened polypeptides to antibody binding.

[0014] In order to achieve the above-mentioned object, according to one aspect of the present invention, a method for screening polypeptides specifically bound by antibodies based on a polypeptide chip is provided, and the screening method comprises: using a polypeptide chip to perform a first screening of polypeptides specifically bound to a first antibody-positive sample and a first antibody-negative sample under closed and non-closed conditions, respectively, to obtain a first seed peptide library under closed conditions and a first seed peptide library under non-closed conditions; taking the intersection of polypeptides in the first seed peptide library under closed conditions and the first seed peptide library under non-closed conditions to obtain a second seed peptide library; under closed conditions, using a polypeptide chip to perform a second screening of polypeptides specifically bound to a second antibody-positive sample and a second antibody-negative sample; to obtain a third seed peptide library; to obtain a fourth seed peptide library by taking the intersection of the third seed peptide library and the second seed peptide library; wherein the first antibody positive sample is one or more sample dilutions containing a single antibody, the second antibody positive sample is one or more serum samples containing a single antibody, the first antibody negative sample is a sample dilution not containing a single antibody, and the second antibody negative sample is a serum sample not containing a single antibody; or the first antibody positive sample is one or more groups of sample dilutions containing mixed antibodies, the second antibody positive sample is one or more groups of serum samples containing mixed antibodies, the first antibody negative sample is a sample dilution not containing mixed antibodies, and the second antibody negative sample is a serum sample not containing mixed antibodies.

[0015] Furthermore, the first antibody positive sample is a plurality of sample dilutions containing a single antibody, the second antibody positive sample is a plurality of serum samples containing a single antibody, the first antibody negative sample is a sample dilution not containing a single antibody, and the second antibody negative sample is a serum sample not containing a single antibody, and the screening method further comprises excluding polypeptides having intersections between a plurality of different single antibodies; preferably, excluding polypeptides having intersections between a plurality of different single antibodies at any one or more of the following times: a. after the first screening under non-blocking conditions; b. after the first screening under blocking conditions; c. after the second screening.

[0016] Furthermore, the first antibody-positive samples are multiple groups of sample dilutions containing mixed antibodies, the second antibody-positive samples are multiple groups of serum samples containing mixed antibodies, the first antibody-negative samples are sample dilutions not containing mixed antibodies, the second antibody-negative samples are serum samples not containing mixed antibodies, and the antibody types contained in any two groups of sample dilutions containing mixed antibodies and / or any two groups of serum samples containing mixed antibodies do not overlap, then the screening method also includes excluding polypeptides that have intersections between multiple groups of different mixed antibodies; preferably, excluding polypeptides that have intersections between multiple groups of different mixed antibodies at any one or more of the following times: a. after the first screening is performed under non-blocking conditions; b. after the first screening is performed under blocking conditions; c. after the second screening is performed.

[0017] Further, under non-blocking conditions and / or under non-blocking conditions, two or more first antibody-positive samples with different antibody concentrations are used for the first screening; preferably, under non-blocking conditions, two or more first antibody-positive samples with different antibody concentrations are used for the first screening, and two first antibody-positive samples with different antibody concentrations are determined for the first screening under blocking conditions; preferably, the concentration of the single antibody contained in the first antibody-positive sample or the concentration of each single antibody in the mixed antibody is 10 ng / mL-1 μg / mL, more preferably 50 ng / mL-500 ng / mL; preferably, two or more second antibody-positive samples with different antibody concentrations are used for the second screening, more preferably, the concentration of the single antibody contained in the second antibody-positive sample or the concentration of each single antibody in the mixed antibody is 10 ng / mL-1 μg / mL, further preferably 50 ng / mL-500 ng / mL.

[0018] Further, the sample diluent is a PBST buffer containing D-mannitol, wherein the mass volume content of D-mannitol in the PBST buffer is 0.5% to 1%; preferably, under the blocking condition, it means that the polypeptide chip is blocked with a blocking solution, and the blocked polypeptide chip is used for screening, and more preferably, the blocking solution comprises the following components: 130 to 137 mM sodium chloride, 2.5 to 2.7 mM potassium chloride, 3.8 to 4.3 mM disodium hydrogen phosphate, 1.2 to 1.4 mM potassium dihydrogen phosphate, 0.05% to 1% Tween-20 v / v, 0.05% to 0.1% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v, and the pH of the blocking solution is 7.2-7.6, more preferably 7.38-7.42; further preferably, the blocking solution is 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20v / v, 0.1% Proclin950v / v, 1% D-mannitol w / v and 0.1% casein w / v, and the pH of the blocking solution is 7.4.

[0019] Further, the antibody is a monoclonal antibody or a polyclonal antibody; preferably, each single antibody in the multiple single antibodies or mixed antibodies is a polyclonal antibody; preferably, each single antibody in the multiple single antibodies or mixed antibodies has the same or different species origin; preferably, the species origin is rabbit, goat, rat, mouse, guinea pig or human; preferably, the multiple single antibodies or mixed antibodies are selected from multiple of the following antibodies: antibodies to human SNRPC / U1C, antibodies to human PCNA, antibodies to human CENPB, antibodies to human NUP210 / gp210, antibodies to human FTCD / 58K Golgi protein, antibodies to HIB, antibodies to SARS-CoV E protein, antibodies to Plasmodium and antibodies to GAD.

[0020] Furthermore, the first screening and the second screening using the polypeptide chip both include sequential incubation of antibody samples and incubation of secondary antibodies, wherein the secondary antibody is fluorescently labeled; preferably, the species origin of the secondary antibody is selected based on the species origin of each single antibody or single antibody in the mixed antibody in the first screening and the second screening steps.

[0021] According to the second aspect of the present application, a method for screening polypeptides that specifically bind to antibodies based on a polypeptide chip is provided, and the screening method comprises: under non-blocking conditions, using a polypeptide chip to perform a first screening of first positive samples of multiple single antibodies or multiple groups of mixed antibodies and corresponding first negative samples for specific binding polypeptides, to obtain a first anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies; wherein the first positive sample is a sample dilution containing a single antibody or a sample dilution containing mixed antibodies, and the first negative sample is a sample dilution not containing a single antibody or mixed antibodies; removing polypeptides that have intersections in the first anti-interference peptide library corresponding to each of the multiple single antibodies or multiple groups of mixed antibodies, to obtain a second anti-interference peptide library corresponding to each single antibody or multiple groups of mixed antibodies.

[0022] Furthermore, the screening method further includes: under closed conditions, using a peptide chip to perform a second screening of specific binding peptides on the first positive sample and the corresponding first negative sample to obtain a third anti-interference peptide library; taking the intersection of the peptides in the second anti-interference peptide library and the third anti-interference peptide library to obtain a fourth anti-interference peptide library.

[0023] Furthermore, the screening method further includes: under closed conditions, using a polypeptide chip to perform a third screening of specific binding polypeptides on the second positive sample and the corresponding second negative sample to obtain a fifth anti-interference peptide library; wherein the second positive sample is a serum sample containing a single antibody or a serum sample containing mixed antibodies, and the second negative sample is a serum sample not containing a single antibody or mixed antibodies; taking the intersection of the fifth anti-interference peptide library and the fourth anti-interference peptide library to obtain a sixth anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies.

[0024] Furthermore, after obtaining the third anti-interference peptide library and / or the fifth anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies, the screening method also includes: removing polypeptides in which multiple single antibodies or multiple groups of mixed antibodies have intersections in each third anti-interference peptide library and / or the fifth anti-interference peptide library.

[0025] Further, each single antibody in the plurality of single antibodies or mixed antibodies is screened for the first time using two or more different antibody concentrations; preferably, the concentration of the single antibody contained in the first positive sample is 10 ng / mL-1 μg / mL, more preferably 50 ng / mL-500 ng / mL; preferably, the sample diluent is a PBST buffer containing D-mannitol, wherein the mass volume content of D-mannitol in the PBST buffer is 0.5% to 1%; preferably, under non-blocking conditions, two or more first positive samples with different antibody concentrations are used for the first screening, and then two first positive samples with different antibody concentrations are determined, and then a second screening is performed under blocking conditions; preferably, each single antibody The third screening is performed using two or more second positive samples with different antibody concentrations; preferably, the concentration of the single antibody contained in the second positive sample is 10 ng / mL-1 μg / mL, more preferably 50 ng / mL-500 ng / mL; preferably, the blocking condition refers to blocking the polypeptide chip with a blocking solution, and using the blocked polypeptide chip for the second screening and the third screening respectively; preferably, 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% Proclin950 v / v, 0.5% to 1% D-mannitol w / v and 0.1% to 1% casein w / v, the pH of the blocking solution is 7.2 to 7.6, more preferably 7.38 to 7.42; further preferably, the blocking solution is 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, and the pH of the blocking solution is 7.4; preferably, the single antibody is a monoclonal antibody or a polyclonal antibody; preferably, each of the multiple single antibodies or mixed antibodies is a polyclonal antibody; preferably, each of the multiple single antibodies or mixed antibodies has the same or different species origin; preferably, the species origin is rabbit, goat, rat, mouse, guinea pig or human; preferably, the multiple single antibodies or mixed antibodies are selected from multiple of the following antibodies: antibodies to human SNRPC / U1C, antibodies to human PCNA, antibodies to human CENPB, antibodies to human NUP210 / gp210, antibodies to human FTCD / 58K Golgi protein, antibodies to HIB, antibodies to SARS-CoV E protein, antibodies to Plasmodium and antibodies to GAD.

[0026] According to the third aspect of the present application, a method for detecting a target antibody in a test sample is provided, the detection method comprising: using a target polypeptide to which the target antibody specifically binds, detecting the test sample and a negative control of the test sample to obtain respective corresponding detection results; if there is a significant difference in the detection results corresponding to the test sample and the negative control, it indicates that the test sample contains the target antibody; the target polypeptide is a polypeptide that specifically binds to the target antibody screened by any of the above screening methods.

[0027] Furthermore, under the condition of blocking the polypeptide chip, the polypeptide chip containing the target polypeptide is used to detect the sample to be tested and the negative control of the sample to be tested, and the detection results of the corresponding target polypeptides are obtained; if there is a significant difference between the detection results of the target polypeptide corresponding to the sample to be tested and the negative control, it indicates that the sample to be tested contains the target antibody; preferably, the sample to be tested is a serum sample diluted with a sample diluent, the negative control is the sample diluent, and the target polypeptide is any multiple polypeptides in the polypeptide set that specifically binds to the target antibody screened by any of the above screening methods; preferably, the target antibody is selected from any one or more of the following: antibodies to human SNRPC / U1C, antibodies to human PCNA, antibodies to human CENPB, antibodies to human NUP210 / gp210, antibodies to human FTCD / 58K Golgi protein, antibodies to HIB, SARS-CoV E protein antibody, malarial parasite antibody and GAD antibody; preferably, the sample diluent is a PBST buffer containing D-mannitol, wherein the mass volume content of D-mannitol in the PBST buffer is 0.5% to 1%; preferably, before the test sample and the negative control of the test sample are tested, the detection method further comprises: using a blocking solution to block the polypeptide chip; preferably, the blocking solution comprises the following components: 130 to 137 mM sodium chloride, 2.5 to 2.7 mM potassium chloride, 3.8 to 4.3 mM disodium hydrogen phosphate, 1.2 to 1.4 mM potassium dihydrogen phosphate, 0.05% to 1% Tween-20 v / v, 0.05% to 0.1% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v, and the pH of the blocking solution is 7.2-7.6, more preferably 7.38-7.42; more preferably, the blocking solution is 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20v / 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.

[0028] Furthermore, under the condition of blocking the polypeptide chip, using the polypeptide chip containing the target polypeptide, the sample to be tested and the negative control of the sample to be tested are detected, including: incubating the sample diluent and the serum sample diluted by the sample diluent with the blocked polypeptide chip to obtain a first incubation product; co-incubating the first incubation product with a fluorescently labeled secondary antibody to obtain a second incubation product; performing fluorescence scanning on the second incubation product to obtain a fluorescent signal of the sample diluent and a fluorescent signal of the serum sample; detecting whether there is a significant difference between the fluorescent signal of the target polypeptide in the sample diluent and the fluorescent signal of the serum sample, if so, it indicates that the target antibody exists in the serum sample; preferably, the secondary antibody is selected according to the species source of the serum sample.

[0029] Furthermore, a prediction model is constructed based on the fluorescence signals of the detected target polypeptide in the target antibody-positive population and the target antibody-negative population using multiple clinical sample cohorts whose presence or absence of the target antibody is known. The prediction model is used to predict whether there is a significant difference between the fluorescence signal of the target polypeptide in the sample diluent and the fluorescence signal of the serum sample, that is, the prediction model outputs whether the serum sample is a target antibody-positive sample.

[0030] The technical scheme of the present invention is applied, by using the advantages of high-density random peptide chips in antigen epitope analysis (compatible with the binding of linear epitope and non-linear epitope antibodies), under the condition that the peptide chip is not blocked, the sample diluent without antibody is used as the background signal, and the peptides specifically bound to the antibodies diluted by the sample diluent are detected, thereby screening out a set of peptide segments with a higher than background signal for each antibody, and these peptide segments have high affinity with the corresponding antibodies, and peptide segments with intersections between the peptide segments of multiple antibodies are removed, thereby obtaining high-affinity peptide segments specific to each antibody. These peptide segments have high sensitivity and specificity when applied to potential drugs (targets), vaccines or antibody detection. Furthermore, the non-specific binding peptides interfered by the blocking and the peptides caused by other impurity proteins in the serum are excluded, so that the polypeptides and their combinations suitable for the detection of antibodies in clinical serum samples can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1 A schematic diagram showing the principle of detecting antibodies with a polypeptide chip provided in an embodiment of the present invention is shown;

[0033] Figure 2 A heat map of detecting single antibodies and mixed antibodies based on a polypeptide chip provided according to an embodiment of the present invention is shown;

[0034] Figure 3 A statistical diagram showing the results of detecting GAD antibodies in clinical serum samples based on a polypeptide chip according to an embodiment of the present invention and the actual results;

[0035] Figure 4 The figure shows the ROC curve analysis diagram of detecting GAD antibodies in clinical serum samples based on a polypeptide chip according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0037] Terminology explanation:

[0038] Antibodies: A type of glycoprotein in blood and tissue fluids, produced by plasma cells generated by proliferation and differentiation of B cells after antigen stimulation. They are mainly found in serum and other body fluids, and can specifically bind to the corresponding antigens. They are important effector molecules that mediate humoral immunity. B cells activated by antigen stimulation can produce 10 11 Copies of a single specific antibody.

[0039] Epitope: also called antigenic determinant, refers to the part of the antigen that is specifically recognized by the antigen receptors TCR and BCR. There are two types of epitope structures, one is linear epitope (continuous in sequence); the other is conformational epitope (adjacent in spatial structure but discontinuous in sequence).

[0040] Polypeptide: In this application, polypeptide or peptide segment has the same meaning and can be used interchangeably, both referring to amino acid sequence fragments.

[0041] Peptide chip: a chip based on substrate materials, which includes features with pre-designed quantity, position and sequence. One feature is a cluster of polypeptides with the same sequence. The polypeptide sequences between features are often different. These features constitute a high-density polypeptide array.

[0042] Peptide chip technology: It is a detection technology based on peptide chips. It uses the contact between a wide variety of peptides on the peptide chip and the sample, and then uses image acquisition technology to collect various characteristic signals on the peptide chip (specifically, it can be expressed as a fluorescent image carrying each characteristic signal), and then outputs the signal intensity of each characteristic in the chip, that is, the peptide chip detection result data. Output sample detection signals, based on the sample detection signal output by the peptide chip detection result data, can realize the analysis of the analyte in the sample bound to the peptide on the peptide chip, the analysis of the sample, etc.

[0043] ROC working curve: a curve that reflects the relationship between sensitivity and specificity. The horizontal axis X-axis is 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 is called sensitivity, also known as the true positive rate. The larger the Y-axis is, the better the sensitivity. According to the position of the curve, the entire graph is divided into two parts. The area under the curve is called AUC (Area Under Curve), which is used to indicate the accuracy of prediction. The higher the AUC value, the higher the prediction accuracy. The closer the curve is to the upper left corner (the smaller the X, the larger the Y), the higher the prediction accuracy.

[0044] Peptide chip antibody detection technology is a cutting-edge technology in the biochip industry. It is mainly based on the peptide-specific capture of antibodies in body fluid samples, thereby achieving high-throughput detection of antibodies in samples. Compared with traditional antibody detection methods, chip detection technology can detect multiple antibody indicators in one experiment, with less sample usage, faster analysis and detection speed, safer and more environmentally friendly detection methods, and can achieve advanced automation and integration. Compared with protein chip antibody detection technology, the production process of peptide chips is more stable, the collected data is more reliable and accurate, and the price is more advantageous.

[0045] For example, a peptide chip (HealtTell V13) used in this application contains more than 130,000 synthetic peptides with known sequences. Each peptide contains 5-13 amino acids. The amino acid combination of the peptide is an unbiased random combination. It measures the overall level of free antibodies in body fluids in accordance with the immunological principles of antigen-antibody competitive binding and amino acid-amino acid interaction. The schematic diagram of the principle of peptide chip detection of antibodies is shown in the figure below. Figure 1 shown.

[0046] As mentioned in the background technology, conformational epitopes recognized by antibodies are difficult to design based on sequence information. The peptide chip (HealthTell's V13 chip) contains 130,000 synthetic peptides of known sequences, whose amino acid combinations cover 99.9% of 4mer amino acid combinations (4mer refers to a collection of polypeptide sequences composed of 4 amino acids, and the adjacent polypeptide sequences differ by one amino acid, for example, MGAS, GAST, ASTC, etc. Similarly, 5mer refers to a polypeptide sequence composed of 5 amino acids, and the adjacent polypeptide sequences differ by one amino acid) and 48.3% of 5mer amino acid combinations, which are more suitable for antibody binding analysis of linear and conformational epitopes.

[0047] However, there are a large number of peptides on the random peptide chip, and the detection sensitivity is high. When the chip is incubated with the antibody and the fluorescent labeled secondary antibody is added, the signal may include the following situations: 1) antigen 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 method of target antigen sequence alignment is not suitable for judging whether the antigen epitope peptide corresponding to the antibody is among many binding signal peptides. In addition, for polyclonal antibodies, the antigen epitope is not unique, and it is crucial to detect as many target antigen epitopes as possible for the sensitivity of the detection of the target antigen corresponding antibody in subsequent complex samples. Furthermore, from the perspective of subsequent clinical antibody detection applications, complex samples such as serum / plasma are usually used, which increases the interference of other proteins and antibodies in the sample, and the signal is complex. The 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 strong specificity and no interference from other proteins in complex samples can be used for the development of subsequent diagnostic kits. To this end, the present application improves the existing method based on the detection principle of random peptide chips to provide an antibody-specific binding peptide segment with strong anti-interference ability for stable detection of antibodies in complex samples. In addition, in the screening process of the antibody-specific binding peptide segment with strong anti-interference ability of the present application, a standardized screening process was developed, which provides guidance for the screening and identification of such antibody-specific binding peptide segments.

[0048] Therefore, the screening method of the present application focuses on eliminating interfering signals step by step, especially simulating the interference of complex signals in serum samples on antibody detection, and defining a set of specific anti-interference peptides for stable detection of antibodies in complex samples.

[0049] In order to develop relevant detection products suitable for one or more antibodies used in auxiliary diagnosis of clinical diseases, the present application utilizes peptide chip technology, under the condition of peptide chip blocking or non-blocking, by using antibody samples under different concentrations and different solution backgrounds for peptide chip detection, to screen out peptide libraries with strong sensitivity and specificity for specific binding to the target antibody. The peptides screened by the method of the present application for specific binding to the antibody (including both linear antigen epitopes and conformational epitopes, wherein the linear epitopes can be determined by comparing with the protein sequence of the corresponding antigen, and the conformational epitopes are not related to the antigen sequence, but can simulate the state of antigen-antibody binding) are compared with the existing antibody detection methods (such as the mainstream IVD antibody detection kit, i.e., ELISA method), not only for samples containing a single antibody, but also for samples containing multiple antibodies, the specificity is also higher (after the minimum detection limit test, the minimum detection limit of the detection method of the peptide chip of the present application can reach 0.05ng / mL, while the minimum detection limit of the reference ELISA is 1.56ng / ml). The polypeptides in these peptide libraries can be used as antigen epitope peptides for detecting corresponding antibodies, and can be used to detect the presence or absence of corresponding antibodies in patient serum samples. On this basis, the applicant proposed the technical solution of the present application.

[0050] In order to more clearly describe the solution of this application, the types of samples involved in this application are described here:

[0051] Positive samples include: sample diluents containing a single antibody or a mixed antibody (such as PBST samples containing a single antibody or a mixed antibody) and serum samples containing a single antibody or a mixed antibody. Serum samples containing a single antibody or a mixed antibody further include: simulated clinical positive serum samples prepared with a single antibody or a mixed antibody + negative serum, and real clinical positive serum samples containing a single antibody or a mixed antibody in a known population cohort.

[0052] Negative samples include: sample diluents without single antibodies or mixed antibodies (such as PBST samples without single antibodies or mixed antibodies) and serum samples without single antibodies or mixed antibodies. The serum samples without single antibodies or mixed antibodies are preferably serum samples from healthy people.

[0053] In a typical embodiment of the present application, a method for screening polypeptides specifically bound by antibodies based on a polypeptide chip is provided, the screening method comprising the following steps:

[0054] Under blocking and non-blocking conditions, a peptide chip is used to perform a first screening of specific binding peptides on the first antibody positive sample and the first antibody negative sample, and a first seed peptide library under blocking conditions and a first seed peptide library under non-blocking conditions are correspondingly obtained;

[0055] Taking the intersection of the first seed peptide library under closed conditions and the first seed peptide library under non-closed conditions to obtain a second seed peptide library;

[0056] Under the condition of blocking, a peptide chip is used to perform a second screening of specific binding peptides on the second antibody positive samples and the second antibody negative samples to obtain a third seed peptide library;

[0057] Taking the intersection of the third seed peptide library and the second seed peptide library to obtain a fourth seed peptide library;

[0058] Wherein, the first antibody positive sample is one or more sample dilutions containing a single antibody, the second antibody positive sample is one or more serum samples containing a single antibody, the first antibody negative sample is a sample dilution not containing a single antibody, and the second antibody negative sample is a serum sample not containing a single antibody; or

[0059] The first antibody positive samples are one or more groups of sample dilutions containing mixed antibodies, the second antibody positive samples are one or more groups of serum samples containing mixed antibodies, the first antibody negative samples are sample dilutions without mixed antibodies, and the second antibody negative samples are serum samples without mixed antibodies.

[0060] In this embodiment, by selecting the intersection of polypeptides that bind to the target antibody to be tested (one or more single antibodies, or one or more groups of mixed antibodies, where the mixed antibody refers to two or more single antibodies contained in a test sample at the same time) under non-blocking and blocking conditions of the polypeptide chip in the sample dilution system, and further taking the intersection with the set of polypeptides that specifically bind to the target antibody to be tested when the target antibody to be tested is in the serum solution system under blocking conditions, the polypeptides that bind to the target antibody to be tested under different interference conditions are excluded, and a set of specifically bound polypeptides with strong anti-interference ability is obtained, which can achieve more accurate detection results when applied to antibody detection in clinical serum samples (complex samples).

[0061] The above scheme is explained below according to the amount of antibodies:

[0062] When the antibody is a single antibody, under the condition that the peptide chip is not blocked or blocked, the antibody diluted with the sample diluent and the sample diluent without the antibody are used as the control of the background signal (that is, under the condition of non-blocking and blocking, the antibody is in the sample diluent system for the first screening), so as to screen out the peptide set with a single antibody higher than the background signal (that is, the signal of the peptide bound by the sample diluent) and the peptide set resistant to the interference of the blocking solution. The intersection of the two is the specific binding peptide set resistant to the interference of the sample diluent and the blocking solution. The intersection is further taken with the peptide set screened by the antibody in the serum system, and the specific peptide set that is also resistant to the interference of other proteins in the serum is obtained. After the step-by-step peptide chip screening process, different background interfering peptides are excluded, so as to obtain the peptide set with strong specificity of the single antibody.

[0063] When the antibody is a plurality of single antibodies (a single antibody here refers to a different antibody, and a plurality of single antibodies refers to a plurality of different single antibodies), each antibody is subjected to the removal of interfering polypeptides under the same three conditions mentioned above, and a collection of polypeptides with strong specificity corresponding to each single antibody can be obtained.

[0064] When the antibody is a mixed antibody of one or more groups of multiple single antibodies, each group of mixed antibodies undergoes the elimination of non-specific binding polypeptides under the above three conditions to obtain a set of specifically binding polypeptides corresponding to the group of mixed antibodies. Mixed antibodies can be a mixture of antibodies with the same or similar biological functions (for example, antibodies belonging to autoimmune diseases, or antibodies belonging to infectious diseases, or antibodies related to tumor diseases), or they can be a mixture of antibodies with different biological functions. Screening for antibody-specific polypeptides in the form of mixed antibody grouping helps to improve screening efficiency and obtain a set of specific binding polypeptides of multiple antibodies at one time.

[0065] As mentioned above, whether it is one or more single antibodies or one or more groups of mixed antibodies, by eliminating non-specific binding polypeptides under the above different interference conditions, a relatively high specificity polypeptide set can be obtained. Taking the screening of multiple single antibodies as an example, the specific binding polypeptides of each single antibody obtained in the above screening step, when used to detect the corresponding single antibody separately, the specific polypeptides screened by the screening method are not only highly specific, but also numerous (including potential linear epitopes and non-linear epitopes), so the detection sensitivity is also higher than that of conventional diagnostic kits. On this basis, in order to further improve the specificity of the screened polypeptide set, when performing specific polypeptide screening for multiple single antibodies or grouped mixed antibodies, the screening method also includes a step of further removing intersections.

[0066] In a preferred embodiment, the first antibody-positive sample is a plurality of sample dilutions containing a single antibody, the second antibody-positive sample is a plurality of serum samples containing a single antibody, the first antibody-negative sample is a sample dilution not containing a single antibody, and the second antibody-negative sample is a serum sample not containing a single antibody, and the screening method further comprises excluding polypeptides that have intersections between a plurality of different single antibodies; preferably, excluding polypeptides that have intersections between a plurality of different single antibodies at any one or more of the following times: a. after performing the first screening under the non-blocking conditions; b. after performing the first screening under the blocking conditions; c. after performing the second screening.

[0067] Removing the polypeptides that intersect between multiple single antibodies helps to further improve the specificity of the polypeptides bound by each single antibody, so that it is not only applicable to the case of detecting the single antibody alone, but also applicable to the case of simultaneous detection with other single antibodies. When detecting with other single antibodies simultaneously, since the polypeptides that intersect between different single antibodies are removed, the occurrence of cross-reactions can be reduced, making the detection results more accurate.

[0068] As for the timing of removing intersections, removing them at any of the three times mentioned above can reduce the occurrence of cross-reactions. Removing intersections at three different times can remove peptides that may cause cross-reactions under different conditions to the greatest extent, thereby obtaining a set of peptides with higher specificity for each single antibody. Removing intersections after the first screening under non-blocking conditions helps to obtain high-affinity peptides specific to each antibody. These peptides have high sensitivity and specificity when applied to potential drugs (targets), vaccines or antibody detection.

[0069] Similarly, when the first antibody-positive samples are multiple groups of sample dilutions containing mixed antibodies, the second antibody-positive samples are multiple groups of serum samples containing mixed antibodies, the first antibody-negative samples are sample dilutions not containing mixed antibodies, the second antibody-negative samples are serum samples not containing mixed antibodies, and the antibody types contained in any two groups of sample dilutions containing mixed antibodies and / or any two groups of serum samples containing mixed antibodies do not overlap, the screening method also includes excluding polypeptides that have intersections between multiple groups of different mixed antibodies; preferably, excluding polypeptides that have intersections between multiple groups of different mixed antibodies at any one or more of the following times: a. after the first screening is performed under non-blocking conditions; b. after the first screening is performed under blocking conditions; c. after the second screening is performed.

[0070] When mixed antibodies are screened in groups, there are two situations. One is that when the same single antibody exists in any two groups of mixed antibodies, the specific binding polypeptide sets screened by different groups can verify each other to a certain extent. For example, the polypeptide sets screened by the mixed antibodies containing A, B, and C and the mixed antibodies containing A and B can verify each other to a certain extent, but it does not mean that the polypeptide set screened by the mixed antibodies containing A, B, and C minus the polypeptide set screened by the mixed antibodies containing A and B is the polypeptide set corresponding to antibody C. The polypeptide set specifically bound by one of the two mixed antibodies cannot be obtained by a simple subtraction operation.

[0071] Another method is that when there is no identical single antibody between any two groups of mixed antibodies, the above preferred embodiment can be used to remove the intersecting peptides between different groups, thereby obtaining a set of highly specific binding polypeptides corresponding to each group of antibodies. The timing for removing the intersecting polypeptides is also the above three timings.

[0072] In a preferred embodiment, under non-blocking conditions and / or under non-blocking conditions, two or more first antibody-positive samples with different antibody concentrations are used for the first screening (to exclude the possibility that there are differences in the sensitivity of polypeptide binding at different concentrations, thereby screening out polypeptides that can be detected at different concentrations); preferably, under non-blocking conditions, two or more first antibody-positive samples with different antibody concentrations are used for the first screening, and two first antibody-positive samples with different antibody concentrations are determined for the first screening under blocking conditions; preferably, the concentration of a single antibody contained in the first antibody-positive sample or the concentration of each single antibody in the mixed antibody is 10 ng / mL-1 μg / mL, more preferably 50 ng / mL-500 ng / mL.

[0073] By first screening peptides that can stably bind to different concentrations of antibodies under sample dilution conditions, and selecting two different antibody concentrations (there are also requirements for the detection sensitivity and stability of the peptides), screening is then performed under closed conditions to reduce the workload, lower the detection cost, and improve the detection efficiency to a certain extent.

[0074] Similarly, in a serum environment, multiple concentrations can also be used to determine the appropriate high and low concentrations, and then further screening can be performed. In a preferred embodiment, two or more second antibody-positive samples with different antibody concentrations are used for the second screening. More preferably, the concentration of the single antibody contained in the second antibody-positive sample or the concentration of each single antibody in the mixed antibody is 10 ng / mL-1 μg / mL, and more preferably 50 ng / mL-500 ng / mL. Within this concentration difference range spanning an order of magnitude, more stably bound polypeptides can be detected.

[0075] The sample diluent can be any buffer system that can maintain the activity of the antibody. In a preferred embodiment, the sample diluent is a PBST buffer containing D-mannitol, wherein the mass volume content of D-mannitol in the PBST buffer is 0.5% to 1%. The sample diluent can provide a stable solution environment under the sample incubation conditions, and avoid interference with the experimental results without affecting the normal binding of the polypeptide-antibody, thereby providing true, stable and effective data for subsequent fluorescence imaging. Preferably, the sample dilution formula is as follows: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM sodium dihydrogen 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, pH 7.4; more preferably, the sample diluent formula is: 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM sodium dihydrogen 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), pH 7.4.

[0076] Preferably, under blocking conditions, it means that the polypeptide chip is blocked with a blocking solution, and the blocked polypeptide chip is used for screening. More preferably, 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, and the pH of the blocking solution is 7.2-7.6, more preferably 7.38-7.42; further preferably, 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 and 0.1% casein w / v, and the pH of the blocking solution was 7.4.

[0077] The effective ingredients in general blocking reagents include BSA, animal serum, Fab fragment single-chain secondary antibodies, etc. Among them, the BSA component is relatively single and may contain bovine IgG, so it can have a strong cross-reaction with secondary antibodies against cows, goats, sheep, horses, etc., which will cause a certain background. Some animal blocking serum may contain sodium azide, so it is not suitable for HRP enzyme-labeled detection systems. The preparation of Fab fragment single-chain secondary antibodies is complicated and expensive, and it is not suitable for large-scale use. That is, the existing blocking solution has potential problems such as easy to cause background signals such as non-specific binding, high cost, and unstable blocking effect.

[0078] The above-mentioned blocking solution provided in the present application can provide a stable solution environment under the incubation conditions of the secondary antibody, and can reduce the non-specific adsorption of the secondary antibody without affecting the normal binding of the polypeptide-antibody, especially the non-specific adsorption of the secondary antibody to the solid phase components other than the polypeptide array on the polypeptide chip, thereby avoiding interference with the experimental results, and is conducive to the subsequent fluorescence imaging to provide real, effective and stable data. In addition, the above-mentioned blocking solution has low cost and stable performance, and has the effect of improving specificity and sensitivity in immunological detection, especially detection or other scientific research using polypeptide array chips.

[0079] The antibodies used in the screening method of the present application may be monoclonal antibodies or polyclonal antibodies; preferably, multiple single antibodies are all polyclonal antibodies (which helps to screen more specifically binding polypeptides); preferably, multiple single antibodies or multiple groups of mixed antibodies have the same or different species origin; preferably, the species origin is rabbit, goat, rat, mouse, guinea pig or human; preferably, multiple single antibodies or multiple groups of mixed antibodies are selected from multiple of the following antibodies: antibodies to human SNRPC / U1C, antibodies to human PCNA, antibodies to human CENPB, antibodies to human NUP210 / gp210, antibodies to human FTCD / 58K Golgi protein, antibodies to HIB, antibodies to SARS-CoV E protein, antibodies to Plasmodium and antibodies to GAD.

[0080] In a preferred embodiment, the first screening and the second screening using the polypeptide chip both include sequential incubation of antibody samples and incubation of secondary antibodies, wherein the secondary antibody is fluorescently labeled; preferably, the species origin of the secondary antibody is selected based on the species origin of each single antibody or single antibody in the mixed antibody in the first screening and the second screening steps, so as to avoid non-specific signals caused by cross-reactions between species, thereby further improving the specificity of the screened polypeptides.

[0081] In a second typical embodiment of the present application, a method for screening polypeptides that are specifically bound by antibodies based on a polypeptide chip is provided, and the screening method comprises the following steps: under non-blocking conditions, using a polypeptide chip to perform a first screening of first positive samples of multiple single antibodies or multiple groups of mixed antibodies and corresponding first negative samples for specific binding polypeptides, to obtain a first anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies; wherein the first positive sample is a sample dilution containing a single antibody or a sample dilution containing a mixed antibody of multiple single antibodies, and the first negative sample is a sample dilution not containing a single antibody or a mixed antibody; removing polypeptides that have intersections in the first anti-interference peptide library corresponding to each of the multiple single antibodies or multiple groups of mixed antibodies, to obtain a second anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies.

[0082] In this embodiment, by using antibodies diluted by sample diluent and sample diluent without antibodies as controls for background signals under the condition that the peptide chip is not blocked (i.e., the antibodies are in the sample diluent system for the first screening under non-blocking conditions), a peptide segment set of each single antibody or each group of mixed antibodies with a signal higher than the background signal (i.e., the signal of the peptide segment bound by the sample diluent) is screened, and these peptide segments have high affinity with the corresponding antibodies, and peptide segments with intersections between the peptide segments of multiple antibodies are removed, thereby obtaining high-affinity peptide segments specific to each single antibody or each group of mixed antibodies (i.e., the second anti-interference peptide library). These peptide segments have high sensitivity and specificity when applied to potential drugs (targets), vaccines or antibody detection.

[0083] When multiple groups of mixed antibodies are used for screening at the same time, it is helpful to screen a large number of antibodies for specific binding peptides and improve the screening efficiency. Unlike screening with a single antibody, the peptides screened by each group of mixed antibody samples are the sum of the peptides bound by multiple antibodies in the mixed antibody group. Therefore, it is suitable for detecting whether there are multiple antibodies in the sample to be tested. For example, when screening a sample to be tested for the presence of a marker antibody for a certain (uncertain) disease, such mixed antibodies can be used for screening in the screening stage. If a positive antibody is detected in a group of mixed antibodies, each single antibody specific binding peptide corresponding to the mixed antibody group can be further used for detection to confirm the positive antibody.

[0084] In order to further improve the specificity of binding of the screened polypeptides to the target antibodies and improve the anti-interference ability, in a preferred embodiment, the screening method further includes: under closed conditions, using a polypeptide chip to perform a second screening of specific binding polypeptides on the first positive samples of multiple single antibodies or multiple groups of mixed antibodies and the corresponding first negative samples to obtain a third anti-interference peptide library corresponding to each antibody; taking the intersection of the polypeptides in the second anti-interference peptide library and the third anti-interference peptide library to obtain a fourth anti-interference peptide library corresponding to each antibody.

[0085] In this preferred embodiment, by blocking the polypeptide chip and placing the antibody in the sample diluent for a second screening, the peptide segments that non-specifically bind to each single antibody or each group of mixed antibodies under non-blocking conditions are further excluded (i.e., peptide segments that non-specifically bind to the blocking solution are excluded), and a peptide segment set significantly higher than the background signal (the background signal here refers to the signal of the peptide segment bound to the sample diluent after the peptide segment that non-specifically binds to the blocking solution is removed) is obtained, and the intersection is further taken with the second anti-interference peptide library screened under the aforementioned non-blocking sample diluent conditions, thereby obtaining a high-affinity peptide segment set (i.e., the fourth anti-interference peptide library) that specifically binds to the corresponding antibody under two different conditions. These peptide segments have a stronger anti-interference ability to blocking conditions, and when used for target antibody detection, the specificity of detection is further improved to a certain extent.

[0086] On the basis of the above, in order to further eliminate other possible interferences in the clinical testing process, in a preferred embodiment, the screening method further includes: under closed conditions, using a polypeptide chip to perform a third screening of specific binding polypeptides on the second positive samples of multiple single antibodies or multiple groups of mixed antibodies and the corresponding second negative samples to obtain a fifth anti-interference peptide library corresponding to each antibody; wherein the second positive sample is a serum sample containing a single antibody, and the second negative sample is a serum sample not containing a single antibody; taking the intersection of the fifth anti-interference peptide library and the fourth anti-interference peptide library to obtain a sixth anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies.

[0087] In this preferred embodiment, by placing the antibody in a serum environment for a third polypeptide chip test, polypeptides bound to other components in the serum environment are further excluded, so that the binding specificity of the peptides in the sixth anti-interference peptide library to the corresponding antibodies is further improved, thereby being suitable for the detection of specific antibodies in complex samples such as serum / plasma.

[0088] In a preferred embodiment, after obtaining the third anti-interference peptide library and / or the fifth anti-interference peptide library corresponding to each antibody, the screening method also includes: removing polypeptides in which multiple single antibodies or multiple groups of mixed antibodies have intersections in each third anti-interference peptide library and / or the fifth anti-interference peptide library.

[0089] Under the above-mentioned blocking conditions, in the step of placing the antibody in the sample diluent for peptide chip detection, and under the blocking conditions, in the step of placing the antibody in simulated serum for peptide chip detection, after screening multiple single antibodies or multiple groups of mixed antibodies to obtain the corresponding third anti-interference peptide library and / or fifth anti-interference peptide library, the peptide segments with intersections in the multiple single antibodies or multiple groups of mixed antibodies can be further removed, and the peptide segments in the third anti-interference peptide library and / or the fifth anti-interference peptide library that are retained have stronger specificity. In an optimal embodiment, both of the above steps include the step of removing the corresponding intersection peptide segments.

[0090] It should be noted that the polypeptides that specifically bind to antibodies in the random polypeptide arrays screened in the above preferred embodiments of the present application simulate the characteristics of antigenic epitopes, including both linear epitopes and conformational epitopes (i.e., non-linear epitopes). In the case of conformational epitopes, they are not necessarily related to the linear sequence of the antigen, but their binding to the antibody may be as strong as or even stronger than that of the original antigen.

[0091] In the above two different embodiments of the screening method, whether it is one or more single antibodies, or a mixed antibody formed by multiple single antibodies, when performing the detection, it is preferred that each single antibody is screened for the first time using two or more first positive samples with different antibody concentrations (to exclude the possibility that there is a difference in the sensitivity of polypeptide binding at different concentrations, thereby screening out polypeptides that can be detected at different concentrations); preferably, the concentration of (any one) single antibody contained in the first positive sample is 10ng / mL-1μg / mL, more preferably 50ng / mL-500ng / mL. Within this concentration difference range spanning an order of magnitude, more stably bound polypeptides can be detected.

[0092] The sample diluent can be any buffer system that can maintain the activity of the antibody. In a preferred embodiment, the sample diluent is a PBST buffer containing D-mannitol, wherein the mass volume content of D-mannitol in the PBST buffer is 0.5% to 1%. The sample diluent can provide a stable solution environment under the sample incubation conditions, and avoid interference with the experimental results without affecting the normal binding of the polypeptide-antibody, thereby providing true, stable and effective data for subsequent fluorescence imaging. Preferably, the sample dilution formula is as follows: 130-137 mM sodium chloride, 2.5-2.7 mM potassium chloride, 3.8-4.3 mM sodium dihydrogen 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, pH 7.4; more preferably, the sample diluent formula is: 137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM sodium dihydrogen 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), pH 7.4.

[0093] The aforementioned polypeptide chip detection at multiple concentrations helps to screen out polypeptides that can stably bind to antibodies at different concentrations. In order to further improve the efficiency of the detection, in a preferred embodiment, under non-blocking conditions, a first screening is performed using first positive samples with more than two different antibody concentrations, and then two first positive samples with different antibody concentrations are determined, and then a second screening is performed under blocking conditions.

[0094] In this preferred embodiment, polypeptides that can stably bind to antibodies of different concentrations are first screened under sample dilution conditions, and two different antibody concentrations, high and low, are selected (there are also requirements for the detection sensitivity and stability of the polypeptide), and then screening is performed under closed conditions to reduce the workload, reduce the detection cost, and improve the detection efficiency to a certain extent.

[0095] Similarly, in a serum environment, multiple concentrations can also be used to determine the appropriate high and low concentrations for further subsequent screening. In a preferred embodiment, each single antibody is screened for the third time using two or more second positive samples with different antibody concentrations; preferably, the concentration of the single antibody contained in the second positive sample is 10ng / mL-1μg / mL, more preferably 50ng / mL-500ng / mL. Within this concentration difference range spanning an order of magnitude, more stably bound polypeptides can be detected.

[0096] In a preferred embodiment, the blocking condition refers to blocking the polypeptide chip with a blocking solution, and using the blocked polypeptide chip for the second screening and the third screening respectively. Before loading the polypeptide chip, the polypeptide chip is first blocked with a blocking solution, which helps to reduce the signal intensity of non-specific binding peptides. The above-mentioned blocking solution can also be a commonly used blocking solution. In order to more effectively reduce the background signal of the high-density polypeptide array, in the preferred embodiment of the present application, the composition of the blocking solution used is different from the composition of the generally used blocking reagent.

[0097] The effective ingredients in general blocking reagents include BSA, animal serum, Fab fragment single-chain secondary antibodies, etc. Among them, the BSA component is relatively single and may contain bovine IgG, so it can have a strong cross-reaction with secondary antibodies against cows, goats, sheep, horses, etc., which will cause a certain background. Some animal blocking serum may contain sodium azide, so it is not suitable for HRP enzyme-labeled detection systems. The preparation of Fab fragment single-chain secondary antibodies is complicated and expensive, and it is not suitable for large-scale use. That is, the existing blocking solution has potential problems such as easy to cause background signals such as non-specific binding, high cost, and unstable blocking effect.

[0098] In a preferred embodiment of the present 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% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v; pH is 7.2-7.6, preferably 7.38-7.42. In a more preferred embodiment of the present 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.

[0099] The above-mentioned blocking solution provided in the present application can provide a stable solution environment under the incubation conditions of the secondary antibody, and can reduce the non-specific adsorption of the secondary antibody without affecting the normal binding of the polypeptide-antibody, especially the non-specific adsorption of the secondary antibody to the solid phase components other than the polypeptide array on the polypeptide chip, thereby avoiding interference with the experimental results, and is conducive to the subsequent fluorescence imaging to provide real, effective and stable data. In addition, the above-mentioned blocking solution has low cost and stable performance, and has the effect of improving specificity and sensitivity in immunological detection, especially detection or other scientific research using polypeptide array chips.

[0100] The antibodies used in the screening method of the present application may be monoclonal antibodies or polyclonal antibodies; preferably, multiple single antibodies are all polyclonal antibodies (which helps to screen more specifically binding polypeptides); preferably, multiple single antibodies or multiple groups of mixed antibodies have the same or different species origin; preferably, the species origin is rabbit, goat, rat, mouse, guinea pig or human; preferably, multiple single antibodies or multiple groups of mixed antibodies are selected from multiple of the following antibodies: antibodies to human SNRPC / U1C, antibodies to human PCNA, antibodies to human CENPB, antibodies to human NUP210 / gp210, antibodies to human FTCD / 58K Golgi protein, antibodies to HIB, antibodies to SARS-CoV E protein, antibodies to Plasmodium and antibodies to GAD.

[0101] In a preferred embodiment, the steps of the first screening, the second screening and the third screening all include sequential incubation of antibody samples and incubation of secondary antibodies, wherein the secondary antibodies are fluorescently labeled; preferably, the species source of the secondary antibodies is selected according to the species source of the corresponding antibodies in the first screening, the second screening and the third screening steps, respectively. The antibody samples for the first screening and the second screening are both sample diluents containing antibodies; the antibody sample for the third screening is serum containing antibodies. The species source of the secondary antibody is selected according to the species source of the corresponding sample antibody, which can avoid nonspecific signals caused by cross-reactions between species, thereby further improving the specificity of the screened polypeptide.

[0102] In a third typical embodiment of the present application, a method for detecting a target antibody in a test sample is provided, the detection method comprising: using a target polypeptide to which the target antibody specifically binds, detecting the test sample and a negative control of the test sample to obtain respective corresponding detection results; if there is a significant difference in the detection results corresponding to the test sample and the negative control, it indicates that the test sample contains the target antibody; the target polypeptide is a polypeptide that specifically binds to the target antibody screened by any of the aforementioned screening methods.

[0103] The polypeptides screened by the present application are a collection of polypeptides that are specifically bound by one or more antibodies. These polypeptides can be selected and prepared into conventional IVD antibody detection kits (such as ELISA kits), and the presence or absence of antibodies in the sample to be tested can be determined by comparing with the negative control. Since the polypeptides screened by the screening method of the present application have the advantages of high sensitivity and strong specificity, not only the sensitivity is higher when detecting samples containing a single antibody, but also the specificity is higher when detecting samples containing multiple antibodies.

[0104] In addition to being prepared into conventional IVD antibody detection kits for antibody detection, the polypeptides screened by the present application can also be prepared into polypeptide chips for antibody detection, or detected using an existing polypeptide chip array containing a target polypeptide that specifically binds to the antibody to be detected (it is only necessary to compare the difference between the target polypeptide of interest in the sample to be tested and the control sample). Depending on whether the sample to be tested contains the target antibody, when the polypeptide set (of one or more antibodies) screened by the present application is used for detection, not only can the presence or absence of the target antibody be detected, but also, if it is present, whether there is one or more than one.

[0105] In a preferred embodiment, under the condition of blocking the polypeptide chip, a polypeptide chip containing the target polypeptide is used to detect the sample to be tested and the negative control of the sample to be tested, and the detection results of the corresponding target polypeptides are obtained; if there is a significant difference between the detection results corresponding to the sample to be tested and the negative control of the target polypeptide, it indicates that the sample to be tested contains the target antibody; preferably, the sample to be tested is a serum sample diluted with a sample diluent, the negative control is the sample diluent, and the target polypeptide is any multiple polypeptides or peptide libraries in the polypeptide set that specifically binds to the target antibody screened by the aforementioned screening method (each peptide library covers multiple antibodies that specifically bind).

[0106] In a preferred embodiment, the target antibody is selected from any one or more of the following: human SNRPC / U1C antibody, human PCNA antibody, human CENPB antibody, human NUP210 / gp210 antibody, human FTCD / 58K Golgi protein antibody, HIB antibody, SARS-CoV E protein antibody, Plasmodium antibody and GAD antibody. The use of a polypeptide chip can simultaneously detect the presence or absence of one or more target antibodies.

[0107] In a preferred embodiment, the sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of D-mannitol in PBST is 0.5% to 1%.

[0108] When using a polypeptide chip for detection, the processing conditions of the polypeptide chip are the same as those for polypeptide screening. In a preferred embodiment, the secondary antibody is selected according to the species source of the serum sample (to improve the specificity of the detection); preferably, before the test sample and the negative control of the test sample are detected, the detection method also includes: using a blocking solution to block the polypeptide chip (to reduce the non-specific binding caused by the blocking solution to improve the specificity of the detection); preferably, the blocking solution includes the following components: 130-137mM sodium chloride, 2.5-2.7mM potassium chloride, 3.8-4.3mM disodium hydrogen phosphate, 1.2-1.4mM potassium dihydrogen phosphate, 0.05%-1% Tween-20v / v, 0.05%-0.1% Proclin950 v / v, 0.5% to 1% D-mannitol w / v and 0.1%-1% casein w / v, the pH of the blocking solution is 7.2 to 7.6, more preferably 7.38 to 7.42; more preferably, the blocking solution is 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20v / 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. As mentioned above, the blocking solution of the present application can reduce the nonspecific adsorption of the secondary antibody, and has good blocking effect, low cost, stable performance, and has the effect of improving detection specificity and sensitivity.

[0109] In a preferred embodiment, under the condition of blocking the polypeptide chip, using the polypeptide chip containing the target polypeptide, the sample to be tested and the negative control of the sample to be tested are detected, including: incubating the sample diluent and the serum sample diluted by the sample diluent with the blocked polypeptide chip to obtain a first incubation product; co-incubating the first incubation product with a fluorescently labeled secondary antibody to obtain a second incubation product; performing fluorescence scanning on the second incubation product to obtain a fluorescent signal of the sample diluent and a fluorescent signal of the serum sample; detecting whether there is a significant difference between the fluorescent signal of the sample diluent and the fluorescent signal of the serum sample for the target polypeptide (only using the target polypeptide for difference judgment helps to reduce the amount of signal processing, improve the signal processing speed and efficiency, and improve the detection efficiency). If so, it indicates that the target antibody exists in the serum sample (to be tested) (as long as there is a target antibody, whether it is one or more, it can be detected).

[0110] It should be noted that in a preferred embodiment of the present application, when a plurality of screened polypeptides or polypeptide sets are used to detect the presence or absence of one or more target antibodies in a sample to be tested, it is more appropriate to use artificial intelligence to process such "big data", such as by establishing a prediction model (such as using a ridge regression method for modeling) to predict the sample to be tested. Specifically, a prediction model can be established by using the difference between the fluorescence signal of the above-mentioned target polypeptide in a positive serum sample cohort and the fluorescence signal in a negative serum sample cohort in a serum / plasma sample in which the antibody positivity is known clinically, and then by inputting the fluorescence signal detected by the sample to be tested in the corresponding target polypeptide into the prediction model, the test result of whether the sample to be tested is antibody positive or negative can be directly output.

[0111] In a preferred embodiment, the above-mentioned 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; wherein, establishing the antibody prediction model includes: obtaining the signal intensity of the target polypeptide detected by the polypeptide chip under closed conditions in the positive serum samples and the negative serum samples based on multiple positive serum samples in which the target antibody is clinically known to be positive and multiple negative serum samples known to be negative, and establishing the antibody prediction model based on the relationship between the signal intensity and the positivity or negativity of the target antibody.

[0112] In a preferred embodiment of the present application, the above clinically known negative serum samples are serum samples of healthy people, which is convenient for distinguishing healthy people from unhealthy people, so as to further distinguish different clinical samples of unhealthy people who carry one or more diseases or are at different stages or courses of the disease.

[0113] The following uses commercial antibodies to further explain in detail the polypeptide screening method of the present application in some more specific examples (gradually increasing interference conditions for polypeptide chip technology detection), which mainly includes the following steps:

[0114] 1) Under non-blocking conditions, the antibody is diluted with sample diluent (preferably at multiple concentrations, or two concentrations, high and low, such as 10 ng / mL-1 μg / mL) and tested on the machine;

[0115] 2) diluting the sample with sample diluent under blocking conditions (preferably multiple concentrations, or high and low concentrations, such as 10 ng / mL-1 μg / mL) and testing on the machine;

[0116] 3) The antibody is mixed with negative serum background under blocking conditions and then diluted (preferably at multiple concentrations, or high and low concentrations, such as 10 ng / mL-1 μg / mL) and tested on a machine;

[0117] Then, by gradually eliminating the blocking solution and serum background interference signals, the peptides specifically bound to the antibody sample were analyzed.

[0118] For example: Experimental design with increasing interference conditions step by step

[0119] Table 1:

[0120]

[0121] It should be noted that the blocking condition means that the sample (i.e., the specific commercial antibody in this application) is incubated with the polypeptide chip with a blocking solution before being added to the polypeptide chip; after incubation, the sample is added to the polypeptide chip for incubation and detection.

[0122] Final concentration of antibody on the machine: the concentration of the antibody in the reaction system when the antibody is tested on a peptide chip;

[0123] Detection background refers to the solution system in which the antibody is located when performing peptide chip detection.

[0124] The testing process is as follows:

[0125] 1. Sample preparation: Dissolve the antibody sample in sample diluent and negative serum to obtain high and low concentration antibody samples, respectively, recorded as [low antibody + sample diluent], [high antibody + sample diluent], [low antibody + serum], [high antibody + serum]. Also prepare [no antibody + sample diluent] and [no antibody + serum] samples as negative controls.

[0126] 2. Sample dilution: Dilute the above sample 1000 times with sample diluent and vortex to mix.

[0127] 3. Sample incubation: Under non-blocking conditions, add 1000-fold diluted [low antibody + sample diluent], [high antibody + sample diluent] samples, [no antibody + sample diluent] negative control samples and blank (cleaning solution), incubate at a constant temperature, and wash the plate; when using blocking solution, add [low antibody + sample diluent], [high antibody + sample diluent], [low antibody + serum], [high antibody + serum] samples, [no antibody + sample diluent] and [no antibody + serum] negative control samples in turn, incubate with blank (cleaning solution) at a constant temperature, and wash the plate.

[0128] 4. Secondary antibody incubation: Add secondary antibodies according to the species of the antibodies (if the antibody is mouse-derived, select anti-mouse secondary antibodies; if the antibody is rabbit-derived, select anti-rabbit secondary antibodies; if the antibody is human-derived, select anti-human secondary antibodies), incubate at a constant temperature, and wash the plate. The negative control secondary antibody is selected according to the species of the antibody on the same detection chip. If there is more than one antibody species on the same detection chip, add a corresponding control.

[0129] 5. Fluorescence imaging: The chip is transferred to an imaging device, which scans the fluorescence signal and produces a high-resolution image.

[0130] 6. Image processing: Convert the images obtained by fluorescence imaging into fluorescence intensity values ​​to obtain the corresponding numerical matrix.

[0131] 7. Preliminary data processing and quality control: Perform logarithmic transformation and standardization on sample values ​​and output a standardized matrix. Perform single sample quality control and system stability quality control.

[0132] Data analysis process:

[0133] 1. Using antibody peptide chip detection experiments with gradually increasing interference conditions, we can mine antibody-specific peptide sets that are not interfered by blocking solution and other serum proteins. The idea of ​​graded peptide analysis is as follows:

[0134] 1) For a single detection antibody, first screen out high-affinity peptides (denoted as Lv1) that can be stably captured by the peptide chip platform;

[0135] 2) For multiple detection antibodies, cross-linked polypeptides are excluded to obtain specific polypeptides for detecting a single antibody (denoted as Lv2);

[0136] 3) The number of specific peptides detected under blocking conditions is lower than that under non-blocking conditions, 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);

[0137] 4) Detection in a blocking and serum environment to obtain a specific binding peptide (denoted as Lv4) that is resistant to double interference (blocking solution and serum environment);

[0138] 5) When the screened polypeptides are used, the polypeptides that specifically bind to multiple antibodies are combined in a combined form (recorded as Lv5) to detect at one time whether the target antibody is present in the serum, and whether the target antibody is one or more.

[0139] 2. The specific steps are as follows:

[0140] (1) By testing the same antibody at different concentrations under non-blocking conditions, the signal intensity of the peptide signal in the sample is obtained, and the peptides that are significantly higher than the signal intensity threshold of the negative control sample [no antibody + sample diluent] are screened as the preliminary seed peptide library, and the false positive signals introduced by the production and experimental links are excluded, and the high-signal peptide set stably captured by each antibody platform - Lv1 peptide set is obtained; the Lv1 peptide sets of each antibody that have been tested are compared with each other, and the common peptides of antibodies of non-identical immunogens or homologous immunogens are excluded, which contain systematic interference signals caused by factors such as antibody preparation and purification methods, and the specific peptide set of each antibody - Lv2 peptide set is output, which can indicate the presence or absence of a specific antibody under non-blocking conditions.

[0141] (2) Analyze the peptide signals detected under high and low concentration blocking conditions of the same antibody, screen the peptide set that is significantly higher than the signal intensity threshold of the negative control sample [no antibody + sample diluent], exclude the false positive signals introduced in the production experiment, and obtain the high signal peptide set that is stably captured by the platform under blocking conditions for each antibody. Then compare the antibodies with each other, exclude the common peptides of antibodies with non-identical immunogens or homologous immunogens, and preliminarily obtain the specific peptide set of each antibody under blocking conditions. Excluding the false positive signals caused by the interaction between the blocking solution components and the antibody, the intersection of the antibody-specific peptides under blocking and non-blocking conditions - Lv3, can indicate the presence or absence of a specific antibody under blocking conditions.

[0142] (3) Analyze the peptide signals detected under blocking conditions of high and low concentrations of the same antibody mixed into the serum background, screen the peptide set that is significantly higher than the signal intensity threshold of the negative control sample [no antibody + serum], exclude the false positive signals introduced by the indicated production experiment link, and obtain the high signal peptide set that the platform stably captures under blocking conditions and in the presence of serum background for each antibody. Then compare the antibodies with each other, exclude the common peptides of antibodies of non-identical immunogens or homologous immunogens, and preliminarily obtain the specific peptide set of each antibody under blocking conditions and in the serum background. In order to exclude false positive signals caused by the interaction between the blocking solution components and the 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 whether a specific antibody exists under blocking conditions and in the serum background.

[0143] The beneficial effects of the present invention are further described below in conjunction with specific examples. It should be noted that the components of the sample diluent used in the following examples are as follows: PBST containing 1% D-mannitol 1, PBST is PBS containing 0.05%-1% Tween 20, wherein the specific formula of 1×PBS (pH 7.9, Teknova) is as follows: 137mM sodium chloride, 2.7mM potassium chloride, 4.3mM disodium hydrogen phosphate, 1.4mM potassium dihydrogen phosphate, 1% Tween-20 v / v, 0.1% Proclin950 v / v, 1% D-mannitol w / v.

[0144] Blocking solution: 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, the pH of the blocking solution is 7.4.

[0145] Example 1: Highly reliable specific binding peptides of antibodies are used to identify antibody components in mixed antibodies

[0146] A total of eight antibodies were included to analyze the high-confidence specific binding peptides of the antibodies by filtering interference information step by step. Four of them were selected for two-antibody, three-antibody, and four-antibody mixed detection analysis, and the interference conditions of the peptide chip technology were gradually increased as above, that is, direct detection under non-blocking conditions, detection under blocking conditions, and detection of antibodies mixed with negative serum samples. The final concentration of the antibody was set to 50ng / ml and 500ng / ml for single-machine detection, and the final concentration of the mixed antibody was set to 50ng / ml.

[0147] A antibody test alone

[0148] 1. Sample preparation

[0149] 1. Antibody sample information confirmation: including pre-treatment requirements, concentration source species, etc. See the table below for details.

[0150] 2. The information of the detection antibodies involved in this application is shown in the following table:

[0151] Table 2:

[0152]

[0153]

[0154] Note:

[0155] PCNA antibody: Proliferating Cell Nuclear Antigen (PCNA) was first discovered and named by Miyachi et al. in the serum of SLE (systemic lupus erythematosus) patients in 1978. It is named because it only exists in normal proliferating cells and tumor cells. PCNA is a protein with a molecular weight of 36KD. It is synthesized and exists in the cell nucleus. It is an auxiliary protein of DNA polymerase δ. PCNA antibody has been used clinically for the specific detection of systemic lupus erythematosus, but its positive rate is very low. The positive rate of IIF method is only 2% to 5%. In recent years, studies have shown that it can be detected in a variety of autoimmune diseases. SLE is still the main detected disease. Systemic lupus erythematosus patients with anti-PCNA positive are more likely to have rashes, Raynaud's phenomenon, neuropsychiatric lupus and kidney involvement, which are related to disease activity.

[0156] CENPB antibody: The centromere protein is composed of three components: centromere protein A (CENPA), centromere protein B (CENPB) and centromere protein C (CENPC); among them, CENPB has a relative molecular mass of 80,000 and is the main target antigen of anti-centromere antibodies. Anti-centromere protein antibodies have long been considered to have high specificity for systemic sclerosis (SSc), especially the CREST subtype, the main clinical manifestations of which include calcification, Raynaud's manifestation, esophageal motility disorder, fingertip sclerosis, and capillary dilation. The positive rate of anti-centromere antibodies in patients with CREST syndrome is 40% to 90%.

[0157] gp210 antibody: Anti-gp210 antibody is a type of anti-nuclear envelope protein antibody that shows nuclear membrane type fluorescent staining. 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). Anti-mitochondrial antibody (AMA) is a specific antibody for diagnosing PBC with a sensitivity of 90%, but 10% of PBC patients are still AMA antibody negative, that is, AMA has certain limitations in the diagnosis of PBC. Anti-gp210 antibody is present in 20% to 47% of AMA-negative PBC patients. For patients with clinical, biochemical and histological manifestations suspected of PBC but AMA negative, or AMA positive but atypical clinical symptoms, and overlapping syndromes (such as overlapping with Sjögren's syndrome), anti-gp210 antibody detection is of great value. In addition, the incidence of cirrhosis in patients with positive gp210 is significantly higher than that in negative patients. Therefore, the positive antibody indicates a poor prognosis for patients and can also be used as a prognostic indicator for PBC patients.

[0158] LC-1 antibody: In 1988, Martini et al. first confirmed the existence of anti-LC-1 antibody in the serum of 6 adult AIH patients using immunofluorescence (IIF) and ID methods. Its target antigen exists in the cytosol of hepatocytes, and the immunoblotting (IB) method detected it as a 58kD to 62kD hepatocyte cytosolic polypeptide protein. Anti-hepatocyte cytoplasmic type 1 antibody (anti-LC-1 antibody) or anti-hepatocyte cytoplasmic antigen type 1 antibody is a specific and sensitive marker antibody for type II autoimmune hepatitis (AIH-II type). The positive rate in AIH-II is 56% to 72%, which is closely related to the diagnosis of autoimmune hepatitis.

[0159] HIB antibody: HIB invasive Haemophilus influenzae type b is a common commensal bacterium in the nasopharynx of children. It is a bacterium that causes an estimated 3 million serious illnesses and an estimated 386,000 deaths each year, mainly through meningitis and pneumonia.

[0160] SARS-Cov-E: The SARS virus causes "atypical pneumonia" and its E protein is an envelope protein. Detection of antibodies to this protein can assist in diagnosing whether the test sample has been infected by the SARS virus.

[0161] GAD antibody: Anti-glutamic acid decarboxylase antibody is suitable for the diagnosis of late-onset autoimmune diabetes in adults. Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme that converts glutamate into the inhibitory neurotransmitter γ-aminobutyric acid (GABA). GAD exists in pancreatic β cells and also synthesizes and secretes GABA. Glutamic acid decarboxylase antibody (GADA) is an immune marker in the early stages of type 1 diabetes and is also used as an indicator for monitoring the efficacy of treatment for patients with type 1 diabetes. The GADA positivity rate in patients with type 1 diabetes and Graves' disease is significantly higher than that in patients with type 1 diabetes without Graves' disease. The GAD level in patients with Graves' disease can be significantly increased. The presence of GADA in non-diabetic patients does not always predict the occurrence of type 1 diabetes.

[0162] Pf: Antibody against Plasmodium falciparum ferredoxin-NADP+ reductase (pfFNR). Plasmodium falciparum is one of the four malarial parasites that parasitize humans and is the pathogen that causes falciparum malaria. Plasmodium falciparum uses humans and female Anopheles mosquitoes as hosts. It undergoes schizont proliferation and the beginning of gamete reproduction in humans. It completes gamete reproduction and spore proliferation in mosquitoes. It develops in liver cells and red blood cells in the human body.

[0163] 3. Sample pretreatment: Dissolve some of the powdered antibodies and then test the concentration together with some antibodies without concentration information. All antibodies to be tested are uniformly diluted to 100μg / ml using sample diluent.

[0164] 4. Prepare the total IgG quality control product (the total IgG sample extracted from the same serum sample by ammonium persulfate precipitation method. In other embodiments, unless otherwise specified, the total IgG quality control product is the same as this).

[0165] 2. Detection of pure antibodies under non-blocking conditions

[0166] Each antibody was diluted to 50 ng / ml and 500 ng / ml with sample diluent for peptide chip detection;

[0167] The secondary antibody for antibody detection is selected according to the source species information (for example, if the antibody is from rabbit, the secondary antibody is anti-rabbit secondary antibody);

[0168] Try to arrange antibodies from the same species 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 adding antibodies);

[0169] The control secondary antibody is selected according to the source species of the detection antibody on the same detection chip. If there is more than one source of antibody species on the same detection chip, corresponding controls are added (i.e., if the detection antibodies on the same detection chip are from rabbit and mouse, control secondary antibodies of rabbit and mouse are set on the chip).

[0170] Detection Operation

[0171] 1. Sample dilution

[0172] a) Dilution of total IgG control

[0173] Use a 5 ml centrifuge tube to dilute IgG, take 16 μL of IgG and add it to 1584 μL of sample diluent to form a 100-fold diluted IgG, and shake to mix.

[0174] b) Antibody dilution

[0175] The antibodies were diluted to 500 ng / ml and 50 ng / ml respectively using the sample diluent.

[0176] 1) Take 2 μL of 100 μg / ml antibody and add it to 398 μL of sample diluent to form a diluted antibody with a concentration of 500 ng / ml (gradient 1), and shake to mix;

[0177] 2) Take 20 μL of gradient 1 and add it to 180 μL of sample diluent to form a diluted antibody with a concentration of 50 ng / ml (gradient 2), and shake to mix.

[0178] 2. Typesetting and sample adding

[0179] According to the sample layout design, dispense 120 μL of 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 dispenser to transfer 90 μL to the corresponding wells of the assay Cassette and seal the film.

[0180] 3. First incubation

[0181] 1) Turn on the thermostatic mixer in advance and stabilize the temperature to 37°C.

[0182] 2) Place the assay cassette on a thermomixer and incubate for 60 min. Oscillate the cassette for 15 s every 6 min.

[0183] 4. First wash

[0184] Stop shaking, tear off the membrane and put it into the plate washer, make sure there is enough liquid in the waste bottle of the plate washer, and wash the plate with an automatic plate washer ( Microplate magnetic washer).

[0185] 5. Add secondary antibody

[0186] Prepare secondary antibody diluent: In a light-proof condition (turn off the light), use a 15ml centrifuge tube to dilute the rabbit secondary antibody (i.e. the rabbit antibody) 1500 times (9μL of secondary antibody is added to 13500μL of secondary antibody diluent). Then, according to the well position information in the layout table, use a dispenser to take 40μL / well and add it to the corresponding wells of the corresponding Cassette, and seal the film.

[0187] It should be noted that in other embodiments, the corresponding secondary antibody and its corresponding dilution conditions are selected according to the species of the antibody, such as mouse or human. For example, the human secondary antibody (i.e., the antibody of human antibody) can be diluted 1500 times (4 μL of secondary antibody is added to 6 ml of secondary antibody diluent), and the mouse secondary antibody (i.e., the antibody of mouse antibody) can be diluted 3000 times (2 μL of secondary antibody is added to 6 ml of secondary antibody diluent).

[0188] 6. Second incubation

[0189] Seal the cassette and incubate it on a thermomixer for 60 min. Oscillate the cassette for 15 s every 6 min.

[0190] 7. Second wash

[0191] Stop shaking, tear off the membrane and put it into the plate washer, make sure the waste liquid bottle of the plate washer has enough capacity, and use an automatic plate washer to wash the plate ( Microplate magnetic washer).

[0192] 8. Chip removal

[0193] 1) Prepare the operating area in the biosafety cabinet, including the chip centrifuge, low cotton absorbent pad, 90% isopropyl alcohol spray bottle, MilliQ water basin with chip slot and handle, MilliQ hydration box, clean chip storage box. Carefully take out each chip in the assay cassette, one at a time, and place it on the chip slot.

[0194] 2) Chips are dried and packed in boxes.

[0195] 9. Assemble the chip in the imaging cassette.

[0196] 10. Fluorescence Imaging

[0197] The Imaging System uses a robotic arm to transfer the imaging cassette to the barcode scanning position. After the imaging cassette and chip barcode scanning is completed, the imaging cassette is transferred to Image Xpress for imaging. The system controls the experimenter through the Venus software to guide the experimenter to perform imaging operations, and the operator needs to set the exposure time and the number of imaging cassettes. When imaging begins, the operator can continue to add imaging cassettes that need to be imaged.

[0198] The imaging process generates high-resolution TIFF images for each array using a Molecular Devices Image Xpress 4 imager. Gridding Software extracts feature intensity data from the TIFF image files.

[0199] When the imaging cassette is assembled, the cassette is transferred to the cassette hotel. Next, the automated imaging process will begin.

[0200] The specific data preprocessing process is as follows:

[0201] 1) Extract the fluorescence intensity value of the feature and output a GPR5 data file and a corner images file. The GPR5 file contains all the information of a sample and the fluorescence intensity information of all features.

[0202] 2) Extract the characteristic fluorescence intensity information from the GPR5 data files of all samples to generate the original fluorescence intensity (FG, foreground) data matrix. Then logarithmically transform the data of each sample to obtain the LFG (log-transferred foreground) data matrix, and then subtract the median of the LFG data matrix of each sample from each value in the LFG data matrix of each sample to obtain the NLFG (normalized and log-transferred foreground) data matrix. This step will also generate a sample chip information file, which includes information such as the sample array position and the chip number used.

[0203] 3) Quality Control

[0204] The samples and system were quality controlled using Health Tell's own quality control method and were found to be qualified.

[0205] 3. Pure antibody detection under closed conditions

[0206] Under blocking conditions, the antibody was diluted with sample diluent to a final concentration of 50 ng / ml and 500 ng / ml for peptide chip detection;

[0207] The secondary antibody for antibody detection is selected according to the source species information;

[0208] Try to arrange antibodies from the same species on the same chip, and add one well of pure sample dilution to each detection chip as a control with an antibody concentration of 0;

[0209] The control secondary antibody is selected according to the species of the detection antibody on the same detection chip. If there is more than one source of antibody species on the same detection chip, corresponding controls should be added.

[0210] Detection Operation

[0211] 1. Sample dilution:

[0212] a) Dilution of total IgG control

[0213] Use a 1.5 ml centrifuge tube to dilute IgG, take 16 μL of IgG and add it to 784 μL of sample diluent to form a 50-fold diluted IgG, and shake to mix.

[0214] b) Antibody dilution

[0215] Use sample diluent to dilute the antibody to 1000ng / ml and 100ng / ml respectively.

[0216] 1) Take 2 μL of 100 μg / ml antibody and add it to 18 μL of sample diluent to form a diluted antibody with a concentration of 10 μg / ml (gradient 1), seal the membrane, shake and mix, and centrifuge (4000 rpm for 1 min);

[0217] 2) Take 15 μL of the antibody in gradient 1 and add it to 135 μL of the sample diluent to form a diluted antibody with a concentration of 1000 ng / ml (gradient 2), seal the membrane, shake and mix, and centrifuge (4000 rpm for 1 min);

[0218] 3) Take 12 μL of gradient 2 and add it to 108 μL of sample diluent to form a diluted antibody with a concentration of 100 ng / ml (gradient 3), seal the membrane, shake and mix, and centrifuge (4000 rpm 1 min)

[0219] c) Preparation of blocking solution

[0220] Take 2 ml of 1% casein and add it to 18 ml of sample diluent, mix well and set aside.

[0221] 2. Typesetting and sample adding

[0222] 1) Sealing of the chip

[0223] Use a dispenser to dispense 45 μL of the prepared blocking solution into the wells of the Cassette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate in an incubator at 37°C for 1 hour.

[0224] 2) Blocking and loading of Cassette (chip)

[0225] According to the sample layout design, use a pipette to transfer 45 μL of 1000 ng / ml diluted antibody and 100 ng / ml diluted antibody to the corresponding wells of the Cassette.

[0226] 3. First incubation

[0227] Seal the Cassette, place it on the incubation module of the automated instrument, and incubate for 1 hour (for specific operations, refer to the first incubation under non-blocking conditions).

[0228] 4. First wash

[0229] Tear off the membrane of Cassette, place in automatic plate washer, and wash the plate (for specific operation, refer to the first plate washing under non-blocking conditions).

[0230] 5. Add secondary antibody

[0231] Under light-proof conditions (turn off the lights), use a 15ml centrifuge tube to dilute the human secondary antibody 1500 times (4μL of secondary antibody added to 6ml of secondary antibody diluent), dilute the mouse secondary antibody 3000 times (2μL of secondary antibody added to 6ml of secondary antibody diluent), and use a 50ml centrifuge tube to dilute the rabbit secondary antibody 1500 times (9μL of secondary antibody added to 13500μL of secondary antibody diluent). Then, according to the well position information in the layout table, use a dispenser to take 40μL and add it to the corresponding wells of the corresponding Cassette.

[0232] 6. Second incubation

[0233] Seal the cassette and place it on a thermomixer for incubation for 60 min. Oscillate the cassette for 15 s every 6 min.

[0234] 7. Second wash

[0235] Stop shaking, tear off the membrane and put it into the plate washer, make sure the waste liquid bottle of the plate washer has enough capacity, and use an automatic plate washer to wash the plate ( Microplate magnetic washer).

[0236] 8. Chip removal

[0237] 1) Prepare the operating area in the biosafety cabinet, including the chip centrifuge, low cotton absorbent pad, 90% isopropyl alcohol spray bottle, MilliQ water basin with chip slot and handle, MilliQ hydration box, clean chip storage box. Carefully take out each chip in the assay cassette, one at a time, and place it on the chip slot.

[0238] 2) Chips are dried and packed in boxes.

[0239] 9. Assemble the chip in the imaging cassette.

[0240] 10. Fluorescence Imaging

[0241] The Imaging System uses a robotic arm to transfer the imaging cassette to the barcode scanning position. After the imaging cassette and chip barcode scanning is completed, the imaging cassette is transferred to Image Xpress for imaging. The system controls the experimenter through the Venus software to guide the experimenter to perform imaging operations, and the operator needs to set the exposure time and the number of imaging cassettes. When imaging begins, the operator can continue to add imaging cassettes that need to be imaged.

[0242] The imaging process generates high-resolution TIFF images for each array using a Molecular Devices Image Xpress 4 imager. Gridding Software extracts feature intensity data from the TIFF image files.

[0243] When the imaging cassette is assembled, the cassette is transferred to the cassette hotel. Next, the automated imaging process will begin.

[0244] 4. Antibodies mixed with negative serum for detection under closed conditions

[0245] Under blocking conditions, antibodies were added to negative serum at 50 μg / ml and 500 μg / ml, respectively, and then the serum was diluted 1000 times for peptide chip detection (in actual operation, the serum can be diluted first and then the antibody is added according to the final concentration on the machine. The final concentration on the machine is the concentration of the antibody in the system during the peptide chip detection reaction. The specific operation takes into account the consumption of serum and samples. The serum and antibody samples can be diluted separately first to achieve a 1000-fold dilution of the background serum in the reaction system on the machine, and the antibody concentration is 50 ng / ml and 500 ng / ml.)

[0246] The secondary antibody for antibody detection is selected according to the source species information;

[0247] Antibodies from the same species should be placed on the same chip as much as possible, and a negative serum diluted 1:1000 should be added to each chip as a control with an antibody concentration of 0;

[0248] The control secondary antibody is selected according to the species of the antibody detected by the same detection chip. If there is more than one source of antibody species on the same detection chip, corresponding controls should be added.

[0249] Detection Operation

[0250] 1. Sample dilution:

[0251] a) Dilution of total IgG control

[0252] Use a 1.5 ml centrifuge tube to dilute IgG, take 16 μL of IgG and add it to 784 μL of sample diluent to form a 50-fold diluted IgG, and shake to mix.

[0253] b) Serum dilution

[0254] Use a 50 ml centrifuge tube, take 60 μL of serum sample and add it to 29940 μL of sample diluent to form a 500-fold diluted serum, and shake to mix.

[0255] c) Antibodies are mixed into serum for dilution

[0256] Use 500-fold diluted serum to dilute the antibody to 1000 ng / ml and 100 ng / ml respectively. The dilution steps are as follows:

[0257] 1) Take 2 μL of 100 μg / ml antibody and add it to 18 μL of sample diluent to form a diluted antibody with a concentration of 10 μg / ml (gradient 1), seal the membrane, shake and mix, and centrifuge (4000 rpm for 1 min);

[0258] 2) Take 10 μL of the antibody in gradient 1 and add it to 90 μL of 500-fold serum to form a diluted antibody with a concentration of 1000 ng / ml (gradient 2), seal the membrane, shake and mix, and centrifuge (4000 rpm for 1 min);

[0259] 3) Take 10 μL of gradient 2 and add it to 90 μL of 500-fold serum to form a diluted antibody with a concentration of 100 ng / ml (gradient 3), seal the membrane, shake and mix, and centrifuge (4000 rpm for 1 min).

[0260] d) Preparation of blocking solution

[0261] Take 2 ml of 1% Casein and add it to 18 ml of sample diluent, mix well and set aside.

[0262] 2. Typesetting and sample adding

[0263] 1) Sealing of the chip

[0264] Use a dispenser to dispense 45 μL of the prepared blocking solution into the wells of the Cassette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate in an incubator at 37°C for 1 hour.

[0265] 2) Blocking and loading of Cassette (chip)

[0266] According to the sample arrangement design, 45 μL of 1000 ng / ml diluted antibody and 100 ng / ml diluted antibody diluted with 500 times serum were transferred to the corresponding wells of Cassette using a dispenser.

[0267] 3. First incubation

[0268] Seal the Cassette, place it on the incubation module of the automated instrument, and incubate for 1 hour (for specific operations, refer to the first incubation under non-blocking conditions).

[0269] 4. First wash

[0270] Tear off the membrane of Cassette, place in automatic plate washer, and wash the plate (for specific operation, refer to the first plate washing under non-blocking conditions).

[0271] 5. Add secondary antibody

[0272] Under light-proof conditions (turn off the lights), use a 15ml centrifuge tube to dilute the human secondary antibody 1500 times (4μL of secondary antibody added to 6ml of secondary antibody diluent), dilute the mouse secondary antibody 3000 times (2μL of secondary antibody added to 6ml of secondary antibody diluent), and use a 50ml centrifuge tube to dilute the rabbit secondary antibody 1500 times (9μL of secondary antibody added to 13500μL of secondary antibody diluent). Then, according to the well position information in the layout table, use a dispenser to take 40μL and add it to the corresponding wells of the corresponding Cassette.

[0273] 3. Second incubation

[0274] Seal the cassette and place it on a thermomixer for incubation for 60 min. Oscillate the cassette for 15 s every 6 min.

[0275] 4. Second plate washing

[0276] Stop shaking, tear off the membrane and put it into the plate washer, make sure the waste liquid bottle of the plate washer has enough capacity, and use an automatic plate washer to wash the plate ( Microplate magnetic washer).

[0277] 5. Imaging and scanning (For specific operations, please refer to the chip disassembly under non-sealed conditions, chip assembly in the imaging cassette, and fluorescence imaging)

[0278] 5. Data Analysis

[0279] According to the analysis ideas and specific steps of eliminating interference conditions step by step, the specific binding peptides of eight antibodies were obtained. See the table below:

[0280] Table 3:

[0281] Antibody Code Number of Lv4 peptides RNPC 55 PCNA 30 CENPB 65 gp210 57 LC-1 31 HIB 201 SARS-Cov-E 80 Pf 508 GAD 339

[0282] Table 4:

[0283]

[0284]

[0285]

[0286] B. Mixed antibody detection

[0287] Hybrid Antibody Test Design:

[0288] Table 5:

[0289]

[0290]

[0291] In addition, add one well of total IgG quality control product to each detection chip, and perform the same operation for blocking / non-blocking.

[0292] a. In the absence of blocking solution

[0293] Each antibody component of the mixed antibody is diluted with the same sample diluent to prepare a mixed antibody sample for peptide chip detection.

[0294] The final concentration of each antibody component in the sample was 50 ng / ml.

[0295] Select anti-rabbit secondary antibodies according to the species information of each antibody source. Add pure sample diluent to each detection chip as a control with an antibody concentration of 0.

[0296] b. In the presence of blocking fluid

[0297] Each antibody component of the mixed antibody is diluted with the same sample diluent to prepare a mixed antibody sample for peptide chip detection.

[0298] The final concentration of each antibody component in the sample was 50 ng / ml.

[0299] Select anti-rabbit secondary antibodies according to the species information of each antibody source. Add pure sample diluent to each detection chip as a control with an antibody concentration of 0.

[0300] c. In the presence of blocking fluid

[0301] Each antibody component of the mixed antibody was added to the same negative serum sample at 50 μg / ml, and then the serum was diluted 1000 times to prepare a mixed antibody sample with serum background for peptide chip detection.

[0302] Select the 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.

[0303] C. Antibody-specific binding peptides for analysis of antibody components in mixed antibodies

[0304] Heat map of mixed antibody detection is shown in Figure 2 .exist Figure 2In the figure, all antibody Lv4 signal peptides of mixed antibodies 1-4 and control antibodies 5-8 are used as heat maps. Each row in the figure represents a peptide, and each column represents a test antibody sample. The first 8 columns represent single antibodies 1 to 8 respectively; the last three columns are mixed antibody samples, which are antibody 1, 2 mixed samples, antibody 1, 2, 3 mixed samples, and antibody 1, 2, 3, 4 mixed samples. The brightness of each band is the relative signal intensity of a single peptide in a single sample. As shown in the figure, antibodies 1 to 8 have their own positive peptides, and the signal peptide from one antibody only shows a high signal level in the antibody. The specific peptide signals of each antibody component exist in the mixed sample at the same time. And the detection signal level at the same concentration is equivalent to the detection signal level of the antibody alone, and no abnormal signal is generated due to antibody mixing.

[0305] The above results show that after gradually eliminating interference (including the removal of cross-peptides between multiple antibodies under different conditions, and the intersection of peptide sets detected by the same antibody under different conditions), the antibodies and antibody combinations have the advantage of high specificity.

[0306] Example 2: High-confidence specific binding peptides of antibodies are used to identify the presence of specific antibodies in clinical samples

[0307] A. Antibody peptide chip detection and analysis:

[0308] Screening of GAD antibody-specific binding polypeptides: This was performed together with the polypeptide chip detection of other single antibodies in Example 1. The main process is as follows:

[0309] (1) Peptide chip detection

[0310] a. In the absence of blocking solution

[0311] The antibody was diluted with sample diluent and then tested on a peptide chip. The final concentration of the antibody was 50 ng / ml and 500 ng / ml.

[0312] Select anti-rabbit secondary antibody according to the species information of the antibody source. Add pure sample diluent to the detection chip as a control with an antibody concentration of 0.

[0313] b. In the presence of blocking fluid

[0314] The antibody was diluted with sample diluent and then tested on peptide chip. The final concentration of the antibody was 50ng / ml and 500ng / ml.

[0315] Select anti-rabbit secondary antibody according to the species information of the antibody source. Add pure sample diluent to the detection chip as a control with an antibody concentration of 0.

[0316] c. In the presence of blocking fluid

[0317] The antibody to be tested was added to the negative serum sample at 50 μg / ml, and the serum was diluted 1000 times to prepare the antibody sample with serum background for peptide chip detection.

[0318] Select the anti-rabbit secondary antibody according to the species information of the antibody source. Add a well of negative serum diluted 1:1000 to the detection chip as a control with an antibody concentration of 0.

[0319] d. Scanning imaging and data analysis (For specific operations, please refer to the chip disassembly under non-sealed conditions, chip assembly in the imaging cassette, and fluorescence imaging)

[0320] After scanning and imaging, data processing is performed, and analysis is performed according to the analysis ideas and specific steps of eliminating interference conditions step by step to obtain the target antibody-specific binding peptides (used to test the following confirmed clinical serum samples to verify the detection effect of the peptides obtained here).

[0321] B. Clinical peptide chip detection:

[0322] Sample collection: Serum samples from clinically diagnosed type 1 diabetes patients and healthy controls (see the table below) were tested for the presence of GAD antibodies and IA2A antibodies by radioimmunoassay.

[0323] Table 6:

[0324]

[0325] Peptide chip detection: Clinical serum sample detection under closed conditions

[0326] Under closed conditions, each serum sample was diluted with sample diluent and then tested on a peptide chip. The overall sample dilution factor was 1000 times.

[0327] Add one well of pure sample dilution to each detection chip as a control;

[0328] Detection Operation

[0329] 1. Sample dilution:

[0330] a) Dilution of total IgG control

[0331] Use a 1.5 ml centrifuge tube to dilute IgG, take 16 μL of IgG and add it to 784 μL of sample diluent to form a 50-fold diluted IgG, and shake to mix.

[0332] b) Sample dilution

[0333] Dilute the serum sample 500 times using sample diluent

[0334] 1) Take 2 μL of serum sample and add it to 48 μL of sample diluent to form a diluted sample with a dilution factor of 25 (gradient 1), seal the film, shake and mix, and centrifuge (4000 rpm 1 min);

[0335] 2) Take 5 μL of sample dilution solution of gradient 1 and add it to 95 μL of sample dilution solution to form a diluted sample with a dilution degree of 500 (gradient 2), seal the film, shake and mix, and centrifuge (4000 rpm for 1 min);

[0336] c) Preparation of blocking solution

[0337] Take 2 ml of 1% Casein and add it to 18 ml of sample diluent, mix well and set aside.

[0338] 2. Typesetting and sample adding

[0339] 2) Sealing of the chip

[0340] Use a dispenser to dispense 45 μL of the prepared blocking solution into the wells of the Cassette, seal the membrane, mix at 600 rpm for 20 seconds, and then incubate in an incubator at 37°C for 1 hour.

[0341] 3) Blocking and loading of Cassette (chip)

[0342] According to the sample arrangement design, 45 μL of the 500-fold diluted sample was transferred to the corresponding well of the Cassette using a pipette.

[0343] 3. First incubation

[0344] Seal the Cassette, place it on the incubation module of the automated instrument, and incubate for 1 hour (for specific operations, refer to the first incubation under non-blocking conditions).

[0345] 4. First wash

[0346] Tear off the membrane of Cassette, place in automatic plate washer, and wash the plate (for specific operation, refer to the first plate washing under non-blocking conditions).

[0347] 5. Add secondary antibody

[0348] In a light-proof condition (turn off the lights), use a 15 ml centrifuge tube to dilute the human secondary antibody 1500 times (4 μL of secondary antibody is added to 6 ml of secondary antibody diluent), and use a dispenser to take 40 μL and add them to the corresponding wells of the Cassette.

[0349] 6. Second incubation

[0350] Seal the cassette and place it on a thermomixer for incubation for 60 min. Oscillate the cassette for 15 s every 6 min.

[0351] 7. Second wash

[0352] Stop shaking, tear off the membrane and put it into the plate washer, make sure the waste liquid bottle of the plate washer has enough capacity, and use an automatic plate washer to wash the plate ( Microplate magnetic washer).

[0353] 8. Imaging and scanning (For specific operations, please refer to the chip disassembly under non-sealed conditions, chip assembly in the imaging cassette, and fluorescence imaging)

[0354] 9. Data processing

[0355] C.GAD specific binding peptide collection is used to identify whether GAD antibodies are positive in clinical samples:

[0356] The GAD antibody (rabbit polyclonal antibody) specific binding polypeptide set found in step A of this embodiment was used to train the test results of serum samples of patients with positive GAD test antibodies in the diabetes cohort mentioned in this embodiment and the test results of serum samples of healthy people (the test results here refer to the results after the aforementioned data processing flow of converting from FG to LFG and then processing to NLFG), and the ridge regression model was used for modeling (parameter alpha = 1000). The obtained model was tested in the test results of serum samples of patients with multiple antibody tests including GAD positive (and not only GAD positive) and the test results of serum samples of another batch of healthy people.

[0357] In this example, the peptide segments of Lv1 to Lv4 were modeled according to the above ideas. The prediction results of the model established by using the peptide segment set of Lv1 are as follows: Figure 3 and Figure 4 As shown. The prediction results of the peptide sets from Lv2 to Lv4 after modeling are similar to those of Lv1 (not shown). When the peptide set of Lv4 is used for modeling prediction, the processing speed is faster because the number of peptides is relatively smaller. In addition, for the peptide set of Lv4, the model prediction method is not used to detect the presence of the target antibody. When a detection method similar to conventional ELISA is used, since the number of peptides in the peptide set of Lv4 is small and the specificity is higher, the detection result of the sample to be tested can be determined by direct comparison with the negative sample.

[0358] It should be noted that the test results of serum samples from the population cohort with positive GAD antibodies alone (16 cases) and the test results of serum samples from the healthy population (24 cases) were used as the training set (40 cases in total), and then the test results of the remaining samples were used as the test set (including the test results of 13 samples from the healthy population, and the test results of 24 serum samples from diabetic patients who were positive in GAD tests and at least one of the IA2A and ZnT8 tests, a total of 37 cases) to verify the predictive accuracy of the constructed model (in the case of samples containing more than one GAD antibody positive).

[0359] Figure 3 The statistical results are shown (accuracy 35 / 37=95%). Figure 4 The performance of the GAD antibody specific binding peptide set determined by ROC analysis is shown. All GAD antibody specific binding peptide sets were selected for comprehensive analysis, and the accuracy of distinguishing GAD antibody positive and GAD antibody negative (non-diabetic) in the validation set (i.e., test set) was 97%, and the AUC was 0.98. This result shows that the specific binding peptides screened by the antibody peptide chip detection analysis are of practical significance in the application of detecting whether there are specific antibodies in the clinical cohort.

[0360] From the above examples, it can be seen that the method for screening polypeptides that specifically bind to antibodies based on polypeptide chips provided in this application uses the unique advantages of high-density random polypeptide chip technology platforms (such as HealthTell's V13 chip containing 130,000 polypeptides) in antigen epitope analysis---compatible with the binding of linear epitopes and nonlinear epitope antibodies, and uses experimental means and analytical ideas to eliminate possible background interference step by step, to obtain highly sensitive and highly specific polypeptide sets and combinations, and the corresponding peptides can be used for antibody detection. During detection, the antibody-specific binding polypeptide binds to the antibody in the blood, and after incubation with the fluorescently labeled secondary antibody, the fluorescence value is detected in the microplate reader to fully and unbiasedly reflect the antibody spectrum in the blood.

[0361] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. Sequence Listing <110> Zhuhai Carbon Cloud Intelligent Technology Co., Ltd. <120> Method for screening polypeptides specifically bound by antibodies based on polypeptide chips and application of the polypeptides screened <130> PN153005SZTY <160> 85 <170> SIPOSequenceListing 1.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(10) <223> Antibody specifically binds 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> Synthetic peptide that specifically binds to the antibody against human SNRPC / U1C <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> Synthetic peptide that specifically binds to the antibody against human SNRPC / U1C <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> Synthetic peptide that specifically binds to the antibody against human SNRPC / U1C <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> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <400> 5 Ala Ala Lys Arg Asp Asp Ala Leu Asp 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <400> 6 Ala Ala Lys Arg Asp Asp Ala Leu Asp 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(9) <223> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <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> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <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> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <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> Synthetic peptide that specifically binds to antibodies against human SNRPC / U1C <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> Synthetic sequence, antibody specific binding to 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 peptide that specifically binds 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 peptide that specifically binds 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 peptide that specifically binds 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 peptide that specifically binds 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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> Synthetic peptide that specifically binds to antibodies 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to the antibody against 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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <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 peptide that specifically binds to human GAD antibody <400> 85 Phe His Glu Pro Asn Gln Leu Lys Arg Asp 1 5 10

Claims

1. A method for screening polypeptides specifically bound by antibodies based on a polypeptide chip, characterized in that: The screening method comprises: Under blocking and non-blocking conditions, the peptide chip is used to perform a first screening of specific binding peptides on the first antibody positive sample and the first antibody negative sample, respectively, to obtain a first seed peptide library under blocking conditions and a first seed peptide library under non-blocking conditions; Taking the intersection of the polypeptides in the first seed peptide library under the closed condition and the first seed peptide library under the non-closed condition to obtain a second seed peptide library; Under closed conditions, using the peptide chip to perform a second screening of specific binding peptides on the second antibody positive sample and the second antibody negative sample to obtain a third seed peptide library; Taking the intersection of the third seed peptide library and the second seed peptide library to obtain a fourth seed peptide library; wherein the first antibody-positive sample is one or more sample dilutions containing a single antibody, the second antibody-positive sample is one or more serum samples containing a single antibody, the first antibody-negative sample is a sample dilution not containing the single antibody, and the second antibody-negative sample is a serum sample not containing the single antibody; or The first antibody-positive sample is one or more groups of sample dilutions containing mixed antibodies, the second antibody-positive sample is one or more groups of serum samples containing mixed antibodies, the first antibody-negative sample is a sample dilution not containing the mixed antibodies, and the second antibody-negative sample is a serum sample not containing the mixed antibodies.

2. The screening method according to claim 1, characterized in that The first antibody-positive sample is a plurality of sample dilutions containing a single antibody, the second antibody-positive sample is a plurality of serum samples containing a single antibody, the first antibody-negative sample is a sample dilution not containing the single antibody, and the second antibody-negative sample is a serum sample not containing the single antibody. The screening method further includes excluding polypeptides having intersections between the plurality of different single antibodies.

3. The screening method according to claim 2, characterized in that Excluding polypeptides that overlap between multiple different single antibodies at any one or more of the following times: a. After the first screening is performed under the non-blocking conditions; b. After the first screening is performed under the closed conditions; c. After performing the second screening.

4. The screening method according to claim 1, characterized in that The first antibody-positive samples are multiple groups of sample dilutions containing mixed antibodies, the second antibody-positive samples are multiple groups of serum samples containing mixed antibodies, the first antibody-negative samples are sample dilutions not containing the mixed antibodies, the second antibody-negative samples are serum samples not containing the mixed antibodies, and the antibody types contained in any two groups of sample dilutions containing mixed antibodies and / or any two groups of serum samples containing mixed antibodies do not overlap, then the screening method further includes excluding polypeptides that have intersections between multiple groups of different mixed antibodies.

5. The screening method according to claim 4, characterized in that Excluding polypeptides that overlap between multiple groups of different mixed antibodies at any one or more of the following times: a. After the first screening is performed under the non-blocking conditions; b. After the first screening is performed under the closed conditions; c. After performing the second screening.

6. The screening method according to any one of claims 1 to 5, characterized in that Under the non-blocking condition and / or under the non-blocking condition, two or more first antibody-positive samples with different antibody concentrations are used to perform the first screening.

7. The screening method according to claim 6, characterized in that The first antibody-positive samples with two or more different antibody concentrations are used for the first screening under the non-blocking condition, and two first antibody-positive samples with two different antibody concentrations are determined for the first screening under the blocking condition.

8. The screening method according to claim 6, characterized in that The first antibody-positive sample is a plurality of sample dilutions containing a single antibody or a plurality of groups of sample dilutions containing mixed antibodies. The concentration of the single antibody contained in the first antibody-positive sample or the concentration of each single antibody in the mixed antibodies is 10 ng / mL to 1 μg / mL.

9. The screening method according to claim 8, characterized in that The concentration of the single antibody contained in the first antibody-positive sample or the concentration of each single antibody in the mixed antibody is 50 ng / mL-500 ng / mL.

10. The screening method according to claim 6, characterized in that The second screening is performed using two or more samples positive for the second antibody at different antibody concentrations.

11. The screening method according to claim 10, characterized in that The concentration of the single antibody contained in the second antibody-positive sample or the concentration of each single antibody in the mixed antibody is 10 ng / mL-1 μg / mL.

12. The screening method according to claim 11, characterized in that The concentration of the single antibody contained in the second antibody-positive sample or the concentration of each single antibody in the mixed antibody is 50 ng / mL-500 ng / mL.

13. The screening method according to any one of claims 1 to 5, characterized in that The sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of the D-mannitol in the PBST buffer solution is 0.5% to 1%.

14. The screening method according to claim 13, characterized in that The term "under blocking conditions" refers to blocking the polypeptide chip with a blocking solution, and using the blocked polypeptide chip for screening.

15. The screening method according to claim 14, characterized in that 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% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v, and the pH of the blocking solution is 7.2-7.

6.

16. The screening method according to claim 15, characterized in that The pH of the blocking solution is 7.38-7.

42.

17. The screening method according to claim 16, characterized in that The blocking solution comprises 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.

18. The screening method according to claim 13, characterized in that The antibody is a monoclonal antibody or a polyclonal antibody.

19. The screening method according to claim 18, characterized in that Each single antibody in the plurality of single antibodies or the mixed antibodies is a polyclonal antibody.

20. The screening method according to claim 18, characterized in that Each single antibody in the plurality of single antibodies or the mixed antibodies may be derived from the same or different species.

21. The screening method according to claim 20, characterized in that The species origin is rabbit, goat, rat, mouse, guinea pig or human.

22. The screening method according to claim 18, characterized in that The plurality of single antibodies or the plurality of mixed antibodies are selected from the following antibodies: antibodies against human SNRPC / U1C, antibodies against human PCNA, antibodies against human CENPB, antibodies against human NUP210 / gp210, antibodies against human FTCD / 58K Golgi protein, antibodies against HIB, antibodies against SARS-CoV E protein, antibodies against Plasmodium and antibodies against GAD.

23. The screening method according to claim 18, characterized in that The first screening and the second screening using the polypeptide chip both include sequential incubation of antibody samples and incubation of secondary antibodies, wherein the secondary antibody is fluorescently labeled.

24. The screening method according to claim 23, characterized in that The species origin of the secondary antibody is selected according to the species origin of each of the single antibodies or the single antibodies in the mixed antibodies in the first screening and the second screening steps.

25. A method for screening polypeptides specifically bound by antibodies based on a polypeptide chip, characterized in that: The screening method comprises: Under non-blocking conditions, a polypeptide chip is used to perform a first screening of specific binding polypeptides on first positive samples of multiple single antibodies or multiple groups of mixed antibodies and corresponding first negative samples to obtain a first anti-interference peptide library corresponding to each of the single antibodies or each group of mixed antibodies; wherein the first positive sample is a sample diluent containing a single antibody or a sample diluent containing the mixed antibody, and the first negative sample is a sample diluent not containing the single antibody or the mixed antibody; The polypeptides with intersections in the first anti-interference peptide library corresponding to each of the multiple single antibodies or multiple groups of mixed antibodies are removed to obtain a second anti-interference peptide library corresponding to each of the single antibodies or multiple groups of mixed antibodies.

26. The screening method according to claim 25, characterized in that The screening method further comprises: Under closed conditions, using a peptide chip to perform a second screening of specific binding peptides on the first positive sample and the corresponding first negative sample to obtain a third anti-interference peptide library; The intersection of the polypeptides in the second anti-interference peptide library and the third anti-interference peptide library is taken to obtain a fourth anti-interference peptide library.

27. The screening method according to claim 26, characterized in that The screening method further comprises: Under closed conditions, a peptide chip is used to perform a third screening of specific binding peptides on the second positive sample and the corresponding second negative sample to obtain a fifth anti-interference peptide library; wherein the second positive sample is a serum sample containing a single antibody or a serum sample containing the mixed antibody, and the second negative sample is a serum sample not containing the single antibody or the mixed antibody; The intersection of the fifth anti-interference peptide library and the fourth anti-interference peptide library is taken to obtain a sixth anti-interference peptide library corresponding to each of the single antibodies or each group of the mixed antibodies.

28. The screening method according to claim 27, characterized in that After obtaining the third anti-interference peptide library and / or the fifth anti-interference peptide library corresponding to each single antibody or each group of mixed antibodies, the screening method further includes: removing polypeptides in which there are intersections among multiple single antibodies or multiple groups of mixed antibodies in each of the third anti-interference peptide libraries and / or the fifth anti-interference peptide libraries.

29. The screening method according to claim 27 or 28, characterized in that The first screening is performed using two or more different antibody concentrations for each of the plurality of single antibodies or the mixed antibodies.

30. The screening method according to claim 29, characterized in that The concentration of the single antibody contained in the first positive sample is 10 ng / mL-1 μg / mL.

31. The screening method according to claim 30, characterized in that The concentration of the single antibody contained in the first positive sample is 50 ng / mL-500 ng / mL.

32. The screening method according to claim 29, characterized in that The sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of the D-mannitol in the PBST buffer solution is 0.5% to 1%.

33. The screening method according to claim 29, characterized in that Under the non-blocking condition, the first positive samples with two or more different antibody concentrations are used for the first screening, thereby determining the first positive samples with two different antibody concentrations, and then the second screening is performed under the blocking condition.

34. The screening method according to claim 29, characterized in that The third screening is performed on the second positive samples with two or more different antibody concentrations for each single antibody.

35. The screening method according to claim 29, characterized in that The concentration of the single antibody contained in the second positive sample is 10 ng / mL-1 μg / mL.

36. The screening method according to claim 35, characterized in that The concentration of the single antibody contained in the second positive sample is 50 ng / mL-500 ng / mL.

37. The screening method according to claim 29, characterized in that The "under blocking conditions" refers to blocking the polypeptide chip with a blocking solution, and using the blocked polypeptide chip to perform the second screening and the third screening respectively.

38. The screening method according to claim 29, characterized in that 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% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v, and the pH of the blocking solution is 7.2-7.

6.

39. The screening method according to claim 38, characterized in that The pH of the blocking solution is 7.38-7.

42.

40. The screening method according to claim 39, characterized in that The blocking solution comprises 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.

41. The screening method according to claim 29, characterized in that The single antibody is a monoclonal antibody or a polyclonal antibody.

42. The screening method according to claim 29, characterized in that Each single antibody in the plurality of single antibodies or the mixed antibodies is a polyclonal antibody.

43. The screening method according to claim 29, characterized in that Each single antibody in the plurality of single antibodies or the mixed antibodies may be derived from the same or different species.

44. The screening method according to claim 43, characterized in that The species origin is rabbit, goat, rat, mouse, guinea pig or human.

45. The screening method according to claim 29, characterized in that The plurality of single antibodies or mixed antibodies are selected from a plurality of the following antibodies: antibodies against human SNRPC / U1C, antibodies against human PCNA, antibodies against human CENPB, antibodies against human NUP210 / gp210, antibodies against human FTCD / 58K Golgi protein, antibodies against HIB, antibodies against SARS-CoV E protein, antibodies against Plasmodium and antibodies against GAD.

46. ​​A method for detecting a target antibody in a sample to be tested, characterized in that: The detection method comprises: Using the target polypeptide specifically bound by the target antibody, the sample to be tested and the negative control of the sample to be tested are tested to obtain respective corresponding test results; If there is a significant difference between the test results of the sample to be tested and the negative control, it indicates that the sample to be tested contains the target antibody; The target polypeptide is a polypeptide that specifically binds to the target antibody and is screened by the screening method described in any one of claims 1 to 24, or the target polypeptide is a polypeptide that specifically binds to the target antibody and is screened by the screening method described in any one of claims 25 to 28.

47. The detection method according to claim 46, characterized in that Under the condition of blocking the polypeptide chip, using the polypeptide chip containing the target polypeptide, the sample to be tested and the negative control of the sample to be tested are tested to obtain the detection results of the target polypeptide corresponding to each of them; If there is a significant difference between the detection results of the target polypeptide in the test sample and the negative control, it indicates that the test sample contains the target antibody.

48. The detection method according to claim 47, characterized in that The sample to be tested is a serum sample diluted with a sample diluent, the negative control is the sample diluent, and the target polypeptide is any multiple polypeptides in the polypeptide set that specifically binds to the target antibody screened by the screening method according to any one of claims 1 to 24.

49. The detection method according to claim 47, characterized in that The target antibody is selected from any one or more of the following: antibodies against human SNRPC / U1C, antibodies against human PCNA, antibodies against human CENPB, antibodies against human NUP210 / gp210, antibodies against human FTCD / 58K Golgi protein, antibodies against HIB, antibodies against SARS-CoV E protein, antibodies against Plasmodium and antibodies against GAD.

50. The detection method according to claim 47, characterized in that The sample diluent is a PBST buffer solution containing D-mannitol, wherein the mass volume content of the D-mannitol in the PBST buffer solution is 0.5% to 1%.

51. The detection method according to claim 47, characterized in that Before testing the sample to be tested and the negative control of the sample to be tested, the detection method further comprises: blocking the polypeptide chip with a blocking solution.

52. The detection method according to claim 47, characterized in that: 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% Proclin950 v / v, 0.5%-1% D-mannitol w / v and 0.1%-1% casein w / v, and the pH of the blocking solution is 7.2-7.

6.

53. The detection method according to claim 52, characterized in that: The pH of the blocking solution is 7.38-7.

42.

54. The detection method according to claim 52, characterized in that: The blocking solution comprises 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.

55. The detection method according to claim 47, characterized in that Under the condition of blocking the polypeptide chip, using the polypeptide chip containing the target polypeptide, detecting the sample to be tested and the negative control of the sample to be tested includes: Incubating the sample diluent and the serum sample diluted by the sample diluent with the blocked polypeptide chip to obtain a first incubation product; Co-incubating the first incubation product with a fluorescently labeled secondary antibody to obtain a second incubation product; Performing fluorescence scanning on the second incubation product to obtain a fluorescence signal of the sample diluent and a fluorescence signal of the serum sample; Detect whether there is a significant difference between the fluorescence signal of the target polypeptide in the sample diluent and the fluorescence signal of the serum sample. If there is a significant difference, it indicates that the target antibody exists in the serum sample.

56. The detection method according to claim 55, characterized in that The secondary antibody was from the same species as the serum sample.

57. The detection method according to claim 55, characterized in that A prediction model is constructed using the fluorescence signals of the target polypeptide detected in a target antibody-positive population and a target antibody-negative population using a plurality of clinical sample cohorts in which the presence or absence of the target antibody is known. The prediction model is used to predict whether there is a significant difference between the fluorescence signal of the target polypeptide in the sample diluent and the fluorescence signal of the serum sample, that is, the prediction model is used to output whether the serum sample is a target antibody-positive sample.

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